Review of Quarantine Disease Risks Related to

Review of Quarantine Disease Risks Related to Bovine Semen

A Report for the Australian Quarantine and Inspection Service

David B. Adams Animal and Plant Health Branch Bureau of Resource Sciences August 1995

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Table of Contents summary........................................................................................................................5 introduction.................................................................................................................12 Animal disease lists of the OIE...........................................................................................................13 Standards for preventing the transmission of infectious disease by bovine semen............................14 OIE.................................................................................................................................................14 Australia.........................................................................................................................................15

Treatment of bovine semen to reduce risk.................................................................15 Antimicrobial agents...........................................................................................................................15 Other treatments .................................................................................................................................16

preliminary considerations in the transmission of disease agents by semen...........17 Biological factors................................................................................................................................17

the assessment of risk.................................................................................................21 the risk of transmission via bovine semen of individual disease agents in oie list a23 Category 1 - Diseases where transmission via semen has been demonstrated...................................37 Foot and Mouth Disease................................................................................................................37 Bluetongue.....................................................................................................................................39 Category 2 - Diseases where causative organisms have been demonstrated in semen......................45 Rinderpest......................................................................................................................................46 Lumpy skin disease........................................................................................................................46 Category 3 - Diseases with a presumptive risk of transmission by bovine semen.............................47 Rift Valley Fever............................................................................................................................47 Peste de petits ruminants................................................................................................................49 Contagious bovine pleuropneumonia.............................................................................................49 Category 4 - Diseases with risk associated only with extrinsic contamination. ................................49 Vesicular stomatitis........................................................................................................................49 Category 5 - Diseases with no appreciable risk of transmission by bovine semen............................50

the risk of transmission via bovine semen of individual disease agents in oie list b51 diseases of Multiple species.............................................................................................51 Category 1 - Diseases where transmission via semen has been demonstrated...................................56 Leptospirosis..................................................................................................................................56 Category 2 - Diseases where causative organisms have been demonstrated in semen......................57 Paratuberculosis.............................................................................................................................57 Category 3 - Diseases with a presumptive risk of transmission by bovine semen.............................59 Aujeszky’s disease.........................................................................................................................59 Heartwater......................................................................................................................................60 Q fever............................................................................................................................................61 Rabies.............................................................................................................................................62 Category 4 - Diseases with risk associated only with extrinsic contamination..................................62 Anthrax ..........................................................................................................................................62 Category 5 - Diseases with no appreciable risk of transmission by bovine semen............................63 Screw-worm flies (Cochliomyia hominivorax and Chrysomyia bezziana) and echinococcosishydatidosis.....................................................................................................................................63

diseases of Cattle..............................................................................................................64 Category 1 - Diseases where transmission via semen has been demonstrated...................................74 Bovine brucellosis .........................................................................................................................75 Bovine genital campylobacteriosis................................................................................................76 Bovine tuberculosis........................................................................................................................77 Bovine virus diarrhoea/Mucosal disease........................................................................................78 Infectious bovine rhinotracheitis/infectious pustular vulvovaginitis.............................................80 Trichomoniasis...............................................................................................................................81

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Category 2 - Diseases where causative organisms have been demonstrated in semen......................82 Enzootic bovine leukosis...............................................................................................................82 Category 3 - Diseases with a presumptive risk of transmission by bovine semen.............................83 Bovine malignant catarrh...............................................................................................................84 Bovine spongiform encephalopathy...............................................................................................84 Haemorrhagic septicaemia.............................................................................................................86 Theileriosis.....................................................................................................................................86 Trypanosomiasis............................................................................................................................88 Category 4 - Diseases with risk associated only with extrinsic contamination..................................89 Anaplasmosis.................................................................................................................................89 Babesiosis.......................................................................................................................................90 Dermatophilosis.............................................................................................................................91 Category 5 - Diseases with no appreciable risk of transmission by bovine semen............................91 Cysticercosis..................................................................................................................................91

other disease agents with material risk of transmission via bovine semen..............93 Bacteria ..............................................................................................................................................93 Haemophilus somnus.....................................................................................................................93 Mycoplasma spp, Ureoplasma spp and other mollicutes...............................................................93 Rickettsia............................................................................................................................................94 Chlamydiosis..................................................................................................................................94 Lyme borreliosis............................................................................................................................94 Viruses................................................................................................................................................95 Akabane disease.............................................................................................................................95 Ephemeral fever.............................................................................................................................95 Herpes viruses not specified in OIE List B....................................................................................95 Other viruses..................................................................................................................................96

conclusions..................................................................................................................97 The evolving disease picture .........................................................................................................97 Categories of risk...........................................................................................................................97 The role of Approved Artificial Breeding Centres........................................................................98 Regional, herd and donor freedom from disease...........................................................................98 Diagnosis of disease and infection in semen donor bulls..............................................................99 Diagnostic tests for disease agents in semen.................................................................................99 Non-sperm cells in semen..............................................................................................................99 Recommendations.............................................................................................................................100

references..................................................................................................................102

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Table of Boxes

Table of Tables

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SUMMARY 1.

The present report reviews the information available in the scientific literature on the possible transmission in bovine semen of the causative agents of diseases in Lists A and B of the Office International des Épizooties. Its purpose is to aid in the assessment and containment of disease risks associated with the import and export of bovine semen. In all, the risk of transmitting disease agents in semen is higher than for embryo transfer. However, the disease risk from imported semen may not be less than that from imported animals in all circumstances.

2.

Various standards have been adopted around the world to prevent the transmission of disease agents in bovine semen. Some are voluntary whereas others are imposed officially. The framework for international trade in bovine semen is provided by the OIE and is based upon three grades of freedom from a particular disease. These are regional freedom, herd freedom and freedom in an individual bull and its semen. OIE lists of communicable diseases and accredited artificial breeding centres are important parts of this framework. List A diseases have the potential for very serious and rapid spread irrespective of national borders, serious socio-economic or public health consequences and major importance in the international trade in livestock and livestock products. List B diseases have socio-economic or public health importance within countries and significance in the international trade in livestock and livestock products. List A and B diseases must be reported under the OIE International Zoo-Sanitary Code. List C diseases, not covered in the present review, do not require reporting. They are classified as having important socio-economic and/or sanitary influence at the local level. Approved artificial insemination or breeding centres are a critical component in disease control in the international trade in bovine semen. The standards under which they should operate and their accreditation for the export of bovine semen are described in appendices to the OIE International Health Code. These include the requirements for admitting animals to artificial breeding centres and the routine testing for disease thereafter. The hygienic collection and processing of semen is also described. The management and approval of artificial breeding centres in Australia is covered in two official documents; "Minimum Health Standards for Stock Standing at Licensed or Approved Artificial Breeding Centres in Australia" (3rd Edition, 1988) and "Code of Practice for Australian Livestock Artificial Breeding Centres" (1988).

3.

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Treatment of bovine semen with antimicrobial agents to control microbial contaminants is mentioned in the Code of Practice for Australian Livestock Breeding Centres and is reviewed in the present report. A combination of lincomycin/spectinomycin, tylosin and gentamycin in whole semen and nonglycerolated whole milk or egg-based diluent is effective against Mycoplasmas and Ureoplasmas, Haemophilus somnus and Campylobacter fetus subsp. venerealis without damaging the viability and fertilising capacity

of spermatozoa. New agents such as photosensitive protoporphyrins and antibodies produced by monoclonal methods have been tested for their use in controlling disease agents in semen but have been found either to be ineffective or work on them has not progressed. In summary, antimicrobial agents may not be effective against every pathogen found in semen. They can supplement not replace other more decisive measures for controlling the risk of transmitting disease agents in bovine semen. 4.

A set of biological ideas is described in the review as part of the framework for assessing the disease risk presented by semen. These are:  Semen may be contaminated by microbes within the male reproductive tract, intrinsic contamination, or during the process of ejaculation and collection, extrinsic contamination.  A blood-testis barrier operates similar to the blood-brain barrier and intrinsic contamination may occur mainly through the epididymis, ductus deferens or accessory glands.  Semen may be intrinsically contaminated by disease agents either residing in the tissues of the male reproductive tract or present in blood, for example when viraemia occurs in viral diseases or bacteraemia occurs in bacterial disease.  Intrinsic contamination of semen may be facilitated by inflammatory processes in the male reproductive tract, for example in seminal vesiculitis and bulbourethritis.  Disease agents may pass into semen more readily if they are associated with migratory cells such as monocytes/macrophages or lymphocytes. Examples are the retrovirus of bovine leukaemia, and the bacteria, Brucella abortus and Mycobacterium bovis. Knowledge about the presence of nonsperm cells such as lymphocytes, monocytes, neutrophils etc. in semen is scarce but could aid in the general assessment of risk.  The disappearance of libido and mating capacity as a result of illness may curtail the collection of semen during the hazardous stages of many infectious diseases. However, some diseases are characterised by the carrier state, latent or inapparent infection or related phenomena. In such instances, the risk of contamination for semen may be persistent. Bovine pestivirus is a relevant example here.  It is not known whether different methods for the collection of semen produce different risk. Ejaculation produced by transrectal stimulation of the accessory glands of the male reproductive falls under suspicion since it may lead to the presence of blood cells in semen and microbial contamination from this source. The preparation of spermatozoa directly from testis, for example after euthanasia of valuable animals, must be regarded as having high risk for disease transmission because of the presence of non-sperm cells.  Artificial insemination bypasses the defence mechanisms against disease transmission that apply to natural mating.  Semen ejaculates are diluted up to 100-fold with buffer containing antibiotics before being cryopreserved. Dilution itself and the use of antibiotics may reduce the risk of disease transmission but cannot substitute for having disease-free semen in the first place.

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 Storage of semen at ultra-low temperatures is likely to favour the survival of most disease agents. 5.

The diseases in OIE Lists A and B are placed in five categories for present purposes. Category 1 contains diseases where transmission by artificial insemination has been established. Category 2 contains diseases where the causative agent has been found in semen but where transmission remains unclear. Category 3 contains diseases with a presumptive risk of transmission derived from the set of biological considerations outlined earlier. Category 4 contains diseases where contamination of semen is possible only during the stages of collection and processing. Category 5 contains diseases where the risk of contamination is virtually inconceivable.

6.

Separate sections of the review consider OIE list A diseases, list B diseases that affect multiple species and list B diseases that affect cattle. Diseases are analysed individually in each of these sections. For convenience, the results of these analyses are provided in tables at the start of each section. Each analysis commences with a brief profile of the particular disease and its causative agent, then deals with factors relevant to the risk of transmission by semen and ends with a summary of the risk and measures for its containment. The report goes beyond its strict terms of reference and considers some viral, bacterial and rickettsial diseases that are not in the OIE lists but which have been canvassed in the international arena for their risk potential.

6.

List A Diseases

Foot and mouth disease and bluetongue are the only List A diseases where transmission via semen has been observed (Category 1). The risk from foot and mouth disease can be contained by accepting semen only from where regional freedom from this disease exists. In some parts of the world, bovine semen is accepted on the basis of freedom from infection in donor bulls. The risk from bluetongue can be contained by ensuring that donor bulls in bluetongue-free areas are serologically tested for disease-freedom twice per year. In endemic areas, donor bulls may be seropositive or seronegative but must not have viraemia or active infection when semen is being collected. For this reason, bulls in endemic areas should be tested each week by virological and serological methods and semen should be withheld until freedom from infection has been confirmed. Rinderpest and lumpy skin disease are two List A diseases where causative agents have been observed in semen but transmission by artificial insemination is unclear (Category 2). Regional freedom from rinderpest should be a prerequisite for the acceptance of bovine semen. Rinderpest virus is said to be destroyed by glycerol which is present in some semen diluents. The period of contamination risk for semen may be greatest when donor bulls show clinical disease. Bovine semen cannot be accepted from places where lumpy skin disease occurs. The vaccination of donor bulls with vaccines prepared from sheep or goats strains of the causative agent, a capripox virus, would create an additional risk to be borne by sheep and goats, not cattle. 7

Rift Valley fever, peste de petits ruminants and contagious bovine pleuropneumonia (CBPP) are diseases with a presumptive risk of transmission in bovine semen (Category 3). Viraemia occurs in Rift Valley fever and virus can be found in milk, saliva and other secretions. The causative agent of CBPP can be found in urine. Peste de petits ruminants could be expected to behave like a related disease, rinderpest, and can be transmitted experimentally to calves. Regional freedom from these three diseases is a required for the acceptance of bovine semen. Vesicular stomatitis is the only list A disease classified as having a risk for semen associated only with the possibility of extrinsic contamination (Category 4). Semen should not be accepted from bulls with clinical vesicular stomatitis. Sheep and goat pox, African horse sickness, African swine fever, classical swine fever, swine vesicular disease, fowl plague and Newcastle disease do not include cattle in their species range. As a consequence, they are classified as diseases with no appreciable risk of transmission in bovine semen (Category 5). 7.

OIE List B diseases affecting multiple species of animals

Leptospirosis is the only list B disease affecting multiple species of animals where transmission by semen has been demonstrated (Category 1). Antibiotics in semen and diluent can protect against transmission of leptospirosis but semen should only be accepted from donors which are accredited as free from infection. The serological assays for leptospirosis require improvement. The causative organisms of paratuberculosis have been found in semen which places it in Category 2. However, experimental evidence is against transmission by artificial insemination. Nevertheless, herd freedom in artificial centres is required before semen can be accepted. Identification of organisms in faeces and the complement fixation test for specific antibody are adequate screening tests but the absorbed ELISA (enzyme linked immunosorbent assay) and gamma interferon assay may have increased sensitivity and specificity. Aujeszky's disease, heartwater, Q fever and rabies all have a presumptive risk of transmission in bovine semen (Category 3). Herd freedom is required for Aujeszky's disease before semen can be accepted. This requires that semen donor bulls have no direct or indirect contact with pigs, particularly respiratory aerosols from pigs. Serum antibody can be detected by serum immunodiffusion and ELISA tests. Semen from areas where heartwater is endemic should only be accepted from clinically healthy bulls without infection with Cowdria ruminantium. Semen cannot be accepted from bulls infected with Coxiella burnetii and serological testing of bulls before entry to artificial breeding centres and as a routine thereafter is required in areas where Q fever is found. Rabies has a slight presumptive risk of transmission in bovine semen. This risk would be covered by surveillance of semen donors in areas where rabies occurs. A with-holding period of semen of four weeks would cover the possibility of semen contamination during the immediate preclinical period of the disease. Anthrax may be a risk for extrinsic contamination of semen (Category 4) in places where this disease is endemic, "anthrax belts". This risk would be contained by the hygienic collection of semen. 8

The nature of echinococcosis/hydatidosis and screw-worm flies rule out possible intrinsic or extrinsic contamination of bovine semen. 8.

OIE List B diseases affecting cattle

Bovine brucellosis, bovine genital campylobacteriosis, bovine tuberculosis, bovine virus diarrhoea/mucosal disease, infectious bovine rhinotracheitis/infectious vulvovaginitis and trichomoniasis are proven risks for transmission in bovine semen (Category 1). Bovine brucellosis and bovine tuberculosis are particularly important since these diseases have been the subject of major eradication campaigns in Australia.. Herd freedom from infection from these two diseases should be required for the acceptance of semen. The same applies to Bovine virus diarrhoea/mucosal disease (bovine pestivirus)and infectious bovine rhinotracheitis/infectious vulvovaginitis (bovine herpesvirus-1) which are becoming increasingly important. Freedom from infection in donor bulls is required for bovine genital campylobacteriosis and trichomoniasis. This can be provided satisfactorily through herd freedom. The screening tests for all these diseases and their causative agents are described in the body of the report. The bovine leukaemia virus, the causative agent of enzootic bovine leukaemia has been demonstrated in semen (Category 2) but experimental results indicate no transmission by artificial insemination. Absolute protection against transmission, however, can be obtained by using seronegative bulls only and this should be a requirement for the acceptance of semen. Presumptive risk of transmission by semen (Category 3) applies to bovine malignant catarrh, bovine spongiform encephalopathy (BSE), haemorrhagic septicaemia, theileriosis and trypanosomiasis. Risk from bovine malignant catarrh would be contained by keeping semen donor bulls well separated from sheep and wildebeest. Although there is epidemiological evidence that BSE is not transmitted by semen, the presumptive risk status is suggested until the results of definitive transmission experiments have been completed. The crucial factor is the absence of exposure in semen donors to the causative agent of BSE. Accordingly, semen collected from donor bulls born since the ban on feeding ruminant derived protein to animals was introduced in great Britain in 1988 would be acceptable. Semen from bulls born before that time would also be acceptable if it could been demonstrated that they had never been fed ruminant derived protein. Donor freedom as determined by serial swabs of naso-pharyngeal secretions would be required for the acceptance of semen from areas where haemorrhagic septicaemia occurs. As for theileriosis, bulls should be confirmed free of infection before entry to artificial breeding centres and during the time they provide semen. The same applies to trypanosomiasis which can establish in regions without the tsetse fly vector. Babesiosis, anaplasmosis and dermatophilosis are diseases where risk comes from the possibility of extrinsic contamination during the collection of semen (Category 4). The source of risk for intraerythrocytic parasites like Babesia and Anaplasma spp is extravasated blood. For dermatophilosis the source is skin lesions. Semen donor bulls should be diagnosed as free of these three infections to remove any risk. Examination 9

of blood smears is required for Babesia and Anaplasma spp. Clinical inspection is required for dermatophilosis. The use of blood-derived vaccines for controlling babesiosis and anaplasmosis brings a risk of infection by bovine pestivirus, bovine herpesvirus-1 or bovine leukaemia virus and the possibility that these agents could contaminate semen. There is no appreciable risk of transmitting cysticercosis by bovine semen. 9.

Other diseases

A group of bacterial, viral and rickettsial diseases outside OIE lists A and B are considered in the report because their possible spread via bovine semen is likely to come under scrutiny on the international scene. The bacteria are Haemophilus somnus, Mycoplasma spp, and Ureoplasma spp. All these organisms are killed by the antibiotic cocktail used in semen diluent (lincomycin/spectinomycin, tylosin and gentamycin). Chlamydia psittaci has a presumptive risk of transmission in bovine semen. Donor freedom can be determined by tests such as ELISAs and fluorescent antibody to detect the organism rather than antibodies. Infection can be removed from donor bulls by antibiotic treatment. The virus of Akabane disease does not enter the semen of experimentally infected bulls and is not regarded as a risk. Ephemeral fever, however, has a presumptive risk of transmission via bovine semen since the causative agent is found in neutrophils during the febrile stage of the disease. Accordingly, semen collected from seropositive bulls is safe , as is semen from donors remaining seronegative during and after the collection period. Herpesvirus-4 has been found in bulls with orchitis but its spread via semen has not been demonstrated and the situation needs to be monitored. Paravaccinia virus and parainfluenza type-3 virus have been found in bovine semen but their importance and possible spread by artificial insemination is not clear. 10.

Recommendations

The following recommendations are taken from the Conclusions section at the end of the report. (1)

The “Code of Practice for Australian Livestock Artificial Breeding Centres” and Minimum Health Standards for Stock Standing at Licensed or Approved Artificial Breeding Centres in Australia which were both published in 1988 should be revised in form and content to account for progress in the area. -

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Since that time, knowledge on the risk of diseases that may be transmitted in bovine semen has advanced. In addition, the OIE International Animal Health Code which describes the accreditation of artificial insemination centres for export

-

purposes and methods for the hygienic collection and processing of semen was published in 1992. The two documents published in 1988 overlap in their coverage and could be merged into a single publication. More explicit guidance on the diagnosis and control of disease is required. In addition, the coverage of infectious disease in these documents is no longer complete.

(2)

The official disease status of countries is an important guide in protocol formulation. However, the latest information on the status of particular diseases in countries being considered for the import of semen requires direct contact with animal health officers in the country concerned.

(3)

Approved artificial breeding centres are central to the management of disease in the international trade in bovine semen. Upgraded protocols for approving and routine inspection of such centres are required domestically. They will also be a guide to what should be expected in artificial breeding centres in countries being considered for the import of bovine semen.

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INTRODUCTION Artificial insemination can increase the rate of genetic change in populations of animals and is an option for the husbandry of cattle in most parts of the world. The technology arises because bovine semen can be stored indefinitely at the temperature of liquid nitrogen (-196ºC) and can be conveniently transported, virtually anywhere, in this cryopreserved state. This long-term storage and ease of transport brings two unwelcome consequences. One is the possibility for disseminating undesirable genetic characters. The other is the risk of transmitting infectious disease which is dealt with here. The present report has been prepared for the Australian Quarantine and Inspection Service to aid in the assessment of risks associated with the import and export of bovine semen (see Box 1 for terms of reference). It reviews present knowledge on the transmission via cattle semen of the disease agents set out in List A and List B of the International Animal Health Code (Office International des Épizooties, 1992) and gives a commentary on the risks associated with each agent. Earlier reviews of the subject (FAO, 1981; Sellers, 1983; Hare, 1985; Singh, 1988a and b; Afshar and Eaglesome, 1990; Eaglesome and Garcia, 1992; Eaglesome, Garcia and Stewart, 1992; Parsonson, 1993; Philpott, 1993) are built upon and extended with more recent findings. It is a counterpart to reports by Parsonson (1989) and Gard (1994) on the risk of disease transmission in embryos. Box 1- Terms of reference 1.

Review literature on information on transmission of OIE List A and List B disease agents in cattle semen.

2.

Comment on risk re each agent.

3.

Produce report suitable for inclusion in international discussions and correspondence.

The report sets its course by outlining a set of epidemiological considerations which provide a basis for the assessment of risk. These considerations arise from the biology of bovine semen, the technology used in its preservation, the procedures used for insemination and the nature of the disease agents and the host-parasite relationship. The report next moves to issues arising from the International Animal Health Code, the procedures used in artificial breeding centres in Australia and the possibilities for and implications of the treatment of bull semen with antimicrobial agents. It then addresses its major task where each disease agent is considered in turn. Present attention is directed to the disease agents in OIE list A and B. However, other disease agents have been described in semen (Afshar and Eaglesome, 1990; Eaglesome, Garcia and Stewart, 1992) and are mentioned for future reference. New diagnostic methods for detecting either the disease agents themselves or the antibodies they elicit have been a focus of laboratory effort in recent times. These methods are based mainly on DNA technology (for example, polymerase chain 12

reactions) or enzyme technology; for example, enzyme linked immunosorbent assays (ELISAs) of various sorts. They are not a focus for the present report but may be referred to incidentally where they have contributed to relevant knowledge on a given disease agent. The present report has implications for the maintenance of genetic diversity in major domestic animals such as cattle and for the preservation of rare and endangered species. It is crucial that the international efforts in this regard (see FAO, 1992: National Research Council, USA, 1993) are safeguarded against the presence of disease agents in preserved germ plasm such as semen or embryos (see Gard, 1994) by procedures and protocols based on the best possible knowledge. Animal disease lists of the OIE. Three lists of animal diseases are used by the OIE (International Office of Epizootics) in conjunction with the FAO (Food and Agriculture Organization) and the WHO (World Health Organization) and are described in the 1991 Yearbook of OIE (Anonymous, 1992). List C is not dealt with in the present report. It contains "communicable diseases with important socio-economic and/or sanitary influence at the local level". List C diseases differ from those in List A and List B which must be reported as a requirement under the OIE International Zoo-Sanitary Code. List A diseases are "communicable diseases which have the potential for very serious and rapid spread, irrespective of national borders, which are of serious socioeconomic or public health consequence and which are of major importance in the international trade in livestock and livestock products". List B contains "communicable diseases which are considered to be of socio-economic and/or public health importance within countries and which are significant in the international trade of livestock and livestock products". The diseases covered in the present report are shown in Table 1. Table 1 OIE List A Diseases and List B Diseases of multiple species or cattle. List A Diseases Foot and mouth disease Vesicular stomatitis Swine vesicular disease Rinderpest Peste des petits ruminants Contagious bovine pleuropneumonia Lumpy skin disease Rift Valley fever Bluetongue Sheep pox and goat pox African horsesickness 13

List B Diseases of Multiple Species Anthrax Aujeszky’s disease Echinococcosishydatidosis Heartwater Leptospirosis Q fever Rabies Paratuberculosis Screwworm (Cochliomyia hominivorax)

List B Diseases of Cattle Anaplasmosis Babesiosis Bovine brucellosis Bovine genital campylobacteriosis Bovine tuberculosis Cysticercosis (C. bovis) Dermatophilosis Enzootic bovine leukosis Haemorrhagic septicaemia Infectious bovine rhinotracheitis/infectious

Hog cholera (classical swine fever) Fowl plague Newcastle disease

pustular vulvovaginitis Theileriosis Trichomoniasis Trypanosomiasis Bovine malignant catarrh Bovine spongiform encephalopathy Bovine virus diarrhoea* *Bovine virus diarrhoea is not mentioned in List B as described in the OIE International Animal Health Code (1992) but is given as a List B disease in the OIE Manual of Recommended Diagnostic Techniques and Requirements for Biological Products for Lists A & B Diseases, Volume II (1990##). Standards for preventing the transmission of infectious disease by bovine semen Various standards have been adopted throughout the world to prevent the spread of disease agents in bovine semen. Some are self-regulatory, for example those of Certified Semen Services of the USA, whereas others are imposed officially - for example Directive 88/407/EEC of the European Union. The standards for bovine semen set out in the International Animal Health Code (mammals, birds and bees) of the Office International des Épizooties (1992) and codes of practice used in Australia provide useful orientation for the present analysis of risk. OIE The framework for international trade in semen developed by OIE consists of three grades of disease-freedom. Regional freedom comes first and is followed by, herd freedom then freedom from specific disease an in individual donor animal and its gametes. An appendix (4.2.1.1.) in the OIE International Animal Health Code (1992) deals with the artificial insemination centres, their accreditation for export purposes and bovine semen. The requirements for admitting semen donors and teaser bulls into artificial insemination centres and the program of routine and prescribed tests to maintain disease freedom are described. Prescribed and routine tests are directed to bovine tuberculosis, bovine brucellosis, campylobacteriosis and trichomoniasis. The diluents used as semen extenders are considered as are protocols for the storage of semen. Another appendix (4.2.1.2.) of the Code deals with the hygienic collection and handling of fresh and preserved bovine semen and with methods for the measurement of contamination by common microflora. The procedures are directed at minimising or eliminating extrinsic contamination of semen as a means of spreading disease.

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Australia In Australia, centres for the collection of bovine semen operate under conditions set out in two documents prepared under the auspices of the Animal Health Committee of the Standing Committee of Agriculture and Resource Management. The first is "Minimum Health Standards for Stock standing at Licensed or Approved Artificial Breeding Centres in Australia" (3rd Edition, 1988) which describes requirements for quarantine facilities and management, the introduction of animals onto licensed or approved premises, the regular monitoring for freedom from disease and the reporting of results of examination and testing for freedom from disease. Appendices (1) catalogue the hereditary defects in cattle and other species, (2) describe techniques for collecting bovine preputial scrapings and vaginal mucus for the diagnosis of campylobacteriosis and trichomoniasis, (3) set out pro formas for stock health declarations and the certification of artificial breeding centres and (4) list the licensed/approved artificial centres in Australia at the time of printing. The second document applying to the collection of bovine semen in Australia is the "Code of Practice for Australian Livestock Artificial Breeding Centres" (1988). The sections relevant to bovine semen and disease deal with (1) the contingency of exotic disease, (2) the testing of animals before entry to an artificial breeding establishment, (3) the provision of hospital facilities, (4) a set of precautionary practices including the management of working dogs and horses, (5) recommended practices for the hygienic collection and handling of fresh and preserved bovine semen, (6) guidelines for the control of infectious bovine rhinotracheitis, (7) Mycoplasma and Ureoplasma infection in bulls, and (8) the control of wild birds and avian tuberculosis. TREATMENT OF BOVINE SEMEN TO REDUCE RISK Antimicrobial agents Treatment of bovine semen with antimicrobial agents to control microbial contaminants is recommended in the Australian code of practice mentioned earlier and has been reviewed by Eaglesome, Garcia and Stewart (1992). An effect has been reported against one of the disease agents in OIE list A and B, Campylobacter fetus, but the treatment was not bactericidal. For this reason, antimicrobial treatment can only be used to support other more decisive measures for controlling the possible transmission of pathogens in bovine semen. Antimicrobial treatment of semen has a history dating from 1941 when it was realised that bull semen contained bacteria and that semen diluents would support microbial growth. The elimination of Campylobacter fetus was an early aim which met with limited success since the glycerol in semen diluents protected the organism against the action of antibiotics such as streptomycin (Brinley-Morgan, Melrose and Stewart, 1959). Better results have now been reported (Shisong, Redwood and Ellis, 1990). Eaglesome, Garcia and Stewart (1992) describe the characteristics required by antimicrobial agents, alone or in combination, for suppressing microbial growth. These are: (1) effectiveness against microorganisms at the concentration used and 15

under the circumstances used, (2) no effect on the viability or fertilising capacity of spermatozoa, and (3) no unfavourable interactions with other substances in the semen-diluent mixture. These authors list reports where certain antimicrobial agents did not express these characteristics. They then describe combinations of antibiotics which are recommended for use because these characteristics have been accredited. A combination of lincomycin/spectomycin, tylosin and gentamycin in both whole semen and non-glycerolated whole milk or egg yolk based diluent has been shown to be effective against Mycoplasma bovis, M. bovigenitalium, Ureoplasma spp., Haemophilus somnus and Campylobacter fetus subsp. venerealis without damaging viability (Lorton et al., 1988a) or the capacity to fertilise (Lorton et al., 1988b). This mixture has been adopted as a standard by Certified Semen Services (CSS) which is a self-regulatory association for artificial insemination enterprises in the USA. For this standard to work, the CSS has other requirements. These include the use of an accredited diluent, that contact between antibiotics and raw semen must be for less than 3 minutes, a cooling time for semen and nonglycerol fraction to 5°C of less than two hours and that glycerol cannot be added until the semen-diluent mixture has fallen below 5°C. In summary, treatment with antimicrobial agents is effective against only a small number of the pathogens likely to be found in bovine semen. It can supplement not replace the other more decisive measures for controlling the risk of transmitting disease agents in bovine semen. Other treatments New agents are being tested for the elimination of contaminants from bovine semen. Photosensitive protoporphyrins plus irradiation under appropriate sources of light have been studied (Eaglesome et al., 1994). These were effective against Mycoplasma bovigenitalium, Mycoplasma canadense and Ureoplasma diversum. Only one agent was effective against Leptospira pomona. None were effective against Campylobacter fetus, Leptospira hardjo or Mycoplasma bovis. Other agents suggested for controlling microbial contamination in semen include antibodies produced by monoclonal methods, anti-idiotypic antibodies and greater use of the antimicrobial substances already present in semen (Eaglesome and Garcia, 1992). Such ideas are easily conceived from available technology. The difficulty lies in accrediting their efficacy in use and understanding their limitations from the outset. For example, microbes are not destroyed by antibody-binding alone.

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PRELIMINARY CONSIDERATIONS IN THE TRANSMISSION OF DISEASE AGENTS BY SEMEN Biological factors A set of biological considerations can be applied to the risk of transmitting the disease agents in OIE List A and B via bovine semen. They shed light on where hazards lie and where control procedures might be applied. As for control procedures, risk from many of the disease agents in OIE List A and B can be addressed by the design and management of artificial breeding centres, the examination of bulls before admission to breeding centres, regular monitoring of bulls for freedom from disease and the hygienic collection and handling of semen. Certain other disease agents will require special attention and testing. The epidemiological considerations shown in Box 2 are put forward in support of subsequent discussion of individual disease agents. They supplement the general approach which has been to seek evidence on the presence of disease agents in semen or the transmission of disease by semen or both. Significant drawbacks to this general approach include technical problems in the isolation of viruses and other pathogens from semen or the unknown immune status of animals in which test transmissions of disease are attempted. Semen can be toxic for cell cultures, unweaned mice or fertile eggs and can have antiviral activity itself (Kahrs, Gibbs and Larsen, 1980). The Cornell semen test (Schultz et al., 1982) and variants sidestep some of the problems of virus isolation by testing pooled semen samples in live animals. Box 2 - Biological factors influencing the presence of disease agents in stored bovine semen 1.

Extrinsic and intrinsic microbial contamination of semen Disease agents may be present in semen before it is ejaculated or may arrive in semen as a result of contamination during the process of ejaculation and semen collection. This is referred to respectively as intrinsic or extrinsic contamination (Afshar and Eaglesome, 1990). Extrinsic contamination includes organisms found in faeces (examples are bovine corona virus and Mycobacterium paratuberculosis) or on the prepuce (examples are Campylobacter fetus and Tritrichomonas foetus). Extrinsic contamination can be addressed to a large extent by cleanliness during semen collection, general veterinary oversight and the good order with which artificial breeding centres are managed. Protocols and codes of practice for the management of artificial breeding centres and semen collection provide guidance in this regard.

2.

Semen composition and the blood-testis barrier Semen is composed of seminal plasma, spermatozoa and other cells. Seminal plasma is formed in the epididymis and the accessory glands; the prostate, seminal vesicles, bulbo-urethral glands and the ampullae. Infectious agents

17

may be found in association with either the spermatozoa, seminal plasma or other cells depending upon their biology. There is a blood-testis barrier which functions similarly to the blood-brain barrier and restricts the inward passage to the testis of certain substances including antibody (Setchell, 1991). Accordingly, intrinsic contamination may occur mainly through the epididymis, ductus deferens or accessory glands rather than the testis. 3.

The process of intrinsic contamination of semen via the reproductive organs Intrinsic contamination of semen can occur from disease agents either residing in the tissues of the male reproductive tract or present in the general circulation. There is a risk of intrinsic contamination of semen whenever disease agents are present in blood, for example during viraemia in viral diseases or bacteraemia in bacterial diseases. Entry to semen requires that disease agents pass across epithelial surfaces and then into the collecting duct system of the reproductive tract. Passage of free disease agent would entail either adaptations for this purpose or a change in the permeability of membrane barriers; for example, during an inflammatory response. Examples of adaptations allowing passage across membranes, however, are difficult to find. Alternatively, passage could occur in association with motile host cells which will also be increased in number and activity during an inflammatory response. As a consequence, the risk of transmission of disease agents in bovine semen will be increased by inflammation of the testis, epididymis or accessory glands such as the seminal vesicles or bulbo-urethral glands.

4.

Inflammation in the reproductive organs Seminal vesiculitis is a complex syndrome which occurs in 1-10% of bulls and decreases semen quality (McCauley, 1980). It is associated with increased numbers of leucocytes in semen and, accordingly, will heighten the risk of transmitting pathogens. A wide range of micro-organisms has been implicated including bacteria, viruses, chlamydiae and mycoplasmas. Bacteria include Brucella abortus, Corynebacterium pyogens, Streptococcus spp., Pseudomonas spp., Escherichia coli, Proteus mirabilis, Mycobacterium paratuberculosis and Actinobacillus actinoides. Bulbourethritis was found in 1.2% of bulls in an abattoir study in tropical Australia (Campero, Ladds and Thomas, 1988) and organisms such as Mycobacterium paratuberculosis, Pseudomonas aeruginosa, Corynebacterium pyogenes , Haemophilus somnus and Mycoplasma and Ureoplasma spp have been isolated from these glands. The risk related to seminal vesiculitis or bulbourethritis would be reduced by prompt responses to signs of these diseases in semen-donors and by a program of regular physical examination which includes special attention to the reproductive tract and palpation of the seminal vesicles and other secretory glands.

18

5.

Cell-associated microbes The properties of most disease agents found as intrinsic contaminants in semen means that they will occur in association with non sperm cells rather than spermatozoa or seminal plasma. For example, viruses such as the retrovirus of bovine leukosis occur in association with lymphocytes (Johnson and Kaneene, 1992). Organisms such as Brucella bovis and Mycobacterium bovis are obligate intracellular parasites and will occur in semen in association with such cells. Organisms such as Babesia and Anaplasma spp are also obligate intracellular parasites but of red blood cells and will become a concern only when semen is contaminated with such cells. The presence of non-sperm cells such as erythrocytes or leucocytes can be regarded as an essential factor in predisposing for the transmission of disease agents via bovine semen.

6.

Non-sperm cells such as leucocytes in semen Non-sperm cells were believed not to occur in the bovine semen in the absence of an inflammatory response and it has been difficult to find quantitative information on their presence. They are likely to be found in small numbers as part of normal physiological behaviour in the absence of disease and could also occur as the result of simple injury; for example to the prepuce or penis. As for particular cell types, lymphocytes migrate across epithelial linings in both directions and will be found in semen as a result of this behaviour. Macrophages can be found in the epididymal lumen where they are involved with the removal of aged spermatozoa. If more information were available on the numbers and types of non-sperm cells found in bovine semen in different situations, measures of them could provide general guidance on the risk of transmitting pathogens in bovine semen. Direct counts or methods based on more recent technology would be possible. Results would supplement primary diagnostic information about semen obtained by detecting or isolating organisms or their antigen or antibody indicators.

7.

Latent infection, the carrier state and related phenomena Latent infection, chronic infection, inapparent infection and the carrier state are similar phenomena. They occur with many infectious diseases particularly those caused by viruses. Bovine pestivirus is a relevant example (Radostitis and Littlejohns, 1988). DNA viruses are of concern as a class because of their long-term relationship with the host via nuclear DNA. This leads to a tendency for producing persistent and latent infection (Truyen et al., 1995; Sleigh, Pennington and Lucas, 1992). Bovine herpes virus 1, the causative agent of infectious bovine rhinotracheitis is an example here (Barnard and Collett, 1994). The issue for semen is the possible transmission of disease via non-sperm cells which contain virus.

19

8.

Method of semen collection Semen may be collected for artificial insemination by the artificial vagina, electroejaculation or manual stimulation of the ampullae and vesicular glands through the rectal wall. Transrectal manual stimulation can lead to damage and the presence of blood in semen (Lucas and Roberts, 1982). As a consequence, this method of ejaculation presents a significant risk for disease transmission, particularly with erythrocyte-associated disease agents. In exceptional circumstances such as accidents when valuable semen-donors are unlikely to recover, spermatozoa can also be prepared directly from dissected testes. This procedure is used with threatened species of animals kept in zoos. Since non-sperm cells cannot be removed completely, spermatozoa collected by dissection from the testis are considered to carry a significant general risk for the transmission of disease and may bring to light unknown pathogens. The apparently new viruses that emerge from time to time (Truyen et al , 1995) are relevant here. There are differences in the plasma of semen collected with the artificial vagina or by electro-ejaculation but reports of differences in the risk of disease transmission have not been found.

9.

Site of insemination of females Semen used for artificial insemination is introduced through the cervix and deposited in the posterior part of the uterus. As a result, the portal of entry for disease agents is intrauterine and the defence mechanisms in the lower reproductive tract are bypassed. Horizontal infection of the dam or vertical infection of the offspring or both is possible with contaminated semen. Modified immunity against pathogens in the uterus at the time of insemination and conception could be speculated as a complicating factor but has not been demonstrated. Immunosuppression is one of the suggested but not accredited mechanisms for allowing a pregnant female to tolerate the developing embryo and foetus (Roitt, 1992). However, there is little indication that it extends to pathogens.

10.

Dilution of semen Bulls typically ejaculate 4-6 ml volume of semen at a sperm concentration of 1.0 x 109 to 1.2 x 109 per ml (Gardner, 1991). After semen is diluted and stored, cows are inseminated with 10 - 15 x 106 spermatozoa in a volume of 0.25 - 1.0 ml. Accordingly, a typical ejaculate can be divided into about 500 aliquots and diluted 100 fold. Some of the early literature implied that since this process of dilution and the diluent employed destroyed some of the contaminating microorganisms it would also help destroy pathogens. This proposition has not been tested and cannot be invoked as a factor mitigating the risk of disease transmission by stored semen.

20

11.

Effect of cryopreservation Conditions of cryostorage for the long-term preservation of viable spermatozoa could favour the survival of many of the disease agents in OIE lists A and B but may be harmful to others. The effect of cryopreservation on pathogens in semen was reviewed by FAO in 1982. The present discussion takes the standpoint that cryopreservation will favour the survival of a given pathogen unless there is definitive evidence for the opposite effect.

12.

Disease risk of cryopreserved semen versus cryopreserved embryos The risk of disease transmission through semen differs from that for embryos and is considered to be higher (Singh, 1988). The process of fertilisation and the presence of an intact zona pellucida excludes disease agents from embryos. In addition, the embryo can be washed free of many pathogens. Similar procedures for decontamination cannot be applied to semen although antimicrobial substances are sometimes added as will be discussed. Embryo transfer is the preferred method for artificial breeding if eliminating the risk of transmitting virtually all known and unknown pathogens is the aim.

13.

Breeding value versus disease risk The full potential for genetic improvement brought about by artificial insemination is realised only when the breeding value of semen donors is assessed by the performance of their progeny in the environment in which production occurs. A sound foundation in population genetics and statistical methods is available for progeny and performance testing procedures (see Nicholas, 1987). This point is relevant for addressing possible arguments that the genetic merit of a given bull outweighs the risk of transmitting a given disease via its semen. Assertions about the genetic merit of an individual animal or strain or breed of animal may now be insupportable in view of the possibility of scientifically defensible estimates of breeding value. However, estimates of breeding value themselves require scrutiny because they may not apply to the new environment or to the population of animals in which semen will be used for artificial breeding.

THE ASSESSMENT OF RISK The risk of bovine semen transmitting the causative agents of disease in OIE lists A and B falls into five categories for present purposes. Similar approaches have been used to classify the potential risk of transmitting various diseases by embryo transfer (International Embryo Transfer Society; quoted by Philpott, 1993) or by artificial insemination in the sheep and goat (Bane, 1981). Primary criteria for classifying risk are evidence for the transmission of a particular disease by artificial insemination or the isolation of a particular disease agent from semen, or both. These are supplemented by the epidemiological ideas described in Box 2 above. Important factors here are (1) whether contamination of semen is likely 21

to be intrinsic from within the reproductive tract or extrinsic from skin, the environment and so on and (2) whether the organism is present when there are no signs of disease as during convalescence or in the carrier state. The presence of clinical disease implies the presence of organism in the environment and is a clear risk for extrinsic contamination if illness does not prevent the collection of semen. Silent or symptomless infection or the carrier state is more insidious and requires special attention. The probability of whether a disease agent might enter Australia in bovine semen and the probability of its domestic establishment are the principal considerations in the final evaluation of risk (AQIS, 1991) with disease establishment being subordinate to disease entry. These two considerations are not used as criteria for the categories of risk in the present paper. The possible impact of a disease agent after introduction and establishment also has a biological basis and this is considered where necessary. Four categories of risk are used in the present report as follows: .

Category 1 : This contains diseases where material risk has been concluded from experimental transmission of disease by semen or artificial insemination or from unequivocal epidemiological analyses of the disease in nature.

.

Category 2: This contains diseases where causative organisms have been isolated from semen but transmission by artificial insemination has not been demonstrated.

.

Category 3: This contains diseases with a presumptive risk based on the ideas in Box 2. Examples are cell-associated viruses or bacteria or obligate parasites of erythrocytes.

.

Category 4: This contains diseases in List A and B where there is no presumptive risk but where extrinsic contamination of semen cannot be ruled out if the disease is in progress.

.

Category 5: This contains diseases where risk is is inconceivable. Examples are diseases with a distinct species specificity such as Newcastle disease and fowl plague and diseases caused by certain metazoan pests and parasites such as the screw worm fly Cochliomyia hominivorax or the tapeworms of Echinococcus spp.

22

THE RISK OF TRANSMISSION VIA BOVINE SEMEN OF INDIVIDUAL DISEASE AGENTS IN OIE LIST A Table 2 shows the OIE List A diseases which infect multiple species of animals and summarises properties relevant to the risk of transmission via bovine semen. These diseases are grouped according to categories of risk in both table 2 and the ensuing discussion.

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Table 2 - Summary of the risk of transmission by bovine semen of OIE List A diseases which can infect multiple species of animals Disease

24

Causative organism

Species range

Infectivity of/ presence in semen

R es ist an ce to en vi ro n m en t an d ul tr alo w te m pe ra tu re s

Commentary on risk

Category 1 - Diseases where transmission via semen has been demonstrated

25

Foot and mouth disease

26

An aphthovirus in the family Picornoviridae which occurs as seven distinct types: A, O, C, SAT1, SAT2, SAT3 and ASIA 1.

All cloven footed mammals (plus the elephant and some species of rodents in south America)

Presence of virus demonstrated in semen. Transmission by semen demonstrated

Vi rus in se me n or epi the liu m un aff ect ed by sto rag e at -5 0° C for 32 0 da ys an d 11 ye ars res pe cti

Vaccination of donor bulls against foot and mouth disease complicates risk. Bovine semen should only be accepted from unvaccinated semen donors born, raised and maintained where there is regional freedom from the disease.

Bluetongue

An arbovirus in the Orbivirus genus of the family Reoviridae which occurs as 24 serotypes. Vectors are Culicoides spp insects.

Bluetongue disease is most severe in sheep. Moderate disease occurs in goats and infection in cattle is usually inapparent.

Bluetongue virus is found inconsistently in the semen of infected bulls. Early demonstration of transfer by artificial insemination not repeated.

Category 2 - Diseases where causative organisms have been demonstrated in semen

27

Re sis tan tvir us iso lat ed aft er 25 ye ars fro m gly cer olox ala teph en ol pre ser ve d blo od.

Programs suggested for managing the risk of bluetongue virus in bovine semen in international trade are based on the demonstration of donor freedom from the bluetongue virus which depend upon close surveillance of semen donors for viraemia. Risk from semen is considered lower than that with imported live animals

Rinderpest

28

A virus in the genus Morbillivirus in the Paramyxoviridae.

Chiefly affects cattle but some wild ungulates may be mildly susceptible.

Identified in semen by work in 1899 but not corroborated. Likelihood of presence in semen has strong indirect support.

Ri nd erp est vir us sai d to be de str oy ed by gly cer ol ma y not sur viv e in se me n dil ue nt.

Regional freedom from rinderpest is a prerequisite for acceptance of bovine semen. Contamination risk extends beyond presence of clinical disease in semen donors. Risk may dissipate as antibody titre in blood declines.

Disease

Causative organism

Species range


Category 2 - Diseases where causative organisms have been demonstrated in semen (continued)

29


Commentary on risk

Lumpy skin disease

A virus in the Capripox genus of the Poxviridae.

Cattle

Virus found in semen of infected bulls during inapparent infection.

Category 3 - Diseases with a presumptive risk of transmission by bovine semen

30

Pr ese rve d at te mp era tur es les s tha n -6 0° C.

Semen can only be regarded as safe if it comes from regions where lumpy skin disease does not exist. The use in semen donors of live virus vaccines prepared from sheep or goat strains would create an additional risk.

Rift Valley fever

31

Mosquito-borne virus in the Phlebovirus genus of the Bunyaviridae.

Occurs in cattle, sheep and goats and a range of other species including humans. Inapparent infection in horses, pigs, camels dogs and cats.

Viraemia occurs during clinical disease and virus is present in low concentrations in milk, saliva and other secretions.

Vi rus res ist ant to se ver al mo nth s sto rag e in ser um at 4° C. Sta ble for lon g per iod s in ser um at -6 0° C.

Regional freedom from Rift Valley fever is a prerequisite for the acceptance of semen. Carrier state is not a characteristic of Rift Valley fever but period of risk for semen could overshoot the duration of clinical disease in semen donor bulls.

Peste de petits ruminants

Virus in the genus Morbillivirus of the Paramyxo-viridae - related to the Rinderpest virus.

Sheep, goats and related ruminants.

Same factors as for Rinderpest apply. Peste de petits ruminants can occur experimentally in cattle.

Contagious bovine pleuropneumonia (CBPP)

A mollicute bacterium Mycoplasma mycoides , subspecies mycoides, type SC.

Cattle

Causative organisms are found in urine with the possibility of contaminating semen

32

As for the Ri nd erp est vir us. Vi abi lit y ma int ain ed by sto rag e at -6 0° C.

As for Rinderpest.

Transmission of CBPP ruled out by the use of disease free donor bulls raised and maintained in the absence of the disease.

Disease

Causative organism

Species range


Category 4 - Diseases with risk associated only with extrinsic contamination

33


Commentary on risk

Vesicular stomatitis

34

A virus in the genus Vesiculovirus in the family Rhabdoviridae.

Cattle, pigs, horses and mules.

Causative organisms liable to cause extrinsic contamination of semen present only when disease is present.

Vi rus ha s an en vel op e an d ma y be les s res ist ant to fre ezi ng an d tha wi ng tha n vir us es wit ho ut

Semen should not be accepted from bulls with clinical vesicular stomatitis.

Category 5 - Diseases with no appreciable risk of transmission by bovine semen Sheep pox and goat pox

A virus in the Capripox genus of the Poxviridae.

African horse sickness

An arbovirus in the Orbivirus genus of the Reoviridae.

African swine fever

A virus of the family Iridoviridae

35

Highly contagious in sheep and goats. The most severe of the poxes in animals NOT CATTLE Natural infection of domestic livestock occurs only in equine species. Odd occurrence in the dog. Porcine species are the only natural hosts.

Disease

Causative organism

Species range


Category 5 - Diseases with no appreciable risk of transmission by bovine semen (continued)

36


Commentary on risk

Hog cholera (classical swine fever) Swine vesicular disease

A virus in the Pestivirus genus of the Togaviridae.

Fowl plague (Avian influenza)

An influenza virus in the Orthomyxo-viridae

Newcastle disease

A virus in the Paramyxovirus genus of the Paramyxo-viridae

A virus in the Enterovirus genus of the Picornaviridae.

The pig is the only natural host. Pigs are the only livestock species affected. The disease is restricted to birds. The disease is restricted to birds but infection in humans can occur

Category 1 - Diseases where transmission via semen has been demonstrated. Foot and Mouth Disease This acute highly infectious disease of cloven-hoofed animals is of particular importance to Australia where the major threat it poses is the subject of specific policies. The disease is caused by an aphthovirus, a single-stranded RNA virus in the family Picornaviridae, which occurs as seven antigenically distinct types: A, O, C, South African Territories (SAT) 1, SAT 2, SAT 3 and Asia 1. The epidemiology of disease caused by these strains can differ. However, the clinical picture is uniform and the following analysis of the risk related to semen applies to all strains. The transmission of foot and mouth disease virus in frozen bovine semen is a real risk because two major conditions are satisfied. Firstly, the virus survives virtually indefinitely at ultra-low temperatures. Virus concentrations in infected tongue epithelium were virtually unaffected by

37

storage at -50°C for 11 years (Cottral et al., 1966) and were unaffected in infected semen stored at the same temperature for 320 days (Cottral et al.,1968).Secondly, the virus can be found in the semen of infected animals from where it can transmit to other animals via insemination. The transmission of foot and mouth disease by insemination is well established. Cottral et al.(1968) determined the minimum amount of foot3.4 and mouth disease virus to infect cows or heifers by insemination as 10 mouse LD . In one laboratory study of the intrinsic contamination 50 1.5 4.7 of semen, virus at titres of 10 to 10 mouse LD per ml of semen was found up to four days before the onset of clinical signs in 50 6.4 1.4 experimentally infected bulls (Sellers et al., 1968). The titre rose to 10 mouse LD per ml of semen when lesions appeared and was 10 50 mouse LD per ml five days later. Trace amounts of virus were isolated from semen 37 days after the appearance of lesions. 50 The source of virus for intrinsic contamination is viraemia. However, virus may also multiply in the the skin around the preputial orifice and lead to extrinsic contamination of semen during ejaculation. Virus found at this site in vaccinated bulls indicated that vaccine protected against systemic but not localised infection (Sellers et al., 1969 and 1977). A research group on foot and mouth disease of the European Commission (Donaldson and Sellers,1983) summed up the risk of transmission as coming (1) from semen obtained from infected bulls before clinical disease appears, (2) from vaccinated bulls with systemic not local protection and (3) from semen from vaccinated bulls in contact with the disease. These ideas generated a set of safety guidelines for the importation of semen when donors are vaccinated (see Box 3). Box 3 - Guidelines suggested for the exclusion of foot and mouth disease virus from imported bovine semen (Donaldson and Sellers, 1983) (1) An adequate period (30 days) following vaccination before semen collection to ensure that the vaccine contains no live virus. (2) A probang sample* from the donor bull at the time of semen collection, and a confirmation that no clinical signs of the disease have occurred in the stud for the subsequent 30 days. (3) Samples of ejaculates are cultured for virus and confirmed negative before semen is distributed. A confirmed declaration of freedom from the disease at least three months before semen collection and at least one month after collection is necessary under all circumstances. 38

*Refers to a sample of oropharyngeal fluid obtained with the probang cup which is an accepted method for the detection of persistent viral infection in carrier animals (Sutmoller and Gaggero, 1965). The carrier state is an important epidemiological feature of foot and mouth disease (Salt, 1993). However, virus does not pass into the salivary secretions of carriers (Wittman and Eissner, 1966) and is, thus unlikely to pass into the secretions of the reproductive tract and be present in semen. The importation and use of semen from donors vaccinated against foot and mouth disease is untenable for Australia because of the unexplored nature of the carrier state in this disease and the strain-specificity of vaccines, where a vaccine against one strain will not protect against another strain. The carrier state has been recognised as a major epidemiological feature of foot and mouth disease for a long time (Waldmann et al. , 1931; quoted by Salt, 1993) and understanding about it has advanced. However, the underlying immunological mechanisms remain speculative (Salt, 1993) and the picture of the phenomenon as it occurs in nature appears to be incomplete. Cattle can act as carriers post-infection for periods reported as 15 months (Sellers, 1971) or 2.5 years (Hedger, 1968). The carrier state in the African buffalo (Synceros caffer) can be important in the cycle of transmission of foot and mouth disease to cattle in parts of Africa (Hedger, 1970; Hedger and Condy, 1985). Vaccinated cattle that showed no clinical signs themselves were shown to transmit infectious doses of virus for up to seven days after experimental challenge infection (Donaldson and Kitching, 1989). It is not clear whether the form of inapparent infection following vaccination and challenge infection is the same as the carrier state observed in the field. Nevertheless, the persistence of foot-and-mouth virus in cattle must be regarded as a major feature of this host-parasite relationship and a major factor in risk assessment. In summary, the risk of transmitting foot and mouth disease with fresh or cryopreserved bovine semen is proven. Vaccination of donor bulls does not remove the risk but complicates it. Given the consequences of introducing foot and mouth disease into Australia, bovine semen can only be accepted from unvaccinated semen donors born, raised and maintained where there is regional freedom from the disease. Bluetongue

39

Bluetongue virus has been found in bovine semen under some conditions but not others. As a consequence, standards for the control of bluetongue are an important issue for the international trade in bovine semen (Walton and Osburn, 1992; Roberts, Lucas, and Bell,1993; BarrattBoyes and MacLachlan, 1995). Progress in the area has come from an understanding of the circumstances under which bluetongue virus occurs in bovine semen and through the development of direct and indirect methods for diagnosing contamination. Bluetongue itself is an arthropod-borne viral disease of ruminants (see reviews by Verwoerd and Erasmus, 1994, and Barratt-Boyes and MacLachlan, 1995) characterised by inflammation, haemorrhages and ulceration of the mouth and mouth cavity, feet and mucous membranes in general. There can be lung involvement. The clinical course is variable ranging from peracute to chronic and the mortality rate extends from 2 to 30%. Bluetongue is a disease problem for sheep not cattle or goats where inapparent infections are usual. Infections in sheep can also be inapparent and the severity of disease depends upon the serotype of virus involved. The causative agent is a virus in the genus Orbivirus of the family Reoviridae. It has a genome of double-stranded RNA and occurs as 24 serotypes with complex cross-relationships (Gorman, 1992). Replication in the mammalian body occurs primarily in the endothelial cells of capillaries and other small blood vessels (Verwoerd and Erasmus, 1994). The mode of transmission of the bluetongue virus in nature is an important determinant of risk with implications firstly for its possible introduction to a new area as a contaminant of bovine semen and secondly for its establishment and propagation. The virus is a classic example of an arbovirus where transmission between susceptible ruminants occurs through the bites of blood-sucking insects and the virus multiplies in both insects and ruminants. Reservoirs of bluetongue virus for infecting sheep and causing disease result primarily from a propagative cycle involving cattle and insect vectors. Bluetongue virus is found only where its vectors, Culicoides spp insects, are found and its seasonal incidence reflects both the activity of these vectors and how efficient they are at transmitting infection (Doyle, 1992). Bluetongue disease is seen in most countries of Africa, the Middle East, the Indian subcontinent, the USA and Mexico and occurs predominantly in sheep and goats. Bluetongue virus, as opposed to clinical disease, occurs in South East Asia, Papua New Guinea and northern South America. The situation for bluetongue virus in Australia is shown in Box 4. Box 4 - The bluetongue virus in Australia Reservoirs of bluetongue virus are present in Australia in several species of Culicoides but the disease does not occur in the field. The four main vectors identified in Australia are the biting midges Culicoides brevitarsis, C. wadai, C. fulvus and C. actoni (Standfast, Muller and Dyce, 40

1992). C. brevitarsis has the widest distribution of the four species and is the determinant of the bluetongue endemic area in Australia. Its habitat range arises from climatic factors, a predilection for cattle rather than sheep and a requirement for cattle dung in the breeding cycle. To date, eight serotypes of the bluetongue virus have been identified in Australia: namely; 1, 3, 9, 15, 16, 20, 21 and 23 (Doyle, 1992). However, surveys of serum obtained from cattle (Ward, Flanagan and Baldock, 1995) and sheep and goats (Flanagan et al., 1995) in the bluetongue endemic area of north and central Queensland indicate the active presence of only two serotypes, 1 and 21. The absence of clinical disease in these areas infers that both serotypes are relatively benign for sheep. Experimental infections with five of the remaining serotypes (3, 9, 15, 16 and 23) have produced disease in sheep (Johnson, Hoffmann, and Flanagan, 1992). No clinical bluetongue has been recorded in sheep in the field in Australia. The epidemiology of bluetongue infection argues strongly against any prospect of transmission of the virus by direct contact between animals or by the aerosol or oral routes. As a result, it rules out the establishment of the disease in areas where potential vectors do not exist. The formal possibility that transmission might occur apart from insects has been alluded to in connection with international trade in bovine semen (Roberts, Lucas and Bell, 1993). Such transmission cannot be refuted by either epidemiological reasoning alone or by transmission experiments, which can never cover all circumstances, However, the possibility runs against the natural history of bluetongue infection and is not supported by scarcity of virus in the secretions and excretions of infected animals (Verwoerd and Erasmus, 1994). Semen is the significant exception. Bluetongue virus has been found on occasions in bovine semen and an understanding of the circumstances involved provides a basis for managing the risk of contamination. Initial concern was roused by a bull which was infected in utero at 60 days gestation and had a viraemia which continued for 11 years (Luedke, 1977; Luedke and Walton, 1981). The absence of serum antibody during this period suggests the action of immunological unresponsiveness. Evidence since that time indicates that bluetongue virus is shed sporadically into semen only when donors are viraemic and that the concentration of virus in semen is always less than in blood (Breckon et al., 1980; Bowen et al., 1983; Parsonson et al., 1981, 1987a and 1987b). One study with regular testing of 29 bulls over a span of five years (Gard et al. 1989), showed no clear sign of bluetongue virus in semen in spite of 18 episodes of infection. The longest period of viraemia in this study was 29 days compared with other observations of 35 days and 7 to 106 days (Howard, 1982, quoted in Sellers, 1983). If bluetongue virus does not replicate in the reproductive tract, viraemia is a logical prerequisite for the intrinsic contamination of semen. However, there is one report of pathological changes in the reproductive tract of bulls suggesting the localisation of virus (Foster et al. 1980). During viraemia, bluetongue virus is closely associated with erythrocytes or leucocytes (Hoff and Trainer, 1974) rather than being free in plasma

41

(Lawman, 1979, quoted in Verwoerd and Erasmus, 1994). Entry into semen is believed to occur by the infiltration of infected blood cells into the reproductive tract (Howard, 1982, quoted in Sellers, 1983). Bluetongue infection occurs in cows when contaminated semen is introduced into the cervix or uterus but is not associated with signs of disease in either the cow or the conceptus (Bowen and Howard, 1984; Parsonson et al., 1987a and 1987b). Furthermore, bluetongue infections set up by artificial insemination do not appear to extend to embryos (Schlafer et al., 1990). Transplacental transmission to the foetus is uncommon and appears to arise when infection occurs later in pregnancy (Luedke et al., 1977; Luedke and Walton, 1981; Groocock et al., 1983; Parsonson et al. 1987b). In one study, bluetongue virus could not be isolated at birth when bovine foetuses were inoculated at 125 days gestation (MacLachlan et al., 1984). The conclusion here is that contaminated semen could set up infections without disease in cows but not developing calves and that such infections could be a problem when and where potential vectors are active and small ruminants are at risk. Guidelines for the management of bluetongue are not described in the Australian health standards for artificial breeding centres (DPIE, 1988). However, model guidelines have been suggested (see Box 5) and require analysis. An initial consideration is whether either regional, herd or donor freedom from disease should be the basis for international standards for exchange of bovine semen. If regional freedom is opted for, that band of the world between latitudes 40°N and 35°S could be excluded indefinitely since it is unlikely that vectors and the reservoir of bluetongue virus could ever be eliminated. Box 5 - Guidelines suggested for the exclusion of bluetongue virus from bovine semen used in the international exchange of genetic material 1.

From MacLachlan, Gard and Team (1992)

“2.

Bulls in artificial insemination (AI) centres located in BLU*-free areas should be serologically tested semiannually. Seronegative bulls entering from BLU-endemic areas should be serologically tested prior to arrival, and after pre-entry quarantine of at least 35 days. After this they should be tested semiannually.

3.

Semen from seropositive bulls in AI centres in BLU-free areas should be withheld from export until serologic and virus detection tests on their blood have indicated continued freedom from infection.

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4.

For AI centres in endemic areas, either seropositive or seronegative bulls are acceptable as donors. However, semen should be withheld from export until serologic and virus detection tests on their blood confirm continued freedom from infection during the period of semen donation. Serologic and virologic testing of blood should be carried out particularly during the vector season to confirm the bull’s virusfree status; weekly testing would be a logical minimal requirement for virus detection during the vector season.

5.

All virologic and serologic testing must be done with the most specific and sensitive tests available.”

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2.

From Alexander, Portugal and Team (1992)

“Bulls entering artificial insemination centres (AI) centers in BLU-free areas should be tested serologically upon entry to the center and (if from BLU-endemic areas) again at 35 days after entry. If they are seronegative, their semen should be cleared for export, and they should then be tested serologically, semiannually. Seropositive bulls should have virus detection tests conducted on their blood to establish their freedom from infection before their semen is cleared for export. They should be tested semiannually. For AI centers in endemic areas, either seronegative or seropositive animals should be acceptable. However, semen should be with-held from export until serologic and virus detection tests from whole blood samples both pre- and post-semen collection have been shown to be negative. Regular testing should be carried out particularly during the vector season to confirm the bulls’ virus-free status. The frequency of such testing should relate to the level of risk of infection and may be as frequent as weekly where there is a high level of risk.” 3.

From Roberts, Lucas and Bell (1993)

“(a)

The population of the semen collection centre in which the donor bull has been resident during the last vector season should be seronegative to relevant orbiviruses;

(b)

in periods of proven vector inactivity, the donor bull should remain seronegative for 28 days after semen collection;

(c)

during periods of vector activity or when a vector free season does not occur, a blood sample taken from the donor on the day of semen collection showed no evidence of viraemia when injected into a fully susceptible sheep or into chicken embryos in addition to the negative serological test at (b) above.”

*BLU: bluetongue The scheme of MacLachlan et al. (1992) in Box 5 is recommended since it covers the possibility of bluetongue in both endemic and nonendemic areas and employs virus detection tests for semen as well as serology for antibody. The scheme of Roberts et al (1993) is partly based on herd freedom which is not relevant in practice to an arthropod-borne disease. It contains the same elements as the MacLachlan et al. scheme.

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However, weekly testing for virus during the vector season is regarded as sufficient since semen collected between tests can be destroyed if necessary. The scheme of Alexander et al (1992) is similar to that of MacLachlan et al (1992). In their proposal for managing bluetongue for the international exchange of bovine germplasm, MacLachlan et al. (1992) recommended that “virologic and serologic testing must be done with the most specific and sensitive tests available”. Serological tests accredited in Australia (Gard and Kirkland, 1992) are complement fixation (CF), agar gel immunodiffusion (AGID), competition ELISA (c-ELISA) and virus neutralisation. Virus neutralisation tests are used for type-specific as opposed to group-specific antibodies which can be difficult to interpret when animals have been exposed to several serotypes. The other tests are directed to group-specific antibodies. CF tests have relatively low specificity and sensitivity and have been superseded by AGID tests in international regulations. However, the c-ELISA has higher specificity and sensitivity (Hübschle et al., 1981; Lunt et al. 1988) and is recommended. In summary, the bluetongue virus has been established as a contamination risk in bovine semen. Artificial insemination with contaminated semen can lead to infection in cows but not the conceptus. Satisfactory programs have been suggested for managing this risk in the international trade in bovine semen (MacLachlan et al., 1992). These programs cover artificial breeding centres in endemic and non-endemic areas. They are based on donor freedom from disease agents and ultimately depend upon surveillance for viraemia in donor bulls. Routine physical examination of bulls is suggested as an important component of these programs for managing contamination risk. Scrotal and rectal palpation for gross changes in the male reproductive system could aid against the outside possibility of intrinsic contamination of semen as a result of proliferation of bluetongue virus in the locality rather than from viraemia. Questions remain about methods of semen collection and the presence of bluetongue virus in semen. Ejaculation stimulated by transrectal massage of male reproductive organs and the stripping of seminal fluid from seminal vesicles may cause local trauma and favour the entry of cell-associated virus into semen (see page 6). It is not known whether electro-ejaculation may have a similar effect since no accounts of the nonsperm cells in bovine semen can be found in the scientific literature. Accordingly, collection of semen with the artificial vagina is recommended as an additional procedure in risk management. Spermatozoa collected from dissected testes should be excluded from international trade because freedom from blood and endothelial cells which could contain virus cannot be assured. Category 2 - Diseases where causative organisms have been demonstrated in semen.

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Rinderpest Rinderpest is an acute disease of cattle and other cloven-hoofed animals characterised by fever, erosive stomatitis and gastroenteritis, diarrhoea and dehydration. It has appeared as catastophic epidemics and pandemics and is usually transmitted by contact. The disease has occurred in many parts of the world. It has been eradicated from most of Africa except for a contracting equatorial area (see Rossiter, 1994) and remains endemic in India. The causative organism of rinderpest belongs to the genus Morbillivirus of the Paramyxoviridae and has a genome composed of single-stranded antisense RNA. Rinderpest is classified as a disease where the organism is known to be present in semen but where transmission by insemination has not been confirmed (Hare, 1985). Early work (Nicolle and Bey, 1899) is cited as evidence for the presence of the Rinderpest virus in semen (Scott, 1964; Hare, 1985). The nature of the evidence and the methods used to obtain it are not known. However, the likelihood of the virus being present in semen as a result of intrinsic contamination in the reproductive tract has other support. Infected animals have a viraemia (Leiss and Plowright, 1964) and fragments of viral RNA can be demonstrated in peripheral blood lymphocytes up to three months after clinical disease (Barrett, 1987). During infection, the virus can move into various body fluids and has been found in ocular, nasal, oral and vaginal secretions (Scott, 1964; Wafula, Rossiter, Wamwayi and Scott, 1989). Virus enters these secretions before the onset of fever and excretion continues for nine to ten days and wanes as the titre of antibody rises (Leiss and Plowright, 1964; Forman, Rowe and Taylor, 1983). Virus could enter semen during the same period and as a result of the same mechanisms. In summary, early work demonstrating the presence of rinderpest virus in semen is supported by the viraemia which occurs during this disease and the observation that virus is excreted in other external secretions. The risk of intrinsic contamination of bovine semen by rinderpest virus extends beyond the presence of clinical disease in semen donors. Risk dissipates as the titre of specific antibody in plasma increases. Rinderpest virus is relatively fragile and usually survives for only a few hours outside the host. It is destroyed by glycerol (Rossiter, 1994) and would not survive in semen diluent containing this substance. Lumpy skin disease Lumpy skin disease is an acute to subacute viral disease of cattle that occurs in sub-Saharan Africa and Madagascar (see review by Davies, 1991). It is marked by fever, multiple nodular swellings in the skin and necrotic lesions of mucous membranes in the respiratory tract and mouth. The disease does not have a high mortality in adult cattle but results in prolonged debilitation and production losses. Biting insects are believed 46

to have major reponsibility for transmission of the causative agent which is a a member of the Capripox genus of the Poxviridae and has a genome composed of double-stranded linear DNA. Bovine herpesviruses 2 and 3 have been isolated from lumpy skin disease lesions but do not cross-protect against the disease. Although lumpy skin disease is usually spread mechanically by biting insects, the causative agent has a genome composed of double-stranded DNA which raises the prospect of its spread via semen in association with cells. There is evidence of its transmission by various body fluids (Barnard et al., 1994b). For example, saliva has been incriminated as a result of transmission when drinking troughs were shared with diseased cattle. The virus can be transmitted to suckling calves through milk. Finally, the virus has been found in the semen of infected bulls (Prozesky and Barnard, 1982) and, importantly, when infection was inapparent (Woods, 1988). Viraemia can be detected for 2-4 days after infection but the virus may be found for up to 11 days in saliva and 22 days in semen (Davies, 1991). In summary, lumpy skin disease fits into risk category 2 because the causative agent has been isolated from semen and, importantly, from bulls with inapparent infection. As a consequence, semen can only be regarded as safe if it comes from regions where the disease does not occur. Vaccination is practised to control the disease. However, vaccines consist either of an attenuated strain of the initial Neethling virus or are derived from sheep and goat pox viruses. It is not clear whether the first produces a sterile immunity. Semen from bulls vaccinated the second way would be unacceptable because of the possible transmission of sheep or goat pox to susceptible populations of sheep and goats in Australia Category 3 - Diseases with a presumptive risk of transmission by bovine semen. Rift Valley Fever Rift Valley fever is a mosquito-borne viral disease of economic importance in cattle, sheep and goats which appears as a generalised haemorrhagic fever accompanied by necrotic hepatitis and foetal abnormalities (see reviews by Shimshony and Barzilan, 1983; Swanepoel and Coetzer, 1994). The disease occurs in a range of other species including man. Camels, horses, pigs, dogs, cats, African monkeys, rabbits and guinea pigs are resistant to disease but sustain inapparent infections. Rift Valley Fever occurs mainly in southern Africa but outbreaks have been recorded in Egypt and the Sudan and there is serological evidence of its occurrence in Madagascar. The causative agent belongs to Phlebovirus genus of the Bunyaviridae and has a genome composed of single-stranded antisense RNA. The following set of ideas indicate that Rift Valley fever has a potential for transmission via bovine semen. Virus particles are plentiful in the blood of infected livestock and are available for possible haematogenous contamination of semen. Although transmission during epidemics 47

usually occurs through the bites of insects such as mosquitoes, midges and flies (Davies and Highton, 1980), virus has been found in low concentrations in milk, saliva, nasal secretions and other body fluids (Shimshony and Barzilai, 1983; Jouan et al. 1989) where it becomes available for contact transmission including the extrinsic contamination of semen. In general, the viraemia persists only as long as the illness (Swanepoel, Struthers, Erasmus, Shepherd and McGillivray, 1986). However, the virus can be found in the spleen and other organs of sheep up to 21 days after infection (Yedloutschnig, Dardiri and Walker, 1981). The same could hold for cattle. Here it may extend the period of risk related to semen beyond the disappearance of clinical signs of disease. Finally, the virus of Rift Valley fever is resistant to several months storage in serum at 4°C and is stable in serum for prolonged periods of storage at -60°C. It is therefore likely to survive in deep-frozen semen. In summary, there is a presumptive risk of transmission of Rift Valley fever in bovine semen. Viraemia occurs during clinical disease and virus is found in body fluids indicating the possibility of intrinsic contamination of semen. Although the carrier state is not a characteristic of Rift Valley fever, the period of risk for a semen donor bull could overshoot the duration of clinical disease.

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Peste de petits ruminants Peste des petits ruminants is a disease of sheep and goats and related ruminants and characterised by a syndrome of stomatitis, pneumonitis and enteritis. It occurs in northern part of sub-Saharan Africa, the Sudan, Ethiopia, the Arabian peninsula, India and Pakistan. The causative agent is related to the Rinderpest virus and belongs to the genus Morbillivirus of the Paramyxoviridae, As such, it has a genome composed of singlestranded antisense RNA. Peste des petits ruminants does not occur in cattle in nature (Taylor, 1984) but is discussed here because the causative virus grows readily in some lines of bovine kidney cells and the oral lesions of the disease have been recorded in young calves (Rossiter and Taylor, 1994). The latter observation raises the possibility that cattle might act as a reservoir of infection for sheep and goats. Accordingly, peste des petits ruminants is a disease with a strong presumptive risk of transmission via semen and is placed in Category 3. Contagious bovine pleuropneumonia Contagious bovine pleuropneumonia (CBPP) is an acute to chronic respiratory disease of cattle characterised by a fibrinous pneumonia, serofibrinous pleuritis and walled-off lesions in the lung which give rise to a chronic, subclinical carrier state. It is a particular concern because it has been eradicated from Australia. The causative agent is Mycoplasma mycoides, subspecies mycoides, type SC. CBPP is classified as a disease with a presumptive risk of transmission via semen because the causative organisms may be found in the urine of cattle with severe disease (Masiga, Windsor and Read, 1972) and there is a possibility for extrinsic contamination of semen. However, the overwhelmingly predominant route of transmission of Mycoplasma mycoides, subspecies mycoides, type SC is by droplet infection from clinically or subclinically infected cattle. Aerosols may spread for more than 20 metres ensuring rapid spread of the disease in a susceptible population (Schneider, van der Lugt and Hübschle, 1994). Transmission of CBPP via semen would be ruled out by the use of disease free donor bulls raised and maintained in the absence of the disease. Category 4 - Diseases with risk associated only with extrinsic contamination. Vesicular stomatitis 49

Vesicular stomatitis is a viral disease of cattle, pigs, horses and mules characterised by vesicular lesions in the oral mucosa and coronary band of the feet reminiscent of foot and mouth disease. The disease causes production loss and the causative agent is a member of the Vesiculovirus genus of the Rhabdoviridae with a genome composed of single-stranded antisense RNA. The method of transmission of vesicular stomatitis is not clear and there is little evidence that the disease transmits between animals (Wilks, 1994). Insects may be involved in a mechanical way and milking machines may spread teat lesions. Viraemia is characteristically absent in cattle and horses ruling out haematogenous entry into semen. For this reason, vesicular stomatitis virus is unlikely to be spread by semen and the agent is placed in category 4. However, semen should not be accepted from animals with the disease because of possible extrinsic contamination. Category 5 - Diseases with no appreciable risk of transmission by bovine semen. Several of the disease agents in List A have a distinct species-specificity and can be disregarded for the risk of their transmission via bovine semen under the conditions for collection, processing and storage set out in the OIE International Animal Health Code (1992). These are Newcastle disease, avian influenza, African horse sickness, African swine fever, swine vesicular disease and hog cholera (classical swine fever).

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THE RISK OF TRANSMISSION VIA BOVINE SEMEN OF INDIVIDUAL DISEASE AGENTS IN OIE LIST B DISEASES OF MULTIPLE SPECIES Table 3 shows the OIE List B diseases which infect multiple species of animals and summarises properties relevant to the risk of transmission via bovine semen. These diseases are grouped according to categories of risk in both Table 3 and the ensuing discussion.

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Table 3 Summary of the risk of transmission by bovine semen of the OIE List B diseases which can infect multiple species of animals Disease

Causative organism

Species range

Presence in semen

Resistance to ultra-low temperatures

Commentary on risk

Pathogenic strains survive well in semen and semen diluents. Present in cryopreserved semen.

Antibiotic cocktail in semen diluent has been demonstrated to protect against transmission of disease. Serological surveillance of semen donors should continue but the assay methods in current use have shortcomings.

Category 1 - Diseases where transmission via semen has been demonstrated Leptospirosis

The spirochaete bacterium, Leptospira interrogans which occurs in 26 serogroups.

Most warm-blooded species and some poikilotherms.

Present in semen during natural and experimental infection. Transmitted by artificial insemination and natural mating.

Category 2 - Diseases where causative organisms have been demonstrated in semen. Paratuberculosis

The acid-fast, Gram positive bacterium, Mycobacterium paratuberculosis which occurs as a cattle strain and two sheep strains.

Cattle, sheep, goats and deer.

Has been found in the semen of bulls. There is evidence against transmission by contaminated semen.

Mycobacterium paratuberculosis can remain infective on pasture for about a year and indefinitely at ultra-low temperature.

Transmission by semen is unlikely but risk can be controlled absolutely by screening of bulls before thay enter AI centres and by surveillance thereafter. Identification of organisms in faeces or complement fixation test for serum antibody is considered sufficient. However, the absorbed ELISA and gamma interferon assay increase sensitivity and specificity.

Disease

Causative organism

Species range

Presence in semen

Resistance to ultra-low

Commentary on risk

52

temperatures Category 3 - Diseases with a presumptive risk of transmission by bovine semen Aujeszky’s disease

Suid herpesvirus-1 (SVH-1), a member of the subfamily alphaherpesvirinae.

Occurs chiefly in pigs but can occur in most other mammalian species.

Heartwater

The tick-borne rickettsia, Cowdria ruminantium.

Cattle, sheep, goats and wild ruminants.

Q fever

The tick-borne rickettsia Coxiella burnetii.

Disease

Causative organism

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SVH-1 is found in the saliva and semen of acutely infected pigs but transmission by contaminated pig semen not demonstrated. The similar pathogenesis of SVH-1 in cattle raises risk of SVH-1 in bovine semen. Hypothetical risk is related to present of organism in leucocytes and the cell-assisted passage into semen.

Virus is inactivated in contaminated meat stored at -18ºC for 35 days but survives at -80ºC and -196ºC.

Cattle become infected usually by being bitten or nuzzled by infected pigs. Risk arises when cattle have direct contact with pigs or respiratory aerosols from pigs or feed and water contaminated by pigs. Serum antibody can be detected by immunodiffusion and ELISA tests.

Cowdria ruminantium can remain infective in spleen homogenates or blood after cryopreservation at -76ºC for two years.

Q fever occurs in man but Coxiella burnetii exists subclinically in sheep, cattle and goats

Hypothetical risk is the possible entry of Coxiella burnetii into semen by the same mechanism that allows entry into milk.

Coxiella burnetii maintains infectivity for long periods in tick faeces. Preserved at ultra-low temperatures.

Three-host ticks (Amblyomma spp) may be required for transmission and propagation of disease in new area. However, absence of known vectors should not justify use of semen at risk from contamination with Cowdria ruminantium. Semen from areas where Cowdria ruminantium is endemic should only be accepted from seronegative bulls according to the indirect fluorescent antibody test. A low risk of transmission by bovine semen but it is unlikely that such transmission would propagate Coxiella burnetii in new area without the vector. Semen donor bulls should be free of Coxiella burnetii as determined by serology (ELISA or complement fixation).

Species range

Presence in semen


Commentary on risk

Category 3 - Diseases with a presumptive risk of transmission by bovine semen (continued) Rabies

A lyssavirus in the family Rhabdoviridae.

Affects all warm blooded animals.

Hypothetical mechanism for contamination of semen is the same as that causing presence of virus in saliva. Virus can be present in saliva for a short period before onset of clinical signs.

Virus is fragile away from host and survives for only a few hours in dried saliva. However, virus survives at -196ºC.

There is a slight presumptive risk of virus being present in the semen of infected bulls for several days before symptoms of disease appear. Risk of transmission via semen would be contained by surveillance and maintenance of health in semen donors and a with-holding period of semen of 4 weeks.

Category 4 - Diseases with risk associated only with extrinsic contamination. Anthrax

The Gram positive spore-forming bacterium, Bacillus anthracis

Most herbivores susceptible; carnivores relatively resistant with omnivores in between.

Possible but unlikely because of the acute and peracute nature of the disease which would remove mating capacity of bulls before the period of risk.

Anthrax spores are extremely resistant and appear to persist indefinitely in endemic areas. Preserved at ultra-low temperatures

Hygienic collection of semen and vaccination of bulls should contain any risk of semen contamination by Bacillus anthracis if artificial breeding centres are located in endemic areas or “anthrax belts”.

Disease

Causative organism

Species range

Presence in semen


Commentary on risk

54

Category 5 - Diseases with no appreciable risk of transmission by bovine semen Echinococcosishydatidosis

The tapeworms, Echinococcus granulosus, E. multilocularis, E. oligarthus and E. vogeli.

Adult tapeworms reside in gut of dogs and other carnivores. Larval stages (hydatid cysts) occur in omnivores and herbivores.

Screwworm

The screw worm flies Cochliomyia hominivorax and Chrysomyia bezziana

Most warm-blooded animals.

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Presence in semen of ova requires gross contamination with dog or other faeces and is incompatible with semen preservation and viability Presence in semen incompatible with semen collection and storage procedures

Larval stage killed by freeze-thawing.

Risk is inconceivable.

Killed by freethawing.

Risk is inconceivable.

Category 1 - Diseases where transmission via semen has been demonstrated. Leptospirosis Leptospirosis occurs in all species of domestic livestock, many species of wild mammals and some cold-blooded vertebrates but not birds (Hunter and Herr, 1994). It appears as a range of syndromes including septicaemia, haemolytic anaemia, interstitial nephritis, mastitis, abortions, stillbirths and reproductive failure. It is caused by a pathogenic spirochaete, Leptospira interrogans, which exists in some 26 serogroups containing almost 200 serovars. The disease has a world-wide distribution and wild animals such as rats, mice, pigs, foxes and jackals provide a source of infection for livestock with specific serovars. Leptospirosis is an important zoonosis and transmission by bovine semen has been demonstrated. However, the best course of action for containing the risk has not been settled (see review by Eaglesome and Garcia, 1992). The following points help clarify the nature of the risk of transmitting leptospirosis in bovine semen. Firstly, the causative organism has been isolated from the semen of bulls after natural (Rodina and Balashov, 1971; Kiktenko et al., 1976) and experimental infection (Sleight et al. 1964). Secondly, leptospirosis can be transmitted in cattle by coitus and artificial insemination (Roberts, 1958; Sleight and Williams, 1961). Thirdly, the pathogenic serovars of Leptospira survive well in semen and semen diluents. Fourthly, infection can be inapparent or subclinical for both Leptospira pomona and Leptospira hardjo with organisms being shed into urine and contaminating semen via the urethra. Leptospiruria persists for longer than 450 days in cattle with experimental infections with Leptospira pomona (Doherty, 1967). Control of leptospirosis in artificial breeding centres for cattle is under active discussion (Eaglesome and Garcia, 1992). In Australia, cattle require negative results at two sequential agglutination lysis tests (microscopic agglutination tests) against Leptospira hardjo and Leptospira pomona at an interval of 4 to 8 weeks before admission to an artificial breeding centre. Treatment with streptomycin (at least one dose parenterally at 25 mg/kg liveweight) is a further requirement. Cattle may be accepted if they show a stable or falling antibody titre after antibiotic treatment. Further serological testing is required each year for surveillance. In addition, vaccination is recommended for consideration in some situations. Unfortunately, the serological diagnosis of leptospirosis is not clearcut. The microscopic agglutination test is relatively insensitive and inconsistent and cannot differentiate between vaccinates and chronic leptospirosis carriers (Ellis et al., 1981; Thiermann, 1984; Hathaway et al., 1986). As a result, the vaccination of bulls with multivalent vaccines is ruled out as a practical measure for preventing leptospiral infection or controlling leptospiruria. ELISA tests are available (Terpstra, 1989) but these have the same deficiencies as the microscopic agglutination test. The observation that seropositive bulls may not necessarily transmit leptospirosis via their semen (Gale and Kingscote, 1989) has raised concern about the exclusion of bulls from artificial breeding centres on grounds of circulating antibody alone. However, a more important concern for the serological screening of semen donors is that tests 56

such as the microscopic agglutination test are occasionally negative for infected animals (Chappel, 1994). In summary, there is a demonstrated risk of transmission of leptospirosis in bovine semen. However, the risk from cryopreserved semen is regarded as containable because antibiotics in semen diluent have been demonstrated to protect against the transmission of disease (Sleight, 1965). Dilution of semen itself and reduction of the possible dose of infective organisms may also have a protective impact (Eaglesome and Garcia, 1992). To guard against leptospirosis it is recommended that semen diluents contain antibiotics and that processing procedures for semen be specified (see page 4, Treatment of bovine semen to reduce risk). Serological testing as specified in the Australian health standards for artificial breeding centres (DPIE,1988) should continue as an interim measure but the test procedures in current use have shortcomings which can only be redressed by immunological research. The arguments against vaccination of donor bulls arise from difficulties in differentiating vaccinates from infected animals by serological methods rather than on the protective immunity given to semen donors (Eaglesome and Garcia, 1992). Vaccination cannot be disregarded as a possible control measure for leptospirosis in semen donor bulls and requires critical evaluation. Particular attention to the biology of the immune response may shed light on possible diagnostic tests for distinguishing vaccinates from infected animals. Category 2 - Diseases where causative organisms have been demonstrated in semen. Paratuberculosis Paratuberculosis or Johne’s disease (JD) is a chronic infectious enteritis of cattle, deer, sheep and goats characterised by persistent diarrhoea and progressive emaciation (see review by Chiodini et al., 1984). The principal lesion is thickening and corrugation of the intestinal wall. The causative agent is the acid-fast Grampositive bacterium, Mycobacterium paratuberculosis, and cattle may be infected with the cattle strain or either of the two sheep strains. Paratuberculosis occurs in most countries including Australia but has not been observed in Western Australia or Queensland. Mycobacterium paratuberculosis has been isolated from the testes, prostate and bulbourethral glands, and seminal vesicles of bulls and was found in the undiluted semen of one animal (Larsen and Kopecky, 1970). A further study demonstrated the organism in small numbers on repeated sampling from a single infected bull (Larsen et al., 1981). However, there is evidence against transmission occurring by artificial insemination with contaminated semen. An uncorroborated set of experiments with large numbers of M. paratuberculosis in doses used for transmission (Merkal et al.,1982), showed that organisms are eliminated from the uterus of cows when installed before arrival of the zygote, but remain viable and can be recovered from the uterus, but not the foetus, when installed after the zygote arrives. Apparently, uterine 57

infection does not interfere with attachment of the blastocyst nor with subsequent pregnancy. Transmission of M. paratuberculosis by artificial insemination, particularly with diluted semen, is regarded as highly unlikely (Chiodini et al., 1984; Eaglesome, Garcia and Stewart, 1992; Philpott, 1993). However, since the supporting experimental evidence has not been corroborated and because of the incidence of congenital infection (McQueen and Russell, 1979; Pearson and McClelland, 1955), discretion is necessary and minimum health standards (DPIE, 1988) are required. For this reason, bulls should be accredited against M. paratuberculosis before entry to artificial breeding centres and use as semen donors. Regular surveillance is required thereafter. Accreditation against M. paratuberculosis is complex and depends upon the the nature of paratuberculosis in cattle and the methods currently available for diagnosing infection. The crux is to recognise that infection with M. paratuberculosis can occur without disease. Accordingly, the following discussion takes the line that accreditation for the purpose of managing the risk of transmitting M. paratuberculosis in semen rests on the detection of clinically ill animals and animals that shed organisms in their faeces. The natural history of paratuberculosis supports the idea that symptomless carriers which do not shed M. paratuberculosis in their faeces will not be a risk. These animals will not have the organism in their semen either as a result of entry in the reproductive tract or by contamination during the process of collection. For M. paratuberculosis in cattle, resistance to infection as opposed to disease increases with age (Doyle, 1953: Larsen et al., 1975) and adult cattle are difficult to infect (Rankin, 1962). It is believed that organisms establishing during the first few weeks of life when animals are susceptible are the source of clinical disease later on. Faeces containing M. paratuberculosis is the principal source of infection (Larsen, 1972) and this usually occurs by mouth, perhaps from soiled teats (Julian, 1975). Ingested M. paratuberculosis enter the body via Peyer’s patches (Payne and Rankin, 1961) where they are held in check by phagocytes (Zubrick and Czuprynski, 1987) until other precipitating factors intervene and disease appears. The factors that precipitate clinical paratuberculosis themselves are unclear but poor protein/energy or mineral nutrition, high milk production, and parasitism have been implicated (Chiodini et al., 1984). The result is an incubation period ranging from 6 months to 15 years with clinical disease appearing most commonly at 3-5 years of age (Huchzermeyer et al., 1994). Many infected animal never show clinical disease and the organism can be shed in faeces before symptoms appear. Once underway, paratuberculosis is progressive and numbers of M. paratuberculosis organisms increase in line with the developing granulomatous inflammation of gut. As the pace of disease quickens, organisms are released from senescent macrophages and incite an antibody response, particularly IgG1 (Abbass and Riemann, 1988), or enter the gut lumen. A cell-mediated response can occur but specific immunological unresponsiveness (tolerance) is a feature of the disease. Eventually, infected macrophages enter the bloodstream and the possibility of spread to other tissues, including the reproductive tract, arises. According to this sequence of events, which 58

has been put together from the accounts of Chiodini et al. (1984) and Huchzermeyer et al. (1994), the earliest situation for entry of M. paratuberculosis into semen occurs when organisms are shed in faeces and there is an antibody response in blood. Accreditation against paratuberculosis requires diagnostic testing of bulls both before and after they become semen donors. There is a wide range of tests to choose from (Stephens, 1987). Two relatively recent tests, the absorbed ELISA and the interferon gamma assay, have both been trialled in Australia (Ridge et al., 1991; Billman-Jacobe et al., 1992). They have higher specificities and sensitivities than tests previously available and are recommended for this reason. However, given the argument that intrinsic contamination of semen is likely only when M. paratuberculosis is shed in faeces and antibody appears in blood, faecal culture for the organism and complement fixation tests for antibody remain satisfactory screening tests for semen donor bulls. In summary, M. paratuberculosis has been found in the semen of infected bulls. However, experiments indicate that transmission by artificial insemination with infected semen is unlikely. The risk of transmission by this route can be controlled absolutely by screening of bulls for infection with M. paratuberculosis before they become semen donors and by surveillance testing thereafter. Detection of organisms in faecal cultures or the complement fixation test for antibody are considered adequate screening tests. Nevertheless, newer tests such as the absorbed ELISA and gamma interferon assay are recommended for their greatly improved sensitivity and specificity. The transfer of bovine genestocks between countries by cryopreserved bovine semen is considered safer for the control of paratuberculosis than the transfer of live animals. Category 3 - Diseases with a presumptive risk of transmission by bovine semen. Aujeszky’s disease Aujeszky's disease or pseudorabies is a viral disease primarily of pigs which affects the nervous, reproductive and respiratory systems. It has a wide species range and occurs sporadically in cattle. Its broad geographical distribution includes the United States, Britain, Europe, Asia, North Africa and South America, New Zealand, but not Australia. The causative agent is suid herpes virus 1 (SVH-1), a member of the subfamily alphaherpesvirinae, which has a genome composed of supercoiled double stranded DNA. Because DNA viruses, and herpes viruses in particular, establish themselves longterm in the nucleus of host cells, suid herpes virus 1 has a hypothetical potential for intrinsic contamination of bovine semen. However, the nature of the disease (Maré, 1994) makes the risk of such contamination virtually non-existent in artificial breeding centres where semen donors have no physical association with pigs. Points relevant to the risk of transmitting Aujeszky's disease via bovine semen arise from the mode of transmission of suid herpes virus 1, differences in the disease between cattle and pigs, and the carrier state that occurs in pigs. Acutely infected pigs excrete virus in nasal discharges, respiratory aerosols, saliva and semen (Maré, 1994). 59

Infection of susceptible pigs usually occurs under conditions of direct contact either through inhalation of aerosols or by ingestion of contaminated food and water. Carrier pigs may transmit infection to their offspring during pregnancy. The portal of entry of virus in all species is through the intact respiratory epithelium or through abraded skin and cattle usually become infected by being bitten or nuzzled by infected pigs. Mortalities approaching 100% are seen in infected susceptible newborn piglets but these reduce to about 5% at five months of age. In contrast, the disease is regarded as being 100% fatal for cattle but a non-fatal case has been recorded (Hagermoser and Moss, 1978). A carrier state is important for propagating Aujeszky's disease in pigs and transmitting the virus to "dead-end" hosts such as cattle. Pigs become carriers after recovery from the disease or after symptomless infection as adults. Vaccinated pigs exposed to infection can become carriers (Mock, Crandall and Mesfin, 1981; Wittman, Ohlinger and Rziha, 1983). Until vaccines are available which can prevent the carrier state, vaccination can be regarded as a control procedure of last resort in pigs. Since most cattle die after exposure to suid herpes virus 1, the carrier state is not a feature of infection in this species. However, cattle can be vaccinated with inactivated virus (Biront et al ., 1982: Van Oirschot , De Leew, P.W. and Tiessink, J.W., 1985). Given the nature of herpes viruses and the experience with pigs, vaccination can be expected to predispose towards the carrier state and create circumstances for the transmission of Aujeszky's disease via semen. It is not known whether the virus is shed in the semen of carrier pigs. In summary, the risk of transmission of suid herpes virus 1 via bovine semen is possible in regions where Aujeszky's disease is recorded. The risk arises when cattle have direct contact with pigs or are exposed to respiratory aerosols from pigs or feed or water contaminated by pigs. Risk will be increased by vaccination with inactivated vaccines which will presdispose to the carrier state. Antibody to suid herpes virus in serum can be detected by enzyme-linked immunosorbent assays and various immunodiffusion tests (Goyal et al., 1987; Van Oirschot and De Waal, 1987). Heartwater Heartwater is an acute tick-borne rickettsial disease of cattle, sheep, goats and wild ruminants characterised by high fever, nervous signs, hydropericardium, hydrothorax, oedema of the lungs and brain and death. It occurs in most of sub-Saharan Africa except for Namibia, Burkino Faso, Guinea and Sierra Leone. It also occurs in Madagascar, São Tomé, Réunion, Mauritius, Zanzibar and some islands in the Caribbean. Heartwater is regarded as a major obstacle to livestock improvement by the introduction of high producing animals in much of Africa (Bezuidenhout et al., 1994). The causative agent is Cowdria ruminantium, a member of the tribe Ehrlichieae in the family Rickettsiaceae of the order Rickettsiales. Heartwater is transmitted in nature by three-host ticks of the genus Amblyomma and occurs only when these vectors are present. Transmission by semen is not mentioned in reviews of the disease (Blood and Radostits, 1989: Bezuidenhout et al., 1994) and 60

the possibility does not appear to have been pursued experimentally. However, transmission by semen is a hypothetical possibility for two reasons. Firstly, the organism is carried in neutrophils (Logan et al. 1987) which could migrate into seminal fluid under the influence of inflammatory responses occurring in the male reproductive tract. Secondly, the organism can be experimentally transmitted from animal to animal with blood or spleen homogenates which can remain infective after cryopreservation at -76°C for two years (Neitz, 1968). In summary, there is a risk of transmission of heartwater via preserved bovine semen. The extent of the risk is not known but is likely to be slight and to occur only when semen donors show clinical signs of disease. Vaccination with live virulent organisms is practised as a form of control. However, it is not accompanied by a sterile immunity and may not reduce the possibility for intrinsic contamination of semen in vaccinated semen donors. Three-host ticks (Amblyomma spp) are essential for the propagation of heartwater disease in any new area. Accordingly, the introduction of heartwater organisms in semen is unlikely to lead to the establishment of disease where vector ticks are not found. However, the reported absence of intermediate hosts from a region should not be used to rationalise the introduction of bovine semen likely to contain Cowdria ruminantium. Semen from areas where Cowdria ruminantium is endemic should only be accepted from bulls that are seronegative according to the indirect fluorescent antibody test recommended by the OIE as the standard test for international trade. Q fever Q fever is an acute, febrile, systemic disease mainly of man caused by the tick-borne rickettsia., Coxiella burnetii. The causative organism can exist subclinically in sheep, cattle and goats but abortions have been associated with heavy infections in sheep and goats (Blood and Henderson, 1989). Q fever has public health importance (Stevenson and Hughes, 1988) as a zoonotic disease. There is indicative evidence that Coxiella burnetii can be transmitted through bovine semen. The organism is maintained in nature by cycles such as that occurring in Australia in the bandicoot and at least six species of ticks (Albiston, 1965). Infection occurs through tick bites or by the inhalation or ingestion of dusts containing tick faeces and the organism which maintains its infectivity in such conditions. Humans can also be infected by the ingestion of unpasteurised infected cow’s or goat’s milk (Woodward, 1987). It is possible that the mechanisms allowing passage of Coxiella burnetii into milk will also allow intrinsic contamination of bovine semen. In summary, there is a risk of transmitting the disease agent of Q fever in bovine semen but it is highly unlikely that such transmission would propagate the disease in cattle in a new area where vectors are not found. Extrinsic contamination is possible through rickettsial laden dust particularly from the hair coat of bulls containing dried tick faeces. Intrinsic contamination is also possible from systemic infection through the same mechanism that allows Coxiella burnetii to enter milk. Semen donor bulls must be free of Coxiella burnetii as determined by serology. Complement fixation, micro-agglutination, immunofluorescence tests and ELISAs are available.

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Rabies Rabies is a viral disease of all warm blooded animals which affects the nervous system and is almost invariably fatal. It occurs in Europe (except for Britain and Ireland), the Americas, the Middle East, Africa and most of Asia, but not Australia, Japan, Singapore, some of the Indonesian islands, Papua New Guinea or New Zealand and other Pacific Islands. The causative agent is a lyssavirus in the family Rhabdoviridae and has a genome composed of single stranded, antisense RNA. Rabies is usually spread by the bites of infected animals and is not listed as a disease where transmission by semen is a tangible risk. However, since the disease has outstanding implications for public health and is of veterinary importance, the risk will be explained. Key factors against transmission of rabies in bovine semen are the highly neurotropic behaviour of the virus and the high fatality rate which removes the possibility of a carrier state and subsequent "silent" shedding of the virus. The virus is fragile away from the host, is susceptible to most disinfectants and survives for only a few hours in dried saliva. The usual means of transmission is through saliva and oral secretions carried in the bite of infected animals. Spread from the site of infection occurs through the peripheral nerves to the spinal cord, eventually ascending to the brain. Descending migration of virus down cranial nerves explains its presence in the cornea, saliva and mouth secretions. Virus does not enter the bloodsteam and intrinsic contamination of semen by virus passing from blood to the reproductive system is improbable. However, intrinsic contamination could occur during the course of disease through virus descending peripheral nerves and entering the secretions of accessory glands in the male reproductive system. The presence of virus in saliva up to five days before the onset of clinical signs of rabies (Blood and Radostits, 1989) provides a guide as to when the virus might occur in semen. In summary, the transfer of rabies virus in semen has not been demonstrated experimentally and is not observed in nature. However, there is a slight presumptive risk of virus being present in the semen of infected bulls for several days before symptoms of disease appear. The risk of transmitting rabies in bovine semen would be contained by surveillance and maintenance of health in donors and a withholding period for semen of four weeks. Category 4 - Diseases with risk associated only with extrinsic contamination. Anthrax Anthrax is an acute to peracute disease characterised by septicaemia (de Vos, 1994). It affects a wide range of wild and domestic animals, particularly herbivores. The disease is found worldwide and is often enzootic to particular areas, “anthrax belts”, where soil conditions favour survival of the disease agent. Anthrax is caused by a Gram-positive sporeforming rod, Bacillus anthracis. The disease is not listed as a 62

contender for transmission via bovine semen in reviews of this subject. However, since anthrax is a zoonosis (Stevenson and Hughes, 1988), the risk requires clarification. Anthrax is usually transmitted by contact with infected animals, animal products or soil but disease can occur after inhalation or ingestion of B. anthracis spores. The portal of entry is either through intact mucous membranes or through breaks in skin and mucous membranes. Organisms are contained in phagocytes during the early stage of disease but then proliferate extracellularly at an explosive rate. Death from septicaemia is rapid and mediated by lethal toxins produced by the organism. No carrier state exists after recovery from the disease and the organism does not survive unless spore-formation is induced by contact with air. In summary, the acute nature of anthrax and the absence of the carrier state are barriers to the intrinsic contamination of bovine semen with B. anthracis. Extrinsic contamination through spores in dust is possible if artificial breeding centres are located in known “anthrax belts”. However, the hygienic collection of semen should prevent this risk. Category 5 - Diseases with no appreciable risk of transmission by bovine semen. Screw-worm flies (Cochliomyia hominivorax and Chrysomyia bezziana) and echinococcosis-hydatidosis Transmission via bovine semen of the screw-worm flies (Cochliomyia hominivorax and Chrysomyia bezziana) or echinococcosis-hydatidosis is inconceivable in view of the life history and morphology of the metazoan organisms involved. In addition, the causative agents do not survive freezing to -20°C and subsequent thawing.

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DISEASES OF CATTLE Table 4 shows the OIE List B diseases which specifically infect cattle and summarises properties relevant to the risk of transmission via bovine semen. These diseases are grouped according to categories of risk in both Table 4 and the ensuing discussion.

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Table 4 Summary of the OIE List B diseases which are specific for cattle and the factors influencing risk of transmission by semen Disease

Causative organism

Host range

Presence in semen

Resistance of causative organism to ultra-low temperatures

Commentary on risk

Herd freedom from infection is called for. Indirect haemolysis tests and ELISAs are more sensitive than agglutination and complement fixation assays and are the serological tests of choice for detecting infected bulls. Regardless of serological results, bulls with palpable lesions in testes, seminal vesicles or other organs in the reproductive systems should be excluded. • Freedom from infection must be demonstrated in donor bulls and supported by herd freedom. Identification of C. fetus venerealis in preputial smegma and washings is the recommended

Category 1 - Diseases where transmission via semen has been demonstrated Bovine brucellosis

The Gram-negative intracellular bacterium Brucella bovis.

Mainly cattle but can infect humans, horses, dogs and some other species.

Transmission of Brucella bovis by artificial insemination has been demonstrated.

Survival for more than 800 days in water at -40°C has been demonstrated.

Bovine genital campylobacteriosis

The Gram-negative microaerophilic rod, Campylobacter fetus subspecies venerealis.

C. fetus venerealis is an obligate parasite of the genitalia of male and female cattle.

Transmission of C. fetus venerealis by artificial insemination is a long established risk.

C. fetus venerealis survives in cryopreserved semen with infectivity intact.

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•

•

Disease

screening test. A mixture of the antibiotics, tylosin, gentamycin, lincomycin, and spectinomycin in semen diluent aids in control but should not be relied upon as the only method. Care must be taken with the method of adding glycerol which counteracts the action of antibiotics. Vaccination can be used to eradicate C. fetus venerealis from AI centres.

Causative Host range Presence in Resistance of causative organism to organism semen ultra-low temperatures Category 1 - Diseases where transmission via semen has been demonstrated (continued)

Commentary on risk

Bovine tuberculosis

Semen donor bulls must be free of infection with M. bovis and herd freedom provides the best guarantee. Negative results to tests must be present before entry to AI centres and bulls must be negative to routine tests thereafter. Sensitivities from 66-95% have been

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The acid -fast bacterium, Mycobacterium bovis.

Cattle, goats and pigs are the most susceptible species but horses and sheep are resistant. M. bovis causes severe disease in humans.

Artificial insemination with M. bovis contaminated semen will lead to infection.

M. bovis survives in cryopreserved semen with infectivity intact.

Bovine virus diarrhoea/ mucosal disease

A virus in the Pestivirus genus of the Flaviviridae

Cattle, other ruminants, pigs.

Semen produced by symptomless carrier animals may contain virus and transmission by artificial insemination has been demonstrated.

Remains infective in cryopreserved semen.

Disease

Causative organism

Host range

Presence in semen

Resistance of causative organism to ultralow temperatures

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observed with intradermal tests. The interferon gamma assay on whole blood may have greater sensitivity. The detection of infected animals may be improved by using skin tests plus the gamma interferon assay. Herd freedom required in AI centres based on the identification and removal of infected bulls. Virological tests are preferred for the detection of infected animals because serological tests are compromised by the carrier animals which are immunotolerant. The poor sensitivity of earlier virological detection methods has been improved by antigen capture ELISA tests and reverse transcription polymerase chain reaction(RT-PCR) tests.

Commentary on risk

Category 1 - Diseases where transmission via semen has been demonstrated (continued) Infectious bovine Bovine herpesvirus-1 Cattle Regarded as the most rhino(BHV-1). common pathogen in tracheitis/ infectious bovine semen. pustular Transmission by vulvovaginitis artificial insemination verified. Virus shed in semen of asymptomatic carriers.

Trichomoniasis

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The flagellated protozoan parasite Tritrichomonas foetus.

Cattle

Transmission of T. foetus by artificial insemination is well documented.

Virus remains infective in cryopreserved semen. Stable for 30 days in culture medium at 4ºC.

Preservation of T. foetus for 5 years at -79°C was an early discovery.

The use of seronegative bulls in AI centres has been regarded as best option for removing BHV1 from semen. Pre-entry and routine tests of bulls by virological means can rid AI centres of virus but risk of re-entry remains because of ubiquity of the virus. ELISA tests have yet to be standardised between laboratories. A polymerase chain reaction (PCR) method has been developed for the direct detection of virus in semen. When it is accredited, it could change the approach towards obtaining BHV-1 free semen. Herd freedom required. Freedom from infection with T. foetus is required in semen donor bulls. The direct microscopic detection of T. foetus in preputial washings from bulls can be used for screening and surveillance. The present Australian requirements of

negative results to four tests a week apart before bulls are admitted to AI centres would contain risk. Herd freedom preferred. Category 2 - Diseases where causative organisms have been demonstrated in semen Enzootic bovine The bovine Cattle Results of leukosis leukaemia virus, a transmission studies retrovirus in the have been variable. Oncovirinae No transmission subfamily of the found by most work. Retroviridae.

Disease

Causative organism

Host range

Presence in semen

Category 3 - Diseases with a presumptive risk of transmission by bovine semen Bovine malignant A herpes virus, Disease in cattle but Presumptive risk is a catarrh alcephaline herpes reservoir in sheep and consequence of virus 1. other ruminants such possible migration of as the wildebeest. virus-containing migratory cells into semen.

Bovine spongiform

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A proteinaceous

Cattle mainly but

Epidemiological

Survival at temperatures for the cryopreservation of semen.


Virus survives at the temperatures used for the cryopreservation of semen.

Causative agent resistant to heat. Preserved at

Absolute protection against transmission in semen of bovine leukosis would be obtained by using seronegative bulls only. ELISA tests for antibody is more sensitive than agar gel immunodiffusion. A test for tumour-associated antigen is under development. Commentary on risk

Risk would occur only during the incubation period of disease and would be contained by a withholding period for semen. Since the caustive agent is spread from sheep to cattle, the risk would be contained by keeping semen donor bulls away from sheep. Until results of definitive

encephalopathy (BSE)

infective particle or prion related to the causative agent of scrapie in sheep,

captive wild ruminants and cats were infected by ingesting contaminated meat and bone meal.

evidence indicates no spread via artificial insemination. Presumptive risk is proposed until the results of definitive transmission experiments are available.

the ultra-cold temperatures used for the storage of bovine semen.

Haemorrhagic septicaemia

The bacterium, Pasteurella multocida.

Cattle and buffaloes.

Bacteria enter saliva, milk and urine during disease and could enter semen via the same mechanism.

Preserved at the ultra-cold temperatures used for the storage of bovine semen.

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transmission studies are available, precautions should operate for the trade in bovine semen. Freedom from exposure to BSE in donor bulls is required. Semen from bulls born since the 1988 feed ban in the U.K. would be acceptable as would semen collected up to 1981-2 before the appearance of the disease. Semen from bulls born before the feed ban would be acceptable if it could be demonstrated that they have never been fed ruminant derived protein. Presumptive risk of transmission of P. multocida via semen unlikely to be realised because mating capacity in bulls would be abolished early in infection by fever and depression. Risk controlled completely by ensuring that donor bulls are not carriers as detected by isolating P. multocida in nasopharyngeal secretions.

Disease

Causative organism

Host range

Presence in semen


Commentary on risk

Category 3 - Diseases with a presumptive risk of transmission by bovine semen (continued) Theileriosis

Intracellular protozoan parasites of genus Theileria (family Theileridae, order Piroplasmia of class Sporozoa). The Theileria parva complex causes East Coast fever, Corridor disease and Zimbabwe theileriosis. T. annulata causes Mediterranean theileriosis.

Disease occurs in cattle and requires vector ticks to transmit disease and maintain the parasite in nature. Rhipicephalus appendiculatus and 15 other species vector for Theileria parva. Hyalomma species involved for Theileria annulata.

Hypothetical risk related to the schizont life stage in lymphocytes and the possibility that these migratory cells may carry organism into semen. However, there is no epidemiological evidence for semen being contaminated.

Organism maintained in blood stored at -79° C or -196°C for laboratory studies.

Trypanosomiasis

Extracellular protozoan parasites of genus Trypanosoma (suborder Trypansomatina, order Kinetoplastida, class Zoomastigophora, phylum Sarcomastigophora).

Trypanosomes in question are T. congolense, T. vivax,T. evansi and T.brucei which infect cattle (causing nagana), sheep, goats, pigs, horses and donkeys. T. brucei severe in humans. Tsetse flies (Glossina

Hypothetical risk occurs because trypanosomes can be found outside the bloodstream and thus could invade semen. In addition, tsetse fly vector may not be obligatory. Mechanical transmission by other

Organism maintained in blood stored at -79° C or -196°C for laboratory studies.

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To contain presumptive risk, bulls must be confirmed free of infection before entry to artificial breeding centres and during the time they provide semen. Diagnosis of freedom can be made with smears of peripheral blood and fine needle aspirates of superficial lymph nodes. The presenceof piroplasms indicates infection and the presence of hyperplastic lymphocytes should raise suspicion. Presumptive risk of transmission of the African trypanosomiases occurs only where these diseases are endemic. Semen should only be accepted from bulls maintained in or derived from such areas if freedom from infection can be demonstrated.

Disease

Causative organism

spp) are vector.

biting flies possible.

Host range

Presence in semen

Category 4 - Diseases with risk associated only with extrinsic contamination Anaplasmosis The rickettsiae, Cattle and other Risk to semen is Anaplasma centrale herbivores such as through and Anaplasma antelope, deer, elk contamination with marginale; obligate and camels. red blood cells as a parasites of red blood Transmission in result of trauma to cells. Australia via one-host reproductive tract or tick Boophilus penis and prepuce microplus. Blood is and subsequent infective and bleeding. anaplasmosis can be transmiited during husbandry operations like castration and eartagging.

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Organism maintained in blood stored at temperature of liquid nitrogen for laboratory studies.

However, diagnosis of trrypanosomiasis can be difficult. Blood smears alone are too insensitive. An antigen-trapping ELSIA shows promise but has not been fully accredited. Commentary on risk

*

Risk from anaplasmosis itself is associated with the presence of red blood cells in semen and contamination by extravasated blood. Semen donors should be diagnosed free of anaplasmosis for this reason.

*

Blood-derived vaccines are used against anaplasmosis. These raise the possiblility of infection with bovine pestivirus,

Babesiosis

Protozoan parasites of bovine red blood cells, Babesia bovis and Babesia bigemina (family Babesiida, suborder Piroplasmorina, order Eucoccidiodora, class Sporoasida, phylum Apicomplexa.

Cattle and buffalo. Babesias as a group are relatively host specific.

Risk to semen is through contamination with red blood cells as a result of trauma to reproductive tract or penis and prepuce and subsequent bleeding.

Organism maintained in blood stored at temperature of liquid nitrogen for laboratory studies.

*

*

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bovine herpesvirus-1 and the bovine leukaemia virus and the risk these organisms have for semen. Risk from babesiosis itself is associated with the presence of red blood cells in semen and contamination by extravasated blood. Semen donors should be diagnosed free of anaplasmosis for this reason. Blood-derived vaccines are used against babesiosis. These raise the possiblility of infection with bovine pestivirus, bovine herpesvirus-1 and the bovine leukaemia virus and the risk these organisms have

for semen.

Disease

Causative organism

Host range

Presence in semen

Resistance of causative organism to ultra-low temperatures

Commentary on risk

Category 4 - Diseases with risk associated only with extrinsic contamination (continued). Dermatophilosis

The pleomorphic bacterium, Dermatophilus dermatonomus (family Dermatophilaceae of the order Actinomycetales).

Mainly in herbivores but Dermatophilus dermatonomus will affect other species.

Possible only by contamination from lesions on skin during collection of semen.

Viable at storage temperatures for semen.

Extrinsic contamination of semen would require the presence of the obvious skin lesions of dermatophilosis. Dermatophilosis is readily controlled by antibiotics such as long-cating oxytetracycline or penicillin and streptomycin or dihydrostreptomycin in combination.

C. bovis killed by freeze-thawing. Cestode eggs may remain infective after cryopreservation.

Gross contamination by human or dog faeces would destroy semen for any possible use.

Category 5 - Diseases with no appreciable risk of transmission by bovine semen. Cysticercosis (C. bovis)

The intermediate stage of the tapeworm, Taenia saginata. Adult tapeworms reside in gut of dogs and humans.

C. bovis occurs in cattle only

Possible only through gross contamination with dog or human faeces containing ova.

Category 1 - Diseases where transmission via semen has been demonstrated.

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Bovine brucellosis The disease, bovine brucellosis, is characterised by abortion late in pregnancy which leads to a high rate of infertility and by inflammation of the testes and other organs in the reproductive tract of bulls (Nicoletti, 1980). It is distributed worldwide but has been eradicated from Australia, Canada, Israel, Japan, Austria, Switzerland, Denmark, Finland, Norway, Sweden, New Zealand and certain Pacific islands. The causative agent is the intracellular bacterium, Brucella abortus; a small, Gram-negative, non-sporulating and non-encapsulated coccobacillus. Brucella abortus has zoonotic importance, causing undulant fever in people. Transmission of the disease by artificial insemination has been demonstrated (Bendixen and Blom, 1947; Manthei, Detray and Goode, 1950) but natural service is not considered an important means of spread (Rankin, 1965). Infection usually occurs orally through licking or nuzzling infected foetal membranes, foetal fluids, lochia or other genital secretions or by consuming contaminated feed or water. The organism is shed irregularly in milk and may be found in urine and faeces of infected animals. B. abortus produces a fibrotic and necrotising orchitis and epididymitis with granuloma formation and a fibrinpurulent and necrotic inflammation of seminal vesicles (Ladds, 1985). Organisms have been isolated from the ampulla and seminal vesicles of an infected bull (Plant et al., 1976). The Brucellae are intracellular parasites (Enright, 1990) and their presence in mobile cells such as macrophages can be expected to enhance their migration into tissues of the male reproductive tract and semen. In summary, the transmission of bovine brucellosis by artificial insemination is a proved risk. Accordingly, semen should only be accepted from brucellosis free animals maintained in a brucellosis free herd to protect against both intrinsic and extrinsic contamination. The approach for demonstrating freedom from brucellosis in the Australian health standards for artificial breeding centres (DPIE, 1988) calls for negative results on an approved test with serum collected at 4 and 8 weeks before bulls are admitted to artificial breeding centres. Retesting of bulls in centres is required annually. The testing program of OIE is similar. The difficulty of diagnosing infection with B. abortus in bulls (Plant et al., 1976) has implications for demonstrating disease freedom in semen donors. For example, bulls with clinical signs of brucellosis such as orchitis, epididymitis, ampullitis or vesiculitis may not show positive (Plant et al., 1976) to OIE recommended serological tests such as agglutination or complement fixation (OIE, 1990). Indirect haemolysis tests (Plackett et al. 1980) and enzyme-linked immunosorbent assays (Cargill, Lee and Clarke, 1985; Sutherland, 1984 and 1985) detect a greater number of infected animals, in other words are more sensitive, than serum agglutination, complement fixation or the rose

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bengal test. However, such tests may be less specific. It is not clear whether the problem of serological diagnosis in bulls is biological rather than technical. A deficiency in antibody production as a result of infection in immunologically privileged sites may be responsible. If this is so, increases in the sensitivity of diagnostic tests may not be gainful. The problem of demonstrating freedom from brucellosis in bovine semen donors is partly answered in the Australian minimum health standards for artificial breeding centres (DPIE, 1988) with the ambit requirement that all sires be examined to show freedom from evidence of infectious disease. In countries where bovine brucellosis has not been eradicated, semen should not be accepted from bulls which show orchitis or epididymitis on palpation or vesiculitis, ampullitis, vesiculitis or bulbo-urethritis on rectal examination. At the moment, serological evidence of disease freedom, regardless of the test employed, is not sufficiently credible to take precedence over physical examination in such circumstances. At the same time, however, serological evidence of brucellosis should disqualify a semen donor in the absence of clinically obvious physical changes. Bovine genital campylobacteriosis Bovine genital campylobacteriosis is an infectious venereal disease characterised by disrupted oestrous cycles, death and resorption of embryos, decreased fertility and increased intercalving intervals, and, on some occasions, by abortions (see reviews by Dekeyser, 1986; Schutte et al., 1994). The disease occurs wherever cattle are raised throughout the world. The causative agent is bacterial; the Gram-negative, microaerophilic rod, Campylobacter fetus subspecies venerealis which inhabits the genital tract of both cows and bulls. Campylobacter fetus subspecies fetus is less common and causes sporadic abortions in cattle. A related organism, Campylobacter jejuni, has been implicated in abortions in cattle but is more commonly associated with infection of the gut. Transmission of bovine campylobacteriosis by artificial insemination is a confirmed risk which was recognised early in the history of this technology (Willett et al., 1955; Hughes, 1956; Bartlett, 1981). Under natural conditions, bulls become infected by serving infected cows but some transmission occurs through contact with contaminated bedding (Dekeyser, 1986). The result is the carrier state with no noticeable pathological changes. Bulls over four years of age are more susceptible than younger bulls, perhaps because of changes in the mucosa of penis and prepuce (Samuelson and Winter, 1966). The immune response against C. fetus induced by infection is feeble and coincides with a long survival of organisms in the penis and prepuce (Hoffer, 1981). The risk of transmitting C. fetus in semen can be contained by detecting carrier bulls before their entry to artificial breeding establishments and use as semen donors and by regular testing thereafter (DPIE, 1988). Identification of C. fetus in preputial smegma and washings is the

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diagnostic test used for screening (Hum and McInnes, 1993). Vaccination of bulls has been used to eradicate campylobacteriosis from artificial breeding centres (Clark et al., 1974 and 1979). Antibiotic treatment of semen has had a history as a control method against C. fetus (Eaglesome, Garcia and Stewart, 1992) which includes the discovery that glycerol abolished the efficacy of streptomycin (Brinley Morgan et al., 1959). The protocols for collection, handling, preservation and storage of semen of Certified Semen Services (quoted in Parsonson, 1993) use an antibiotic mixture of tylosin, gentamycin, lincomycin and spectinomycin and require that glycerol is not added as an extender component until after the semen and non-glycerol extender has cooled to 5ºC. Bovine tuberculosis Tuberculosis is a chronic bacterial infection characterised by the formation of distinctive granulomas known as tubercles in any organ of the body, a vigorous cell-mediated hypersensitivity, and progressive wasting which ultimately results in death. The disease occurs in all domestic species except sheep and has been recorded in all countries. The causative organism of bovine tuberculosis is the acid-fast bacterium, Mycobacterium bovis. The organism has zoonotic importance and people can die before infection is diagnosed (Grange and Yates, 1994). Mycobacterium tuberculosis, the causative organism of human tuberculosis, can infect cattle but is not maintained within this species and is a minor cause of disease. Bovine tuberculosis has particular importance to Australia because a rigorous eradication campaign has been successful (Tweddle and Livingstone, 1994). Re-introduction of the disease would undo the benefits obtained for public health, trade and agricultural production. Mycobacterium bovis is usually spread by the inhalation of infected droplets from the respiratory tract. However, other routes of infection are possible and include gut, skin, the teat canal and the reproductive system. Venereal transmission from the penis and prepuce of bulls can occur (Thoen et al., 1977) and artificial insemination with preserved semen is a proved risk. Roumy (1966) describes a history where a bull which had been used as a semen donor for over 1,000 cows was found to have generalised tuberculosis on slaughter. Tuberculosis with lesions of internal organs established in more than 10% of semen recipients. The presence of viable Mycobacterium bovis in mobile phagocytes is part of the natural history of infection and would facilitate passage of organisms into semen, accounting for intrinsic contamination. In summary, the transmission of bovine tuberculosis via artificial insemination with preserved semen is a demonstrated risk. However, it is a risk that can be effectively contained by procedures already in operation (OIE, 1992: DPIE, 1988). Semen should only be accepted from tuberculosis free animals maintained in a tuberculosis free herd to protect against both intrinsic and extrinsic contamination.

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As to the demonstration of freedom from bovine tuberculosis, minimum health standards for artificial breeding centres in Australia (DPIE, 1988) call for an approved test with negative results before bulls are admitted to artificial breeding centres and retesting at intervals of three years again with an approved test and negative results. The testing program of OIE requires a negative result to the intradermal test with “mammalian” tuberculin and routine tests at least every 12 months. The question of the approved test requires clarification because tests of cell-mediated hypersensitivity other than skin tests are now available. Skin tests such as the single intradermal test and comparative intradermal test are durable and well established. However, they have a variable sensitivity (proportion of true positive reactors) ranging from a low of 66% (Wood et al, 1991) to a cluster around 77-95% (Monaghan et al., 1994). An in vitro assay for the cytokine1 , bovine gamma-interferon, in incubated whole blood (the IFN- assay; Wood and Rothel, 1994) is now available. This has an increased sensitivity compared with the single intradermal test, 94% versus 66% (Wood, et al., 1991), but a similar specificity (proportion of true negative reactors). Some infected animals were positive to the IFN- assay but negative on skin test while other infected animals were positive to the skin test but negative to the IFN- assay. As a result, it may be beneficial to use the two tests in combination. Bovine virus diarrhoea/Mucosal disease Bovine virus diarrhoea and mucosal disease are two disease syndromes of cattle caused by the same virus which is found throughout the world (see reviews by Radostits and Littlejohns, 1988; Harkness and van der Lugt, 1994). Bovine virus diarrhoea is a minor disease which lasts for a few days and has a high morbidity but low mortality. By contrast, mucosal disease has a low morbidity but is invariably fatal. It arises when cattle infected with a non-cytopathic strain of the virus in utero are challenged with a cytopathic strain during adult life. The most striking lesions of both syndromes occur in the gut but the virus has a strong affinity for lymphoreticular tissue. The causative agent (bovine pestivirus) is a member of the Pestivirus genus of the family Flaviviridae (Wengler, 1991) with a genome composed of single-stranded message-sense RNA and a tendency to appear in semen. 1 Cytokines or interleukins are soluble products of macrophages or T-lymphocytes, usually protein, which influence the differentiation of themselves or other cells and which are important regulators of the immune response.

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Bovine pestivirus is now recognised as a cause of production-limiting disease characterised by embryonic death, abortions and stillbirths if infection occurs in the first 125 days of pregnancy, foetal abnormalities if infection occurs between 125 and 180 days and small weak calves if infection occurs thereafter. Persistently infected calves grow poorly and up to 45% may either die or be culled in the first two years of life (quoted in Harkness and van der Lugt, 1994). Infections early in foetal life lead to specific immunological unresponsiveness and bring about an epidemiologically significant carrier state with a lifelong viraemia. One result is carrier bulls that excrete virus into semen in the absence of serum antibody. Bovine pestivirus is a significant contaminant of bovine semen (Afshar and Eaglesome, 1990; Philpott, 1993) where its presence may be facilitated by the migration of infected lymphocytes. It was the only virus found repeatedly at infective concentrations in bovine semen in a survey which used the Cornell semen test (Schultz et al.,1982). Complications are created by the existence of the carrier state in immunotolerant donor bulls. Semen produced by carrier animals without clinical signs may contain virus and infection can be transmitted from this source by natural mating or artificial insemination (Coria and McClurkin, 1978; Barlow et al., 1986; Bolin et al., 1987; Meyling and Jensen, 1988). Infected cattle also shed large amounts of virus in oral and nasal secretions and urine. As a result, carrier bulls can also spread the virus to uninfected herdmates in artificial breeding centres (Meyling and Jensen, 1988: Paton et al.,1989) but these latter animals shed less virus in their semen during their transient infections. Contamination by bovine pestivirus is an issue for the international and domestic trade in bovine semen. It is not currently covered in the Australian health standards for stock at artificial breeding establishments (DPIE, 1988). According to the behaviour of bovine pestivirus in cattle, its control in artificial breeding centres must be based upon herd freedom and involve the identification and disposal of persistently infected bulls (Afshar and Eaglesome, 1990). Herd freedom can also form the basis for international standards. Virological methods for the direct detection of viraemia in infected cattle may have an advantage over serological tests because of the immunotolerance produced by infection during foetal life. Antigen capture ELISA tests (Shannon et al (1991), as set out by Kirkland and MacKintosh (1992), and reverse transcription-polymerase chain reactions (RT-PCR; Hertig et al., 1991) are major advances over earlier methods for viral detection which may have had problems of sensitivity (Afshar and Eaglesome, 1990). RT-PCR appears to be more sensitive than antigen capture ELISA (Horner et al., 1995). However, the ELISA was suggested as being the most suitable for large-scale testing for the diagnosis and control of pestivirus infections. It could be used for the screening of bulls before entry to artificial breeding centres and for surveillance of all bulls thereafter.

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Nevertheless, serological tests are valuable and could still contribute to testing for freedom from pestivirus in bulls. Semen from clinically healthy bulls which are seropositive by virus neutralisation or ELISA would be acceptable. Seronegative bulls are the diagnostic problem since this could result from no exposure or from immunotolerance in carrier animals. Accordingly, seronegative bulls should be submitted to virological examination before their semen could be regarded as acceptable. Seronegative bulls that seroconvert at retesting after 2 to 4 weeks could be regarded as safe from then on. Given the ubiquitous nature of bovine pestivirus, vaccination has been suggested for its control in artificial breeding centres but has some shortcomings. Present live vaccines may damage the health of cattle through immunosuppression and subsequent potentiation of other infections (Roth and Kaeberle, 1983; Wray and Roeder, 1987). Killed vaccines appear to be safe (Harkness and van der Lugt, 1994). Advances are being made in the molecular biology of bovine pestivirus (Ridpath et al., 1994) and an effective sub-unit vaccine is likely to be under consideration. Infectious bovine rhinotracheitis/infectious pustular vulvovaginitis BHV-1 and bovine pestivirus are foreshadowed as latent disease problems of significant magnitude in the present context. Both viruses have a potential for widespread dissemination by artificial insemination with virus-contaminated bovine semen. In addition, both viruses can be highly pathogenic under circumstances such as intensive husbandry in feedlots. Infectious bovine rhinotracheitis and infectious pustular vulvovaginitis are two syndromes caused by bovine herpes virus type 1 (BHV-1) which has other effects including abortion, conjunctivitis, meningoencephilitis, and generalised infection of neonates (see reviews by Gibbs and Rweyemanu, 1977, and Barnard and Collett, 1994). BHV-1 is an alphaherpes virus and has a genome composed of double-stranded DNA. It has the same attribute as other herpesviruses and produces latent infections, in this case in the trigeminal ganglia (Homan and Easterday, 1980). Latent infections neither damage the host nor incite potentially protective immune responses. They have important implications for the potential spread of the BHV-1 by bovine semen. BHV-1 has world-wide distribution and is regarded by some authorities as the most common viral pathogen found in bovine semen (Afshar and Eaglesome, 1990). The virus has been observed repeatedly in bovine semen (Spradbrow, 1968; Chapman et al., 1979; Elezhary et al., 1980) and infection has been transmitted experimentally by artificial insemination (Schlafer et al., 1990). Shedding of BHV-1 virus in the semen can occur in bulls with latent infections and without clinical signs (Afshar and Eaglesome, 1990). Bulls with latent infections can have a range of titres of specific antibody in blood (Straub, 1991).

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Vaccination of bulls with live or killed anti-BHV vaccines has been put forward as a way of reducing viral contamination of semen. However, field strains of BHV-1 can infect cattle after vaccination with killed vaccine (Frerichs et al., 1982) and modified virus from live vaccines can be shed into semen (Gregerson and Wagner, 1985). Treatment of semen with hyperimmune serum has been proposed as a control measure (Schultz et al., 1988) but has not yet been accredited for use. The use of seronegative bulls in artificial breeding centres is regarded as the best option for eliminating BHV-1 from semen (Afshar and Eaglesome, 1990; Parsonson, 1993). Programs of pre-entry testing and routine testing of bulls by serological and virological means have successfully eradicated BHV-1 from artificial breeding centres (Lucas, 1986; Corkish, 1988) but the ubiquity of the virus makes re-introduction a continuing threat. Serological tests employed in successful programs for controlling BHV-1 were serum neutralisation or enzyme-linked-immunosorbent assays (ELISAs). ELISAs, however, have yet to be standardised between laboratories (Philpott, 1993). A more basic problem with tests for antibody is the possibility that latent BHV-1 infections may not stimulate the production of specific antibody. The solution is the development of highly sensitive tests for the presence of BHV-1 or its antigens in semen. An assay which employs the polymerase chain reaction has recently been described for this purpose (Wiedmann et al., 1993). It could revolutionise the approach towards virus-free semen and requires further clarification. The assay is directed against a portion of the glycoprotein IV gene and avoids the difficulties presented for other virological methods by cytotoxic agents in bovine semen. It could be used for the screening of bulls before entry to artificial breeding centres and for surveillance of all bulls thereafter. Trichomoniasis Trichomoniasis is an infectious venereal disease of cattle characterised by disrupted oestrous cycles, infertility, early abortion and pyometra. The disease occurs throughout the world and can be highly prevalent in extensive grazing systems. The causative organism is the flagellated protozoan parasite Tritrichomonas foetus previously known as Trichomonas foetus which has a predilection for the female genital tract. Transmission of trichomoniasis by artificial insemination was confirmed very early in the life of this technology (Bartlett et al., 1947; Joyner and Miller, 1952; Bartlett et al., 1953; Kendrick, 1953; Gabel et al., 1956). It was also discovered early that the infectivity of Tritrichomonas foetus can be preserved by storage at -79ºC (Blackshaw and Beattie, 1955) for up to 5 years (Levine and Anderson, 1966) and in semen diluent

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containing milk (McWade and Williams, 1954). As a consequence, the potential for transmission of trichomoniasis in preserved bovine semen is well recognised. Under natural circumstances, transmission of trichomoniasis occurs during coitus (Clark et al., 1977) from reservoirs of the organism in the mucous membranes of penis and prepuce (Parsonson et al., 1974). Bull to bull transmission can occur as a result of pseudocopulation. Female to female and mechanical transmission by biting flies is virtually insignificant (Clark et al., 1977). Bulls younger than three years appear to have an inbuilt resistance to infection despite exposure whereas infection establishes readily in older bulls and becomes chronic. Recovery without treatment has been described (Clark et al., 1971). In summary, the transmission of trichomoniasis by preserved bovine semen is a proved risk but one which can be contained by procedures already specified for use in artificial breeding centres (DPIE, 1988; OIE, 1992). Containment depends on the exhaustive screening of bulls before they enter artificial breeding establishments for use as semen donors. It is supplemented by regular testing to ensure that freedom from trichomoniasis is maintained. Direct microscopic demonstration of motile T. foetus organisms in preputial washings from bulls is the primary diagnostic test used for screening and surveillance (Vaughan, 1993; Schutte et al., 1994). In Australia, negative results are required from a sequence of four examinations made a week apart. Microscopic examination of sedimented preputial washings can be supported by culture methods to detect T. foetus (Clark et al., 1971; Reece et al., 1983). The succesful eradication of trichomoniasis from artificial breeding establishments by strategies based on the diagnostic tests just mentioned (Clark et al., 1983) is a measure of their reliability. It should be emphasised, however, that success depends upon rigorous adherence to protocols for the collection of preputial washings such as that described by Schutte et al., (1994). Freedom from trichomoniasis in semen donor bulls means that artificial insemination with their semen can be used as a control and eradication measure against this disease in the field . Category 2 - Diseases where causative organisms have been demonstrated in semen. Enzootic bovine leukosis Enzootic bovine leukosis is a viral lymphoid neoplastic disease which occurs in most countries of the world including Australia. Infection is chronic and primarily of B-lymphocytes and prevalence in cattle herds can reach 60 to 90%. Malignant lymphoma and persistent lymphocytosis,

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however, appear in only a relatively small number of infected animals (Johnson and Kaneene, 1992). The causative agent is a retrovirus, the bovine leukaemia virus which belong to the Oncovirinae subfamily of the Retroviridae. Enzootic bovine leukosis is transmitted horizontally from animal to animal not vertically via the germ line (Johnson and Kaneene, 1992). The virus is not found in the genome of ova or spermatozoa (Callahan et al., 1976; Kettmann et al., 1980). Horizontal transmission occurs mainly within herds of cattle and does not involve neighbouring herds indicating that direct contact and prolonged exposure is required for transmission. The virus is cell-associated with B-lymphocytes and is believed to be transmitted through body fluids that contain relatively high numbers of these cells; for example, blood , colostrum and, to a variable extent, milk. In all, the risk of transmission of virus via bovine semen depends upon the presence of lymphocytes. As for observations on risk factors, virus can be transmitted though the uterus (Van Der Maaten and Miller, 1977). However, results of transmission studies with semen and artificial insemination have been variable. No transmission via semen was found by Baumgartner et al. (1978), Miller and Van Der Maaten (1979), Kaja and Olson (1982), Schultz et al. (1982) and Monke (1986). In contrast, a study by Lucas et al. (1980) showed seroconversion in sheep following intraperitoneal injection with semen from a seropositive bull. Bovine leukosis virus was identified in lymphocytes from these sheep. Semen was collected from the bull in question by transrectal massage of of the genital tract which may have facilitated the entry of blood and leukocytes into semen. In summary, the risk of transmitting the bovine leukosis virus in frozen bovine semen can be regarded as acceptable even for seropositive donor bulls. The qualifications are that semen is collected by the artificial vagina, not by transrectal palpation or electroejaculation and has a normal spermogram. In addition, there should be no local disease in the reproductive tract such as seminal vesiculitis, ampullitis or urethritis which would favour the entry of virus-containing lymphocytes into semen. Sensitive assays for non-sperm cells in semen appear to be unavailable but could define that component of risk related to the presence of lymphocytes. Absolute protection against the risk of transmission of bovine leukosis would be obtained by restricting semen to that obtained from seronegative bulls. In this regard, enzyme linked immunosorbent assays have been demonstrated as more sensitive than the older agar gel immunodiffusion test (Portetelle and Mammerickx, 1986). A test for tumour associated antigen is in the offing and may be superior to assays of antibody in the detection of diseased animals. Category 3 - Diseases with a presumptive risk of transmission by bovine semen.

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Bovine malignant catarrh Malignant catarrhal fever or bovine malignant catarrh is a peracute or acute disease of cattle and deer characterised grossly by profuse mucopurulent ocular and nasal discharges, keratoconjunctivitis with corneal opacities and enlargement of peripheral lymph nodes (Barnard, van der Lugt and Mushi, 1994). The pathognomonic lesion is a necrotising vasculitis. The disease appears sporadically and is generally fatal. It occurs in most countries of the world. The causative agent is a herpes virus (Alcephaline herpes virus 1) with a genome composed of DNA. Malignant head catarrh is not discussed in recent reviews of diseases transmitted via bovine semen (Afshar and Eaglesome, 1990; Philpott, 1993). Nevertheless, there is a discernible risk of transmission by this route which could be realised under some limited circumstances. Risk arises through the general behaviour of herpes and other DNA viruses and is a consequence of the migration of virus-bearing non-sperm cells into semen as discussed under the heading of biological considerations (see page 6). Malignant head catarrh does not usually spread between cattle and disease derives from a reservoir of virus in sheep and wildebeest with inapparent infections (Plowright, 1968). Virus is not detectable in lesions but is found in association with cells (Mushi and Rurangirwa, 1981), particularly medium lymphocytes (Rossiter, 1985); an observation highly relevant to present lines of thought on risk. Virus is found in low concentrations in ocular and nasal secretions of infected animals but semen does not appear to have been studied. Clinical disease would cancel libido and rule out collection of semen with the artificial vagina. However, the disease has an incubation period of three to seven or more weeks and contamination of semen could be a possibility during this time. In summary, the causative agent of malignant head catarrh is a herpes virus and intrinsic contamination of semen is made possible by the migration of virus-bearing non-sperm cells. Risk would occur only during the incubation period of the disease. Since malignant catarrh is spread to cattle from a reservoir in sheep, the risk of spreading the disease agent in bovine semen would be removed by keeping semen donors separated from sheep. For example, it is recommended in Africa that cattle and wildebeest, which harbour the virus, be separated by 1,000 metres (Barnard, van der Lugt and Mushi, 1994). Bovine spongiform encephalopathy Epidemiological evidence indicates that bovine spongiform encephalopathy (BSE) is not transmitted through bovine semen (Wilesmith, 1994). However, the disease has a long onset and definitive transmission experiments will not yield their results for some time. In the meantime, BSE

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is classified as a disease with a presumptive risk of transmission in bovine semen and should be written into protocols for the international exchange of germ plasm. BSE is a progressive and invariably fatal disease of the nervous system that first appeared in cattle in Great Britain in 1986 and 138, 444 cases have been confirmed there in the ensuing eight years. The disease has also occurred in Ireland, Switzerland, Portugal and France. The risk to people of BSE is regarded as remote (SEAC, 1995). The pathological changes in BSE are similar to those of Kuru and Creutzfeldt-Jakob disease in man, mink encephalopathy, and scrapie in sheep. As for scrapie in sheep, the causative agent of BSE is named as a proteinaceous infective particle or prion (Prusiner, 1992). This nomenclature, however, is not used universally and the biology of prions is far from clear. BSE in British cattle appears to have resulted from exposure to a scrapie-like agent or prion present in the meat and bone meal used as a protein supplement for rations. Exposure began in 1981- 1982 and corresponds to a change in the rendering process which allowed the causative agent to survive. The initial source of infection is likely to have been sheep but the epidemic was accelerated when infected bovine tissue was used as a feedstuff. A ban on the use of ruminant-derived protein in ruminant feedstuffs in 1988 has curtailed the incidence of BSE in animals born since that time. Clinical observations that the BSE epidemic in Great Britain was sustained solely by the ingestion of contaminated feedstuffs do not rule out the possibility of other forms of transmission. However, other indirect lines of evidence run against the possible presence of the BSE-agent in bovine semen. The closely-related scrapie-agent was not detected in the semen, testis or seminal vesicles of diseased rams (Palmer, 1959; Hadlow et al. 1982). So far, transmission studies have identified the BSE-agent only in the brain tissue of affected cattle (Fraser et al., 1992). The incidence of BSE in the offspring of infected semen donors and in the cows at risk from insemination appears to be no greater than that in the rest of the cattle population in Great Britain (Kimberlin, 1992). In summary, there is no clear evidence that the BSE-agent appears in bovine semen. However, until the results of definitive transmission studies are available, precautions against BSE should operate for the trade in bovine semen. They can be based upon freedom from exposure to the BSE-agent and, therefore, freedom from BSE in donor bulls. Thus, semen from bulls born since the feed ban in 1988 would have an acceptable risk. Bulls born before the feed ban would be also acceptable for semen if it can be demonstrated that they have never been fed ruminant derived protein..

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Haemorrhagic septicaemia Haemorrhagic septicaemia is an acute bacterial septicaemic disease of cattle and buffaloes caused by two specific serotypes of Pasteurella multocida (see review by De Alwis, 1992). It occurs in southern and south-east Asia, the Near and Middle East and in all regions of Africa. The disease differs from other pasteurelloses, except fowl cholera, where bacterial infection is a necessary but insufficient cause of disease. Transmission of P. multocida occurs by aerosol and by direct and indirect contact with infected animals. Bacteria are excreted in saliva, urine, faeces and milk and organisms can be detected in saliva within 12 hours of experimental infection (Bastianello and Nesbit, 1994). Animals that have recovered from the disease can act as carriers and up to 40% of some populations carry P. multocida in the nasopharynx Bastianello and Nesbit, 1994, op.cit.). The presence of P. multocida in saliva, milk and urine during disease places haemorrhagic septicaemia into the category of diseases with a presumptive risk of transmission via semen. However, the chance of this risk being realised is virtually non-existent because the mating capacity of donor bulls would be abolished early in infection as an accompaniment to fever and depression. Extrinsic contamination is possible in view of the carrier state but would be prevented by hygienic procedures for collection of semen. Risk here would be removed by ensuring that semen donors were not carriers as detected by isolation of the organism from nasopharyngeal secretions. Theileriosis Theileriosis refers to a group of tick-borne infections, mainly in ruminants, with protozoan parasites of the genus Theileria (family Theileriidae, order Piroplasmia, class Sporozoa, phylum Apicomplexa). Theileria parva occurs in east, central and southern Africa as three subspecies or types (see review by Lawrence, de Vos and Irvin, 1994). SubspeciesTheileria parva parva causes East Coast fever, a disease of cattle characterised by high mortality rates (up to 90% in susceptible animals), fever, pulmonary oedema, wasting, lymph node enlargement and panleukopenia. Corridor disease, caused by Theileria parva lawrenci , and Zimbabwe theileriosisis, caused by Theileria parva bovis, are both similar to East Coast fever except that death may occur more rapidly in infected animals. Theileria annulata causes an important disease of cattle in North Africa, southern Europe and Asia which has a pathogenesis similar to East Coast fever (reviewed by Pipano, 1995). Other species are Theileria mutans , which has a variable pathogenicity, and Theileria taurotragi and Theleria velifera which are considered non-pathogenic.

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The theilerioses behave in an essentially similar manner and are classified as diseases with a presumptive risk of transmission in bovine semen because the infective stage of the parasite for cattle is found in lymphocytes. These cells migrate into all organs including those of the reproductive system where they could lead to intrinsic contamination of semen. There is no evidence, however, that the theilerioses are transmitted by artificial insemination or natural mating or that organisms may be found in semen. The conjectural risk requires explanation from knowledge of the biology of Theileria species. Ticks are necessary for the transmission of the theilerioses under natural conditions and these diseases are maintained by a cattle-tick-cattle cycle. The three-host tick, Rhipicephalus appendiculatus, is the major species involved with theTheileria parva complex but fifteen other species can be vectors. About 15 Hyalomma species ticks may transmit Theileria annulata. The transmission cycle begins when immature ticks ingest intra-erythrocytic merozoites (piroplasms) with a blood meal from infected cattle. A phase of proliferation then occurs within the tick. When mature ticks feed on cattle they liberate the infective stage of the parasite (the sporozoite) in their saliva. These enter lymphocytes where they undergo a proliferative phase and can be observed as schizonts. Merozoites are formed and these enter erythrocytes where they are observed as piroplasms and can infect immature ticks to continue the cycle. Thus, piroplasms contained in erythrocytes are infective for ticks and schizonts contained in lymphocytes are infective for cattle. Sources of infection for ticks may be clinically ill or convalescent cattle or persistent carriers of the organism. Piroplasms of T. parva parva remain in the blood of cattle for over a year after recovery from clinical disease (Young et al., 1986). Theileriosis can be transferred between cattle by spleen or lymph node cells or by blood if sufficient numbers of infected lymphocytes are 2 8 present. Depending upon circumstances, the infective dose may range from 10 to 10 lymphocytes (Lawrence et al., 1994). However, no quantitative observations of cells other than spermatozoa in bovine semen can be found in the scientific literature to confirm or refute the possibility that lymphocytes could be present in enough numbers to pose a threat. It is relevant that sporozoites of T. parva parva, the infective stage for cattle found in lymphocytes, can be maintained viable at -70°C or in liquid nitrogen for long periods (Cunningham et al., 1973). In summary, there is a presumptive risk of transmission of theileriosis by bovine semen. For this reason, bulls must be confirmed free of infection before entry to artificial breeding centres and during the period they provide semen. Diagnosis of freedom can be made with smears of peripheral blood and fine needle aspirates of superficial lymph nodes. The presence of piroplasms indicates infection and the presence of hyperplastic lymphocytes should raise suspicion.

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Trypanosomiasis Trypanosomiasis is the name given to infection by protozoan parasites of the genusTrypanosoma which belong to the suborder Trypanosomatina, order Kinetoplastida, class Zoomastigophorea of phylum Sarcomastigophora (Levine et al., 1980). The species relevant to the present context are blood-borne parasites transmitted by biting flies, particularly the tsetse fly, Glossina spp, in which they undergo a cycle of development and maturation (see reviews by Stephen, 1986; Connor, 1994). These trypanosomes are Trypanosoma congolense and Trypanososma vivax which are severely pathogenic for cattle, Trypanosoma evansi , which is moderately pathogenic, and Trypanosoma brucei , which is regarded as mildly pathogenic for cattle. The diseases they cause are called nagana and are associated with anaemia, tissue damage and modulation or suppression of the immune response. Circulating immune complexes may be important in their pathogenesis. Although Trypanosoma equiperdum, which causes dourine in horses, is transmitted by coitus, there is no clinical or experimental evidence for the transmission by natural mating or artificial insemination of the four species which are important in cattle. Nevertheless, these organisms are regarded as having a presumptive risk of transmission in bovine semen for two main reasons. The first reason is that the location of trypanosomes in blood provides an opportunity for their invasion of reproductive organs and subsequent appearance in semen. Trypanosomes can be found outside the bloodstream. For example, Trypanosoma congolense recirculates in lymph (Akol and Murray, 1986) and can be isolated from the central nervous system (Masake et al., 1984). Trypanosoma vivax has been found in a variety of tissues including the central nervous system, eye and myocardium (Masake, 1980). There is a relatively lower parasitaemia in infections with Trypanosoma evansi or Trypanosoma brucei but more invasion of tissues (Goodwin, 1970). The second reason for a presumption of risk is that the absence of tsetse fly vectors may not prevent the establishment of trypanosomiasis in a new area if it were to be introduced in semen. Trypanosomes can be transmitted mechanically by biting flies such as the buffalo fly, March flies and stable flies (Connor, 1994). Trypanosoma vivax is reported to have established in Mauritius and parts of South America where it is likely to be transmitted mechanically (Wells, 1972). Other factors relevant to the risk of transmission by bovine semen are that trypanosomes can be cryopreserved with their viability intact and that infection may be subclinical (Connor, 1994). Accordingly, the fail-safe of illness and low libido will not prevent the collection of semen that could be infective.

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In summary, there is a presumptive risk of transmission of pathogenic trypanosome species via bovine semen. The risk occurs only where these trypanosomiases are endemic. Semen should only be accepted from bulls maintained in or derived from such areas if freedom from infection can be demonstrated. Unfortunately, the diagnosis of trypanosomiasis in individual animals can be difficult. Blood smears alone are too insensitive. An antigen-trapping ELISA shows promise of being sensitive and specific (Nantylya and Lindquist, 1989) but has not been fully accredited (Connor, 1994). Two trypanosome species have been recorded in domestic animals in Australia (Callow, 1984). Both are classified as nonpathogenic (Mansfield, 1977). The first is the large trypanosome,Trypanosoma theileria , in cattle which has tabanid flies as its vector. The other is Trypanosoma melophagium in sheep which has the ked as its vector. Disease from T. theileria is rarely observed (Connor, 1994). Sheep keds have disappeared from the Australian environment and may have taken T. melophagium with them. Category 4 - Diseases with risk associated only with extrinsic contamination. Anaplasmosis Bovine anaplasmosis is an arthropod-borne disease characterised by fever, anaemia and jaundice (see reviews by Callow, 1984; Potgieter and Stoltsz, 1994). The causative agents are two rickettsial organisms with Anaplasma marginale considered to be more pathogenic than Anaplasma centrale. A. marginale and A. centrale are parasites of erythrocytes and risk comes from the possibility of semen being contaminated with infected blood. Anaplasmosis is maintained in a cycle that requires transmission by ticks. At least 20 species of ticks are involved throughout the world. The increasing distribution of the disease through the world reflects the presence of these vectors. Various ticks have been investigated in Australia but the one-host tick Boophilus microplus is the most important. Blood is infective and anaplasmosis can be spread mechanically during routine procedures such as castration, ear-tagging and dehorning and by biting flies. Transplacental transmission as a result of infection during pregancy has been recorded (Potgieter and van Rensburg, 1987). However, there is no indication of spread through artificial insemination or natural mating or of the presence in semen of the causative organisms. Anaplasma organisms can be cryopreserved in liquid nitrogen without loss of infectivity (Callow, 1984).

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Risk for semen from bovine anaplasmosis comes in two ways. First is the possibility of contamination by parasitised red bloods cells resulting from trauma during the process of semen collection or from pathological changes in the reproductive system. Because anaplasmosis may be spread mechanically the risk may be greater than for other intra-erythrocytic parasites such as Babesia spp which are discussed next. Bulls may carry anaplasma organisms without showing clinical disease. The second source of risk comes from vaccination against anaplasmosis. Bulls in AI centres could become infected with cell-associated viruses, such as bovine herpes virus 1, bovine leukosis virus or bovine pestivirus, if they receive vaccine prepared from the blood of splenectomised animals. Vaccines for use in bulls that may eventually become semen donors must be confirmed as free of these important viruses. The possibility of semen being contaminated by blood is a presumptive risk for all diseases where the causative agent is found in this body fluid. At the moment the general problem cannot be defined because a sensitive test has not been accredited for the detection of blood in bovine semen. In addition, it is not clear whether different methods for collecting semen may lead to the presence of blood in semen. Transrectal massage of accessory glands requires particular clarification in this regard. Babesiosis Bovine babesiosis is a sub-acute to acute tick-borne disease characterised by a range of signs including fever, inappetance, dullness, weakness, ataxia, haemoglobinuria, jaundice, and anaemia (see reviews by Callow, 1984; de Vos and Potgieter 1994). The major causative agents are the protozoan parasites of bovine red blood cells, Babesia bovis and Babesia bigemina (family Babesiidae, suborder Piroplasmorina, order Eucoccidiorida, class Sporoazoasida of the phylum Apicomplexa) and this gives rise to the possibility of semen being contaminated with infected blood. In Australia, Babesia bovis is regarded as more pathogenic than Babesia bigemina. Other babesias of cattle are B. divergens , which is considered as pathogenic, and B. major in Britain and parts of Europe. Two minor species are Babesia occultans in southern Africa (Gray and de Vos, 1981) and Babesia ovata in Japan (Minami and Ishihara, 1980). Babesia spp are spread in Australia by the one-host tick Boophilus microplus in which these protozoans undergo a cyle of proliferation. Other ticks may be involved in transmission elswhere in the world and the distribution of these vectors determines the distribution of bovine babesiosis. There is no evidence for mechanical transmission by biting flies or for the presence of Babesia spp in semen or transmission by artificial insemination or natural mating. In addition, there is no likelihood of babesiosis establishing in regions with no vectors. Babesia organisms can be preserved by storage at -196°C (Dalgliesh and Mellors, 1974).

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Bovine babesiosis has two implications for the disease-free status of semen. First is the minor possibility of contamination by parasitised red bloods cells resulting from trauma during the process of semen collection or from pathological changes in the reproductive system. Bulls may carry babesia infections without clinical signs of disease. The second implication is the same as that for anaplasmosis. Bulls could be infected with cell-associated viruses such as bovine herpes virus 1, bovine leukosis virus or bovine pestivirus if they receive vaccine prepared from the blood of splenectomised animals. Blood-derived vaccines against babesiosis must be accredited as free of these viruses if they are used at any stage in the life of bulls destined to become semen donors. Issues related to the possible contamination of semen by parasitised or infected blood cells are dealt with in the section on anaplasmosis. Dermatophilosis Dermatophilosis is a skin disease produced by infection with the pleomorphic bacterium, Dermatophilus congolensis, which belongs to the family Dermatophilaceae of the order Actinomycetales (see review by van Tonder and Horner 1994). It occurs worldwide, mainly in herbivores but may affect other species. The disease is characterised by an exudative and proliferative dermatitis with the formation of scabs. There is no prior reason for supposing that D. congolensis may be an intrinsic contaminant of semen. D. congolensis is transmitted by direct contact between infected and uninfected animals but can be spread mechanically by insects or on plants etc. Successful infection usually occurs when defence mechanisms of skin such as sebaceous secretions have been breached. Extrinsic contamination of semen would require the presence of obvious dermatophilic skin lesions either on the semen donor or other animals in the artificial breeding establishment. Dermatophilosis is readily controlled by antibiotics such as penicillin and streptomycin or dihydrostreptomycin in combination, or by long-acting oxytetracycline. Category 5 - Diseases with no appreciable risk of transmission by bovine semen. Cysticercosis

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Risk of transmission via bovine semen does not apply to cysticercosis caused by the tapeworm Cysticercus bovis because of the life history and morphology of the causative organism. In addition, the eggs produced by the primary parasite in the gut of humans and the cyst stage in the intermediate host are killed by freezing to -20°C and subsequent thawing.

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OTHER DISEASE AGENTS WITH MATERIAL RISK OF TRANSMISSION VIA BOVINE SEMEN The following disease agents do not come under Lists A and B of the OIE and, thus, fall outside the strict terms of reference for the present report. However, their possible spread via cryopreserved bovine semen is likely to come under scrutiny on the international scene. Some of the following organisms are well recognised by the artificial breeding industry as contaminants of semen that possess a disease risk. Bacteria Haemophilus somnus Haemophilosis in cattle is characterised by septicaemia, thromboembolic meningoencephalitis, pneumonia, myocarditis, arthritis, mastitis and reproductive disease (reviewed by Harris and Janzen, 1989; Kitching and Bishop, 1994). It has been recorded in Australia (Patterson et al., 1984), South America, Europe, Africa, Japan , New Zealand and North America where it is an important cause of mortality in beef feedlots. The causative organism is the microaerophilic Gram-negative coccobacillus, Haemophilus somnus, which belongs to the family Pasteurellaceae. Haemophilus somnus has a recognised risk of transmission via contaminated bovine semen (Eaglesome et al., 1992; Philpott, 1993). It can be isolated from the reproductive tract of cows and bulls without clinical disease (Humphrey et al.,1982b) and can be present in semen (Humphrey et al., 1982a; Krogh et al., 1983). Haemophilus somnus is eliminated by the antibiotics used in semen diluents (lincomycin/spectinomycin, gentamycin and tylosin; Shin et al., 1988). The concern that Haemophilus somnus inside non-sperm cells may be protected against antibiotic action (Eaglesome et al., 1992) requires resolution. Mycoplasma spp, Ureoplasma spp and other mollicutes Various surveys of bovine semen have found a high incidence of contamination with various mycoplasma and ureoplasma species (Eaglesome, Garcia and Stewart, 1992). These organisms have been isolated from healthy animals and animals with signs of reproductive disease; for example orchitis, epididymitis and seminal vesiculitis in bulls and vulvitis in cows. The granular vulvitis, endometritis and salpingitis ascribed to Ureoplasma diversum has been reproduced by experimental infection (Doig et al., 1980a and 1980b). The disease ascribed to Mycoplasma bovigenitalium has been reproduced experimentally in bulls. Ureoplasmosis has been proposed as one of the potentially most important infectious causes of infertility in dairy cattle (Doig and Ruhnke, 1986a). However, the importance of genital mycoplasmosis as a reproduction disease in cattle requires clarification (Doig and Ruhnke, 1986b). Some strains of Mycoplasma bovigenitalium

can suppress sperm mobility in the absence of disease and are important for this reason. Mycoplasma and ureoplasma contamination is semen can be controlled by hygiene during collection and processing and by the use of antibiotics in diluent. A combination of lincomycin, spectinomycin, gentamycin and tylosin is reported to be effective in controlling Mycoplasma bovis, M. bovigenitalium, Ureoplasma spp. H. somnus and C. fetus venerealis in cryopreserved bovine semen (Shin, 1988). Rickettsia Chlamydiosis Chlamydiosis occurs throughout the world in a wide range of mammals, birds and amphibians (see review by Pienaar and Schutte, 1994). The causative agent is the obligate intracellular parasite, Chlamydia psittaci. Chlamydiae differ from other rickettsias in being dependent on host cells for their energy requirements and having a distinctive cycle of development. Reticulated or initial bodies multiply in the cytoplasm of host cells and are non-infectious. Elementary bodies are released from host cells and are infectious. Chlamydiosis is characterised by pneumonia, abortions and genital infections in people. In cattle, chlamydiosis due to antigenic type 1 C. psittaci is characterised by abortion, seminal vesiculitis, pneumonia and enteritis. Type 2 C. psittaci is associated with sporadic bovine encephalomyelitis, polyarthritis and conjunctivitis (Shewen, 1986). C. psittaci can be isolated from the semen of clinically and subclinically infected bulls (Storz et al., 1968; Storz et al., 1976) and can be transmitted experimentally by contaminated semen (Bowen et al., 1978). Under natural conditions, however, venereal transmission of chlamydiosis is believed to be insignificant compared to the faecal-oral route (Pienaar and Schutte, 1994). Furthermore, some reviewers regard the role of contaminated semen in transmitting C.psittaci as being unknown (Shewen, 1986). On balance, an indicative risk of transmission by semen must be assigned to C.psittaci and semen from infected bulls should be rejected. Tetracycline treatment of sufficient length has been suggested for removing infection and preventing the carrier state (Shewen, 1986). Diagnosis of infection is best based on detection of the organism rather than specific antibodies. Enzyme linked immunosorbent assays (ELISAs)and fluorescent antibody assay are available for this purpose (Timms, 1993). ELISAs are are also available for the detection of specific antibody and are more convenient than previous complement-fixation tests. Lyme borreliosis A significantly large proportion of an admixture of the causative organism of Lyme borreliosis, Borrelia burgdorferi, has been demonstrated to survive in bovine semen

stored at -196°C for 12 weeks (Kumi-Diaka and Harris, 1995). However, there is no indication that this organism is found in the semen of infected bulls. Viruses Akabane disease Akabane disease is caused by an arbovirus in the Simbu group of the Bunyaviridae and has a genome composed of single-stranded antisense RNA. It has been considered in reviews on the viruses found in bovine serum. However, the virus could not be identified in the semen of experimentally infected bulls (Parsonson et al., 1981) and is not regarded as a risk. Ephemeral fever The causative agent of ephemeral fever is an arbovirus in the family Rhabdoviridae and has a genome composed of single-stranded antisense RNA. Like Akabane virus, it has been considered in reviews on the viruses found in bovine semen. However, experimental observations show that cows inseminated with virus-contaminated semen did not become infected but remained susceptible to infection by the usual route of transmission (Parsonson and Snowdon, 1974). These results suggest that ephemeral fever virus may not be a disease risk in semen. However, virus is found in blood neutrophils during the febrile phase of disease (Young and Spradbrow, 1980) and this points to a presumptive risk. Moreover, since bovine ephemeral fever is regarded as an important exotic disease in North America and Europe, care should be taken with semen during periods when ephemeral fever is occurring in a region. The clinical disease associated with ephemeral fever is likely to curtail libido and limit the possible collection of semen when the risk of intrinsic contamination is present. Viraemia in experimentally infected cattle appears the day before fever commences and persists for one to two days after clinical signs disappear (St George, 1994). Bulls can be subclinically infected with ephemeral fever but are refractory to reinfection after clinical or subclinical infection. Accordingly, semen from seropositive bulls is safe, as is semen from donors remaining seronegative during and after the collection period. Herpes viruses not specified in OIE List B Herpes viruses are the causative agents of the infectious bovine rhinotracheitis/pustular vulvovaginitis syndrome and malignant head catarrh which are considered elsewhere in this report. However, another member of the family, bovine herpesvirus 4 (see review by Goyal and Naeem, 1992) requires consideration as a disease risk in bovine semen. It is associated with abortions, metritis, mammary pustular dermatitis, and respiratory and enteric infections but usually occurs as a subclinical infection. It has been associated with orchitis but its spread via semen has not been corroborated (Afshar and Eaglesome, 1990). No action except for monitoring the situation is recommended at the moment.

Other viruses There is a single report of the presence of paravaccinia virus, the cause of pseudocowpox in cattle and milkers' nodules in people, in the semen of two bulls (Johnston and Deas, 1971). However, it is not considered important in other reviews on possible pathogens in bovine semen. Parainfluenza type 3 (PI-3) virus has been found infrequently in the semen of bulls. The most recent report (Abraham and Alexander, 1986) occurred in bulls after vaccination with a live vaccine. The importance of this virus to the artificial insemination industry is not clear and requires further investigation (Afshar and Eaglesome, 1990).

CONCLUSIONS The following conclusions emerge from the analysis and review of the quarantine risks associated with bovine semen. They may either reinforce the continuing need for practices already in place or suggest refinements or additions. They lead to a list of recommendations which follows and is also reproduced in the summary. The evolving disease picture Disease risk from bovine semen is unlikely to remain static. The emergence of new disease agents and changes in virulence, pathogenicity and antibiotic resistance have to be included in the overall approach to risk management. For example, bovine pestivirus (bovine viral diarrhoea virus) and bovine herpes virus (the causative agent of infectious bovine rhinotracheitis) are now recognised as significant risks for transmission by bovine semen but are not listed for special attention in the 1988 Code of Practice for Australian Livestock in Artificial Breeding Centres. Progressive changes in Australia's cattle population and cattle husbandry should be considered as a necessary component of disease risk. Australia has successfully eradicated bovine tuberculosis and brucellosis. This has provided a national comparative advantage for animal husbandry. However, it has also led to a cattle population which is uniquely susceptible to these pathogens because acquired immunity is now quiescent. Intensive lot-feeding of cattle is now an integral part of Australia's beef industry and has different disease considerations compared with extensive grazing. The disease potential of semen-borne viruses and bacteria such as the bovine pestivirus, bovine herpesvirus-1 and Haemophilus somnus may be greater for cattle destined for feedlots than cattle maintained at pasture. Haemophilus somnus is an example of a disease not in OIE lists A and B but which should be considered in disease protocols. Other diseases in this category are those caused by mollicutes (Mycoplasmas and Ureoplasmas) and the chlamydias. Categories of risk Five categories of risk were used in the present review as a starting point for analysis. Category 1 contained diseases for which transmission by artificial insemination has been demonstrated. Category 2 contained diseases where causative organisms have been isolated from semen. Both categories depend upon the limited pool of knowledge that is available. Reliance on them for establishing the complete picture of risk would be incorrect. They are likely to contain the error of excluding diseases where risk has not been formally demonstrated. For this reason, diseases with a presumptive risk of transmission (Category 3) require critical consideration. It must also be recognised that the disease risk from bovine semen is borne directly by those who use it and indirectly by those who produce it. The impact here comes as a result of the response in the marketplace. Disease transmitted by contaminated semen may

be expressed later in life, for example when animals produced by artificial insemination are in feedlots. The role of Approved Artificial Breeding Centres The general risk for bovine semen ranks higher than that for embryos which are protected by the zona pellucida and can be washed by tested methods. However, the safety of semen relative to the importation of live animals depends upon the disease in question. Since contaminated semen can be distributed widely, it has a potential for causing multiple outbreaks of disease on a wide front. Exposure from an imported bull used for natural mating would be more limited. The withholding period for semen before release into Australia can be regarded as comparable to maintaining animals in quarantine and allowing time for the expression of an infectious disease under incubation. Quarantine stations, however, are under Australia's direct control and they allow the direct opportunity for the application of diagnostic tests. Certified artificial breeding (AB) centres are an essential part of the Australian strategy for controlling disease associated with semen. AB centres occupy a similar place in the sanitary codes of the OIE for the hygienic processing of semen in the control of disease. Since AB centres are relied upon to perform a surveillance function for disease agents in semen comparable with quarantine stations for disease agent in live animals, their facilities, standards and procedures must be equal to the task and must be auditable. Record-keeping, surveillance for disease and procedures for maintaining health and hygiene, hygienic processing of semen, reliable control and labelling of semen stock, and the rapid and reliable reporting of disease incidents are essential elements. AB centres will become more important as the basis of disease control in the international trade in bovine semen. As a consequence, they should have inbuilt mechanisms for continually improving their function as experience grows. Hazard analysis and critical control points (HACCP) systems used in the food processing industry could be adapted for bovine semen (Eaglesome et al., 1992). Regional, herd and donor freedom from disease Three categories of disease freedom underlie measures for disease control in the international trade in animals and animal products, including semen. These are regional freedom, herd freedom and freedom in the individual semen donor bull from infection or disease. All will lead to semen that is free from a given disease agent. Regional freedom is the most satisfactory and most easily instituted guarantee of the disease-freedom of bovine semen since it can be based on the official disease status of countries. New and sensitive diagnostic tests are available or at various stages of development for detecting the presence of disease organisms. It is more logical to apply these to programs for producing disease freedom in individual donors and the whole herd at

AB centres rather than using them to screen semen without reference to the health status of the donor. Disease freedom in donors eliminates the problem of sampling error that is inherent in the direct testing of semen. In the same vein, treatment of semen with antibiotics or with hyperimmune serum cannot replace the mainstay of donor freedom from disease. Antibiotics may be bacterostatic rather than bactericidal and antibiotic resistance is always a possibility. Hyperimmune semen may be insuffient to kill contaminating organisms and the accreditation of such procedures may be more involved than achieving herd and donor freedom. Donor clinical freedom will not be sufficient to underwrite the safety of semen for disease agents like bovine pestivirus or bovine herpesvirus-1 which can transmit from animal to animal with relative ease without the display of clinical signs. Herd freedom from these agents and measures to prevent their introduction such as pre-entry testing of new animals are necessary. For diseases like bluetongue where transmission occurs through highly mobile and uncontrollable vectors, a rigorous program for ensuring donor freedom from infection is the only option possible. Diagnosis of disease and infection in semen donor bulls Diagnosis of disease starts with physical examination and examination of the environment. It is then supported by other procedures. Since bulls with clinical signs of disease must be considered a prima facie risk for contaminated semen, regardless of cause or whether mating capacity is eliminated, all disorders of animals in AB centres should be diagnosed and recorded. A general withholding period for semen is required to cover the possibility that semen collected during the incubatory phase of an undiagnosed clinical disease could be released prematurely. Diseases of the male reproductive organs require special attention. Bulls with palpable lesions of the testis, epididymis, seminal vesicles or other secretory organs in the reproductive tract should not be used as semen donors without a substantiated diagnosis of cause. Diagnostic tests for disease agents in semen Diagnostic tests are based on the identification of an organism with virological and other microbiological methods or immunological methods which include the identification of specific antibody, antigens, nucleic acids and their products or both and skin tests of various sorts. There is a general trend away from serology for identifying infection partly because of the inherent shortcomings of serology and partly because technology is now available as a replacement. Examples are tests for antigens, fragments of organisms or whole infectious organisms such as antigencapture ELISA tests and tests based on the PCR reaction. Innovative diagnostic tests must be accurate, robust and accredited for their sensitivity and specificity before they replace tests already in use. The OIE has listed diagnostic tests which are acceptable to its member countries. Non-sperm cells in semen

Disease organisms enter semen intrinsically as a result of (1) local infection in the male genital organs or (2) general infection when organisms are present in blood. Many disease organisms, for example Mycobacterium bovis, Brucella bovis, bovine herpesvirus-I and bovine pestivirus are carried from blood into semen in association with migratory cells such as lymphocytes, monocytes/macrophages or neutrophils. A local inflammatory response may not be necessary for passage into semen but may enhance it. The bluetongue virus is closely attached to erythrocytes and contamination may only occur when infected erythrocytes are extravasated into semen for one reason or another. Given the possible role of non-sperm cells in the process of contamination with disease organisms, the scarcity of information about their presence in semen is surprising. Normal values for erythrocytes and other cells is semen collected by various methods are not available. Accordingly, it has not been shown quantitatively whether methods of semen collection such as transrectal stimulation of secretory glands leads to the entry of more non-sperm cells into seminal fluid. The counting of non-sperm cells in semen could be an important general test for the possibility of contamination with disease organisms. However, such tests have not been developed or adapted for use. The cyanmethaemoglobin test may not be sensitive enough to pick up significant levels of contamination with red blood cells. However, assays for erythrocyte-specific enzymes may be suitable. Similarly, direct counts of non-sperm cells may not be sufficiently sensitive to detect significant levels of contamination and alternatives are required. Recommendations (1)

The “Code of Practice for Australian Livestock Artificial Breeding Centres” and Minimum Health Standards for Stock Standing at Licensed or Approved Artificial Breeding Centres in Australia which were both published in 1988 should be revised in form and content to account for progress in the area. -

(2)

Since that time, knowledge on the risk of diseases that may be transmitted in bovine semen has advanced. In addition, the OIE International Animal Health Code which describes the accreditation of artificial insemination centres for export purposes and methods for the hygienic collection and processing of semen was published in 1992. The two documents published in 1988 overlap in their coverage and could be merged into a single publication. More explicit guidance on the diagnosis and control of disease is required. In addition, the coverage of infectious disease in these documents is no longer complete.

The official disease status of countries is an important guide in protocol formulation. However, the latest information on the status of particular

diseases in countries being considered for the import of semen requires direct contact with animal health officers in the country concerned. (3)

Approved artificial breeding centres are central to the management of disease in the international trade in bovine semen. Upgraded protocols for approving and routine inspection of such centres are required domestically. They will also be a guide to what should be expected in artificial breeding centres in countries being considered for the import of bovine semen.

REFERENCES