CLINICAL AND SUBCLINICAL CYTAUXZOON FELIS INFECTIONS IN DOMESTIC CATS FROM A RECENTLY IDENTIFIED ENDEMIC REGION.

Cytauxzoon felis is a tick-transmitted intraerythrocytic apicomplexan infecting felids in the southeastern and midwestern United States. Bobcats (Lynx rufus) are the natural wildlife reservoir of C. felis, where in enzootic areas prevalence can reach 100%. Domestic cats can be subclinically infected with C. felis or can develop cytauxzoonosis. Two studies have documented the presence of C. felis in domestic cats in Illinois; these studies have shown a limited number of cases submitted to specialty labs. During 2014-2018, we obtained blood samples collected by veterinary staff from 514 cats that were apparently healthy and 74 cats that were suspected of cytauxzoonosis. These samples were screened using a sensitive, nested PCR to detect the presence of C. felis DNA. We herein document frequent occurrences of cytauxzoonosis (8-15 cases/year from 4 veterinary clinics) and 12.5% prevalence of subclinical infections in southern Illinois, a locality showing a sharp increase in cases of cytauxzoonosis. Our results suggest a high risk of cytauxzoonosis in southern Illinois, despite only recently being recognized in the area. We found no specific risk factors for cytauxzoonosis or subclinical infections in this location. In addition, cases of cytauxzoonosis occur every month of the year (with the highest frequency in summer) and therefore tick prevention should be used in domestic cats in enzootic regions throughout the year.

Molecular detection of piroplasmids in mammals from the Superorder Xenarthra in Brazil.

Xenarthra mammals can be found from southern North America to southern South America, including all Brazilian biomes. Although it has been shown that Xenarthra mammals can play a role as reservoirs for several zoonotic agents, few studies investigate the diversity of piroplasmids (Apicomplexa: Piroplasmida) in this group of mammals. Taking into account that piroplasmids can cause disease in animals and humans, understanding the prevalence and diversity of piroplasmids in Xenarthra mammals would contribute to conservation efforts for this group of animals as well as to infer risk areas for transmission of emergent zoonosis. The present study aimed to investigate the occurrence and molecular identity of piroplasmids in free-living mammals of the Superorder Xenarthra from four Brazilian states (Mato Grosso do Sul, São Paulo, Rondônia, and Pará). For this, DNA was extracted from blood or spleen samples from 455 animals. A nested PCR based on the 18S rRNA gene was used as screening for piroplasmids. Of the 455 samples analyzed, 25 (5.5%) were positive. Additionally, PCR assays based on 18S rRNA near-complete, cox-1, cox-3, hsp70, cytB, ß-tubulin genes and the ITS-1 intergenic region were performed. Five out of 25 positive samples also tested positive for ITS-1-based PCR. The phylogenetic analysis positioned three 18S rRNA sequences detected in Priodontes maximus into the same clade of Babesia sp. detected in marsupials (Didelphis albiventris, Didelphis marsupialis, and Monodelphis domestica) and Amblyomma dubitatum collected from opossums and coatis in Brazil. On the other hand, the 18S rRNA sequence obtained from Dasypus novemcinctus was closely related to a Theileria sp. sequence previously detected in armadillos from Mato Grosso State, grouping in a subclade within the Theileria sensu stricto clade. In the phylogenetic analysis based on the ITS-1 region, the sequences obtained from Myrmecophaga tridactyla and Tamandua tetradactyla were placed into a single clade, apart from the other piroplasmid clades. The present study demonstrated the molecular occurrence of Piroplasmida in anteaters and Babesia sp. and Theileria sp. in armadillos from Brazil.

Multiple piroplasm parasites (Apicomplexa: Piroplasmida) in northeastern populations of the invasive Asian longhorned tick, Haemaphysalis longicornis Neumann (Ixodida: Ixodidae), in the United States.

Piroplasms, which include the agents of cattle fever and human and dog babesiosis, are a diverse group of blood parasites of significant veterinary and medical importance. The invasive Asian longhorned tick, Haemaphysalis longicornis, is a known vector of piroplasms in its native range in East Asia and invasive range in Australasia. In the USA, H. longicornis has been associated with Theileria orientalis Ikeda outbreaks that caused cattle mortality. To survey invasive populations of H. longicornis for a broad range of piroplasms, 667 questing H. longicornis collected in 2021 from 3 sites in New Jersey, USA, were tested with generalist piroplasm primers targeting the 18S small subunit rRNA (395­515 bp, depending on species) and the cytochrome b oxidase loci (1009 bp). Sequences matching Theileria cervi type F (1 adult, 5 nymphs), an unidentified Theileria species (in 1 nymph), an undescribed Babesia sensu stricto ('true' Babesia, 2 adults, 2 nymphs), a Babesia sp. Coco (also a 'true Babesia', 1 adult, 1 nymph), as well as Babesia microti S837 (1 adult, 4 nymphs) were recovered. Babesia microti S837 is closely related to the human pathogen B. microti US-type. Additionally, a 132 bp sequence matching the cytochrome b locus of deer, Odocoileus virginanus, was obtained from 2 partially engorged H. longicornis. The diverse assemblage of piroplasms now associated with H. longicornis in the USA spans 3 clades in the piroplasm phylogeny and raises concerns of transmission amplification of veterinary pathogens as well as spillover of pathogens from wildlife to humans.

Differential gene expression response to acute and chronic Cytauzxoon felis infection in domestic cats (Felis catus).

Cytauxzoonosis is a severe tick transmitted protozoan disease of domestic cats, caused by Cytauxzoon felis. The disease is characterized by acute onset of high fever, depression, lethargy, inappentence, anorexia, icterus, dehydration, hemolytic anemia, and alteration of immune response. The aim of our study was to further detail the immune response of domestic cats to C. felis infection by comparing the differential expression of feline immune transcriptional elements during acute and chronic cytauxzoonosis. True single molecule sequencing (tSMS) was used to analyze the whole genome of acutely and chronically infected C. felis cats, focusing on the analysis of genes involved on the immune response. Two C. felis donor cats were infested with Amblyomma americanum nymphs, which after repletion were collected and kept in humidity chambers until they molted. The resulting A. americanum were randomly selected to infest three C. felis naïve principal cats. Infection of these cats was confirmed by nested PCR of the 18S rRNA C. felis gene and clinical signs. RNA was extracted from whole blood at different time points and used for tSMS analyses, the results revealed overexpression in transcripts involved in type I interferon signaling, cellular and cytokine responses during the acute stage of infection, while cell cycle, and metabolic processes were downregulated. Genes involved in cell adhesion increased their expression in the chronic infected cats, whereas inflammatory and apoptotic related genes were downregulated. This study provided information on the host immune response to C. felis in domestic cats, demonstrating that inflammatory, apoptotic, and cell adhesion are some of the pathways altered during acute and chronic infection.

Host oxidative stress in piroplasmosis: A review in domestic animals.

Piroplasmida is an order of economically important blood parasites, including Babesia, Theileria, and Cytauxzoon, transmitted to mammals by ticks. Oxidative stress is a state in which the balance between oxidants and antioxidants is disturbed so that antioxidants cannot compensate for the harmful effects of oxidants. Due to the high concentration of oxygen and hemoglobin in red blood cells (RBCs), these are among the first cells exposed to oxidative damage. When RBCs are exposed to oxidative stress, their hemoglobin is oxidized, and lipid peroxidation leads to membrane instability, deformation, hemolysis, and anemia. Oxidative stress has a fundamental role in the pathogenesis of these parasites. In the present review article, we collected studies on the oxidative stress caused by Piroplasmida in domestic animals. What is obtained from the total review of studies conducted on piroplasmosis revealed that in these infections, the host faces oxidative stress, and the resultant oxidative injury plays a fundamental role in their pathogenicity. Further studies are needed to be carried out on the more precise role of oxidative stress, the use of more sensitive diagnostic biomarkers, and the possible therapeutic role of antioxidant agents in piroplasmosis.

Sharing of living area and exposure of domestic and wild canids to ticks, Rickettsia parkeri and Rangelia vitalii in the Atlantic Forest of southern Brazil.

Spotted fever caused by the bacterium Rickettsia parkeri, and canine rangeliosis caused by the protozoan Rangelia vitalii, are emerging or re-emerging tick-borne diseases in Brazil, where the main tick vectors are Amblyomma ovale and Amblyomma aureolatum, respectively. In the present study, we evaluated exposure to R. parkeri and R. vitalii infection among domestic (Canis lupus familiaris) and wild (Cerdocyon thous) canids sharing living areas within an Atlantic Forest area of southern Brazil. Four different domestic dogs and three different wild canids were captured. Most of the ticks collected from either domestic or wild canids were identified as A. ovale and A. aureolatum. DNA of R. parkeri was amplified from 14% of the A. ovale specimens. Two domestic dogs and two wild canids were seroreactive to R. parkeri antigens with high endpoint titers (>1024). Rangelia vitalii DNA was detected in two wild canids; one of them was resampled 93 days later, again infected by R. vitalii. We report exposure/infection of domestic dogs and wild canids to R. vitalii and/or R. parkeri-infected ticks in an Atlantic Forest area shared by both canid species, indicating that they also shared the same populations of the tick vectors, A. aureolatum and A. ovale. While A. ovale, A. aureolatum, R. parkeri, R. vitalii and C. thous, are all native to the Atlantic Forest, further studies are needed to evaluate the impact of an exotic canine species - C. lupus familiaris - on the enzootic cycles of R. vitalii and R. parkeri, as well as the possible role of domestic dogs in emergence and re-emergence of R. parkeri-spotted fever in humans and canine rangeliosis, respectively.

Molecular pathogen screening of louse flies (Diptera: Hippoboscidae) from domestic and wild ruminants in Austria.

BACKGROUND: Hippoboscid flies (Diptera: Hippoboscidae), also known as louse flies or keds, are obligate blood-sucking ectoparasites of animals, and accidentally of humans. The potential role of hippoboscids as vectors of human and veterinary pathogens is being increasingly investigated, but the presence and distribution of infectious agents in louse flies is still unknown in parts of Europe. Here, we report the use of molecular genetics to detect and characterize vector-borne pathogens in hippoboscid flies infesting domestic and wild animals in Austria. METHODS: Louse flies were collected from naturally infested cattle (n = 25), sheep (n = 3), and red deer (n = 12) across Austria between 2015 and 2019. Individual insects were morphologically identified to species level and subjected to DNA extraction for molecular pathogen screening and barcoding. Genomic DNA from each louse fly was screened for Borrelia spp., Bartonella spp., Trypanosomatida, Anaplasmataceae, Filarioidea and Piroplasmida. Obtained sequences of Trypanosomatida and Bartonella spp. were further characterized by phylogenetic and haplotype networking analyses. RESULTS: A total of 282 hippoboscid flies corresponding to three species were identified: Hippobosca equina (n = 62) collected from cattle, Melophagus ovinus (n = 100) from sheep and Lipoptena cervi (n = 120) from red deer (Cervus elaphus). Molecular screening revealed pathogen DNA in 54.3% of hippoboscids, including infections with single (63.39%), two (30.71%) and up to three (5.90%) distinct pathogens in the same individual. Bartonella DNA was detected in 36.9% of the louse flies. Lipoptena cervi were infected with 10 distinct and previously unreported Bartonella sp. haplotypes, some closely associated with strains of zoonotic potential. DNA of trypanosomatids was identified in 34% of hippoboscids, including the first description of Trypanosoma sp. in H. equina. Anaplasmataceae DNA (Wolbachia spp.) was detected only in M. ovinus (16%), while < 1% of the louse flies were positive for Borrelia spp. and Filarioidea. All hippoboscids were negative for Piroplasmida. CONCLUSIONS: Molecular genetic screening confirmed the presence of several pathogens in hippoboscids infesting domestic and wild ruminants in Austria, including novel pathogen haplotypes of zoonotic potential (e.g. Bartonella spp.) and the first report of Trypanosoma sp. in H. equina, suggesting a potential role of this louse fly as vector of animal trypanosomatids. Experimental transmission studies and expanded monitoring of hippoboscid flies and hippoboscid-associated pathogens are warranted to clarify the competence of these ectoparasites as vectors of infectious agents in a One-Health context.

A novel variant of Babesia sp. (Piroplasmida) as a hemoparasite in procellariiform seabirds.

Procellariiformes includes pelagic seabirds that only use land for breeding; and also, these sites mostly occur in insular habitats. These peculiar habits make the investigation of hemoparasites a challenging issue. Thus, the data on the blood parasites of Procellariiformes are still scarce. In the order Piroplasmida, 16 species of Babesia have been described in terrestrial birds and seabirds. However, there is no register for Babesia spp. in procellariiform seabirds. Hence, the objective of this survey was to investigate the occurrence of Babesia spp. in these seabirds. A total of 220 tissue samples from 18 different seabird species were analyzed; the samples comprised blood and fragments of liver and spleen. The samples were obtained from live rescued animals and carcasses found along the southern coast of Brazil. Polymerase chain reaction (PCR) was conducted, followed by phylogenetic analysis. Only one blood sample yielded a positive result, from an adult female Thalassarche chlororhynchos (Atlantic yellow-nosed albatross). The sequence obtained showed the highest identity with sequences of Babesia spp. of birds from the South Pacific, and the isolate was named Babesia sp. strain Albatross. In the phylogenetic analysis, the sequence was grouped within the Babesia sensu stricto group, and further still into a subgroup including Babesia spp. of the Kiwiensis clade (parasites from birds). The phylogenetic analysis also showed that Babesia sp. strain Albatross clustered apart from the Peircei group, a clade that includes Babesia spp. from seabirds. As far as it is known, this is the first report of Babesia sp. in procellariiform seabirds. Babesia sp. strain Albatross may constitute a novel variant of tick-borne piroplasmids associated with the Procellariiformes order.

HIGH PREVALENCE OF CYTAUXZOON FELIS IN BOBCATS (LYNX RUFUS) ACROSS OKLAHOMA AND OCCURRENCE IN WEST TEXAS, USA.

Cytauxzoonosis is a fatal tick-borne disease in domestic cats caused by infection with the apicomplexan Cytauxzoon felis. Bobcats are the natural wild-vertebrate reservoirs for C. felis, and infections are typically subclinical and chronic in this species. The present study was done to determine the prevalence and geographic distribution of C. felis infection in wild bobcats from Oklahoma and the occurrence in northwestern Texas. Tongue samples from 360 bobcats were collected from 53 counties in Oklahoma and 13 samples from three counties in Texas. For DNA extracted from each tongue sample, a probe-based droplet digital PCR assay was performed targeting the C. felis mitochondrial gene cytochrome c oxidase subunit III (cox3). Prevalence of C. felis infection was calculated for each county sampled, and data from individual counties were combined according to geographic regions and compared using chi-square tests. Overall prevalence of C. felis in bobcats from Oklahoma was 80.0% (95% confidence interval [CI], 75.6-83.8). The prevalence of infection was >90% for bobcats from central, northeastern, south-central, and southeastern regions of Oklahoma, but <68% for bobcats from northwestern and southwestern regions. Bobcats from central counties in Oklahoma were 25.693 times more likely to be infected with C. felis compared to all other bobcats sampled from the state. Higher prevalence estimates of C. felis in bobcats appeared to be in counties where known tick vectors are most common. Occurrence of C. felis in bobcats from northwestern Texas was 30.8% (95% CI, 12.4%-58.0%) based on 13 samples. Results of this study support the utilization of bobcats as sentinel animals to identify geographic areas with risk of C. felis infection to domestic cats.

Degrade to survive: the intricate world of piroplasmid proteases.

Piroplasmids of the genera Babesia, Theileria, and Cytauxzoon are tick-transmitted parasites with a high impact on animals and humans. They have complex life cycles in their definitive arthropod and intermediate vertebrate hosts involving numerous processes, including invasion of, and egress from, host cells, parasite growth, transformation, and migration. Like other parasitic protozoa, piroplasmids are equipped with different types of protease to fulfill many of such essential processes. Blockade of some key proteases, using inhibitors or antibodies, hinders piroplasmid growth, highlighting their potential usefulness in drug therapies and vaccine development. A better understanding of the functional significance of these enzymes will contribute to the development of improved control measures for the devastating animal and human diseases caused by these pathogens.

Molecular detection of Theileria annulata, Theileria mutans and Theileria velifera but no evidence of Theileria parva infected or vaccinated cattle in Nigeria despite extensive transboundary migrations.

The extensive livestock management system predominant in Nigeria necessitates active disease surveillance for the early detection and prompt control of transboundary animal diseases. Theileriae are obligate intracellular protozoa which infect both wild and domestic bovidae throughout much of the world causing East Coast Fever (Theileria parva), Tropical or Mediterranean theileriosis (Theileria annulata) or benign theileriosis (Theileria mutans; Theileria velifera). This study aimed to detect and characterize Theileria spp. infecting cattle in Nigeria using conventional PCR and sequencing approach. Five hundred and twenty-two DNA samples obtained from different cattle blood samples were subjected to PCR targeting the 18S rRNA gene of piroplasmida and specifically, the p104 kDa and Tp1 genes for the evidence of infection or vaccination respectively, with T. parva. A total of 269 out of 522 (51.5%) of the cattle tested PCR- positive for DNA of piroplasmida. Nucleotide sequence and phylogenetic analyses showed that the cattle were infected with T. annulata, T. mutans and T. velifera. Piroplasmida DNA was associated with sex (ꭓ2 = 7.2; p = 0.007), breed (ꭓ2 = 115; p = 0.000002) of animals and the state where the samples were collected (ꭓ2 = 78.8; p = 0.000002). None of the samples tested positive for T. parva DNA or showed evidence of vaccination (Tp1 gene). This is the first report on the molecular detection and characterization of T. annulata in the blood of cattle from Nigeria. Continuous surveillance of Nigerian cattle for East Coast Fever (ECF) is encouraged considering the recent report of the disease in cattle in the neighboring country, Cameroon, where unregulated transboundary cattle movement into Nigeria has been observed.

Expanding the universe of Piroplasmids: morphological detection and phylogenetic positioning of putative novel piroplasmids in black-eared opossums (Didelphis aurita) from southeastern Brazil, with description of "South American Marsupialia Group" of Piroplasmida.

The growing proximity of wildlife to large urban niches arouses greater interest in understanding wild reservoirs in the epidemiology of diseases of importance to animal and human health. The aim of the present study was to investigate the presence of piroplasmids in opossums rescued from the metropolitan region of Rio de Janeiro state, Brazil. Blood and bone marrow samples were collected from 15 Didelphis aurita and subjected to DNA extraction and PCR using primers for the 18S rRNA, cox1, cox3, and hsp70 genes of piroplasmids. Clinical and hematological evaluation of the animals was also performed. Five (33.3%) of the 15 opossums tested positive for piroplasms in the nested PCR based on the 18S rRNA, and in two animals, it was possible to observe intra-erythrocytic structures compatible with merozoites. One of the positive animals showed clinical signs of infection such as jaundice, fever, and apathy. Anemia, low level of plasma protein, leukocytosis, and regenerative erythrocyte signs were observed in positive animals. Phylogenetic analysis based on both 18S rRNA and cox-3 genes demonstrated that the piroplasmids detected in D. aurita formed a unique sub-clade, albeit related to piroplasmids previously detected in Didelphis albiventris and associated ticks from Brazil. This study proposes the novel Piroplasmida Clade, namely "South American Marsupialia Group," and reinforces the need for new clinical-epidemiological surveys to understand the dynamics of these infections in didelphids in Brazil.

Molecular Survey of Rodent-Borne Infectious Agents in the Ferlo Region, Senegal.

Zoonotic pathogens are responsible for most infectious diseases in humans, with rodents being important reservoir hosts for many of these microorganisms. Rodents, thus, pose a significant threat to public health. Previous studies in Senegal have shown that rodents harbour a diversity of microorganisms, including human pathogens. Our study aimed to monitor the prevalence of infectious agents in outdoor rodents, which can be the cause of epidemics. We screened 125 rodents (both native and expanding) from the Ferlo region, around Widou Thiengoly, for different microorganisms. Analysis, performed on rodent spleens, detected bacteria from the Anaplasmataceae family (20%), Borrelia spp. (10%), Bartonella spp. (24%) and Piroplasmida (2.4%). Prevalences were similar between native and the expanding (Gerbillus nigeriae) species, which has recently colonised the region. We identified Borrelia crocidurae, the agent responsible for tick-borne relapsing fever, which is endemic in Senegal. We also identified two other not-yet-described bacteria of the genera Bartonella and Ehrlichia that were previously reported in Senegalese rodents. Additionally, we found a potential new species, provisionally referred to here as Candidatus Anaplasma ferloense. This study highlights the diversity of infectious agents circulating in rodent populations and the importance of describing potential new species and evaluating their pathogenicity and zoonotic potential.

Transmission of Cytauxzoon felis by injection of Amblyomma americanum salivary glands.

BACKGROUND: Cytauxzoonosis is a life-threatening disease of cats, caused by the tick-borne piroplasmid hemoparasite, Cytauxzoon felis. Current experimental models for cytauxzoonosis rely on either tick transmission or direct injection of infected cat tissues. These models require researchers to directly work with infected ticks or use cats with acute cytauxzoonosis. To improve the feasibility and accessibility, there is a need to establish sharable resources among researchers. In related piroplasmid parasites, sporozoite-based inoculums are routinely produced from tick salivary glands, cryopreserved and distributed to other investigators and facilities. For these parasites, sporozoites have been the basis for vaccine development and in vitro cultivation, both of which remain lacking for C. felis research. If infectious sporozoites can be similarly isolated for C. felis, it would significantly broaden our capabilities to study this parasite. Aims of this study was to determine if C. felis sporozoites inoculums collected from the salivary glands of Amblyomma americanum ticks were capable of inducing cytauxzoonosis in naïve cats. MATERIALS AND METHODS: A. americanum nymphs were acquisition-fed on a donor cat chronically infected with C. felis and allowed to molt to adults. Four groups of adult ticks (n = 50/group) were either stimulation-fed for 4 days on naïve cats or were heated at 37 °C for 4 days. After these treatments, salivary glands (SG) of each group of ticks were collected to create inoculums. Infectivity of these inoculums was then tested by subcutaneous injection into naïve cats. RESULTS: The two naïve cats used for stimulation feeding and as controls both developed cytauxzoonosis, indicating these groups of ticks were capable of producing infectious sporozoites. Of the 2 cats that were injected with SGs from the stimulation-fed ticks, one cat developed cytauxzoonosis and C. felis infection was confirmed by both light microscopy and PCR. The other cat did not develop cytauxzoonosis and only had equivocal evidence of infection. Neither cat injected with SGs from the heated ticks developed cytauxzoonosis. One of these cats had equivocal evidence of infection and one had no evidence of infection. CONCLUSION: This study validates the feasibility of collecting infectious sporozoites from C. felis-infected ticks that can be used to infect naïve cats. While this model requires further optimization, it has the potential to expand resources to study C. felis and further advance research in this field.

Identifying suitable habitat for Ixodes scapularis (Acari: Ixodidae) infected with Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), Babesia microti (Piroplasmida: Babesiidae), and Borrelia miyamotoi (Spirochaetales: Spirochaetaceae) to guide surveillance efforts in the eastern United States.

Understanding the distribution of infected ticks is informative for the estimation of risk for tickborne diseases. The blacklegged tick, Ixodes scapularis (Acari: Ixodidae), is the primary vector for 7 medically significant pathogens in United States. However, knowledge of the ranges of these pathogens in host-seeking ticks is incomplete, particularly for those occurring at low prevalence. To aid in prioritizing costly field sampling efforts, we estimated ranges of suitable habitat for Anaplasma phagocytophilum, Babesia microti, and Borrelia miyamotoi in the eastern United States based on existing county-level surveillance records. The resulting suitability maps were compared against those developed previously for Bo. burgdorferi s.s., which shares similar ecology but has been detected in a greater number of counties. The overall accuracy of the habitat suitability models was high (AUC ≥ 0.92) for all 4 pathogens. The most important predictors were related to temperature and moisture. The upper midwestern and northeastern states were predicted to be highly suitable for all 4 pathogens. Based on our models, we prioritized sampling in 431, 275, and 539 counties currently lacking pathogen records that our models classified as suitable for A. phagocytophilum, Ba. microti, and Bo. miyamotoi, respectively. As a second-tier priority, we identified 311 (A. phagocytophilum), 590 (Ba. microti), and 252 (Bo. miyamotoi) counties, based on high suitability scores for Bo. burgdorferi. Our models can be used to improve cost-effectiveness of field sampling efforts aimed at improving accuracy and completeness of pathogen distribution maps.

Apicomplexan haemoparasites in domestic cats in Romania.

BACKGROUND: Apicomplexan haemoparasites are protozoans that infect a variety of domestic and wild animal species, as well as humans. Data regarding haemoprotozoans in domestic cats are limited; therefore, the aim of this study was to assess the occurrence of Babesia spp., Cytauxzoon spp., and Hepatozoon spp. in domestic cats in Romania using molecular tools. METHODS: Blood samples from 371 domestic cats were screened for the presence of piroplasmids. All samples that yielded a visible band in agarose gels were subsequently tested by specific assays targeting the 18S rDNA of Babesia spp., Cytauxzoon spp., and Hepatozoon spp. Moreover, nested PCR assays targeting mitochondrial genes of Babesia spp. were used for screening of all Babesia spp. 18S rDNA-positive samples. RESULTS: From the total number of sampled cats, 19.4% were positive in the PCR assay targeting piroplasmids. Babesia spp. were identified in 15.1% of cats, while 0.5% were positive for Hepatozoon spp. Molecular analyses confirmed the presence of Babesia canis. No samples were positive for Cytauxzoon spp. CONCLUSIONS: The high infection rates of domestic cats with Babesia spp. and the need for species differentiation highlight the importance of mitochondrial genes as targets for molecular protocols.

Cytauxzoonosis.

Cytauxzoon felis is a hematoprotozoan parasite with a complex life cycle involving a tick-vector and a mammalian host. The mammalian hosts are all felidae but in the bobcat reservoir host, the parasite typically causes only a brief, self-resolving illness followed by a prolonged subclinical infection. In domestic cats, however, infection often leads to an acute febrile illness characterized by severe morbidity and mortality. Diagnosis is based on microscopic identification of parasites or molecular testing. Treatment for ill cats is expensive, difficult, and often unsuccessful. Prevention is quite possible and depends on avoidance of feeding by vector ticks.

Piroplasmid infection is not associated with clinicopathological and laboratory abnormalities in cats from Midwestern Brazil.

Feline piroplasmids include the genera Babesia spp., Cytauxzoon spp., and Theileria spp. In Brazil, there are few reports regarding these hemoprotozoans; however, clinicopathological and molecular data are scarce. This study aimed to characterize the clinical relevance of these parasites through hematological, biochemical, and molecular approaches. For this purpose, 166 cats from Brasilia, Federal District, Midwestern Brazil, were screened using a quantitative polymerase chain reaction (qPCR) for piroplasmids based on the LSU4 mitochondrial gene, which resulted in an overall prevalence of 36/166 (21.7%). Twelve of 166 samples (7.2%) were positive for C. felis, while 19/166 (11.4%) were positive for Babesia vogeli. No samples tested positive for Theileria spp. Babesia vogeli and Cytauxzoon spp. LSU4 sequences showed identities of 97-100% and 99.3%, respectively, to US isolates. The hematological and biochemical findings did not differ significantly between the cats that tested positive and negative for piroplasmids. Although the lack of abnormalities in clinical and laboratory parameters does not eliminate the possibility that these cats were sick and recovered, it may suggest that the Brazilian strain of Cytauxzoon spp. is not as pathogenic as that from the USA, despite the high molecular identity with North American isolates.

Molecular epidemiological study on ticks and tick-borne protozoan parasites (Apicomplexa: Cytauxzoon and Hepatozoon spp.) from wild cats (Felis silvestris), Mustelidae and red squirrels (Sciurus vulgaris) in central Europe, Hungary.

BACKGROUND: Among live wild mammals adapted to urban and peri-urban habitats in Europe, members of the families Felidae, Mustelidae and Sciuridae deserve special attention as pathogen reservoirs because all of these families include members that are kept as pets. We report here the results of our study on two important groups of tick-borne protozoan parasites in ticks and tissues of wild cats, mustelids and red squirrels. METHODS: DNA was extracted from the tissues of carnivores (wild cats, mustelids; n = 16) and red squirrels (n = 4), as well as from ixodid ticks (n = 89) collected from these hosts. These DNA extracts were screened for piroplasms and Hepatozoon spp. using conventional PCR analysis and sequencing. In addition, 53 pooled samples of 259 questing Haemaphysalis concinna ticks were evaluated for the presence of Hepatozoon DNA, followed by phylogenetic analyses. RESULTS: One wild cat was found to be coinfected with Cytauxzoon europaeus and a new genotype of Hepatozoon felis, and two additional wild cats were infected with H. felis from a different phylogenetic group. In mustelids, Hepatozoon martis and two further Hepatozoon genotypes were detected. The latter clustered separately, close to others reported from eastern Asia. In addition, Hepatozoon sciuri was detected in red squirrels. Morphologic and molecular analyses verified eight tick species. One wild cat was infected with a H. felis genotype that was significantly different from that in Ixodes ricinus females infesting this cat. Only three pools of questing H. concinna nymphs tested positive for Hepatozoon, one of which contained H. martis. CONCLUSIONS: This study provides the first evidence of the occurrence of any Cytauxzoon species and of three Hepatozoon species in Hungary. In addition to H. martis, two further mustelid-associated Hepatozoon genotypes were detected, one of which was new in terms of phylogenetic and broader geographical contexts. This may be the first indication that H. felis genotypes from both of its phylogenetic groups occur in Europe. This also appears to be the first evidence of H. felis and C. europaeus coinfection in felids in Europe, and of autochthonous H. felis infection in wild cats north of the Mediterranean Basin. New tick-host associations were also observed in this study. Based on the results, H. felis and H. martis might survive transstadially in I. ricinus and H. concinna, respectively.

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.