The identification of novel single nucleotide polymorphisms to assist in mapping the spread of Bacillus anthracis across the Southern Caucasus

Anthrax is common as a zoonotic disease in the southern Caucasus area including parts of Turkey and Georgia. In this region, population genetics of the etiological agent Bacillus anthracis comprises, where known, the major canonical single nucleotide polymorphism (canSNP) groups A.Br.Aust94 and A.Br.008/009 of the pathogen’s global phylogeny, respectively. Previously, isolates of B. anthracis from Turkey have been genotyped predominantly by multi locus variable number of tandem repeat analysis (MLVA) or canSNP typing. While whole genome sequencing is the future gold standard, it is currently still costly. For that reason we were interested in identifying novel SNPs which could assist in further distinguishing closely related isolates using low cost assay platforms. In this study we sequenced the genomes of seven B. anthracis strains collected from the Kars province of Eastern Anatolia in Turkey and discovered new SNPs which allowed us to assign these and other geographically related strains to three novel branches of the major A-branch canSNP-group (A.Br.) Aust94. These new branches were named Kafkas-Geo 1–3 and comprised isolates from the Kars region and the neighboring republic of Georgia suggesting a common ancestry. The novel SNPs identified in this study connect the population genetics of B. anthracis in the South Caucasus and Turkey and will likely assist efforts to map the spread of the pathogen across this region

The use of germinants to potentiate the sensitivity of Bacillus anthracis spores to peracetic acid

Elimination of Bacillus anthracis spores from the environment is a difficult and costly process due in part to the toxicity of current sporicidal agents. For this reason we investigated the ability of the spore germinants L-alanine (100 mM) and inosine (5 mM) to reduce the concentration of peracetic acid (PAA) required to inactivate B. anthracis spores. While L-alanine significantly enhanced (p = 0.0085) the bactericidal activity of 500 ppm PAA the same was not true for inosine suggesting some form of negative interaction. In contrast the germinant combination proved most effective at 100 ppm PAA (p = 0.0009). To determine if we could achieve similar results in soil we treated soil collected from the burial site of an anthrax infected animal which had been supplemented with spores of the Sterne strain of B. anthracis to increase the level of contamination to 104 spores/g. Treatment with germinants followed 1 h later by 5000 ppm PAA eliminated all of the spores. In contrast direct treatment of the animal burial site using this approach delivered using a back pack sprayer had no detectable effect on the level of B. anthracis contamination or on total culturable bacterial numbers over the course of the experiment. It did trigger a significant, but temporary, reduction (p < 0.0001) in the total spore count suggesting that germination had been triggered under real world conditions. In conclusion, we have shown that the application of germinants increase the sensitivity of bacterial spores to PAA. While the results of the single field trial were inconclusive, the study highlighted the potential of this approach and the challenges faced when attempting to perform real world studies on B. anthracis spores contaminated sites

Caenorhabditis elegans predation on Bacillus anthracis: Decontamination of spore contaminated soil with germinants and nematodes

Remediation of Bacillus anthracis-contaminated soil is challenging and approaches to reduce overall spore levels in environmentally contaminated soil or after intentional release of the infectious disease agent in a safe, low-cost manner are needed. B. anthracis spores are highly resistant to biocides, but once germinated they become susceptible to traditional biocides or potentially even natural predators such as nematodes in the soil environment. Here, we describe a two-step approach to reducing B. anthracis spore load in soil during laboratory trials, whereby germinants and Caenorhabditis elegans nematodes are applied concurrently. While the application of germinants reduced B. anthracis spore load by up to four logs depending on soil type, the addition of nematodes achieved a further log reduction in spore count. These laboratory based results suggest that the combined use of nematodes and germinants could represent a promising approach for the remediation of B. anthracis spore contaminated soil

Human exposure to naturally occurring Bacillus anthracis in the Kars region of Eastern Türkiye

Environmental contamination with Bacillus anthracis spores poses uncertain threats to human health. We undertook a study to determine whether inhabitants of the anthrax-endemic region of Kars in eastern Türkiye could develop immune responses to anthrax toxins without recognised clinical infection. We measured anti-PA and anti-LF IgG antibody concentrations by ELISA in serum from 279 volunteers, 105 of whom had previously diagnosed anthrax infection (100 cutaneous, 5 gastrointestinal). Of the 174 without history of infection, 72 had prior contact with anthrax-contaminated material. Individuals were classified according to demographic parameters, daily working environment, and residence type. All villages in this study had recorded previous animal or human anthrax cases. Stepwise regression analyses showed that prior clinical infection correlated strongly with concentrations at the upper end of the ranges observed for both antibodies. For anti-PA, being a butcher and duration of continuous exposure risk correlated with high concentrations, while being a veterinarian or shepherd, time since infection, and town residence correlated with low concentrations. For anti-LF, village residence correlated with high concentrations, while infection limited to fingers or thumbs correlated with low concentrations. Linear discriminant analysis identified antibody concentration profiles associated with known prior infection. Profiles least typical of prior infection were observed in urban dwellers with known previous infection and in veterinarians without history of infection. Four individuals without history of infection (two butchers, two rural dwellers) had profiles suggesting unrecognised prior infection. Healthy humans therefore appear able to tolerate low-level exposure to environmental B. anthracis spores without ill effect, but it remains to be determined whether this exposure is protective. These findings have implications for authorities tasked with reducing the risk posed to human health by spore-contaminated materials and environments

Efficacy of pomades containing different percentages of enilconazole in the treatment of bovine dermatophytosis

Background - Enilconazole is a broad-spectrum topical antimycotic agent used for the management of bovine dermatophytosis

Immunohistochemical and Molecular Detection of Mannheimia spp. and Pasteurella spp. in Sheep with Pneumonia in Kars Province - Turkey

In this investigation, presence of Mannheimia haemolytica and Pasteurella multocida in sheep brought to Kafkas University Faculty of Veterinary Medicine between 2011 and 2013 with a suspicion of pneumonia was studied by immunohistochemical and bacteriological methods. Study materials were 100 sheep lungs. After routine histopathological investigations lungs were immunohistochemically stained with antibodies prepared against both of the agents. In bacteriological investigations, tissue samples were inoculated into 7% sheep blood agar and Mc Conkey agar and incubated for 24-48 h in aerobic conditions, and the suspected colonies were evaluated for Mannheimia and Pasteurella species. In histological investigations, pneumonias were classified as 28% fibrinous bronchopneumonia, 21% prulent bronchopneumonia, 20% acute-catharal bronchopneumonia, 18% interstitial pneumonia, 11% verminous pneumonia and 2% adenomatous pneumonia. In bacteriological investigations Mannheimia haemolytica and Pasteurella multocida was detected in 19 and 3 cases, respectively, these numbers were 17 and 2, respectively in immunohistochemical investigations. The results of PCR analysis for both Mannheimia haemolytica and Pasteurella multocida paralleled with the findings of microbiological culture. The results of the study showed that Mannheimia haemolytica is an important cause of pneumonia in sheep in Kars, and Pasteurella multocida, although with lesser importance can cause pneumonia in this species

Protection of farm goats by combinations of recombinant peptides and formalin inactivated spores from a lethal Bacillus anthracis challenge under field conditions

Background: Bacillus (B.) anthracis, the causal agent of anthrax, is effectively controlled by the Sterne live spore vaccine (34F2) in animals. However, live spore vaccines are not suitable for simultaneous vaccination and antibiotic treatment of animals being at risk of infection in an outbreak situation. Non-living vaccines could close this gap. Results: In this study a combination of recombinant protective antigen and recombinant Bacillus collagen-like antigen (rBclA) with or without formalin inactivated spores (FIS), targeted at raising an immune response against both the toxins and the spore of B. anthracis, was tested for immunogenicity and protectiveness in goats. Two groups of goats received from local farmers of the Kars region of Turkey were immunized thrice in three weeks intervals and challenged together with non-vaccinated controls with virulent B. anthracis, four weeks after last immunization. In spite of low or none measurable toxin neutralizing antibodies and a surprisingly low immune response to the rBclA, 80% of the goats receiving the complete vaccine were protected against a lethal challenge. Moreover, the course of antibody responses indicates that a two-step vaccination schedule could be sufficient for protection. Conclusion: The combination of recombinant protein antigens and FIS induces a protective immune response in goats. The non-living nature of this vaccine would allow for a concomitant antibiotic treatment and vaccination procedure. Further studies should clarify how this vaccine candidate performs in a post infection scenario controlled by antibiotics

Effectiveness of the Local Application of 1% Tioconazole in the Treatment of Bovine Dermatophytosis

This study consisted of twenty-five dermatophytotic cattle, aged 1.5-11 months obtained from Kars province and its surrounding, diagnosed as dermatophytosis following clinical and microbiologically. All animals were divided into two groups; trial group (n=15) and control group (n=10). The skin lesions of the trial animals were treated using a pomade containing 1% tioconazole once daily for a period of 5 days. In 12 of the trial animals the amount of keratinized tissue found in the dermatophytosis lesions decreased significantly following the 3rd application and had disappeared completely after the 5th application. On the other hand, the remaining 3 animals in the trial group were applied the medicament seven times due to the persistence of keratinized tissue in the lesions. In all of the animals administered with tioconazole, new hair growth in the site of the lesions resumed in the 3rd-4th weeks and complete recovery occurred within 7-8 weeks. In conclusion, 1% tioconazole, used for the first time for the treatment of bovine dermatophytosis in this study was found to be rather effective owing to its ease of application and strong therapeutic effect