A cross-sectional serosurvey of SARS-CoV-2 and co-infections in stray cats from the second wave to the sixth wave of COVID-19 outbreaks in Spain.

Severe Acute Respiratory Syndrome Coronavirus 2 is the causative agent of Coronavirus Disease 2019 in humans. Among domestic animals, cats are more susceptible to SARS-CoV-2 than dogs. The detection of anti-SARS-CoV-2 antibodies in seemingly healthy cats and/or infected cats which are in close contact with infected humans has been described. The presence of animals that tested positive by serology or molecular techniques could represent a potential transmission pathway of SARS-CoV-2 that can spill over into urban wildlife. This study analyses the seroprevalence variation of SARS-CoV-2 in stray cats from different waves of outbreaks in a geographical area where previous seroepidemiological information of SARS-CoV-2 was available and investigate if SARS-CoV-2-seropositive cats were exposed to other co-infections causing an immunosuppressive status and/or a chronic disease that could lead to a SARS-CoV-2 susceptibility. For this purpose, a total of 254 stray cats from Zaragoza (Spain) were included. This analysis was carried out by the enzyme-linked immunosorbent assay using the receptor binding domain of Spike antigen and confirmed by serum virus neutralization assay. The presence of co-infections including Toxoplasma gondii, Leishmania infantum, Dirofilaria immitis, feline calicivirus, feline herpesvirus type 1, feline leukemia virus and feline immunodeficiency virus, was evaluated using different serological methods. A seropositivity of 1.57% was observed for SARS-CoV-2 including the presence of neutralizing antibodies in three cats. None of the seropositive to SARS-CoV-2 cats were positive to feline coronavirus, however, four SARS-CoV-2-seropositive cats were also seropositive to other pathogens such as L. infantum, D. immitis and FIV (n = 1), L. infantum and D. immitis (n = 1) and L. infantum alone (n = 1).Considering other pathogens, a seroprevalence of 16.54% was detected for L. infantum, 30.31% for D. immitis, 13.78%, for T. gondii, 83.86% for feline calicivirus, 42.52% for feline herpesvirus type 1, 3.15% for FeLV and 7.87% for FIV.Our findings suggest that the epidemiological role of stray cats in SARS-CoV-2 transmission is scarce, and there is no increase in seropositivity during the different waves of COVID-19 outbreaks in this group of animals. Further epidemiological surveillances are necessary to determine the risk that other animals might possess even though stray cats do not seem to play a role in transmission.

Serological evidence of SARS-CoV-2 and co-infections in stray cats in Spain.

A new coronavirus known as SARS-CoV-2 emerged in Wuhan in 2019 and spread rapidly to the rest of the world causing the pandemic disease named coronavirus disease of 2019 (COVID-19). Little information is known about the impact this virus can cause upon domestic and stray animals. The potential impact of SARS-CoV-2 has become of great interest in cats due to transmission among domestic cats and the severe phenotypes described recently in a domestic cat. In this context, there is a public health warning that needs to be investigated in relation with the epidemiological role of this virus in stray cats. Consequently, in order to know the impact of the possible transmission chain, blood samples were obtained from 114 stray cats in the city of Zaragoza (Spain) and tested for SARS-CoV-2 and other selected pathogens susceptible to immunosuppression including Toxoplasma gondii, Leishmania infantum, feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV) from January to October 2020. Four cats (3.51%), based on enzyme-linked immunosorbent assay (ELISA) using the receptor binding domain (RBD) of Spike antigen, were seroreactive to SARS-CoV-2. T. gondii, L. infantum, FeLV and FIV seroprevalence was 12.28%, 16.67%, 4.39% and 19.30%, respectively. Among seropositive cats to SARS-CoV-2, three cats were also seropositive to other pathogens including antibodies detected against T. gondii and FIV (n = 1); T. gondii (n = 1); and FIV and L. infantum (n = 1). The subjects giving positive for SARS-CoV-2 were captured in urban areas of the city in different months: January 2020 (2/4), February 2020 (1/4) and July 2020 (1/4). This study revealed, for the first time, the exposure of stray cats to SARS-CoV-2 in Spain and the existence of concomitant infections with other pathogens including T. gondii, L. infantum and FIV, suggesting that immunosuppressed animals might be especially susceptible to SARS-CoV-2 infection.

Clinical leishmaniosis in a domestic ferret (Mustela putorius furo) treated with miltefosine plus allopurinol: Serological and clinical follow-up.

The published information on the treatment of mustelid leishmaniosis is extremely scarce because there are only two case reports available. In one case, a domestic ferret (Mustela putorius furo) was treated with a combination of meglumine antimoniate plus allopurinol and, in the other case, a therapeutic regimen with allopurinol was administrated to a Eurasian otter (Lutra lutra). This article describes for the first time a combined therapeutic protocol with miltefosine (2 mg/kg once a day during 28 days per os), and allopurinol (10 mg/kg twice a day PO sine die) in a domestic ferret with splenomegaly, lymphadenomegaly and a facial pyogranulomatous dermatitis, with a moderate level of antibodies to Leishmania infantum.

A cross-sectional study of Leishmania infantum infection in stray cats in the city of Zaragoza (Spain) using serology and PCR.

BACKGROUND: Feline leishmaniosis is a vector-borne parasitic disease caused by Leishmania spp. Leishmania infection in dogs is prevalent in the Mediterranean basin, but in other animals, such as cats, it could also play a role in the epidemiology of the disease. Information on the geographical distribution and epidemiological features of L. infantum infection in cats is scarce, particularly in urban stray cats living in regions where canine leishmaniosis is endemic. As diagnosis can be challenging, combining different serological and molecular methods is a useful approach. Our aim was to investigate the prevalence of infection of L. infantum in apparently healthy stray cats in an endemic region of Spain (Zaragoza city) using serological and molecular methods, and to compare the results of the different techniques. METHODS: The prevalence of Leishmania infection was studied in stray cats captured in urban and peri-urban areas of Zaragoza. Blood was collected from each animal for serology and molecular analysis. Three serological methods, namely the immunofluorescent antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and western blot (WB), were used to detect L. infantum antibodies and a real-time PCR (qPCR) assay was used to detect L. infantum DNA. The results were analyzed by Fisher's exact test and Cohen's kappa statistic (κ) to assess the level of agreement between the diagnostic techniques. RESULTS: Serological analysis of blood samples from 180 stray cats revealed 2.2% (4/179) Leishmania infection positivity by IFAT, 2.8% (5/179) by ELISA and 14.5% (26/179) by WB. Leishmania DNA was detected by qPCR in 5.6% (10/179) of the cats. Sixteen cats (8.9%) tested positive by only one serological technique and four tested positive by all three serological methods used. The overall rate of infected cats (calculated as the number of cats seropositive and/or qPCR positive) was 15.6%, and only two cats tested positive by all the diagnostic methods. A significant association was found between male cats and a positive qPCR result. Comparison of the techniques revealed a fair agreement in seropositivity between blood qPCR and IFAT (κ = 0.26), blood qPCR and ELISA (κ = 0.24), WB and ELISA (κ = 0.37) and WB and IFAT (κ = 0.40). The highest agreement between seropositive results was between IFAT and ELISA (κ = 0.89), and the lowest was between blood qPCR and WB (κ = 0.19). The prevalence of the feline leukemia virus antigen was 4.49% (8/178 cats) and that of the feline immunodeficiency virus (FIV) antibody was 6.74% (12/178), while co-infection with both retroviruses was observed in one female cat (1/178). Leishmania ELISA and IFAT seropositivity were statistically associated with FIV status by the chi-square test. CONCLUSIONS: The results obtained in this study, using serological tests and qPCR, indicate the existence of L. infantum asymptomatic infection in apparently healthy stray cats in the city of Zaragoza, an endemic area in Spain.

Treatment and follow-up of a domestic ferret (Mustela putorius furo) with clinical leishmaniosis caused by Leishmania infantum.

Leishmania infantum infection including treatment and follow up in domestic animals other than dogs and cats has not been described at this moment. This article describes the anti-Leishmania treatment and follow-up of a ferret (Mustela putorius furo) with leishmaniosis. A combined therapeutic protocol established for the patient, not yet approved for ferrets, was a combination of meglumine antimoniate plus allopurinol. A follow-up was established monthly during the first year in order to monitor the health condition of the patient. Six months after commencing allopurinol therapy, xanthine crystalluria was observed in urine sediment with no other urine alterations detected by urine analysis. The ferret worsened progressively with diarrhoea and weight loss after cohabiting with another ferret diagnosed with cryptosporidiosis. Cryptosporidium parvum was isolated in faecal samples from the patient detected by three different methods including Ziehl-Neelsen staining, a qualitative test to detection of C. parvum antigens and finally a specific molecular analysis to characterize the species. To the best of the authors´ knowledge, this is the first report providing information about anti-Leishmania protocol therapy used and follow-up in a domestic ferret with clinical leishmaniosis. Veterinarians practicing in endemic areas should be aware of this infection in ferrets at risk and their susceptibility especially when immunosuppressive conditions are present.

Evaluation of the performance of three serological tests for diagnosis of Leishmania infantum infection in dogs using latent class analysis / Avaliação da realização de três testes sorológicos para diagnóstico de infecção por Leishmania infantum em cães utilizando análise de classe latente

Abstract Canine leishmaniasis (CanL) is a disease caused by Leishmania infantum. Serological methods are the most common diagnostic techniques used for the diagnosis of the CanL. The objective of our study was to estimate the sensitivity and specificity of one in-house ELISA kit (ELISA UNIZAR) and three commercially available serological tests (MEGACOR Diagnostik GmbH) including an immunochromatographic rapid test (FASTest LEISH®), an immunofluorescent antibody test (MegaFLUO LEISH®) and an enzyme-linked immunosorbent assay (MegaELISA LEISH®), using latent class models in a Bayesian analysis. Two hundred fifteen serum samples were included. The highest sensitivity was achieved for FASTest LEISH® (99.38%), ELISA UNIZAR (99.37%), MegaFLUO LEISH® (99.36%) followed by MegaELISA LEISH® (98.49%). The best specificity was obtained by FASTest LEISH® (98.43%), followed by ELISA UNIZAR (97.50%), whilst MegaFLUO LEISH® and MegaELISA LEISH® obtained the lower specificity (91.94% and 91.93%, respectively). The results of present study indicate that the immunochromatographic rapid test evaluated FASTest LEISH® show similar levels of sensitivity and specificity to the quantitative commercial tests. Among quantitative serological tests, sensitivity and specificity were similar considering ELISA or IFAT techniques.

Resumo A leishmaniose canina (Lcan) é uma causada pela Leishmania infantum. Os métodos sorológicos são as técnicas diagnósticas mais utilizadas para o diagnóstico da leishmaniose canina. O objetivo do nosso estudo foi estimar a sensibilidade e a especificidade de um kit ELISA interno (ELISA UNIZAR) e de três testes sorológicos disponíveis comercialmente, feitos pelo mesmo fabricante (MEGACOR Diagnostik GmbH), incluindo um teste rápido imunocromatográfico (FASTest LEISH®), um teste de anticorpos imunofluorescentes (Megafluo LEISH®) e um ensaio de imunoabsorção enzimática (Megaelisa LEISH®), utilizando-se modelos de classe latentes numa análise bayesiana. Foram incluídas duzentas e quinze amostras de soro. A maior sensibilidade foi alcançada para Fastest LEISH® (99,38%), ELISA UNIZAR (99,37%), Megafluo LEISH® (99,36%) seguida por Megaelisa LEISH® (98,49%). A melhor especificidade foi obtida por FASTest LEISH® (98,43%), seguida por ELISA UNIZAR (97,50%), enquanto Megafluo LEISH® e Megaelisa LEISH® obtiveram a menor especificidade (91,94% e 91,93%, respectivamente). Os resultados do presente estudo indicam que o teste rápido imunocromatográfico, avaliado por FASTest LEISH® mostra níveis similares de sensibilidade e especificidade aos testes comerciais quantitativos incluídos. Entre os testes sorológicos quantitativos, a sensibilidade e a especificidade foram semelhantes, considerando-se as técnicas de ELISA ou IFI.