Genomic Analysis of ESBL-Producing E. coli in Wildlife from North-Eastern Germany.

Antimicrobial resistance (AMR) is a serious global health threat and extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales are a major contributor. This study aimed to gain a deeper insight into the AMR burden of wild animals. In total, 1595 fecal samples were collected by two systematic searches in Mecklenburg-Western Pomerania, north-east Germany. Samples were screened for ESBL-carrying Escherichia (E.) coli and isolates found were further analyzed using antimicrobial susceptibility testing and whole-genome sequencing. We found an estimated prevalence of 1.2% ESBL-producing E. coli in wild boar and 1.1% in wild ruminants. CTX-M-1 was the most abundant CTX-M type. We also examined fecal samples from wild boar and wild ruminants using shotgun metagenomics to gain insight into the resistome in wild animals. The latter revealed significantly lower normalized counts for AMR genes in wildlife samples compared to farm animals. The AMR gene levels were lower in wild ruminants than in wild boar. In conclusion, our study revealed a low prevalence of ESBL-producing E. coli and a low overall AMR gene burden in wild boar and wild ruminants, probably due to the secluded location of the search area.

Evaluation of the Baermann-Wetzel method for detecting lungworm larvae in wild ruminants from faecal samples.

Lungworms can exert a negative impact on wild ruminant fitness; for this reason, the diagnosis of the associated diseases is an important prevention measure. The Baerman-Wetzel technique is the most usual method for the diagnosis of bronchopulmonary nematodes and is based on the active migration and movement of their first-stage larvae. Pulmonary tissue samples are frequently used for the post-mortem diagnosis of these parasites, but this kind of sample is not always available and easy to obtain. Faecal samples represent a more accessible choice for parasite monitoring. This work aimed to evaluate the agreement between the results obtained by the Baermann-Wetzel technique when samples of lung parenchyma or faeces from wild ruminants are used. A good level of agreement as well as a similar sensitivity between the two types of sample were observed, validating the use of faecal samples as a less invasive and cost-effective alternative for the monitoring of lungworm in wild ruminant populations.

Re-emergence of bluetongue virus serotype 4 in Iberian ibex (Capra pyrenaica) and sympatric livestock in Spain, 2018-2019.

Between early October and mid-December 2018, mortalities were detected in Iberian ibex (Capra pyrenaica) populations in southern Spain. In the same region and period, bluetongue virus (BTV) circulation was also reported in sentinel and clinically affected domestic ruminant herds. Molecular analyses confirmed BTV serotype 4 (BTV-4) infection in eight Iberian ibexes from six hunting areas, and in 46 domestic ruminants from seven herds in close proximity to affected hunting estates. Histopathological analyses revealed vascular changes in several organs, pneumonia, lymphoid depletion, inflammatory mononuclear cell infiltrate and fibrosis as the most frequently observed lesions in the affected Iberian ibexes. Epidemiological and laboratory results indicate that BTV-4 was the main aetiological agent involved in outbreaks detected in Iberian ibex populations during the study period. Sequence analyses indicated that the BTV-4 strain detected in Iberian ibex had high homology (99.4%-100%) with strains isolated in livestock during the same period, and with previous isolates (≥98.9%) from Spain and Mediterranean Basin countries. Further studies are warranted to determine the impact of BTV-4 on the health status of Iberian ibex populations after the outbreaks. The inclusion of this species in the surveillance programme may be useful for early detection of BTV, especially in epidemiological scenarios at the wildlife-livestock interface.

Non-homogeneous distribution of steroids in fecal pellets: An example in brown brocket deer (Mazama gouazoubira) with progesterone metabolites.

Measuring reproductive hormones in feces has become an important tool in the endocrine characterization of wild animals' reproduction. However, several factors may influence its success, such as fecal collection and storage techniques, knowledge of steroid hormone metabolism, the extraction procedure, immunoassay selection, inherent factors, and the distribution of steroid hormones in the feces. It is known that the distribution of these hormones in the feces is not homogeneous, and prior to the extraction of the steroidal metabolites, homogenization of the feces is recommended. Hormonal analysis is based on only a small fraction of the feces, which in theory should be representative of the total. In the case of cervids and other ruminants, feces consist of pellets. Here, the concentration of the steroid metabolites of each pellet was measured in order to evaluate the distribution of the fecal progesterone metabolites concentration in 10 pellets/fecal mass from five female Mazama gouazoubira. There were large variations in fecal progesterone metabolites concentrations between the pellets of the same feces/female, showing the following amplitude variations: Animal A: 112%; Animal B: 164%; Animal C: 115%; Animal D: 62%; Animal E: 108%. These results show the importance of adequate homogenization prior to steroid metabolite extraction.

Wide exposure to Coxiella burnetii in ruminant and feline species living in a natural environment: zoonoses in a human-livestock-wildlife interface.

Assessment of the role of wild and domestic hosts as potential reservoirs of misdiagnosed zoonoses, such as Q fever by Coxiella burnetii, is an important public health issue today both for wildlife conservation and management of disease in human-livestock-wildlife interface. This study used ELISA, an indirect antibody, to research (2003-2013) C. burnetii infection in seven free-living wild and domestic ruminant species and in European wildcats (Felis silvestris). The animals studied were 0 European wildcats, 21 Spanish ibex (Capra pyrenaica), 314 red deer (Cervus elaphus), 556 fallow deer (Dama dama), 211 European mouflon (Ovis aries musimon), eight roe deer (Capreolus capreolus), 407 bovines (Bos taurus) and 3739 sheep (Ovis aries). All the animals shared the same habitat in the Serranía de Cuenca Natural Park (Castile-La Mancha, Spain). The study area is an example of human-domestic-wildlife interface where people and domestic animals live in close proximity to wildlife. Observed C. burnetii seropositive frequencies were: 33·3% European wildcats, 23·8% Spanish ibex, 22·5% domestic sheep 1·5% red deer, 1·4% European mouflon, 0·24% cattle, 0·18% fallow deer and 0% roe deer. The study found a wide C. burnetii prevalence of previous and present exposure in wild and domestic ruminant hosts in the Serranía de Cuenca Natural Park and reports the first evidence of C. burnetii exposure in free-living European wildcats.

Effect of shortening the prefreezing equilibration time with glycerol on the quality of chamois (Rupicapra pyrenaica), ibex (Capra pyrenaica), mouflon (Ovis musimon) and aoudad (Ammotragus lervia) ejaculates.

The present study reports the effect of shortening the prefreezing equilibration time with glycerol on the quality of frozen-thawed ejaculated sperm from four Mediterranean mountain ungulates: Cantabrian chamois (Rupicapra pyrenaica), Iberian ibex (Capra pyrenaica), mouflon (Ovis musimon) and aoudad (Ammotragus lervia). Ejaculated sperm from these species were divided into two aliquots. One was diluted with either a Tris-citric acid-glucose based medium (TCG-glycerol; for chamois and ibex sperm) or a Tris-TES-glucose-based medium (TTG-glycerol; for mouflon and aoudad sperm), and maintained at 5°C for 3h prior to freezing. The other aliquot was diluted with either TCG (chamois and ibex sperm) or TTG (mouflon and aoudad sperm) and maintained at 5°C for 1h before adding glycerol (final concentration 5%). After a 15min equilibration period in the presence of glycerol, the samples were frozen. For the ibex, there was enhanced (P<0.05) sperm viability and acrosome integrity after the 3h as compared with the 15min equilibration time. For the chamois, subjective sperm motility and cell membrane functional integrity were less (P<0.05) following 15min of equilibration. In the mouflon, progressive sperm motility and acrosome integrity was less (P<0.05) when the equilibration time was reduced to 15min. For the aoudad, the majority of sperm variables measured were more desirable after the 3h equilibration time. The freezing-thawing processes reduced the sperm head size in all the species studied; however, the equilibration time further affected the frozen-thawed sperm head variables in a species-dependent fashion. While the equilibration time for chamois sperm might be shortened, this appears not to be the case for all ungulates.

Experimental studies of microbial populations and incidence of zoonotic pathogens in the faeces of red deer (Cervus elaphus).

UNLABELLED: Wild animals can serve as hosts, amplifiers or reservoirs for various zoonotic diseases. Most species of deer in highly fragmented agricultural landscapes, search out maximum cover from intrusive human activity. Hence, the likelihood of zoonosis transmission is likely to increase the more humans and wildlife interact. In our study, we conducted a comparative analysis of bacteria isolated from the faeces of red deer (Cervus elaphus) living in their natural environment in south-western Poland and brought in from Hungary and Slovakia under a species reintroduction programme. The faecal bacterial flora from 120 specimens of deer were examined, with particular attention to potentially pathogenic agents. We isolated 458 micro-organisms, of which 13 (2·84%) were identified as EHEC (Enterohaemorrhagic Escherichia coli) strains, and of these one strain, produced the Shiga toxin. No strain was identified as having ESBL (Extended-Spectrum Beta-Lactamase) resistance. Other bacteria that are important in terms of the health of humans and animals included Yersinia enterocolitica (4, 0·67%) and Staphylococcus aureus (4, 0·67%), but without methicillin resistance, and Listeria monocytogenes (8, 1·75%). Of all the micro-organisms 138 (30·13%) were bacteria of the genus Enterococcus, including 12 (2·62%) of the species Enterococcus faecium. The results of the study indicate that red deer may play an important role in the environmental maintenance of zoonotic pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: A particularly important factor in the epidemiology of bacterial infections is the introduction of pathogens posing a risk to other animals and humans into the soil, plants and especially water, as contaminants together with faeces. Our study presents screening of potentially pathogenic bacteria in different populations of deer that were displaced under reintroduction programmes. Based on our own research and the literature data, it seems that wild ruminants play an important role in the maintenance of zoonotic pathogens and information about zoonoses from red deer will become increasingly important as deer populations continue to grow, especially in Europe.