Detection of SARS-CoV-2 RNA in a Zoo-Kept Red Fox (Vulpes vulpes).

Many different animal species are susceptible to SARS-CoV-2, including a few Canidae (domestic dog and raccoon dog). So far, only experimental evidence is available concerning SARS-CoV-2 infections in red foxes (Vulpes vulpes). This is the first report of SARS-CoV-2 RNA detection in a sample from a red fox. The RT-qPCR-positive fox was zoo-kept together with another fox and two bears in the Swiss Canton of Zurich. Combined material from a conjunctival and nasal swab collected for canine distemper virus diagnostics tested positive for SARS-CoV-2 RNA with Ct values of 36.9 (E gene assay) and 35.7 (RdRp gene assay). The sample was analysed for SARS-CoV-2 within a research project testing residual routine diagnostic samples from different animal species submitted between spring 2020 and December 2022 to improve knowledge on SARS-CoV-2 infections within different animal species and investigate their potential role in a One Health context. Within this project, 246 samples from 153 different animals from Swiss zoos and other wild animal species all tested SARS-CoV-2 RT-qPCR and/or serologically negative so far, except for the reported fox. The source of SARS-CoV-2 in the fox is unknown. The fox disappeared within the naturally structured enclosure, and the cadaver was not found. No further control measures were undertaken.

Evaluation of a Revised Point-of-Care Test for the Detection of Feline Leukaemia p27 Antigen and Anti-p15E Antibodies in Cats.

The first point-of-care (PoC) test (v-RetroFel®; modified version 2021) determining the presence of FeLV p27 antigen and FeLV anti-p15E antibodies has become recently commercially available to identify different feline leukaemia virus (FeLV) infection outcomes. This study aimed to assess this PoC test's performance concerning FeLV p27 antigen and FeLV anti-p15E antibody detection. Sensitivity, specificity, positive and negative predictive values (PPV, NPV) were assessed after ten minutes (recommended) and 20 min (prolonged) incubation times. The test results were evaluated as either positive or negative. Serum samples from 934 cats were included, originating from Italy (n = 269), Portugal (n = 240), Germany (n = 318), and France (n = 107). FeLV p27 antigen and anti-p15E antibodies were measured by reference standard ELISAs and compared to the PoC test results. The PoC test was easy to perform and the results easy to interpret. Sensitivity and specificity for FeLV p27 antigen were 82.8% (PPV: 57.8%) and 96.0% (NPV: 98.8%) after both, ten and 20 minues of incubation time. Sensitivity and specificity for anti-p15E antibodies were 31.4% (PPV: 71.6%) and 96.9% (NPV: 85.1%) after ten minutes incubation time; sensitivity was improved by a prolonged incubation time (20 min) to 40.0% (PPV: 76.3%), while specificity remained the same (96.9%, NPV: 86.7%). Despite the improved sensitivity using the prolonged incubation time, lower than ideal sensitivities for both p27 antigen and especially anti-p15E antibodies were found, indicating that the PoC test in its current version needs further improvement prior to application in the field.

Prospective Investigation of Feline Leukemia Virus Infection in Stray Cats Subjected to a Trap-Neuter-Return Program in Switzerland.

Feline leukemia virus (FeLV) remains a serious concern in some countries despite advances in diagnostics and vaccines. FeLV-infected cats often have reduced lifespans due to FeLV-associated diseases. The infection is transmitted through social interactions. While Northern European countries have reported a decrease in FeLV among pet cats, Switzerland's rates remain stagnant at 2.7% (2016/17: 95% CI 1.4-5.2%). Research on FeLV in Swiss stray cats has been lacking, even though these animals could serve as a virus reservoir. Sampling stray cats that do not receive regular veterinary care can be challenging. Collaboration with the Swiss Network for Animal Protection (NetAP) allowed for the prospective collection of saliva samples from 1711 stray cats during a trap-neuter-return program from 2019 to 2023. These samples were tested for FeLV RNA using RT-qPCR as a measure for antigenemia. Viral RNA was detected in 4.0% (95% CI 3.1-5.0%) of the samples, with 7.7% (95% CI 4.9-11.3%) in sick cats and 3.3% (95% CI 2.4-4.4%) in healthy ones. We identified three geographically independent hotspots with alarmingly high FeLV infection rates in stray cats (up to 70%). Overall, including the previous data of privately owned cats, FeLV-positive cats were scattered throughout Switzerland in 24/26 cantons. Our findings underscore welfare concerns for FeLV infections among stray cats lacking veterinary attention, highlighting the potential risk of infection to other free-roaming cats, including those privately owned. This emphasizes the critical significance of vaccinating all cats with outdoor access against FeLV and developing programs to protect cats from FeLV infections.

Long-term surveillance of the feline leukemia virus in the endangered Iberian lynx (Lynx pardinus) in Andalusia, Spain (2008-2021).

Feline leukemia virus (FeLV) infection is considered one of the most serious disease threats for the endangered Iberian lynx (Lynx pardinus) Over 14 years (2008-2021), we investigated FeLV infection using point-of-care antigen test and quantitative real-time TaqMan qPCR for provirus detection in blood and tissues in lynxes from Andalusia (Southern Spain). A total of 776 samples from 586 individuals were included in this study. The overall prevalence for FeLV antigen in blood/serum samples was 1.4% (5/360) (95% CI: 0.2-2.6), FeLV proviral DNA prevalence in blood samples was 6.2% (31/503) (95% CI: 4.1-8.6), and FeLV proviral DNA in tissues samples was 10.2% (34/333) (95% CI: 7-13.5). From a subset of 129 longitudinally sampled individuals, 9.3% (12/129) PCR-converted during the study period. Our results suggest that FeLV infection in the Andalusian population is enzootic, with circulation of the virus at low levels in almost all the sampling years. Moreover, since only one viremic individual succumbed to the infection, this study suggests that lynxes may therefore control the infection decreasing the possibility of developing a more aggressive outcome. Although our results indicate that the FeLV infection in the Iberian lynx from Andalusia tends to stay within the regressive stage, continuous FeLV surveillance is paramount to predict potential outbreaks and ensure the survival of this population.

Serological and Molecular Investigation of SARS-CoV-2 in Horses and Cattle in Switzerland from 2020 to 2022.

Horses and cattle have shown low susceptibility to SARS-CoV-2, and there is no evidence of experimental intraspecies transmission. Nonetheless, seropositive horses in the US and seropositive cattle in Germany and Italy have been reported. The current study investigated the prevalence of antibodies against SARS-CoV-2 in horses and cattle in Switzerland. In total, 1940 serum and plasma samples from 1110 horses and 830 cattle were screened with a species-specific ELISA based on the SARS-CoV-2 receptor-binding domain (RBD) and, in the case of suspect positive results, a surrogate virus neutralization test (sVNT) was used to demonstrate the neutralizing activity of the antibodies. Further confirmation of suspect positive samples was performed using either a pseudotype-based virus neutralization assay (PVNA; horses) or an indirect immunofluorescence test (IFA; cattle). The animals were sampled between February 2020 and December 2022. Additionally, in total, 486 bronchoalveolar lavage (BAL), oropharyngeal, nasal and rectal swab samples from horses and cattle were analyzed for the presence of SARS-CoV-2 RNA via reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Six horses (0.5%; 95% CI: 0.2-1.2%) were suspect positive via RBD-ELISA, and neutralizing antibodies were detected in two of them via confirmatory sVNT and PVNA tests. In the PVNA, the highest titers were measured against the Alpha and Delta SARS-CoV-2 variants. Fifteen cattle (1.8%; 95% CI: 1.0-3.0%) were suspect positive in RBD-ELISA; 3 of them had SARS-CoV-2-specific neutralizing antibodies in sVNT and 4 of the 15 were confirmed to be positive via IFA. All tested samples were RT-qPCR-negative. The results support the hypotheses that the prevalence of SARS-CoV-2 infections in horses and cattle in Switzerland was low up to the end of 2022.

Prevalence of Different Courses of Feline Leukaemia Virus Infection in Four European Countries.

Prevalence of progressive feline leukaemia virus (FeLV) infection is known to still be high in cats in Europe, especially in Southern Europe, but the prevalence of other outcomes of FeLV infection has not been determined in most countries. The present study aimed to investigate the prevalence of progressive, regressive, abortive, and focal infection in four European countries, two with a high (Italy, Portugal) and two with a low expected prevalence (Germany, France). Blood samples of 934 cats (Italy: 269; Portugal: 240; France: 107; Germany: 318) were evaluated for the p27 antigen, as well as anti-whole virus, anti-SU, and anti-p15E antibodies by enzyme-linked immunosorbent assay (ELISA) in serum and for proviral DNA by quantitative polymerase chain reaction (qPCR) in whole blood. Positive p27 antigen ELISA results were confirmed by reverse transcriptase-qPCR (RT-qPCR) detecting viral RNA in saliva swabs and/or blood. The outcome of FeLV infection was categorised as progressive (antigen-positive, provirus-positive), regressive (antigen-negative, provirus-positive), abortive (antigen- and provirus-negative, antibody-positive), and focal (antigen-positive, provirus-negative) infection. Overall FeLV prevalence was 21.2% in Italy, 20.4% in Portugal, 9.5% in Germany, and 9.3% in France. Prevalence of progressive, regressive, abortive, and focal infection in Italy was 7.8%, 4.5%, 6.3%, and 2.6%; in Portugal 3.8%, 8.3%, 6.7%, and 1.7%; in Germany 1.9%, 1.3%, 3.5%, and 2.8%; in France 1.9%, 3.7%, 2.8%, and 0.9%, respectively. In conclusion, overall FeLV prevalence is still very high, especially in Southern European countries. Therefore, testing, separation of infected cats, and vaccination are still important measures to reduce the risk of FeLV infection.

Long-term follow-up of cats in complete remission after treatment of feline infectious peritonitis with oral GS-441524.

OBJECTIVES: Feline infectious peritonitis (FIP), a common disease in cats caused by feline coronavirus (FCoV), is usually fatal once clinical signs appear. Successful treatment of FIP with oral GS-441524 for 84 days was demonstrated recently by this research group. The aim of this study was to evaluate the long-term outcome in these cats. METHODS: A total of 18 successfully treated cats were followed for up to 1 year after treatment initiation (9 months after completion of the antiviral treatment). Follow-up examinations were performed at 12-week intervals, including physical examination, haematology, serum biochemistry, abdominal and thoracic ultrasound, FCoV ribonucleic acid (RNA) loads in blood and faeces by reverse transciptase-quantitative PCR and anti-FCoV antibody titres by indirect immunofluorescence assay. RESULTS: Follow-up data were available from 18 cats in week 24, from 15 cats in week 36 and from 14 cats in week 48 (after the start of treatment), respectively. Laboratory parameters remained stable after the end of the treatment, with undetectable blood viral loads (in all but one cat on one occasion). Recurrence of faecal FCoV shedding was detected in five cats. In four cats, an intermediate short-term rise in anti-FCoV antibody titres was detected. In total, 12 cats showed abdominal lymphadenomegaly during the follow-up period; four of them continuously during the treatment and follow-up period. Two cats developed mild neurological signs, compatible with feline hyperaesthesia syndrome, in weeks 36 and 48, respectively; however, FCoV RNA remained undetectable in blood and faeces, and no increase in anti-FCoV antibody titres was observed in these two cats, and the signs resolved. CONCLUSIONS AND RELEVANCE: Treatment with GS-441524 proved to be effective against FIP in both the short term as well as the long term, with no confirmed relapse during the 1-year follow-up period. Whether delayed neurological signs could be a long-term adverse effect of the treatment or associated with a 'long FIP syndrome' needs to be further evaluated.

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.

Fatal gastritis and enterocolitis due to concurrent Helicobacter pylori and Campylobacter jejuni infection in a captive cheetah (Acinonyxjubatus).

A 3.5-year-old female cheetah (Acinonyx jubatus) died after a 10-day history of anorexia, regurgitation and diarrhoea despite symptomatic therapy. At gross post-mortem examination, the stomach was blood-filled with mucosal thickening and multifocal ulcerations. The intestinal mucosa was thickened and reddened, and the intestinal lumen was filled with dark red to black pasty content. Gastric histological lesions were compatible with gastritis due to Helicobacter infection, which was confirmed by polymerase chain reaction. Histology of the intestines revealed a severe necrotizing neutrophilic enterocolitis with abundant intralesional curved to spiral bacteria, corresponding to Campylobacter jejuni, which were subsequently isolated from both small and large intestinal contents. No other intestinal pathogens were detected despite thorough investigations. These findings suggest that C. jejuni may have played an aetiological role in the enterocolitis. Such an association has not been previously reported in non-domestic felids.

Field Performance of a Rapid Test to Detect Progressive, Regressive, and Abortive Feline Leukemia Virus Infections in Domestic Cats in Australia and Germany.

Different feline leukemia virus (FeLV) infection outcomes are possible in cats following natural exposure, such as progressive infections (persistent viremia), regressive infections (transient or no viremia followed by proviral persistence) and abortive infections (presence of only antibodies). Laboratory-based testing is currently required for categorization of infection outcomes in cats. The aim of this study was to evaluate the field performance of a novel, rapid, combination point-of-care (PoC) test kit commercially available in Europe (v-RetroFel®Ag/Ab; 2020-2021 version) to determine different FeLV infection outcomes by concurrent detection of FeLV antigen (p27) and antibodies against FeLV transmembrane envelope protein (p15E). A secondary aim was to evaluate the performance of the same test kit (v-RetroFel®FIV) to determine positive/negative feline immunodeficiency virus (FIV) infection status by the detection of antibodies to FIV capsid protein (p24) and transmembrane glycoprotein (gp40). Two cohorts of domestic cats were recruited and tested with v-RetroFel® using plasma or serum, including cats in Australia (n = 200) and cats in Germany (n = 170). Results from p27 antigen PoC testing, proviral DNA PCR, and neutralizing antibody testing or testing for antibodies against non-glycosylated surface unit envelope protein (p45) were used to assign cats to groups according to different FeLV infection outcomes. Testing with a laboratory-based FeLV p15E antibody ELISA was also performed for comparison. In the first cohort, v-RetroFel®Ag/Ab correctly identified 89% (109/122) FeLV-unexposed cats and 91% (21/23) progressive infections, but no regressive (0/23) or abortive (0/32) infections. In the second cohort, v-RetroFel®Ag/Ab correctly identified 94% (148/158) FeLV-unexposed cats and 100% (4/4) progressive infections, but no regressive (0/2) and only 17% (1/6) abortive infections. There was test agreement between v-RetroFel®Ab and the p15E laboratory ELISA in 58.9% of samples. As a secondary outcome of this study, the sensitivity and specificity of v-RetroFel®FIV testing in cohort 1 were 94.7% (18/19) and 98.3% (178/181), and in cohort 2, 30.0% (3/10) and 100.0% (160/160), respectively. Prior history of FIV vaccination did not produce any false-positive FIV results. In conclusion, v-RetroFel®Ag/Ab (2020-2021 version) was unable to accurately determine different FeLV infection outcomes in the field. Improvements of the test prior to application to field samples are required.

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.

Clinical Follow-Up and Postmortem Findings in a Cat That Was Cured of Feline Infectious Peritonitis with an Oral Antiviral Drug Containing GS-441524.

This is the first report on a clinical follow-up and postmortem examination of a cat that had been cured of feline infectious peritonitis (FIP) with ocular manifestation by successful treatment with an oral multicomponent drug containing GS-441524. The cat was 6 months old when clinical signs (recurrent fever, lethargy, lack of appetite, and fulminant anterior uveitis) appeared. FIP was diagnosed by ocular tissue immunohistochemistry after enucleation of the affected eye. The cat was a participant in a FIP treatment study, which was published recently. However, 240 days after leaving the clinic healthy, and 164 days after the end of the 84 days of treatment, the cured cat died in a road traffic accident. Upon full postmortem examination, including histopathology and immunohistochemistry, there were no residual FIP lesions observed apart from a generalized lymphadenopathy due to massive lymphoid hyperplasia. Neither feline coronavirus (FCoV) RNA nor FCoV antigen were identified by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunohistochemistry, respectively, in any tissues or body fluids, including feces. These results prove that oral treatment with GS-441524 leads to the cure of FIP-associated changes and the elimination of FCoV from all tissues.

Detection of Feline Coronavirus Variants in Cats without Feline Infectious Peritonitis.

(1) Background: This study aimed to detect feline coronavirus (FCoV) and characterize spike (S) gene mutation profiles in cats suffering from diseases other than feline infectious peritonitis (FIP) using commercial real-time reverse transcription polymerase chain reaction (RT-qPCR) and reevaluating results by sequencing. (2) Methods: In 87 cats in which FIP was excluded by histopathology and immunohistochemistry, FCoV 7b gene and S gene mutation RT-qPCR was performed prospectively on incisional biopsies and fine-needle aspirates of different organs, body fluids, and feces. Samples positive for S gene mutations or mixed FCoV underwent sequencing. (3) Results: In 21/87 cats, FCoV RNA was detectable. S gene mutations were detected by commercial RT-qPCR (and a diagnostic algorithm that was used at the time of sample submission) in at least one sample in 14/21 cats (66.7%), with only mutated FCoV in 2/21, only mixed in 1/21, and different results in 11/21 cats; in the remaining 7/21 cats, RNA load was too low to differentiate. However, sequencing of 8 tissue samples and 8 fecal samples of 9 cats did not confirm mutated FCoV in any of the FCoV RNA-positive cats without FIP. (4) Conclusions: Sequencing results did not confirm results of the commercial S gene mutation RT-qPCR.

Fecal Feline Coronavirus RNA Shedding and Spike Gene Mutations in Cats with Feline Infectious Peritonitis Treated with GS-441524.

As previously demonstrated by our research group, the oral multicomponent drug Xraphconn® containing GS-441524 was effective at curing otherwise fatal feline infectious peritonitis (FIP) in 18 feline coronavirus (FCoV)-infected cats. The aims of the current study were to investigate, using samples from the same animals as in the previous study, (1) the effect of treatment on fecal viral RNA shedding; (2) the presence of spike gene mutations in different body compartments of these cats; and (3) viral RNA shedding, presence of spike gene mutations, and anti-FCoV antibody titers in samples of 12 companion cats cohabitating with the treated cats. Eleven of the eighteen treated FIP cats (61%) were shedding FCoV RNA in feces within the first three days after treatment initiation, but all of them tested negative by day 6. In one of these cats, fecal shedding reoccurred on day 83. Two cats initially negative in feces were transiently positive 1-4 weeks into the study. The remaining five cats never shed FCoV. Viral RNA loads in feces decreased with time comparable with those in blood and effusion. Specific spike gene mutations linked to systemic FCoV spread were consistently found in blood and effusion from treated FIP cats, but not in feces from treated or companion cats. A new mutation that led to a not yet described amino acid change was identified, indicating that further mutations may be involved in the development of FIP. Eight of the twelve companion cats shed FCoV in feces. All but one of the twelve companion cats had anti-FCoV antibodies. Oral treatment with GS-441524 effectively decreased viral RNA loads in feces, blood, and effusion in cats with FIP. Nonetheless, re-shedding can most likely occur if cats are re-exposed to FCoV by their companion cats.

A Pre- and Within-Pandemic Survey of SARS-CoV-2 RNA in Saliva Swabs from Stray Cats in Switzerland.

Cats have been shown to be highly susceptible to SARS-CoV-2, and transmission within the species has been demonstrated experimentally. In cats undergoing natural SARS-CoV-2 infections, human-to-animal transmission was mostly suspected. It can be postulated that, in stray cats with no or only minimal contact with humans, SARS-CoV-2 may pose a minor risk. The current study investigated the prevalence of active SARS-CoV-2 infections in Swiss stray cats using quantitative reverse transcriptase real-time polymerase chain reaction (RT-qPCR). Saliva swabs from 1405 stray cats were collected in 14 Swiss cantons. The animals were sampled between February 2019 and February 2020 (pre-COVID-19 cohort: 523 cats) and between February 2020 and August 2021 (COVID-19 cohort: 882 cats). All the samples were tested by RT-qPCR, amplifying the envelope (E) gene and, in case of positive or inconclusive results, the RNA-dependent RNA polymerase (RdRp) gene of SARS-CoV-2. No SARS-CoV-2 viral RNA could be detected in any of the tested saliva swab samples. Our findings support the assumption that SARS-CoV-2 infections in stray cats are not highly prevalent in Switzerland. Nevertheless, the monitoring of stray cats and other susceptible animal species is necessary, since the "One Health" approach has been recognized as being essential to successfully fight the COVID-19 pandemic.

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.

First molecular evidence of Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum' infections and its association with epidemiological factors in dogs from Cuba.

Haemotrophic mycoplasmas (haemoplasmas) are unculturable, epicellular, cell wall-less gram-negative bacteria distributed worldwide, which infect several mammalian species. In dogs, Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum' have been reported as causative agents of infectious anaemia, especially in splenectomised or immunocompromised animals. The present cross-sectional study aims to assess the prevalence, risk factors, and molecular characterization of canine haemoplasmas in Cuba. A total of 391 dog blood samples and 247 tick samples were tested for the presence of canine haemoplasmas by species-specific quantitative TaqMan® real-time PCR assays. Overall, 17.9% (70/391; 95% CI: 14.1-21.7) blood samples were PCR-positive for at least one canine haemoplasmas species, where 15.1% (59/391; 95% CI: 11.5-18.7) for Mycoplasma haemocanis, 4.4% (17/391; 95% CI: 2.3-6.4) for 'Candidatus Mycoplasma haematoparvum', and 1.5% (6/391; 95% CI: 0.3-2.8) were co-infected. All collected ticks were identified morphologically as Rhipicephalus sanguineus sensu lato, and none of the tested tick samples was found PCR-positive for the presence of Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum'. Risk factors for canine haemoplasmas species infection included the presence of tick infestation, crossbreeding and living in kennels, while no association was found with the occurrence of anaemia. Phylogenetic analyses based on the 16S rRNA gene sequences of Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum' revealed >99% identity to other isolates distributed worldwide, indicating low genetic variability amongst these canine haemoplasmas species. To the best of the authors´ knowledge, this is the first molecular evidence of Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum' infections in dogs from Cuba.

Investigation on haplotypes of ixodid ticks and retrospective finding of Borrelia miyamotoi in bank vole (Myodes glareolus) in Switzerland.

The current status of tick species, important tick-borne bacteria and protozoan parasites is well-documented in Switzerland. However, reports on the genetic diversity and geographical relationships of tick species in this country appear to be in part lacking or outdated. Thus, the aim of this study was to collect ticks from various host species in southern Switzerland, to compare them in a geographical context and to screen in these samples rare tick-borne pathogens hitherto not reported or having low prevalence in Switzerland. In 2019-2020 altogether 177 ixodid ticks were collected from the vegetation, as well as from humans (n = 17), dogs (n = 23), cats (n = 41), red deer (n = 8), a European rabbit and a European hedgehog at 25 locations in three cantons of south Switzerland. Tick species were identified morphologically, followed by DNA extraction and comparison of mitochondrial haplotypes with molecular-phylogenetic methods. Tick DNA extracts, as well as sixty-two rodent liver or spleen tissue DNA extracts (representing six species) available from 2005 to 2006 were screened for trypanosomes, Occidentia massiliensis and Borrelia miyamotoi. Morphologically, three tick species were identified: Ixodes ricinus (n = 170), Rhipicephalus sanguineus sensu lato (n = 6) and I. hexagonus (n = 1). In contrast to companion animals (dogs, cats) immature ticks (larvae and nymphs) predominated on humans, which was a highly significant association (P < 0.0001). Molecular comparison of the cytochrome c oxidase subunit I (cox1) gene with GenBank data established the species as R. sanguineus sensu stricto and confirmed I. hexagonus, both showing 99.8-100% sequence identity to conspecific ticks from northern Italy. Seventy-nine specimens morphologically identified as I. ricinus revealed high 16S rRNA gene haplotype diversity and represented two phylogenetic groups. Two I. ricinus haplotypes from Switzerland belonged to the same haplogroup with I. inopinatus from Spain, Germany and Austria as well as with I. ricinus reported from a broad geographical range of Europe (including Italy, the Netherlands, Poland, Latvia and Sweden). All 141 tick DNA extracts (from five R. sanguineus s.l., 135 I. ricinus and one I. hexagonus) and 62 rodent tissue DNA extracts were negative for trypanosomes and O. massiliensis. However, B. miyamotoi was identified in a bank vole (Myodes glareolus) and three ticks by sequencing. From Switzerland, this is the first report of tick haplotypes that are phylogenetically closely related to I. inopinatus. However, based on their morphology, both specimens are considered as I. ricinus. These results highlight the importance that the identification of I. inopinatus should be based on coherent morphologic and molecular properties. This is also the first report of rodent-borne B. miyamotoi in Switzerland. Taking into account the year of collection (2005), in a chronological order this might be the first indication of B. miyamotoi in any rodent species in Europe.

Curing Cats with Feline Infectious Peritonitis with an Oral Multi-Component Drug Containing GS-441524.

Feline infectious peritonitis (FIP) caused by feline coronavirus (FCoV) is a common dis-ease in cats, fatal if untreated, and no effective treatment is currently legally available. The aim of this study was to evaluate efficacy and toxicity of the multi-component drug Xraphconn® in vitro and as oral treatment in cats with spontaneous FIP by examining survival rate, development of clinical and laboratory parameters, viral loads, anti-FCoV antibodies, and adverse effects. Mass spectrometry and nuclear magnetic resonance identified GS-441524 as an active component of Xraphconn®. Eighteen cats with FIP were prospectively followed up while being treated orally for 84 days. Values of key parameters on each examination day were compared to values before treatment initiation using linear mixed-effect models. Xraphconn® displayed high virucidal activity in cell culture. All cats recovered with dramatic improvement of clinical and laboratory parameters and massive reduction in viral loads within the first few days of treatment without serious adverse effects. Oral treatment with Xraphconn® containing GS-441524 was highly effective for FIP without causing serious adverse effects. This drug is an excellent option for the oral treatment of FIP and should be trialed as potential effective treatment option for other severe coronavirus-associated diseases across species.