Clostridium perfringens type D infects ruminants and causes the enterotoxemia disease by ε-toxin. A mutated ε-toxin gene lacking toxicity was designed, synthesized, and cloned into the pT1NX vector and electroporated into Lactobacillus casei competent cells to yield LC-pT1NX-ε recombinant strain. BALB/c mice, immunized orally with this strain, highly induced mucosal, humoral, and cell-mediated immune responses and developed a protection against 200 MLD/ml of the activated ε-toxin. This study showed that the LC-pT1NX-ε could be a promising vaccine candidate against the enterotoxemia disease.

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The aim of the present study was to determine the prevalence of Coxiella burnetii antibodies in small ruminants in Southeast Iran. A total of 368 small ruminant blood samples (241 caprine blood samples and 127 ovine blood samples) were collected from January to May of 2011 in Southeast Iran. A commercial ELISA test kit was employed to identify specific antibodies against C. burnetii in the sheep and goats. Seropositivity in the examined counties ranged from 17.1% to 39.2%. Of the animals tested, 97 animals (26.4%), including 43 sheep (33.9%) and 54 goats (22.4%), had antibodies to C. burnetii. The results of the current study reveal the high prevalence of antibody positivity in small ruminants in Southeast Iran. Thus, sheep and goats are important reservoirs in this area. Additionally, we performed a logistic regression to the identify risk factors for positivity and concluded that age was an important risk factor (P<0.001).

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Background: There are several protocols to extract DNA from Lactobacillus spp. In the case of L. casei it is harder because of its especial and thick cell wall. Objectives: In this study, nine DNA extraction protocols (by lysozyme treatment) were evaluated and compared in two categories (traditional and kit-based protocols) and an improved method was presented. Materials and Methods: DNA quantity and quality was determined by spectrophotometry, agarose gel electrophoresis and polymerase chain reaction (PCR). Results:The results revealed that the yield of extracted DNA differed by each protocol (5.8 -17.1 μg/100 μL), but provided appropriate DNA for PCR amplification. The modified protocol offered the best total DNA extraction method when both quality (DNA purity; 1.54 μg) and quantity (DNA yield; 17.1 μg) were considered. Conclusions: We suggest this protocol for effective and inexpensive DNA isolation from L. casei for downstream biological processes such as PCR.

Background Clostridium perfringens (C. perfringens), a major enteric pathogen, can lead to both clinical and subclinical disease in broiler chickens. 1 This bacterium was divided to five types (A, B, C, D and E) based on the presence of major toxins (α, β, ε and ι). It produced some important minor toxins such as enterotoxin, beta 2 , necrotic enteritis toxin B (NetB), TpeL and perfringolysin O (PFO). 2 C. perfringens is responsible for causing necrotic enteritis (NE) of poultry, especially by type A and rarely type C. 3 C. perfringens type A is the most frequently isolated clostridial type from NE cases. 4,5 NE is an economically important disease with severe gastro-intestinal signs in commercial broiler farms and was reported for the first time by Parish. 6 Two forms of NE were described: clinical and subclinical. 7 Clinical NE, primarily in young chickens (between two to six weeks), is characterized by severe necrosis in the mucosa of proximal jejunum and associated with high mortality rates. 8 Subclinical NE is led to a decreased performance (reduced growth, reduced feed efficiency) without mortality, due to the extensive mucosal damage. 9 Keyburn et al discovered a pore forming toxin of C. per-fringens which they named NetB and the encoding gene, netB and recognized this gene in C. perfringens isolates recovered from chickens. They showed the relationship between presence of netB gene and NE outbreaks and reported that NetB is critical to the development of NE, in chickens. 10 To our knowledge, there were not published data about NE outbreaks and responsible toxins for causing this disease in organic broiler farms. Objectives This study was firstly aimed to genotype the pathogenic C. perfringens isolates in organic broiler farms and secondly to assess the presence of netB gene among them and its occurrence with respect to the disease NE. Materials and Methods Sampling A total of 103 intestinal samples of broiler chickens, clinically suspected to NE, were obtained from eight organic farms. Samples were collected aseptically in plastic bags in the post-mortem examination of chickens and quickly transported to the laboratory in ice-cooled containers.

The alpha-toxin is one of the virulence factors of Clostridium perfringens for gas gangrene in humans and animals or necrotic enteritis in poultry. The C-terminal domain of this toxin ( cpa ) was synthesized and cloned into pT1NX vector to construct the pT1NX-alpha plasmid. This surface-expressing plasmid was electroporated into Lactobacillus casei ATCC 393, generating the recombinant L. casei strain expressing alpha-toxoid (LC-α strain). Expression of this modified alpha-toxoid was confirmed by SDS-PAGE, immunoblotting, and direct immunofluorescence microscopy. BALB/c mice, immunized orally by the recombinant LC-α strain, elicited mucosal and significantly humoral immune responses (p< 0.05) and developed a protection against 900 MLD/mL of the standard alpha-toxin. This study showed that this recombinant LC-α strain could be a promising vaccine candidate against gas gangrene and necrotic enteritis.

There are few practical protocols to extract efficient plasmid DNA from the difficult-to-lyse bacterium, Lactobacillus casei. This is related to production of a large amount of exopolysaccharide coat and its special physiological characteristics. In this study, we optimized a protocol to extract efficient plasmid DNA from a recombinant L. casei strain. Different extraction methods were evaluated in three classes of conventional, kit-based, and combined protocols. The quantity and quality of the extracted plasmid DNA were determined by spectrophotometry, agarose gel electrophoresis, and PCR. Results revealed that the yield of the extracted plasmids differed for each protocol and conventional protocols showed higher plasmid yields. We suggested an effective, inexpensive protocol to extract plasmid DNA from the recombinant L. casei for downstream biological processes.