Barbara Willi

Prevalence, Risk Factor Analysis, and Follow-Up of Infections Caused by Three Feline Hemoplasma Species in Cats in Switzerland

ABSTRACT Recently, a third novel feline hemotropic Mycoplasma sp. (aka hemoplasma), “Candidatus Mycoplasma turicensis,” in a cat with hemolytic anemia has been described. This is the first study to investigate the prevalence, clinical manifestations, and risk factors for all three feline hemoplasma infections in a sample of 713 healthy and ill Swiss cats using newly designed quantitative real-time PCR assays. “Candidatus Mycoplasma haemominutum” infection was detected in 7.0% and 8.7% and Mycoplasma haemofelis was detected in 2.3% and 0.2% of healthy and ill cats, respectively. “Candidatus Mycoplasma turicensis” was only detected in six ill cats (1.1%); three of them were coinfected with “Candidatus Mycoplasma haemominutum.” The 16S rRNA gene sequence of 12 Swiss hemoplasma isolates revealed >98% similarity with previously published sequences. Hemoplasma infection was associated with male gender, outdoor access, and old age but not with retrovirus infection and was more frequent in certain areas of Switzerland. “Candidatus Mycoplasma haemominutum”-infected ill cats were more frequently diagnosed with renal insufficiency and exhibited higher renal blood parameters than uninfected ill cats. No correlation between hemoplasma load and packed cell volume was found, although several hemoplasma-infected cats, some coinfected with feline immunodeficiency virus or feline leukemia virus, showed hemolytic anemia. High M. haemofelis loads (>9 × 105 copies/ml blood) seem to lead to anemia in acutely infected cats but not in recovered long-term carriers. A repeated evaluation of 17 cats documented that the infection was acquired in one case by blood transfusion and that there were important differences among species regarding whether or not antibiotic administration led to the resolution of bacteremia.

Identification, Molecular Characterization, and Experimental Transmission of a New Hemoplasma Isolate from a Cat with Hemolytic Anemia in Switzerland

ABSTRACT 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.

Concurrent Infections with Vector-Borne Pathogens Associated with Fatal Hemolytic Anemia in a Cattle Herd in Switzerland

ABSTRACT Bovine anaplasmosis is a vector-borne disease that results in substantial economic losses in other parts of the world but so far not in northern Europe. In August 2002, a fatal disease outbreak was reported in a large dairy herd in the Swiss canton of Grisons. Diseased animals experienced fever, anorexia, agalactia, and depression. Anemia, ectoparasite infestation, and, occasionally, hemoglobinuria were observed. To determine the roles of vector-borne pathogens and to characterize the disease, blood samples were collected from all 286 animals: 50% of the cows were anemic. Upon microscopic examination of red blood cells, Anaplasma marginale inclusion bodies were found in 47% of the cows. The infection was confirmed serologically and by molecular methods. Interestingly, we also found evidence of infections with Anaplasma phagocytophilum, large Babesia and Theileria spp., and Mycoplasma wenyonii. The last two species had not previously been described in Switzerland. Anemia was significantly associated with the presence of the infectious agents detected, with the exception of A. phagocytophilum. Remarkably, concurrent infections with up to five infectious vector-borne agents were detected in 90% of the ill animals tested by PCR. We concluded that A. marginale was the major cause of the hemolytic anemia, while coinfections with other agents exacerbated the disease. This was the first severe disease outbreak associated with concurrent infections with vector-borne pathogens in alpine Switzerland; it was presumably curtailed by culling of the entire herd. It remains to be seen whether similar disease outbreaks will have to be anticipated in northern Europe in the future.

Phylogenetic Analysis of “Candidatus Mycoplasma turicensis” Isolates from Pet Cats in the United Kingdom, Australia, and South Africa, with Analysis of Risk Factors for Infection

ABSTRACT Two hemotropic mycoplasmas have been recognized in cats, Mycoplasma haemofelis and “Candidatus Mycoplasma haemominutum.” We recently described a third feline hemoplasma species, designated “Candidatus Mycoplasma turicensis,” in a Swiss cat with hemolytic anemia. This isolate induced anemia after experimental transmission to two specific-pathogen-free cats and analysis of the 16S rRNA gene revealed its close relationship to rodent hemotropic mycoplasmas. The agent was recently shown to be prevalent in Swiss pet cats. We sought to investigate the presence and clinical importance of “Candidatus Mycoplasma turicensis” infection in pet cats outside of Switzerland and to perform the molecular characterization of isolates from different countries. A “Candidatus Mycoplasma turicensis”-specific real-time PCR assay was applied to blood samples from 426 United Kingdom (UK), 147 Australian, and 69 South African pet cats. The 16S rRNA genes of isolates from different countries were sequenced and signalment and laboratory data for the cats were evaluated for associations with “Candidatus Mycoplasma turicensis” infection. Infections were detected in samples from UK, Australian, and South African pet cats. Infection was associated with the male gender, and “Candidatus Mycoplasma haemominutum” and M. haemofelis coinfection. Coinfected cats exhibited significantly lower packed cell volume (PCV) values than uninfected cats. Phylogenetic analyses revealed that some Australian and South African “Candidatus Mycoplasma turicensis” isolates branched away from the remaining isolates. In summary, “Candidatus Mycoplasma turicensis” infection in pet cats exists over a wide geographical area and significantly decreased PCV values are observed in cats coinfected with other feline hemoplasmas.

Worldwide Occurrence of Feline Hemoplasma Infections in Wild Felid Species

ABSTRACT While hemoplasma infections in domestic cats are well studied, almost no information is available on their occurrence in wild felids. The aims of the present study were to investigate wild felid species as possible reservoirs of feline hemoplasmas and the molecular characterization of the hemoplasma isolates. Blood samples from the following 257 wild felids were analyzed: 35 Iberian lynxes from Spain, 36 Eurasian lynxes from Switzerland, 31 European wildcats from France, 45 lions from Tanzania, and 110 Brazilian wild felids, including 12 wild felid species kept in zoos and one free-ranging ocelot. Using real-time PCR, feline hemoplasmas were detected in samples of the following species: Iberian lynx, Eurasian lynx, European wildcat, lion, puma, oncilla, Geoffroys cat, margay, and ocelot. “Candidatus Mycoplasma haemominutum” was the most common feline hemoplasma in Iberian lynxes, Eurasian lynxes, Serengeti lions, and Brazilian wild felids, whereas “Candidatus Mycoplasma turicensis” was the most prevalent in European wildcats; hemoplasma coinfections were frequently observed. Hemoplasma infection was associated with species and free-ranging status of the felids in all animals and with feline leukemia virus provirus-positive status in European wildcats. Phylogenetic analyses of the 16S rRNA and the partial RNase P gene revealed that most hemoplasma isolates exhibit high sequence identities to domestic cat-derived isolates, although some isolates form different subclusters within the phylogenetic tree. In conclusion, 9 out of 15 wild felid species from three different continents were found to be infected with feline hemoplasmas. The effect of feline hemoplasma infections on wild felid populations needs to be further investigated.

Feline leukemia virus and other pathogens as important threats to the survival of the critically endangered Iberian lynx (Lynx pardinus)

Background The Iberian lynx (Lynx pardinus) is considered the most endangered felid species in the world. In order to save this species, the Spanish authorities implemented a captive breeding program recruiting lynxes from the wild. In this context, a retrospective survey on prevalence of selected feline pathogens in free-ranging lynxes was initiated. Methodology/ Principal Findings We systematically analyzed the prevalence and importance of seven viral, one protozoan (Cytauxzoon felis), and several bacterial (e.g., hemotropic mycoplasma) infections in 77 of approximately 200 remaining free-ranging Iberian lynxes of the Doñana and Sierra Morena areas, in Southern Spain, between 2003 and 2007. With the exception of feline immunodeficiency virus (FIV), evidence of infection by all tested feline pathogens was found in Iberian lynxes. Fourteen lynxes were feline leukemia virus (FeLV) provirus-positive; eleven of these were antigenemic (FeLV p27 positive). All 14 animals tested negative for other viral infections. During a six-month period in 2007, six of the provirus-positive antigenemic lynxes died. Infection with FeLV but not with other infectious agents was associated with mortality (p<0.001). Sequencing of the FeLV surface glycoprotein gene revealed a common origin for ten of the eleven samples. The ten sequences were closely related to FeLV-A/61E, originally isolated from cats in the USA. Endogenous FeLV sequences were not detected. Conclusions/Significance It was concluded that the FeLV infection most likely originated from domestic cats invading the lynxs habitats. Data available regarding the time frame, co-infections, and outcome of FeLV-infections suggest that, in contrast to the domestic cat, the FeLV strain affecting the lynxes in 2007 is highly virulent to this species. Our data argue strongly for vaccination of lynxes and domestic cats in and around lynxs habitats in order to prevent further spread of the virus as well as reduction the domestic cat population if the lynx population is to be maintained.

Molecular Investigations of Rickettsia helvetica Infection in Dogs, Foxes, Humans, and Ixodes Ticks

ABSTRACT 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.

In vivo transmission studies of 'Candidatus Mycoplasma turicensis' in the domestic cat

The natural transmission routes of the three feline haemotropic mycoplasmas – Mycoplasma haemofelis, ‘Candidatus Mycoplasma haemominutum’, and ‘Candidatus Mycoplasma turicensis’ (CMt) – are largely unknown. Since CMt has been detected in the saliva of infected cats using PCR, we hypothesised that direct transmission via social or aggressive contact may occur. The aim of this study was to evaluate this transmission route. CMt-positive saliva and blood samples were obtained from three prednisolone-treated specific pathogen-free (SPF) cats that were infected intraperitoneally with CMt. Five SPF cats were inoculated with CMt-positive saliva or blood subcutaneously to mimic cat bites, and five cats were inoculated orally with blood or oronasally with saliva to mimic social contact. Blood samples were monitored for CMt infection using quantitative real-time PCR and for seroconversion using a novel western blot assay. Neither oronasal nor subcutaneous inoculation with CMt-positive saliva led to CMt infection in the recipient cats, as determined by PCR, independent of prior prednisolone treatment. However, when blood containing the same CMt dose was given subcutaneously, 4 of the 5 cats became PCR-positive, while none of the 5 cats inoculated orally with up to 500 μL of CMt-positive blood became PCR-positive. Subsequently, the latter cats were successfully subcutaneously infected with blood. All 13 CMt-exposed cats seroconverted. In conclusion, CMt transmission by social contact seems less likely than transmission by aggressive interaction. The latter transmission may occur if the recipient cat is exposed to blood from an infected cat.

Real-Time PCR Investigation of Potential Vectors, Reservoirs, and Shedding Patterns of Feline Hemotropic Mycoplasmas

ABSTRACT Three hemotropic mycoplasmas have been identified in pet cats: Mycoplasma haemofelis, “Candidatus Mycoplasma haemominutum,” and “Candidatus Mycoplasma turicensis.” The way in which these agents are transmitted is largely unknown. Thus, this study aimed to investigate fleas, ticks, and rodents as well as saliva and feces from infected cats for the presence of hemotropic mycoplasmas, to gain insight into potential transmission routes for these agents. DNA was extracted from arthropods and from rodent blood or tissue samples from Switzerland and from salivary and fecal swabs from two experimentally infected and six naturally infected cats. All samples were analyzed with real-time PCR, and some positive samples were confirmed by sequencing. Feline hemotropic mycoplasmas were detected in cat fleas and in a few Ixodes sp. and Rhipicephalus sp. ticks collected from animals but not in ticks collected from vegetation or from rodent samples, although the latter were frequently Mycoplasma coccoides PCR positive. When shedding patterns of feline hemotropic mycoplasmas were investigated, “Ca. Mycoplasma turicensis” DNA was detected in saliva and feces at the early but not at the late phase of infection. M. haemofelis and “Ca. Mycoplasma haemominutum” DNA was not amplified from saliva and feces of naturally infected cats, despite high hemotropic mycoplasma blood loads. Our results suggest that besides an ostensibly indirect transmission by fleas, direct transmission through saliva and feces at the early phase of infection could play a role in the epizootiology of feline hemotropic mycoplasmas. Neither the investigated tick nor the rodent population seems to represent a major reservoir for feline hemotropic mycoplasmas in Switzerland.

Description of outcomes of experimental infection with feline haemoplasmas: Copy numbers, haematology, Coombs' testing and blood glucose concentrations

The aim of this study was to compare blood copy, haematological and glucose values between cats experimentally infected with either Mycoplasma haemofelis (Group HF: 10 cats), ‘Candidatus M. haemominutum’ (Group HM: 3 cats) or ‘Candidatus M. turicensis’ (Group TU: 3 cats). Blood samples were collected regularly up to 85 days post-infection (DPI) for haemoplasma real-time quantitative PCR, haematology, Coombs’ testing and blood glucose measurement. Statistical analysis was performed using a general linear model (ANOVA) appropriate for a repeated measures experiment with significance set as P < 0.05. Cats in Group TU had significantly lower blood copy numbers than cats in Group HF (P < 0.001) and HM (P < 0.001). All Group HF cats developed anaemia (often severe), macrocytosis and evidence of erythrocyte-bound antibodies whereas Groups HM and TU cats did not. Group HF had significantly lower PCVs, haemoglobin concentrations and red blood cell counts, and significantly higher mean cell volumes, than Groups HM and TU. In Group HF, erythrocyte-bound antibodies reactive at 4 °C (both IgM and IgG) appeared between 8 and 22 DPI and persisted for two to four weeks, whereas those reactive at 37 °C (primarily IgG) appeared between 22 and 29 DPI and persisted for one to five weeks. In most cats antibodies appeared after the fall in haemoglobin started. Although Group TU had significantly lower glucose concentrations than Groups HF (P = 0.006) and HM (P = 0.027), mean blood glucose concentrations remained within the reference range in all groups. This study demonstrates that M. haemofelis infection, in contrast to ‘Candidatus M. haemominutum’ and ‘Candidatus M. turicensis’ infection, can result in a severe macrocytic anaemia and the development of cold and warm reactive erythrocyte-bound antibodies.