First molecular detection of Francisella tularensis in turtle (Testudo graeca) and ticks (Hyalomma aegyptium) in Northwest of Iran.

Francisella tularensis, causative agent of tularemia, is a contagious zoonotic ailment. This study was aimed to molecularly detect F. tularensis in tortoise blood (n = 100) and ticks (n = 100) collected in the West Azerbaijan province, Iran suing a 16SrRNA gene of the Francisella genus through employment of the Nested-PCR technique. The identified ticks were s Hyalomma aegyptium by morphological analysis. Seven percent (with a 95% CI: 3.5%-13.75%) of animal blood samples yielded positive results for the presence of the Francisella. Meanwhile, the Francisella was identified in tick samples at a rate of fifteen percent (15%) (with a 95% CI: 9%-23%). The samples containing positive results were specifically classified as F. tularensis subsp. holarctica. The samples were taken from ticks belonging to the H. aegyptium species that were gathered in Oshnavieh, southern part of West Azerbaijan province, Iran. This research was aimed to validate the existence of F. tularensis in ticks found within the West Azerbaijan province. Consequently, it is vital to acknowledge the potential of these ticks to transmit the bacteria to both livestock and humans through tick bites in this specific area.

Identification of Salmonella carriers by amplification of FimA, Stn and InvA genes and bacterial culture methods in fecal samples of buffalo.

Salmonellosis is one of the most important bacterial diseases in human and animals. Rapid diagnosis and sub sequence accurate treatment of Salmonella carriers help reduce the salmonellosis in human and livestock animals. In this study, 420 fecal samples were taken during year 2019 from buffalo in the Urmia, Khoy and Piranshahr regions in west Azerbaijan province, Iran. Samplings were carried out in different seasons. Presence of Salmonella invasion genes (FimA, Stn and InvA) were evaluated by polymerase chain reaction. The bacterial culture and biochemical tests were performed on feces samples for isolation of bacterium Salmonella; however, all samples were negative in culture method. PCR findings showed that, 50 (11.90%) fecal samples were positive to the genes. The analysis of results showed that frequency of salmonellosis outbreak in different parts of west Azerbaijan province followed a similar pattern and the incidence of salmonellosis according to forecast in the warm seasons (spring and summer) was more than in cold seasons (autumn and winter). The prevalence of Salmonella in buffalo's feces based on warm and cold seasons were 32 (64.00%) and 18 (36.00%), respectively. The results showed significant difference between cold and warm season in the prevalence of salmonellosis. Therefore, the application of molecular technics is essential for the prevention and treatment of salmonellosis. The results also showed that specificity of PCR method was better than culture method for detection of Salmonella in feces sample.

Biocontrol of pigeon tick Argas reflexus (Acari: Argasidae) by entomopathogenic fungus Metarhizium Anisopliae (Ascomycota: Hypocreales).

The pigeon tick Argas reflexus is a pathogen-transmitting soft tick that typically feeds on pigeons, but can also attack humans causing local and systemic reactions. Chemical control is made difficult due to environmental contamination and resistance development. As a result, there is much interest in increasing the role of other strategies like biological control. In this study, the efficacy of three strains (V245, 685 and 715C) of entomopathogenic fungus Metarhizium anisopliae for biological control of three life stages of pigeon tick A. reflexus including eggs, larvae, engorged and unfed adults was investigated under laboratory conditions. Five concentrations of different strains of M. anisopliae ranging from 10(3) to 10(7) conidia/ml were used. All fungal strains significantly decreased hatchability of A. reflexus eggs. Strain V245 was the most effective strain on the mortality of larval stage with nearly 100% mortality at the lowest concentration (10(3) conidia/ml) at 10 days post-inoculation. The mortality rate of both engorged and unfed adult ticks were also increased significantly exposed to different conidial concentrations compared to the control groups (P < 0.05) making this fungus a potential biological control agent of pigeon tick reducing the use of chemical acaricides.

Phylogenetic and molecular analysis based on genes 16S-rRNA, OMPA and POMP to identify Chlamydia abortus infection occurrence at the milk samples of goats and sheep in west Azerbaijan of Iran.

BACKGROUND AND OBJECTIVES: Enzootic abortion in sheep and goats, also called ovine enzootic abortion (OEA) or enzootic abortion of ewes (EAE), is caused by Chlamydia abortus. The disease has a major economic impact as it represents the most important cause of lamb loss in sheep in parts of Europe, North America and Africa. This serious and potentially life-threatening zoonosis can also affect pregnant women after contact with lambing ewes, leading to severe febrile illness in pregnancy and loss of the foetus. MATERIALS AND METHODS: The present study was conducted to the Phylogenetic and Molecular Analysis based on Genes 16S-rRNA, OmpA and POMP of C. abortus in milk samples collected from sheep and goats in West Azerbaijan province, Iran. During 2018, a total number of 360 milk samples were collected from sheep (n = 180) and goats (n = 180) of different regions of the province. All milk samples were subjected to DNA extraction and examined by PCR. RESULTS: Among 360 milk samples collected from sheep and goats, 31 (8.611%; 95% CI=6.13-11.96) were positive for Chlamydia spp. The helicase, 16S-rRNA and ompA genes were examined and resulted in 8, 31, 31 of positive samples respectively. The accession numbers have been deposited in GenBank (NCBI) (MT367602 and MT367603). CONCLUSION: Phylogenetic analysis based on the gene of helicase showed that most of the isolates shared similarity > 99.97%.

Prevalence of Coxiella burnetii in milk collected from buffalo (water buffalo) and cattle dairy farms in Northwest of Iran.

The present study was conducted to determine the prevalence of C. burnetii in raw milk samples collected from water buffalos and cattle in Northwest of Iran (West Azerbaijan Province). A total number of 840 milk samples were randomly collected from buffalos and cattle belonged to three different geographical regions in west Azerbaijan (the map is necessary). The milk samples were collected seasonally during 2018 and the age of animals were recorded. All the milk samples were subjected to DNA extraction. Nested-PCR was used to detect C. burnetii based on the transposable gene IS1111. The results showed that 16.9% (95% CI: 14.5%-19.6%) of the examined milk samples (19.3% buffalo and 14.6% cattle samples) were positive for C. burnetii. There was a significant difference in C. burnetii shedding in milk between different age groups in cattle but not in buffalos (p value <0.05). The shedding of C. burnetii in milk was highly prevalent in summer (31.1%) (p < 0.05, 95% CI: 26.1%-38.4%). There were significant regional and seasonal variations in the prevalence of C. burnetii in the examined milk samples. It was concluded that buffalo population in west Azerbaijan should be considered as an important factor in the epidemiology of Q fever and consequently in public health.

Molecular identification and phylogenetic analysis of Lactobacillus and Bifidobacterium spp. isolated from gut of honeybees (Apis mellifera) from West Azerbaijan, Iran.

Polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) and phylogenetic analysis were used for molecular identification of lactic acid bacteria (LABs) isolated from Apis mellifera. Eighteen honeybee workers were collected from three different apiaries in West Azerbaijan. LABs from the gut of honeybees were isolated and cultured using routine biochemical procedures. Genomic DNA was extracted from LABs and a fragment of 1540 bp in size of 16S rRNA gene was amplified. PCR products were digested using HinfI endonuclease and digested products with different RFLP patterns were subjected to nucleotide sequencing and phylogenetic analysis. The results revealed that Lactobacillus and Bifidobacteria spp. are were the most abundant LABs in honeybee gut. Phylogenetic analysis showed that both Lactobacillus and Bifidobacterium were closely clustered with high similarity percentage with the same bacteria isolated from honeybees' gut elsewhere. It was concluded that LABs isolated from honeybees had low sequence divergence in comparison with LABs isolated from other sources such as dairy products.

Multimeric recombinant M2e protein-based ELISA: a significant improvement in differentiating avian influenza infected chickens from vaccinated ones.

Killed avian influenza virus (AIV) vaccines have been used to control H5N1 infections in countries where the virus is endemic. Distinguishing vaccinated from naturally infected birds (DIVA) in such situations however, has become a major challenge. Recently, we introduced the recombinant ectodomain of the M2 protein (M2e) of H5N1 subtype as a novel tool for an ELISA based DIVA test. Despite being antigenic in natural infection the monomer form of the M2e used in ELISA had limited antigenicity and consequently poor diagnostic capability. To address this shortcoming, we evaluated the use of four tandem copies of M2e (tM2e) for increased efficiency of M2e antibody detection. The tM2e gene of H5N1 strain from Indonesia (A/Indonesia/CDC540/2006) was cloned into a pMAL- p4x expression vector and expressed in E.coli as a recombinant tM2e-MBP or M2e-MBP proteins. Both of these, M2e and tM2e antigens reacted with sera obtained from chickens following live H5N1 infection but not with sera from vaccinated birds. A significantly stronger M2e antibody reaction was observed with the tM2e compared to M2e antigen. Western blotting also supported the superiority of tM2e over M2e in detection of specific M2e antibodies against live H5N1 infection. Results from this study demonstrate that M2e tetramer is a better antigen than single M2e and could be more suitable for an ELISA based DIVA test.

Biocontrol of pigeon tick Argas reflexus (Acari: Argasidae) by entomopathogenic fungus Metarhizium Anisopliae (Ascomycota: Hypocreales)

The pigeon tick Argas reflexus is a pathogen-transmitting soft tick that typically feeds on pigeons, but can also attack humans causing local and systemic reactions. Chemical control is made difficult due to environmental contamination and resistance development. As a result, there is much interest in increasing the role of other strategies like biological control. In this study, the efficacy of three strains (V245, 685 and 715C) of entomopathogenic fungus Metarhizium anisopliae for biological control of three life stages of pigeon tick A. reflexus including eggs, larvae, engorged and unfed adults was investigated under laboratory conditions. Five concentrations of different strains of M. anisopliae ranging from 10³ to 10(7) conidia/ml were used. All fungal strains significantly decreased hatchability of A. reflexus eggs. Strain V245 was the most effective strain on the mortality of larval stage with nearly 100 percent mortality at the lowest concentration (10³ conidia/ml) at 10 days post-inoculation. The mortality rate of both engorged and unfed adult ticks were also increased significantly exposed to different conidial concentrations compared to the control groups (P < 0.05) making this fungus a potential biological control agent of pigeon tick reducing the use of chemical acaricides.