Susceptibility testing of Prototheca bovis isolates from cases of bovine mastitis using the CLSI reference broth microdilution method and the Sensititre YeastOne colorimetric panel.

A total of 62 Prototheca bovis isolates from cases of bovine mastitis were tested for susceptibility to different antifungal compounds by the Clinical and Laboratory Standards Institute (CLSI) reference microdilution method and a commercial colorimetric microdilution panel (Sensititre YeastOne). All isolates displayed low susceptibility to echinocandins (MICs > 8 µg/ml for anidulafungin, caspofungin, and micafungin), flucytosine (MIC > 64 µg/ml), and the azoles enilconazole and fluconazole (MICs > 4 and > 64 µg/ml, respectively). Moreover, 45.2, 32.3, and 1.6% of isolates had MICs > 4 µg/ml for ketoconazole, terbinafine, and voriconazole, respectively, when tested by the CLSI method. In contrast, all isolates were more susceptible to the polyene compounds amphotericin B and nystatin, and itraconazole, posaconazole, and ravuconazole (MICs ≤ 2 µg/ml, in all cases). Comparison of the results obtained in the CLSI and Sensititre methods showed excellent essential agreement (EA) for azoles (98.4% for itraconazole and posaconazole, and 100% for voriconazole) and moderate EA for amphotericin B (72.6%), when MICs were read after 48 and 24 h of incubation, respectively. In contrast, much lower EA values were obtained in some cases when the MICs for both techniques were determined after 48 h of incubation (e.g., 9.7% for amphotericin B and 69.4% for posaconazole). Therefore, the CLSI broth microdilution method and the Sensititre YeastOne panel can be used indistinctly for susceptibility testing of P. bovis isolates against azoles but not against amphotericin B until further optimization of the test conditions. LAY SUMMARY: The antifungal susceptibility of Prototheca bovis isolates was analyzed. All tested isolates displayed low susceptibility to echinocandins, flucytosine, and some azoles. Excellent agreement of the results of two different test methods was obtained for azoles, but not for the polyene amphotericin B.

Seasonal and spatial occurrence of zoonotic Salmonella serotypes in griffon vultures at farmland environments: Implications in pathogen pollution and ecosystem services and disservices.

Livestock production is a large source of microbial, pharmaceutical, and antimicrobial pollution worldwide. Vultures are one group of birds with particularly high exposure to food-borne pathogens due to frequent consumption of infected livestock carcasses. The potential origin and spatial-temporal shedding patterns of livestock-adapted Salmonella serotypes of zoonotic importance were evaluated in adult and nestling Griffon vultures (Gyps fulvus). We specifically assessed the exposure source and subsequent elimination of Salmonella-infected carcasses (ecosystem services) or transmission back to livestock (ecosystem disservices) by vultures, thus contributing respectively to disease mitigation or amplification in natural and farmed environments. The results show a seasonal high occurrence and turnover of a high variety of serotypes, especially swine-adapted ones isolated at high frequency. This suggests that vultures can be reservoirs and long-distance carriers of faecal Salmonella shed in supplementary feeding stations and breeding colonies. Contrary to their conservation purposes, feeding stations can act as Salmonella hotspots and reservoirs. However, a role for vultures in the transmission back to food-producing animals seems impossible because they do not come into contact at indoor farms, while transmission to free-ranging ruminants was not supported by the presence of ruminant-adapted serotypes in the vultures. Therefore, vultures do not promote disservices associated with the re-infection of livestock with Salmonella, but can provide quantitatively important ecosystem services by removing carrion contaminated with these and other zoonotic pathogens potentially affecting their health. Sanitary vigilance of the farms authorised to provide food for avian scavengers should avoid the disposal of swine and poultry carcasses with Salmonella and the antibiotics used to treat it. Extensive free-ranging livestock and their carcasses exploited in the countryside should be a priority for the conservation of vultures and their ecological function as cleaners and disease mitigators.

Culture- and molecular-based detection of swine-adapted Salmonella shed by avian scavengers.

Salmonella can play an important role as a disease agent in wildlife, which can then act as carriers and reservoirs of sanitary importance at the livestock-human interface. Transmission from livestock to avian scavengers can occur when these species consume contaminated carcasses and meat remains in supplementary feeding stations and rubbish dumps. We compared the performance of PCR-based detection with conventional culture-based methods to detect Salmonella in the faeces of red kites (Milvus milvus) and griffon vultures (Gyps fulvus) in central Spain. The occurrence of culturable Salmonella was intermediate in red kites (1.9%, n=52) and high in griffon vultures (26.3%, n=99). These proportions were clearly higher with PCR-based detection (13.5% and 40.4%, respectively). Confirmation cultures failed to grow Salmonella in all faecal samples positive by the molecular assay but negative by the initial conventional culture in both scavenger species, indicating the occurrence of false (non-culturable) positives by PCR-based detection. This suggests that the molecular assay is highly sensitive to detecting viable Salmonella in cultures, but also partial genomes and dead or unviable bacteria from past infections or contamination. Thus, the actual occurrence of Salmonella in a particular sampling time period can be underestimated when using only culture detection. The serovars found in the scavenger faeces were among the most frequently isolated in pigs from Spain and other EU countries, especially those generally recognized as swine-adapted monophasic variants of S. Typhimurium. Because the studied species obtain much of their food from pig carcasses, this livestock may be the primary source of Salmonella via direct ingestion of infected carcasses and indirectly via contamination due to the unsanitary conditions found in supplementary feeding stations established for scavenger conservation. Combining culture- and molecular-based detection is encouraged to understand the epidemiology and impact of Salmonella in wildlife populations.