Recently, a gammaherpesvirus was described in domestic cats (FcaGHV1). The goal of the present study was to investigate the presence of FcaGHV1 in Swiss domestic cats and analyze potential risk ...factors. Blood samples from 881 cats presented to veterinarians in all Swiss cantons and from 91 stray cats and neoplastic tissue samples from 17 cats with lymphoma were evaluated. FcaGHV1 was detected by real-time PCR targeting the glycoprotein B gene, followed by sequencing. Blood samples were also tested for feline hemoplasmas, feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV). The molecular prevalence of FcaGHV1 was 6.0% (95% confidence interval (CI), 4.5-7.8%) in cats presented to veterinarians and 5.5% (95% CI, 1.8-12.4%) in stray cats. FcaGHV1 PCR-positive cats originated from 19/26 Swiss cantons. Factors significantly associated with FcaGHV1 detection included male sex, age >3 years, nonpedigree status and co-infection with FIV and hemoplasmas. Moreover, FeLV viremia tended to be associated with FcaGHV1 detection. High FcaGHV1 blood loads were found more frequently in FeLV-viremic cats and less frequently in hemoplasma-infected cats than in uninfected cats. Clinical information was unavailable for most of the 881 cats, but leukemia, carcinoma and cardiomyopathy were reported in FcaGHV1-positive cats. None of the tissue samples from the 17 cats with lymphoma tested positive for FcaGHV1. Sequence analyses revealed homogeneity among the Swiss isolates and >99.7% identity to published FcaGHV1 sequences. In conclusion, FcaGHV1 is present in Switzerland with a similar prevalence in cats presented to veterinarians and in stray cats. The pathogenic potential of FcaGHV1 needs further evaluation.
Recently, there has been a growing interest in hemotropic mycoplasmal species (also known as the hemoplasmas), the causative agents of infectious anemia in several mammalian species. In felids, two ...different hemoplasma species have been recognized: Mycoplasma haemofelis (formerly Haemobartonella felis) and "Candidatus Mycoplasma haemominutum." Recently developed molecular methods have allowed sensitive and specific identification and quantification of these agents in feline blood samples. In applying these methods to an epidemiological study surveying the Swiss pet cat population for hemoplasma infection, we discovered a third novel and unique feline hemoplasma isolate in a blood sample collected from a cat that had exhibited clinical signs of severe hemolytic anemia. This agent was readily transmitted via intravenous inoculation to two specific-pathogen-free cats. One of these cats was immunocompromised by the administration of methylprednisolone acetate prior to inoculation, and this cat developed severe anemia. The other immunocompetent cat showed a moderate decrease in packed cell volume. Additionally, an increase in red blood cell osmotic fragility was observed. Sequencing of the entire 16S rRNA gene of the new hemoplasma isolate and phylogenetic analysis showed that the isolate was most closely related to two rodent hemotropic mycoplasmal species, M. coccoides and M. haemomuris. A quantitative real-time PCR assay specific for this newly discovered agent was developed, which will be a prerequisite for the diagnosis of infections with the new hemoplasma isolate.
Rickettsia helvetica, a tick-borne member of the spotted-fever-group rickettsiae, is a suspected pathogen in humans; however, its role in animals is unknown. The aims of this study were to establish ...a R. helvetica-specific real-time TaqMan PCR assay and apply it to the analysis of tick vectors (to determine potential exposure risk) and blood samples from Canidae and humans (to determine prevalence of infection). The newly designed 23S rRNA gene assay for R. helvetica was more sensitive than a published citrate synthase gene (gltA) assay for several rickettsiae. Blood samples from 884 dogs, 58 foxes, and 214 human patients and 2,073 ticks (Ixodes spp.) collected from either vegetation or animals were analyzed. Although the maximal likelihood estimate of prevalence was 12% in unfed ticks and 36% in ticks collected from animals, none of the 1,156 blood samples tested PCR positive. Ticks from cats were more frequently PCR positive than ticks from dogs. Sequencing of the 23S rRNA and/or the gltA gene of 17 tick pools confirmed the presence of R. helvetica. Additionally, Rickettsia monacensis, which has not been previously found in Switzerland, was identified. In conclusion, R. helvetica was frequently detected in the tick population but not in blood samples. Nevertheless, due to the broad host range of Ixodes ticks and the high rate of infestation with this agent (i.e., R. helvetica was 13 times more frequent in unfed ticks than the tick-borne encephalitis virus), many mammals may be exposed to R. helvetica. The PCR assay described here represents an important tool for studying this topic.
•We evaluated pre-analytical conditions and two RT-qPCR assays for the FCV detection.•Addition of a viral transport medium increases FCV stability in the sample.•FCV-detection after cell culture is ...more sensitive than direct RT-qPCR from swabs.•A combination of different RT-qPCR assays increases sensitivity of FCV detection.
Feline caliciviruses (FCVs) are non-enveloped RNA viruses that exhibit high genetic variation. Two reverse transcription quantitative polymerase chain reaction (RT-qPCR) FCV assays (S1 and S2) were evaluated using samples from 300 field cats. The direct detection of FCV in swabs and after propagation in cell culture, as well as the influence of storage conditions, was assessed. FCV-RNA detectability on dry swabs was similar after storage at either 4°C or −20°C, but viral burdens were maintained for a longer time period when viral transport medium was used. A total of 97 (32%) samples was considered FCV PCR-positive. Of these, 81% and 77% tested positive directly from swabs using S1 and S2, respectively; 84% and 81% tested positive after enrichment in cell culture, respectively. Combined detection by RT-PCR directly from swabs and after VI was most sensitive (up to 96%). Neither of the methods alone were able to detect all FCV-positive samples. In conclusion, clinical samples should be collected in viral transport medium, stored at ≤4°C and processed as soon as possible. The combination of cell culture with RT-qPCR or detection directly from swabs using a combination of different RT-qPCR assays is recommended to reach a high sensitivity of FCV detection.
•First molecular report and characterization of Mycoplasma haemocanis and ‘Candidatus mycoplasma haematoparvum’ isolates in dogs (Canis lupus familiaris) from cuba.•Overall, PCR results revealed ...15.1% (59/391; 95% CI: 11.5–18.7) positive dogs 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) showed dual infection.•All tested rhipicephalus sanguineus sensu lato tick samples were PCR-negative, but the occurrence of tick infestation was significantly associated with canine haemoplasma infection in dogs.•DNA sequencing and phylogenetic analysis based on 16S rRNA gene sequences revealed low genetic variability amongst Mycoplasma haemocanis and ‘Candidatus mycoplasma haematoparvum’ isolates 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.
Concomitantly with an outbreak of fatal anaplasmosis in a cattle herd in Switzerland in 2002, we detected two bovine hemoplasma species in diseased animals: Mycoplasma wenyonii (formerly ...Eperythrozoon wenyonii) and a second, novel bovine hemoplasma species later designated "Candidatus Mycoplasma haemobos" (synonym, "Candidatus Mycoplasma haemobovis"). The second species was characterized by a shorter 16S rRNA gene. The aims of the present study were to provide a detailed molecular characterization of this species, to develop specific quantitative real-time PCR assays for the two bovine hemoplasma species, and to apply these assays in order to evaluate the prevalence and clinical significance of the hemoplasmas. Sequencing of the near-complete 16S rRNA gene of the second hemoplasma revealed that it was 94% identical to that of Mycoplasma haemofelis, an anemia-inducing feline hemoplasma species, but less than 85% identical to that of the bovine hemoplasma M. wenyonii. Using the newly developed assays, a total of 159 animals from the anaplasmosis outbreak were reexamined. In addition, we tested 57 clinically ill and 61 healthy Swiss cattle, as well as 47 calves. Both hemoplasmas were highly prevalent in adult cattle but occurred rarely in calves. Animals from the herd with the fatal anemia outbreak were more frequently infected with M. wenyonii and exhibited higher M. wenyonii blood loads than animals with unrelated diseases and healthy animals. Coinfections may increase the pathogenicity and clinical significance of bovine hemoplasmosis.
Hemotropic mycoplasmas (hemoplasmas) are the causative agents of infectious anemia in several mammalian species. Their zoonotic potential has recently been substantiated by the identification of a ...feline hemoplasma isolate in an immunocompromised human patient. Although species-specific diagnostic molecular methods have been developed, their application as screening tools is limited due to the species diversity of hemoplasmas. The goals of this study were to develop a universal hemoplasma screening assay with broad specificity based on the SYBR green PCR principle, to compare the assay with hemoplasma-specific TaqMan PCR, and to analyze potential tick vectors and human blood samples to address the zoonotic potential. The newly developed PCR assay based on the 16S rRNA gene amplified feline, canine, bovine, porcine, camelid, and murine hemoplasmas, as well as Mycoplasma penetrans and Mycoplasma pneumoniae. The lower detection limit for feline and canine hemoplasmas was 1 to 10 copies/PCR. The assay exhibited 98.2% diagnostic sensitivity and 92.1% diagnostic specificity for feline hemoplasmas. All 1,950 Ixodes ticks were PCR negative, suggesting that Ixodes ticks are not relevant vectors for the above-mentioned hemoplasma species in Switzerland. None of the 414 blood samples derived from anemic or immunocompromised human patients revealed a clear positive result. The SYBR green PCR assay described here is a suitable tool to screen for known and so-far-undiscovered hemoplasma species. Positive results should be confirmed by specific TaqMan PCR or sequencing.
Feline Infectious Peritonitis (FIP)—the deadliest infectious disease of young cats in shelters or catteries—is induced by highly virulent feline coronaviruses (FCoVs) emerging in infected hosts after ...mutations of less virulent FCoVs. Previous studies have shown that some mutations in the open reading frames (ORF) 3c and 7b and the spike (S) gene have implications for the development of FIP, but mainly indirectly, likely also due to their association with systemic spread. The aim of the present study was to determine whether FCoV detected in organs of experimentally FCoV infected healthy cats carry some of these mutations. Viral RNA isolated from different tissues of seven asymptomatic cats infected with the field strains FCoV Zu1 or FCoV Zu3 was sequenced. Deletions in the 3c gene and mutations in the 7b and S genes that have been shown to have implications for the development of FIP were not detected, suggesting that these are not essential for systemic viral dissemination. However, deletions and single nucleotide polymorphisms leading to truncations were detected in all nonstructural proteins. These were found across all analyzed ORFs, but with significantly higher frequency in ORF 7b than ORF 3a. Additionally, a previously unknown homologous recombination site was detected in FCoV Zu1.
Two hemoplasma species are known in dogs: Mycoplasma haemocanis (Mhc) and ‘Candidatus Mycoplasma haematoparvum’ (CMhp). Although their transmission routes are poorly understood, Rhipicephalus ...sanguineus has been suggested as a potential tick vector. The aim of the present study was to assess the prevalence, risk factors, and clinical importance of canine hemoplasmas in countries with a Mediterranean climate where R. sanguineus is highly prevalent using TaqMan real-time PCR, and to molecularly characterize the identified isolates. DNA (canine glyceraldehyde-3-phosphate dehydrogenase) was successfully amplified from all samples collected from 850 dogs in Italy, Spain, and Portugal, and 82 (9.6%) were PCR-positive for canine hemoplasmas (43 Mhc, 34 CMhp and 5 co-infected). The hemoplasma sample prevalence was significantly higher in Portugal (40%) than in Italy (9.5%) and Spain (2.5%). Risk factors for infection included living in kennels, young age, crossbreeding, and mange infection. No association was found with anemia. Phylogenetic analyses of the 16S rRNA and RNase P genes revealed >99% identity to other European isolates. In conclusion, canine hemoplasma infections were readily encountered in Mediterranean countries. The climate and living conditions seemed to influence canine hemoplasma prevalence. The clinical importance of canine hemoplasma infections appeared to be low, but the infection stage of the presented dogs was unknown.
Feline parvovirus (FPV) causes severe gastroenteritis and leukopenia in cats; the outcome is poor. Information regarding specific treatments is lacking. Class A CpG oligodeoxynucleotides (CpG-A) are ...short single-stranded DNAs, stimulating type I interferon production. In cats, CpG-A induced an antiviral response in vivo and inhibited FPV replication in vitro. The aim was to prospectively investigate the effects of CpG-A on survival, clinical score, hematological findings, antiviral response (cytokines), viremia, and fecal shedding (real-time qPCR) in cats naturally infected with FPV. Forty-two FPV-infected cats were randomized to receive 100 µg/kg of CpG-A (
= 22) or placebo (
= 20) subcutaneously, on admission and after 48 h. Blood and fecal samples were collected on admission, after 1, 3, and 7 days. All 22 cats showed short duration pain during CpG-A injections. The survival rate, clinical score, leukocyte and erythrocyte counts, viremia, and fecal shedding at any time-point did not differ between cats treated with CpG-A (50%) and placebo (40%). Antiviral myxovirus resistance (
) gene transcription increased in both groups from day 1 to 3 (
= 0.005). Antibodies against FPV on admission were associated with survival in cats (
= 0.002). In conclusion, CpG-A treatment did not improve the outcome in cats with FPV infection. FPV infection produced an antiviral response.
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