Biochemical and molecular characterization of Campylobacter fetus isolates from bulls subjected to bovine genital campylobacteriosis diagnosis in Spain.

BACKGROUND: Bovine genital campylobacteriosis (BGC) is caused by Campylobacter fetus subsp. venerealis (Cfv) including its biovar intermedius (Cfvi). This sexually transmitted disease induces early reproductive failure causing considerable economic losses in the cattle industry. Using a collection of well-characterized isolates (n = 13), C. fetus field isolates (n = 64) and saprophytic isolates resembling Campylobacter (n = 75) obtained from smegma samples of breeding bulls, this study evaluated the concordance of the most used phenotypic (H2S production in cysteine medium and 1% glycine tolerance) and molecular (PCR) methods for the diagnosis of BGC and assessed possible cross-reactions in the molecular diagnostic methods. RESULTS: Characterization at the subspecies level (fetus vs. venerealis) of C. fetus isolated from bull preputial samples using phenotypic and molecular (PCR targeting nahE and ISCfe1) methods showed moderate concordance (κ = 0.462; CI: 0.256-0.669). No cross-reactions were observed with other saprophytic microaerophilic species or with other Campylobacter species that can be present in preputial samples. Whole genome sequencing (WGS) of discrepant isolates showed 100% agreement with PCR identification. For the differentiation of Cfv biovars, comparison of the H2S test (at 72 h and 5 days of incubation) and a PCR targeting the L-cysteine transporter genes showed higher concordance when H2S production was assessed after 5 days (72 h; κ = 0.553, 0.329-0.778 CI vs. 5 days; κ = 0.881, 0.631-1 CI), evidencing the efficacy of a longer incubation time. CONCLUSIONS: This study confirmed the limitations of biochemical tests to correctly identify C. fetus subspecies and biovars. However, in the case of biovars, when extended incubation times for the H2S test (5 days) were used, phenotypic identification results were significantly improved, although PCR-based methods produced more accurate results. Perfect agreement of WGS with the PCR results and absence of cross-reactions with non-C. fetus saprophytic bacteria from the smegma demonstrated the usefulness of these methods. Nevertheless, the identification of new C. fetus subspecies-specific genes would help to improve BGC diagnosis.

Fatal leptospirosis in endangered Barbary macaques (Macaca sylvanus) kept in captivity: Assessing the role of sympatric rodents.

Between December 2020 and January 2021, an outbreak of acute mortality in endangered Barbary macaques (Macaca sylvanus) kept in captivity was detected in a zoo in Spain. The main findings observed in the two fatally affected animals at post-mortem evaluation were jaundice, renal tubular necrosis and interstitial nephritis. Leptospira spp. infection was confirmed by real time PCR (qPCR) in different tissues in both individuals. Analyses of secY gene from a positive individual showed 100% homology with a previously published sequence corresponding to Leptospira interrogans serovar Copenhageni. Free-living sympatric brown rats (Rattus norvegicus) from the affected zoo were also analyzed, and showed a prevalence and seroprevalence of Leptospira spp. of 18.2% (4/22; 95% CI: 2.1-34.3) and 41.9% (26/62; 95% CI: 29.7-54.2), respectively. We detected seropositive sera to five different serovars of Leptospira spp. (Copenhageni, Grippotyphosa, Pomona, Canicola and Hardjo) in the rodent population, with L. Copenhageni being the predominant one. This study describes for first time an outbreak of fatal leptospirosis in captive non-human primates in Europe. Our results show that Barbary macaques, an endangered species, are highly susceptible to Leptospira spp. infection, with sympatric wild rodents being the most likely reservoir animals involved in transmission in this outbreak. Our results suggest that rodent control could be an effective measure for minimizing exposure to Leptospira spp. in zoological collections. Given the potential implications for conservation, animal and public health, non-human primates and rodents should be included in surveillance programs for Leptospira spp. in zoos.

First national survey of residues of active substances in honeybee apiaries across Spain between 2012 and 2016.

This nationwide monitoring aimed to investigate the prevalence of residues of plant protection products (PPPs) and veterinary medicine products (VMPs) based on random selection of apiaries of Apis mellifera. For a three-year period (2012, 2013 and 2016), this study targeted 306 PPPs, VMPs and other active substances in 442 samples of bee bread honeycomb (BBHC) and 89 samples of honeybees collected from up to 177 apiaries. The results indicate that honeybees were most often exposed to residues of coumaphos, tau-fluvalinate, chlorfenvinphos, and acrinathrin, with a prevalence from a maximum of 98.8% to 49.4% in BBHC samples. Residues of coumaphos, tau-fluvalinate, amitraz (DMF + DMPF), carbendazim and orthophenylphenol were also frequently detected, from a maximum of 55.1% to 13.5% of the honeybee samples. Neonicotinoid residues, namely clothianidin and thiamethoxam, whose outdoor uses in crops are completely banned in EU, were not detected. Imidacloprid was found in 3.4% to 13.3% of samples during 2013 and 2016, respectively. Imidacloprid exceeded its acute toxicity (LD50) value for honey bees in two samples of BBHC. Fipronil was detected in 0.5% of the samples during 2013. The diversity of active substances found (% of different residues analyzed) ranged from 33.9% to 37.2% in BBHC from 2012, 2013 to 2016, and was of 26.5% in honeybees in 2016. In at least 54% of the samples, the total residue load was in the range of 200 to 1500 µg·kg-1. Up to 50% of BBHC samples were positive for one or two residues. No toxic residues for honeybees were detected in up to 88.8% of bee samples. This systematic surveillance of active substances assisted the evaluation of which target pesticides to look for and provided support to the competent authorities in the bee health decision-making.

Prevalence and antimicrobial resistance of Campylobacter from wild birds of prey in Spain.

Wild birds have been identified as a relevant reservoir of Campylobacter spp., therefore, a potential source of infection in humans and domestic animals. The objective of this study was to determine the occurrence of Campylobacter spp. on birds of prey in Spain. In addition, antibiotic resistance profiles of the isolates were evaluated. A total of 689 specimens of 28 raptor species were analyzed, with a resulting individual prevalence of 7.5%. C. jejuni was the most frequently isolated species (88.5%), followed by C. coli and C. lari (3.8% each). The occurrence of Campylobacter was significantly higher (p < 0.05) in nocturnal birds of prey (15.3%), in spring season (12.2%) and in carnivorous species (9.4%). Isolates displayed a remarkable resistance to nalidixic acid (69.9%), ciprofloxacin (69.9%), and tetracycline (55.6%), and a low resistance to streptomycin (6.7%). Our findings highlight the importance of birds of prey as reservoirs of Campylobacter strains and their significant role as carriers of antimicrobial resistance.

Overview of spatio-temporal distribution inferred by multi-locus sequence typing of Taylorella equigenitalis isolated worldwide from 1977 to 2018 in equidae.

The accurate identification of Taylorella equigenitalis strains is essential to improve worldwide prevention and control strategies for contagious equine metritis (CEM). This study compared 367 worldwide equine strains using multilocus sequence typing according to the geographical origin, isolation year and equine breed. The strains were divided into 49 sequence types (STs), including 10 described for the first time. Three major and three minor clonal complexes (CCs), and 11 singletons, were identified. The genetic heterogeneity was low (0.13 STs/strain) despite the wide diversity of geographical origins (n = 16), isolation years (1977-2018) and equine breeds (n = 18). It was highest outside Europe and in the 1977-1997 period; current major STs and CCs already existed before 1998. Previous data associated the major CC1 with the first CEM outbreaks in 1977-1978 in the United Kingdom, Australia and the United States, and revealed its circulation in France. Our study confirms its circulation in France over a longer period of time (1992-2018) and its distribution in Spain and Germany but not throughout Europe. In addition to CC1, relationships between non-European and European countries were observed only through ST4, ST17 and ST30. Within Europe, several STs emerged with cross-border circulation, in particular ST16 and ST46 from the major complexes CC2 and CC8. These results constitute a baseline for monitoring the spread of CEM outbreaks. A retrospective analysis of a higher number of strains isolated worldwide between 1977 and the early 2000s would be helpful to obtain an exhaustive picture of the original CEM situation.

Drinking water as the source of Campylobacter coli infection in grandparent heavy breeders.

The aim of the present study was the molecular identification of a common source of infection of Campylobacter coli in two grandparent breeder farms. Campylobacter jejuni and C. coli were isolated from well water and cloacal swabs from grandparent chickens. Colonies were genotyped using restriction fragment length polymorphism-flaA gene, pulsed field gel electrophoresis and multi-locus sequence typing. The same genotype of C. coli was found in both farms and in the well from which drinking water was supplied to the farms. The well water was epidemiologically linked as the source of C. coli infection. The molecular identification for epidemiological source-tracking of C. coli in breeder farms could aid in combating the colonization of this pathogen and therefore to reduce their incidence in human campylobacteriosis.