ASSOCIATED PROFILES OF VIRULENCE GENE MARKERS IN CLOSTRIDIUM PERFRINGENS STRAINS ISOLATED FROM HEALTHY AND DISEASED BROILER CHICKENS WITH NECROTIC ENTERITIS

Dept. of Poultry Diseases,

Faculty of Vet. Med., Assiut Univ., Assiut-Egypt

Associated Profiles of Virulence Gene Markers in Clostridium perfringens Strains Isolated from Healthy and Diseased Broiler Chickens with

Necrotic Enteritis

(With 3 Tables and 4 Figures)

By

M.A. Mohamed; Sylvia O. Ahmed*

and T.Y. Abdel Motelib

* Dept. Animal Hygiene and Zoonosis, Fac.of Vet. Med.,

Assiut University, Assiut, Egypt.

(Received at 12/9/2009)

البروفيل المصاحب لدلالات جينات الضرواة لعترات الکلوستيريديم بيرفيرنجينز المعزولة من دجاج التسمين السليم والمصاب بمرض الالتهاب المعوى النخرى

مؤمن عبد العظيم محمد ، سيلفيا أسامة أحمد ، طلبة يونس عبد المطلب

تعتبر الکلوستيريديم بيرفيرنجينز المسببة لمرض الالتهاب المعوى النخرى من المخاطر الکبيرة لصناعة دجاج التسمين لما تسببه من احداث وفيات عالية ونقص فى معدل النمو بالاضافة الى الاصابة بالتسمم المعوى فى الانسان. وجد أن أفراز السموم من ميکروبات الکلوستيريديم بيرفيرنجينز من العوامل الاساسية لأحداث المرض. أجرى هذا البحث لمعرفة نسبة تواجد الکلوستيريديم بيرفيرنجينز فى دجاج التسمين السليم والمصاب بالالتهاب المعوى النخرى،  وکذلک  مدى  تواجد  الانواع  المختلفة  لهذا  لميکروب  عن  طريق  الکشف عن  دلالات  الجينات  السمية (alpha (cpa), beta (cpb), epsilon (etx), iota (iAp)) وقد شملت الدراسة أيضا مدى انتشار ثلاث من أهم جينات السموم لميکروب الکلوستيريدم وهم (cpe, cpb2 and netB). تم فحص عدد 56 عينة مجمعة (n=4)  من امعاء وکبد الطيور المصابة و 27 عينة مجمعة من المسحات الشرجية لدجاج التسمين السليم.أوضحت نتائج العزل وجود عدد 47 عترة من الکلوستيريديم بيرفيرنجينز من دجاج التسمين المصاب بنسبة83,9% و14 عترة بنسبة51,85% من الدجاج السليم.وللتفريق بين أنواع الکلوستيريديم المعزولة تم استخدام تفاعل البلمرة المتسلسل المتعدد حيث ثبت ناجحه فى التفريق بين أنواع الکلوتيريديم المختلفة واختصاره للوقت وتوفيره للنفاقات اذا اتخدمت الطرق التقليدية(Mouse Toxin Neuratlization Test). وجد أن کل العترات تحمل جين cpa مما دل على أن جميع العترات من النوعA. وفى خطوة تعتبر الاولى من نوعها فى مصر تم تحديد البروفيل الخاص بجينات الضرواة للعترات المعزولة من                الC. perfringens. وجد أن البروفيل الجينى بالنسبة لجين التسمم المعوى cpe هو عدم امتلاک اى من العترات المصاحبة للدجاج السليم أو المصاب لهذا الجين. على العکس وجد أن جين السموم الثقبيةnetBعالي التواجد فى عترات الکلوستيريديم بيرفيرنجينز المعزولة من الطيور المصابة عنها فى الطيور السليمة مما يعطى دلالة على ايجابية مشارکة هذا الجين فى أحداث المرض. أعطيت هذة الدراسة نظرة جديدة على التکوين الجينى لعترات الکلوستيريدم المعزولة من دجاج التسمين بأسيوط الذى بدوره سيساعد على فهم کيفية التغلب على هذا المرض والسيطرة عليه عن طريق عمل اللقاحات المناسبة لمنع انتشاره بين الطيور والانسان.

SUMMARY

Clostridium perfringens induced necrotic enteritis (NE) and subclinical disease have become important threats to poultry health and is one of the main causes of losses in broiler flocks due to high mortalities and reduction in growth rate as well as enterotoxemias in domestic animals and humans . The mechanism of virulence of C. perfringens, a bacterium causing necrotic enteritis in birds, results largely from its ability to produce toxins. A study was set up to look the rate of carriage of C. perfringens among broilers with different heath status, incidence rate of the different toxin genotypes of C. perfringens in healthy and diseased birds and, lastly, the relative abundance of cpe, cpb2 and netB virulence genes. Broiler chickens from healthy flocks were sampled by cloacal swabs, while intestinal and liver samples of birds suffering from necrotic enteritis were analysed. A total of 47 (83.9%) isolates was obtained from 56 examined pooled samples (n=4) of broiler chickens with clinical problems and 14 (51.85%) isolates were obtained from 27 pooled samples (n=4) from broiler chickens without clinical problems. Seroneutralization with mice or guinea pigs has been used to type each toxin, but this conventional method has some disadvantages. Therefore, we used a multiplex PCR (m-PCR) assay with primers amplifying fragments of alpha (cpa), beta (cpb), epsilon (etx), iota (iap), for genotyping of isolated C. perfringens strains. All 61 broiler chicken     C. perfringens isolates were shown to carry the cpa gene but not cpb, etx and iA genes, signifying that all isolates represented type A. For the first time the associated profiles of the following virulence genes [ cpe, cpb2 (beta-2 toxin) and the newly described pore forming toxin gene (netB)] were determined among Egyptian isolated C. perfringens strains. None of the isolates contained the enterotoxin gene that may indicate the enterotoxin of C. perfringens does not play important role in the occurrence of Necrotic enteritis in broiler chickens. netB was almost always found in outbreak isolates, suggesting a positive correlation of netB toxin gene with the diseased status that may explain its role in the pathogenesis of the disease. Whereas cpb2 was found in healthy and diseased bird isolates that suggest there is a weak or no relationship between beta2 toxin and necrotic enteritis in birds. So this study throw novel insights into the presence and distribution of C. perfringens types as well as virulence-associated genes in field strains, which will help us to understand the pathogenesis of disease in poultry and more comprehensively characterize C. perfringens in future studies to put a suitable strategy for prevention and control.

Key words: Clostridium perferingens, broiler chickens, virulence and m-PCR.

Introduction

Enteric diseases are an important concern to the poultry industry because of lost productivity, increased mortality, reduced welfare of birds and the associated contamination of poultry products for human consumption (Dahyia et al., 2006).

One of the most important enteric diseases that affected broilers is necrotic enteritis (NE). NE was first described by Parish in 1961 (Parish, 1961), and since then it has been reported to occur in almost all poultry-producing countries. NE in chickens gets in two forms, as an acute or chronic enterotoxemia (Van Immerseel et al., 2004). The acute disease results in significant levels of mortality whereas the chronic disease leads to loss of productivity and welfare concerns. It has been estimated that the disease costs the international poultry industry in excess of $US 2 billion per year based on average damage of $0.05 per bird (McReynolds et al., 2004).

NE is an enteric disease that is caused predominantly by Clostridium perfringens type A and to a lesser extent by type C strains (Songer, 1996). Mucosal damage inducing factors such as coccidiosis (parasitism), high fiber litters, dietary changes, poor hygienic and housing conditions are considered predisposing factors for NE. The NE is thought to occur when these factors create a favorable environment for C. perfringens to multiply rapidly into much higher numbers in the small intestine, leading to the production of potent lethal toxins that damage the intestine. Moreover, excessive uses of growth promoting antibiotics enhance C. perfringens to induce NE and sub clinical infections, important threats to poultry health (Songer, 1996).

C. perfringens in poultry constitutes a risk for transmission to humans through the food chain. C. perfringens is one of the most frequently isolated bacterial pathogens in foodborne disease outbreaks in humans, after some other pathogens such as Campylobacter and Salmonella (Buzby and Roberts, 1997).

The pathogenicity of the organism is associated with several toxins which are used for toxin typing of the bacteria. The alpha (α), beta (β), iota (ι) and epsilon (ε) are the major lethal toxins produced by the organism that are closely related to its virulence (Petit et al., 1999).       α-toxin (phospholipase C) is commonly produced by all 5 types. It can hydrolyze lecithin into phosphorylcholine and diglyceride, which leads to tissue damage. β-toxin is produced by C. perfringens type b and type C strains and it causes a necrotic enteritis characterized by haemorrhagic mucosal ulceration or superficial mucosal necrosis of the small intestine in animals (types B and C) (Smedley III et al., 2004).  

Clostridium perfringens is classified into five types (A–E) on the basis of their ability to produce major lethal toxins. Within these five types, all C. perfringens produce α toxin. In addition, type B strains produce β and ε toxins, type C produces β toxin, type D produces ε toxin and type E produces ι toxin. In addition to the major lethal toxins, a minority of C. perfringens strains produce a C. perfringens enterotoxin (CPE), which is responsible for the symptoms of common C. perfringens type A food poisoning. Different meats, including poultry meat, have been frequently reported as the most common food vehicles (Eisgruber and Hauner 2001; McClane 2001).

The classification of C. perfringens isolates into toxigenic types has been traditionally performed by toxin neutralization test in mice. This procedure consumes a lot of antisera and experimental animals. Additionally it is time consuming. Nowadays, with the advent of highly efficient detection chemistries, sensitive instrumentation and optimized assays the number molecular techniques such as polymerase chain reaction (PCR) have been used to type C. perfringens (Gkiourtzidis       et al., 2001; Baums et al., 2004; Gholamiandekhordi et al., 2006) according to the presence of toxin genes.

The virulence factors which lead to the development of NE have yet to be fully understood. Based on experimental studies, early investigators suggested that alpha toxin is the major toxin involved in causing NE (Fukata et al., 1988), and there are recent reports that vaccination against alpha toxin is at least partially protective against the disease (Lovland et al., 2004; Copper et al., 2009). However, Keyburn  et al. (2006) have reported that alpha toxin knockout mutants of            C. perfringens were able to produce necrotic enteritis in an experimental model.

Keyburn et al. (2008) reported the discovery of pore forming toxin of C. perfringens which they named NetB. netB gene was only identified in strains recovered from chickens with NE and not found in isolates from chickens which did not have NE. Keyburn et al. (2008) also mentioned that netB knockout mutants failed to produce NE in chickens, while such mutants complemented with the wild type netB gene did cause NE. Clearly, the identification of the toxin responsible for NE would represent a major advance in knowledge, so there remains a need to examine isolates from other countries like Egypt for the presence of netB.

Prophylaxis and control of necrotic enteritis is essential in broiler flocks, and the primary approach to this has been vaccination. For this to be successful, the vaccine must be based upon strains which are clinically relevant. Thus, identification of C. perfringens toxinotypes using a robust genetic test for the presence of toxin-encoding genes is critical for both epidemiological studies and for development and effective use of preventative measures, including vaccination.

However, there are no published data on molecular typing of     C. perfringens in broilers in Assiut, Egypt.  So, we report here for the first time in Egypt esp. in Assiut and Upper Egypt, testing of a multiplex polymerase chain reaction (PCR) assay for the simultaneous identification of the C. perfringens toxin genotyping as well as molecular analysis of the virulence genes among this pathogen and the its association with the disease to undertake the development of control measures, especially for the formulation of cost effecting vaccine.

Materials and Methods

History of broiler chickens and sample collection:

Cases of enteric disorders compatible with necrotic enteritis in 21 to 32 days old broiler chickens reared in different poultry farms in Assiut province, Egypt occurred in the months of September 2009 to Novemeber 2009 were investigated. These farms previously exposed to enteric infection, mainly with coccidiosis.

A daily mortality rate of 1.5 to 2% was recorded. The mortality peak was terminated by a 3-5 day antibiotic treatment with amoxicillin. In every case, postmortem was performed as soon as possible after death. All the internal organs were thoroughly examined and any macroscopic and gross lesions observed were recorded.

Intestinal contents esp. duodenum (n=168) and gall bladder of affected liver samples (n=56) from diseased birds as well as 108 samples of cloacal swabs from healthy birds were collected for microbiological investigation.

Culture of C. perfringens

Pool samples (n=4) were inoculated in sterile cooked meat medium broth (Difco), then overlaid with mineral oil and incubated at 37°C for 24hr.

The inoculums from each cooked meat broth media were seeded onto 5% citrated sheep blood agar containing 12 mg kanamycin sulphate and 30,000 U polymyxin B sulphate/Liter then incubated anaerobically with an anaerobic gas-pack system (BBL Microbiology Systems Cockeysville, Md.; Div. Becton Dickinson and Co.) for 48hr at 37°C. Pure colonies were obtained by plating subcultures on Brucella agar and incubating them anaerobically for 24 hours.

 The identity of isolates was confirmedbybacterial colonies characteristics (size, shape, color and patterns of haemolysis on blood agar produced a double zone of hemolysis), motility test, Gram’s and malachite spore staining, litmus milk test, gelatinase, lecithinase and fermentation of glucose and lactose (Holt et al., 1994).

Detection of Clostridium perferingens types by Multiplex PCR of toxin genes:

DNA extraction

A liquots (300 uL) of stored culture suspensions were transferred to microfuge tubes, boiled for 10 min in a low power microwave, centrifuged at 13,000 xg for 5 min (Ferrarezi et al., 2008), and DNA precipitated with 1mL absolute ethanol for 3 h at - 60C. After centrifugation, pellets were dissolved in 50 uL ultrapure water (Ultra Pure DistilledWater, Invitrogen) and incubated at 56C for 20 min. An aliquot (5 uL) of this extracted DNA was used as template in PCR genotyping.

Multiplex PCR assay

Two multiplex PCR were used: one for detection of cpa, cpb1, cpetx, and cpi genes and one for detection of cpb2, cpe and netB genes. PCR Primers, and references to the sequences from which they were derived, are presented in Table 1.

The multiplex PCR assay was performed by a modification of a previously described method (Songer and Bueschel, 1999). Briefly, each 50 uL reaction contained 25 μl 2×QIAGEN Multiplex PCR Kit (Qiagen) containing HotStart Taq polymerase, multiplex PCR buffer, dNTP mix and 3 mM MgCl₂, a final concentration of 0.4 μM for each primer      (1μl of each primer), 8 μl sterile RNase-free water and 5 μl of test or control DNA.

Amplification was carried out in a Techne Cyclogene Thermocycler, using a program composed of 5 min at 94°C, 35 cycles consisting of 1 min at 94°C as initial denaturation, 1 min at 55°C, and 1 min at 70°C), and a final incubation for 10 min at 72°C as final elongation cycle. Negative (ultrapure water) controls were included. PCR products were examined by electrophoresis in a 1.2% agarose gel with 5 μL/100 mL of ethidium bromide and visualized by UV transillumination, then documented with a gel documentation system (BioRad, Munich, Germany). Molecular weight marker (1 kb Plus DNA Ladder) was added to agarose gels before electrophoresis.

Table 1: Target toxin gene, oligonucleotide primer sequences and length of amplification products of the C. perfringens multiplex PCR.

^aDefinition of Clostridium genotype is based upon amplification of a toxin gene fragment. Type A has cpa alone, type B has cpa, cpb, and etx, type C has cpa and cpb, type D has cpa and etx, and type E has cpa and iA.

Results

Clinical signs and postmortem lesions

During appearance of necrotic enteritis, broiler chickens showed severe depression, decreased appetite, ruffled feathers and diarrhea tinged with blood prior to death. Post mortem examination showed that birds were dehydrated, mid-small intestine (Fig. 1), where the enteric mucosa was abnormally thickened like yellow brownish diphtheritic membrane (Fig. 1), and varying degrees of haemorrhages. Focal necrosis and hemorrhages on the liver surface was also noticed in some chickens. Rest other organs appeared apparently healthy.

Culture of Clostridium perfringens

A total of 56 pooled samples (n=4) from diseased birds            (42 intestinal pooled samples and 14 gall bladder of affected liver samples) were examined for the presence of C. perfringens. 47 (83.9%) isolates of C. perfringens out of 56 examined pooled samples were isolated and identified by colonies characteristics on blood agar as shown in fig.2 as well as biochemical tests as described in Bergey’s manual (Holt et al., 1994) like positive fermentation of glucose, lactose, sucrose, and maltose; hydrolysis of gelatin; production of lecithinase.

Bacteriological investigation was carried out also on 27 pooled samples of cloacal swabs from healthy birds for the presence of            C. perfringens, 14 (51.85%) C. perfringens strains were isolated and identified (Table 2).

Table 2: Number of positive samples for C. perfringens isolates examined from broiler chickens.

^a Number of examined samples

^b Number of positive samples

^cNot subjected for examination

Toxin typing by PCR

All 61 isolates which had been tentatively identified as              C. perfringens from diseased and healthy broiler chickens, were examined for the presence of cpa, cpb, iA, etx, cpb2 and cpe which encode respectively for alpha toxin, beta toxin, iota toxin, epsilon toxin, beta2 toxin and enterotoxin of C. perfringens, by using a multiplex PCR method.

 In all C. perfringens isolated from diseased and healthy birds harboringthe cpa gene of 900 bp fragment (Fig. 3), so all the isolateswere confirmed as C. perfringens type A. However, the cpb, etx, iap and cpe were not detected in any of the examinedisolates (Table 2).

A 27/61 (44.3%) were positive for the presence of cpb2           (b2 toxin) gene of 200 bp fragment (Fig.4). Its presence is particularly high among isolates from affected liver (50%) (Table 3).

Of the 61 C. perfringens isolates which had been recovered from diseased and healthy chickens, 37 (60.6%) were netB positive           (Fig. 4 and Table 3) 35 out of 47 (74.4%) were positive for netB gene. Conversely, 2 out of 14 (14.3%) isolates from healthy chickens, were netB positive (Table 3).

Table 3: Analysis of virulence factors associated with C. perfringens isolates from healthy and diseased broiler chickens