Exploring the Prevalence and Resistance of Campylobacter in Urban Bird Populations.

The increasing urbanization of ecosystems has had a significant impact on wildlife over the last few years. Species that find an unlimited supply of food and shelter in urban areas have thrived under human presence. Wild birds have been identified as amplifying hosts and reservoirs of Campylobacter worldwide, but the information about its transmission and epidemiology is still limited. This study assessed the prevalence of Campylobacter in 137 urban birds admitted at a wildlife rescue center, with 18.8% of individuals showing positive. C. jejuni was the most frequent species (82.6%), followed by C. coli and C. lari (4.3% each). The order Passeriformes (33.3%) showed significant higher presence of Campylobacter when compared to orders Columbiformes (0%) and Ciconiiformes (17.6%), as well as in samples collected during the summer season (31.9%), from omnivorous species (36.8%) and young individuals (26.8%). Globally, Campylobacter displayed a remarkable resistance to ciprofloxacin (70.6%), tetracycline (64.7%), and nalidixic acid (52.9%). In contrast, resistance to streptomycin was low (5.8%), and all the isolates showed susceptibility to erythromycin and gentamycin. The results underline the importance of urban birds as reservoirs of thermophilic antimicrobial-resistant Campylobacter and contribute to enhancing the knowledge of its distribution in urban and peri-urban ecosystems.

Oropharyngeal Trichomonosis Due to Trichomonas gypaetinii in a Cinereous Vulture ( Aegypius monachus) Fledgling in Spain.

A juvenile Cinereous Vulture ( Aegypius monachus) fledgling was found disorientated on the roof of a building in Madrid City, Spain, in October 2016. A veterinary examination revealed multiple plaques distributed throughout the oropharyngeal cavity. Lesions were located under the tongue and at the choanal slit, hard palate, and esophagus opening and ranged from 2 to 7 mm, coalescing in areas up to 2 cm, with a yellowish color of the surface. Motile trichomonad trophozoites were detected in fresh wet mount smears from the lesions. Sequence analysis of the internal transcribed spacer (ITS)1/5.8S/ITS2 and small subunit ribosomal RNA confirmed that Trichomonas gypaetinii was the etiologic agent. Microbiologic cultures did not reveal any pathogenic bacteria or fungi. The animal recovered successfully after treatment with metronidazole and trimethoprim-sulfamethoxazole and was later released in a suitable habitat. Avian trichomonosis lesions caused by T. gypaetinii have not been reported.

Colonization with Enterobacteriaceae-Producing ESBLs, AmpCs, and OXA-48 in Wild Avian Species, Spain 2015-2016.

Antibiotic resistance is a global threat of complex and changeable epidemiology. The role of wild birds in the dissemination of antibacterial resistance might be underestimated. We studied the cloacal colonization by cefotaxime-resistant Enterobacteriaceae in 668 wild birds in Spain. Eighty-eight wild birds (13.2%) of 28 species carried cefotaxime-resistant isolates; 58 of them (8.7%) carried extended-spectrum ß-lactamases (ESBLs) and 15 (2.5%) plasmid-mediated AmpCs of the blaCIT family. The 58 ESBLs belonged to the CTX-M-1 group (63.9%), CTX-M-9 group (23%), and SHV-group (13.1%). Pulsed field gel electrophoresis (PFGE) analysis of the Escherichia coli isolates revealed a high degree of genetic diversity since 44 different PFGE patterns were observed among the 54 cefotaxime-resistant isolates analyzed. Two clusters were detected with a genetic linkage >90%: Cluster 1 included nine CTX-M-15-producing isolates of ST23, and Cluster 2 included four isolates producing plasmid mediated AmpC of the CIT family of ST744. In addition, five birds were colonized by OXA-48- and CTX-M-15-producing isolates: three Klebsiella pneumoniae (isolated from Eurasian eagle-owl, lesser kestrel, and common buzzard), one E. coli (common buzzard), and one Enterobacter cloacae (cattle egret). Also, an mcr-1-positive and CIT-producing E. coli isolate colonized a black vulture. By multilocus sequence typing, the three OXA-48-producing K. pneumoniae isolates belonged to the high-risk human clones ST11 (two) and ST15 (one); the OXA-48-producing E. coli belonged to ST23, and the mcr-1-positive E. coli belonged to ST162. The diversity of eating patterns and migratory habits of the multiple avian species, capable of carrying multiresistant bacteria as observed in this study, may contribute to their global dissemination from human sources.

Impact of human-associated Escherichia coli clonal groups in Antarctic pinnipeds: presence of ST73, ST95, ST141 and ST131.

There is growing concern about the spreading of human microorganisms in relatively untouched ecosystems such as the Antarctic region. For this reason, three pinniped species (Leptonychotes weddellii, Mirounga leonina and Arctocephalus gazella) from the west coast of the Antartic Peninsula were analysed for the presence of Escherichia spp. with the recovery of 158 E. coli and three E. albertii isolates. From those, 23 harboured different eae variants (α1, ß1, ß2, ε1, θ1, κ, ο), including a bfpA-positive isolate (O49:H10-A-ST206, eae-k) classified as typical enteropathogenic E. coli. Noteworthy, 62 of the 158 E. coli isolates (39.2%) exhibited the ExPEC status and 27 (17.1%) belonged to sequence types (ST) frequently occurring among urinary/bacteremia ExPEC clones: ST12, ST73, ST95, ST131 and ST141. We found similarities >85% within the PFGE-macrorrestriction profiles of pinniped and human clinic O2:H6-B2-ST141 and O16:H5/O25b:H4-B2-ST131 isolates. The in silico analysis of ST131 Cplx genomes from the three pinnipeds (five O25:H4-ST131/PST43-fimH22-virotype D; one O16:H5-ST131/PST506-fimH41; one O25:H4-ST6252/PST9-fimH22-virotype D1) identified IncF and IncI1 plasmids and revealed high core-genome similarities between pinniped and human isolates (H22 and H41 subclones). This is the first study to demonstrate the worrisome presence of human-associated E. coli clonal groups, including ST131, in Antarctic pinnipeds.

Seroprevalence of Leptospirosis, Brucellosis, and Q Fever in a Wild Red Deer (Cervus elaphus) Population Kept in a Fenced Reserve in Absence of Contact with Livestock.

Wildlife health is of interest for public and animal health because wild animals have been identified as important sentinels for the surveillance for zoonotic pathogens. This work investigated Brucella spp., Coxiella burnetii, and Leptospira spp. infection seroprevalence in a free-ranging red deer population. The study was conducted in a fenced reserve with controlled hunting activity in central Spain with animals that did not have any contact with livestock. Sampling was performed at two time points before and 5 years after the implementation of new management measures, including a reduction in the red deer population in the reserve. In addition, the presence of Leptospira DNA was tested in placental and fetal samples from seropositive pregnant animals. Antibodies against Brucella and Coxiella were not detected in any sample. The seroprevalence of Leptospira was 9.4% (13/137) in the first sampling for serovars Canicola and Panama. Five years later, the prevalence rose to 38.5% (97/252) with Pomona, the only serovar detected. Animals older than 2 years (50%; 70/140) were more likely to be Pomona seropositive than animals ≤2 years old (25.2%; 27/107; p < 0.001). Leptospira DNA was not detected in any sample tested. In conclusion, wild red deer in this area without contact with livestock seem not to play an important role in Brucella spp. and C. burnetii maintenance. The high seroprevalence of Leptospira spp. serogroup Pomona could indicate a risk for people with narrow contact with these animals, but the carrier status was not assessed. Consequently, it is unknown if red deer would represent a risk for human infection. Considering that wild boar could be the source of infection to red deer, the role of wild boar in the spread of leptospirosis and the risk for human infection should be investigated.

Effect of Feed Form and Whole Grain Feeding on Gastrointestinal Weight and the Prevalence of Campylobacter jejuni in Broilers Orally Infected.

Two independent trials were carried out to evaluate the effect of feed form, whole wheat (WW) and oat hulls (OH) addition on gastrointestinal (GIT) weight and Campylobacter jejuni colonization in orally infected birds. In Trial 1, there were six treatments factorially arranged with two feed forms (mash vs pellets), and three levels of WW from 1-21/22-42d: 0/0, 7.5/15%, 15/30%. Broilers were allocated in cages (3 birds/cage, 12 cages/treatment). In Trial 2, there were three treatments: a mash diet, a mash diet including WW (7.5% from 1-21 and 15% from 22-42d), and a third treatment including also 5%OH. Broilers were allocated in floor pens (1 pen with 30 birds/treatment). At 14d, all broilers in Trial 1 or 3 broilers/pen in Trial 2 were orally challenged with 1.5 x 105 cfu of C. jejuni ST-45 /. In Trial 1, birds fed pelleted diets consumed 13.5% more feed, gained 31% more weight, and presented 12.9% better feed conversion for the whole trial (P<0.05). Pelleting decreased the relative weight of GIT and gizzard and increased the relative weight of proventriculus (P<0.05). Mash diets decreased pH in the gizzard (P<0.05). Inclusion of WW decreased the relative weight of proventriculus, increased gizzard weight, and reduced pH in the gizzard (P<0.05). At 21d of age, mash tended to reduce C. jejuni compared to pellets (7.85 vs 8.27 log10cfu/g; P = 0.091) and WW inclusion at 7.5/15% reduced C. jejuni colonization when compared to lower and higher inclusion (P<0.05). In Trial 2, birds fed T3 (WW+OH) showed 1.38 log10cfu/g less than birds fed Control diet (P<0.05). In conclusion, despite of the clear morphological changes in the GIT derived of FF and WW inclusion, no clear reductions in C. jejuni populations in the ceca were observed. However, WW and OH inclusion to mash diets significantly reduced cecal C. jejuni colonization at 42 days.

Fluorescent microbead-based immunoassay for anti-Erysipelothrix rhusiopathiae antibody detection in cetaceans.

A fluorescent microbead-based immunoassay (FMIA) for detection of anti-Erysipelothrix rhusiopathiae antibodies in pigs was adapted for use in cetaceans. The FMIA was validated and adjusted using serum samples from 10 vaccinated captive bottlenose dolphins Tursiops truncatus collected between 1 and 13 mo after immunization. The technique was then used to analyze specimens from 15 free-ranging cetaceans stranded alive on the Valencian Mediterranean coast between 2006 and 2014: 11 striped dolphins Stenella coeruleoalba, 3 Risso's dolphins Grampus griseus and 1 bottlenose dolphin Tursiops truncatus. One of these wild animals was confirmed to have died from E. rhusiopathiae septicemia, but no anti-E. rhusiopathiae antibodies were detected in its serum, pericardial fluid or milk samples. Another free-ranging individual, which lacked any signs or lesions that might be indicative of E. rhusiopathiae infection, showed high fluorescence intensity similar to that measured in captive dolphins at 6-13 mo after vaccination. These results suggest that this animal underwent an E. rhusiopathiae infection several months before stranding. The findings in the present study suggest that FMIA can be useful for detecting anti-E. rhusiopathiae antibodies in cetaceans, and its application to free-ranging animals is particularly interesting because of the great value of these specimens. Furthermore, the FMIA can be multiplexed to allow the determination of up to 100 analytes per sample in a single well, thereby reducing the cost, time and sample volume needed.

Isolation of a point mutation associated with altered expression of the CmeABC efflux pump in a multidrug-resistant Campylobacter jejuni population of poultry origin.

The objective of this study was to investigate the antibiotic resistance phenotype of Campylobacter jejuni isolates from a poultry flock of broiler production in Spain. Isolates were characterised by RFLP-PCR of the flaA gene and multilocus sequence typing. Minimum inhibitory concentrations of quinolones, aminoglycosides, ß-lactams, tetracyclines, phenicols, macrolides and lincosamides were determined by Etest. Determinants of resistance and the regulatory region of the cmeABC operon were investigated in all isolates by PCR detection and sequencing. Expression of the CmeABC efflux pump was investigated by quantitative RT-PCR and accumulation assay. Based on their molecular markers, two different populations of C. jejuni were identified: one resistant to quinolones, ß-lactams and tetracyclines, considered multidrug-resistant (MDR); and another resistant only to tetracyclines. Both populations possessed the tetO gene, previously associated with tetracycline resistance. The blaOXA-61 gene was also present in both populations, although only the MDR population showed ß-lactamase activity. In addition, MDR isolates possessed the Thr86Ile mutation in the gyrA gene responsible for quinolone resistance. Moreover, sequencing of the regulatory region of the cmeABC operon revealed the presence of the C-32→T mutation in the MDR isolates, which was accompanied by an increase in cmeA mRNA levels compared with the non-mutant population. In conclusion, this is the first report of the mutation C-32→T in the cmeABC operon in C. jejuni isolates of veterinary origin. This mutation is associated with overexpression of the CmeABC efflux pump in a MDR population and is possibly related to enhanced tolerance to antimicrobials that favours the development of resistance.

Health impact evaluation of alternative management systems in vicuña (Vicugna vicugna mensalis) populations in Peru.

To determine the impact of farming over vicuña population in Peru, serum samples were collected from 207 vicuñas (126 captive vicuñas and 81 free-ranging vicuñas) and 614 domestic South American camelids (571 alpacas and 43 llamas), in ten Andean communities at the Salinas y Aguada Blanca reserve, province of Arequipa, southern Peru. Samples were tested for the presence of leptospirosis, foot and mouth disease (FMD), bovine viral diarrhea (BVD), bovine herpesvirus type 1 (BHV-1), brucellosis, bluetongue disease (BT), paratuberculosis, and neosporosis. Serological results showed that 1.9% (4/207) of vicuñas, 18.6% (106/571) of alpacas, and 23.3% (10/43) of llamas were positive to one or more Leptospira serovars. One percent of vicuñas (2/207) and 2.4% of domestic camelids (15/614) had Neospora caninum antibodies tested by ELISA, but only two vicuñas and two alpacas were confirmed by Western blot. Epidemiological evaluation found an association of leptospirosis to sex and age (p < 0.001), with female subjects older than 2.5 years at higher risk of infection. Interestingly, antibodies against Leptospira serovars were only found in captive vicuñas. This is the first study where health status of free-ranging and captive vicuñas has been compared. Results indicate minimal to nil presence of FMD, BVD, BHV-1, brucellosis, BT, paratuberculosis, and neosporosis allied to health disorders in our sample. The detection of seropositive animals against Leptospira, however, unveils the likely significance of leptospirosis in wild and domestic South American camelids, the impact of mixed husbandry over vicuña population and the risk to human health.

Antimicrobial susceptibilities of Campylobacter jejuni and Campylobacter coli strains isolated from two early stages of poultry production.

Our aim was to monitor the resistance of Campylobacter isolates from two initial stages of broiler production in 5 grandparent breeder broiler farms (GPBFs) and 12 parent breeder broiler farms (PBFs) in which no antimicrobials were used during the study. Susceptibility tests were carried out for 805 strains (697 Campylobacter jejuni and 108 Campylobacter coli) against nalidixic acid, ciprofloxacin, erythromycin, amoxicillin, amoxicillin plus clavulanic acid, tetracycline, gentamicin, and chloramphenicol using the disk-diffusion method. Quinolone resistance was the most abundant overall (74.9%) and at each stage of production. The second largest resistance was for tetracycline with 48.2%. The resistance against amoxicillin plus clavulanic acid, gentamicin, and chloramphenicol was not found. The percentages of resistance and multidrug-resistant (MDR) isolates were always higher in the PBFs than in the GPBFs. However, pan-susceptible populations (total 10.3%) were isolated in our survey. C. coli isolates were more resistant to tetracycline and erythromycin (96.3% and 23.1%, respectively) than for C. jejuni (40.7% and 0%, respectively) and were more MDR (33.3% vs. 11.9%). In conclusion, as other authors have shown, even in the absence of antibiotic pressure, relatively high rates of quinolone resistance are found in Campylobacter. However, a decrease in quinolone resistance has been observed compared to other studies in Spain [i.e., 99%; Saenz et al. Antimicrob. Agents Chemother. 2000;44(2):267-271]. MDR, fluoroquinolone-, macrolide-, and tetracycline-resistant Campylobacter populations are issues of concern in public health.

Differences in the prevalence of Tritrichomonas foetus infection in beef cattle farmed under extensive conditions in northern Spain.

Bovine trichomonosis (BT) is a sexually transmitted disease of cattle caused by infection with Tritrichomonas foetus. In a recent study, T. foetus infection was detected in 41.5% of herds of an endangered beef breed, the Asturiana de la Montaña (AM), which is farmed under extensive, mountain pastoral systems in northern Spain. The objective of this study was to determine the prevalence of this pathogen in the more production-centred Asturiana de los Valles (AV) beef breed farmed in the same region, and to identify potential associated management risk factors. Infection was detected in a significantly smaller number (5.2%) of AV herds, despite the fact that both populations share the same ecological niche. Communal grazing was not identified as significant risk factor and study results suggest the prevalence of BT is likely to vary considerably depending on how the cattle are managed.

Detection of a novel genotype of Cryptosporidium in Antarctic pinnipeds.

A study was conducted to investigate the presence of Cryptosporidium and Giardia in Antarctic marine mammals. A total of 270 faecal samples from different species of pinnipeds from different locations in the South Shetland Islands and Antarctic Peninsula were analysed by immunofluorescence microscopy and PCR. Cryptosporidium was detected by PCR in three samples from Southern elephant seals (Mirounga leonina) and 2 Weddell seals (Leptonychotes weddellii). However, no oocysts were observed in any of the samples by immunofluorescence microscopy. Molecular characterisation of the isolates, using the 18S rDNA, the HSP70 and the COWP loci, revealed the presence of a Cryptosporidium sp., previously reported from an Antarctic Southern elephant seal, in the elephant seals and a novel genotype in Weddell seals. Giardia could not be detected in any of the samples analysed.

Detection of Toxoplasma gondii antibodies in Antarctic pinnipeds.

The presence of Toxoplasma gondii antibodies was investigated in Antarctic marine mammals. Two hundred and eleven sera from different species of pinnipeds collected in years 2007, 2010 and 2011 from different locations in the South Shetland Islands and Antarctic Peninsula were analysed using a commercially available agglutination test kit. The presence of antibodies (titres ≥ 1:25) against T. gondii was detected in a total of 28 animals (13.3%). Amongst animal species, percentages of detection were higher in Southern elephant seals (Mirounga leonina) (76.9%; 10/13) followed by Weddell seals (Leptonychotes weddellii) (41.9%; 13/31). Antibodies were also found in 4 of 165 (2.4%) Antarctic fur seals (Arctocephalus gazella) and 1 of 2 Crabeater seals (Lobodon carcinophaga). Highest titres (1:100-1:800) were also observed in Southern elephant seals and Weddell seals. To the best of our knowledge this is the first report on the detection of antibodies against T. gondii in Antarctic marine mammals.