Development of a new multiplex quantitative PCR for the detection of Glaesserella parasuis, Mycoplasma hyorhinis, and Mycoplasma hyosynoviae.

Glaesserella parasuis, Mycoplasma hyorhinis, and Mycoplasma hyosynoviae are important porcine pathogens responsible for polyserositis, polyarthritis, meningitis, pneumonia, and septicemia causing significant economic losses in the swine industry. A new multiplex quantitative polymerase chain reaction (qPCR) was designed on one hand for the detection of G. parasuis and the virulence marker vtaA to distinguish between highly virulent and non-virulent strains. On the other hand, fluorescent probes were established for the detection and identification of both M. hyorhinis and M. hyosynoviae targeting 16S ribosomal RNA genes. The development of the qPCR was based on reference strains of 15 known serovars of G. parasuis, as well as on the type strains M. hyorhinis ATCC 17981T and M. hyosynoviae NCTC 10167T . The new qPCR was further evaluated using 21 G. parasuis, 26 M. hyorhinis, and 3 M. hyosynoviae field isolates. Moreover, a pilot study including different clinical specimens of 42 diseased pigs was performed. The specificity of the assay was 100% without cross-reactivity or detection of other bacterial swine pathogens. The sensitivity of the new qPCR was demonstrated to be between 11-180 genome equivalents (GE) of DNA for M. hyosynoviae and M. hyorhinis, and 140-1200 GE for G. parasuis and vtaA. The cut-off threshold cycle was found to be at 35. The developed sensitive and specific qPCR assay has the potential to become a useful molecular tool, which could be implemented in veterinary diagnostic laboratories for the detection and identification of G. parasuis, its virulence marker vtaA, M. hyorhinis, and M. hyosynoviae.

Identification of Glaesserella parasuis and Differentiation of Its 15 Serovars Using High-Resolution Melting Assays.

Glaesserella parasuis is the etiological agent of Glässer's disease, which is associated with polyserositis and arthritis and has a significant impact on the economy of the pig production industry. For the optimal surveillance of this pathogen, as well as for the investigation of G. parasuis-associated diseases, it is crucial to identify G. parasuis at the serovar level. In this work, we designed and developed new high-resolution melting (HRM) approaches, namely, the species-specific GPS-HRM1 and two serovar-specific HRM assays (GPS-HRM2 and GPS-HRM3), and evaluated the sensitivity and specificity of the assays. The HRM assays demonstrated good sensitivity, with 12.5 fg-1.25 pg of input DNA for GPS-HRM1 and 125 fg-12.5 pg for GPS-HRM2 and GPS-HRM3, as well as a specificity of 100% for the identification of all recognized 15 G. parasuis serovars. Eighteen clinical isolates obtained between 2014 and 2022 in Switzerland were tested by applying the developed HRM assays, which revealed a heterogeneous distribution of serovars 2, 7, 4, 13, 1, and 14. The combination with virulence marker vtaA (virulence-associated trimeric autotransporters) allows for the prediction of potentially virulent strains. The assays are simple to execute and enable a reliable low-cost approach, thereby refining currently available diagnostic tools.

Development of a novel high resolution melting assay for identification and differentiation of all known 19 serovars of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory infectious disease responsible for global economic losses in the pig industry. From a monitoring perspective as well as due to the different courses of disease associated with the various serovars, it is essential to distinguish them in different herds or countries. In this study, we developed a novel high resolution melting (HRM) assay based on reference strains for each of the 19 known serovars and additional 15 clinical A. pleuropneumoniae isolates. The novel HRM comprises the species-specific APP-HRM1 and two serovar-specific HRM assays (APP-HRM2 and APP-HRM3). APP-HRM1 allowed polymerase chain reaction (PCR) amplification of apxIV resulting in an A. pleuropneumoniae specific melting curve, while nadV specific primers differentiated biovar 2 from biovar 1 isolates. Using APP-HRM2 and APP-HRM3, 13 A. pleuropneumoniae serovars can be determined by inspecting the assigned melting temperature. In contrast, serovar 3 and 14, serovar 9 and 11, and serovar 5 and 15 have partly overlapping melting temperatures and thus represent a challenge to accurately distinguish them. Consequently, to unambiguously ensure the correct assignment of the serovar, it is recommended to perform the serotyping HRM assay using a positive control for each serovar. This rapid and user-friendly assay showed high sensitivity with 1.25 fg-125 pg of input DNA and a specificity of 100% to identify A. pleuropneumoniae. Characteristic melting patterns of amplicons might allow detecting new serovars. The novel HRM assay has the potential to be implemented in diagnostic laboratories for better surveillance of this pathogen.

Draft Genome Sequences of Two Clinical Actinobacillus pleuropneumoniae Serotype 19 Strains from Pigs in Switzerland.

Actinobacillus pleuropneumoniae serotype 19 is a very recently described new serotype with a novel type II capsule synthesis locus. Here, we report the draft genome sequences of two Actinobacillus pleuropneumoniae serotype 19 strains with a serogroup 3/6/8/12/15 O-antigen locus that were isolated in 2018 and 2021 from two different pig farms in Switzerland.

Mycobacterium microti: Not Just a Coincidental Pathogen for Cats.

Public interest in animal tuberculosis is mainly focused on prevention and eradication of bovine tuberculosis in cattle and wildlife. In cattle, immunodiagnostic tests such as the tuberculin skin test or the interferon gamma (IFN-γ) assay have been established and are commercially available. Feline tuberculosis is rather unknown, and the available diagnostic tools are limited. However, infections with Mycobacterium tuberculosis complex members need to be considered an aetiological differential diagnosis in cats with granulomatous lymphadenopathy or skin nodules and, due to the zoonotic potential, a time-efficient and accurate diagnostic approach is required. The present study describes 11 independent cases of Mycobacterium microti infection in domestic cats in Switzerland. For three cases, clinical presentation, diagnostic imaging, bacteriological results, immunodiagnostic testing, and pathological features are reported. An adapted feline IFN-γ release assay was successfully applied in two cases and appears to be a promising tool for the ante mortem diagnosis of tuberculosis in cats. Direct contact with M. microti reservoir hosts was suspected to be the origin of infection in all three cases. However, there was no evidence of M. microti infection in 346 trapped wild mice from a presumptive endemic region. Therefore, the source and modalities of infection in cats in Switzerland remain to be further elucidated.

Population structure, genetic diversity and pathotypes of Streptococcus suis isolated during the last 13 years from diseased pigs in Switzerland.

Streptococcus (S.) suis is a globally important swine pathogen, which comprises certain zoonotic serotypes. In this study, a detailed characterization of 88 porcine S. suis isolates was performed by analyzing capsular (cps) types, multilocus sequence typing (MLST) and investigation of the minimum core genome (MCG). In order to focus on the virulence potential of presumable invasive disease-associated S. suis isolates, virulence-associated gene profiles were assessed followed by screening a chosen subset of S. suis strains with a molecular pathotyping tool. Results showed a high genetic variability within this strain collection. In total, seventeen cps types were identified with a predominance of cps type 9 (15.9%) and 6 (14.8%). MLST revealed 48 sequence types (STs) including 41 novel ones. The population structure of S. suis was heterogenous and isolates belonged to eight different clonal complexes (CCs) including CC28 (9.1%), CC1109 (8%), CC13/149 (6.8%), CC1237 (5.7%), CC1 (3.4%), CC17 (3.4%), CC87 (2.3%), and CC1112 (1.1%), whereas a significant portion of isolates (60.2%) could not be assigned to any described CCs. Virulence-associated markers, namely extracellular protein factor (epf), muramidase-released protein (mrp), and suilysin (sly), showed a link with STs rather than with cps types. With this study an expanded knowledge about the population structure and the genetic diversity of S. suis could be achieved, which helps to contribute to an optimal public health surveillance system by promoting a focus on strains with an increased virulence and zoonotic potential.

Mycobacterium nebraskense infection in a dog in Switzerland with disseminated skin lesions.

BACKGROUND: Cutaneous disseminated mycobacteriosis is rare in dogs. To the best of the authors' knowledge, the slowly growing mycobacterial species Mycobacterium nebraskense has not been described before in this species. OBJECTIVE: Description of clinical features, laboratory analyses and treatment regimen of this unusual case. ANIMAL: A 9-year-old female-spayed West Highland white terrier dog presented with progressive nodules and ulcerations on both sides of the thorax and the rostral aspect of the chest. METHODS AND MATERIALS: Investigations involved histopathological examination of skin biopsies (including special stains for fungi, bacteria and mycobacteria), standard and mycobacterial culture (including susceptibility testing), 16S/23S rRNA sequencing and BLAST similarity searching. RESULTS: Ziehl-Neelsen staining of decontaminated biopsy material revealed acid-fast bacteria morphologically consistent with mycobacteria. Treatment with clarithromycin and marbofloxacin achieved partial resolution. A change in the treatment regimen to pradofloxacin and azithromycin resulted in rapid deterioration of skin lesions. Final healing occurred with the addition of prednisolone at an anti-inflammatory dose. The results of mycobacterial culture and susceptibility testing were received 10 and 12 months, respectively, after the first presentation of the dog. Therapy was stopped after 16 months without recurrence of skin lesions. CONCLUSIONS AND CLINICAL IMPORTANCE: This case is noteworthy for the description of a new mycobacterial species contributing to disseminated panniculitis in a dog and for the difficulties experienced in the lengthy empirical treatment of slowly growing nontuberculous mycobacterial infections. The addition of prednisolone to induce complete healing raises the question of whether the mycobacterial infection was primary or whether it occurred secondarily to an ongoing sterile panniculitis.

Assessment of the Prevalence of Extended-Spectrum ß-Lactamase-Producing Enterobacteriaceae in Ready-to-Eat Salads, Fresh-Cut Fruit, and Sprouts from the Swiss Market.

Ready-to-eat (RTE) prepacked salads and fruit have been successfully marketed for the last decade in Switzerland and are increasingly important as a component of everyday diets. To determine whether extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae are present in RTE salads, fresh-cut fruit, and sprouts on the Swiss market, samples of 238 mixed and unmixed RTE produce from a large production plant and 23 sprout samples from two sprout farms were analyzed. Further, four samples from the production plant's recycled wash water, which is used for crop irrigation, were analyzed. Twelve (5%) of the 238 RTE products and one of the recycled wash water samples yielded ESBL-producing Enterobacteriaceae. Strain identification and PCR analysis of the blaESBL genes revealed Kluyvera ascorbata isolated from a tomato sample harboring a blaCTX-M-2-like gene; multidrug-resistant (MDR) Enterobacter cloacae detected in a chives sample imported from Spain harboring the clinically important bla(CTX-M-15) gene; and 10 Serratia spp. isolated from mixed salads (bla(FONA-2) and bla(FONA-2)-like genes were found in 6 [60%] and bla(FONA-4)-like and bla(FONA-5)-like genes were each found in 2 [20%] of the isolates). The recycled wash water sample tested positive for one extraintestinal pathogenic MDR Escherichia coli B2:ST131 harboring bla(CTX-M-27) and for one MDR E. coli A:ST88 containing bla(CTX-M-3). None of the sprout samples tested positive for ESBL-producing Enterobacteriaceae. Overall, the majority of the Enterobacteriaceae detected in Swiss RTE produce were environmental strains producing minor ESBLs. The detection of an isolate producing a clinically important ESBL in a single sample and of an international circulating pathogenic strain (B2:ST131) in recycled wash water highlights the importance of surveillance of fresh produce and of recycled wash water that will be reused for irrigation purposes.