Among pathogenic bacteria, different species of the Clostridia genus
produce the most variable type of toxins which are considered as the main virulence
determinants of these bacteria (Popoff and Bouvet, 2009).
Clostridium perfringens, an anaerobic gram-positive sporeforming bacterium
is ubiquitous in the intestinal flora of human and animals and known to be the
most widely distributed pathogen in nature. C. perfringens produce a
large range of potent toxins and enzymes that are responsible for severe diseases
in humans and other animals (Popoff and Bouvet, 2009).
In the last decade, a great effort has been made to understand the mechanism
of action, structure and function of these toxins.
Some toxins are known as potent virulence factors while the implication of
other toxins in pathogenicity is questionable (Rood, 1998).
In a commonly used classification scheme, C. perfringens is divided into
five toxinotypes (A-E) based on the production of four major toxins (alpha,
beta, epsilon and iota) where each type carries a different combination of the
toxin genes however, this bacterium also produces at least 13 other toxins such
as C. perfringens Enterotoxin (CPE), theta toxin, beta2 toxin and netB
toxin (Keyburn et al., 2008; Petit
et al., 1999). Specific C. perfringens toxin types are associated
with particular human and animal diseases. C. perfringens is often found
in the intestinal tract of healthy birds but it can cause outbreaks of disease
in many species of poultry and especially in broiler and turkey flocks (Songer,
1996). Clostridiosis occurs both as an acute clinical disease called as
Necrotic Enteritis (NE) causing high mortality and as a subclinical disease
with focal necrosis in the intestine (subclinical NE) or C. perfringens-associated
hepatitis with cholangio-hepatitis or fibrinoid necrosis in the liver (Cooper
and Songer, 2009). NE is the most clinically dramatic bacterial enteric
disease of poultry induced by C. perfringens which affects industrial
poultry worldwide and is a global problem (Cooper and Songer,
2009). Much of the current research on NE has focused on finding a definitive
toxin that is responsible for causing disease. The majority of C. perfringens
isolates from poultry belong to toxin type A but a few belong to type C (Cooper
and Songer, 2009). The majority of the chicken strains are toxinotype A,
meaning that they carry the cpa gene encoding alpha toxin (Petit
et al., 1999). For a long time, it was believed that this alpha toxin
was the major virulence factor involved in NE (Fukata et
al., 1988). Recently, the role of C. perfringens alpha toxin
in NE is disputed. A cpa knockout mutant from a virulent C. perfringens
chicken strain was still capable of inducing necrotic lesions in the gut of
experimentally infected broilers (Keyburn et al.,
2006). In addition, almost 2 years ago, the netB toxin and its encoding
gene (netB) were first identified in an Australian strain of C. perfringens
type A that was isolated from a chicken suffering from NE (Keyburn
et al., 2008). Its importance in NE was shown when a netB mutant
C. perfringens strain did not cause NE in an experimental chicken model
and virulence was restored when a functional netB gene was introduced
back into the mutant strain (Keyburn et al., 2008).
The critical importance of netB for the development of necrotic enteritis is
still under discussion as occasionally isolates that lack the netB gene
can be found in birds suffering from NE and NE has been reproduced with netB
negative isolates (Abildgaard et al., 2010; Cooper
and Songer, 2010). However, the exact role of this toxin in pathogenicity
of NE still needs to be elucidated. Various epidemiological studies of C.
perfringens strains from varied geographical locations have been published
but very few studies from Asian countries have been published on C. perfringens-induced
NE in poultry and very little knowledge of the C. perfringens genetic
profile in Asia is available. In the present study, for the first time in Asia,
we analyzed the C. perfringens isolates from poultry flocks in a single
PCR assay in order to determine the presence of netB gene and examined
its occurrence with respect to NE in chickens.
MATERIALS AND METHODS
Bacterial isolates and bacteriological procedures: About 79 isolates of C. perfringens type A collected during the period of 2005-2008 and kept in the laboratory in 50% glycerol at -70°C were used for this study. The collection consisted of 36 isolates obtained from six NE-positive flocks (broiler) and 43 strains obtained from four NE-negative flocks (two broiler, one layer and one broiler breeder). Toxinotypes of C. perfringens isolates were determined by Multiplex PCR in the previous study. The frozen C. perfringens isolates were cultivated in Brain Heart Infusion (BHI) and incubated anaerobically at 37°C for 24-36 h.
Samples were sub-cultured anaerobically in blood agar plates containing 7%
defibrinated sheep blood, Tryptose Sulfite Cycloserine agar (TSC) and Tryptose
Sulfite Neomycin agar (TSN). The identity of the isolates was confirmed by characteristic
colony morphology, hemolytic pattern, gram staining and biochemical tests as
previously described (Quinn et al., 1994). All culture
media and additives used in this study were provided from Merck (Germany). Due
to recent discovery of netB gene, there was no netB-positive standard
strain of C. perfringens for using in PCR. However, the first identified
netB-positive isolate was kept and used in all PCR reaction sets.
Single PCR for netB gene: To extract bacterial DNA, a single
colony of each C. perfringens isolate grown on blood agar plate for overnight
at 37°C suspended into 100 μL distilled water in a clean 1.5 mL microtube,
boiled for 10 min and centrifuged for 10 min at 10000xg. The supernatants were
carefully removed and used as template DNA. The concentration of DNA was determined
by Biophotometer (Eppendorff, Germany) and adjusted to approximately 50 ng for
each PCR reaction. To detect netB gene previously developed forward (5-GCTGGTGCTGGAATAAATGC-3)
and reverse (5-TCGCCATTGAGTAGTTTCCC-3) primers were used (Keyburn
et al., 2008). Amplification reactions were carried out in a 25 μL
reaction volume containing: 2 μL 10xPCR buffer, 2.5 mM MgCl2,
0.2 mM dNTPs mixture, 2.5 units of Taq DNA polymerase, 0.1 μM of each primers,
dH2O and 5 μL of template DNA solution. Negative and positive
controls were included in all PCR reaction sets. Amplification was programmed
in a thermocycler (Gradient Mastercycler, Eppendorff, Germany) as follows: 94°C
for 2 min followed by 35 cycles of 94°C for 30 sec, 55°C for 30 sec,
72°C for 60 sec and a final extension at 72°C for 12 min (Keyburn
et al., 2008). The amplification products were detected by gel electrophoresis
(Apelex, France) in 1.5% agarose gel in 1xTAE buffer, stained with 0.5 μg
mL-1 EtBr. Amplified bands were visualized and photographed under
UV transillumination (UVP Visi-Doc-It System, UK). The primers and other materials
used in PCR reaction were provided by Cinnagen (Tehran, Iran).
DNA sequencing of netB PCR products: Four C. perfringens isolates (ATBS61IR, ATBS65IR, ATBS94IR and ATBS120IR) obtained from separate diseased flocks were selected and their relevant PCR amplified products for netB were purified using the Gene JET Gel Extraction Kit (Fermentas Life Science, Germany) according to the manufacturers instructions and submitted for automated sequencing in both directions at the Geneservice, Source BioScience (Cambridge, England) using PCR primers as sequencing primers. The sequence data were submitted to GenBank under the accession numbers GU453172 and GU581173-5.
RESULTS AND DISCUSSION
Single PCR for netB gene: The presence of the netB gene
was examined in all isolates by single PCR (Fig. 1). The netB
gene was detected in 19/36 (52.8%) isolates from diseased flocks but not in
any isolates from healthy flocks.
||Agarose gel (1.5%) electrophoresis results of the PCR assay
for the detection of a 384 bp fragment of the netB gene in C.
perfringens. Lanes M: Size marker (GeneRuler 50 bp DNA Ladder, Fermentas);
Lane 1: Negative control; Lanes 2-3: netB-positive C. perfringens
isolates from birds suffering from NE
All isolates from a single flock showed an identical pattern except in flock
no. 4 in which one isolate was negative for netB gene but the rest were
positive (Table 1).
Sequence analysis of netB PCR products: Comparison of four Iranian C.
perfringens isolates sequences by Blastn (http://www.ncbi.nlm.nih.gov/Blast)
at the nucleotide level revealed 100% identity to each other and to the netB
sequences of C. perfringens strains available in GenBank.
The recently discovered netB toxin gene was exclusively found in NE-associated flocks in the study although, all C. perfringens isolates from three NE-positive flocks were netB negative (Table 1).
Since the discovery of netB in 2008, the presence of the netB gene have
been screened among a wide variety of C. perfringens isolates from Australia,
Canada, United States and some European countries (Chalmers
et al., 2008; Johansson et al., 2010;
Keyburn et al., 2009; Martin
and Smyth, 2009). However, this study for the first time reports the presence
of netB gene among C. perfringens isolates from Asia. Similar
to the findings, the netB gene has not been detected in all isolates
from definitive cases of NE (Abildgaard et al.,
2010; Chalmers et al., 2008; Johansson
et al., 2010; Keyburn et al., 2009;
Martin and Smyth, 2009).
Despite the first report on the detection of netB gene among isolates
from diseased chickens, the presence of this gene among C. perfringens
isolates from healthy chickens was also confirmed in later studies (Abildgaard
et al., 2010; Chalmers et al., 2008;
Johansson et al., 2010; Keyburn
et al., 2008, 2009; Martin
and Smyth, 2009).
|| Characteristics of Clostridium perfringens isolates
of this study
Chalmers et al. (2008) found the presence of
netB gene only in isolates associated with NE outbreaks in Canada but
not in any isolates from healthy birds which correspond with the findings in
this study. In another North American survey, it was found that the majority
of C. perfringens isolates from chickens with clinical signs of NE carried
the netB gene (58.3%) whereas only a small percentage (8.6%) of isolates
from healthy birds carried this gene (Martin and Smyth,
2009). The highest percentage (>90%) of netB gene positive among
C. perfringens isolates from diseased chickens was reported in a single
broiler flock in Sweden. However, in the same flock, 25% of the isolates from
apparently healthy birds were also netB positive (Johansson
et al., 2010). Possibly, the reason for the detection of a high percentage
of netB positive cases was that all isolates were from a single flock which
represents the dominant C. perfringens population in the chicken organ
lesions. Recently, Keyburn et al. (2009) studied
various C. perfringens isolates from NE-positive and NE-negative flocks
from three continents and found 70% (31/44) and 3.6% (2/55) netB positive isolates,
respectively. In an Italian study, 107 C. perfringens isolates were tested
for the presence of netB gene and it was found that 27% (29/107) of isolates
were netB positive in which 93% (27/29) of isolates had been were originated
from birds affected by NE and other intestinal disorders (Drigo
et al., 2009). A recent Danish study showed a prevalence of approximately
50 and 60% of netB gene among isolates from NE-associated and healthy
flocks, respectively (Abildgaard et al., 2010).
The Danish study was the first and the only one that reported the presence of
netB gene in isolates from healthy chickens more than that of in isolates
from NE-positive chickens.
The presence of netB gene has also been shown in one non-poultry related
C. perfringens isolate. Martin and Smyth (2009)
reported isolation of the first netB-positive C. perfringens from the
liver abscesses in a cow died with gastrointestinal disease. This non-poultry
isolate was shown to cause lesions characteristic of avian NE in a chicken disease
model (Smyth and Martin, 2010). This finding was in contrast
to another recent study which demonstrated the failure of a non-avian netB-positive
C. perfringens isolate to effectively colonize birds following challenge
with a high titre of infective inoculum. It was suggested that the overall deficiency
in poultry colonization factors may be the reason for this failure (Cooper
et al., 2010).
The presence of toxin genes in C. perfringens isolates does not solely
determine the clinical importance of the isolates and there are some predisposing
factors that have been associated with the selection of toxigenic C. perfringens
and consequently, the development of disease. Not all C. perfringens strains
isolated from birds that clearly displayed signs of necrotic enteritis were
netB positive and in one particular study two C. perfringens isolates
that were negative for netB gene produced NE in 6.7 and 16.7% of inoculated
birds (Cooper and Songer, 2010). These results imply
that although there is a clear association between the presence of netB toxin
gene and development of NE, there may be other (yet to be determined) virulence
factors that are produced by these netB-negative disease-producing isolates.
While it is possible that NE may results from the interaction of several toxic
molecules, further investigation on this issue appears to be necessary. It is
noteworthy that all netB-positive isolates identified in the study were cpb2-positive
too. A recent study has revealed that in several NE-positive isolates the netB
gene is part of a large potential pathogenicity locus (Lepp
et al., 2010). The identification of pathogenicity loci may lead
to the discovery of additional virulence factors that are involved in the pathogenesis
of disease (Lepp et al., 2010).
This study demonstrated the presence of netB gene in more than half of the isolates from diseased flocks but not in any isolates from healthy flocks. However, netB can not be the only C. perfringens virulence factor involved in avian NE, since not all C. perfringens isolates from birds with NE contain the netB gene. According to the information obtained from the study and other investigations, netB toxin may not be an obligate requirement for poultry C. perfringens virulence and at least the presence of netB may not be essential for the disease process in all C. perfringens isolates. Although, the role of netB in the induction of NE needs further investigation. In addition, comparison of four Iranian C. perfringens isolates sequences revealed 100% identity to each other and to the netB sequences of C. perfringens strains available in GenBank.
Future investigations should focus on the ability of toxin production by this gene, experimental studies in a disease model to investigate the disease producing capabilities of the netB positive and negative strains recovered from cases of NE and also the netB negative strains recovered from healthy chickens, vaccination with various toxoids and subsequent challenge for opening significant opportunities for the development of novel vaccines against NE in poultry, study on the regulatory mechanisms involved in the expression of netB toxin, identification the netB in C. perfringens isolates from species other than chicken, considering the roles of external and predisposing factors in pathogenesis of NE and identification new genes for toxins or related enzymes in future as was as the case for C. perfringens netB.
This research was funded by a grant (7508049/6/7) from the Research Council of the University of Tehran. The researchers have special thanks to Prof. J. Glenn Songer, Department of Veterinary Science and Microbiology, The University of Arizona for his helpful suggestions and comments at different stages of this research.