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Coxiella burnetii causes query (Q) fever, an important zoonotic disease with worldwide significance. The role of environment in the ecology of C. burnetti, and its influence on seroconversion in animals has not been elucidated in Pakistan. We carried out a cross-sectional study in Punjab province to (1) determine the prevalence and distribution of C. burnetii in soil using an ISIIII gene-based real time-polymerase chain reaction (RT-PCR) assay, (2) analyze association between the occurrence of C. burnetii in soil and its predictors i.e. soil characteristics (macro- and micro-nutrients) and several likely risk factors including the seroconversion in small ruminants at places where its genome had or had not been detected, and (3) predict homology and genetic diversity of the identified strains using sequences originated from different hosts worldwide. A total of 2425 soil samples from nine districts of Punjab province were processed. C. burnetii DNA was detected in 47 samples (1.94%, 95% CI: ±0.55) originating from 35 villages of studied districts (7.22%, 95% CI: ±2.30). The highest prevalence was found in Attock (7.11%, 95% CI: ±3.36), followed by Lahore (4.83%, 95% CI: ±3.49), Sahiwal (4.70%, 95% CI: ±2.6), Dera Ghazi Khan (2.33%, 95% CI: ±2.02), Faisalabad (1.35%, 95% CI: ±1.18) and Sheikhupura (0.68%, 95% CI: ±0.94). The odds of detecting bacterial DNA in soil was increased with a unit increase in organic matter [2.511 (95% CI: 1.453-4.340), p=0.001] and sodium [1.013 (95% CI: 1.005-1.022), p=0.001], whereas, calcium [0.984 (95% CI: 0.975-0.994), p=0.002] and potassium [0.994 (95% CI: 0.990-0.999), p=0.011] had protective effect where a unit increase in each analyte decreased odds for its occurrence by 1.0% approximately. Likewise, for categorical variables (risk factors), the odds of detecting C. burnetii were higher at locations >500m away from a main road [1.95 (95% CI: 1.06-3.78), p=0.04]. The enzyme-linked immunosorbent assay (ELISA) revealed an increased prevalence of antibodies in sheep (17.9%, 95% CI: ±5.54) compared with goats (16.4%, 95% CI: ±4.34). When determining the association between soil DNA and C. burnetii antibodies in small ruminants, the odds of detecting these antibodies were significant in sheep at the livestock barns [2.81 (95% CI: 1.20-7.37), p=0.02]. The IS1111 gene-based sequence analysis revealed a clustering of the DNA into two distinct groups with much genetic divergence (0.76-68.70%): the first group that contained sequences from Lahore district clustered with human and buffalo origin isolates, whereas the second group that contained the sequences from the remaining study districts clustered with goat-, rodent- and human-origin isolates. This study provides the first evidence of the presence of C. burnetii in the environment in Punjab province, Pakistan. Future studies are needed to ascertain the bacteria's molecular epidemiology over a wide geographical area, type the isolates, and evaluates the potential risks to human populations, particularly farmers and veterinarians.

The present study was designed to investigate the antagonistic activity of Bacillus licheniformis BL350-2 against mycotoxigenic strains of Aspergillus and Penicillium. In vitro coincubation for 5 days indicated Aspergillus westerdijkiae BA1 as the most sensitive strain, with a growth inhibition of 62%, followed by A. carbonarius MG7 (60%), Penicillium verrucosum MC12 (53%), A. niger MC05 (50%), A. flavus CM5 (49%), A. parasiticus SB01 (47%), and A. ochraceus MD1 (44%). Likewise, the majority of the tested strains on exposure to bacterial volatiles showed complete inhibition of mycotoxin synthesis. In vivo assays on maize ears resulted in 88% reduction in A. flavus CM5 growth and complete inhibition of fungal sporulation and aflatoxin accumulation. The GC–MS-based volatile profile showed 3-methyl-1-butanol as the most abundant compound. The findings of the present study advocate that B. licheniformis BL350-2 is suitable as a biocontrol agent against mycotoxigenic fungi, at least during storage of cereal grains.

Mycotoxins are important contaminants of food and feed. In this study, low fermenting yeast (Lachancea thermotolerans) and its derivatives were applied against toxigenic fungi and their mycotoxins. A. parasiticus, P. verrucosum and F. graminearum and their mycotoxins were exposed to yeast volatile organic compounds (VOCs) and cells, respectively. VOCs reduced significantly the fungal growth (up to 48%) and the sporulation and mycotoxin synthesis (up to 96%). Very interestingly, it was shown that even 7 yeast colonies reduced Fusarium’s growth and the synthesis of its mycotoxin, deoxynivalenol (DON). Moreover, decreasing yeast nutrient concentrations did not affect the inhibition of fungal growth, but reduced DON synthesis. In addition, inactivated yeast cells were able to remove up to 82% of the ochratoxin A (OTA). As an application of these findings, the potentialities of the VOCs to protect tomatoes inoculated with F. oxysporum was explored and showed that while in the presence of VOCs, no growth was observed of F. oxysporum on the inoculated surface areas of tomatoes, in the absence of VOCs, F. oxysporum infection reached up to 76% of the tomatoes’ surface areas. These results demonstrate that the application of yeasts and their derivatives in the agriculture and food industry might be considered as a very promising and safe biocontrol approach for food contamination.