NECROTIC ENTERITIS ON BROILERS IN SÃO PAULO STATEABSTRACT
The occurrence of the necrotic enteritis disease on a broiler farm in the state of São Paulo is reported. Mortality reached 10% of the 86,000-bird population in a 3-to-4-day period. Major symptoms were depression, diarrhea and leg-related problems. The necropsy revealed, macroscopically, necrotic lesions in · the liver, spleen and small intestine. Bacteriological examinations allowed the isolation of Clostridium perfringens from intestine and liver, where lethal toxin was found when inoculated intraperitoneally in mice. The histopathological examination revealed necrotic lesions in the intestine and liver. Literature data compared to those findings led to its being characterized as a necrotic enteritis outbreak. lt should be mentioned that those data are the first reference of the disease in Brazil.
KEY WORDS:
avian necrotic enteritis; broiler chickens;
Clostridium perfringens
RESUMO
Relata-se a ocorrência de enfermidade em granja de frangos de corte do Estado de São Paulo. A criação, com 86.000 aves, registrou em um período de 3 a4 dias mortalidade de 10% dos lotes. As aves afetadas apresentavam depressão, diarréia e dificuldade de locomoção. A necrópsia revelou, macroscopicamente, áreas necróticas no figado, baço e intestino delgado. O exame bacteriológico das amostras dos animais possibilitou o isolamento de Clostridium perfringens, a partir dos intestinos e figado, nos quais também se determinou a presença de toxina, letal para camundongos quando inoculada intraperitonealmente. O exame anatomopatológico revelou lesões necróticas nos intestinos e figado. Dados de revisão de literatura comparados com esses achados permitiram caracterizar o surto como de Enterite Necrótica. Deve-se salientar que os referidos dados constituem-se na primeira notificação da enfermidade no país.
PALAVRAS-CHAVE:
enterite necrótica aviária; frangos de corte;
Clostridium perfringens
INTRODUCTION
After the first description of necrotic enteritis in birds by PARISH (1961a) in England, other cases were reported showing similar occurrences in other countries such as Australia (NAIRN & BAMFORD, 1967; GARDINER, 1967; HUNGERFORD, 1969); the United States (HELMBOLDT & BRYANT, 1971; BERKHOFF, 1975); Canada (LONG et al., 1974) and India (KWATRA & CHAUDHURY, 1976).
Several authors carried out studies to reproduce the disease experimentally (PARISH, 1961c; NAIRN & BAMFORD, 1967; AL-SHEIKHLY & TRUSCOTT, 1977; TRUSCOTT & AL-SHEIKHLY, 1977; COWEN et al., 1987) as well as its pathology andetiology (McDONEL, 1974;AL-SHEIKHLY & TRUSCOTF,c:1977). Such studies definitely showed the participation of Clostridium perfringens as the agent responsible for such infection.
The purpose of the present study was to investigate the cause ofthe disease which brought considerable lasses to broiler breeding in the state of São Paulo; and in light of the results to add necrotic enteritis to the national avian pathologies, a disease not reported in Brazil till the present moment.
MATERIALS AND METHODS
As a result of the data obtained by anamnesis and the examination of 13 ill and dead birds of an 86,000- bird broiler farm, Hubbard race, the research started aiming at identifying in the laboratory the aetiologic agent responsible for the occurrence.
Intestinal content samples (small, large and cecum) and liver fragments of dead birds euthanized in the final phase of the disease were processed in an attempt to isolate the bacterial agent and also to detect the toxin, following, in this case, the sarne laboratorial sequence as for the botulinum toxin, since both agents belong to the sarne bacterial genus (BALDASSI, 1986).
To detect toxin, the samples of intestinal contents and liver were put in contact with gelatine phosphate buffer, pH 6.2, in a ratio of 1:2 (w/v) and kept under refrigeration for 12 to 16 hours. After this period, the material was centrifuged at 2,000 rpm in a refrigerated centrifuge for 30 minutes. Pure supematants, heated at 100ºC for 15 minutes and treated with trypsin at 1%, were each one inoculated, in a 0.5 mL intraperitonial dose, to a group of 3 albino mice weighing between 18 to 25g. All the groups were observed for 96 hours. Sediments were initially cultivated at 37°C for 48 hours in Tarozzi medium and from there they were cultivated in sheep-blood agar plates which were kept in anaerobic jars in the sarne conditions mentioned above.
The bacterial growth was then examined and isolated in tubes with peptone broth and yeast extract pre-reduced (HOLDEMAN & MOORE, 1975) and subcultivated in the sarne medium added with carbohydrates for biochemical identification. Carbohydrates used were: glucose, fructose, lactose, maltose, salicin, sucrose and starch. A test was carried out for lecithinase and lipase presence, as well as nitrate-nitrite reduction tests and índole production (KONDO et al., 1988).
Fragments of liver, heart and intestine were previóusly fixed in formalin solution at 10%, put in paraffin blocks, cut in 5 micrometer slices and stained by hematoxylin ofHarris and eosin (Me MANUS & MOWRY, 1965).
RESULTS
Historical
Anamnesis revealed the problem in three poultry houses of which two had 25,000 and one 11,000 chicks, totaling 8,552 deaths. Animals affected were, on the average, 29 days old and were debilitated, with diarrhea and leg-related problems, evolving to death within 3 to 4 days. In those affected lots, the average weight ofthe birds was 2.115g at 49 days of age for a feed conversion of 2.32g.
Necropsy findings
The intestinal mucosa, besides being thickened, showed disseminated punctiform ulcerations. The cecum was full of bloody feces and showed a congestedmucosa (Fig. 1). The liverclearly showed necrotic areas of various dimensions.
Histopathology
Heart: Interstitial diffuse myocarditis.
Liver: Focal necrotic hepatitis and passive congestion (Fig. 2).
Intestine: Several fragments ofthe small intestine examined showed a well-preserved histologic structure in almost all its extension, from the mucosa surface up to serosa. lt was clear, however, that especially the apex of some villosities were totally destroyed, with lesions characterized by necrosis with cellular debris and a great quantity of strongly basophilic bacillus. Furthermore the presence of Eimeria spp oocysts was noticed in the submucosa, nearthe mucosa epithelium, with a round morphology, some of them in ellipsis, and with numerous smaller and punctiform formations in their interior. lt should be mentioned that those oocysts, in most cases, were in the villosities with necrotic apexes. On the other hand, villosities morphologically perfect were also carriers of such oocysts. There was also the presence of a discrete-to-moderate inflammatory infiltrate in the connective tissue of the villus submucosa, characterized by the predominance of mononuclear cells where some heterophilae were observed (Figs. 3, 4, and 5).
Bacteriology
Printings prepared from intestinal wall and stained by the Gram method revealed numerous Grampositive bacillus morphologically similar to the Clostridium genus.
The anaerobic culture presented, in ali examined materials, the sarne type of colony as that in the optical microscopy, suggesting the sarne agent observed in ali the samples.
The tests for biochemical characterization were positive for glucose, fructose, lactose, maltose, su.crose, and negative for salicin. Nitrate was reduced to nitrite, starch was hydrolysed and there was no índole production. The lecithinase test was positive while the lipase was negative.
Toxicity test
Results of intestinal contents extracts and liver showed the presence of lethal toxin for mice, which had been inactivated by heating andactivated by treatment with trypsin, demonstrating lethality in a shorter period than that observed without treatment, although there has been no quantitative measurement of this.
DISCUSSION AND CONCLUSIONS
Necrotic enteritis has been associated with enterotoxemia caused by Cl. perfringens. However, it seems difficult to make the morphological differentiation between necrotic enteritis and ulcerative enteritis DAVIS et al. (1971). Therefore, according to BERKHOFF (1975), the identification of the bacteria responsible for those two entities can be of great value to a definite confirmation of the diagnosis. Bacteria isolated in intestine and liver of birds who perished with intestinal necrotic lesion showed, in this study, morphological and biochemical characteristics that allowed their classification as C/. perfringens.
Clostridium perfringens can elaborate soluble "poisons" (Me DONEL, 1980), called toxins, that play an important role not only in the cellular metabolism of the infected organism but also in the indirect production oflesion in the host. There is evidence that the alpha toxin, which is also classified as enterotoxin, is the most powerful Me (LENNAM, 1962).
It should be mentioned that the nondetection of the toxin does not exclude thé possibility of an enterotoxemia. The toxin originallypresent, especially in the case of the beta toxin, might have been inactivated by enzyme action, and as it is recommended by some authors (STERNE & BATTY, 1975) that the investigation might continue through culture, especially when the direct smear ofintestinal contents shows a great concentration of clostridia.
Some authors (PARISH, 1961b; NAIRN & BAMFORD, 1967; LONG et al., 1974) suggest that the aviannecrotic enteritis is an enterotoxic condition caused by the "type C" Cl. perfringens. Others, however, reproduced the disease with toxin obtained from culture of the "type A" Cl. perfringens (ALSHEIKHLY & TRUSCOTT, 1977). A occurrence of Cl. perfringens has also been reported by NAIRN & BAMFORD (1967) but without the type identification.
The presence of double hemolisys in sheep-blood agar and the positive lecithinase activity (Me FARLANE et al., 1941) of the isolated bacterial strain as well as the lethality obtained with inoculation tests show unquestionable evidence of alpha-toxin presence.
HAUSCHILD & HILSHEIMER (1971) suggested that the enterotoxin of the Cl. perfringens was resistant to the treatment with trypsin and, ten years later, it was demonstrated that this digestion might elevate up to 3 times the toxin titer demo_nstrated by intraperitoneally inoculation into mice GRANUM et al. (1981).
Macroscopic and microscopic lesions in examined birds suggest a necrotic enteritis, and characterize the chronic form of the disease. Histological alterations caused by the enterotoxin are characterized by remarkable epithelial cells desquamation, mainly at the upper part of intestinal villosities (Me DONEL, 1974; Me DONEL & DUCAN, 1975), as shown bythe histologic section of the small intestine, representative of the outbreak here reported (Figs. 4 and 5).
In order to investigate the factors that explained the presence of C/. perfringens in the intestine, ALSHEIKLY & TRUSCOTT (1977) studied the role of toxins experimentally and concluded that even the slightest damage produced by coccidia, worms, fibrous feed or any other cause of intestinal lesion associated with the presence of enough quantities of this agent will favor the occurrence of the disease in natural conditions. Of the above-mentioned situations, infection by coccidia seems to be the most significant in providing a favorable environment for the C/. perfringens to set, multiply, produce the toxin and cause the disease, having in field conditions an important role in the occurrence and seriousness of the necrotic enteritis outbreaks (AL-SHEIKHL Y & AL-SAIEG, 1980).
HELMBOLDT & BRYANT (1971) infer that these agents hav a part in the predisposition of the establishment of the disease by a decrease in intestinal motility.
Antibiotics or their residues administered in feed as growth promoters do not necessarily control this condition (MILLER, 1983).
Although considered sporadic by some authors (MAXEY & PAGE, 1977; COWEN etal., 1987) and even though SIMON (1977) believes that the occurrence is more frequent than reported, because it is improperly diagnosed, necrotic enteritis is adisease ofimportance forpoultry breeding dueto the economic losses it brings. The productive rate of the farm prior to the present outbreak showed an average weight of 2,200g for 49-day-old birds with feed conversion of 2.11g. Therefore, it shows that the average loss in weight was about 4%, significantly affecting the feed conversion which represented 10% more in feeding consumption.
Therefore the occurrence of necrotic enteritis is characterized which according to SIMON (1977) must involve the confirmation of the diagnosis by isolating and identifying the causal agent and proving the presence of pathognomonic histopathological lesions of the disease.
Macroscopic aspect of the cecum of a bird with necrotic enteritis showing mucosa edema with strong hemorrhage.
Microphotograph of histological section of the liver of a bird with necrotic enteritis showing the necrotic focal area and a synosoidal dilation. (H.E. staining, amplified 66X).
Microphotograph of histological section of the small intestine of a bird with necrotic enteritis showing necrosis with inflammatory infiltrate at the villus apex, with a great number of bacillus. The Eimeria spp oocyst at the submucosa is clear (arrow). (H.E. staining, amplified 66X).
Amplified detail of the previous figure, showing bacillus at the necrotic villus apex. (H.E. staining, amplified 660X).
REFERENCES
- AL-SHEIKHLY, F. & TRUSCOTT, R.B. The pathology of necrotic enteritis of chickens following infusion of crudetoxinsofClostridium perfringens into duodenum. Avian Dis., v.21, p.241-255, 1977.
- AL-SHEIKHLY, F. & TRUSCOTT, R.B. The interaction of Clostridium perfringens and its toxins in the production of necrotic enteritis of chickens. Avian Dis., v.21, p.256-263, 1977.
- AL-SHEIKHLY, F. & AL-SAIEG, A. Role of coccidia in the occurrence ofnecrotic enteritis of chickens. Avia. Dis., v.24, p.324-333, 1980.
- BALDASSI, L. Isolamento de bactérias do gênero Clostridium e detecção de toxina botulínica a partir de materiais obtidos de bovinos com suspeita clínica de botulismo. São Paulo, 1986. 59p. [Dissertação (Mestrado) - Faculdade de Saúde Pública USP].
- BERKHOFF, G.A. Ulcerative enteritis-Clostridial antigens. Am.J. Vet. Res., v.36,p.583-585, 1975.
- COWEN, B.S.; SCHWARTZ, L.D.; WILSON, R.A.; AMBRUS, S.I. Experimentally inducednecrotic enteritis in chickens. Avian.Dis., v.31, p.904-906, 1987.
- DAVIS, R.B.; BROWN, J.; DAWE, D.L. Quailbiological indicators in differentiation of ulcerative and necrotic enteritis of chickens. Poult. Sei., v.50, p.737-740, 1971.
- GARDINER, M.R. Clostridial infections in poultry in westem Australia. Aust. Vet. J., v.43, p.359-360, 1967.
- GRANUM, P.E.; WHITAKER, J.R.; SKJELKVALE, R. Trypsin activation of enterotoxin from Clostridium perfringens type A. Fragmentation and some physicochemical properties. Biochem.Biophys. Acta, v.668, p.325-332, 1981.
- HAUSCHILD, A.H.W. & HISHEIMER, R. Purification and characteristicsof the enterotoxin of Clostridium perfringens type A. Can. J. Microbial., v.17, p.1425-1433, 1971.
- HELMBOLDT, C.F. & BRYANT, E.S. The pathology ofnecrotic enteritis in domestic fowl. Avian Dis., v.15, p.775-780, 1971.
- HOLDEMAN, L.V. & MOORE, W.E.C. Anaerobic laboratory manual. 3.ed. Virginia: Virginia Polytechnic Institute and State University, Blacksburg, 1975. 324 p.
- HUNGERFORD, T.G. Diseases ofpoultry including caged birds and pigeons. 4.ed. Sydney: Angus and Robertson, 1969. 462p.
- KONDO, F.; TOTTORI, J.; SOKI, K. Ulcerative enteritis in broiler chickens caused by Clostridium colinum and in vitro activity of 19 antimicrobial agents in tests on isolates. Poult. Sei., v.67, p.1424-1430, 1988.
- KWATRA, M.S. & CHAUDHURY,B. Apresumptive diagnosis of avian necrotic enteritis irt the fowl (Gallus gallus domesticus) from Assam (India). Avian Dis., v.20, p.401-406, 1976.
- LONG, J.R.; PETTIT, J.R.; BARNUM, D.A. Necrotic enteritis irt broiler chickens. II Pathology and proposed pathogenisis. Can. J. Comp. Med., v.38, p.467-474, 1974.
- MAXEY, B.W. & PAGE, R.K. Efficacy of lincomycin feed medication for the control of necrotic enteritis in broiler-type chickens. Poult. Sei., v.56, p.1909-1913, 1977.
- Me DONEL, J. L. In vivo effects of Clostridium perfringens enteropathogenic factors on the rat ileum. Jnfect. Immun., v.10,p.1156-1162, 1974.
- Me DONEL, J.L. & DUCAN, C.L. Histopathological effect of Clostridium perfringens enterotoxin in rabbitileum. Jnfect. Immun., v.12, p.1214-1218, 1975.
- Me DONEL, J.L. Clostridium perfringens toxins (type A, B, C, D, E). Pharmacol. & Ther., v.10, p.617-655, 1980.
- McFARLANE, R.G.;DAKLEY, C.L.; ANDERSON, C.G. Haemolysis and the production of opalescence irt serum and lecithovitellirt by the alpha toxirt of Clostridium welchii. J. Pathol. Batteriol., v. 52, p. 99-103, 1941.
- McLENNAM, J.D. Thehistotoxicclostridial infections of man. Bacteriol. Rev., v. 26, p. 177-276, 1962.
- McMANUS, J.F.A. & MOWRY, R. W. Staining methods histologic and histochemical. 3 ed. New York: Harper & Row, 1965. 423 p.
- MILLER, C.R. Necrotic enteritis control with virginiamycin. In: WESTERN POULTRY DISEASE CONFERENCE, 32., 1983. Davis, CA. Proceedings. Davis, CA: Cooperative Extension Service, Univ. Califomia, 1983. p.67-68.
- NAIRN, M. E. & BAMFORD, V. W. Necrotic enteritis of broiler chickens m westem Australia. Aust. Vet. J., v. 43, p. 49-54, 1967.
- PARISH, W. E. Necrotic enteritis in the fowl (Gallus gallus domesticus) I - Histopathology of the disease and isolation of a strain of Clostridium welchii. J. Comp.Pathol., v. 71, p. 377-393, 1961a.
- PARISH, W. E. Necrotic enteritis in the fowl. II. Examination ofthe causal Clostridium welchii. J. Comp. Pathol., v.71, p. 394-404, 1961b.
- PARISH, W. E. Necrotic enteritis in the fowl. III-Theexperimental disease. J. Comp. Pathol., v.71, p.405-413, 1961c.
- SIMON, M. S. Necrotic enteritis in commercial poultry-a technical review. MSD - Ag. Vet. 1977. 26p.
- STERNE, M. & BATTY, I. Pathogenic clostridia. Boston: Butterworths. 1975. 168p.
- TRUSCOTT, R. B. & AL-SHEIKHLY, F. Reproduction and treatment of necrotic enteritis in broilers. Am. J. Vet. Res., v.38, p.857-861, 1977.
