The Genetic Landscape of Antimicrobial Resistance Genes in Enterococcus cecorum Broiler Isolates.

Enterococcus cecorum is associated with bacterial chondronecrosis with osteomyelitis (BCO) in broilers. Prophylactic treatment with antimicrobials is common in the poultry industry, and, in the case of outbreaks, antimicrobial treatment is needed. In this study, the minimum inhibitory concentrations (MICs) and epidemiological cutoff (ECOFF) values (COWT) for ten antimicrobials were determined in a collection of E. cecorum strains. Whole-genome sequencing data were analyzed for a selection of these E. cecorum strains to identify resistance determinants involved in the observed phenotypes. Wild-type and non-wild-type isolates were observed for the investigated antimicrobial agents. Several antimicrobial resistance genes (ARGs) were detected in the isolates, linking phenotypes with genotypes for the resistance to vancomycin, tetracycline, lincomycin, spectinomycin, and tylosin. These detected resistance genes were located on mobile genetic elements (MGEs). Point mutations were found in isolates with a non-wild-type phenotype for enrofloxacin and ampicillin/ceftiofur. Isolates showing non-wild-type phenotypes for enrofloxacin had point mutations within the GyrA, GyrB, and ParC proteins, while five amino acid changes in penicillin-binding proteins (PBP2x superfamily) were observed in non-wild-type phenotypes for the tested ß-lactam antimicrobials. This study is one of the first that describes the genetic landscape of ARGs within MGEs in E. cecorum, in association with phenotypical resistance determination.

Bayesian evaluation of sensitivity and specificity of blood culture media and hypoglycemia in sepsis-suspected calves.

BACKGROUND: Sepsis is a life-threatening condition for which critically important antimicrobials are often indicated. The value of blood culture for sepsis is indisputable, but appropriate guidelines on sampling and interpretation are currently lacking in cattle. OBJECTIVE: Compare the diagnostic accuracy of 2 blood culture media (pediatric plus [PP] and plus aerobic [PA]) and hypoglycemia for bacteremia detection. Estimate the contamination risk of blood cultures in critically ill calves. ANIMALS: One hundred twenty-six critically ill calves, 0 to 114 days. METHODS: Retrospective cross-sectional study in which the performance of PP, PA and hypoglycemia to diagnose sepsis was assessed using a Bayesian latent class model. A Cox proportional hazards model was used to compare time to positivity (TTP). Potential contamination was descriptively analyzed. Isolates were considered relevant when they were; member of the Enterobacterales, isolated from both blood cultures vials, or well-known, significant bovine pathogens. RESULTS: The sensitivities for PP, PA, and hypoglycemia were higher when excluding assumed contaminants; 68.7% (95% credibility interval = 30.5%-93.7%), 87.5% (47.0%-99.5%), and 61.3% (49.7%-72.4%), respectively. Specificity was estimated at 95.1% (82.2%-99.7%), 94.2% (80.7%-99.7%), and 72.4% (64.6%-79.6%), respectively. Out of 121 interpretable samples, 14.9% grew a presumed contaminant in PA, PP, or both. There was no significant difference in the TTP between PA and PP. CONCLUSIONS AND CLINICAL IMPORTANCE: PA and PP appear to outperform hypoglycemia as diagnostic tests for sepsis. PA seems most sensitive, but a larger sample size is required to verify this. Accuracy increased greatly after excluding assumed contaminants. The type of culture did not influence TTP or the contamination rate.

Antimicrobial Resistance in Escherichia coli and Its Correlation with Antimicrobial Use on Commercial Poultry Farms in Bangladesh.

Antimicrobial resistance is a global concern, posing risks to human and animal health. This research quantified antimicrobial resistance (AMR) in E. coli isolates from poultry fecal and environmental samples in Bangladesh and explored their association with antimicrobial use (AMU). We screened 725 fecal and 250 environmental samples from 94 conventional broilers and 51 Sonali farms for E. coli presence using MALDI-TOF mass spectrometry. AMU data were collected at flock levels, expressed as treatment incidence (TI), while minimum inhibitory concentrations (MIC) for 14 antibiotics were determined on five fecal E. coli isolates per farm and on all environmental isolates. MIC results were interpreted using human clinical breakpoints and EUCAST epidemiological cut-off values (ECOFFs). Acquired resistance against commonly used antimicrobial agents such as ciprofloxacin, tetracycline and ampicillin, was extremely high and predominantly clinically relevant. There was a moderate correlation between fecal and environmental antibiotic resistance index (ARI), but there was no significant correlation between AMU and AMR, suggesting that the observed AMR prevalence is unrelated to current AMU in poultry, but may be due to high historical AMU. A high level of multidrug resistance, including against critically important antimicrobials, was found in both farm types. Therefore, an AMR/AMU surveillance program is urgently needed in the poultry production sector of Bangladesh.

The Diversity and Zoonotic Potential of Staphylococcus pseudintermedius in Humans and Pet Dogs in Trinidad and Tobago.

Staphylococcus pseudintermedius is an opportunistic pathogen that is frequently isolated from canines. It is of escalating interest because of its increasing antimicrobial resistance and zoonotic potential. Although many published articles are available that describe isolates obtained from diseased dogs and humans, this study focused on isolates obtained from healthy dogs and their owners who presented at clinics for routine veterinary care and utilized whole genome sequencing-based analyses for strain comparisons. A total of 25 humans and 27 canines were sampled at multiple sites, yielding 47 and 45 isolates, respectively. Whole genome sequence analysis was performed. We detected mostly new sequence types (STs) and a high diversity. Strains carried few antimicrobial resistance genes and plasmids, albeit three MRSP strains were found that belonged to two internationally distributed STs. The virulence content did not provide insights toward a tendency to colonization of humans but supported that there may be differences in the surface proteins between carrier strains and those causing pyoderma. We identified 13 cases in which humans were infected with strains from the dog they owned.

Quantitative and qualitative analysis of antimicrobial usage and biosecurity on broiler and Sonali farms in Bangladesh.

Antimicrobial use (AMU) is a major contributing factor to the rising threat of antimicrobial resistance (AMR) in human and animals. To control AMR, indiscriminate antibiotic use needs to be restricted, preventive measures such as biosecurity must be prioritized and proper action plans must be implemented. This study aimed to quantify and associate AMU and biosecurity status of broiler and Sonali farms in Bangladesh. Data on all antimicrobial treatments administered during a batch production cycle and antimicrobials purchased over a year were collected from 94 conventional broiler and 51 Sonali (cross-breed) farms from the northern and southeast regions of Bangladesh. Flock-level AMU was quantified using Treatment Incidence (TI) per 100 days based on the Defined Daily Dose (TIDDDvet) expressing the number of days per 100 animal-days at risk that the flock receives a standard dose of antimicrobials. The biosecurity status (external and internal) of these farms was assessed by means of the Biocheck.UGent scoring system and the correlation between biosecurity and TIDDDvet were assessed by Pearson's correlation coefficients. Median flock TIDDDvet was 60 and 58 for broilers and Sonali flocks, indicating that the birds were treated around 60% and 58% of their lifetime with an antimicrobial dose, respectively. Minimum and maximum values of TIDDDvet ranged from 18 -188 and 31-212 in broilers and Sonali, respectively. Fluoroquinolones, sulfonamides, tetracyclines and aminopenicillins were the most frequently used antimicrobial classes. The mean external and internal biosecurity scores were 39% and 61% for broilers and 44% and 61% for Sonali, respectively. There was a statistically significant difference in the external biosecurity score in broiler farms in the two regions (p ≤ 0.001), whereas, the internal biosecurity score was borderline not statistically significantly different (p = 0.065). The biosecurity score was negatively correlated with AMU in broiler and sonali farms both for external (R2 =-0.38; -0.36) and internal biosecurity (R2 =-0.33; -0.32), respectively. As most of the farmers treated their birds with antimicrobials for a very large part of the production, it could be concluded that there is a high overuse of antimicrobials both in broiler and Sonali poultry production in Bangladesh. This study also highlighted a low level of farm biosecurity practices. Overuse of antimicrobials and low level of biosecurity practice may be due to a lack of knowledge, ignorance, avoid loss and/or lack of monitoring by governmental agencies. Therefore, urgent action is required to increase awareness and biosecurity levels and to reduce AMU in these production systems.

Prevalence and Molecular Characterization of Methicillin-Resistant Staphylococci (MRS) and Mammaliicocci (MRM) in Dromedary Camels from Algeria: First Detection of SCCmec-mecC Hybrid in Methicillin-Resistant Mammaliicoccus lentus.

Dromedary camels are an important source of food and income in many countries. However, it has been largely overlooked that they can also transmit antibiotic-resistant bacteria. The aim of this study was to identify the Staphylococcaceae bacteria composition of the nasal flora in dromedary camels and evaluate the presence of methicillin-resistant Mammaliicoccus (MRM) and methicillin-resistant Staphylococcus (MRS) in dromedary camels in Algeria. Nasal swabs were collected from 46 camels from seven farms located in two different regions of Algeria (M'sila and Ouargla). We used non-selective media to determine the nasal flora, and antibiotic-supplemented media to isolate MRS and MRM. The staphylococcal isolates were identified using an Autoflex Biotyper Mass Spectrometer (MALDI-TOF MS). The mecA and mecC genes were detected by PCR. Methicillin-resistant strains were further analysed by long-read whole genome sequencing (WGS). Thirteen known Staphylococcus and Mammaliicoccus species were identified in the nasal flora, of which half (49.2%) were coagulase-positive staphylococci. The results showed that four out of seven farms were positive for MRS and/or MRM, with a total of 16 isolates from 13 dromedary camels. The predominant species were M. lentus, S. epidermidis, and S. aureus. Three methicillin-resistant S. aureus (MRSA) were found to be ST6 and spa type t304. Among methicillin-resistant S. epidermidis (MRSE), ST61 was the predominant ST identified. Phylogenetic analysis showed clonal relatedness among M. lentus strains, while S. epidermidis strains were not closely related. Resistance genes were detected, including mecA, mecC, ermB, tet(K), and blaZ. An SCCmec type VIII element was found in a methicillin-resistant S. hominis (MRSH) belonging to the ST1 strain. An SCCmec-mecC hybrid element was detected in M. lentus, similar to what was previously detected in M. sciuri. This study highlights that dromedary camels may be a reservoir for MRS and MRM, and that they contain a specific set of SCCmec elements. This emphasizes the need for further research in this ecological niche from a One Health perspective.

Whole genome sequencing to study antimicrobial resistance and RTX virulence genes in equine Actinobacillus isolates.

Actinobacillus equuli is mostly associated with disease in horses and is most widely known as the causative agent of sleepy foal disease. Even though existing phenotypic tools such as biochemical tests, 16S rRNA gene sequencing, and Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) can be used to identify members of the Actinobacillus genus, these methods struggle to differentiate between certain species and do not allow strain, virulence, and antimicrobial susceptibility typing. Hence, we performed in-depth analysis of 24 equine Actinobacillus isolates using phenotypic identification and susceptibility testing on the one hand, and long-read nanopore whole genome sequencing on the other hand. This allowed to address strain divergence down to the whole genome single nucleotide polymorphism (SNP) level. While lowest resolution was observed for 16S rRNA gene classification, a new multi-locus sequence typing (MLST) scheme allowed proper classification up to the species level. Nevertheless, a SNP-level analysis was required to distinguish A. equuli subspecies equuli and haemolyticus. Our data provided first WGS data on Actinobacillus genomospecies 1, Actinobacillus genomospecies 2, and A. arthritidis, which allowed the identification of a new Actinobacillus genomospecies 1 field isolate. Also, in-depth characterization of RTX virulence genes provided information on the distribution, completeness, and potential complementary nature of the RTX gene operons within the Actinobacillus genus. Even though overall low prevalence of acquired resistance was observed, two plasmids were identified conferring resistance to penicillin-ampicillin-amoxicillin and chloramphenicol in one A. equuli strain. In conclusion our data delivered new insights in the use of long-read WGS in high resolution identification, virulence gene typing, and antimicrobial resistance (AMR) of equine Actinobacillus species.

Long-term follow-up of Mycoplasma hyopneumoniae-specific immunity in vaccinated pigs.

Mycoplasma hyopneumoniae is the primary agent of enzootic pneumonia in pigs. To minimize the economic losses caused by this disease, M. hyopneumoniae vaccination is commonly practiced. However, the persistence of M. hyopneumoniae vaccine-induced immunity, especially the cell-mediated immunity, till the moment of slaughter has not been investigated yet. Therefore, on two commercial farms, 25 pigs (n = 50) received a commercial bacterin intramuscularly at 16 days of age. Each month, the presence of M. hyopneumoniae-specific serum antibodies was analyzed and the proliferation of and TNF-α, IFN-γ and IL-17A production by different T cell subsets in blood was assessed using recall assays. Natural infection with M. hyopneumoniae was assumed in both farms. However, the studied pigs remained M. hyopneumoniae negative for almost the entire trial. Seroconversion was not observed after vaccination and all pigs became seronegative at two months of age. The kinetics of the T cell subset frequencies was similar on both farms. Mycoplasma hyopneumoniae-specific cytokine-producing CD4+CD8+ T cells were found in blood of pigs from both farms at one month of age but decreased significantly with increasing age. On the other hand, T cell proliferation after in vitro M. hyopneumoniae stimulation was observed until the end of the fattening period. Furthermore, differences in humoral and cell-mediated immune responses after M. hyopneumoniae vaccination were not seen between pigs with and without maternally derived antibodies. This study documents the long-term M. hyopneumoniae vaccine-induced immune responses in fattening pigs under field conditions. Further research is warranted to investigate the influence of a natural infection on these responses.

Predictive Power of Long-Read Whole-Genome Sequencing for Rapid Diagnostics of Multidrug-Resistant Brachyspira hyodysenteriae Strains.

Infections with Brachyspira hyodysenteriae, the etiological agent of swine dysentery, result in major economic losses in the pig industry worldwide. Even though microbial differentiation of various Brachyspira species can be obtained via PCR, no quick diagnostics for antimicrobial susceptibility testing are in place, which is mainly due to the time-consuming (4 to 7 days) anaerobic growth requirements of these organisms. Veterinarians often rely on a clinical diagnosis for initiating antimicrobial treatment. These treatments are not always effective, which may be due to high levels of acquired resistance in B. hyodysenteriae field isolates. By using long-read-only whole-genome sequencing and a custom-trained Bonito base-calling model, 81 complete B. hyodysenteriae genomes with median Q51 scores and 99% completeness were obtained from 86 field strains. This allowed the assessment of the predictive potential of genetic markers in relation to the observed acquired resistance phenotypes obtained via agar dilution susceptibility testing. Multidrug resistance was observed in 77% and 21% of the tested strains based on epidemiological cutoff and clinical breakpoint values, respectively. The predictive power of genetic hallmarks (genes and/or gene mutations) for antimicrobial susceptibility testing was promising. Sensitivity and specificity for tiamulin [tva(A) and 50SL3N148S, 99% and 67%], valnemulin [tva(A), 97% and 92%), lincomycin (23SA2153T/G and lnuC, 94% and 100%), tylvalosin (23SA2153T/G, 99% and 93%), and doxycycline (16SG1026C, 93% and 87%) were determined. The predictive power of these genetic hallmarks is promising for use in sequencing-based workflows to speed up swine dysentery diagnostics in veterinary medicine and determine proper antimicrobial use. IMPORTANCE Diagnostics for swine dysentery rely on the identification of Brachyspira species using molecular techniques. Nevertheless, no quick diagnostic tools are available for antimicrobial susceptibility testing due to extended growth requirements (7 to 14 days). To enable practitioners to tailor antimicrobial treatment to specific strains, long-read sequencing-based methods are expected to lead to rapid methods in the future. Nevertheless, their potential implementation should be validated extensively. This mainly implies assessing sequencing accuracy and the predictive power of genetic hallmarks in relation to their observed (multi)resistance phenotypes.

Sepsis and survival in critically ill calves: Risk factors and antimicrobial use.

BACKGROUND: Sepsis is a life-threatening disease for which critically important antimicrobials (CIA) frequently are used. Diagnostic and therapeutic guidelines for sepsis and critically ill calves are largely lacking. OBJECTIVES: Identify factors associated with mortality in critically ill calves and describe bacteria obtained from blood cultures of critically ill calves with sepsis and their antimicrobial resistance. ANIMALS: Two-hundred thirty critically ill calves, mainly Belgian Blue beef cattle. METHODS: Retrospective cohort study. Logistic regression, survival analysis, and decision tree analysis were used to determine factors associated with mortality. RESULTS: Of the critically ill calves, 34.3% had sepsis and 61.3% died. The final survival model indicated that calves with sepsis (hazard risk [HR]: 1.6; 95% confidence interval [CI]: 1.0-2.5; P = .05), abnormal behavior (HR: 2.3; 95% CI: 1.3-4.0; P = .005), and hypothermia (HR: 0.82; 95% CI: 0.72-0.95; P = .01) had a significantly higher mortality risk. In a second survival model, hypothermia (HR: 0.87; 95% CI: 0.78-0.96; P = .004) and hypoglycemia (HR: 2.2; 95% CI: 1.5-3.3; P < .001) were risk factors for mortality. Decision tree analysis emphasized the importance of behavior, hypochloremia, hypoglycemia, hyperkalemia, and lung ultrasonography for mortality risk. Escherichia coli (30.6%) was most frequently isolated from blood cultures, of which 90.9% were multidrug resistant. Inappropriate use of antimicrobials was frequent for penicillin, amoxicillin, and sulfamethoxazole/trimethoprim, but less for CIA. CONCLUSIONS AND CLINICAL IMPORTANCE: Many critically ill calves have sepsis, which increases mortality risk. Bacteria involved are often resistant to first-intention antimicrobials but less resistant to CIA. The other identified risk factors for mortality can support therapeutic decision-making.

Dynamics of subclinical pneumonia in male dairy calves in relation to antimicrobial therapy and production outcomes.

Quick thoracic ultrasonography (qTUS) is increasingly used as an on-farm method to diagnose clinical and subclinical pneumonia in dairy calves. The primary objective of this prospective cohort study was to describe dynamics of lung consolidation in a purchase-dependent production system for male dairy calves in relation to antimicrobial therapy and respiratory diagnostics. In addition, we studied the association of cured and uncured pneumonia with average daily gain (ADG) and cold carcass weight (CCW). The third objective was to determine the effects of arriving with lung consolidation on the probability of developing chronic unresponsive pneumonia and reduced performance. A total of 295 male dairy calves were intensively followed by qTUS and clinical scoring on 7 strategic occasions (wk 1, 2, 3, 4, 6, 8, and 12) during the production cycle. Of the calves, 17.6% (52/295) arrived with a lung consolidation ≥1 cm. At the first outbreak of respiratory disease (wk 1 after arrival), this incidence had risen to 30.8%. Initial therapy with tulathromycin and subsequently doxycycline appeared ineffective, resulting in a increase to 43.8% of calves having pneumonia in wk 4. At the start of the first outbreak (wk 1), the majority (86.8%) of the pneumonia cases were subclinical. At wk 4, the outbreak became more clinical, and treatment with amoxicillin resulted in a cure risk of 52.7%. Culture and nanopore sequencing diagnostics on nonendoscopic broncho-alveolar lavage (nBAL) samples identified bovine respiratory syncytial virus and Mycoplasma bovis as the dominant agents in the first outbreak. The isolated M. bovis strain showed mutations associated with macrolide resistance. The second outbreak was characterized by a Pasteurella multocida superinfection and isolation of multiple M. bovis strains from nBAL diagnostic testing. Evaluated over the complete observation period, 83.4% of the calves developed consolidations ≥1 cm on qTUS. Of these calves, 53.9% (135/246) were cured by antimicrobial therapy. Chronic pneumonia (≥30 subsequent days of pneumonia) was seen in 13.9% of the animals (n = 41). Calves with uncured or chronic pneumonia had a lower ADG (992 ± 174 g/d and 930 ± 146 g/d, respectively) compared with calves that never developed pneumonia (ADG = 1,103 ± 156 g/d). In contrast, calves that did fully cure trended toward a lower ADG than calves that never developed pneumonia, but differences were no longer significant. Also, the effect of uncured pneumonia was no longer significant for CCW. Calves with lung consolidation upon arrival had a lower ADG (981 ± 159 g/d vs. 1,045 ± 159 g/d) and were more likely to develop chronic pneumonia [odds ratio = 4.2; 95% confidence interval = 2.1-8.6] compared with calves without consolidation upon arrival. Animals with chronic pneumonia, in turn, had a lower CCW than animals without chronic pneumonia (10.3 ± 4.4 kg; 95% confidence interval: 1.6-19.1 kg). This study documents the consequences of subclinical pneumonia upon arrival and pneumonia developed later in the production cycle on production outcomes in a veal calf setting. Both qTUS and nBAL diagnostics provide important information, offering potential for better control and prevention of bovine respiratory disease in dairy calves.

Different local, innate and adaptive immune responses are induced by two commercial Mycoplasma hyopneumoniae bacterins and an adjuvant alone.

Introduction: Enzootic pneumonia still causes major economic losses to the intensive pig production. Vaccination against its primary pathogen, Mycoplasma hyopneumoniae, is carried out worldwide to control the disease and minimize clinical signs and performance losses. Nonetheless, the effects of both infection with, and vaccination against Mycoplasma hyopneumoniae on the innate and adaptive immune responses remain largely unknown. Therefore, we conducted a study in which piglets were injected once with a commercial bacterin V1 or V2, or the adjuvant of V1 (A) to investigate their effect on local, innate and adaptive immune responses. Methods: Three weeks after vaccination, piglets were challenge infected with M. hyopneumoniae and euthanized four weeks later to assess vaccine efficacy via macroscopic and microscopic evaluation of lung lesions. Blood and broncho-alveolar lavage fluid (BAL) samples were collected to measure antibody responses, cellular immunity, BAL cytokine levels and BAL M. hyopneumoniae DNA load as well as cytokine secretion by monocytes. Results: After vaccination, proliferation of antigen-specific CD3+ T cells and a higher percentage of TNF-α+ CD8+, and TNF-α+ and TNF-α+IFN-γ+ CD4+CD8+ T cells was seen in V1, while proliferation of or a significant increase in cytokine production by different T cell subsets could not be observed for animals from V2. Interestingly, LPS-stimulated blood monocytes from V1 and A secreted less IL-10 on D7. After challenge, higher levels of IgA, more IL-10 and less IL-1ß was detected in BAL from V1, which was not observed in V2. Animals from A had significantly more IL-17A in BAL. The macroscopic lung lesion score and the M. hyopneumoniae DNA load at euthanasia was lower in V1, but the microscopic lung lesion score was lower in both vaccinated groups. Discussion: In conclusion, these results indicate that the two commercial bacterins induced different local and adaptive immune responses, that the adjuvant alone can reduce anti-inflammatory innate immune responses, and that both vaccines had a different efficacy to reduce Mycoplasma-like lung lesions and M. hyopneumoniae DNA load in the lung.

Zoonotic pathogens linked with hedgehog diphtheric disease.

Hedgehog diphtheric disease (HDD), an ulcerative skin disease with a high fatality rate, is an emerging threat to European hedgehogs (Erinaceus europaeus). We explored the potential role of a panel of zoonotic pathogens in the presumed multifactorial nature of HDD in 188 hedgehogs from 3 wildlife rescue centres in Belgium. As expected, and with a prevalence of 67% in 57 hedgehogs with skin lesions, characteristic of HDD, the occurrence of Corynebacterium ulcerans was strongly associated with the disease. Remarkably, with a prevalence of 42% in affected animals, infections with Borrelia burgdorferi sensu lato were 3.92 times more likely to be detected in HDD (95% confidence interval: 1.650-9.880; p = .0024). Overall, 40 hedgehogs tested positive for the B. burgdorferi sensu lato complex, including Borrelia afzelii (n = 30), Borrelia bavariensis (n = 7) and Borrelia spielmanii (n = 7). Other widely occurring pathogens included Salmonella (prevalence of 19%, with three pulsed-field gel electrophoresis profiles) and Leptospira sp. (prevalence of 11%, including Leptospira interrogans and Leptospira borgpetersenii), but these were not associated with the occurrence of HDD. These findings show that hedgehogs in Belgium represent a significant reservoir of multiple zoonotic bacteria, of which toxigenic C. ulcerans and B. burgdorferi sensu lato are associated with widespread hedgehog skin pathology and mortality.

Presence of Methicillin Resistant Staphylococcus aureus (MRSA) in Feces of the Small Indian Mongooses (Urva auropunctata) on Saint Kitts and Nevis, West Indies.

Although, historically, Methicillin-Resistant Staphylococcus aureus (MRSA) was restricted to humans, since 2005 these strains emerged in livestock and wildlife. Therefore, a One Health approach was applied to analyze the diversity and characteristics of S. aureus strains isolated from the invasive species of mongoose (Urva auropunctata) in St. Kitts. Fecal samples collected from these animals (n = 81) were cultured on selective agar. The isolated S. aureus strains were identified using MALDI-TOF and further characterized by whole genome sequence analysis. The fecal microbiome study identified the presence of S. aureus in 5 animals. Both MSSA (n = 3) and MRSA (n = 2) strains were identified. The two MRSA isolated were nearly identical ST5 SCCmec IVa (2B) strains. The two MSSA isolated were a new ST7434, pertaining to clonal complex 30, and the other belonged to ST5, but unrelated to the MRSA ST5. The SCCmec IVa (2B) is, however, the main SCCmec in human MRSA of different STs identified in St Kitts, indicating potential horizontal transmission events. In conclusion, a new type of MSSA, ST7434, was found and MRSA ST5 t002 SCCmec IVa (2B) found its way into wildlife on a small Caribbean Island. Further One Health studies are necessary to determine the role of MRSA in wildlife.

Influence of parity and reproductive stage on the prevalence of Mycoplasma hyopneumoniae in breeding animals in belgian farrow-to-finish pig herds.

BACKGROUND: Dam-to-piglet transmission plays an important role in the epidemiology of enzootic pneumonia on farms. Although Mycoplasma hyopneumoniae (M. hyopneumoniae) infections in breeding animals are often subclinical, their control could have a positive effect on M. hyopneumoniae infection levels in fattening pigs. This study investigated the presence of M. hyopneumoniae in the breeding population of ten Belgian farrow-to-finish farms suspected by the herd veterinarian to be M. hyopneumoniae infected. Gilt vaccination against M. hyopneumoniae prior to first insemination was practiced on nine of the ten farms. At four different time points in the reproductive cycle 20 animals were sampled on each farm, namely 30-40 days of gestation, 75-85 days of gestation, 3-5 days after farrowing, and 1-3 days after weaning. In total, tracheobronchial swabs and blood samples were collected from 344 gilts and 456 sows (n = 80/farm). Swabs were analysed for the presence of M. hyopneumoniae DNA using nested PCR and M. hyopneumoniae-specific antibodies were detected in serum with a commercial ELISA. Generalized linear mixed models with farm as random factor were used to test the effect of time point in the reproductive cycle and parity on M. hyopneumoniae PCR prevalence and seroprevalence. RESULTS: M. hyopneumoniae PCR prevalence ranged between 0% and 43.8% at the farm level and the seroprevalence between 32.5% and 93.8%. Gilts were significantly more M. hyopneumoniae PCR positive than sows at the 2-4th parity (P = 0.02) and > 4th parity (P = 0.02). At 30-40 days of gestation, significantly more breeding animals were PCR positive as compared to 75-85 days of gestation (P = 0.04), 3-5 days after farrowing (P = 0.02) and 1-3 days after weaning (P = 0.02). Gilts had significantly more often M. hyopneumoniae-specific antibodies than sows (P = 0.03). CONCLUSIONS: M. hyopneumoniae PCR prevalence varied a lot between farms and due to gilt vaccination the number of animals with M. hyopneumoniae-specific antibodies was high on most farms. Gilts were more often M. hyopneumoniae PCR positive than sows and positive animals were mostly found at 30-40 days of gestation. This emphasizes the importance of a sufficiently long quarantine period and proper gilt acclimation practices before introducing gilts to the sow herd.

Genome-Wide Association Study Reveals Genetic Markers for Antimicrobial Resistance in Mycoplasma bovis.

Mycoplasma bovis causes many health and welfare problems in cattle. Due to the absence of clear insights regarding transmission dynamics and the lack of a registered vaccine in Europe, control of an outbreak depends mainly on antimicrobial therapy. Unfortunately, antimicrobial susceptibility testing (AST) is usually not performed, because it is time-consuming and no standard protocol or clinical breakpoints are available. Fast identification of genetic markers associated with acquired resistance may at least partly resolve former issues. Therefore, the aims of this study were to implement a first genome-wide association study (GWAS) approach to identify genetic markers linked to antimicrobial resistance (AMR) in M. bovis using rapid long-read sequencing and to evaluate different epidemiological cutoff (ECOFF) thresholds. High-quality genomes of 100 M. bovis isolates were generated by Nanopore sequencing, and isolates were categorized as wild-type or non-wild-type isolates based on MIC testing results. Subsequently, a k-mer-based GWAS analysis was performed to link genotypes with phenotypes based on different ECOFF thresholds. This resulted in potential genetic markers for macrolides (gamithromycin and tylosin) (23S rRNA gene and 50S ribosomal unit) and enrofloxacin (GyrA and ParC). Also, for tilmicosin and the tetracyclines, previously described mutations in both 23S rRNA alleles and in one or both 16S rRNA alleles were observed. In addition, two new 16S rRNA mutations were possibly associated with gentamicin resistance. In conclusion, this study shows the potential of quick high-quality Nanopore sequencing and GWAS analysis in the evaluation of phenotypic ECOFF thresholds and the rapid identification of M. bovis strains with acquired resistance. IMPORTANCE Mycoplasma bovis is a leading cause of pneumonia but also causes other clinical signs in cattle. Since no effective vaccine is available, current M. bovis outbreak treatment relies primarily on the use of antimicrobials. However, M. bovis is naturally resistant to different antimicrobials, and acquired resistance against macrolides and fluoroquinolones is frequently described. Therefore, AST is important to provide appropriate and rapid antimicrobial treatment in the framework of AMR and to prevent the disease from spreading and/or becoming chronic. Unfortunately, phenotypic AST is time-consuming and, due to the lack of clinical breakpoints, the interpretation of AST in M. bovis is limited to the use of ECOFF values. Therefore, the objective of this study was to identify known and potentially new genetic markers linked to AMR phenotypes of M. bovis isolates, exploiting the power of a GWAS approach. For this, we used high-quality and complete Nanopore-sequenced M. bovis genomes of 100 isolates.

Evaluation of Nanopore Sequencing as a Diagnostic Tool for the Rapid Identification of Mycoplasma bovis from Individual and Pooled Respiratory Tract Samples.

Rapid identification of Mycoplasma bovis infections in cattle is a key factor to guide antimicrobial therapy and biosecurity measures. Recently, Nanopore sequencing became an affordable diagnostic tool for both clinically relevant viruses and bacteria, but the diagnostic accuracy for M. bovis identification is undocumented. Therefore, in this study Nanopore sequencing was compared to rapid identification of M. bovis with matrix-assisted laser desorption ionization-time of flight mass spectrometry (RIMM) and a triplex real-time PCR assay in a Bayesian latent class model (BLCM) for M. bovis in bronchoalveolar lavage fluid (BALf) samples obtained from calves. In practice, pooling of samples is often used to save money, but the influence on diagnostic accuracy has not been described for M. bovis. Therefore, a convenience sample of 17 pooled samples containing 5 individual BALf samples per farm was analyzed as well. The results for the pooled samples were compared with those for the individual samples to determine sensitivity and specificity. The BLCM showed good sensitivity (77.3% [95% credible interval, 57.8 to 92.8%]) and high specificity (97.4% [91.5 to 99.7%]) for Nanopore sequencing, compared to RIMM (sensitivity, 93.0% [76.8 to 99.5%]; specificity, 91.3% [82.5 to 97.0%]) and real-time PCR (sensitivity, 94.6% [89.7 to 97.7%]; specificity, 86.0% [76.1 to 93.6%]). Sensitivity and specificity of pooled analysis for M. bovis were 85.7% (95% confidence interval, 59.8 to 111.6%) and 90.0% (71.4 to 108.6%%), respectively, for Nanopore sequencing and 100% (100% to 100%) and 88.9% (68.4 to 109.4%) for RIMM. In conclusion, Nanopore sequencing is a rapid, reliable tool for the identification of M. bovis. To reduce costs and increase the chance of M. bovis identification, pooling of 5 samples for Nanopore sequencing and RIMM is possible.

Widespread Disease in Hedgehogs (Erinaceus europaeus) Caused by Toxigenic Corynebacterium ulcerans.

Toxin-producing Corynebacterium ulcerans, a causative agent of diphtheria in humans, was isolated from 53 hedgehogs in Belgium during the spring of 2020. Isolates showed low levels of acquired antimicrobial drug resistance. Strain diversity suggests emergence from an endemic situation. These findings stress the need for raising public awareness and improved wildlife disease surveillance.

Identification and Antimicrobial Resistance of Dermatophilus congolensis from Cattle in Saint Kitts and Nevis.

Dermatophilosis is a form of dermatitis caused by the bacterium Dermatophilus congolensis. The disease usually presents as localized purulent dermatitis, crusty hair masses or widespread matting of the hair. This condition is most common in domestic ruminants; but it can also affect other wild animals and humans. Antimicrobial therapy is used in many regions to treat clinical dermatophilosis with varying results. In this study, we aimed to assess the antimicrobial susceptibility of D. congolensis isolates. Fifty-two isolates were obtained from animals showing clinical signs of the disease at farms in St. Kitts. The isolates were then confirmed as D. congolensis by phenotypic tests, PCR and MALDI-TOF Mass Spectrometry. Furthermore, minimum inhibitory concentrations (MIC) of 16 antimicrobial agents were determined, using the broth microdilution method. Although most antimicrobials showed MICs in line with published values, the tetracycline results displayed a clear bimodal distribution over the tested range, with most isolates showing low MICs and 6 isolates much higher values (+/- 100-fold increase). These results indicate the presence of acquired tetracycline resistance in D. congolensis on the island of St. Kitts. Whether the current observation has implications for efficacy of treating the disease must be confirmed in further research.

Transfer of Mycoplasma hyopneumoniae-specific cell mediated immunity to neonatal piglets.

Mycoplasma hyopneumoniae is the primary agent of enzootic pneumonia in pigs. Although cell mediated immunity (CMI) may play a role in protection against M. hyopneumoniae, its transfer from sows to their offspring is poorly characterized. Therefore, maternally-derived CMI was studied in piglets from vaccinated and non-vaccinated sows. The potential influence of cross-fostering before colostrum ingestion on the transfer of CMI from dam to piglets was also investigated. Six M. hyopneumoniae vaccinated sows from an endemically infected herd and 47 of their piglets, of which 24 piglets were cross-fostered, were included, as well as three non-vaccinated control sows from an M. hyopneumoniae-free herd and 24 of their piglets. Vaccinated sows received a commercial bacterin intramuscularly at 6 and 3 weeks prior to farrowing. The TNF-α, IFN-γ and IL-17A production by different T-cell subsets in blood of sows, colostrum and blood of piglets was assessed using a recall assay. In blood of sows cytokine producing T-cells were increased upon M. hyopneumoniae vaccination. Similarly, M. hyopneumoniae-specific T-cells were detected in blood of 2-day-old piglets born from these vaccinated sows. In contrast, no M. hyopneumoniae-specific cytokine producing T-cells were found in blood of piglets from control sows. No difference was found in M. hyopneumoniae-specific CMI between cross-fostered and non-cross-fostered piglets. In conclusion, different M. hyopneumoniae-specific T-cell subsets are transferred from the sow to the offspring. Further studies are required to investigate the role of these transferred cells on immune responses in piglets and their potential protective effect against M. hyopneumoniae infections.