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A Polymerase Chain Reaction Assay for the Detection of Leptospira spp. in Bovine Semen Saad A. Masri, Phuong T. Nguyen, S. Pamela Gale, Chris J. Howard, and Suk-Chan Jung

ABSTRACT

RESUME

A rapid and specific method for the detection of pathogenic Leptospira spp. in bovine semen using the polymerase chain reaction (PCR) is described. The primers used were derived from an EcoRl/BamHl fragment that hybridized strongly to chromosomal DNA from the hardjobovis serovar. Three different extraction methods were evaluated in this study: phenol-chloroform extraction method, proteinase K (PK) in 1% SDS, followed by phenol-chloroform, and phenol-chloroform followed by 1% cetyltrimethylammonium bromide (CTAB). A PCR product of approximately 500 base pairs (bp) in length was obtained when DNA from pure Leptospira culture was used as a template for PCR, regardless of the DNA extraction method used. The product was consistent with that predicted from the gene sequence. However, in semen seeded in vitro, as well as in semen from infected bulls, a PCR product was obtained only when the leptospiral DNA was extracted from the specimen using the CTAB method. In contrast, other methods used for DNA extraction did not generate suitable templates for the PCR procedure. This is the first PCR protocol developed to detect Leptospira in bovine semen. The PCR protocol provided a direct and unequivocal demonstration that Leptospira can be detected in semen of infected animals. The CTAB method was also used successfully in detecting Leptospira in the urine of infected animals. The PCR procedure was shown to be more sensitive than either the fluorescent antibody test (FAT) or culture for detecting the organism in urine.

Une methode rapide et specifique utilisant la reaction d'amplification en chaine par la polymerase pour la detection de Leptospira spp. dans la semence bovine est decrite. Les amorces utilisees ont ete obtenues a partir d'un fragment EcoRlI BamHl qui se liait fortement 'a l'ADN chromosomal du serovar hardjobovis. Trois me'thodes differentes d'extraction ont ete evaluees : la me'thode au phenol-chloroforme, celle 'a la protease K (PK) dans du SDS 1% suivi de phenol-chloroforme, et phenolchloroforme suivi d'un traitement au bromure de cetyltrimethylammonium 'a 1% (BCTA). Un produit d'ACP d'une longueur d'environ 500 paires de bases a ete obtenu lorsque de l'ADN d'une culture pure de Leptospira etait utilise comme matrice pour la reaction d'ACP, et ce independamment de la methode d'extraction utilisee. Le produit e'tait conforme 'a ce qui avait ete predit a partir de la sequence du gene. Toutefois, a partir de semence inoculee in vitro, de meme qu'a partir de semence de taureau infecte, un produit d'ACP n'a ete' obtenu que lorsque l'ADN des leptospires etait extrait a l'aide de la methode BCTA. II s'agit du premier protocole de reaction d'ACP developpe pour detecter des leptospires dans la semence bovine. Ce protocole a permis de demontrer de facon directe et sans equivoque que des leptospires peut etre detecte dans la semence d'animaux infectes. La me'thode BCTA a permis egalement de detecter des leptospires dans l'urine d'animaux infectes. La methode d'ACP s'est averee plus sensible que l'epreuve d'immunofluorescence ou la culture pour detecter le microorganisme dans l'urine.

INTRODUCTION

Cattle destined for artificial insemination (Al) centres in Canada must be free of antibody to Leptospira spp. serovars hardjo, grippotyphosa, canicola, pomona, sejroe and icterohaemorrhagiae at a final serum dilution of 1:100 in the microscopic agglutination test (MAT). The naming of leptospires has traditionally been based on a relatively complicated system of serological interactions. Recently, the classification of leptospires has been changed to a DNA-based system. However, the new classification often does not reflect the previous serological classification of the same isolate. It is not yet clear how to handle the problem of describing serological reactions under the new classification system when the isolate involved has not been identified. In addition, there will be some confusion in reviewing older literature which uses the serological naming system. The old naming system continues to be used in regulatory health certificates and Al requirements. For purposes of this paper, it should be noted that the use of hardjo indicates a serological response that could be due to infection with either L. borgpetersenii serovar hardjobovis or L. interrogans serovar hardjoprajitno. Where previous work is cited, the nomenclature used in the original paper is retained. The experimental procedures described herein use a field isolate that has been identified as L. borgpetersenii serovar hardjobovis. This is also the serovar that was used for serological testing, and since hardjobovis was confirmed to be the infecting serovar, the serological reactions in the experimentally infected bulls are described as being due to hardjobovis. The assumption made in screening (Traduit par docteur Serge Messier) animals using the above criteria is

Animal Diseases Research Institute, Agriculture and Agri-Food Canada, P.O. Box 640, Lethbridge, Alberta T1J 3Z4 (Masri, Nguyen, Gale, Howard); Veterinary Research Institute, Rural Development Administration, Anyang, South Korea (Jung). Received April 17, 1995.

Can J Vet Res 1997; 61: 15-20

15

of leptospires in semen would allow the use of semen from valuable animals which have serum antibody to 1 or more leptospiral serovars, but which may not have leptospires in their semen. There are several options for direct detection of leptospires. These include culture, the use of labelled antibody stains (fluorescein, biotin, etc.), silver stain, the use of DNA or RNA probes, or the use of a polymerase chain reaction (PCR) technique. Culture of leptospires is difficult and costly. Leptospires are fastidious organisms that can take up to 6 mo or more to adapt to in vitro conditions, even in experienced hands. All staining methods require time-consuming examination of individual slides for organisms with morFigure 1. Amplification by PCR of Figure 2. Amplification by PCR of 50 PL of phology typical of leptospires, and the Leptospira DNA from pure culture diluted in raw semen samples with 3 different methods sensitivity of detection varies widely 50 pL semen. DNA from 10 000 organisms (as described in Materials and Methods). depending upon the experience of the was amplified using the external primers Semen was seeded with 10 000 leptospires. technician and the type of sample, (lane 3) or the internal primers (lane 4). PCR Lane 2, PCR results from DNA extracted products of approximately 500 and 450 base from Leptospira-free semen using the CTAB among other factors. Nucleic acid pairs are shown in lanes 3 and 4, respec- method. Lane 3, PCR results from DNA probes can be very sensitive and spetively. For a negative control, water was extracted from seeded semen by the phenol- cific, but have had a limited applicaadded to raw, undiluted semen. pGem chloroform method. Lane 4 shows PCR tion in diagnostic laboratories. PCR results from Leptospira seeded semen using marker (Promega, USA) is shown in lane 1. the proteinase K method. Lane 5, PCR technology, however, is well-suited results from DNA extracted from seeded for analysis of multiple samples and semen using the CTAB method. pGem can have very good sensitivity and that animals which have an antibody marker is in lane 1. specificity. The technology is readily titre of less than 1:100 are not carriers available, easy to use and does not of those serovars, and the semen from require specialized containment facilithese animals is safe for artificial sary to have contact with contami- ties, such as required for radioisotopes. insemination. However, the associa- nated urine. The presence of an antiThis study was undertaken to tion between serum antibody titre, body titre does not necessarily indi- develop a PCR method for detection infectious status and ability to trans- cate an ability to transmit infection. of leptospires in semen. mit infection is not clear (1-6). Gale and Kingscote demonstrated the Animal species that act as reservoir failure of a bull, with a significant and hosts for a leptospiral serovar appear persistent antibody titre to hardjo, to MATERIALS AND METHODS to have low serum antibody titres to transmit the infection through either that serovar, as compared with the direct contact with susceptible breedincidence of infection. This is the case ing females or via Al of susceptible OLIGONUCLEOTIDE PRIMERS Primers used for detection of Lepwith hardjobovis infection of cattle. breeding females not in direct contact had been borgpetersenii serovar hardtospira leptospires Although One study demonstrated that antibody (1). titres to hardjo occurred in only 27% identified in this bull's urine in the jobovis were derived from a 3800 bp of cattle from which leptospires were month prior to the start of the experi- EcoRl/BamHl fragment of the hardisolated at slaughter (6). Studies ment, no leptospires were seen during jobovis genome. This fragment which specifically examine the pres- the 9 wk course of the experiment. hybridized to only 11 out of 31 difference of leptospires in semen are not The animal was not being treated with ent serovars (Masri, unpublished numerous; however, leptospires have antibiotics during the experimental data). The fragment was cloned into pUC-13 according to established probeen isolated from the testes of cattle period. Since an animal with leptospirosis cedures (10) and sequenced using the and pigs (2,7) and from bull semen (8). Venereal transmission of lep- may not have a serum titre, it would Sequenase sequencing kit (US Biotospirosis has been shown to occur seem that direct detection of lep- chemicals, Cleveland, Ohio, USA). where contact was limited to coitus or tospires in semen is the most practical The designations and sequences of the to artificial insemination (9). This way to ensure that semen used for Al primers used were: DH79, 5'CAT suggests that transmission can occur will not transmit leptospirosis. In GAA AGC GGC AAG AGG AGC 3' venereally, and it would not be neces- addition, a method for direct detection and RH79, 5'CTT CAC GTC GGC

r~

16

TCT AGA CAA 3'for the external primers and DHN, 5'TCA CGG AAG TTT ACG GAT 3' and RHN 5'CGA TAT TCA TAC AA 3' for the internal nested primers. SEMEN

Fresh semen was collected by electroejaculation from a bull resident in the specific pathogen free (SPF) herd at the Animal Diseases Research Institute in Lethbridge, Alberta. This herd is one from which no case of leptospirosis has ever been diagnosed. The animals are free of antibody titre to a number of Leptospira serovars, including hardjobovis. CULTURES

Log-phase cultures of a field isolate identified as serovar hardjobovis were used for experiments. The organism was grown in T80/40LH medium, prepared as previously described (11), except that the final concentration of superoxide dismutase (SOD) used was 0.001 g/L. Leptospira were enumerated using a Petroff-Hauser chamber.

Figure 3. Amplification by PCR, using the external primers, of leptospiral DNA extracted from 50 pL of semen obtained from bull #1 infected with serovar hardjobovis. PCR results from a control uninfected bull in lane 2, the preinfection semen from bull #1 is shown in lane 3. PCR results from day 7 (lane 4), day 10 (lane 5), day 14 (lane 6), day 21 (lane 7), day 28 (lane 8), day 35 (lane 9), and day 42 (lane 10). The pGem marker is in lane 1.

samples were collected for the fluorescent antibody test (FAT) and culture. Sera were tested for antibodies to hardjobovis by the microscopic agglutination test (MAT Standard Protocol for Agriculture Canada). Urine samples were examined by FAT using polyclonal antiserum against hardjobovis and cultured in T80/40LH medium.

In vitro SEEDING OF SEMEN WITH Leptospira Semen volumes of 5, 10, 25, 50, 75 and 100 pL were diluted to a 100 pL volume with 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (TE buffer). Approximately 500 to 1000 leptospires in 5 pL medium were added to each sample. In addition, to study DNA EXTRACTION FROM SEMEN the sensitivity of the PCR assay, 10An equal volume of TE buffer confold serial dilutions of leptospira taining 2% Triton X-100 (Sigma stock culture containing 107 organ- Chemicals, St. Louis, Missouri, USA) isms/mL were made in 50 pL of was added to each 100 ,L sample, the semen. samples were vortexed, and then they were heated at 94°C for 10 min. SamINFECTION OF ANIMALS ples were then centrifuged at 14 000 X coninfected bulls were by Two g in a microcentrifuge for 5 min. The junctival sac inoculation of approxi- supernatant was collected and the mately 0.5 mL (approximately 107 DNA released by 1 of 3 extraction organisms) of a log-phase culture of methods. In Method 1 (PC Method), the hardjobovis in semisolid medium(12). Blood, urine, and semen samples were supernatant was extracted with phetaken from each bull before infection nol-chloroform and centrifuged at (day 0), and on days 7, 10, 14, 21, 28, 13 000 X g for 10 min. The aqueous 35, and 42 after infection. Semen was phase was harvested and precipitated collected via electroejaculation. Urine with an equal volume of isopropanol. was collected after administration of The precipitate was collected by cen10 mL of an intravenous diuretic trifugation, then dried and resus(Lasix, Hoechst Canada Inc, Mon- pended in 5 pL of TE buffer. In treal). The animals were killed after Method 2 (PK Method), the supercollection of semen, blood and urine natant from the first centrifugation samples on day 42, after which kidney was digested with 50 pg of proteinase

K (Boehringer-Mannheim) in the presence of 1% SDS for 45 min at 37°C. The samples were then processed as described for the PC Method (Method 1). In Method 3 [CTAB Method, based on the protocol described by Ausubel et al (13)], phenol-chloroform extraction of the supernatant was followed by the addition of NaCl (0.5% final concentration) and cetyltrimethylammonium bromide (Sigma Chemicals, St. Louis, Missouri, USA) (CTAB, 1% final concentration) to the resulting aqueous phase. The mixture was then incubated at 65°C for 10 min, an equal volume of chloroform-isoamyl alcohol (24:1) was added, and the final mixture was centrifuged at 13 000 X g for 10 min. The supernatant was collected and precipitated with isopropanol as in Method 1 above. DNA EXTRACTION FROM URINE

Urine samples (10 mL) from the experimentally infected bulls were centrifuged at 2000 X g for 10 min at 4°C in a Sorvall RC5C centrifuge. The urine was then decanted and the supernatant centrifuged again at 13 000 X g for 15 min at 4°C to collect the leptospires. The pelleted organisms were resuspended in 100 ,L of water. Prior to heating at 95°C for 10 min, 10 ,uL of 10% Triton X100 and 5% Chelex

17

11 l

.

l

-

dTTP (Pharmacia LKB Biotechnology Inc.); 1 pM of each primer; 2.5 units of Thermus aquaticus (Taq) DNA X:=X; X B M (Gibco/BRL Canada polymerase Ltd.); and 5 pL of DNA template in | 0 TE_ buffer. Amplification was performed in a Thermolyne Bio-oven for 4725.2'... .~ 30 cycles of 20 s at 940C, 20 s at 550C .* t 30and 1 min at 720C with a final extenQ| ; _ ; of 5 min at 720C. Aliquots of 15 to ~~~~~~~~~~sion 20 pL from the reaction mixture were analyzed by electrophoresis in 2%

agarose gels containing 0.25 pg/mL ethidium bromide. Gels were electrophoresed for 45 min at 100 V. The bands were visualized by ultraviolet transillumination at a wave length of 340 nm and the gel was photographed with Polaroid type 55 film. To increase the sensitivity of the PCR -assay, a 2nd round of amplification Wo w Mwas performed using the nested Figure 4a. Amplification by PCR of Leptospira DNA in 50 pL semen using external primers. primers. For this, 15 pL of the 1st PCR results from 10-fold serial dilutions are shown in lanes 4 to 10, lane 4 (10-), 5 (1o-2), round of amplification was used as a 6 (10-3), 7 (10-4), 8 (10-i). The result from Leptospira-free semen is in lane 3. Water as a nega- template and the parameters were kept tive control is in lane 2. The pGem marker is in lane 1. The lower limit of detection was

as before. Sensitivity of the assay was

500-5000 organisms.

assessed by performing the assay on quadruplicate samples of 10-fold dilution of leptospires in semen from 107 leptospires/mL of semen through to 10'1leptospires/mL of semen. FLUORESCENT ANTIBODY TECHNIQUE

Urine was spun at 12 000 X g for 30 min. Smears were prepared from the pellet, air-dried, and fixed in cold acetone for 10 mmn. Polyclonal antiserum was applied to the slide and t e preparation incubated at room temperature for 45 min. The slides were 0 ~washed with PBS and blotted. A Cappel goat anti-rabbit fluorescein-conjugated antiserum (Organon Teknika Corporation, West Chester, Pennsylvania, USA) was applied. The slides were incubated in the dark for 40 min at 30°C, washed with PBS for 10 min, and counterstained with Erichrome Black (Sigma Chemicals) (1:1000 Figure 4b. Amplification by PCR of Leptospira DNA in semen using the internal nested dilution) and incubated for 10 mm at primers. Fifteen pL samples from the 1st PCR product were used as a template for the 2nd room temperature. The slides were round of amplification using the internal primers. The PCR results from the 10-fold dilutions then examined under a Leitz Diaplan ........

are shown in lanes 4 to 8 as in Fig. 3a, lane 9 is a dilution of 10-7. PCR result from Leptospirafree semen is in lane 3. The result using water as a negative control is in lane 2. The pGem marker is in lane 1. The lower limit of detection was 50-500 organisms.

(Bio-Rad Laboratories, Mississauga, Ontario) were added to the suspension (14). The DNA was then extracted using the CTAB method (Method 3). 18

fluorescent microscope.

MICROSCOPIC AGGLUTINATION TEST

The microscopic agglutination test (MAT) was performed according to the Standard Methods Protocol used PCR ASSAY Reaction mixtures of 100 ,L con- by Agriculture and Agri-Food tained 2.5 mM MgCl2; 10 mM Tris- Canada. Briefly, an initial serum diluHCI (pH 8.3); 50 mM KCI; 0.2 mM of tion of 1:50 is made and 50 PL is each of dATP, dCTP, dGTP, and placed in a well of a microtitre plate.

An equal volume of a standardized antigen is added and the plate is incubated for 1-2 h at room temperature. The wells are examined using a dark field microscope equipped with a long working distance objective lens. Any sample with 25% or more agglutination is titrated starting at a final serum dilution at 1:50. The titre is reported as the last well with >50% agglutination. For this study, a titre of 1:50 or greater was considered to represent a seroconversion.

RESULTS As predicted from the gene sequence, PCR products of approximately 500 bp for the external primers and 450 bp for the internal (nested) primers, were obtained from semen samples to which serovar hardjobovis organisms had been added in vitro, (Fig. 1, lane 3 and 4). The specificity of the assay was determined by hybridization of biotin-labelled PCR product to hardjobovis DNA (data not shown). No PCR product was obtained from semen in the absence of leptospiral DNA (Fig. 1, lane 2 ). In contrast to the results obtained using Method 3 (CTAB method), no PCR product was obtained from DNA extracted from semen samples using Method 1 (PC method) or Method 2 (PK method). This was true for semen samples seeded in vitro as well as semen samples from infected bulls (Fig. 2 lanes 2, 3 and 4,and Fig. 3

respectively). To determine the sensitivity of the PCR assay for detecting leptospires in semen, 10-fold dilutions of a stock culture containing 107 organisms/mL were made in semen. The PCR assay detected a concentration of 500-5000 organisms when the external primers were used, and 50-500 organisms when the nested primers were used (Fig. 4a and 4b, respectively). To determine if the amount of semen from which the DNA was extracted would have an inhibitory effect on the PCR assay, various volumes of semen were inoculated with a constant number of leptospires. The PCR assay was sensitive enough to detect leptospiral DNA in the presence of 100 pL of semen as demonstrated in Figure 5. Both bulls experimentally infected with hardjobovis showed seroconver-

TABLE I. A comparison of the PCR assay of urine and semen as compared to culture, fluorescent antibody test (FAT), and the microscopic agglutination test (MAT) Animal Bull #1

Bull

Test method PCR (urine)

Days postinfection 0 -

PCR(semen) Culture (urine) Culture (kidney) N/A FAT (urine) FAT (kidney) N/A MAT (serum) -

PCR(urine) PCR (semen) #2 Culture (urine) Culture (kidney) N/A FAT(urine) FAT (kidney) N/A MAT (serum) a Postmortem bNot Applicable c Not tested

7 -

10 +

14 -

+

+

N/A N/A -

N/A N/A -

21 +

28 +

35 +

+

+

N/A

N/A

+ +

+ +

+

+

N/A 1:50

N/A -

N/A N/A -

N/A N/A -

42 + + + N/A + N/A -

+ + -

+ N/A N/A

+ + + N/A + N/A

1:200

1:100 N/T

-

-

+

-

+

+

+

+

N/A N/A -

N/A N/A -

N/A +

N/A 1:100

N/A N/A N/A N/A 1:100 1:200

PMa N/Ab N/A N/A -

N/A -

N/Tc N/A N/A N/A -

N/A +

Figure 5. Amplification by PCR of DNA from Leptospira serovar hardjobovis in varying amounts of semen. Volumes of 5, 10, 25, 50, 75, and 100 pL (lanes 4 to 9, respectively) were seeded with 10 000 Leptospira organisms. The DNA was amplified using the external pair of primers. Water as a negative control is shown in lane 2. Control semen with no Leptospira is in lane 3. The pGem marker is shown in lane 1.

sion, although bull # 1 had a titre of 1:50 on only one occasion. Infection was confirmed in both bulls by isolation of leptospires from urine samples, as well as positive FAT and PCR tests of urine and positive PCR of semen. The 1st culture-positive sample was collected at 28 d postinfection. Leptospires could be detected in urine by PCR in samples collected 10 d postinfection, several days before they could

be detected by FAT. Detection by both PCR and FAT was intermittent. Leptospires were detected in semen samples by PCR on day 7 postinfection in both bulls and all subsequent semen samples were positive (Table 1). FAT tests on the kidneys from both bulls were positive. Cultures from kidney samples from bull # 1 were negative; however, cultures from kidney samples from bull # 2 were positive (Table 1). 19

DISCUSSION

substances present in semen, such as polysaccharides, that are inhibitors Bulls are denied entry into Al cen- for the PCR reaction. tres when they have a serum titre of PCR has been used previously to 1:100 or more. The MAT is unable to detect leptospires in urine (15,16), in distinguish between natural infection human clinical samples (17), and and vaccine titre. Based on current from pathogenic leptospires in human entrance criteria, bull #1 would qual- serum (18). There is no previous ify for entrance to an Al centre and report of the detection of Leptospira bull #2 would not. However, bull #1 in semen. was positive more often by tests In the future, the PCR method might demonstrating the presence of lep- have application in approving semen tospires or leptospiral DNA (FAT, for use for Al purposes. As well, it culture, and PCR) than bull #2. Bull could have application in routine diag#2 would have qualified for entrance nostic cases. Present diagnostic methonly in the 1st 10 d postinfection. ods are not widely used, and lepThese results suggest that serological tospirosis is likely underdiagnosed as titre is not a good indicator of infec- a consequence. Further studies should tion and reaffirms what other studies include development of primers that have shown (1-5). detect specific serovars, development The PCR test was able to detect of primers with increased sensitivity, leptospires in urine earlier than cul- evaluation of the risk of transmission ture or FAT, suggesting that it is a of leptospirosis via commercial more sensitive detection method. As extended semen, and development of expected, detection in urine was inter- PCR protocols for other tissues. mittent with all methods. If leptospires are not being shed in large ACKNOWLEDGMENTS numbers or are being shed intermittently, one would expect variation in The authors thank Dr. E. Golsteyn numbers of leptospires per sample, and thus variation in the ability to Thomas, Dr. V. Gannon, and Dr. detect the organism. Detection of lep- W.D.G. Yates for reviewing this tospires in semen is much more con- manuscript. We would like also to thank sistent, possibly because of a higher Dr. W. Lees for his help in collecting concentration of leptospires per semen and other clinical samples. semen sample as compared to urine sample. Semen was not tested by FAT REFERENCES or culture. Based on past experience in this laboratory, semen slides stained for FAT have too much background for reliable evaluation. Culture of semen was not done due to the high probability of losing such cultures to contamination. This points out one of the advantages of detection by PCR, the test can be run on samples that are not easily handled by traditional test methods. Using pure Leptospira culture, DNA extracted by all 3 extraction methods produced good PCR product (data not shown). However, in semen, only the CTAB method for DNA extraction produced DNA templates that gave PCR product (Fig. 2). The CTAB method appears to remove

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