Formalin and ferric chloride inactivated Pasteurella multocida type a adjuvanted with bacterial DNA and alum as a new vaccine candidate in sheep pasteurellosis.

The aim of the present study was to investigate humoral and cellular immune responses in sheep inoculated with inactivated P. multocida antigen with alum and bacterial DNA adjuvant by identifying IgG and cytokines from serum and cell culture. Sheep were immunized with iron and formalin-inactivated antigens at an interval of 2 weeks. These immunogens were mixed with alum adjuvant and P. multocida type A DNA (AbDNA). After injection and blood sampling, the serum antibody titer and cellular immune responses (IL-4, IFN-γ, and TNF-α) on serum samples and lymphocyte cell were tested by ELISA. The ELISA results showed a higher antibody titer in the bDNA adjuvant group compared to the alum adjuvant group and the control group. In general, the level of IgG in the serum of immunized animals was significantly increased compared to the control group. The peak antibody titer (1.794) was observed on the 28th day of injection in the IIV-AbDNA group. After immunization, inactivation with iron and bDNA adjuvant increased cytokine production compared to other experimental and control groups. High levels of lymphocyte and serum titers of IL-4, IFN-γ, and TNF-α were also obtained in the IIV-AbDNA group. The findings showed that killed P. multocida type A antigens formulated with bacterial DNA as an adjuvant are candidates for new immunogens against P. multocida infections in sheep. The inactivation of bacteria with iron also enhanced proper immune responses.

Isolation of the various serotypes of Mannheimia haemolytica and preparation of the first vaccine candidate in Iran.

BACKGROUND: Mannheimia haemolytica is one of the main agents of domestic pneumonic mannheimiosis, but a proper vaccine has not been explored in IRAN. METHODS AND RESULTS: 362 lung and nasal samples from sick domestic animal were detected by culture and PCR methods. Totally, 71 M. haemolytica isolates were identified in three main serotypes (A1, A2, and A6). Serotypes A2 (38/71; 54%) and A1 (25/71; 39%) were the most frequently detected, whereas the A6 serotype was detected with a frequency of less than 1% (1/71; 1%) and 7 isolates remained unknown (7/71; 10%). Subsequently, M. haemolytica vaccinal strain was developed and then formalin-killed vaccine was prepared. It provided the best protection against mannheimiosis in sheep which was proved by indirect ELISA. CONCLUSIONS: Our results suggest that the efficacy and safety of vaccine strain are remarkable and may serve as a new therapeutic target in mannheimiosis.

Pasteurella multocida inactivated with ferric chloride and adjuvanted with bacterial DNA is a potent and efficacious vaccine in Balb/c mice.

PURPOSE: Pasteurella multocida (P. multocida) is a principal pathogen of domestic animals and an opportunistic pathogen of humans. It is the causative agent of pneumonia and haemorrhagic septicaemia in cattle, sheep and goats, fowl cholera in chickens and progressive atrophic rhinitis in swine. In this study, we investigated the humoral and cellular immune responses and protective immunity conferred by an iron-inactivated vaccine with bacterial DNA (IIV+bDNA) as an adjuvant in mice. METHODOLOGY: P. multocida was grown in BHI broth, inactivated with formalin and FeCl3 and adjuvanted with alum and bDNA. Mice were immunized with two whole-cell inactivated vaccine doses 2 weeks apart. The animals were challenged 4 weeks after booster immunization. Immunogens (vaccines and bDNA) posed no safety problems when mice were injected subcutaneously (s/c) with these preparations. The serum antibody titres were tested by ELISA. At 28 days post immunization, cell-mediated immunity responses were determined. The responses were measured by assay of IL-6 and IL-12 in lymphocyte spleen culture supernatants. RESULTS: ELISA results showed that the levels of antibodies in iron inactivated with bDNA adjuvant groups were higher than in the formalin inactivated with alum adjuvant vaccine group. The protection rate of IIV+bDNA adjuvant vaccine was superior to that of the other vaccines and it protected 100 % of the challenge group mice. Following immunization, bDNA promoted increased production of interleukins compared to the control groups. CONCLUSION: These studies indicate that bDNA is effective as an immune adjuvant, and along with stimulatory bDNA represent promising new humoral and cellular immune enhancers for vaccination applications. In addition, this vaccine is able to provide long-term protection against infection.

Genotypic analysis of virulence genes and antimicrobial profile of diarrheagenic Escherichia coli isolated from diseased lambs in Iran.

The aim of the present study was to determine the analysis of virulence genes and antimicrobial profile of diarrheagenic Escherichia coli isolated from diseased lambs. Two hundred ninety E. coli isolates were recovered from 300 rectal swabs of diarrheic lambs and were confirmed by biochemical tests. The pathotype determination was done according to the presence of genes including f5, f41, LTI, STI, bfp, ipaH, stx 1 , stx 2 , eae, ehlyA, cnf 1 , cnf 2 , cdIII, cdIV, and f17 by PCR method. Sixty-six isolates (23.72%) possessed the STI gene and categorized into entrotoxigenic E. coli (ETEC). Nine isolates (3.1%) and five isolates (1.72%) were positive for the cnf1 and cnf2 genes which categorized into necrotoxic E. coli (NTEC). Hundred and seventeen isolates (40.34%) harbored stx 1 and/or stx 2 and classified as Shiga toxin-producing E. coli (STEC). Thirteen isolates (4.48%) were assigned to atypical entropathogenic E. coli (aEPEC) and possessed eae gene. Two isolates (0.68%) were positive for ipaH gene and were assigned to entroinvasive E. coli (EIEC). Statistical analysis showed a specific association between eae gene and STEC pathotype (P < 0.0001). The most prevalent resistance was observed against lincomycin (96.5%) and the lowest resistance was against kanamycine (56.02%), respectively. The high prevalence of STEC and ETEC indicates that diarrheic lambs represent an important reservoir for humans. ETEC may play an important role for frequent occurrence of diarrhea in lambs observed in this region. Due to high antibiotic resistance, appropriate control should be implemented in veterinary medicine to curb the development of novel resistant isolates.

Prevalence of O157:H7 and non-O157 E. coli in Iranian domestic sheep.

The aim of the present study was the isolation of both E. coli O157 and non-O157 in sheep. Verotoxins (VT) 1, 2 and eae genes were tested for this propose. Sheep faces are an important source of Shiga toxin-producing Escherichia coli (STEC). Escherichia coli O157:H7 is a highly virulent food-borne pathogen and threat to public health. Rectal swab samples from sheep were collected during 2009-2010. Conventional plating and Polymerase Chain Reaction (PCR) were carried out according to virulence factors (Stx1, Stx2 and eaeA).There significant differences between prevalence of STEC and session were observed. It was at highest in spring and late summer. Six (3.92%) sheep carcasses were contaminated by E. coli O157:H7.Only six samples were positive by PCR specific for the VT2 gene and produced verocytotoxin VT2, whereas all isolates were negative for the presence of VT1 and eae virulence genes considered. Geographical variations and season may be influenced in the prevalence rate. The composition of the gastrointestinal flora may be changed by different diet and, therefore O157 STEC rate in sheep and lamb was different. Iranian sheep indicated as a natural host of E. coli O157 strains therefore, may be potentially pathogenic for humans. This is the first report of E. coli O157 detection from sheep in Iran.

Development of a disperse dye immunoassay technique for detection of antibodies against Neospora caninum in cattle.

In this study a disperse dye immunoassay method was standardized and evaluated for detection of antibodies against Neospora caninum in cattle. Sera from 150 cattle with a recent history of abortion were collected and tested by commercial ELISA kit and a standardized in-house dye immunoassay system. The positivity rate for the sera used in this study was 34.6% for the disperse dye immunoassay (DDIA) compared to 32% obtained by ELISA kit. This study showed no significant difference between DDIA and ELISA. The results indicated that the DDIA provide an economic, simple, rapid and robust test for detection of N. caninum infection in cattle.

First molecular detection of group A rotavirus in urban and hospital sewage systems by nested-RT PCR in Shiraz, Iran.

BACKGROUND: Group A rotaviruses are the most significant cause of acute gastroenteritis in children worldwide. Rotaviruses are shed in high numbers and dispersed widely throughout bodies of water in the environment. This represents a significant health hazard for humans, mainly due to the stability of the viruses during wastewater treatment processes. This study was conducted to evaluate the prevalence of rotaviruses, to determine G genotypes of circulating rotaviruses and to assess the efficiency of rotavirus removal in urban and hospital sewage treatment plants in Shiraz, Iran. MATERIALS AND METHODS: During the period from October 2010 to June 2011, a total of sixty sewage samples from urban and hospital sewage disposal systems were collected by Grab Sampling in Shiraz, Iran. All the samples were concentrated in pellet form and two-phase methods and then group A rotaviruses were investigated with enzyme immunoassays (EIA). Rotavirus-positive specimens were genotyped by the nested RT-PCR and by using different types of specific primers. RESULTS: In total, rotaviruses were identified in 25% (15 cases) of sewage samples, representing 73.33% (11 cases) of influent and 26.67% (4 cases) of effluent systems. The frequency of rotavirus detection in autumn, winter and spring was 46.67%, 33.33% and 20%, respectively (P= 0.004). The most common circulating genotype was G1 (73.33%), followed by G1G4 (20%) and non-typeable (6.67%), respectively. CONCLUSIONS: The high prevalence of rotaviruses in urban and hospital sewage systems highlights the importance of environmental surveillance as a tool to detect new genotypes and to investigate the epidemiology of rotaviruses circulating in the community.

Prevention by rat amniotic fluid of adhesions after laparatomy in a rat model.

BACKGROUND: Adhesion formation after abdominal surgery (especially multiple operations) is still a major cause of morbidities such as infertility, pain, bowel obstruction, difficult reoperation and other complications. Our aim was to investigate the ability of rat amniotic fluid to prevent adhesion formation after laparatomy in rats. METHODS: This experimental trial was conducted in 20 pregnant rats randomly assigned to two groups of 10 animals each. Measurable serosal and intestinal injuries were created with a standard technique. Rats in both groups underwent laparatomy and hysterectomy. Then amniotic fluid was poured into the abdominal cavity of animals in the case group. All animals were operated on 4 weeks after initial surgery to assess adhesions, which were scored by an examiner who was blind to the animals' group assignment. RESULTS: The frequency of severe adhesions was 30% in the control group and 0% in the intervention group. Total adhesion scores were significantly lower in the amniotic fluid treatment group than in the control group (P = 0.002). CONCLUSIONS: Rat amniotic fluid can decrease the likelihood of post-operative intraperitoneal adhesion formation.

Detection of enterotoxigenic K99 (F5) and F41 from fecal sample of calves by molecular and serological methods.

Enterotoxigenic Escherichia coli (ETEC) is one of the major causes of neonatal calf diarrhea. Almost all ETEC bacteria are known to adhere to receptors on the small intestinal epithelium via their fimbriae, (F5 (K99) and F41).This study was undertaken to investigate the phenotypic and genotypic screening of virulence genes in E. coli K99 and F41. During January 2008 to December 2009, 298 diarrheic neonatal calves at 1-30 days old were studied by multiplex PCR, isolation, and serological grouping. Of the 298 diarrheic samples, 268 E. coli were isolated, of which 16 samples (5.3%) were positive for having the F5 (K99) fimbrial gene by PCR while all of the E. coli isolates also carried F41 fimbrial genes. Twenty-five percent of the isolates were proven not to be toxigenic as they did not possess the STa enterotoxin gene.

Standardization of the outbred BALB/c mice as a suitable animal model for Besnoitia caprae studies.

The Apicomplexan parasite Besnoitia caprae infects wild and domestic goats. Knowledge on Besnoitia caprae specially an optimized animal model is sparse. Experimental infections with tachyzoites of BC-Pars obtained from BALB/c mice were conducted in outbred mice to determine the infectivity and LD50 of Besnoitia caprae. Six groups of five mice were intraperitoneally infected with 12.5 × 10(3), 25 × 10(3), 5 × 10(4), 1 × 10(5) and 2 × 10(5) tachyzoites and a control inoculum of DMEM, respectively. Although morbidity and mortality were observed in all groups, two mice in the 12.5 × 10(3) group showed alopecia and skin lesions on 60 days post-infection (DPI). Histopathological and molecular examination of skins confirmed B. caprae infection. The LD50 was calculated as 25 × 10(3.2) tachyzoites per mouse. The results indicate that outbred BALB/c mice can be used as a suitable model of besnoitiosis and to screen candidate treatments and to test the efficacy of vaccines for besnoitiosis.

Detection and frequency of Stx2 gene in Escherichia coli O157 and O157:H7 strains isolated from sheep carcasses in Shiraz-Iran.

Enterohaemorrhagic Escherichia coli constitute a subset of serotypes (E. coli O157 and some other serogroups) of Shiga toxin-producing E. coli firmly associated with severe human illnesses like bloody diarrhoea and haemolytic uraemic syndrome. Escherichia coli O157:H7 is a zoonotic pathogen. They rarely cause disease in animals, live in the intestines of healthy sheep and ruminants are recognized as their main natural reservoir, so they can contaminate meat during slaughtering practices. The purpose of this study was epidemiological survey on the occurrence of E. coli O157:H7 in healthy sheep in Shiraz-Iran. Polymerase Chain Reaction (PCR) assay was developed to detect the Stx2 gene the only bacterial factor that has been associated with more severe disease. During a period of 7 months (December 2005 to June 2006), 153 slaughtered sheep at Shiraz slaughterhouse, were randomly selected and examined for surface carriage of E. coli O157:H7 by conventional plating and Stx2 gene detection by PCR technique. E. coli O157:H7 was found in 6(3.92%) of 153 sheep. The bacteria were isolated from 5(3.34%) of 114 and 1(2.63%) of 38 sheep two or under two and more than 2 years old, respectively (p = 0.5). The contamination rate might vary depending on season, age and infection time. The higher frequency for younger animals may be due to differences in the composition of the gastrointestinal flora resulting from differences in diet. This is the first report of the presence of E. coli O157:H7 in sheep from Iran.