Molecular evidence of hepatozoonosis in tigers of Vidarbha region of Maharashtra State of India.

BACKGROUND: Hepatozoonosis has been reported in many species around the world. Few incidences have been reported in various species of wild felids. Tigers are endangered large cats and are protected under the Wildlife Protection Act, 1972 under Schedule I. The study was carried out to estimate the positivity rate of hepatozoonosis in tigers of the Vidarbha region of Maharashtra, India. METHODS: Blood (n = 21) or tissue samples (n = 5) were collected from 26 wild captured / zoo-born or dead tigers during the quarantine period/post-mortem examination. Blood smear examination along with Polymerase Chain Reaction (PCR) studies were conducted for the detection of hepatozoonosis. All the amplicons from the positive samples were purified and sequenced, and the sequences were subjected to nBLAST analysis to detect the species of Hepatozoon. The sequences were deposited into public domain database of National Center for Biotechnology Information (NCBI) and accession numbers were allotted. A phylogenetic study was undertaken to understand the evolutionary lineage of the pathogen. Tissue distribution studies were carried out on tissue samples received during post mortem. A clinical case in a tiger cub was managed and sub-clinical cases were monitored for relapse. Age-wise, sex-wise, region-wise and captive time-wise positivity rate was estimated. The data was analyzed using statistical tools. RESULTS: A total of 12 tigers were found positive for H. felis during the screening. A clinical case was diagnosed and successfully treated. The age group of 0-3 years reported a positivity rate of 66.66%, and all the cases found positive were reported between the age group of 0-7 years. Males reported a positivity rate of 58.33 per cent, while females reported 35.71%. Taboba and Andhari Tiger Reserve of the state had a positivity rate of 52.94 per cent. However, the statistical analysis for blood parameters and positivity rate by 't' test and Chi-squared test were found to be non-significant. CONCLUSIONS: An overall positivity rate of 46.15% indicates the wide distribution of hepatozoonosis among wild tigers of the Vidarbha region of Maharashtra, India, which is strategically important considering the gene flow and migration of tigers. Hepatozoonosis can progress to clinical outcomes in young animals and require veterinary intervention. Molecular tools and phylogenetic studies can supplement important data on circulating species of Hepatozoon in the field. Further studies on the clinical management and epidemiology of the infection in wild felids will comprehend the cause of wildlife conservation.

First identification of Cytauxzoon manul in Eurasian lynx (Lynx lynx) in northwestern China.

BACKGROUND: Multiple species of the genera Cytauxzoon and Hepatozoon can infect wild felines, but the diversity of these and other apicomplexan parasites in Eurasian lynx is scarcely known. The aim of this study was to detect Cytauxzoon and Hepatozoon species with molecular methods in Eurasian lynxes and their ticks in northwestern China. METHODS: DNA was extracted from the heart, liver, spleen, lung, and kidney samples of three Eurasian lynxes as well as from their five ixodid ticks. These DNA samples were screened with polymerase chain reactions (PCRs) for Cytauxzoon with the partial cytochrome b gene (CytB), cytochrome c oxidase subunit I gene (COI), and small subunit ribosomal RNA gene (18S rRNA), and Hepatozoon with three different fragments of small subunit ribosomal RNA gene (18S rRNA). PCR products were sequenced, aligned, and phylogenetically analyzed. RESULTS: One adult female of Eurasian lynx (#1, adult female) was co-infected with Cytauxzoon manul and Hepatozoon felis genotype I, while an adult male lynx (#2) was infected with C. manul. Interestingly, H. felis genotype I was both detected in a male cub (#3) and two out of five infesting Hyalomma asiaticum ticks. CONCLUSIONS: For the first time, Cytauxzoon manul is reported here from Eurasian lynx. In addition, H. felis has not been known to occur in this host species in China and Central Asia. Thus, the findings of this study extend our knowledge on the geographical distribution and host range of these haemoprotozoan parasites. Moreover, this is also the first evidence of C. manul and H. felis co-infection in Eurasian lynx.

First report of Hepatozoon felis infection in a domestic cat (Felis catus) in Iran.

A 6-year-old male domestic short-haired cat (Felis catus) was presented with weakness, anorexia, fever, icterus, a painful abdomen, ruffled hair and a tick infestation, and it had no prior surgery. Laboratory analysis revealed left-shifted neutrophils, thrombocytopenia, low albumin content and high serum bilirubin concentration as well as activities of hepatic enzymes including alanine aminotransferase and aspartate aminotransferase. Azotemia and increased serum levels of creatinine and urea were also recorded. In Giemsa-stained blood smear, Hepatozoon gamonts were observed within neutrophils. Species-specific polymerase chain reaction assay was used to amplify an approximately 590 bp fragment of 18S rRNA gene and confirmed Hepatozoon felis infection. The cat was treated with imidocarb dipropionate and doxycycline and recovered completely. Six-month follow-up showed no recurrence. This study reveals the presence of H. felis in Iran and it should be considered in differential diagnosis in febrile and icteric cats. To the authors' knowledge, this is the first description of H. felis infection in a cat in Iran.

Phylogenetic analysis and haplotype networking of Hepatozoon felis infecting wild animals in Gir National Park, Gujarat, India.

The present study was aimed to determine the phylogenetic relationship, haplotype network, and demographic dynamics of H. felis infecting the endangered Asiatic lions in Gir National Park, Gujarat, India, on the basis of partial 18S rRNA gene. The phylogenetic analysis based on the partial 18S rRNA gene sequences of H. felis exhibited the presence of two distinct genotypes of H. felis (HfG1 and HfG2) infecting the Indian wild felids, viz., the Asiatic lion, Royal Bengal tiger, and Indian leopard. The HfG1 and HfG2 genotypes exhibited 97.6-100% and 99.7-100%, and 96.9-98.7% nucleotide identity within and between themselves, respectively. The HfG1 genotype exhibited a higher genetic diversity as compared to HfG2. A total of 22 molecular signatures were identified in the 18S rRNA gene between these genotypes. Further, analysis of a total 67 sequences of H. felis (13 different host species from 13 countries of Africa, South America, Asia, and Europe) that were downloaded from GenBankTM, generated 30 haplotypes. Among all the haplotypes, Hap_17 (h=12) was the most frequent followed by Hap_12 (h=09) and Hap_4 (h=05). Out of 13 location-wise populations, India (h=12) shared the highest number of haplotypes followed by Japan (h=08), and the least number of haplotypes were found in Hungary (h=02). Population dynamics study involving neutrality tests and mismatch distribution, and genetic differentiation indices, revealed the presence of phylogeographic population structure and a constant population size indicating a uniform gene flow among the populations worldwide. In conclusion, a high genetic diversity along with the presence of two distinct genotypes of H. felis were observed on the basis of 18S rRNA gene sequence analysis.

Molecular prevalence and genetic diversity of Hepatozoon spp. in stray cats of Izmir, Türkiye.

Hepatozoon spp. are an apicomplexan protozoan parasites that infect vertebrates including mammals, marsupials, birds, reptiles, and amphibians. Among Hepatozoon species, H. canis and H. felis are causative agents of hepatozoonosis in dogs and cats, respectively and have veterinary importance. This study aimed to determine the prevalence of Hepatozoon spp. in stray cats living in Izmir and investigate genetic diversity among positive samples. To achieve this aim, the prevalence of Hepatozoon spp. 18S rRNA gene was screened by PCR in DNA samples extracted from blood samples of stray cats (n = 1012). Then, Hepatozoon-positive samples were sequenced and the generated data were used for species identification, phylogenetic and haplotype analyses. According to the results, among the samples screened, 2.37 % (24/1012) of them were found to be Hepatozoon-positive, and of these positive samples, 18 (18/24; 75 %) were successfully sequenced. BLAST and phylogenetic analyses revealed that all of these samples were H. felis. Also, phylogenetic analysis showed that H. felis samples were genotype I. Within H. felis samples isolated from cats living in different countries/regions, 9 haplotypes were detected and among these haplotypes, H-1 was found to be prevalent (n = 20 H. felis isolates). In conclusion, this study showed that the prevalence of Hepatozoon spp. was low in stray cats analyzed. Also, H. felis genotype I was predominant in comparison to other Hepatozoon species.

Hepatozoon species infecting domestic cats from countries of the Mediterranean basin.

Tick-borne diseases (TBDs) are caused by pathogens of human and veterinary concern representing a major public health issue worldwide. Although feline medicine has progressed much in the recent decades, data on feline TBDs (FeTBDs) remain scant. Therefore, this study aimed to assess the prevalence of apicomplexan parasite infections, associated risk factors and clinical-hematological abnormalities in domestic feline populations from countries of the Mediterranean basin. Blood and serum samples from cats (n = 600) living in France, Greece, Israel, Italy, Portugal and Spain were collected along with animal data (i.e., age, sex, breed, housing conditions and geographical origin), clinical signs and laboratory blood test parameters. Cats were grouped according to their age as kitten (up to one year), young (between one and six years), mature (between seven and ten years) and senior (older than ten years). Blood samples were tested for Hepatozoon spp. and piroplasmids by conventional PCR targeting 18S rRNA gene. The overall prevalence of Hepatozoon spp. infection was 14.5%, being significantly higher in cats from Greece (30%) and Portugal (23%), followed by Spain (15%), Israel (15%) and France (4%). Cats from Italy scored negative. Hepatozoon felis was identified in 86 animals, with three different sequence types and H. silvestris was detected in one shelter cat from Portugal. No piroplasmid DNA was amplified. The risk of Hepatozoon spp. infection was related to feline geographical provenience, housing condition and age. No statistical correlation was reported with any clinical signs, while increased alanine aminotransferase (ALT) activity was the only laboratory abnormality significantly associated (p = 0.03) with the infection. Data suggest a high circulation of H. felis, and only occasionally of H. silvestris, within domestic feline populations in the Mediterranean basin, mainly in shelter or free roaming and young cats with asymptomatic or subclinical infection.

Hepatozoonosis of Dogs and Cats.

Hepatozoon canis and Hepatozoon americanum are tick-borne infections of dogs transmitted by different tick species, with dissimilar geographic distributions, target organs, and clinical syndromes. H canis is transmitted mostly by the brown dog tick Rhipicephalus sanguineus sensu lato, affects hemolymphoid organs, is associated with anemia and other hematologic abnormalities, and is widely prevalent globally, whereas H americanum is transmitted by the Gulf Coast tick Amblyomma maculatum, causes severe myositis, and is an emerging parasite in the southern United States. Treatment of these 2 infections decreases the parasitic load without elimination. Domestic cats are infected with 3 Hepatozoon species.

Cytauxzoon sp. and Hepatozoon spp. in Domestic Cats: A Preliminary Study in North-Eastern Italy.

Knowledge on the presence of Cytauxzoon sp. and Hepatozoon spp. in Italy is scant and mostly limited to a few areas of Northern and Southern regions, respectively. The present study updated the current epidemiological scenario by investigating the occurrence of these protozoa in domestic cats from three broad regions of North-Eastern Italy. Blood samples from cats at risk of vector-borne diseases were processed by PCR to detect Cytauxzoon and Hepatozoon DNA. Blood smears were observed for haemoparasite inclusions. The influence of cat individual data (e.g., provenance, management, indoor/outdoor lifestyle) on the prevalence of haemoprotozoan infections was statistically evaluated. Among 158 cats, Cytauxzoon and Hepatozoon DNA were detected in 6 (3.8%) and 26 (16.5%) animals, respectively. No Hepatozoon gamonts were detected in blood smears, whereas all Cytauxzoon PCR-positive samples were microscopically positive, though with low levels of parasitaemia. Two species of Hepatozoon were identified, Hepatozoon felis (n = 10) and Hepatozoon silvestris (n = 16). Hepatozoon silvestris prevalence values were significantly (p < 0.05) higher in the region Friuli Venezia Giulia and in stray cats. Cytauxzoon sp. was detected in 6/39 (15.4%) stray cats from Friuli Venezia Giulia (Trieste province). These data add new information on the occurrence of these neglected protozoa in domestic cats' populations.

First record of Hepatozoon spp. in domestic cats in Greece.

Feline hepatozoonosis is an emerging disease of domestic and wild felids though there is limited knowledge of this infection, e.g. regarding geographical distribution and parasite species involved. The present study evaluated microscopically and molecularly the occurrence of Hepatozoon spp. in domestic cats from insular (Crete, Mykonos and Skopelos) and continental (prefectures of Attica and Thessaloniki) Greece. Out of 282 cats examined, 72 (25.5 %) scored positive by PCR for Hepatozoon spp. and of them, 9 (12.5 %) showed gamonts on the blood smear microscopic examination. Sequences obtained from 35 of the amplicons proved the presence of two haplotypes of Hepatozoon felis. One, herein called H1 (34/35 amplicons) resulted 100 % identical with H. felis from Italy and isolates from other continents, and ∼98 % similar with a H. felis isolate causing severe clinical signs in Austria. The haplotype H2, found in a cat in Skopelos, had ∼94 % identity with H1, with H. felis isolates from Italy, Israel, Spain, a ∼92 % identity with the isolate from Austria, and ∼94-98 % with isolates from South Africa. These are the first records of H. felis in cat populations from Greece and indicate that the infection may be present at high prevalences in different regions of the country. Furthermore, the results of the molecular and phylogenetic analysis support a recent hypothesis indicating the existence of a species-complex classification for H. felis. Further studies aiming at elucidating the genetic make-up of Hepatozoon populations and possible variations in terms of geographic distribution and clinical relevance are necessary. The importance of a continuous epizootiological monitoring is crucial for the establishment of preventative and control measures protecting the health of cats living in or travelling to enzootic areas.

Molecular detection of tick-borne pathogens in caracals (Caracal caracal) living in human-modified landscapes of South Africa.

BACKGROUND: Wild carnivores living alongside humans and domestic animals are vulnerable to changes in the infectious disease dynamics in their populations. The aims of this study were to determine the prevalence and diversity of selected tick-borne pathogens (TBPs) of veterinary and/or zoonotic concern in wild populations of caracals (Caracal caracal) occurring in human-modified landscapes in South Africa. Using molecular techniques, we screened 57 caracal blood samples for infection by rickettsial bacteria and piroplasms in three regions of South Africa: rangeland in the Central Karoo (n = 27) and Namaqualand (n = 14) as well as the urban edge of the Cape Peninsula (n = 16) of South Africa. To characterise pathogen identity, we sequenced the 18S rRNA and 16S rRNA genes from positive samples and analysed sequences within a phylogenetic framework. We also examine the diversity of potential tick vectors. RESULTS: All individuals tested were infected with at least one tick-borne pathogen. Pathogens included Hepatozoon felis, Babesia felis, Babesia leo and a potentially novel Babesia species. An Anaplasma species previously described in South African domestic dogs was also found in 88% of urban edge caracals. Higher rates of co-infection characterised urban edge caracals (81% vs 15% and 0% in the two rangeland populations), as well as a greater incidence of mixed infections. Host attached tick species include Haemaphysalis elliptica, an important pathogen vector among carnivore hosts. CONCLUSIONS: This study confirms the occurrence of previously undocumented tick-borne pathogens infecting free-ranging caracals in human-modified landscapes. We identify clear differences in the pathogen profiles among our study populations and discuss the likely health costs to caracals living adjacent to urban areas.

Molecular Characterization of Hemoparasites and Hemoplasmas Infecting Domestic Cats of Southern India.

In the present study, 111 blood samples were collected from apparently healthy cats belonging to four districts of Kerala, southern India, and they were investigated for the presence of hemoparasites and hemoplasmas by light microscopic examination and polymerase chain reaction (PCR). The microscopic examination of the Giemsa-stained blood smears did not reveal any parasites/organisms. However, PCR followed by nucleotide sequencing could detect 10 (9.01%) out of 111 samples infected with Hepatozoon felis, 3 (2.70%) with Cytauxzoon spp., and 10 (9.01%) with Mycoplasma spp. None of the samples revealed amplicons specific for the Babesia spp. and Trypanosoma evansi. The phylogenetic analysis of 18S ribosomal RNA (rRNA) gene sequences of H. felis revealed the existence of two different populations of H. felis circulating in the blood of infected cats. The phylogenetic tree was constructed based on 18S rRNA gene sequences of Cytauxzoon spp. and revealed that these isolates formed a distinct clade and do not cluster with any of the isolates from other countries. Among the 10 samples positive for Mycoplasma spp. infections, 7 were detected positive for Candidatus Mycoplasma haemominutum, two for Mycoplasma haemofelis, and one for Candidatus Mycoplasma turicensis. Phylogenetic analysis of 16S rRNA gene sequences of Mycoplasma spp. showed no distinct geographical grouping of the sequences. The sequences of M. haemofelis, Candidatus M. haemominutum, and Candidatus M. turicensis identified in the study clustered along with their respective isolates from around the world. To the best of our knowledge, this study forms the first report of molecular detection of Cytauxzoon spp. and Candidatus M. turicensis in cats from India.

First autochthonous case of clinical Hepatozoon felis infection in a domestic cat in Central Europe.

Three different Hepatozoon (Apicomplexa, Hepatozoidae) species have been described infecting domestic cats in Europe (i.e. H. felis, H. canis and H. silvestris), however, reports on clinical hepatozoonosis are uncommon and treatment protocols are not clearly defined. A six-year-old male European short-hair cat from Austria presented poor general condition, lethargy, anorexia, icterus, a painful abdomen, fever, ruffled hair and a tick infestation, and it had never left Austria. Laboratory tests revealed leukopenia, thrombocytopenia and increased serum levels of symmetric dimethylarginine (SDMA) and bilirubin. In May Grünwald-Giemsa-stained blood smears, structures resembling Hepatozoon gamonts were observed inside neutrophil granulocytes. A PCR targeting a fragment of the 18S rRNA gene of Hepatozoon spp. and DNA sequencing allowed the diagnosis of H. felis-DNA in blood samples. The cat was treated with imidocarb dipropionate (6 mg/kg body weight, repeated after 14 days) and doxycycline monohydrate (5 mg/kg body weight twice a day, p.o., for four weeks) and recovered completely. A broad haematological and biochemical laboratory control after six months showed all evaluated parameters under normal ranges. Coinfection with other feline pathogens (i.e. feline leukaemia virus, feline immunodeficiency virus, feline Coronavirus, Leishmania and Dirofilaria immitis) could not be detected. This study reveals the presence of H. felis in Austria and provides more evidence on the geographical distribution and pathogenicity of this parasite for domestic cats. To the authors' knowledge, this is the first autochthonous case of feline hepatozoonosis in Central Europe.

High prevalence of vector-borne pathogens in domestic and wild carnivores in Iraq.

Vector-borne diseases (VBDs) of domestic and wild carnivores are of major public health concern both in industrialized and developing countries, especially in poor socioeconomic settings. War-torn areas specifically suffer from absence of veterinary surveillance of VBDs, resulting in lack of scientific knowledge on this topic. To investigate occurence and prevalence of several vector-borne pathogens (VBPs) in some carnivore species from Iraq, blood samples (n = 397) were obtained from 190 canids [97 stray dogs (Canis familiaris), 55 jackals (Canis aureus) and 38 red foxes (Vulpes vulpes)] and 207 stray cats (Felis catus) collected during a feral animal control and zoonotic disease surveillance program in several United States military bases in Iraq. The presence of Babesia spp., Hepatozoon spp., Ehrlichia spp., Anaplasma spp., Dirofilaria spp. and Leishmania spp. DNA was molecularly investigated. Out of 397 animals tested, 176 (44.3%; 95% CI: 39.5-49.2%) were positive for at least one pathogen with the highest prevalence in foxes (73.7%; 95% CI: 58-85%), followed by jackals (54.5%; 95% CI: 41.5-67%), dogs (38.1%; 29.1-48.1%) and cats (39.1%; 95% CI: 32.7-45.9%). Up to five pathogens were diagnosed in dogs. Hepatozoon canis was the most prevalent VBP in jackals (49.1%; 95% CI: 36.4-61.9%), foxes (47.3%; 95% CI: 32.5-62.7%) and dogs (33%; 95% CI: 24.4-42.8%), whereas Hepatozoon felis was the only species detected in cats (39.1%; 95% CI: 32.7-45.9%). A species of Babesia related to but different from Babesia lengau and designated as Babesia sp. MML was detected in six foxes (15.8%; 95% CI: 7.4-30.4%) and in one jackal (1.8%; 95% CI: 0.3-9.6%). This finding suggested the existence of a new species in the genus Babesia as inferred by molecular and phylogenetical analysis. Further, Babesia vulpes was identified only in two foxes (5.3%; 95% CI: 1.5-17.3%). All samples were negative for Leishmania spp. and Ehrlichia spp. Co-infection with H. canis and Babesia spp. was the most prevalent (5/176, 2.8%, i.e., 4 foxes and 1 jackal), followed by H. canis and Dirofilaria immitis (1/176, 1.3%, i.e., in 1 jackal), H. canis and Dirofilaria repens or Acanthocheilonema reconditum (1/176, 1.3%, i.e., in one dog, each). Data presented fill gaps into knowledge of VBPs in dogs, cats and wild canids in Iraq, indicating that different pathogens circulate amongst animal populations living in the same areas, possibly sharing the same tick vectors. Large-scale surveys are urgently needed to further assess VBPs distribution in Iraq and establish preventative strategies in domestic animals to minimize the risk of infection for animals and humans.

Pathogenic and endosymbiont apicomplexans in Ctenocephalides felis (Siphonaptera: Pulicidae) from cats in Jerusalem, Israel.

This study was conducted to determine the prevalence of pathogenic and endosymbiont apicomplexans in the cat flea, Ctenocephalides felis (Bouché) infesting 185 stray cats in Jerusalem, Israel using PCR assay and sequencing approach. Two pathogens, Hepatozoon felis and Babesia vogeli and an endosymbiont Steinina ctenocephali were detected in 1.9%, 0.2% and 5.8% of 685 C. felis evaluated respectively. There was a significant association (p < 0.05) between the prevalence of H. felis and the sex of cats hosting the fleas as well as the season of sampling but not for age or health status of the cats or sex of the fleas tested. Prevalence of S. ctenocephali was significantly (p < 0.001) associated with season, being higher in the warm season. This report represents the first molecular detection of S. ctenocephali in C. felis. Further studies to determine the potential role of C. felis in the epidemiology of H. felis and B. vogeli are warranted.

Anaplasma phagocytophilum, Bartonella spp., haemoplasma species and Hepatozoon spp. in ticks infesting cats: a large-scale survey.

BACKGROUND: Ticks derived from cats have rarely been evaluated for the presence of pathogens. The aim of this study was to determine the prevalence of Anaplasma phagocytophilum, Bartonella spp., haemoplasma species and Hepatozoon spp. in ticks collected from cats in the UK. METHODS: Five hundred and forty DNA samples extracted from 540 ticks collected from cats presenting to veterinarians in UK practices were used. Samples underwent a conventional generic PCR assay for detection of Hepatozoon spp. and real-time quantitative PCR assays for detection of Anaplasma phagocytophilum and three feline haemoplasma species and a generic qPCR for detection of Bartonella spp. Feline 28S rDNA served as an endogenous internal PCR control and was assessed within the haemoplasma qPCR assays. Samples positive on the conventional and quantitative generic PCRs were submitted for DNA sequencing for species identification. RESULTS: Feline 28S rDNA was amplified from 475 of the 540 (88.0%) ticks. No evidence of PCR inhibition was found using an internal amplification control. Of 540 ticks, 19 (3.5%) contained DNA from one of the tick-borne pathogens evaluated. Pathogens detected were: A. phagocytophilum (n = 5; 0.9%), Bartonella spp. (n = 7; 1.3%) [including Bartonella henselae (n = 3; 0.6%) and Bartonella clarridgeiae (n = 1; 0.2%)], haemoplasma species (n = 5; 0.9%), "Candidatus Mycoplasma haemominutum" (n = 3; 0.6%), Mycoplasma haemofelis (n = 1; 0.2%), "Candidatus Mycoplasma turicensis" (n = 1; 0.2%), Hepatozoon spp. (n = 2; 0.4%), Hepatozoon felis (n = 1; 0.2%) and Hepatozoon silvestris (n = 1; 0.2%). CONCLUSION: These data provide important information on the prevalence of tick-borne pathogens in ticks infesting cats, with the identification of haemoplasma species, A. phagocytophilum, H. felis and Bartonella spp. (including B. henselae and B. clarridgeiae). This study also documents the first report of H. silvestris in ticks collected from domestic cats.

Molecular characterization of Hepatozoon felis in Rhipicephalus sanguineus ticks infested on captive lions (Panthera leo).

Hepatozoon spp. are protozoan parasites that infect a wide range of domestic and wild animals. The infection occurs by ingestion of an infected tick. This study was carried out to detect and characterize Hepatozoon spp. in ticks collected from captive lions (Panthera leo) in Thailand based on the partial 18S rRNA gene sequence. A total of 30 ticks were collected and identified as Rhipicephalus sanguineus. The collected ticks were separated into 10 tick pools by sex and life stages. Of the 10 tick pools examined, only one (10%) was found to be infected with the Hepatozoon species. Sequencing and phylogenetic analysis showed a clustering of the partial 18S rRNA gene sequence like that of H. felis from the GenBank database. This is the first report of H. felis in R. sanguineus ticks collected from captive lions in Thailand. Our results indicated that R. sanguineus may be a possible vector of feline Hepatozoon in Thailand.

Three different Hepatozoon species in domestic cats from southern Italy.

Three species of Hepatozoon, namely, Hepatozoon felis, Hepatozoon canis and Hepatozoon silvestris may affect domestic and/or wild felids. Although hepatozoonosis has been documented in a wide range of mammal species, data on cats are limited. To investigate the occurrence of these pathogens in cats, blood samples were collected from animals living in three provinces of southern Italy (Bari, Lecce, and Matera), and molecularly analysed by PCR amplification and sequencing of segments of the 18S rRNA gene. Out of 196 blood samples collected, Hepatozoon spp. DNA was amplified in ten cats (5.1%, CI: 3%-9%), with the majority of infected animals from Matera (8/34, 23.5%) and one each from the other two provinces. BLAST analysis revealed the highest nucleotide identity with sequences of H. canis, H. felis and H. silvestris deposited in GenBank. Results of this study indicate that these three species of Hepatozoon infect domestic cats in Italy. This is the first report of H. silvestris infection in a domestic cat.

Molecular detection of Hepatozoon spp. and Cytauxzoon sp. in domestic and stray cats from Madrid, Spain.

BACKGROUND: Different species of apicomplexan protozoans of the genera Hepatozoon and Cytauxzoon can infect domestic cats, but their epidemiology and clinical relevance are not fully understood. The aim of this study was to assess the molecular prevalence of Hepatozoon spp. and Cytauxzoon spp. and to identify associated risk factors and clinical and laboratory abnormalities in a population of cats from Madrid, Spain. METHODS: Six hundred and forty-four client-owned and stray cats from Madrid, Spain, were included in this study. DNA samples were analyzed by two polymerase chain reaction (PCR) tests to detect a partial sequence of the 18S rRNA gene of Hepatozoon spp. and Cytauxzoon spp. In order to evaluate possible associations between infection by these protozoans and epidemiological or clinical parameters, data were collected related to: the season of sample collection, age, gender, spayed/neutered status, breed, living area, lifestyle, outdoor access, contact with other animals, prey on wild animals, history of tick or flea infestation, travel history, ectoparasiticide treatment, previous blood transfusion, previous tetracycline administration in the last 60 days, Feline Leukemia virus (FeLV) and Feline Immunodeficiency virus (FIV) status, positivity to other vector-borne diseases, the presence or absence of clinical signs and hematological or biochemical alterations. RESULTS: DNA of Hepatozoon spp. and Cytauxzoon sp. was amplified from the blood of 10 (1.6%) and 8 (1.2%) cats, respectively. Previous treatment with tetracyclines in the last 60 days, previous administration of blood transfusion, a decrease in haematocrit and an increase in creatinine were associated with Hepatozoon spp. infection. Cytauxzoon sp. infection was more frequent in samples collected during the winter months and in cats living in rural areas. This infection was associated with a FIV-positive status. Some of the cats that were positive for Hepatozoon spp. or Cytauxzoon sp. had been exposed to other vector-borne pathogens, such as Ehrlichia canis and Bartonella henselae. CONCLUSIONS: Our results indicate that cats from Madrid, central Spain, are infected with Hepatozoon spp. and Cytauxzoon sp., although with a low prevalence. Further studies are needed to determine the virulence of these agents in Spanish cats.

Prevalence study and risk factor analysis of selected bacterial, protozoal and viral, including vector-borne, pathogens in cats from Cyprus.

BACKGROUND: Feline infectious agent studies are lacking in Cyprus. The aims of this study were to determine the prevalence and risk factors for various feline infectious agents, including feline vector-borne pathogens (FVBP), in cats from Cyprus. METHODS: A cross-sectional, descriptive, multicentre study was performed on 174 feline samples [138 owned and 36 shelter-feral, including both healthy (43) and non-healthy (131), cats] from private veterinary clinics from all six districts of Cyprus. Real-time quantitative polymerase chain reaction (qPCR) assays were used to detect Mycoplasma haemofelis (Mhf), "Candidatus Mycoplasma haemominutum" (CMhm) and "Candidatus Mycoplasma turicensis" (CMt). The population was tested for four FVBP including Bartonella henselae and Leishmania spp. using qPCR, while conventional PCR assays were used to detect Ehrlichia/Anaplasma spp. and Hepatozoon spp. Serological assays were performed to detect Leishmania infantum antibodies, feline leukaemia virus (FeLV) antigen and feline immunodeficiency virus (FIV) antibodies. Statistical analysis was performed to test associations and possible risk factors between variables and infectious agents. RESULTS: Ninety-six (55.2%) of the 174 cats were PCR-positive for at least one infectious agent. Forty-six cats (26.4%) were haemoplasma positive, including 13 (7.5%) for Mhf, 36 (20.7%) for CMhm and 12 (6.9%) for CMt. Sixty-six cats (37.9%) were positive for Hepatozoon spp., while 19 (10.9%) were positive for B. henselae, four (2.3%) for Leishmania spp. and one (0.6%) for Ehrlichia/Anaplasma spp. Sequencing revealed the presence of Hepatozoon felis, L. infantum and Anaplasma platys. Of the 164 cats that underwent retroviral serology, 10 (6.1%) were FeLV-positive and 31 (18.9%) were FIV-positive, while L. infantum serology was positive in 7 (4.4%) of the 160 cats tested. Multivariable logistic regression revealed significant associations for various infectious agents including L. infantum with each of Hepatozoon spp. and CMt infection. CONCLUSIONS: A high prevalence of infectious agents was found in cats from Cyprus with Mhf, CMhm, CMt, L. infantum, B. henselae, H. felis, A. platys, FeLV and FIV infections reported for the first time. The significant associations between different pathogens provide a better understanding of similarities in the epidemiology of these pathogens and interactions between them.

Hepatozoon silvestris sp. nov.: morphological and molecular characterization of a new species of Hepatozoon (Adeleorina: Hepatozoidae) from the European wild cat (Felis silvestris silvestris).

Based on morphological and genetic characteristics, we describe a new species of Hepatozoon in the European wild cat (Felis silvestris silvestris), herein named Hepatozoon silvestris sp. nov. The study also provides the first data on the occurrence of H. felis in this wild felid. Hepatozoon meronts were observed in multiple cross-sections of different organs of four (44%) cats. Additionally, extracellular forms, resembling mature gamonts of Hepatozoon, were found in the spleen and myocardium of two cats. Furthermore, tissues of six animals (67%) were positive by PCR. Hepatozoon felis was identified infecting one cat (11%), whereas the 18S rRNA sequences of the remaining five cats (56%) were identical, but distinct from the sequences of H. felis. Phylogenetic analyses revealed that those sequences form a highly supported clade distant from other Hepatozoon spp. Future studies should include domestic cats from the areas where the wild cats positive for H. silvestris sp. nov. were found, in order to investigate their potential role to serve as intermediate hosts of this newly described species. Identification of its definitive host(s) and experimental transmission studies are required for elucidating the full life cycle of this parasite and the possible alternative routes of its transmission.