A. Criado-Fornelio

Molecular studies on Babesia, Theileria and Hepatozoon in southern Europe: Part I. Epizootiological aspects

Molecular epizootiology of piroplasmids (Babesia spp., Theileria spp.) and Hepatozoon canis was studied in mammals from southern Europe (mainly from Spain, but also from Portugal and France). Partial amplification and sequencing of the 18s rRNA gene was used for molecular diagnosis. In some particular cases (B. ovis and B. bovis) the complete 18s rRNA gene was sequenced. Blood samples were taken from domestic animals showing clinical symptoms: 10 dogs, 10 horses, 10 cows, 9 sheep and 1 goat. In addition, DNA samples were isolated from blood of 12 healthy dogs and from spleen of 10 wild red foxes (Vulpes vulpes). The results of the survey were the following: Piroplasmid infections: Approximately from 50 to 70% of wild or domestic mammals (symptomatic) were infected. Piroplasmids detected in ruminants were:COW: B. bovis, T. annulata and Theileria sp. (type C). Sheep and goat: B. ovis. Piroplasmids present in canids were: Babesia canis vogeli, Babesia canis canis, Theileria annae and B. equi. The only piroplasmid found in asymptomatic dogs was B. equi. Piroplasmids found in horse were: B. equi and B. canis canis.H. canis infections in canids: H. canis was absent of domestic dog samples, whereas all foxes studied were infected by this protozoa. Genetic analysis showed that most of piroplasmid and Hepatozoon isolates from southern Europe matched unambigously with previously described species, as demonstrated by the high level sequence identity between them, usually between 99 and 100%. Minor differences, usually detected in hypervariable regions of 18s rRNA gene are probably due to strain variations or rare genetic polymorphisms. A possible exception was B. bovis, which shows a relatively lower degree of homology (94%) with regard to other B. bovis isolates from several countries. The same is true for B. ovis, that showed a 94% identity with regard to Babesia sp. from South African cow and a 92% with rapport to B. bovis from Portugal.

Presence of Mycoplasma haemofelis, Mycoplasma haemominutum and piroplasmids in cats from southern Europe: a molecular study

Clinical symptoms produced by Mycoplasma spp. and piroplasmids in cats are sometimes similar. Diagnosis of these pathogens is difficult by microscopic procedures and molecular methods have been used as an alternative. We present in this work, the development of new molecular procedures for diagnosis of the aforementioned organisms, together with a molecular characterization of isolates found in southern European cats.A single PCR-RFLP procedure was designed for diagnosis of Mycoplasma spp. and a seminested PCR-RFLP was designed for diagnosis of piroplasmids. The 16S or 18S rRNA genes of isolates found in clinical samples were partially sequenced in all positive cases. Mycoplasma spp. was detected in 9 (30%) out of 30 symptomatic cats from Spain. Sequencing indicated that 66.6% of these isolates can be ascribed to Mycoplasma haemofelis and only 33.3% to Mycoplasma haemominutum. Partial 16S rRNA sequences obtained in Spanish isolates were very similar to those previously published from the UK and the USA. The presence of piroplasmids (Babesia and Theileria spp.) was studied in 16 cats from Spain (n=13) and Portugal (n=3). Animals analyzed were 10 cats with immunosuppressive viral infection (either FeLV or FIV), 5 asymptomatic cats and 1 cat with Babesia-compatible symptoms. Asymptomatic cats were all PCR-negative. Partial sequencing of 18S rRNA gene demonstrated that the Babesia-symptomatic cat was infected with Babesia canis canis whereas 3 (30%) out of the 10 cats with immunosuppressive viral infection were coinfected with piroplasmids (1 with B. canis canis, 1 with Theileria annae, and 1 with B. canis canis and T. annae both).

NEW MOLECULAR DATA ON MAMMALIAN HEPATOZOON SPECIES (APICOMPLEXA: ADELEORINA) FROM BRAZIL AND SPAIN

Molecular techniques were used to examine the phylogenetic relationships among Hepatozoon species isolated from 13 foxes and 15 opossums from Brazil, and from 15 dogs, 20 foxes, 45 rodents, and 330 domestic cats from Spain. Hemogregarine infection was confirmed by amplification of the 18S rRNA gene and later sequencing. No hemogregarine infections were found in opossums. The prevalence of Hepatozoon in canids ranged from 26.6% (symptomatic domestic dogs) to 90% (Spanish foxes). Four different H. canis genotypes were detected, as well as an H. americanum-related protozoan (97% identical to the USA strain). Two Spanish cats were parasitized by a Hepatozoon species (0.6% prevalence) that showed 96% sequence identity to H. canis. DNA amplification assays performed on Spanish rodents showed 2 bank voles (Clethrionomys glareolus) to be infected by a Hepatozoon species (4.44% prevalence) with 95% sequence identity to Hepatozoon sp. from cats. Phylogenetic analysis showed Hepatozoon to be a monophyletic genus, in which species from carnivorous mammals (Hepatozoon sp. from cats, H. americanum and H. canis) appear as a sister lineage of that of lower vertebrates and rodents. This association suggests that H. americanum evolved in ticks and carnivores (either canids, or felids, or both) rather than in other ectoparasites and other types of mammal.

Molecular studies on Babesia, Theileria and Hepatozoon in southern Europe: Part II. Phylogenetic analysis and evolutionary history

Following a study on molecular epizootiology of Hepatozoon canis and piroplasmids (Babesia spp. and Theileria spp.) in southern Europe, newly obtained sequences of 18s rRNA gene were used for phylogenetic analysis. Partial sequences were analysed in isolates showing high degree of homology (>99%) with previous GenBank entries: H. canis, B. canis vogeli, B. equi (two isolates, Spain1 and Spain2), T. annulata and Theileria sp. The complete gene sequences were used for B. ovis and B. bovis, that showed lower homology (<95%) with rapport to previously reported species or isolates. A first set of phylogenetic trees constructed with partial 18s rRNA sequences showed that most European isolates clustered unambiguously with previously described species, so that minor sequence dissimilarities found are due probably to strain variations. The second set of phylogenetic trees was made using the complete 18s rRNA sequences of 44 species from GenBank and the newly sequenced B. ovis and B. bovis. The analysis revealed for the first time a division of piroplasmids in five clades: (1) B. microti group, with B. rodhaini, B. felis, B. leo, B. microti and T. annae (proposed name for the group, without taxonomic value: Archaeopiroplasmids), (2) Western USA Theilerid-like group (proposed name: Prototheilerids), (3) Theileria group, containing all Theileria species from Bovinae (proposed name: Theilerids), (4) A first group of Babesia species including B. canis and B. gibsoni from canids together with B. divergens and B. odocoilei (proposed name: Babesids), (5) A second group composed mainly by Babesia species from ungulates: B. caballi, B. bigemina, B. ovis, B. bovis and Babesia sp. from cow (proposed name: Ungulibabesids). The bootstrap support obtained with several analytical procedures for this new dicotomy of Babesiidae was always very high. Taking into account the present phylogenetic analysis and additional paleogeographic, parasitological and zoological evidences, two hypothesis on the origin and evolution of piroplasmids groups are presented.

A parasitological survey of wild red foxes (Vulpes vulpes) from the province of Guadalajara, Spain.

An epizootiological survey of leishmaniosis, coccidiosis and parasitic helminths in 67 foxes (Vulpes vulpes) was conducted in Guadalajara (central Spain). Examination for parasitic protozoa revealed prevalences of 74% Leishmania (determined by molecular methods) and 2.9% coccidia oocysts (fecal flotation). Survey of parasitic helminths (fecal flotation/necropsy) demonstrated the presence of nine species, including six nematodes, two cestodes and one trematode. Nematodes were the most common parasites of foxes, followed by cestodes and trematodes. Greater levels of nematodes like Uncinaria, with a free-living stage in its life-cycle, were found in foxes in areas where moist soils were likely to exist, in contrast to areas of semiarid characteristics, where Toxascaris leonina or Trichuris vulpis were predominant. With regard to helminths of importance as human pathogens, trichinoscopy revealed the presence of a relatively high number of foxes (8.9%) infected with Trichinella spiralis. Finally, Toxocara canis infection was less frequent (4.4%) than trichinellosis.

Hemoprotozoa of domestic animals in France: Prevalence and molecular characterization

Very limited information is available on epizootiology of haematozoan infections in French domestic animals. In an attempt to address this issue, prevalence of piroplasmida was studied in carnivores and ruminants, whereas prevalence of Hepatozoon spp. was only investigated in carnivores. In total, 383 animals were included in the survey (namely 116 cats, 108 dogs, 91 sheep and 68 cows). Parasite diagnosis was carried out using molecular methods such as PCR and sequencing of the 18S rRNA gene. In addition, ruminant samples were analyzed with the reverse line blotting technique (RLB). Results of RLB and PCR plus sequencing were in total agreement. In carnivores, haematozoan prevalence was close to 1%. Two cats were infected by H. canis (1.7% prevalence) and one of them was co-infected by Cytauxzoon sp. (0.8%). This represents the first finding of both pathogens in French cats. One dog was infected by H. canis (0.9%) and another by Babesia canis vogeli (0.9%). In ruminants, haematozoan prevalence (piroplasmida) was significantly higher than in carnivores (4.8% in sheep and 8.8% in cow). Theileria ovis was found in 1 sheep, Theileria sp. in 2 sheep, Theileria buffeli in 5 cows and B. major in 1 cow. Evidence presented in this contribution indicates that haematic protozoa are not widely distributed in domestic mammal populations of France.

A molecular survey of Piroplasmida and Hepatozoon isolated from domestic and wild animals in Burgos (northern Spain)

This study reports a molecular survey of Hepatozoon species and of the order Piroplasmida in the Province of Burgos, northern Spain. The diagnostic techniques employed included PCR and the sequencing of the 18S rRNA gene. Eighty-nine blood samples from domestic animals plus 138 blood/coagulated blood samples from wild mammals were examined. Theilerid protozoa were found at relatively high frequencies in bovines (14.6%) and horses (36%). Theileria buffeli, T. sergenti and T. annulata were diagnosed in cows. T. equi was common in horses and T. annae was found in a donkey for the first time. A new piroplasmid was found in the European badger (20%). This appears to be distantly related to both T. annae and a piroplasmid isolated from Lontra canadensis. A moderate prevalence (14%) for T. annae was recorded in red foxes. A species of hepatozoon was found in one bank vole (17%), while 28% of the red foxes examined were found to be infected with H. canis. Twenty-five wild house mice were studied and found not to be carriers of piroplasmids or Hepatozoon species. Wild boars, roe deer, hares, Apodemus sp. and moles were also negative for haematozoan infection. The present study indicates that piroplasmid protozoa are present at a low to moderate frequency in some domestic herbivores in the Burgos area. They also infect certain wild mammalian species, which may act as zoonotic carriers.

Proteolytic enzymes from Trichinella spiralis larvae

Trichinella spiralis larvae infect their hosts by the penetration of small intestine enterocytes. The exact mechanism of penetration is unknown, but the presence of proteolytic enzymes is suspected. In this study, whole worm extracts and excretory-secretory (ES) components were obtained and their proteolytic enzymes examined. Enzymes from worm extracts were capable of hydrolysing azocoll, a general protease substrate in a wide range of pH (2-8), with maximal activity at pH 5. Trichinella spiralis larval enzymes were sensitive to metalloprotease and serine protease inhibitors. Three proteases were identified in worm extracts at molecular weight (MW) 48, 54 and 62 kDa by incorporating a gelatine substrate into a standard or a modified sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) set-up, in which we used low SDS concentration in gel and electrophoresis buffer (0.01%). Intact larvae incubated in a medium containing azocoll showed azocollytic activity. Subsequent analysis of ES products by modified SDS-PAGE in gels containing gelatine demonstrated the presence of three protease of apparent MW 33, 62 and 230 kDa.

Molecular characterization of arthropod-borne hematozoans in wild mammals from Brazil, Venezuela and Spain

A survey of Babesia, Theileria and Hepatozoon was conducted in wild mammals, including the capybara (Hydrochaeris hydrochaeris; n = 14) from Brazil, the jaguar (Panthera onca; n = 2) and crab-eating raccoon (Procyon cancrivorus; n = 4) from Venezuela, and the red deer (Cervus elaphus; n = 70), red squirrel (Sciurus vulgaris; n = 5) and Eurasian pine marten (Martes martes; n = 3) from Spain. Diagnostic procedures included both microscopy and molecular methods (PCR and sequencing of the 18S rRNA gene). Microscopic examination of blood smears revealed no hematozoan infections — unlike the molecular analyses. Nine Brazilian capybaras were found to be infected with Hepatozoon canis (prevalence 64%), two of which were coinfected with a previously unknown babesid (prevalence 14%) loosely related to Theileria equi (90% 18S rRNA gene similarity according to BLAS® analysis). One jaguar and one crab-eating raccoon from Venezuela were infected by H. canis. Four of the red deer were infected with theilerids (5.7% prevalence), two with Theileria sp. and two with T. annulata. One red squirrel and three pine martens were infected with Hepatozoon sp. The isolate form the red squirrel was phylogenetically related to Hepatozoon sp. reported in Spanish bank voles, whereas those infecting the pine martens were related to Hepatozoon felis reported in Spanish cats. In conclusion, the molecular findings show that some non-canid mammals are carriers of H. canis in South America, while red deer may carry T. annulata in Europe. Small mammals in Europe appear to be unlikely hosts of H. canis and H. felis.

Development of fluorogenic probe-based PCR assays for the detection and quantification of bovine piroplasmids

This paper reports two new quantitative PCR (qPCR) assays, developed in an attempt to improve the detection of bovine piroplasmids. The first of these techniques is a duplex TaqMan assay for the simultaneous diagnosis of Babesia bovis and B. bigemina. This technique is ideal for use in South America where bovids harbour no theilerids. The second technique, which is suitable for the diagnosis of both babesiosis and theileriosis worldwide, involves fluorescence resonance energy transfer (FRET) probes. In FRET assays, Babesia bovis, B. divergens, Babesia sp. (B. major or B. bigemina), Theileria annae and Theileria sp. were all identifiable based on the melting temperatures of their amplified fragments. Both techniques provided linear calibration curves over the 0.1fg/microl to 0.01ng/microl DNA range. The assays showed good sensitivity and specificity. To assess their performance, both procedures were compared in two separate studies: the first was intended to monitor the experimental infection of calves with B. bovis and the second was a survey where 200 bovid/equine DNA samples from different countries were screened for piroplasmids. Comparative studies showed that duplex TaqMan qPCR was more sensitive than FRET qPCR in the detection of babesids.