Live attenuated vaccine-based control of necrotic enteritis of broiler chickens

A vaccine for necrotic enteritis (NE) of chickens would reduce the current need to prevent or treat the disease in broiler chickens with antimicrobial drugs. The objective of this study was to understand aspects of immunity to the disease. The first experiment examined the virulence of six strains of Clostridium perfringens isolated from cases of NE in broiler chickens. Using a 5-day experimental oral infection of 2-week-old broiler chickens, four of the six strains were found to be virulent. Pulsed-field gel electrophoresis and PCR showed that virulence was not associated with a plasmid encoding the beta2 toxin gene, cpb2, since this was present in virulent and one of the two avirulent strains. In the second experiment, two virulent and one avirulent strains were tested for their ability to immunize ("infection-immunization") chickens through the oral route. The procedure used experimental infection for 5 days followed by bacitracin treatment for 9 days, and then re-challenge 2 days later with a virulent strain, CP4. Infection-immunization with the virulent isolates protected chickens from subsequent virulent challenge, whereas the infection-immunization with the avirulent isolate did not. In a third experiment, two of four alpha-toxin-negative mutants of CP4 protected birds from experimental NE after oral immunization. These two mutants were also attenuated for virulence. We conclude that it is possible to immunize chickens successfully against NE and that immunogen(s) other than alpha-toxin are important in protective immunity against oral infection.     

Towards the control of necrotic enteritis in broiler chickens with in-feed antibiotics phasing-out worldwide

Poultry production has undergone a substantial increase compared to the livestock industries since 1970.However, the industry worldwide is now facing challenges with the removal of in-feed antibiotics completely or gradually, as the once well-controlled poultry diseases have re-emerged to cause tremendous loss of production. Necrotic enteritis(NE) is one of the most important diseases which costs the industry over two billion dollars annually. In this paper, we review the progress on the etiology of NE and its control through dietary modifications, pre-and probiotics, short chain fatty acids, and vaccination. The other likely measures resulted in the most advances in the toxin characterization are also discussed. Vaccine strategies may have greater potential for the control of NE mainly due to clearer etiology of NE having been elucidated in recent years with the identification of necrotic enteritis toxin B-like(NetB) toxin. Therefore, the use of alternatives to in-feed antibiotics with a better understanding of the relationship between nutrition and NE, and limiting exposure to infectious agents through biosecurity and vaccination, might be a tool to reduce the incidence of NE and to improve gut health in the absence of in-feed antibiotics. More importantly, the combinations of different measures may achieve greater protection of birds against the disease. Among all the alternatives investigated, prebiotics, organic acids and vaccination have shown improved gastrointestinal health and thus, have potential for the control of NE.     

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.     

Perturbations of the ileal mycobiota by necrotic enteritis in broiler chickensOA

Background: Intestinal microbiota is critical for maintaining animal health and homeostasis. However, involvement of the fungal community, also known as the mycobiota, in animal health and disease is poorly understood. This study was aimed to examine the association between the intestinal mycobiota and the severity of necrotic enteritis(NE), an economically significant poultry disease.Methods: A total of 90 day-of-hatch Cobb broilers were infected with Eimeria maxima on d 10, followed by an oral...     

Efficacy of narasin in the prevention of necrotic enteritis in broiler chickens

The efficacy of narasin in the control of necrotic enteritis (NE) was investigated in a floor pen study of 2000 broiler chickens using a Clostridium perfringens feed inoculum challenge model. Treatments were 1) nonmedicated, nonchallenged; 2) nonmedicated, challenged; 3) narasin, nonchallenged; 4) narasin, challenged. Narasin was administered at 70 ppm in the feed from day 0 to trial termination on day 41. Challenge inoculum contained approximately 1 x 10(8) colony-forming units CP/ml and was administered from day 14 to day 16. In the unmedicated groups, challenged birds had significantly (P < 0.05) lower mean body weight and reduced feed efficiency at day 21 and significantly (P < 0.01) higher cumulative NE mortality at day 41 compared with unchallenged. Similarly, among unmedicated birds, those challenged had a significantly (P < 0.01) higher mean NE score on day 17 and significantly (P < 0.05) higher mean huddling scores on days 15-17 than unchallenged. Among challenged birds, those fed narasin had significantly (P < 0.05) higher mean body weight and improved feed efficiency at days 21 and 41 and significantly (P < 0.01) lower cumulative NE mortality at day 41 than unmedicated. Similarly, among challenged birds, those receiving narasin had a lower mean NE score on day 17 (P > 0.05) and significantly (P < 0.05) lower huddling scores on days 16 and 17 than unmedicated. Coccidiosis lesion scores were zero for birds euthanatized from all treatment groups on day 17, suggesting that the beneficial effects of narasin were not due to prevention of coccidiosis. This study thus provides evidence that narasin is effective in the prevention of necrotic enteritis in broiler chickens.     

A framework for assessing the efficacy of antimicrobials in the control of necrotic enteritis in broiler chickens

This review presents a framework for assessing the efficacy of antimicrobials used to control necrotic enteritis (NE) caused byClostridium perfringens (CP) in the context of susceptibility testing and clinical efficacy, and their potential interactions with the intestinal microbiota of poultry. Practitioners have traditionally based their choice of antimicrobial agent on antimicrobial susceptibility testing, but there appears to be a lack of correlation with clinical efficacy for in-feed antimicrobials (particularly bacitracin and virginiamycin). Resistance patterns of CP and antimicrobials have been monitored using epidemiological cutoffs for minimal inhibitory concentration (MIC), which are not intended to guide therapy. Also, most data have been determined using CP isolates from healthy birds (i.e., potentially from commensal strains not known to be clinically relevant). It is believed that NE is caused by specific virulent CP strains (and potentially other bacteria) that proliferate and displace these commensals. The presence of resistant commensals is not necessarily detrimental (and may even be beneficial) if they inhibit the single CP strain dominance effect observed in acute NE. The choice of antimicrobial therapy in a clinical setting should thus be based on a variety of factors, including an accurate diagnosis, results of efficacy studies, prior experience at the premises in question, and interpretation of MIC data, recognizing that it is not necessarily well correlated with clinical efficacy.     

Dietary supplementation of essential oils and lysozyme reduces mortality and improves intestinal integrity of broiler chickens with necrotic enteritis

The objective of this study was to investigate the individual and combined effects of essential oils (EO; comprised of thymol and carvacrol) and lysozyme on experimental NE in broiler chickens. A total of 320 1-day-old chicks were randomly assigned to five treatment groups: no-challenge control (NC), NC + C. perfringens challenge (CC), CC + 120 mg/kg of EO, CC + 100 mg/kg of lysozyme, and CC + 120 mg/kg of EO + 100 mg/kg of lysozyme. The results showed that EO or lysozyme decreased the mortality, alleviated the gut lesions, inhibited the liver Enterobacteriaceae carriage, and increased the villus height of the ileum compared with CC (p < .05), although the proliferation of C. perfringens in the ileum was not inhibited (p > .05). Moreover, EO or lysozyme was found to decrease the ileal concentration of sialic acid and the Mucin2 mRNA expression (p < .05). However, the blend of EO and lysozyme did not display significant effect on the NE-associated mortality or gut damage in contrast with CC (p > .05). In conclusion, these findings suggest the similar protective effects of EO and lysozyme in NE-associated mortality and intestinal impairment, but their blend did not exhibit ameliorative effect.     

Vaccination with recombinant NetB toxin partially protects broiler chickens from necrotic enteritis

NetB toxin from Clostridium perfringens is a major virulence factor in necrotic enteritis in poultry. In this study the efficacy of NetB as a vaccine antigen to protect chickens from necrotic enteritis was examined. Broiler chickens were immunized subcutaneously with purified recombinant NetB (rNetB), formalin treated bacterin and cell free toxoid with or without rNetB supplementation. Intestinal lesion scores and NetB antibody levels were measured to determine protection after mild oral gavage, moderate in-feed and heavy in-feed challenges with virulent C. perfringens isolates. Birds immunized with rNetB were significantly protected against necrotic enteritis when challenged with a mild oral dose of virulent bacteria, but were not protected when a more robust challenge was used. Bacterin and cell free toxoid without rNetB supplementation did not protect birds from moderate and severe in-feed challenge. Only birds immunized with bacterin and cell free toxoid supplemented with rNetB showed significant protection against moderate and severe in-feed challenge, with the later giving the greatest protection. Higher NetB antibody titres were observed in birds immunized with rNetB compared to those vaccinated with bacterin or toxoid, suggesting that the in vitro levels of NetB produced by virulent C. perfringens isolates are too low to induce the development of a strong immune response. These results suggest that vaccination with NetB alone may not be sufficient to protect birds from necrotic enteritis in the field, but that in combination with other cellular or cell-free antigens it can significantly protect chickens from disease.     

Fumonisins affect the intestinal microbial homeostasis in broiler chickens,predisposing to necrotic enteritis

Fumonisins (FBs) are mycotoxins produced by Fusarium fungi. This study aimed to investigate the effect of these feed contaminants on the intestinal morphology and microbiota composition, and to evaluate whether FBs predispose broilers to necrotic enteritis. One-day-old broiler chicks were divided into a group fed a control diet, and a group fed a FBs contaminated diet (18.6 mg FB1+FB2/kg feed). A significant increase in the plasma sphinganine/sphingosine ratio in the FBs-treated group (0.21 ± 0.016) compared to the control (0.14 ± 0.014) indicated disturbance of the sphingolipid biosynthesis. Furthermore, villus height and crypt depth of the ileum was significantly reduced by FBs. Denaturing gradient gel electrophoresis showed a shift in the microbiota composition in the ileum in the FBs group compared to the control. A reduced presence of low-GC containing operational taxonomic units in ileal digesta of birds exposed to FBs was demonstrated, and identified as a reduced abundance of Candidatus Savagella and Lactobaccilus spp. Quantification of total Clostridium perfringens in these ileal samples, previous to experimental infection, using cpa gene (alpha toxin) quantification by qPCR showed an increase in C. perfringens in chickens fed a FBs contaminated diet compared to control (7.5 ± 0.30 versus 6.3 ± 0.24 log10 copies/g intestinal content). After C. perfringens challenge, a higher percentage of birds developed subclinical necrotic enteritis in the group fed a FBs contaminated diet as compared to the control (44.9 ± 2.22% versus 29.8 ± 5.46%).

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0234-8) contains supplementary material, which is available to authorized users.     

Experimentally induced necrotic enteritis in chickens

A 1.3 to 10% incidence of necrotic enteritis was experimentally produced in broiler-type chickens in three of five trials. The incidence range observed was considerably narrower and lower than the 5.6-37.3% range reported in the literature. Clostridium perfringens was inconsistently isolated from the liver and intestine of dosed chickens.     

Dietary hydrolysed yeast cell wall extract is comparable to antibiotics in the control of subclinical necrotic enteritis in broiler chickens

ABSTRACT

1. The aim of this study was to examine the effect of yeast cell wall (YCW) on performance and physiological responses of broiler chickens under subclinical necrotic enteritis challenge.

2. Six treatments in a 2 × 3 factorial arrangement (non-challenged or challenged plus no supplement, YCW or antibiotics (AB)) was used. Each treatment was replicated eight times with 12 birds per replicate. The treatments included: (1) Positive control (PC; no additive, not challenged); (2) Negative control (NC; no additive, with challenge); (3) YCWN = yeast cell wall (2.0 g/kg diet, not challenged; (4) YCWC = yeast cell wall (2.0 g/kg diet, challenged); (5) ABN = zinc bacitracin 50 ppm + Salinomycin 60 ppm, not challenged); (6) ABC = zinc bacitracin 50 ppm + Salinomycin 60 ppm, challenged).

3. Eimeria challenge at 9 d of age did not affect feed intake (FI), body weight gain (BWG), FCR or liveability at 10 d. The BWG and FCR at 10 d were greater (P < 0.05) in birds fed YCW or AB (AB) diets relative to the PC or NC groups. On 24 and 35 d, FI, BWG, FCR and flock uniformity (28 d) were greater (P < 0.05) in the challenged groups fed YCW or AB diets compared to NC group.

4. Supplementation with YCW ameliorated the negative effects of NE on liver, spleen and bursa weight of birds.

5. Necrotic enteritis challenge decreased (P < 0.05) caecal Lactobacillus and Bifidobacterium spp. counts, and increased ileum lesion score and caecal Clostridium perfirngens counts. This was reversed by the addition of either YCW or AB.

6. Supplementation with YCW and AB resulted to a greater (P < 0.05) dressing percentage and meat yield (35 d).

7. The results indicated that YCW plays a vital role in improving the physiological response and performance of broiler chickens under subclinical necrotic enteritis challenge.     

In vitro lecithinase activity and sensitivity to 22 antimicrobial agents of Clostridium perfringens isolated from necrotic enteritis of broiler chickens

Viable Clostridium perfringens ranging from 10(5) to 10(8) g-1 was detected in all of 88 intestinal content specimens of necrotic enteritis in broiler chickens. In vitro lecithinase activity and sensitivity to 22 antimicrobial agents were determined for the 88 isolates. The activities of lecithinase in the culture filtrate of isolates were estimated to be 0.5 to 4.0 AE ml-1 as alpha-antitoxin equivalent. With reference to antimicrobial activity penicillins and cephazolin showed excellent activity and no resistance; peptides, of the agents used as growth promoters, showed that all except bacitracin had low minimum inhibitory concentration levels (1.6 micrograms ml-1 or less) against this organism; polyethers of monensin, salinomycin and lasalocid were generally adequate in low concentrations while there was a high level of resistance to three tetracyclines in 90 per cent of the strains and all isolates were insusceptible to streptomycin of the aminoglycoside antibiotics.     

An economic analysis of the impact of subclinical (mild) necrotic enteritis in broiler chickens

Costs to broiler producers associated with subclinical (mild) necrotic enteritis (SNE) were estimated using published information on impacts on body weight and feed conversion rate (FCR) associated with SNE and costs and revenues associated with broiler production. Estimates were expressed in U.S. dollars from the perspective of poultry producers. SNE was estimated to result in a 12% reduction in body weight and a 10.9% increase in FCR compared with healthy birds. For the purposes of this analysis, we considered scenarios involving hypothetical flocks of 20,000 birds raised to final body weights ranging from 4.63 to 7.94 lb. The incidence of SNE was assumed to occur at 20% based on the literature. For flocks raised for the length of time required to reach these target weights, SNE resulted in a loss to producers ranging from US$878.19 to US$1480.52 per flock. When feed costs required to obtain SNE flocks having a total live body weight equal to equivalent healthy flocks at market age were calculated, the increased cost to producers ranged from US$370.49 to US$739.38 per flock. SNE has the potential to cause a significant negative economic impact in broiler flocks. Strategies to reduce the incidence of SNE may help to increase the profitability of broiler production.     

Molecular detection and characterization of Clostridium perfringens toxin genes causing necrotic enteritis in broiler chickens

Tropical Animal Health and Production - A total of 464 samples comprising of cloacal swabs from necrotic enteritis suspected live birds (191), intestinal scrapings from dead birds with symptoms of...     

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.     

蛋鸡坏死性肠炎的诊治

1发病情况 2001年7月23日新城子区财落镇某养鸡户饲养135日龄蛋鸡1200只,从130日龄开始发病,3～4天后出现死亡.     

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.     

一起杂交肉鸡坏死性肠炎的诊治体会

8月份以来,我省平原县某地饲养的杂交肉食鸡出现了以饲料消化不良、粪便变稀、变绿、略带红色或桔红色等特征的疾病,当作大肠杆菌病、球虫病进行治疗,效果较差。后来笔者经过临床剖检及实验室诊断,确定为魏氏梭菌引起的肉鸡坏死性肠炎。现将其诊治过程报道如下。1发病情况山东省平原县的几处养鸡场(户),共发病肉杂鸡9500只,日龄6～20d左右。发病5d内共死亡350只左右,死亡率3.68%。使用环丙沙星、庆大霉素、痢特灵等治疗效果不佳。2临床症状及剖检变化病鸡表现为打喷嚏、甩头、流涕、呼噜,眼睑肿胀,排红色或桔红色粪便,呈现水样腹泻,精神沉郁…