Etiology and pathogenesis of necrotic enteritis

Sporulated oocysts of Eimeria acervulina were administered orally to cage-housed broilers at a dose of 3.5 X 10(5) resulted in mild subclinical coccidiosis. Clostridium perfringens incorporated in feed at a level of 2.5 X 10(8) organisms/g. produced lesions characteristic of necrotic enteritis. Mortality of 8% (7/80) occurred in birds fed a ration inoculated with Cl. perfringens alone. Mortality of 35% (28/80) was observed in birds which received an oral dose of E. acervulina and which were fed simultaneously with a ration containing Cl. perfringens. Birds which were fed an inoculated ration two days after an oral dose of E. acervulina showed 41% (33/80) mortality. Birds which received an inoculated ration for two days before administration of an oral dose of E. acervulina demonstrated 18% mortality (15/80). Birds which were fed an inoculated ration four days after an oral dose of E. acervulina showed 10% mortality. Infection with E. acervulina reduced the pH of intestinal contents with a simultaneous depression in serum protein. A 39% increase in intestinal passage time from 178 to 248 minutes occurred on the fifth day after infection with E. acervulina. These experiments suggest that necrotic enteritis, attributed to proliferation of a toxigenic strain of Cl. perfringens, followed intestinal stasis and minimal lesions induced by mild intestinal coccidiosis.     

Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth

This study tested the hypothesis that a host mucogenic response to an intestinal coccidial infection promotes the onset of necrotic enteritis (NE). A chick NE model was used in which birds were inoculated with Eimeria acervulina and E. maxima and subsequently with Clostridium perfringens (EAM/CP). A second group of EAM/CP-infected birds was treated with the ionophore narasin (NAR/EAM/CP). These groups were compared to birds that were either non-infected (NIF), or infected only with E. acervulina and E. maxima (EAM), or C. perfringens (CP). The impact of intestinal coccidial infection and anti-coccidial treatment on host immune responses and microbial community structure were evaluated with histochemical-, cultivation- and molecular-based techniques. Barrier function was compromised in EAM/CP-infected birds as indicated by elevated CFUs for anaerobic bacteria and C. perfringens in the spleen when compared to NIF controls at day 20, with a subsequent increase in intestinal NE lesions and mortality at day 22. These results correlate positively with a host inflammatory response as evidenced by increased ileal interleukin (IL)-4, IL-10 and IFN-gamma RNA expression. Concurrent increases in chicken intestinal mucin RNA expression, and goblet cell number and theca size indicate that EAM/CP induced an intestinal mucogenic response. Correspondingly, the growth of mucolytic bacteria and C. perfringens as well as alpha toxin production was greatest in EAM/CP-infected birds. The ionophore narasin, which directly eliminates coccidia, reduced goblet cell theca size, IL-10 and IFN-gamma expression, the growth of mucolytic bacteria including C. perfringens, coccidial and NE lesions and mortality in birds that were co-infected with coccidia and C. perfringens. Collectively the data support the hypothesis that coccidial infection induces a host mucogenic response providing a growth advantage to C. perfringens, the causative agent of NE.     

Experimental reproduction of necrotic enteritis in the chicken. 1. Mono- and polyinfections with Clostridium perfringens and coccidia with reference to cage managemen]

Experimental mono-infection and poly-infection, using vegetative germs of a CL. perfringens Type A strain 1663 and its toxin as well as E. acervulina, necatrix or mitis oocysts, were applied to 100 SPF chicken aged seven days. While clostridial or coccidial mono-infection did not cause any loss and only unspecific pathologic-anatomic changes, polyinfection was accompanied by diarrhoea and slower weight increase, with 55.4 per cent of the chicken having been lost three to nine days from the outbreak of the infection. The pathologic-anatomic findings recorded from the chicken with poly-infections included haemorrhagic, ulcerative, and necrotic intestinal inflammations, primarily in the small intestin. The results of these experimental infections indicated a possible correlation between CL. perfringens and coccidia in the pathogenesis of necrotic enteritis.     

Effects of necrotic enteritis challenge on intestinal micro-architecture and mucin profile

1. This study investigated the effect of Eimeria spp./Clostridium perfringens induced necrotic enteritis and traditional antibiotic preventatives on intestinal micro-architecture and mucin profile. 2. A total of 600 Cobb 500 broiler chickens were randomly assigned to the following three groups: (i) unchallenged, (ii) challenged, and (iii) zinc bacitracin/monensin (ZnB/monensin) (n = 25 chickens/pen, 8 pens/group). The challenged and ZnB/monensin chickens were individually inoculated with Eimeria acervulina, E. maxima and E. tenella and C. perfringens type A (EHE-NE18) at 9 and 15 d post-hatch respectively, to induce necrotic enteritis. 3. The challenge procedure significantly decreased villus height, increased villus width and increased crypt depth in the challenged compared to the unchallenged chickens. Zinc bacitracin and monensin maintained villus-crypt structure similar to that of the unchallenged chickens. 4. Mucin profile was not affected by Eimeria spp./C. perfringens challenge as demonstrated by periodic acid-Schiff and high iron diamine-alcian blue pH 2 x 5 staining. Zinc bacitracin and monensin decreased the number of intestinal mucin-containing goblet cells. 5. Lectin histochemistry showed a trend towards greater Arachis hypogea (PNA) reactivity in unchallenged chickens. 6. In summary, Eimeria spp./C. perfringens challenge disrupted intestinal micro-architecture; however, challenge did not appear to affect intestinal mucin profile. Traditional antibiotics, zinc bacitracin and monensin maintained micro-architecture.     

Effects of Eimeria brunetti infection and dietary zinc on experimental induction of necrotic enteritis in broiler chickens.

Broilers infected with Eimeria brunetti and given dietary zinc were examined for experimental induction of necrotic enteritis. Inoculation with sporulated E. brunetti oocysts at 7 days of age was followed by 5 consecutive days of oral inoculation with cultured Clostridium perfringens. Feed was supplemented with zinc at 1000 ppm. Upon necropsy of broilers 6 days after coccidial inoculation, necrotic enteritis was found in 20% (2/10) of birds given both organisms and dietary zinc. Coccidial lesion scores were also highest in that group. Birds infected with E. brunetti and C. perfringens with no dietary zinc had significantly higher coccidiosis lesion scores (P less than 0.05) than groups inoculated with E. brunetti only, regardless of zinc supplementation. Alpha toxin levels in intestinal contents were low in groups infected with both organisms, regardless of zinc supplementation. Zinc was tested for effects of alpha toxin production in vitro. In the mid-log phase (6 hours incubation), a high level of alpha toxin was produced in zinc-supplemented media, but this was lost quickly in the presence of trypsin. Addition of zinc partly protected the toxin from the action of trypsin.     

安徽省部分地区鸡球虫种类及感染情况调查

目的了解安徽省五个县（区）鸡球虫种类及感染情况。方法采用群体采样法分别从个调查点采集新鲜鸡粪，检查粪样。阳性粪样采用饱和盐水漂浮和离心沉淀法进行卵囊分离。显微镜下观察卵囊进行虫种鉴定，并统计各调查点的鸡球虫感染率和感染强度等。结果检查172份鸡的新鲜粪样，得出鸡球虫感染率为100％；共检出7种球虫，经鉴定均隶属于艾美耳属，分别为毒害艾关耳球虫（Eimeria necatrix），堆型艾关耳球虫（E．acervulina），巨型艾关耳球虫（E．maxima），柔嫩艾美耳球虫（E．tenella），和缓艾美耳球虫（E．mitis），哈氏艾美耳球虫（E．hagani）和早熟艾美耳球虫（E．praecox）。结论鸡球虫在安徽省的这5个县（区）普遍存在，且有的调查点感染强度很高。     

Optimized necrotic enteritis model producing clinical and subclinical infection of Clostridium perfringens in broiler chickens

In this study we assessed the roles of Eimeria infection and dietary manipulation (feeding a diet with a high level of fishmeal) in an Australian necrotic enteritis (NE) challenge model in broiler chickens. An experiment was designed to test the hypothesis that Eimeria infection and dietary manipulation, i.e., inclusion of fishmeal in the diet, are necessary to induce NE experimentally. The results showed that the combination of Eimeria administration and fishmeal feeding had a significant effect on induction of clinical and subclinical Clostridium perfringens infection. The majority of the mortality that occurred during the second week of the trial was due to an NE outbreak following the C. perfringens challenge. The mortality rate of the birds was 12.00% for the high-fishmeal (HFM; 500 g/kg) group and 9.33% for the low-fishmeal (LFM; 250 g/kg) group when the birds were subjected to C. perfringens and Eimeria. Fishmeal alone did not induce significant mortality in birds challenged only with C. perfringens but showed a significantly higher C. perfringens count than the non-fishmeal (NFM) control group. Eimeria administration had a significant effect on NE-related mortality but did not have an effect on the C. perfringens count. In accordance with the time course of bird mortality, it can be determined that of the 3 successive days of oral gavage with C. perfringens, the first inoculation was essential for inducing NE, but the third had no additional effect on NE-related mortality. Also, reducing the fishmeal level from 500 to 250 g/kg had no negative impact on the reproducibility of the model. It may be concluded that NE can be consistently induced under experimental conditions by feeding broilers a diet containing 250 g/kg fishmeal, using a single inoculation with low numbers of Eimeria, administering one or two oral C. perfringens inoculations, and maintaining appropriate ambient temperatures and diets.     

Necrotic enteritis in cage-reared commercial layer pullets

Necropsy of five 12-week-old pullets from a flock of 99,300 suffering from an increased mortality rate revealed enlarged, gas-filled intestines, the mucosal surfaces of which had the "dirty turkish towel" appearance typical of necrotic enteritis. Although the pullets had been raised entirely in cages, intestinal scrapings revealed the presence of Eimeria maxima. Histopathological findings were compatible with necrotic enteritis. Clostridium perfringens was isolated by anaerobic culture from the intestines. Mortality returned to normal after bacitracin and amprolium were added to the feed.     

Anticoccidial efficacy of diclazuril against recent field isolates of Eimeria from commercial poultry farms

The anticoccidial efficacy of diclazuril, a novel anticoccidial agent, was titrated in laboratory experiments using recent field isolates of Eimeria. Fifty tests were conducted with six individual species isolates, and seven tests were done with a mixture of the six species. Results were based on intestinal lesion scores at necropsy, droppings scores, and weight gain. Diclazuril at 0.5 ppm was almost completely effective against E. tenella, E. acervulina, and E. mitis. Prevention of E. brunetti was better at 1.0 ppm than at 0.5 ppm. In birds infected with E. mitis. Prevention of E. brunetti was better at 1.0 ppm than at 0.5 ppm. In birds infected with E. maxima, diclazuril at 0.5-1.5 ppm significantly reduced lesion scores and droppings scores and improved weight gain, although lesions were higher than with other species. Oocyst shedding by E. maxima was almost completely prevented by 0.5-1.5 ppm. Lesion scores and droppings scores caused by E. necatrix or mixed infections were greatly reduced by 0.5 ppm of diclazuril, but 1.0 ppm was necessary to obtain full protection of weight gain. Results suggest that 1.0 ppm of diclazuril best prevents coccidiosis caused by six species of coccidia in chickens.     

Relative value of oocyst counts in evaluating anticoccidial activity.

Birds medicated with roxarsone and in another experiment with zoalene in the feed produced higher oocysts counts than unmedicated control birds receiving the same oocyst dose of Eimeria tenella or a mixture of six species (E. tenella, E. necatrix, E. brunetti, E. maxima, E. acervulina, and E. mitvati). These experiments confirm the conclusion that oocyst counts constitute an unsatisfactory and unreliable parameter for judging effectiveness of an anticoccidial even though such increases are a relatively rare occurrence in anticoccidial evaluation experiments.     

The influence of diet on necrotic enteritis in broiler chickens.

Necrotic enteritis was reproduced in broiler chickens by mixing cultures of Clostridium perfringens in the feed. Mortality due to necrotic enteritis was higher among chickens fed rations based on wheat, rye, barley, and oat groats than among chickens fed corn-based rations. Addition of pentosanase to a wheat-based diet did not affect the level of mortality due to necrotic enteritis. Addition of pectin and guar gum to different rations severely reduced growth rate and eliminated necrotic enteritis from test birds. Addition of glucose to a corn-based diet caused a small increase in mortality due to necrotic enteritis.     

Development of protective immunity against Eimeria tenella and E. acervulina in White Leghorn chickens inoculated repeatedly with high doses of turkey coccidia

Repeated inoculation (immunization) of 2-week-old white leghorn chickens with 10(6) oocysts of the turkey coccidia Eimeria adenoeides or E. meleagrimitis partially protected chickens against moderate challenge with E. tenella or E. acervulina oocysts, but not with E. necatrix oocysts. After challenge, mean weight gains of the immunized chickens and the unchallenged controls did not differ significantly, but weight gains of unimmunized chickens were significantly lower. The mean feed-conversion ratio of the immunized challenged chickens was 3.14, as compared with 4.42 for unimmunized challenged control chickens. In general, immunization did not markedly reduce intestinal lesions. Repeated inoculation of chickens with the turkey coccidium E. gallopavonis failed to produce statistically significant protection against challenge with E. tenella, E. acervulina, or E. necatrix, as determined by weight gain, feed-conversion efficiency, and lesion scores. Antibody profiles of individual chickens did not correlate with protection.     

Prior or concurrent exposure to different species of avian Eimeria: effect on sporozoite invasion and chick growth performance.

The effects of prior (immunity) or concurrent administration of Eimeria acervulina or Eimeria tenella on cellular invasion in vivo and in vitro and on growth performance in white leghorn chickens (WLC) were examined. Weight gains of WLC immunized with E. acervulina and challenged with E. tenella were significantly greater than those of nonimmunized chicks challenged with E. tenella (this occurred despite the increased invasion by E. tenella in E. acervulina-immunized chicks that was reported earlier). The weight gains and modest but consistent improvements in intestinal lesion scores, feed conversion ratios, and oocyst shedding in immunized/challenged WLC indicated that E. acervulina conferred a small measure of protection against E. tenella infection that was independent of the effect on invasion. In contrast, immunization of WLC with E. tenella significantly decreased (41%-51%) invasion by E. acervulina as compared with that in nonimmunized WLC but had little effect on chick growth performance. Concurrent inoculation of chicks with E. tenella and E. acervulina had little effect on invasion by E. tenella sporozoites or on subsequent performance of the chicks. In vitro, prior exposure of cultured cells to either of two isolates of E. tenella also caused a significant decrease in invasion by E. acervulina. No gross changes occurred in the culture morphology between the E. tenella-inoculated and noninoculated cultures. Collectively, the data indicate that prior exposure of WLC and cultured cells to single isolates of avian coccidia markedly influenced invasion by other species but had less effect on the growth performance of the birds.     

Oocyst output and transmission rates during successive infections with Eimeria acervulina in experimental broiler flocks

The infection dynamics of Eimeria species determine the clinical manifestation of the disease coccidiosis in poultry flocks, and a better understanding of the dynamics may contribute to improvement of control measures. Our aim was to study the course of infection and the transmission of Eimeria acervulina in groups of broilers by quantifying the transmission rate parameter and oocyst output. Three transmission experiments were carried out with groups of 20 male SPF broilers. At 2 days of age, one bird in each trial was orally inoculated with five sporulated E. acervulina oocysts (D0 post-inoculation, pi). One day after inoculation (D1 pi), the inoculated bird was housed with 19 non-inoculated contact birds. Individual faecal droppings were examined daily from D3-D32 pi to quantify the number of oocysts per gram faeces. The inoculated bird started shedding oocysts at D5 pi and contact birds between D10 and D17 pi. Contact birds that became infected due to oocyst excretion by the inoculated bird were characterized as first generation contact birds (C1). Contact birds excreting from D15 pi onwards (C2) became infected after the first C1 birds had started shedding and were considered to belong to a successive generation of the flock infection. Oocyst output was significantly lower for C1 compared to C2 birds, but the transmission rate parameter remained constant for both infection generations. These results suggest that although oocyst load increases, the transmission rate of E. acervulina remains constant between successive generations of infection in a flock.     

Anticoccidial efficacy of diclazuril in pheasants

Diclazuril, a new anticoccidial drug, was tested in young pheasants artificially infected with the three most common pathogenic species of Eimeria, E colchici, E duodenalis and E phasiani. In two replicate experiments each with 40 birds the mortalities in the infected controls were 50 and 25 per cent. Diclazuril was administered in the feed at dose levels of 1, 2 and 4 ppm from the day before the inoculation of coccidia until the end of the test on day 6 after infection. The 1 ppm dose failed to inhibit the development of the parasite completely, as was shown by a reduction of the weight gain of the birds and the output of a small number of oocysts. Diclazuril at 2 or 4 ppm adequately controlled the infection, with weight gains similar to those of the uninfected controls. At all dose levels, mortality, intestinal lesions and diarrhoea were prevented.     

Influence of coccidiosis on growth rate and feed conversion in broilers after experimental infections with Eimeria acervulina and Eimeria maxima

Under experimental conditions, the effects of subclinical Eimeria (E.) acervulina and E. maxima infections on growth and feed conversion in broilers of different ages were analysed. It was concluded that infection with E. acervulina and E. maxima led to a process which was independent of the age at which the birds were infected. The infection adversely affected growth and feed conversion for 2 to 3 weeks, followed by a recovery period of 2 to 3 weeks when compensatory growth took place. From this study it may be concluded that subclinical coccidiosis in the first weeks of life and in the last week of life of broilers does not lead to appreciable damage on growth and feed conversion. Since coccidiosis cannot be avoided in practice, systems in which broilers contact subclinical coccidiosis either in the first weeks of life or in the last week of life should be aimed for. It is suggested that in a coccidiostat programme an efficient anticoccidiosis agent is particularly desirable in the 3rd and 2nd week before slaughter. Examinations performed one week or less before slaughter can hardly be justified, on the grounds that there is a risk of a negative effect on growth an feed conversion due to subclinical coccidiosis.     

Infection by Salmonella typhimurium, S. agona, S. enteritidis or S. infantis of chicks with caecal coccidiosis

Experimental infections of Salmonella typhimurium, S. agona, S. enteritidis or S. infantis were studied in chicks infected with Eimeria tenella. In all experiments, 4-d old birds were given 5 daily oral doses of approximately 10(4) salmonella organisms per bird. One day before this inoculation one group of birds received a single dose, in the range 1 X 10(4) to 4 X 10(4) sporulated oocysts of E. tenella per os whilst the other group was not inoculated and served as the control. Chicks were examined post morten 7, 10 or 14 d after receiving coccidia. With S. typhimurium infection, the number of salmonellas in the caeca of the E. tenella-infected birds was greater than in those infected only with salmonella. With S. agona and S. enteritidis, the counts of salmonella in the caeca and the numbers of birds positive for salmonella in the caeca and liver were greater in the E. tenella-infected birds. The rate of infection of S. infantis was not increased by caecal coccidiosis.     

Characterization of the antibody response in birds following infection with wild-type and attenuated strains of Eimeria tenella and Eimeria necatrix

Live vaccines containing attenuated parasite strains are increasingly used to control chicken coccidiosis. In this paper antibody responses elicited by infections with wild-type and attenuated strains of Eimeria tenella and Eimeria necatrix were characterized by immunoblotting and ELISA with homologous and heterologous antisera. Few differences between antisera from birds infected with wild and attenuated strains of E. tenella were evident in immunoblots conducted with merozoite antigen preparations from both E. tenella strains, however the reactivity of sera raised in birds infected with the wild-type strain was noticeably more intense. In ELISAs conducted with merozoite antigen preparations, antisera from birds infected with the wild-type strains of E. tenella and E. necatrix consistently produced a significantly higher (P<0.05) antibody response than antisera from birds infected with the attenuated strains. Likewise, avidity ELISAs conducted with the E. tenella strains demonstrated that antibodies in birds infected with the wild-type strain were of significantly higher avidity (P<0.05) than antibodies in birds infected with the attenuated strain. The differences in the antibody responses are probably due to changes in the attenuated strain as a result of selection for precocious development and the less severe tissue damage and inflammation of the intestine resulting from infection with the attenuated strain.     

Evaluation of interspecific hybrids of the chicken, guinea fowl, and Japanese quail for innate resistance to coccidia

Experimental chicken/guinea fowl hybrids, guinea fowl, and chickens were orally inoculated with Eimeria acervulina or E. tenella, which are specific for chickens, or with E. grenieri, which is specific for guinea fowl. No intact oocysts were found in feces within 24 hr of inoculation, suggesting that excystation occurred in the normal and abnormal hosts. No oocysts were found in the feces of hybrids during a 9-day postinoculation period. The guinea fowl passed oocysts of guinea fowl coccidia (E. grenieri) but not those of chicken coccidia, and the chickens passed oocysts of chicken coccidia (E. acervulina and E. tenella) but not those of guinea fowl coccidia. Some asexual development (schizogony) occurred in hybrids inoculated with E. tenella, but sexual development (gametogony) did not. In contrast, quail/chicken hybrids became infected with oocysts of chicken coccidia (E. acervulina, E. tenella, and E. maxima) and quail coccidia (E. bateri) and passed a few oocysts during the normal patent period; control chickens and quails became heavily infected with oocysts of chicken and quail coccidia, respectively.     

Toxinotypes of Clostridium perfringens isolated from sick and healthy avian species.

Currently, the factors/toxins responsible for Clostridium perfringens-associated avian enteritis are not well understood. To assess whether specific C. perfringens' toxinotypes are associated with avian enteritis, the isolates of C. perfringens from 31 cases of avian necrotic or ulcerative enteritis submitted between 1997 and 2005 were selected for retrospective analysis using multiplex PCR. C. perfringens was isolated from chickens, turkeys, quail, and psittacines. The toxinotypes of isolates from diseased birds were compared against the toxinotype of 19 C. perfringens isolates from avian cases with no evidence of clostridial enteritis. All C. perfringens isolates were classified as type A regardless of species or disease history. Although many isolates (from all avian groups) had the gene encoding the C. perfirngens beta2 toxin, only 54% produced the toxin in vitro when measured using Western blot analysis. Surprisingly, a large number of healthy birds (90%) carried CPB2-producing isolates, whereas over half of the cpb2-positive isolates from diseased birds failed to produce CPB2. These data from this investigation do not suggest a causal relationship between beta2 toxin and necrotic enteritis in birds.