Pathomorphological changes in broiler chickens with combined aflatoxin and Eimeria spp. infection Kumar Pradeep3, Rao P.G. Suguna3,*, Sathyanarayana M.L.3, Byregowda S.M.1,3, Puttalakshmamma G.C.2,3 3Department of Veterinary Pathology, Institute of Animal Health and Veterinary BiologicalsKarnataka Veterinary, Animal and Fisheries Sciences University (KVAFSU), Hebbal, Bangalore 1Institute of Animal Health and Veterinary BiologicalsKarnataka Veterinary, Animal and Fisheries Sciences University (KVAFSU), Hebbal, Bangalore 22Department of Veterinary Parasitology, Veterinary College, Karnataka Veterinary, Animal and Fisheries Sciences University (KVAFSU), Hebbal, Bangalore *Corresponding author; e-mail: sugunabg@yahoo.com
Abstract Pathomorphological changes were studied in combined aflatoxin (AF) and Eimeria sp. infection. Aflatoxin B1 was fed to broiler chickens at the rate of 1 ppm for five weeks and sporulated oocysts of mixed sp. of Eimeria orally at the dose rate of 50,000/bird on 21st day of age. Hepatomegaly with petechial haemorrhages in the liver and regression of thymus and bursa were consistent in all the aflatoxin alone fed birds. Histologically, congestion, haemorrhage, fatty changes with biliary hyperplasia in liver and lymphoid cell depletion were observed in lymphoid organs. Distension of the caecum with frank blood was seen in all the Eimeria infected groups in acute phase with histopathological evidences of haemorrhage, mucosal sloughing and numerous developing stages of Eimeria in mucosal epithelial cells. In the combined infection with AF and Eimeria, the lesions were more pronounced with total loss of caecal architecture and severe liver damage. Reconstruction of lost architecture of intestine commenced early in the Eimeria alone fed group but was delayed in the combination group. The gross and histopathological lesions in the caecum of birds challenged with aflatoxin and Eimeria tenella oocysts in combination were more severe in comparison to the Eimeria tenella alone challenged birds and recovery from coccidiosis was delayed in the combination group. Top Keywords Aflatoxin, Caecum, Coccidiosis, Eimeria tenella, Patho-morphology. Top |
INTRODUCTION The tremendous growth of Poultry farming in India has been threatened by different diseases like New Castle disease, Infectious bronchitis, salmonellosis, coccidiosis and disorders especially those related with feed contaminated with aflatoxicosis and ochratoxicosis. Aflatoxicosis is the important feed associated disorder caused by group of aflatoxins produced mainly by Aspergillus flavus and Aspergillus parasiticus. The toxins are immunosuppressive in nature and increase the susceptibility of poultry birds for infections like Coccidiosis1,2. The present work was undertaken to study the effects of aflatoxin B1 and Eimeria tenella oocysts individually and in combination in broiler chicks by the evaluation of patho-morphological changes. The gross and histopathological changes produced in the experimental infection with coccidiosis and aflatoxin alone and in combination have been recorded. |
Top MATERIALS AND METHODS Production of Aflatoxin Aspergillus parascticus, NRRL 2999 culture maintained at the Department of Veterinary Pathology, Veterinary College, Bangalore was used to produce aflatoxin. Aflatoxin was produced by growing toxigenic Aspergillus parascticus, NRRL 2999 strain in sterile polished rice by following the method described by Shotwell et al.3. Aflatoxin thus produced was quantified by using thin layer chromatography technique at research and development laboratory of M/s Tetragon chemie Pvt. Ltd, Bangalore. Isolation of Eimeria oocysts Eimerian oocysts were obtained from the intestinal contents of broiler birds from commercial broiler outlets after sedimentation, concentration and sporulation4. Later the isolated sporulated Eimerian oocysts were propagated in a group of ten birds for experimental inoculation. Based on the anatomical location in the intestine of the propagated birds, oocysts morphology and sporulation time, the sp. of Eimerian oocysts found in the feces of propagated birds were identified as Eimeria tenella predominantly along with Eimeria acervulina. Experimental design Two hundred, day-old broiler chicks were procured from a reputed commercial hatchery and were reared under optimum managemental practices. On day seven, they were weighed individually and were divided into four groups (T1, T2, T3, T4) consisting of 50 chicks each. The birds of T1 served as a control and received aflatoxin free normal diet. Birds of all the groups received normal basal diet up to seven days of age. From eighth day, T1 and T2 birds continued with normal basal diet while T3 and T4 were fed with ration containing AF at 1 ppm dose rate till the end of the experiment. Each bird of T2 and T4 groups received 50,000 sporulated oocysts of mixed sp. of Eimerian parasite consisting predominantly of Eimeria tenella sp. orally on 21st day viz., 0 day post infection (DPI). The T2 group served as a coccidiosis control and T3 group as an aflatoxin control. Six birds from each group were sacrificed on 0, 3, 5, 7, 9, 11, 14 and 21 DPI and were examined for gross pathological changes in organs and representative samples were collected in 10 % neutral buffered formalin for histopathological examination. Top RESULTS Gross changes The gross pathological changes of the caecum, liver, kidneys, heart, thymus, bursa of Fabricius and spleen of different treatments were recorded on day 0 to 21 DPI. Grossly, the caeca of the birds of T2 group were distended with large serosal and mucosal haemorrhages on 5 DPI (Fig.1). Moderate distension of caecum was seen on 7 DPI with firm caecal cores. The recovery from coccidiosis in the form of formation of pasty feces in the caecal lumen was observed from 11 DPI. Regeneration of the lost caecal mucosal lining occurs with an increase in goblet cell activity in the newly proliferating glandular epithelium. Paleness of liver, kidneys and heart were observed on 5 DPI. The caeca of the birds of T4 group were distended with blood clots, mucosal & submucosal components on 5, 7 and 9 DPI. Kidney, heart and lymphoid organs did not reveal any gross changes except for occasional haemorrhages. Histopathological findings Histologically, caeca of birds with coccidial infection (T2 group) revealed classical changes of coccidiosis with severe degree of congestion, haemorrhages, necrosis, desquamation of villous epithelium into lumen, presence of secondary schizonts, merozoites and macrogametes in enlarged glandular epithelial cells as well as freely in lamina propria and submucosa on 5 and 7 DPI (Figs. 2, 3). Cystic dilatation of the glands of lamina propria with flattened lining epithelium and necrotic debris, degenerating oocysts and heterophils in the lumen were evident on 9 DPI with large number of oocysts in the lumen. From 11 DPI, recovery in the form of proliferation of crypt epithelium with stratification and regeneration of mucosal epithelium was observed which was marked on 14 DPI, with reconstruction of the villous structure. Similar lesions with increased severity and total loss of intestinal components up to submucosa during acute phase of infection (5 and 7 DPI) were observed in birds of group T4. The destruction and loss of architecture of caecum persisted with mild proliferative changes on 14 DPI and on 21 DPI. There was no complete restoration of caecal architecture as observed in T2 group birds, although proliferative changes were observed. There was persistence of oocysts in considerable number in lamina propria and submucosa up to 21 DPI (Fig. 4). The liver of aflatoxin fed birds with or without coccidial infection (T3 and T4 groups) revealed congestion, haemorrhages, fatty cysts, focal necrosis and biliary hyperplasia along with periportal infiltration of lymphoid cells. The kidneys showed mild damage to the tubular epithelium with vacuolation only during the later days of infection. Top DISCUSSION Eimeria tenella is known to cause hemorrhagic typhlitis leading to accumulation of blood and blood clots in the caecal pouches causing distension5. Loss of caecal mucosal components along with blood clots into the lumen might result in the formation of firm caecal cores. Regeneration of caecal mucosal lining might result in the pastyness of the feces. Paleness of the visceral organs could be due to excessive loss of blood due to haemorrhage in the caecum6. The prolonged duration of severity of lesions in the caeca could be attributed to the effect of aflatoxin which might have reduced the integrity of the caecal wall thereby inhibiting the initiation of recovery in the form of reduction in hemorrhage, caecal core formation and proliferative changes in mucosa. The recovery was observed from 14 DPI. These findings coincided with the observations of earlier works2,7,8,9. Liver was pale or yellowish, enlarged and friable in the aflatoxin fed treatments (T3 and T4 groups), which were in agreement with the findings of earlier workers10,11. The histopathological findings of caeca were in agreement with the observations of earlier workers9,12,13,14,15. These findings were not dissimilar to the observations recorded by earlier workers1,7,9. The possible reason for the increased severity could be due to loss of tissue integrity on an account of effect in the intestine. The delay in regeneration of caecal architecture could be attributed directly to the combined effect of aflatoxicosis and coccidiosis and increased susceptibility of birds due to aflatoxicosis to coccidiosis as opined by Edds et al.1 and Wyatt et al.2. Rosa et al.10 and Balachandran and Ramakrishnan16 have also noticed the similar findings. The changes in liver and kidneys could be attributed to the effect of aflatoxin on the parenchyma as observed by Rosa et al.10, Shivappa11 and Balachandran and Ramakrishnan16. The lymphoid organs revealed congestion, haemorrhage, sparse cellularity of follicles, lymphocytolytic activity and histiocytosis in birds of T3 and T4 groups which could be accounted for the effect of aflatoxin on immune cells10,11. |
Top ACKNOWLEDGEMENTS Material support and technical help from M/s Tetragon Chemie Pvt. Ltd., Bangalore for carrying out this work is duly acknowledged. |
Top Figures | Fig. 1.: Caecal pouches distended with blood showing hemorrhages on the serosa at 5 DPI in T2 group;
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| | Fig. 2.: Caecum showing haemorrhage in the lamina propria along with second generation merozoites at 5 DPI in T2 group. H&E × 500;
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| | Fig. 3.: Caecum showing macrogametes in the glandular pithelium in the crypt of Leiberkunh at 7 DPI in T2 group. H&E × 1250;
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| | Fig. 4.: Caecum showing large number of intact oocysts in the lamina propria on 21 DPI in T4 group. Note mild regenerative changes in the crypt epithelium. H&E × 500.
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