Lesions Of Necrotic Enteritis Research Articles

Caracterização de coccidiose e avaliação de casos sugestivos de enterite necrótica subclínica em frangos de corte

ABSTRACT: This study performed the characterization of coccidiosis in broilers and evaluated the occurrence of suggestive cases of necrotic enteritis (NE), seeking if there is an association between the diseases in Brazilian flocks. Two hundred and fifty-six birds from 32 flocks were evaluated. Macroscopic and histopathological lesions were graduated for coccidiosis and NE. Intestinal content was investigated by polymerase chain reaction (PCR) for seven species of Eimeria and by selective anaerobic culture for Clostridium perfringens and identification of the NetB gene. Flocks positive for coccidiosis represented 93.8%. Macroscopic lesions of coccidiosis were Grade 1 for E. acervulina (27%); E. tenella (9.7%) and E. maxima (8.9%). Histopathological evaluation showed Grade 1 in duodenum (38.2%); jejunum (21.4%); cecum (9.3%) and ileum (5%). PCR demonstrated positivity for E. tenella (21.9%), E. maxima (18.8%), and E. acervulina (3.1%). Suggestive macroscopic lesions of necrotic enteritis ranged from Grade 1 (16%), 2 (23%) and 3 (10,9%). Histopathology indicated the absence of necrosis, showing only hemorrhage in the mucosa and submucosa, with the presence of Eimeria spp. Clostridium perfringens type A netB+ was not isolated, demonstrating that macroscopic lesions found mostly in the jejunum did not characterize NE, based on histopathology and negativity of the NetB gene. The study suggests that, due to the high occurrence of coccidiosis, many macroscopic findings suggestive of NE are, in fact, attributed to atypical lesions caused by the reproduction of Eimeria spp.

Genetic characterization, antibiogram pattern, and pathogenicity of Clostridium perfringens isolated from broiler chickens with necrotic enteritis

The aims of this investigation were characterization, demonstration of the antibiogram pattern and detection of the pathogenicity of Clostridium perfringens (C. perfringens) strains isolated from broiler chickens in Damietta governorate, Egypt. A total of 357 samples representing 202 intestinal contents and 155 liver samples from freshly dead broiler chickens were collected from 18 broiler farms. Isolates of C. perfringens were identified morphologically, microscopically, and biochemically. Forty-seven C. perfringens isolates were recovered, which represented 20.3% of the intestinal contents and 3.8% of the liver samples. The toxins and virulence genes of C. perfringens were investigated using polymerase chain reaction. All of the toxigenic C. perfringens strains were type A and carried netB, tpeL, cpe, and plc genes. The in vitro antibiogram of C. perfringens strains revealed 100% sensitivity to gentamycin and levofloxacin and 100% resistance to nalidixic acid and ceftriaxone. The isolated C. perfringens strains were highly pathogenic and induced signs and lesions of necrotic enteritis as well as 43.3% mortalities in 20-day-old chicks. In conclusion, C. perfringens is an important pathogen that affects broiler chickens due to the presence of virulence genes and the pathogenicity in the inoculated birds.

Experimental induction of necrotic enteritis with or without predisposing factors using netB positive Clostridium perfringens strains

BackgroundPoultry necrotic enteritis (NE) is an economically important disease caused by C. perfringens. The disease causing ability of this bacterium is linked with the production of a wide variety of toxins. Among them, necrotic enteritis B-like (NetB) toxin is reported to be involved in the pathogenesis of NE; in addition there is some circumstantial evidence that tpeL toxin may enhance virulence, but this is yet to be definitely shown. The situation becomes more complicated in the presence of a number of predisposing factors like co-infection with coccidia, type of diet and use of high protein diet. These co-factors alter the intestinal environment, thereby favoring the production of more toxins, leading to a more severe disease. The objective of this study was to develop a successful animal model that would induce clinical signs and lesions of NE using C. perfringens type G strains obtained from field outbreaks. A separate trial was simultaneously considered to establish the role of dietary factor with coccidial co-infection in NE.ResultsThe results have shown that use of net-B positive C. perfringens without predisposing factors induce moderate to severe NE (Av. Lesion score 1.79 ± 1.50). In a separate trial, addition of fish meal to a feed of C. perfringens challenged birds produced higher number of NE cases (Av. Lesion score 2.17 ± 1.28). However, use of less virulent E. necatrix strain along with fish meal in conjunction with net-B positive strain did not alter the severity of NE lesions in specific pathogen free chicken (Av. Lesion score 2.21 ± 1.13).ConclusionsThis study suggests that virulent C. perfringens type G strains can induce NE lesions in the absence of other predisposing factors. Birds in the clostridia challenged group showed moderate to severe NE lesions. Use of less virulent coccidia strain contributed to a lesser extent in increasing the severity of disease. Maize based diet along with fishmeal (1:1) increased the severity of lesions but statistically it was non-significant. The NE lesions in all experimental groups were found to be present more frequently in the duodenum. In this way, this study provided an effective model for in vivo production of NE in poultry birds.

The cytotoxicity and molecular mechanisms of the Clostridium perfringens NetB toxin.

The necrotic enteritis toxin B-like (NetB) toxin secreted by Clostridium perfringens is a key virulence agent in the pathogenesis of avian necrotic enteritis, a disease that causes significant economic loss to the poultry industry worldwide. NetB was purified from Clostridium perfringens type G (CNEOP004) that was isolated from chickens with necrotic enteritis in Japan. EC50 of this purified NetB toward chicken liver-derived LMH cells was 0.63 µg/ml. In vivo pathogenicity of NetB to chicks produced characteristic lesions of necrotic enteritis. Analysis of the localization of the NetB monomer and oligomer molecules on LMH cells showed that both molecules of the toxin were localized in non-lipid raft regions. Moreover, removal of cholesterol with the cholesterol depletion assay carried out in LMH cells detected both oligomers and monomers of the NetB molecule. These data suggest that the NetB toxin may recognize membrane molecules different from cholesterol in non-raft region. Furthermore, NetB-binding molecules on LMH cell membranes using the toxin overlay assay with immunoblotting showed that protein molecules of different molecular sizes were bound to NetB on non-lipid raft fractions. Further studies are necessary to characterize these protein molecules to examine their specific association with NetB binding and oligomerization.

Impacts of antibiotic reduction strategies on zootechnical performances, health control, and Eimeria spp. excretion compared with conventional antibiotic programs in commercial broiler chicken flocks

Increasing efforts have been made in recent years to reduce antimicrobial use in animal production. The objective of this prospective study was to evaluate, in commercial broiler chicken farms, 2 antibiotic reduction strategies that eliminated the use of antibiotics important for human medicine, in comparison with the conventional use of antibiotics. On 7 broiler chicken farms, a house was allocated to the antibiotic reduction treatments for 6 consecutive flocks, whereas a similar house on the same premises was assigned to the conventional use of antibiotics (CONV) for 6 consecutive flocks. The antibiotic reduction strategies consisted of continuous in-feed use of ionophores (TX1) and continuous in-feed use of ionophores with butyric acid (TX2). In the 84 flocks, zootechnical performance was recorded, lesion scoring at 21 and 28 D of age was performed, and fecal samples were recovered during grow out for Eimeria spp. oocysts counts. There was no statistical difference between TX1, TX2, and CONV for weights at slaughter, feed conversion ratios, average daily gains, age at slaughter, total mortalities, and condemnations. The probability of identifying oocysts in the fecal samples significantly increased with the age of the flock, but there was no significant treatment effect between 7 and 16 D of age. At 19 D of age, the probability of a sample containing oocysts was higher in TX1 than in CONV, but TX2 was not statistically different from TX1 and CONV. Predicted oocysts per gram in CONV flocks were significantly lower between 22 and 34 D of age than in TX1 and TX2 flocks, whereas there were no significant differences between TX1 and TX2 for all ages. Lesion scoring of the gastrointestinal system showed no differences for coccidiosis scores between TX1, TX2, and CONV. No lesions of necrotic enteritis were observed. In conclusion, it was possible to adequately control intestinal diseases and maintain zootechnical performances by relying exclusively on ionophores, when compared with broiler chicken flocks using standard shuttle programs with antibiotic growth promoters.

Prevention of Necrotic Enteritis in Broiler Chickens by Prebiotics and Probiotics VS Control by Antibiotics, in Vivo Study

Clostridium perfringens (C.perfringens) induced necrotic enteritis (NE) has become a worldwide significant problem in broilers’ industry. A total of 100 intestinal samples were collected from broilers’ chickens that demonstrated clinical manifestations and post mortem lesions of Necrotic Enteritis. Bacteriological examination of samples revealed that C. perfringens was isolated from (62/100) 62%. PCR was used to screen the circulating toxigenic subtypes and revealed that 14/20 (70%) of isolates were positive to cpa gene thus belonged to C. perfringens type A. Phenotypic antimicrobial resistance patterns was demonstrated by isolates to lincomycin 51/62(82.26%), ampicillin 45/62 (72.6%), nalidixic acid 43/62(69.35%), and spectinomycin 43/62 (69.35%), respectively. Futhermore, 49/62 (79%) of isolates demonstrated multidrug resistance phenotypes. Experimental study demonstrated better performance indicators in C.perfringens challenged chicks fed on probiotic (Bacillus subtilis) and prebiotic (Mannan Oligosaccharide / MOS) compared to the challenged chicks treated with amoxicillin. In conclusion, the prevalence of multidrug resistant C.perfringens in infected broilers’ flocks, signified the need for applying AMR surveillance and raising awareness on the proper use of antibiotics in broilers’ sector. The promising results of NE prevention associated with feed supplementation by Bacillus subtilis and Mannan Oligosaccharide (MOS) represented an effective alternative to be used in conjunction with good management for prevention of NE rather than its control by antibiotics.

Oral vaccination of broiler chickens against necrotic enteritis using a non-virulent NetB positive strain of Clostridium perfringens type A.

Necrotic enteritis (NE) is a severe disease of chickens and turkeys caused by some strains of Clostridium perfringens type A. The disease is well controlled by the use of in-feed antibiotic growth promoters (AGPs). However, due to worldwide public and regulatory pressure to reduce the use of AGPs inter alia, there is an urgent need to develop non-antibiotic based preventative measures. Vaccination would be a suitable control measure, but currently there is no commercial vaccine. NetB (necrotic enteritis toxin B-like) is a pore-forming toxin produced by C. perfringens that has been reported as an important virulence factor in the pathogenesis of NE. The present study tests a non-virulent NetB producing strain of C. perfringens (nvNetB+), with or without adjuvants, as an orally administered live vaccine. Adjuvants used were Gel 01™, Cholera toxin (CT), Escherichia coli wild type heat-labile holotoxin (LT) and mutant E. coli LT (dmLT) (R192G/L211A). Several vaccine administration regimes were tested. All vaccination regimes elicited serum and mucosal antibody responses to alpha toxin and to secreted proteins of both nvNetB+ and a very virulent NetB positive (vvNetB+) strain (p<0.0001 to p<0.05). In some vaccinated groups, there was milder intestinal pathology upon disease challenge. 55% of birds vaccinated orally at days 2, 12 with nvNetB+ adjuvanted with CT did not develop any lesions of NE by 6 days post challenge, compared to a 100% incidence of NE lesions in the unvaccinated disease challenged group.

Pathological effects of Curcuma zedoaria (shoti) flour feeding in growing broilers

Curcuma zedoaria (shoti), is one of the underexploited natural bounty which is considered as a good source of food and or feed for humans and as well as birds. In the present study, we investigated the effects of shoti meal as a primary source of carbohydrate in the growing broiler diet. The birds were fed with the shoti meal @ 10, 20, 40% raw and 20% autoclaved meal/Kg and fed for 19 days. The diet was estimated to have contributed to 83 g crude protein and 750 g starch/Kg dry flour. The results have shown that although there had been no deaths, however, broilers fed on different levels of flour grew slowly. Gross changes included lesions of necrotic enteritis and varying degrees of alterations in the gut and coxo-femoral joints. Entire articular cartilage was separated from the sub-chondral bone. The mesentery was depleted of fat, so was the abdominal and the fat pad from the thighs. The experimental birds appeared to be leaner than the corresponding control. The liver showed fatty changes and chronic inflammatory changes with non-specific granuloma in the parenchyma. The results suggested that more research should be carried out before considering Curcuma zedoaria as a feed source for the poultry.

EXPERIMENTAL PRODUCTION OF NECROTIC ENTERITIS IN BROILER CHICKENS

The present study was conducted to isolate and identify the Clostridium perfringens, the etiology of necrotic enteritis (NE) from broiler chickens and experimental production of NE with this isolate. A total of 50 samples were collected from jejunum of necropsied birds for isolation and identification of Cl. perfringens. Out of 50 samples, only 4 isolates of Cl. perfringens were isolated and identified (prevalence 8%). In experimental NE, the birds of group A (orally administered with only 0.1 ml (1x103 sporulated Eimeria acervulina oocysts/bird) showed dullness, ruffled feather, vent feather soiled with bloody faeces after 1 week of coccidial challenge. The birds of group B (orally administered 1x103 sporulated Eimeria acervulina oocysts/bird and 1 ml of 2 days old broth culture of Cl. perfringens) showed severe depression, ruffled feathers, bloody faeces with fibrinous cast with 80% prevalence rate and 30% mortality in experimental NE. The birds of group C (orally inoculated with 1 ml of 2 days old broth culture of Cl. perfringens) showed no striking clinical, gross and histopathological lesions. Postmortem changes in small intestine (duodenum) were congestion and haemorrhages specially for birds of group A. The most severe gross lesions comprised of ascites, enlarged liver and heart, intestinal distension, profuse haemorrhage, fibrinous cast, fragile intestinal wall and gas bubble formation in the small intestine (duodenum, jejunum and ileum) of birds of group B. Histologically, birds of group B showed hemorrhage and congestion in liver, heart and intestine, desquamation of intestinal epithelium and intense leukocytic infiltration in intestine, liver and heart. The findings obtained from this study showed that simultaneous coccidial infection enhanced the pathological lesions of NE. DOI = http://dx.doi.org/10.3329/bjvm.v9i1.11207Bangl. J. Vet. Med. (2011). 9(1): 33–41

Pathogenicity markers of Clostridium spp. in commercial turkeys

Since growth promoters ban in Europe, enteritis of different aetiologies (virus, bacteria and protozoa) are increasingly becoming the main cause of economic loss in commercial turkeys production. This study is focused on typing of Clostridium spp. isolated from samples of jejunum and ileum of 82 birds out of 17 turkeys flocks. The birds were 6-day to 104-day old, both male and female, with enteric disorders. The presence of toxin NetB was investigated. Multiplex PCR to detect cpa, cpb1, cpetx, cp1, cpb2 and cpe toxin genes were used for Clostridium typing. No lesions of necrotic enteritis were observed. Clostridium perfringens type A was isolated from 25 enteric samples, Clostridium difficile was found in 4 cases and Clostridium sordelli in one case. Clostridium perfringens was present from 6 to 104 days of age indicating its possible role in the enteric disorders of commercial turkeys. NetB toxin was found in no sample. 3 out of 4 isolates of Clostridium difficile were characterized by the presence of toxin genes.

A study on bacterial dissemination and pathology of experimental intraperitoneal infection of broiler chickens with Pasteurella multocida

Following intraperitoneal inoculcation of 11week - old broiler chicken with 1ml (109cfu/ml) Pasteurella multocida the organisms were disseminated in different organs at specific intervals. The microorganisms presisted in the brain (69.23%) for 19 days postinoculation, in the kidneys and ovary or testis (76.92%) for 23 days, in liver (92.31%), in spleen and lungs (100%) and in heart (84.62%) and for 27 days postinoculation in all of these organs. During the first week postinoculation (acute stage). There were extensive congestion, edema, thrombosis and petichial and eccymotic haemorrhages in the various organs. Infiltration of heterophils, fibrin deposition and acute necrotic areas were also found in different organs such as the brain, meninges, intestine, liver, spleen, kidneys, lungs, heart wall and ovary or testis. During the second week postinoculation (subacute stage), the Infiltration of heterophils, fibrin deposition and necrotic areas in different organs were localized and replaced gradually from the outside by lymphocytes and macrophages to form early pyogranulomas. Also, hyperplasia of peyer's patches and solitary lymphoid nodules in the intestinal wall were predominant. During the 3rd and 4 weeks postinoculation (chronic stage), the early pyogranulomas seen in subacute stage become more localized and chronic, consisting of central areas of necrosis surrounded by lymphocytes, macrophages, and giant cells and on the outside there was fibroblasts proliferation. These chronic granulomas were mostly predominant in liver, lungs, kidney and ovary or testis. Also, lesions of chronic necrotic enteritis were seen in certain cases. The above findings revealed that acute septicemic lesions, followed by pyogranulomas and finally chronic granulomas were persistent lesions associated with Pasteurella multocida infection.

A role for the Clostridium perfringens β2 toxin in bovine enterotoxaemia?

Non-enterotoxigenic type A Clostridium perfringens are associated with bovine enterotoxaemia, but the α toxin is not regarded as responsible for the production of typical lesions of necrotic and haemorrhagic enteritis. The purpose of this study was to investigate the putative role of the more recently described β2 toxin. Seven hundred and fourteen non-enterotoxigenic type A C. perfringens isolated from 133 calves with lesions of enterotoxaemia and high clostridial cell counts (study population) and 386 isolated from a control population of 87 calves were tested by a colony hybridisation assay for the β2 toxin. Two hundred and eighteen (31%) C. perfringens isolated from 83 calves (62%) of the study population and 113 (29%) C. perfringens isolated from 51 calves (59%) of the control population tested positive with the β2 probe. Pure and mixed cultures of four C. perfringens (one α+β2+, one α+enterotoxin+ and two α+) were tested in the ligated loop assay in one calf. Macroscopic haemorrhages of the intestinal wall, necrosis and haemorrhages of the intestinal content, and microscopic lesions of necrosis and polymorphonuclear and mononuclear cell infiltration of the intestinal villi were more pronounced in loops inoculated with the α and β2-toxigenic C. perfringens isolate. These results suggest in vivo synergistic role of the α and β2 toxins in the production of necrotic and haemorrhagic lesions of the small intestine in cases of bovine enterotoxaemia. However, isolation of β2-toxigenic C. perfringens does not confirm the clinical diagnosis of bovine enterotoxaemia and a clostridial cell counts must still be performed.

Clostridium perfringensnecrotizing enteritis of the fowl: A light microscopic, immunohistochemical and ultrastructural study of spontaneous disease

Moderate lesions of spontaneous necrotic enteritis were investigated by light microscopy, immunoperoxidase staining, and electron microscopy. Concurrent coccidial infection was detected in the intestinal mucosa of the majority of diseased birds. Electron microscopy of intestinal epithelium revealed vesiculation and blebbing of the luminal cell membrane, as well as degenerative changes of cytoplasmic organelles. Using immunoperoxidase staining, Clostridium perfringens was identified and shown to constitute the majority of organisms in necrotic tissues, whereas bacteria were never seen in viable tissue. Thus, Clostridium perfringens always appeared to be present in the lesions and to act locally, causing a coagulative type of necrosis. The observed vesiculation of epithelial cell membranes indicates that the hydrolysis of membrane components by bacterial toxins could be important in the pathogenesis of necrotic enteritis.

Influence of Clostridium perfringens and its toxin in germ-free chickens

Twenty-one of 56 germ-free chickens died after receiving an oral inoculation of a broth culture of Clostridium perfringens. At necropsy there was detachment and disruption of the epithelial layer at the tips of villi and sloughed epithelial cells in the duodenum. These are characteristic lesions of necrotic enteritis. Germ-free chickens receiving either purified α-toxin or the supernatant of broth cultures of C perfringens died, but no bird died after receiving supernatant of broth cultures neutralised by anti-α-toxin serum. It was concluded that α-toxin of C perfringens was the cause of death in young germ-free chickens.

A Presumptive Diagnosis of Avian Necrotic Enteritis in the Fowl (Gallus gallus domesticus) from Assam (India)

In postmortem examination of carcasses of White Leghorn chickens, 3 had typical gross lesions of necrotic enteritis affecting the small intestines. The occurrence of avian necrotic enteritis was noticed after an outbreak of coccidiosis had subsided. On the basis of the gross and microscopic examination the disease was differentiated from coccidiosis and ulcerative enteritis.