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Alexandria Journal of Veterinary Sciences AJVS. Vol. 53: 131-137. April 2017 DOI: 10.5455/ajvs.243277

Clinical, Biochemical and Histopathlogical Alteration in Broiler Chickens Experimentally Infected with H9N2 Avian Influenza Virus During Aflatoxicosis Hany F. Ellakany 1, Hanaa A. Elsamadony2, Ahmed A. Elbestawy 1, Heba Allah S. Ismael2 1Department

of Poultry and Fish Diseases, Faculty of Veterinary Medicine, Damanhour University, Egypt 2Animal Health Research Institute, Dokki Lab. (Poultry Dep.), Egypt

Abstract Key words: Broiler, H9N2 Avian Influenza Aflatoxicosis

Correspondence to: [email protected] m


The present study was conducted to evaluate the adverse effects of an interaction between low dietary aflatoxins and H9N2 infection in broiler chickens. They were infected with H9N2 AI virus at the 23 days of age at dose of 10 6 EID50/100 ul/bird and /or fed on diet containing 200ppb aflatoxin with a group kept as a non-treated noninfected negative control. Aflatoxins treated groups exhibit significant lower body weight gain (average530g) than the control group (average 1850g). The clinical signs of H9N2 were more pronounced in the group which received aflatoxin and infected with H9N2 than the group infected with H9N2 alone. Also there was significant increase in serum (AST and ALT) liver enzymes in chickens in this group. Histopathological examination regarding aflatoxins treated groups there was hepatomegaly, hydropic degeneration, hepatocytic vacuolation and necrosis in liver with nephrosis and ureates deposition in kidney. The lesions in trachea and lung in the H9N2 infected groups were more sever in the group receive aflatoxins and infected with H9N2 virus than group infected with H9N2 alone. Thus the aflatoxins severely affect the performance traits in broiler and when combined with H9N2 virus increase the mortalities from 0 in nonaflatoxicated groups to 20% in aflatoxicated groups.


detected in Egypt (Soliman et al., 2012). Moreover, it has been reported that the Egyptian H9N2 strain have the ability to become more pathogenic by acquiring basic amino acids at the HA cleavage site (Abdel- Moneim et al., 2012).

Low pathogenic avian influenza virus H9N2 is becoming a serious threat to poultry. H9N2 is an emerging threat inducing respiratory problems, it has been isolated from different birds from a number of countries and reported to have zoonotic potential (Lin et al., 2000; Alexander, 2003 ; Ahad et al., 2013a.b). H9N2 AIV has been isolated from live bird markets and poultry farms as well as from poultry workers from different parts of India, Pakistan and Egypt (Abdel-Moneim et al., 2012; Afifi et al., 2013).

It is well known that immunosuppression can increase disease susceptibility to various bacteria and viruses in poultry (Subler et al., 2006). Host cell mediated immune response is considered important in the pathogenesis of avian influenza viruses and plays an important role in recovery from viral infection (Wells et al., 1981). One of the immunosuppressive agent that influence the pathogenesis of H9N2 virus in broiler is aflatoxins as reported by (El Miniawy et al., 2014) who found that even very low levels of aflatoxins can disturb the immune system of birds and thus can exacerbate disease outcomes in association with other pathogens.

Egypt is located in the pathway of migratory birds and represents a hinge zone of wild bird migration, where the East Africa–West Asia and Black Sea–Mediterranean flyways overlap and large diversity of species migrating to and from South Africa, Europe, and Central Asia were


Ellakany et al., 2017. AJVS 53:131-137

randomization procedures that approximately equalized the initial body weights among the different groups.

Aflatoxins produced by A. flavus and A.parasiticus in both field and storage. Infection is most common after the kernels have been damaged by insects, birds, mites, hail, early frost, heat and drought stress, windstorms and other unfavourable weather (Jacobsen et al., 1993).Aflatoxins is a potent liver toxin causing hepatocarcinogenesis, hepatocellular hyperplasia, hepatic necrosis, cirrhosis, biliary hyperplasia, and acute liver damage in affected animals. Other effects include mutagenic and teratogenic effects. Large doses of aflatoxins are lethal and chronic exposure to low levels of aflatoxins can result in cancer and immunosuppression (Sharma, 1993).

Group I (control group) received the control ration but no AF and served as negative control. Group II received the aflatoxin containing basal diet (200 ppb). Group III received the aflatoxincontaining diet (200 ppb) and was infected with H9N2 virus. Group IV received the basal diet with no aflatoxins but was infected with H9N2 virus. 2.5. Production of aflatoxin: Aflatoxins were produced by growing standard aflatoxigenic strains on sterile polished rice by the method modified by (West, et al., 1973). Briefly, the rice was cleaned, washed, and autoclaved at 121°C for 15 min, dispensed into 500ml Erlenmeyer flasks, and moistened with distilled water (10 ml/flask). Each flask was injected with 10 ml of a fresh saline spore suspension of Aspergillus flavus containing 108 spores per milliliter and then sealed with a tight cotton cork. The flasks were incubated for 2 days at 15°C, then for another 3 days at 20°C and 3 days at 26°C. The flasks were shaken vigorously every day to prevent clumping of the rice, to ensure a homogenous toxin distribution, and to prevent fungal overgrowth. Finally, the flasks were sterilized by autoclaving to kill the fungus and its spores, and the toxins were restored. The rice was dried and ground into powder in an electric blender. No other mycotoxins were produced in this solid substrate fermentation process. The rice powder was incorporated into the diet of broiler chickens to provide a final concentration of 200 ppb total aflatoxins (AF) (Sakhare et al. 2007). AF were detected quantitatively in rations by using affinity column chromatography (Aflatest 10, Naremco,Springfield, IL, USA) and fluorometry (Sequcia Tuner Model 450 with a 360 nm excitation filter and a 450 nm emission filter) according to the method of (Nabney and Nesbitt ,1965).

So this work was aimed to study the effect of combined H9N2 virus with aflatoxins in commercial broilers chickens. 2. MATERIAL AND METHODS 2.1. Experimental birds One hundred unvaccinated day old Cobb chicks were obtained from commercial hatchery (West Badrashin Association) and were used to carry out this experiment. 2.2 Challenge virus Avian influenza A H9N2virus strain (Accession No. under preparation with NCBI ) was kindly obtained from Department of Poultry Diseases, Faculty of Veterinary Medicine, Damnhour University. 2.3 Asperigellus strain for aflatoxin production Asperigellus flavus strain was obtained from Department of Mycolgy, Animal Health Research Institute , Agriculture Research Center, Ministry of Agriculture. 2.4 Experimental design: The reared day old chicks were weighed and randomly allocated into four main groups (25 chicks each).The chicks were ranked by restricted Table I: Experimental design Group I

Aflatoxin from day 0 to 35 days 200 ppb in basal diet -

H9N2 infection 106 EID50/100μl/bird at 23th day -

Group II



Group III



Group IV




Ellakany et al., 2017. AJVS 53:131-137

clearly in the elevation of liver enzymes and the hepatocytic affection by histopathological examination and these data confirms the data obtained by (Johri and Majumdar, 1990; Verma et al., 2004).

All birds were individually weighed weekly starting from the age of 7days until the end of the experiment. The AF-containing ration (200 ppb) was administered to chicks in groups II and III starting from the first day of age till the end of the experiment at 35 days.

3.2 Mortalities:


On the 23 day after hatching, chicks in (groups III and IV) were infected with 10 6 EID50/100μl/bird H9N2 in diluted allantoic fluid via nasal route. Performance traits, including body weight, body weight gain. Also clinical signs as well as mortality, lesion scoring, biochemical parameters, and pathological examinations were evaluated.

There were no mortalities in the negative control group (I) and the H9N2 infected group(group IV) while there were 20% (5chicks of 25 chicks) mortalities in the group treated with aflatoxins and inoculated with H9N2 virus (group III). 3.3 Post mortem lesions:


There were no characteristic lesions in group IV which infected with H9N2 virus only except for mild sinusitis, tracheal exudates and mild air saculitis which was more pronounced in group III which was infected with H9N2 virus and treated with aflatoxins

3.1 Clinical signs: No abnormal clinical signs were observed in the non-infected non-toxicated negative control group from the one day till the end of experiment at 25 days old.

The aflatoxins treated groups (II and III) showed marked enlargement in liver with abnormal discoloration and kidney enlargement with ureter distended with ureates.

The clinical signs in groups III and IV which were infected with H9N2 virus were restricted to depression, anorexia, and decreased feed consumption, mild respiratory manifestation as sneezing and mild conjunctivitis as reported by (Swayne and halvorson, 2003;Ladaman et al., 2008; Hadipour et al., 2011).

These results reflect the harmful effect and the stress factor of the aflatoxins on the severity of the H9N2 virus infection in broilers which exhibited by the more pronounced clinical signs in the group received both aflatoxins and H9N2 virus (group III) than the group infected only with H9N2 virus (group IV). These results are in agree with ( Lee et al., 2007; Elminiawy et al., 2014; Umar et al., 2015) who reported that presence of aflatoxins induce immunosuppression and thus increase the severity of H9N2 virus infection while infection with H9N2 cause only transient mild sinusitis, conjunctivitis and depression with no mortalities.

These clinical signs were more prominent in the group treated with aflatoxin and inoculated with H9N2 virus (group III) than the group inoculated with the H9N2 virus alone (group IV). Group II treated with the aflatoxins only showed marked decrease in feed consumption with higher feed conversion rate and stunted growth with lower weight gain when compared with control group. The suppressed appetite in aflatoxicosis is due to the impaired liver metabolism consequently to the impaired liver structure which appeared

Table (1): Mean values ± S.E of the average body weight (grams) in control and experimental groups received feed contain 200ppb AFB1 and/or infected with H9N2 AIV Group I Group II Group III Group IV DAY 1 39.2 ± 0.58 40 ± 0.7 40.8 ± 0.58 40.2 ± 0.86 7 DAYS 164 ± 1.18a 120.4 ± 2.13b 123 ± 1.41b 162.2 ± 0.81a 14 DAYS 290 ± 7.41a 148 ± 24.72b 158 ± 18.54b 298 ± 6.63a 21 DAYS 876.4 ± 22.4a 324 ± 72.42b 302 ±52.66b 879 ± 21.93a 28 DAYS 1532 ± 20.83a 482 ± 34.26b 500 ± 41.83b 1020 ± 25.49c 35 DAYS 1850 ± 35.35a 560 ± 36.74b 530 ± 25.49b 1240 ± 43.01c Values have different scripts at the same row are significantly different at P ≤ 0.05 (n = 10)


negative impact on weight gain is irreversible (Leeson et al., 1995).

3.4 Body Weight: The average weight of each group was taken from day one and weekly till the end of the experiment and illustrated in table (1) Aflatoxin was presented from day 0 to 35 days of age at dose of 200ppb Chickens were infected by H9N2 virus at 23 days of age at dose of 106 EID50/ml.

3.5 Serum biochemistry (AST& ALT): The liver enzymes activity (AST-ALT) in the serum samples from chicken groups received feed contain 200 ppb AFB1 and/or infected with 10 6 EID50/mlH9N2 AIV illustrated in table (2). Aflatoxins treated groups (groups II and III) showed significant increase in serum ALT and AST level based on comparison with the control chickens which were statistically significant,[p

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