EFFECT OF AFLATOXIN IN POULTRY

Abstract

The results of the work are summarized as under: I. A total number of 300 samples of finished commercial poultry feeds, obtained from poultry feed mills (75 samples>, wholesale poultry feed dealers (70 samples) and poultry farms (155 samples) were analysed for the presence of aflatoxins and it was found that 126 (42%) samples including 18 (24%) from mills, 19 (27.1%) from dealers and 89 (57.42%) from farms wer-e contaminated with aflatoxins. Extraction of aflatoxin from feed samples was carried out by aqueous acetone method, and chloroform extraction methods. The extracts were qualitatively examined by quick screening method, minicolumn chromatography method and thin layer chromatography methods. All three methods gave comparable results. Purification of extracts was carried out by column chromatography followed by thin layer chromatography. The quantity of aflatoxin in positive samples ranged from 20 microgram/kg to 2000 microgram/kg. The quantity of aflatoxin in majority of the contaminated samples (90.5%) ranged from 20 microgram/kg to 156 microgram/kg feed and only 9.5% of the samples contained higher levels of aflatoxins. Majority of the highly contaminated samples came from commercial poultry farms and were always accompanied with complaints of high mort?litis among the young broiler stocks and production problems in breeder flocks. 1 II. The experimental production of aflatoxin was carried out on long grain rice using a toxigenic strain of Asperaillus parasiticus (FRR-2752) and a semi static incubation method at 28 degree centigrade in an atmospher? of high humidity. The cultures were steamed dried and extracted either by chloroform method or by aqueous acetone method. Long grain rice was found a convenient substrate far experimental production of aflatoxin. In the present study a maximum yield of 803 microgram aflatoxin/g rice was obtained. III. Determination of LD50 of aflatoxin was carried out in 210 one day old Hybred broiler chicks with a average weight of 38 gram each. The chicks were divided into 21 groups labelled from 1 to 21 with 10 birds in each. A single dose of different levels of aflatoxin ranging from 32.82 mg/kg body weight to 0.33 mg/kg body weight was inoculated into crap of chicks in groups 1 to 19, groups 20 acted as solvent control and group 21 as aflataxin free control. The birds were observed for 7 days post inoculation. The birds who have received higher levels of aflatoxin died within few hours of inoculation showing symptoms and lesions of per acute aflatoxicosis. The LD50 ?as calculated by Abbot's probit method and was found to be 9.278 mg/kg b.w. for one day old broiler chicks. IV. effect of a single organs was studied The dose of aflatoxin Bl on immunocompetent in a group of broiler chickens. Day-old 60 Hybred broiler chicks were raised on aflatoxin free feed for 3 weeks and then divided into six groups 2 labelled 1 to 6 with 10 birds in each. A single dose aflatoxin Bl at dose rates of 8, 16, 26, 50 microgram/birds was given to birds in groups of of pure and 100 1 to 5 respectively, the sixth group acted as toxin free control. The birds were maintained on aflatoxin free feed and water ad libitum and observed for 3 week p.i. The bursa of Fabricius, thymus and spleen of each bird was removed and histologically examined. No appreciable histological changes were seen in the organs of birds which have recived 8, 16 and 26 microgram AFB1/bird while reductions in size accompanied with other degenerative changes were seen in thymus, bursa and spleen of birds, which have been injected 50 and 100 microgram AFB1/birds respectively. The normal tissue of these organs was replaced by inflammatory and fibrous tissue. No changes, gross pathological or histological changes were seen in the thymus bursa and spleen of control group birds. v. The Immunomodulatory effects of a single dose of 100 microgram AFB1/bird on the development of immunity against New Castle Disease virus vaccine was studied in broiler and layer chickens. A group of 120 one day-old Hybred broiller chicks were divided into two groups <Exp. I and Exp. II> with 60 birds in each and raised for 3 weeks on aflatoxin free feed. Three randomly selected chicks were bled on first, 7th, 14th and 21st day of age for determination of maternal Haemagglutinin inhibiting (HAI> antibody titres. At 3 weeks of age the broilers were each experimental batch were further subdivided into T, Tl, T2, Kl and K2 groups with 10 birds in each. 3 A batch of layer chicks was raised for 8 weeks on aflataxin free feed and then divided into groups T, Tl, T2, Kl and K2 with 10 birds in each <Experiment III>. Three randomly selected chicks were bled an day one and thereafter weekly far determination of maternal HI antibody titres till 7th week of age. The birds in groups T received vaccine and aflatoxin simultaneously, the birds in groups Tl received vaccine 72 hours before toxin and the birds in groups T2 received toxin fallowed 72 hours later by vaccine. The birds in groups Kl acted as toxin free vaccinated control and the birds in groups K2 acted as toxin free unvaccinated control. The chicks of three experimental batches were maintained on aflatoxin free feed for further 4 weeks. Three randomly selected birds of each group were bled weekly for determination of serum HI antibody titres. At the end of 3rd week post initiation the birds in experiment I and III were challenged with a virulent New Castle Disease Virus while the birds in experiment II were given a booster dose vaccine. All birds of groups K2 <Exp.I and III> died within 72 hours of challenge, while the rest of the birds survived. No death was seen in the birds of Exp. II in the challenge. The survivors of Exp.I,II and III were bled and sacrificed one week after challenge. The sera of birds were examined for HI antibody titres. The results showed that the administration of toxin and vaccine simultaneously depressed the development of HI antibodies significantly. 4 The Immunomodulatary effects of continued feeding of aflatoxin ·on the development of immunity against Pasturella multocida vaccine were studied in layer and broiler chickens (Ex.IV and V). Seventy two one-day old hyline layer chicks were raised for 6 weeks on aflatoxin feed and then divided into groups T, Tl, T2, T3, Kl and K2 with 12 birds in each (Exp. IV). Seventy two Hybred broiler chicks, one-day old <Exp.V> were divided into 6 groups T, Tl, T2, T3, Kl and K2 with 12 birds in each. A toxic feed containing 2.1 microgram of aflatoxin/gram was prepared. The birds in groups T were given toxic feed for 42 days (21 days before and 21 days after vaccination). The birds in group Tl were fed toxic meal for 21 days before vaccination. The birds in group T2 were given toxic feed for 21 days from the day of vaccination. The birds in groups T3 received in single injection of 9.278 mg Aflatoxin/kg body weight on the day of vaccination. The birds in Kl acted as toxin free vaccine control and the birds in group K2 as toxin free vaccine free control. All birds except those in K2 were vaccinated with Pasteurella multocida vaccine an 21st day of age in broilers and 63 days af age in layers and challenged with a virulent Pasteurella multocida organisms 3 weeks post vaccination. Birds of both experiments were shifted to aflatoxin free feed from the day of challenge till the termination of the experiment. One bird each of group T and T3 died in layer chicken. A mortality rate of 11/12 was seen in groups K2 of both experiment. In broiller experiment 6 birds died in group T and 5 birds died in group T3. The experiment was terminated on 7th day post challenge, the survivors were bled and sacrificed. Serum was collected from 4 randomly selected birds on 5 day of vaccination and thereafter weekly till the termination of experiment. The antibody titre were determined by IHA and ELISA test. There was a significant difference <P<0.05) in the mean IHA and ELISA titres of group Kt as compared with the mean titres of groups T, Tl, T2 and T3 in bath experimental groups. The continued feeding of aflatoxin depressed the development of humoral immunity against Pasteurella multacida vaccine. VI. The pathological effects of aflatoxin were studied in 3 week old broiler chicks. A batch of 36 one day?old broiler chicks were raised on aflatoxin free feed for 3 weeks. On 21 days of age the chicks were divided into 3 groups T, Tl and C with 12 birds in each. A single dose of 9.278 mg/kg body weight was injected into the crap of birds in group T? The birds in group Tl received 9.278 mg aflatoxin/bird which was mixed in the feed on which these birds were kept over next 4 weeks. The third group CK> acted as control. The experiment was terminated on 49th day of age by sacrificing the survivors. The internal organs were removed for histological examination. It was observed that susceptibility of birds to aflatoxin decreased with age and law mortalities were observed in experimental groups. The liver in group Tl were enlarged, and the hearts atrophied. Regenerative changes, bile duct hyperplasia and fatty changes were seen in livers of group T. In group T1 the livers were enlarged and showed nodular hyperplasia. Histologically the liver tissues showed acute necrosis, pericellular fibrosis, nuclear disalution 6 bile duct proliferation and lymphoid hyperplasia. Degenerative changes were also seen in heart and kidneys and other organs. No gross or histological changes were present in the organs of control group K. VII. The nutritional effects of aflatoxin an live weight, dressed weight, weight of liver, heart and gizzard and some serum enzymes and bilirubin was studied in a group of 165 broiler chicks. Day old hybred broiler chicks were raised on aflataxin free feed for 3 weeks and divided into 3 groups T, Tl and K with 55 birds in each. Each bird in group T received a single dose of 9.278mg aflataxin/body weight while each bird in group Tl received 9.278 mg aflatoxin which was mixed in feed and offered ad libitum to these birds aver the next 4 weeks. The birds in group K acted as a toxin free control. The experiment was terminated on 28th day p.i by sacrificing the survivors. Five chicks from, each group were bled through cardiac puncture and sacrificed, daily from Oday (21st day age> ta 7th day p.i (28th day age) and thereafter, weekly till the end of the experiment. Serum of these birds was examined far SGOT, SGPT, LDH, Alkaline Phosphatase and Bilirubin. The continued feeding of aflatoxin or administration of a single dose of aflatoxin significantly depressed the live weight, dressed weight and weight of heart, while it signifi'cantly increased the weights of liver and gizzard. Administration of a single dose of aflatoxin or continued feeding of aflatoxin produced significant changes in the enzymic profile. The volume activity of these enzymes and bilirubin were significantly increased in birds of group T with·n 7 24 hours of toxin administration, the values remained higher during the first week and thereafter slowly came down by the end of the 2nd week. In birds of group T1 the volume activity of these enzymes and bilirubin increased gradually during the first week and remained significantly higher till the termination of the experiment. The values in control group remained on baseline levels. VIII. A significant difference CP <0.05) was seen in the total serum protein values of bird which have received a single dose of Aflatoxin (Tl>, the birds which have kept an aflataxin contaminated feed (T2> and the birds which were fed aflatoxin free feed CC>. Much greater depression was seen in the birds which have received a contaminated feed. The single dose group showed a recovery and the serum protein values were comparable with the control group by the end of 6th week. Continued feeding of aflataxin significantly <P<0.05) depressed the serum Fe and serum Calcium levels while it significantly <P <0.05) increased the serum Inorganic Phosphate levels in broilers,as compared with the values of the control group. A single dose of aflataxin also significantly <P <0.05) depressed the total serum Fe and serum Calcium levels and significantly (P <0.05) raised the serum Inorganic Phosphate levels as compared to the control group during the first week post administeratian, but later on showed a recovery. IX. The birds of control group showed a linear increase in weight. The birds which have received 2.lug aflatoxin/g feed did 8 not show a consistant growth pattern, the depression during three weeks post administration was followed by a recovery during the following period. In birds which were given 4.2 ug aflatoxin/g feed showed a depressed growth which was almost linear in character. significant Differences in the growth and growth rates were <P <0.05) between three groups. The feed intake in three groups was significantly (P <0.05) different, being high in the 2.lug group and higher in the 4.2ug group as compared to the control group.· x. The layers consuming 2.1ug aflatoxin per gram feed started laying by the end of 20th week while the laying in controlled birds started by the end of 21st week of life. The production in toxin group remaind significantly depressed throughout the course of experiment. The egg size and the weight of eggs in the aflatoxin group was lower than that of the control group. XI. Te layer chic?e s raised for 1 year on feed containing 2 ug aflatoxin/g fee did not r-eveal any evidence of carcinogenicity. XI I. The livers, ?idneys and breast muscles of broiler chickens in experiment V "immunomodulatory effects of Aflatoxin" and layers of experiment, "Carcinogenic Effects of Aflatoxin" were examined for aflatoxin residues in these tissues. No aflatoxin residues were detected in liver, kidneys and breast muscles of broiler chickens. Aflatoxin at a level of microgram/100 gram tissue was present in the liver of layer 9 1 chickens while it was present at levels lower than 1 microgram/100 gram tissue in kidneys of these birds. No aflatoxin could be detected in the breast muscles of layer chickens. 10