Parasites and zoonotic bacteria in the feces of cats and dogs from animal shelters in Carinthia, Austria

Due to their close associations with humans, dogs and cats can be important reservoirs for zoonotic pathogens. In the current study 200 fecal samples of dogs (n = 70 samples) and cats (n = 130 samples) from animal shelters in Carinthia, southern Austria, were examined for the presence of parasites (fecal flotation and larval migration assay) and selected bacteria. Overall, 17.1% of the canine and 38.5% of the feline samples were positive for parasites (p < 0.001), most commonly Giardia duodenalis (dogs and cats), including potentially zoonotic genotypes revealed by multilocus genotyping, and Toxocara cati (cats). Cryptosporidium (C. felis), Cystoisospora spp. (dogs and cats), hookworms (dog), Trichuris (dog) Capillaria hepatica (cats), taeniids (cat), and Aelurostrongylus abstrusus (cat) were also found. Zoonotic bacteria were detected in 10.5% of the samples, Salmonella enterica (dogs), Campylobacter jejuni (dogs and cats) and Yersinia enterocolitica (cat) and were significantly associated with parasite infections in cats but not in dogs. Samples that were positive for several pathogens were common; especially G. duodenalis and T. cati were frequently found in association with each other, other parasites or bacteria. The spectrum of detected pathogens is comparable to that of other dog and cat populations in central Europe. However, since animals from shelters are frequently rehomed, diagnostic measures, appropriate hygiene and therapy as well as training of shelter staff are recommended to prevent zoonotic transmission of enteropathogens to staff or new owners. The presence of heteroxenic parasites, i.e. Aelurostrongylus abstrusus and Taenia taeniaeformis, and spurious excretion of Ca. hepatica in cats, indicates that these animals preyed on intermediate hosts, and that biosafety measures in pet shelters need to be evaluated for their efficacy in the prevention of pathogen transmission

Sheep Infection Trials with ‘Phase-Locked’ Vpma Expression Variants of Mycoplasma agalactiae—Towards Elucidating the Role of a Multigene Family Encoding Variable Surface Lipoproteins in Infection and Disease

The significance of large multigene families causing high-frequency surface variations in mycoplasmas is not well-understood. Previously, VpmaY and VpmaU clonal variants of the Vpma family of lipoproteins of M. agalactiae were compared via experimental sheep infections using the two corresponding \u27Phase-Locked Mutants\u27. However, nothing is known about the infectivity of the remaining four Vpma expression variants VpmaX, VpmaW, VpmaZ and VpmaV as they were never evaluated in vivo. Here, in vivo infection and disease progression of all six Vpma expressers constituting the Vpma family of type strain PG2 were compared using the corresponding xer1-disrupted PLMs expressing single well-characterized Vpmas. Each of the six PLMs were separately evaluated using the intramammary sheep infection model along with the control phase-variable wildtype strain PG2. Thorough bacteriological, pathological and clinical examinations were performed, including assessment of milk quality, quantity and somatic cell counts. Altogether, the results indicated that the inability to vary the Vpma expression phase does not hamper the initiation of infection leading to mastitis for all six PLMs, except for PLMU, which showed a defect in host colonization and multiplication for the first 24 h p.i. and pathological/bacteriological analysis indicated a higher potential for systemic spread for PLMV and PLMX. This is the first study in which all isogenic expression variants of a large mycoplasma multigene family are tested in the natural host

Retrospective Analysis of the Detection of Pathogens Associated with the Porcine Respiratory Disease Complex in Routine Diagnostic Samples from Austrian Swine Stocks

The diagnostic workup of respiratory disease in pigs is complex due to coinfections and non-infectious causes. The detection of pathogens associated with respiratory disease is a pivotal part of the diagnostic workup for respiratory disease. We aimed to report how frequently certain viruses and bacteria were detected in samples from pigs with respiratory symptoms in the course of routine diagnostic procedures. Altogether, 1975 routine diagnostic samples from pigs in Austrian swine stocks between 2016 and 2021 were analysed. PCR was performed to detect various pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) (n = 921), influenza A virus (n = 479), porcine circovirus type 2 (PCV2) (n = 518), Mycoplasma (M.) hyopneumoniae (n = 713), Actinobacillus pleuropneumoniae (n = 198), Glaesserella (G.) parasuis (n = 165) and M. hyorhinis (n = 180). M. hyorhinis (55.1%) had the highest detection rate, followed by PCV2 (38.0%) and Streptococcus (S.) suis (30.6%). PRRSV was detected most frequently in a pool of lung, tonsil and tracheobronchial lymph node (36.2%). G. parasuis was isolated more frequently from samples taken after euthanasia compared to field samples. PRRSV-positive samples were more likely to be positive for PCV2 (p = 0.001), M. hyopneumoniae (p = 0.032) and Pasteurella multocida (p< 0.001). M. hyopneumoniae-positive samples were more likely to be positive for P. multocida (p< 0.001) and S. suis (p = 0.046), but less likely for M. hyorhinis (p = 0.004). In conclusion, our data provide evidence that lung samples that were positive for a primary pathogenic agent were more likely to be positive for a secondary pathogenic agent

Detection of Atypical Salmonella Infantis Phenotypes in Broiler Environmental Samples

In numerous countries, strict and targeted measures concerning Salmonella monitoring and control are implemented and high quality of surveillance is ensured by obligatory investigation of samples from the primary production level of animals according to EN/ISO standards. Here, 2 phenotypic characteristics of Salmonella exhibited on compulsory media are crucial, namely, motility demonstrated on modified semisolid Rappaport Vassiliadis agar (MSRV), and production of hydrogen sulfide (H2S) on xylose lysine deoxycholate agar (XLD). In the present study, we describe the detection of Salmonella Infantis variants found in broiler environmental samples with major alterations in their growth characteristics on MSRV, XLD, and brilliant green-phenol red-agar (BPLS). The variants proved to be non-motile on MSRV and displayed non-confirming colony appearances on the previously mentioned selective agars. The growth spectrum comprised pinhead sized yellow colonies with small black centers, but also pinpoint sized colorless colonies, both colony types of regular shape. Our work contributes to highlight the finding of S. Infantis variants which possess more than one phenotypic deviation from the "typical" growth characteristics and by this limit the detection power of the actual obligatory used media. IMPORTANCE Salmonellosis caused by non-typhoidal Salmonella serovars is the second most frequently reported zoonotic disease in humans in the EU. The transmission of these agents is mainly via contaminated food of animal origin. In this context, poultry products are the main source of infection. Therefore, continuous and standardized surveillance of the prevalence of such Salmonella serovars at the primary production level is essential. Our findings show the phenotypic heterogeneity of the serovar Infantis and provide growth characteristics of atypical variants. Such variants pass unnoticed official screening methods, resulting in incorrect identification and being underrepresented in epidemiological surveillance programs

Agreement between Clinical Assessment and Laboratory Diagnosis of Ringworm in Calves at Auction Markets

To limit the spread of bovine ringworm, control measures such as movement restrictions are highly recommended. In this context, calves at auction markets in Styria, Austria, displaying skin lesions characteristic for bovine ringworm, are excluded from the auctions. To investigate whether these clinical assessments correspond to laboratory diagnosis, a total of 166 samples taken from skin lesions assigned to the three clinical categories \u27ringworm very likely (v), likely (l) or unlikely (u)\u27 were mycologically examined using microscopy, culture, and nested PCR followed by amplicon sequencing. Further, the relationships of isolated dermatophytes were determined through multi-locus sequence typing (MLST). Overall, a high agreement between clinical assessment and laboratory results were observed with microscopy and nested PCR, providing more consistent results and molecular detection possessing an analytical sensitivity superior to that of cultural isolation (culture 21.7% vs. nested PCR 48.2%). Phylogenetic analyses revealed that most of the isolated dermatophytes belong to a unique Trichophyton verrucosum MLST genotype. In conclusion, clinical assessments were largely confirmed through laboratory diagnosis with nested PCR and sequencing, providing rapid, sensitive, and species-specific detection of dermatophytes in calves at auction markets displaying skin lesions typical for ringworm; this seems to be predominantly caused by a single Trichophyton verrucosum strain

Presence of Equine and Bovine Coronaviruses, Endoparasites, and Bacteria in Fecal Samples of Horses with Colic

Acute abdominal pain (colic) is one of the major equine health threats worldwide and often necessitates intensive veterinary medical care and surgical intervention. Equine coronavirus (ECoV) infections can cause colic in horses but are rarely considered as a differential diagnosis. To determine the frequency of otherwise undetected ECoV infections in horses with acute colic, fresh fecal samples of 105 horses with acute colic and 36 healthy control horses were screened for viruses belonging to the Betacoronavirus 1 species by RT-PCR as well as for gastrointestinal helminths and bacteria commonly associated with colic. Horses with colic excreted significantly fewer strongyle eggs than horses without colic. The prevalence of anaerobic, spore-forming, gram-positive bacteria (Clostridium perfringens and Clostridioides difficile) was significantly higher in the feces of horses with colic. Six horses with colic (5.7%) and one horse from the control group (2.8%) tested positive for Betacoronaviruses. Coronavirus-positive samples were sequenced to classify the virus by molecular phylogeny (N gene). Interestingly, in three out of six coronavirus-positive horses with colic, sequences closely related to bovine coronaviruses (BCoV) were found. The pathogenic potential of BCoV in horses remains unclear and warrants further investigation

Host cell interactions of novel antigenic membrane proteins of Mycoplasma agalactiae

Mycoplasma agalactiae is the main etiological agent of Contagious Agalactia syndrome of small ruminants notifiable to the World Organization for Animal Health. Despite serious economic losses, successful vaccines are unavailable, largely because its colonization and invasion factors are not well understood. This study evaluates the role of two recently identified antigenic proteins (MAG_1560, MAG_6130) and the cytadhesin P40 in pathogenicity related phenotypes.Adhesion to HeLa and sheep primary mammary stromal cells (MSC) was evaluated using ELISA, as well as in vitro adhesion assays on monolayer cell cultures. The results demonstrated MAG_6130 as a novel adhesin of M. agalactiae whose capacity to adhere to eukaryotic cells was significantly reduced by specific antiserum. Additionally, these proteins exhibited significant binding to plasminogen and extracellular matrix (ECM) proteins like lactoferrin, fibrinogen and fibronectin, a feature that could potentially support the pathogen in host colonization, tissue migration and immune evasion. Furthermore, these proteins played a detrimental role on the host cell proliferation and viability and were observed to activate pro-apoptotic genes indicating their involvement in cell death when eukaryotic cells were infected with M. agalactiae. To summarize, the hypothetical protein corresponding to MAG_6130 has not only been assigned novel adhesion functions but together with P40 it is demonstrated for the first time to bind to lactoferrin and ECM proteins thereby playing important roles in host colonization and pathogenicity

Predominant Single Stable VpmaV Expression in Strain GM139 and Major Differences with Mycoplasma agalactiae Type Strain PG2

Although mycoplasmas have a reduced genome and no cell wall, they have important mechanisms for the antigenic variation in surface lipoproteins that modulate their interactions with the host. Mycoplasma agalactiae, the main etiological agent of contagious agalactia, has a multigene family involved in the high-frequency phase variation in surface lipoproteins called variable proteins of M. agalactiae (Vpmas). The Vpma lipoproteins are involved in the immune evasion, colonization, dissemination, and persistence of M. agalactiae in the host. In this paper, we evaluate the Vpma phenotypic profiles of two different strains of M. agalactiae, namely, GM139 and the type strain PG2, to assess possible correlations between Vpma phase variability and the geographic localization, animal origin, and pathogenicity of these two strains. Using monospecific Vpma antibodies against individual Vpmas in immunoblots, we demonstrate that, unlike PG2, which expresses six Vpma proteins with high-frequency phase variation, colonies of GM139 predominantly express VpmaV and do not exhibit any sectoring phenotype for any Vpma. Since VpmaV is one of the most important Vpmas for cell adhesion and invasion, its predominant sole expression in GM139 without high-frequency variation may be the basis of the differential pathogenicity of GM139 and PG2. Additionally, MALDI-ToF MS analysis also demonstrates significant differences between these two strains and their relatedness with other M. agalactiae strains

Mycoplasma agalactiae Vaccines: Current Status, Hurdles, and Opportunities Due to Advances in Pathogenicity Studies

Contagious agalactia (CA) is a serious multietiological disease whose classic etiological agent is Mycoplasma agalactiae and which causes high morbidity and mortality rates in infected herds. CA is classified as a notifiable disease by the World Organization for Animal Health due to its significant worldwide economic impact on livestock, primarily involving goat and sheep farms. The emergence of atypical symptoms and strains of M. agalactiae in wildlife ungulates reestablishes its highly plastic genome and is also of great epidemiological significance. Antimicrobial therapy is the main form of control, although several factors, such as intrinsic antibiotic resistance and the selection of resistant strains, must be considered. Available vaccines are few and mostly inefficient. The virulence and pathogenicity mechanisms of M. agalactiae mainly rely on surface molecules that have direct contact with the host. Because of this, they are essential for the development of vaccines. This review highlights the currently available vaccines and their limitations and the development of new vaccine possibilities, especially considering the challenge of antigenic variation and dynamic genome in this microorganism

Diversity of Staphylococcus aureus associated with mastitis from dairy cows in Rwanda

The objective of the present study was to examine the diversity of Staphylococcus aureus from mastitis milk samples of cows in Rwanda.A total of 1080 quarter milk samples from 279 dairy cows were collected in 80 different farms from all five provinces of Rwanda. In total, 135 S. aureus isolates were obtained and subjected to genotyping (spa typing, DNA microarray, whole-genome sequencing (WGS)), antimicrobial susceptibility testing (AST) and phenotypic profiling by Fourier Transform Infrared (FTIR) spectroscopy (including capsular serotyping).Resistance to penicillin and/or tetracycline was most frequently observed. Ten sequence types (STs) (ST1, ST151, ST152, ST5477, ST700, ST7110, ST7983, ST7984, ST8320, ST97) belonging to seven clonal complexes (CCs) (CC1, CC130, CC152, CC3591, CC3666, CC705, CC97) were detected. The Panton-Valentine leukocidin (PVL) genes (lukF-PV/lukS-PV), the bovine leukocidin genes (lukM/lukF-P83) and the human and bovine toxic shock syndrome toxin gene tst-1 variants were detected. FTIR-based capsular serotyping showed CC-specific differences. Most CC97 (cap5 allele) isolates were primarily nonencapsulated (82%), whereas isolates of CC3591 and CC3666 (cap8 allele) were mostly encapsulated (86.4% and 57.8%, respectively). Our results underline the widespread global distribution of cattle-adapted CC97.The presence of CC3591 and CC3666 in bovine mastitis suggests an important role in cattle health and dairy production in Rwanda. The results of the present study support the need for a rigorous One-Health Surveillance program of the bovine-human interface