Molecular detection of Coxiella burnetii in horse sera in Iran.

Coxiella burnetii is a zoonotic bacterium that can infect a wide range of animals including horses. However, its circulation dynamics in and through horses are still unclear. The aim of this study was to evaluate prevalence of C. burnetii and its genomic characteristics in horse sera samples in the North of Iran (Golestan Province). The samples were collected in 2018 and the age, sex, and breed of each animal were recorded. Nested-PCR was used to detect C. burnetii based on the presence of the transposable gene IS1111. The results showed that 7.50 % (P < 0.05; 95 % CI: 0.5 %-0.12 %) of the examined sera samples were positive for C. burnetii. Based on the resuls, prevalence of C. burnetii in the age groupof < Years 1-5 (p-value <0.05, 95 % CI: 1 %-8 %) was less than the age group of >6 years old (p-value <0.05, 95 %, CI: 7 %-19.8 %). In previous studies, it was concluded that the horses' population in Golestan Province should be considered as an important factor in the epidemiology of Q fever and consequently in public health. Further studies should be implemented to evaluate if horses may be relevant indicators of zoonotic risk in urban and suburban endemic areas.

Molecular Detection and Antimicrobial Resistance Patterns of Shiga Toxigenic Escherichia coli Isolated from Bovine Subclinical Mastitis Milk Samples in Kurdistan, Iran.

Bovine subclinical mastitis is regarded as a devastating disease due to the economic costs imposed on dairy husbandry. Moreover, it is a hazard in the public sector in the cases of zoonotic bacteria because of the potential role of unpasteurized milk and dairy products to propagate the infectious agent to the human food chain. The present study aimed to evaluate the frequency, virulence content, and antimicrobial resistance profile of Shiga toxin-producing Escherichia coli (STEC) strains isolated from bovine subclinical mastitis in Kurdistan Province, West of Iran. A total of 400 bovine subclinical mastitis milk samples recognized in the California Mastitis Test were collected aseptically and analyzed for the presence of E. coli phenotypically and molecularly. The isolates were genotypically screened for stx1, stx2, and eae genes. Furthermore, O157:H7 STEC strain was searched among the isolates in a duplex polymerase chain reaction. The antimicrobial resistance scheme of the isolates was determined using the agar disk diffusion method. In general, 173 (43.25%) E. coli isolates were detected among which 39 (22.54%) isolates were STEC. The frequency of STEC virulence genotypes was stx2 (25 isolates, 64.10%), stx2+eae (6 isolates, 15.38%), stx1+stx2 (6 isolates, 15.38%), and stx1+stx2+eae (2 isolates, 5.12%). In addition, three O157: H7 strains were identified with the genetic content of stx1+stx2+eae (2 isolates) and stx1+stx2 (1 isolate). The most prevalent antimicrobial resistance was observed against streptomycin, tetracycline, and ampicillin. Gentamycin, amoxicillin-clavulanic acid, and trimethoprim-sulfadiazine were the most effective antibiotics against O157 strains, whereas gentamycin, ciprofloxacin, and nitrofurantoin were effective against non-O157 strains. The results revealed the significant role of STEC in bovine subclinical mastitis in the studied region. In addition, the distribution of O157:H7 strain and high prevalence of multidrug resistance among the isolates is a matter of concern. Therefore, there is a potential threat of human infection following the consumption of contaminated milk with STEC in Kurdistan Province, Iran.

Prevalence of C. burnetii DNA in sheep and goats milk in the northwest of Iran.

Q fever is a common zoonotic disease with worldwide distribution. The causative agent of Q fever is Coxiella burnetii, a gram-negative and polymorphic rod bacterium. Sheep and goats are the primary reservoirs of this disease, although a variety of animal species can be infected. The main route of Q fever transmission from animals to humans is the inhalation of contaminated aerosols with C. burnetii. The bacterium is excreted in milk of infected animals and therefore; the consumption of unpasteurized milk and dairy products might be a route of coxiella burnetii transmission from animals to humans. The present study was conducted to determine the prevalence of C. burnetii in milk samples collected from sheep and goats in west Azerbaijan province, Iran. During 2018, a total number of 420 milk samples were collected from sheep (n = 210) and goats (n = 210) of different regions of the province. All milk samples were subjected to DNA extraction and examined by a highly and specific nested-PCR method. The results showed that 51 (12.1%) (95% CI: 9.3%-15.6%) examined samples [sheep; n = 16 (7.6%) and goat; n = 35 (16.6%)] were positive for C. burnetii. The prevalence of C. burnetii in goat milk samples was significantly higher than sheep milk samples (P < 0.05). The shedding of C. burnetii in milk was significantly higher in summer (25%) (P < 0.05, 95% CI: 17.7%-34%) than the other seasons. It was concluded that sheep and goat populations in west Azerbaijan play an important role in the epidemiology of Q fever.

Seroprevalence and Risk Factors of Neospora caninum and Toxoplasma gondii in Small Ruminants in Southwest of Iran.

Toxoplasma gondii and Neospora caninum are Apicomplexan intracellular protozoa with global distribution. Small ruminants play an important role as intermediate hosts for N. caninum and T. gondii, parasites of great public health concern. The main goal of the current survey was to evaluate N. caninum and T. gondii infection rate in sheep and goats of Khuzestan Province, southwest of Iran, using enzyme-linked immunosorbent assay (ELISA). In this cross-sectional study during February-April 2016, whole blood samples were taken randomly from 735 animals from 37 herds. The animals were reared under the traditional husbandry system in different parts of the province. Among 550 sheep and 185 goats, 37 (6.8%) sheep and 20 (10.8%) goats were seropositive for N. caninum and 59 (10.8%) sheep and 37 (20%) goats were seropositive for T. gondii. The incidence rates of mixed infection with N. caninum and T. gondii were 3.2% and 5.4% in sheep and goats, respectively. Seroprevalence rate of N. caninum was significantly higher in goats at T. gondii and abortion (18.2%). Also, a significant correlation was detected between seroprevalence of N. caninum and T. gondii and mixed infection in goats with a history of abortion. This is the first report of IgG antibody production against N. caninumand T. gondii co-infection in small ruminants in Iran. Our findings indicated that neosporosis and toxoplasmosis may be responsible for abortion in small ruminants in this region. Therefore, further investigations are needed to improve sanitary strategies in animals&rsquo; husbandry and launching control programs.

Epidemiological Study of Lumpy Skin Disease Outbreaks in North-western Iran.

Lumpy skin disease (LSD) is a highly contagious transboundary disease of cattle with major economic losses. This study was undertaken to address the emergence and epidemiological features of LSD in four north-western provinces of Iran. These provinces have extensive borders with others country including Iraq, Turkey, Azerbaijan and Armenia. A population of 683 cattle from 91 farms were examined during LSD outbreak in Iran during 2014-2016. The information of the farms including the population size, gender, age, vaccination status, clinical signs and the number of death because of LSD were recorded in the designed questionnaires. A number of 234 blood samples were collected randomly from animals with and without clinical signs of LSD. DNA was extracted from blood samples, and they were used for amplifying a fragment of 434 bp in size coupled with restriction fragment length polymorphism (RFLP) for molecular detection of lumpy skin disease virus (LSDV). The estimated prevalence, cumulative mortality and case fatality were 17.9%, 3.5% and 19.7%, respectively. There was no significant difference in occurrence of the disease between male and female cattle. LSD occurrence in age groups above 5 years old and below 6 months old showed highest and lowest relative frequencies, respectively. Vaccination was significantly decreased the occurrence of clinical disease. The developed PCR-RFLP technique was able to differentiate between LSDV, sheep pox virus (ShPV) and goat pox virus (GPV). It was concluded that LSD was entered into Iran probably from Iraq via uncontrolled animal movements along common land borders between two countries. Developed PCR-RFLP could be used as a rapid and inexpensive method for differentiating Capripoxviruses (CaPVs).

Polymorphisms in the ovine myostatin gene are associated with birth weight but not with weight gain in Iranian Makoei sheep.

Myostatin, a transforming growth factor-beta superfamily member, has been well documented as a negative regulator of muscle growth and development. Myostatin, which has 376 amino acids, is synthesized as a precursor protein. Polymorphism of the myostatin gene in Makoei sheep was investigated by PCR and single-strand conformation polymorphism technique (SSCP). Genomic DNA of 92 sheep was isolated from whole blood. A 417-bp myostatin intron I segment was amplified by standard PCR, using locus-specific primers. Four SSCP patterns, representing four different genotypes, were identified. The frequencies of the genotypes were 0.413, 0.293, 0.130, and 0.163 for AD, AC, AE, and BC, respectively. Allele frequencies were 0.4185, 0.0815, 0.2283, 0.2065, and 0.0652 for A, B, C, D, and E, respectively. Observed heterozygosity was 0.7192. There was significant deviation from Hardy-Weinberg equilibrium for this locus. Analysis of myostatin gene sequences revealed heterozygous SNPs, which were in agreement with results obtained in the SSCP analysis. We concluded that SSCP analysis is a quick, sensitive and reliable technique for determination of DNA polymorphisms. The effect of these genotypes on some traits was investigated, and the AD genotype was found to be associated with birth weight. No phenotypic associations were detected with the other genotypes. No associations of myostatin variants with weight gain were detected. We conclude that polymorphism in the ovine myostatin gene is associated with birth weight, but not with weight gain in Iranian Makoei sheep.

Field bioassay of Metarhizium anisopliae strains to control the poultry red mite Dermanyssus gallinae.

The poultry red mite, Dermanyssus gallinae is one of the most economically deleterious ectoparasite of laying hens worldwide. To evaluate the efficacy of three strains (V245, 3247 and 715C) of entomopathogenic fungus Metarhizium anisopliae with potential as acaricides against D. gallinae, this investigation was carried out in a commercial caged laying poultry farm in Naghedeh, West Azarbaijan of Iran. The parasite infestation already existed in the farm. Sunflower oil suspension of all fungal strains, each in two concentrations (1×10(7) and 1×10(9) conidia/ml) were used separately as spray on hens and cages, and in the control group the cages were only sprayed with sunflower oil and sterile distilled water. For estimating the population rate of mites before and after treatment, special cardboard traps were fixed to cages during a 1-month period. The traps were placed on weeks -1, 0, 1, 2 and 3 and always removed after 1 w. The results showed that the population rates post fungal treatment with the lower concentration were not significantly different compared to the control group. However, the reduction in mite numbers induced by all three strains at the concentration of 1×10(9) conidia/ml was significantly higher than the control (P<0.05). The results revealed that under field conditions, higher concentrations of M. anisopliae will be required for controlling D. gallinae.

Metarhizium anisopliae (Ascomycota: Hypocreales): an effective alternative to chemical acaricides against different developmental stages of fowl tick Argas persicus (Acari: Argasidae).

The fowl bloodsucking tick Argas persicus is of great medical and veterinary importance in tropical and subtropical regions because of its role as vector of certain parasitic, bacterial and viral pathogens. A variety of acaricides are used for the control of tick infestation in poultry, resulting in environmental contamination and the development of resistance. In order to develop an alternative control method, the efficacy of three strains (V245, 685 and 715C) of entomopathogenic fungus Metarhizium anisopliae against different life stages of A. persicus including eggs, larvae, unfed and engorged adult females was evaluated under laboratory conditions. Five concentrations of different strains of M. anisopliae ranging from 10(3) to 10(7)conidia/ml were utilized. The effects of fungal strains on egg hatchability and larva and adult female mortality were significant and dose-dependent compared to the control groups (P<0.05). The mortality rates of larvae ranged from 92% to 100% for two different concentrations (10(3) and 10(4)conidia/ml) of M. anisopliae strains. Treated engorged females were more susceptible than the unfed females reaching mortality rate of 100% at the highest concentration (10(7)conidia/ml) at 18 days post-inoculation. Among strains used in this study, V245 was the most virulent strain regarding the LC(50) values for adult females exposed to fungal conidia. The results demonstrate that the application of M. anisopliae as a biocontrol agent is a promising option in reducing the use of chemical acaricides, resulting in benefits to poultry and the environment.

Molecular typing of Staphylococcus aureus isolated from bovine mastitis based on polymorphism of the coagulase gene in the north west of Iran.

The objective was to investigate the genotypic characteristics and distribution of Staphylococcus aureus in 9 dairy herds of Tabriz and Urmia regions which are located in east and west Azerbaijan provinces, respectively, Iran. In this study 58 S. aureus isolates were recovered from 370 milk samples of cows with clinical and subclinical mastitis. S. aureus isolates were identified on the basis of the cultural and biochemical properties as well as by amplification of the aroA gene specific to S. aureus and then were analyzed using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of the coagulase gene (coa). Amplification of the variable region of the coa gene from these isolates produced five different PCR products ranging in size from approximately 490 bp to 850 bp. To obtain RFLP patterns of the PCR products they were subjected to digestion with restriction endonuclease HaeIII and the fragments separated by gel electrophoresis. Nine coa gene RFLP patterns, numbered I-IX, were observed, with 23 isolates (39.66%) assigned to RFLP pattern I and 14 isolates (24.14%) assigned to RFLP pattern III. Five out of nine patterns were found in both regions and four of nine patterns were only found in one region. The results demonstrated that several variants of the coa gene are present in the studied regions, but only a few of them were predominant, suggesting contagious transmission, a common source, or host adaptation of subset of the population of S. aureus strains. This study also indicated that genetic heterogeneity among S. aureus isolates recovered from bovine mastitis may be exists within and among herds in different regions.

Laboratory evaluation of three strains of the entomopathogenic fungus Metarhizium anisopliae for controlling Dermanyssus gallinae.

The pathogenicity of three strains of the entomopathogenic fungus Metarhizium anisopliae on different life stages of Dermanyssus gallinae was evaluated in the laboratory. All the strains tested were virulent to D. gallinae but pathogenicity varied among the strains. Strain V245 induced a higher mortality rate using different concentrations than other two strains. The estimated median lethal concentration of different strains of M. anisopliae against D. gallinae varied depending on the exposure time of D. gallinae to M. anisopliae. It was concluded that the pathogenicity of the entomopathogenic fungus M. anisopliae on different life stages of D. gallinae was concentration and time dependent.

Rapid differentiation of current infectious bronchitis virus vaccine strains and field isolates in Australia.

OBJECTIVE: Rapid differentiation of vaccine strains of infectious bronchitis virus (IBV) from wild type strains would enhance investigations of disease outbreaks. This study aimed to develop a reverse transcription-polymerase chain reaction (RT-PCR) assay to differentiate between Australian vaccine strains of IBV and field isolates. PROCEDURE: A fragment of 6.5 kilobases that contains the S, M and N genes was amplified by RT-PCR from ten different IBV strains, including vaccine strains and field isolates, and then sequenced. RESULTS: Comparison of the sequences of these strains revealed a deletion of 58 bases in the 3' untranslated region (UTR) of IBV vaccine strains but not in the field isolates. Two primers were designed to amplify a fragment of the 3' UTR that differed in size between the vaccine strains and field isolates. RT-PCR was performed using these two primers to screen 20 IBV strains, including field isolates and the vaccine strains. All strains were correctly identified as either vaccine strains or field isolates. CONCLUSION: This procedure is a rapid, sensitive and inexpensive method for discrimination between most current Australian vaccine strains and field isolates of IBV.