Two high-risk clones of carbapenemase-producing Klebsiella pneumoniae that cause infections in pets and are present in the environment of a veterinary referral hospital.

OBJECTIVES: Infections with carbapenem-resistant Enterobacterales (CRE) are an emerging problem in pets and a major threat to public health. We determined the genetic relationships among carbapenemase-producing Klebsiella pneumoniae (CPKp) strains causing infections in hospitalized pets in a veterinary clinic and those found in the environment. METHODS: WGS was performed with both the Illumina and Nanopore platforms. Searches of genetic features were performed using several databases and bioinformatics tools, and phylogeny was assessed by whole-genome MLST (wgMLST) using SeqSphere and SNP calling with Snippy. RESULTS: WGS analysis of the CPKp strains identified all environmental and almost all animal strains as the high-risk clone ST11, with the exception of two strains that belonged to ST307. All CPKp belonged to novel complex types (CTs) and carried a conjugative 63 kb IncL plasmid encoding the carbapenemase gene blaOXA-48, yersiniabactin and other virulence factors. Although all CPKp ST11 strains carried additional similar IncR plasmids harbouring multiple antimicrobial resistance genes (ARGs), such as the plasmid-mediated blaDHA-1 AmpC gene, some structural variations were observed. The two ST307 strains carried identical 156 kb MDR IncFIB(K) plasmids with several ARGs, including the blaCTX-M-15 ESBL gene. Both wgMLST and cgSNP analysis confirmed that CPKp strains of the same ST were genetically highly related independent of the source of isolation. CONCLUSIONS: This study demonstrated that the clinical CPKp strains were highly related to those contaminating the clinical environment. These findings confirmed nosocomial spread and highlight veterinary hospitals as a source of CPKp, which may further spread to animals, the environment and humans.

Comparison of antimicrobial prescription patterns in calves in Switzerland before and after the launch of online guidelines for prudent antimicrobial use.

BACKGROUND: The increasing threat of bacterial resistance promotes the need for antibiotic stewardship programs to foster responsible antimicrobial use. Therefore, guidelines for prudent use supported by an online stewardship tool (AntibioticScout.ch) were introduced in Switzerland in December 2016. They recommend (with decreasing preference) a first, second or third line antimicrobial for treatment. The objective of this study was to evaluate antimicrobial prescriptions for Swiss calves before (2016) and after (2018) the launch of these guidelines. Cases of calves with pneumonia, diarrhea and otitis from a university hospital and eight private practices in Switzerland were included. Data on anamnesis, clinical findings, diagnostic work-up and treatment were collected. Type and percentages [95% confidence interval] of antimicrobial prescriptions were compared between 2016 and 2018. RESULTS: Of the total number of calves, 88.2% [85.4-90.6] in 2016 (n = 625) and 88.4% [85.7-90.7] in 2018 (n = 655) were treated with antibiotics. The use of highest priority critically important antimicrobials (HPCIAs) decreased from 52.7% [48.6-56.9] in 2016 to 38.0% [34.2-41.9] in 2018; this decrease was found at the university hospital and in private practice and in cases with pneumonia and diarrhea. Particularly the use of fluoroquinolones decreased (2016: 43.1% [39.2-47.2]; 2018: 31.1% [27.6-34.8]). Overall, the number of first line treatments increased from 12.8% [10.4-15.6] in 2016 to 20.2% [17.3-23.4] in 2018. In cases of pneumonia, first line treatments increased (2016: 15.3% [11.6-19.9]; 2018: 26.5% [21.8-31.9]) and third line treatments decreased (2016: 43.5% [38.0-49.3]; 2018: 27.9% [23.1-33.3]); this was seen at the university hospital, whereas in private practice only a decrease of third line treatments was observed. In cases of diarrhea, more second line at the expense of unlisted antimicrobials were prescribed at the university hospital in 2018. Antimicrobial treatment of calves with otitis did not change from 2016 to 2018. CONCLUSIONS: After the introduction of AntibioticScout.ch, more prudent use was observed in the treatment of calves with pneumonia and diarrhea as less HPCIAs, particularly fluoroquinolones, and more first line antimicrobials were prescribed. However, the overall frequency of antimicrobial treatment did not change and the use of HPCIAs was still common in 2018, especially in private practices. Therefore, further antimicrobial stewardship activities are necessary.

Antimicrobial prescriptions in cats in Switzerland before and after the introduction of an online antimicrobial stewardship tool.

BACKGROUND: Antimicrobial stewardship activities are essential to improve prudent antimicrobial use. The aim of the present study was to evaluate changes in antimicrobial prescriptions in cats after the introduction of prudent use guidelines promoted by an online antimicrobial stewardship tool (AntibioticScout.ch) in Switzerland. Data from 792 cats presented to two university hospitals and 14 private practices in 2018 were included and compared to 776 cases from 2016. Cats were diagnosed with acute upper respiratory tract disease (aURTD), feline lower urinary tract disease (FLUTD) and abscesses. Clinical history, diagnostic work-up and antimicrobial prescriptions (class, dosage, duration) were assessed. Type and proportions [95% confidence intervals] of antimicrobial prescriptions were compared between the two evaluation periods and a mixed effects logistic regression model was applied to evaluate compliance with Swiss prudent use guidelines. RESULTS: From 2016 to 2018, the proportion of antimicrobial prescription in all included cases decreased from 75.0% [71.8-78.0] to 66.7% [63.3-69.9]; this decrease was most pronounced for treatments at university hospitals (67.1% [59.5-74.0] to 49.3% [40.9-57.8]) and for cats with FLUTD (60.1% [54.6-65.4] to 48.8% [43.2-54.4]). Use of 3rd generation cephalosporins in private practices declined from 30.7% [26.5-35.1] to 22.1% [18.4-26.2], while overall use of non-potentiated aminopenicillins increased from 19.6% [16.4-23.0] to 27.8% [24.1-31.9]. In cases where antimicrobial therapy was indicated, compliance with guidelines did not increase (33.3% [26.6-40.6] to 33.5% [27.2-40.2]), neither at universities nor in private practices. On the other hand, antimicrobial treatment was more often withheld in cases with no indication for antimicrobial therapy (35.6% [30.1-41.4] to 54.0% [47.6-60.4]); this was found for private practices (26.7% [20.8-33.4] to 46.0% [38.4-53.7]) and for aURTD cases (35.0% [26.5-44.2] to 55.4% [44.7-65.8]). CONCLUSIONS: Overall proportions of antimicrobial prescription, unjustified antimicrobial therapy and, in private practices, use of 3rd generation cephalosporins decreased from 2016 to 2018 for the investigated feline diseases. However, overall compliance with Swiss prudent use guidelines was still low, implying that further efforts are required to foster prudent antimicrobial use in cats.

Shedding of OXA-181 carbapenemase-producing Escherichia coli from companion animals after hospitalisation in Switzerland: an outbreak in 2018.

BackgroundCarbapenem-resistant Enterobacteriaceae pose a serious threat to public health worldwide, and the role of companion animals as a reservoir is still unclear.AimsThis 4-month prospective observational study evaluated carriage of carbapenem-resistant Enterobacteriaceae at admission and after hospitalisation in a large referral hospital for companion animals in Switzerland.MethodsRectal swabs of dogs and cats expected to be hospitalised for at least 48 h were taken from May to August 2018 and analysed for the presence of carbapenem-resistant Enterobacteriaceae using selective agar plates. Resistant isolates were further characterised analysing whole genome sequences for resistance gene and plasmid identification, and ad hoc core genome multilocus sequence typing.ResultsThis study revealed nosocomial acquisition of Escherichia coli harbouring the carbapenemase gene bla OXA-181, the pAmpC cephalosporinase gene bla CMY-42 as well as quinolone resistance associated with qnrS1 and mutations in the topoisomerases II (GyrA) and IV (ParC). The bla OXA-181 and qnrS1 genes were identified on a 51 kb IncX3 plasmid and bla CMY-42 on a 47 kb IncI1 plasmid. All isolates belonged to sequence type ST410 and were genetically highly related. This E. coli clone was detected in 17 of 100 dogs and four of 34 cats after hospitalisation (21.6%), only one of the tested animals having tested positive at admission (0.75%). Two positive animals were still carriers 4 months after hospital discharge, but were negative after 6 months.ConclusionsCompanion animals may acquire carbapenemase-producing E. coli during hospitalisation, posing the risk of further dissemination to the animal and human population and to the environment.

Clinical, serological and echocardiographic examination of healthy field dogs before and after vaccination with a commercial tetravalent leptospirosis vaccine.

BACKGROUND: Leptospirosis is a re-emerging bacterial zoonosis caused by spirochetes of the genus Leptospira. Severe disease has been reported in dogs in Europe despite vaccination with bivalent Leptospira vaccines. Recently, a tetravalent canine Leptospira vaccine (Nobivac® L4) was licenced in Europe. The goal of this study was to investigate clinical signs, microscopic agglutination test (MAT) titres, haematology, blood biochemistry, cardiac (c) Troponin I levels and echocardiography before and after vaccination with this tetravalent vaccine. Forty-eight healthy dogs were prospectively enrolled and vaccinated twice, 3-4 weeks apart (T0 and T1). Before vaccination (T0) and 16-31 days after the second vaccination (T2), MAT (n = 48), haematology (n = 48), blood biochemistry (n = 36) and cTroponin I measurements (n = 29) were performed, and MAT was repeated 347-413 days after the second vaccination (T3, n = 44). Echocardiography was performed before the first and second vaccination (T0 and T1, n = 24). RESULTS: Mild and transient clinical signs within 5 days following the first and second vaccination occurred in 23% and 10% of the dogs, respectively. Before the first vaccination (T0), all dogs showed negative MAT titres for the tested serovars except for Canicola (50% with titres 100-400). At T2, positive MAT titres to the serovars Canicola (100%), Australis (89%), Grippotyphosa (86%), Bratislava (60%), Autumnalis (58%), Copenhageni (42%), Pomona (12%), Pyrogenes (8%) and Icterohaemorrhagiae (2%) were found. Median to high titres (≥ 400) were most common to the serovar Canicola (92%) and less common to the serovars Australis (41%), Grippotyphosa (21%), Bratislava (12%), Autumnalis (4%), Pyrogenes (4%) and Pomona (2%). At T3, positive MAT titres (titre range: 100-400) were found in 2-18% of the dogs to serovars of the vaccine serogroups and in 2-18% of the dogs to the non-vaccine serovars Pomona, Autumnalis, Pyrogenes and Ballum. Haematology, blood biochemistry, cTroponin I levels and echocardiography results did not change significantly following vaccination. CONCLUSIONS: Clinical signs following vaccination with Nobivac® L4 were transient and mild in all cases. Seroconversion differed considerably among individual dogs and among the vaccine serogroups.

Genetic diversity and phenotypic associations of feline caliciviruses from cats in Switzerland.

Feline calicivirus (FCV) is a common viral pathogen in domestic cats worldwide. The variable regions of the capsid (VP1) gene of FCV have one of the highest recorded rates of molecular evolution. Understanding the genetic diversity and phylogeny of FCV is a prerequisite to exploring the epidemiology and pathogenesis of this virus and to the development of efficacious vaccine strategies. In this study, we undertook a nationwide molecular characterization of FCV using for the first time nearly complete capsid (VP1) gene sequences. Sequences from 66 FCV samples were used to investigate the correlation between viral phylogeny and several traits, including geographic origin, signalment, husbandry, FCV vaccination and co-infections. Codon-based nucleotide alignment showed that individual nucleotides and their corresponding amino acid sites were either invariant or highly variable. Using a threshold of 20 % genetic distance in variable region E, FCV samples were grouped into 52 strains, 10 of which comprised two to three samples. Significant associations between FCV phylogeny and host characteristics were found, specifically the pedigree status of the cats, and two well-supported lineages were identified in which the current FCV strain definition was confounded. No correlation between viral genetic distances and geographic distances was evident. The greater resolution of the FCV phylogeny in this study compared to previous studies can be attributed to our use of more conserved regions of the capsid (VP1) gene; nonetheless, our results were still hampered by sequence saturation. The study highlights the need for whole-genome sequences for FCV phylogeny studies.

Passive immunization does not provide protection against experimental infection with Mycoplasma haemofelis.

Mycoplasma haemofelis (Mhf) is the most pathogenic feline hemotropic mycoplasma. Cats infected with Mhf that clear bacteremia are protected from Mhf reinfection, but the mechanisms of protective immunity are unresolved. In the present study we investigated whether the passive transfer of antibodies from Mhf-recovered cats to naïve recipient cats provided protection against bacteremia and clinical disease following homologous challenge with Mhf; moreover, we characterized the immune response in the recipient cats. Ten specified pathogen-free (SPF) cats were transfused with pooled plasma from cats that had cleared Mhf bacteremia; five control cats received plasma from naïve SPF cats. After homologous challenge with Mhf, cats were monitored for 100 days using quantitative PCR, hematology, blood biochemistry, Coombs testing, flow cytometry, DnaK ELISA, and red blood cell (RBC) osmotic fragility (OF) measurement. Passively immunized cats were not protected against Mhf infection but, compared to control cats, showed significantly higher RBC OF and B lymphocyte (CD45R/B220(+)) counts and occasionally higher lymphocyte, monocyte and activated CD4(+) T lymphocyte (CD4(+)CD25(+)) counts; they also showed higher bilirubin, total protein and globulin levels compared to those of control cats. At times of peak bacteremia, a decrease in eosinophils and lymphocytes, as well as subsets thereof (B lymphocytes and CD5(+), CD4(+) and CD8(+) T lymphocytes), and an increase in monocytes were particularly significant in the passively immunized cats. In conclusion, passive immunization does not prevent bacteremia and clinical disease following homologous challenge with Mhf, but enhances RBC osmotic fragility and induces a pronounced immune response.

Retroviral DNA--the silent winner: blood transfusion containing latent feline leukemia provirus causes infection and disease in naïve recipient cats.

BACKGROUND: The feline leukemia virus (FeLV) is a gamma-retrovirus of domestic cats that was discovered half a century ago. Cats that are infected with FeLV may develop a progressive infection resulting in persistent viremia, immunodeficiency, tumors, anemia and death. A significant number of cats mount a protective immune response that suppresses viremia; these cats develop a regressive infection characterized by the absence of viral replication and the presence of low levels of proviral DNA. The biological importance of these latter provirus carriers is largely unknown. RESULTS: Here, we demonstrate that ten cats that received a transfusion of blood from aviremic provirus carriers developed active FeLV infections, some with a progressive outcome and the development of fatal FeLV-associated disease. The infection outcome, disease spectrum and evolution into FeLV-C in one cat mirrored those of natural infection. Two cats developed persistent antigenemia; six cats were transiently antigenemic. Reactivation of infection occurred in some cats. One recipient developed non-regenerative anemia associated with FeLV-C, and four others developed a T-cell lymphoma, one with secondary lymphoblastic leukemia. Five of the ten recipient cats received provirus-positive aviremic blood, whereas the other five received provirus- and viral RNA-positive but aviremic blood. Notably, the cats that received blood containing only proviral DNA exhibited a later onset but graver outcome of FeLV infection than the cats that were transfused with blood containing proviral DNA and viral RNA. Leukocyte counts and cytokine analyses indicated that the immune system of the latter cats reacted quicker and more efficiently. CONCLUSIONS: Our results underline the biological and epidemiological relevance of FeLV provirus carriers and the risk of inadvertent FeLV transmission via blood transfusion and demonstrate the replication capacity of proviral DNA if uncontrolled by the immune system. Our results have implications not only for veterinary medicine, such as the requirement for testing blood donors and blood products for FeLV provirus by sensitive polymerase chain reaction, but are also of general interest by revealing the importance of latent retroviral DNA in infected hosts. When aiming to eliminate a retroviral infection from a population, provirus carriers must be considered.

Lack of cross-protection against Mycoplasma haemofelis infection and signs of enhancement in "Candidatus Mycoplasma turicensis"-recovered cats.

"Mycoplasma haemofelis" and "Candidatus Mycoplasma turicensis" are feline hemoplasmas that induce hemolytic anemia. Protection from homologous re-challenge was recently demonstrated in cats recovered from primary infection. Here, we determined if cats recovered from "Cand. M. turicensis" infection were protected against infections with the more pathogenic M. haemofelis. Ten specified pathogen-free cats were exposed to M. haemofelis. Five of the ten cats had recovered from "Cand. M. turicensis" bacteremia (group A), and five cats were naïve controls (group B). No cross-protection was observed. By contrast, the "Cand. M. turicensis"-recovered cats displayed faster M. haemofelis infection onset (earlier PCR-positive and anemic) than the controls. No "Cand. M. turicensis" was detected in any cat. M. haemofelis shedding was observed in saliva, feces and urine. In both groups, evidence of a Th1 response was observed (high IFN-γ, low IL-4), but IL-10 levels were also high. In group A, total, CD4+ and CD8+ T cells increased within days after M. haemofelis exposure. At times of maximal bacteremia, macrocytic hypochromic anemia, neutropenia, monocytosis and a decrease in leukocyte, eosinophil, and lymphocyte counts and subsets thereof (B- and T-cells, CD4+, CD8+ and CD4+CD25+ cells) were particularly significant in group A. Moreover, an increase in protein concentrations, hypoalbuminemia and a polyclonal hypergammaglobulinemia were observed. Five of ten M. haemofelis-infected cats subsequently cleared bacteremia without antibiotic treatment. In conclusion, the study suggests that a previous hemoplasma infection, even when the cat has ostensibly recovered, may influence subsequent infections, lead to an enhancement phenomenon and other differences in infection kinetics.

Feline calicivirus and other respiratory pathogens in cats with Feline calicivirus-related symptoms and in clinically healthy cats in Switzerland.

BACKGROUND: Cats with feline calicivirus (FCV)-related symptoms are commonly presented to veterinary practitioners. Various clinical manifestations have been attributed to FCV, i.e. upper respiratory tract disease (URTD), oral ulcerations, gingivostomatitis, limping syndrome and virulent systemic disease. Additionally, healthy cats can shed FCV. The aims of this study were 1) to investigate the frequency of FCV in cats with FCV-related symptoms and in healthy cats in Switzerland, 2) to assess risk and protective factors for infection, such as signalment, housing conditions, vaccination, and co-infection with URTD-associated pathogens, and 3) to address the association between clinical symptoms and FCV infection. RESULTS: Oropharyngeal, nasal and conjunctival swabs were collected in 24 veterinary practices from 200 FCV-suspect and 100 healthy cats originating from 19 cantons of Switzerland. The samples were tested for FCV using virus isolation and reverse-transcription real-time quantitative polymerase chain reaction (qPCR) and for feline herpesvirus-1 (FHV-1), Mycoplasma felis, Chlamydophila felis, Bordetella bronchiseptica using real-time qPCR. Within the two populations (FCV-suspect/healthy), the observed PCR prevalences were: FCV 45%/8%, FHV-1 20%/9%, C. felis 8%/1%, B. bronchiseptica 4%/2%, M. felis 47%/31% and any co-infections thereof 40%/14%. Based on multivariable regression models amongst FCV-suspect cats (odds ratio [95% confidence interval]), co-infection with M. felis (1.75 [0.97; 3.14]), group housing (2.11 [1.02; 4.34]) and intact reproductive status (1.80 [0.99; 3.28]) were found to be risk factors for FCV infection. In healthy cats, intact reproductive status (22.2 [1.85; 266.7]) and group housing (46.4 [5.70; 377.7]) were found to be associated with FCV infection. Based on an univariable approach, FCV-suspect cats were found to be significantly less often FCV-positive when vaccinated (0.48 [0.24; 0.94]). Oral ulcerations, salivation, gingivitis and stomatitis, but not classical signs of URTD were significantly associated with FCV infection (all p < 0.001). CONCLUSIONS: FCV was detected in less than half of the cats that were judged FCV-suspect by veterinary practitioners. For a clinical diagnosis, FCV-related symptoms should be revisited. FCV infection was present in some healthy cats, underlining the importance of asymptomatic carriers in FCV epidemiology. To reduce FCV-related problems in multi-cat environments, reduction of group size in addition to the generally recommended vaccination are advocated.

Clinical and molecular investigation of a canine distemper outbreak and vector-borne infections in a group of rescue dogs imported from Hungary to Switzerland.

BACKGROUND: Canine distemper virus (CDV) is a major pathogen of dogs and wild carnivores worldwide. In Switzerland, distemper in domestic dogs is rarely reported. In recent years, the import of dogs from Eastern Europe to Switzerland has steadily increased. In the present study, we describe a distemper outbreak in 15 rescue dogs that were imported from Hungary to Switzerland by an animal welfare organisation. The data on vaccination and medical history were recorded (14 dogs), and the samples were collected to investigate CDV and vector-borne infections (13 dogs) and canine parvovirus infection (12 dogs). The dogs were monitored for six months. RESULTS: One dog was euthanised directly after import. Thirteen dogs showed clinical signs after arrival, i.e., diarrhoea (57 %), coughing (43 %) and nasal and/or ocular discharge (21 %); radiographic findings that were compatible with bronchopneumonia were present in four dogs. CDV infection was diagnosed in 11 dogs (85 %); 10 dogs (91 %) tested PCR-positive in conjunctival swabs. Vector-borne infections (Babesia spp., Leishmania infantum, Dirofilaria immitis) were found in 4 dogs (31 %). Three dogs were hospitalized, and six dogs received ambulatory therapy for up to two months until recovery. None of the dogs developed neurological disease. CDV shedding was detected for a period of up to four months. Because dogs were put under strict quarantine until CDV shedding ceased, CDV did not spread to any other dogs. The CDV isolates showed 99 % sequence identity in the HA gene among each other and belonged to the Arctic-like lineage of CDV. CONCLUSIONS: The present study highlights the imminent risks of spreading contagious viral and vector-borne infections through the non-selective import of sick dogs and dogs with incomplete vaccination from Eastern Europe. CDV shedding was detected for several months after the cessation of clinical signs, which emphasised the roles of asymptomatic carriers in CDV epidemiology. A long-term follow-up using sensitive PCR and strict quarantine measures is of upmost importance in preventing the spread of infection. Dog owners and animal welfare organisations should be educated regarding the importance of complete vaccinations and the impact of dog imports on the spread of viral and vector-borne pathogens.

Plasma procalcitonin kinetics in healthy dogs and dogs undergoing tibial plateau leveling osteotomy.

BACKGROUND: Procalcitonin (PCT) is a well-established biomarker for bacterial infection in human patients. OBJECTIVES: We aimed to analyze the kinetics of plasma PCT (pPCT) in healthy dogs and dogs with canine cranial cruciate ligament (CCL) rupture undergoing tibial plateau leveling osteotomy (TPLO). METHODS: This prospective, longitudinal study included 15 healthy dogs and 25 dogs undergoing TPLO. Hematology, pPCT, and C-reactive protein (CRP) were assessed on 3 consecutive days in healthy dogs and 1 day preoperatively and days 1, 2, 10, and 56 postoperatively. Inter- and intraindividual variability of pPCT were assessed in healthy dogs. Median pPCT concentrations of dogs with CCL rupture preoperatively were compared with healthy controls, and median pPCT concentrations, as well as percentage change post anesthesia, arthroscopy, and TPLO, were compared with baseline. For the correlation analysis, the Spearman rank correlation test was used. RESULTS: Inter- and intraindividual variabilities of pPCT in healthy dogs were 36% and 15%, respectively. Median baseline pPCT concentrations were not significantly different between healthy dogs (118.9 pg/mL; IQR: 75.3-157.3 pg/mL) and dogs undergoing TPLO (95.9 pg/mL; IQR: 63.8-117.0 pg/mL). Plasma PCT concentrations were significantly lower immediately post- than preoperatively (P < 0.001). CRP, WBC, and neutrophil concentrations increased significantly on post-OP day 2 and had normalized by day 10. CONCLUSIONS: These results indicate that CCL rupture, as well as anesthesia, arthroscopy, and TPLO combined, are not associated with increased pPCT concentrations in dogs with uncomplicated recovery. Considering the high intraindividual variability, individual serial measurements rather than a population-based reference interval should be considered.

Detection and Molecular Characterization of the SARS-CoV-2 Delta Variant and the Specific Immune Response in Companion Animals in Switzerland.

In human beings, there are five reported variants of concern of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). However, in contrast to human beings, descriptions of infections of animals with specific variants are still rare. The aim of this study is to systematically investigate SARS-CoV-2 infections in companion animals in close contact with SARS-CoV-2-positive owners ("COVID-19 households") with a focus on the Delta variant. Samples, obtained from companion animals and their owners were analyzed using a real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) and next-generation sequencing (NGS). Animals were also tested for antibodies and neutralizing activity against SARS-CoV-2. Eleven cats and three dogs in nine COVID-19-positive households were RT-qPCR and/or serologically positive for the SARS-CoV-2 Delta variant. For seven animals, the genetic sequence could be determined. The animals were infected by one of the pangolin lineages B.1.617.2, AY.4, AY.43 and AY.129 and between zero and three single-nucleotide polymorphisms (SNPs) were detected between the viral genomes of animals and their owners, indicating within-household transmission between animal and owner and in multi-pet households also between the animals. NGS data identified SNPs that occur at a higher frequency in the viral sequences of companion animals than in viral sequences of humans, as well as SNPs, which were exclusively found in the animals investigated in the current study and not in their owners. In conclusion, our study is the first to describe the SARS-CoV-2 Delta variant transmission to animals in Switzerland and provides the first-ever description of Delta-variant pangolin lineages AY.129 and AY.4 in animals. Our results reinforce the need of a One Health approach in the monitoring of SARS-CoV-2 in animals.

A Risk Factor Analysis of SARS-CoV-2 Infection in Animals in COVID-19-Affected Households.

A higher prevalence of SARS-CoV-2 infections in animals that have close contact with SARS-CoV-2-positive humans ("COVID-19 households") has been demonstrated in several countries. This prospective study aimed to determine the SARS-CoV-2 prevalence in animals from Swiss COVID-19 households and to assess the potential risk factors for infection. The study included 226 companion animals (172 cats, 76.1%; 49 dogs, 21.7%; and 5 other animals, 2.2%) from 122 COVID-19 households with 336 human household members (including 230 SARS-CoV-2-positive people). The animals were tested for viral RNA using an RT-qPCR and/or serologically for antibodies and neutralizing activity. Additionally, surface samples from animal fur and beds underwent an RT-qPCR. A questionnaire about hygiene, animal hygiene, and contact intensity was completed by the household members. A total of 49 of the 226 animals (21.7%) from 31 of the 122 households (25.4%) tested positive/questionably positive for SARS-CoV-2, including 37 of the 172 cats (21.5%) and 12 of the 49 dogs (24.5%). The surface samples tested positive significantly more often in households with SARS-CoV-2-positive animals than in households with SARS-CoV-2-negative animals (p = 0.011). Significantly more animals tested positive in the multivariable analysis for households with minors. For cats, a shorter length of outdoor access and a higher frequency of removing droppings from litterboxes were factors that were significantly associated with higher infection rates. The study emphasizes that the behavior of owners and the living conditions of animals can influence the likelihood of a SARS-CoV-2 infection in companion animals. Therefore, it is crucial to monitor the infection transmission and dynamics in animals, as well as to identify the possible risk factors for animals in infected households.

Massive Spread of OXA-48 Carbapenemase-Producing Enterobacteriaceae in the Environment of a Swiss Companion Animal Clinic.

BACKGROUND: Companion animal clinics contribute to the spread of antimicrobial resistant microorganisms (ARM) and outbreaks with ARM of public health concern have been described. METHODS: As part of a project to assess infection prevention and control (IPC) standards in companion animal clinics in Switzerland, a total of 200 swabs from surfaces and 20 hand swabs from employees were collected during four days in a medium-sized clinic and analyzed for extended spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E), carbapenemase-producing Enterobacteriaceae (CPE), vancomycin-resistant enterococci (VRE), and methicillin-resistant staphylococci (MRS). RESULTS: A total of 22 (11.0%) environmental specimen yielded CPE, 14 (7.0%) ESBL-E, and 7 (3.5%) MRS; MR Staphylococcus aureus were isolated from two (10.0%) hand swabs. The CPE isolates comprised Escherichia coli, Klebsiella pneumoniae, Enterobacter hormaechei, Citrobacter braakii, and Serratia marcescens. Whole genome sequencing revealed that all CPE carried closely related blaOXA-48 plasmids, suggesting a plasmidic spread within the clinic. The clinic exhibited major deficits in surface disinfection, hand hygiene infrastructure, and hand hygiene compliance. CPE were present in various areas, including those without patient contact. The study documented plasmidic dissemination of blaOXA-48 in a companion animal clinic with low IPC standards. This poses a worrisome threat to public health and highlights the need to foster IPC standards in veterinary clinics to prevent the spread of ARM into the community.

Cytauxzoon europaeus infections in domestic cats in Switzerland and in European wildcats in France: a tale that started more than two decades ago.

BACKGROUND: Cytauxzoon spp. infection is believed to be a newly emerging tick-borne disease in felids in Europe, with three species of the haemoparasite having recently been differentiated in wild felids. In Switzerland, rare infections have been documented in domestic cats in the west and northwest of the country, the first of which was in 2014. The aims of the present study were: (i) to characterize a Cytauxzoon spp. hotspot in domestic cats in central Switzerland; (ii) to elucidate the geographic distribution of Cytauxzoon spp. in domestic cats in Switzerland; (iii) to assess suspected high-risk populations, such as stray and anaemic cats; and (iv) to investigate the newly emerging nature of the infection. Cytauxzoon spp. were further differentiated using mitochondrial gene sequencing. METHODS: The overall study included samples from 13 cats from two households in central Switzerland (study A), 881 cats from all regions of Switzerland (study B), 91 stray cats from a hotspot region in the northwest of Switzerland and 501 anaemic cats from across Switzerland (study C), and 65 Swiss domestic cats sampled in 2003 and 34 European wildcats from eastern France sampled in the period 1995-1996 (study D). The samples were analysed for Cytauxzoon spp. using real-time TaqMan quantitative PCR, and positive samples were subjected to 18S rRNA, cytochrome b (CytB) and cytochrome c oxidase subunit I (COI) gene sequencing. RESULTS: In study A, six of 13 cats from two neighbouring households in central Switzerland tested postive for Cytauxzoon spp.; two of the six infected cats died from bacterial infections. In studies B and C, only one of the 881 cats (0.1%; 95% confidence interval [CI]: 0-0.3%) in the countrywide survey and one of the 501 anaemic cats (0.2%; 95% CI: 0-0.6%) tested postive for Cytauxzoon spp. while eight of the 91 stray cats in the northwest of Switzerland tested positive (8.8%; 95% CI: 3.0-14.6%). In study D, Cytauxzoon spp. was detected in one of the 65 domestic cat samples from 2003 (1.5%; 95% CI: 0-4.5%) and in ten of the 34 European wildcat samples from 1995 to 1996 (29%; 95% CI: 14.2-44.7%). The isolates showed ≥ 98.6% sequence identities among the 18S rRNA, CytB and COI genes, respectively, and fell in the subclade Cytauxzoon europaeus based on CytB and COI gene phylogenetic analyses. CONCLUSIONS: The study challenges the newly emerging nature of Cytauxzoon spp. in central Europe and confirms that isolates from domestic cats in Switzerland and European wild felids belong to the same species.

Detection of feline haemoplasma species in experimental infections by in-situ hybridisation.

The aim of this study was to use fluorescence in-situ hybridisation (FISH) to search for the tissues and cell types important in survival and persistence of Mycoplasma haemofelis, "Candidatus Mycoplasma haemominutum" or "Candidatus Mycoplasma turicensis" in infected cats. A 16S rDNA probe for each species was applied to formalin-fixed, paraffin wax-embedded tissues sections collected from experimentally infected cats. Tissues (n = 12) were collected, at necropsy, from ten cats which had been infected with M. haemofelis, and one each with "Ca. M. haemominutum" and "Ca. M. turicensis". M. haemofelis specific hybridisation was present on red blood cells (RBCs) in all tissues from acutely infected cats, but not the majority of tissues from chronically infected cats. "Ca. M. haemominutum" specific hybridisation was present on scattered RBCs within the spleen and liver. Specific probe hybridisation was not detected in any of the "Ca. M. turicensis" infected tissues. Haemoplasmas were detected on the surface of RBCs only and not any other cell type. Additionally, FISH was limited by sensitivity and could not detect the lower numbers of organisms present in tissues of cats chronically infected with M. haemofelis. Occasional organisms were detected in cats acutely infected with "Ca. M. haemominutum" but not "Ca. M. turicensis".

Chronic "Candidatus Mycoplasma turicensis" infection.

"Candidatus Mycoplasma turicensis" infects felids. The pathogenesis of "Candidatus M. turicensis" chronic infection is poorly understood. The goals of the present study were to (1) induce reactivation of the infection in chronic carrier cats by attempted immunosuppression, (2) identify potential tissue sequestration using real-time TaqMan® PCR and (3) monitor the humoral immune response by DnaK enzyme-linked immunosorbent assay (ELISA). Ten specified pathogen-free cats that had ostensibly recovered from experimental "Candidatus M. turicensis" infection were used: five cats (group 1) received high dose methylprednisolone (attempted immunosuppression), while five cats served as untreated controls (group 2). Besides weekly blood samples, tissue samples were collected from bone marrow, kidney, liver and salivary glands at selected time points. The cats in group 1 had significantly lower lymphocyte counts and higher blood glucose levels after methylprednisolone administration than the controls. After methylprednisolone administration one blood and three tissue samples from cats in group 1 tested PCR-positive; before the administration, only one sample was positive. All other samples tested PCR-negative. All cats stayed seropositive; the antibody levels of the cats in group 1 showed a significant transient decrease after methylprednisolone administration. This is the first study to report the presence of "Candidatus M. turicensis" in tissues of chronically infected cats and the persistence of anti-feline hemoplasma antibodies in the absence of detectable bacteremia. Methylprednisolone administration did not lead to a significant reactivation of the infection. Our results enhance the knowledge of "Candidatus M. turicensis" infection pathogenesis and are clinically relevant to the prognosis of hemoplasma-infected cats.

Hand Hygiene Evaluation Using Two Different Evaluation Tools and Hand Contamination of Veterinary Healthcare Workers in a Swiss Companion Animal Clinic.

Hand hygiene (HH) is the most important measure to prevent nosocomial infections. HH compliance in companion animal clinics has been reported to be poor. The present study compared an online application with the WHO evaluation form to assess the WHO five moments of HH in a Swiss companion animal clinic. In 202 hand swabs from 87 staff members, total viable count (TVC) before and after patient contact was evaluated and the swabs were tested for selected antimicrobial resistant microorganisms of public health importance. HH compliance (95% confidence interval) was 36.6% (33.8-39.5%) and was similar when assessed with the two evaluation tools. HH differed between hospital areas (p = 0.0035) and HH indications (p < 0.0001). Gloves were worn in 22.0% (18.0-26.6%) of HH observations and were indicated in 37.2% (27.3-48.3%) of these observations. Mean TVC before patient contact was lower (0.52 log CFU/cm2) than after patient contact (1.02 log CFU/cm2) but was similar before patient contact on gloved and ungloved hands. Three hand swabs (1.5% (0.4-4.3%)) were positive for methicillin-resistant Staphylococcus aureus. Gloving should not be regarded as a substitute for HH. Overall, HH in companion animal medicine should urgently be fostered.