The Recent Evolution of a Maternally-Inherited Endosymbiont of Ticks Led to the Emergence of the Q Fever Pathogen, Coxiella burnetii.

Q fever is a highly infectious disease with a worldwide distribution. Its causative agent, the intracellular bacterium Coxiella burnetii, infects a variety of vertebrate species, including humans. Its evolutionary origin remains almost entirely unknown and uncertainty persists regarding the identity and lifestyle of its ancestors. A few tick species were recently found to harbor maternally-inherited Coxiella-like organisms engaged in symbiotic interactions, but their relationships to the Q fever pathogen remain unclear. Here, we extensively sampled ticks, identifying new and atypical Coxiella strains from 40 of 58 examined species, and used this data to infer the evolutionary processes leading to the emergence of C. burnetii. Phylogenetic analyses of multi-locus typing and whole-genome sequencing data revealed that Coxiella-like organisms represent an ancient and monophyletic group allied to ticks. Remarkably, all known C. burnetii strains originate within this group and are the descendants of a Coxiella-like progenitor hosted by ticks. Using both colony-reared and field-collected gravid females, we further establish the presence of highly efficient maternal transmission of these Coxiella-like organisms in four examined tick species, a pattern coherent with an endosymbiotic lifestyle. Our laboratory culture assays also showed that these Coxiella-like organisms were not amenable to culture in the vertebrate cell environment, suggesting different metabolic requirements compared to C. burnetii. Altogether, this corpus of data demonstrates that C. burnetii recently evolved from an inherited symbiont of ticks which succeeded in infecting vertebrate cells, likely by the acquisition of novel virulence factors.

Evolutionary changes in symbiont community structure in ticks.

Ecological specialization to restricted diet niches is driven by obligate, and often maternally inherited, symbionts in many arthropod lineages. These heritable symbionts typically form evolutionarily stable associations with arthropods that can last for millions of years. Ticks were recently found to harbour such an obligate symbiont, Coxiella-LE, that synthesizes B vitamins and cofactors not obtained in sufficient quantities from blood diet. In this study, the examination of 81 tick species shows that some Coxiella-LE symbioses are evolutionarily stable with an ancient acquisition followed by codiversification as observed in ticks belonging to the Rhipicephalus genus. However, many other Coxiella-LE symbioses are characterized by low evolutionary stability with frequent host shifts and extinction events. Further examination revealed the presence of nine other genera of maternally inherited bacteria in ticks. Although these nine symbionts were primarily thought to be facultative, their distribution among tick species rather suggests that at least four may have independently replaced Coxiella-LE and likely represent alternative obligate symbionts. Phylogenetic evidence otherwise indicates that cocladogenesis is globally rare in these symbioses as most originate via horizontal transfer of an existing symbiont between unrelated tick species. As a result, the structure of these symbiont communities is not fixed and stable across the tick phylogeny. Most importantly, the symbiont communities commonly reach high levels of diversity with up to six unrelated maternally inherited bacteria coexisting within host species. We further conjecture that interactions among coexisting symbionts are pivotal drivers of community structure both among and within tick species.

Prevalence of tick-borne pathogens in ticks collected from the wild mountain ungulates mouflon and chamois in 4 regions of France.

Ticks are major vectors of various pathogens of health importance, such as bacteria, viruses and parasites. The problems associated with ticks and vector-borne pathogens are increasing in mountain areas, particularly in connection with global climate change. We collected ticks (n = 2,081) from chamois and mouflon in 4 mountainous areas of France. We identified 6 tick species: Ixodes ricinus, Rhipicephalus bursa, Rh. sanguineus s.l., Haemaphysalis sulcata, H. punctata and Dermacentor marginatus. We observed a strong variation in tick species composition among the study sites, linked in particular to the climate of the sites. We then analysed 791 ticks for DNA of vector-borne pathogens: Babesia/Theileria spp., Borrelia burgdorferi s.l., Anaplasma phagocytophilum, A. marginale, A. ovis, and Rickettsia of the spotted fever group (SFG). Theileria ovis was detected only in Corsica in Rh. bursa. Babesia venatorum (2 sites), Borrelia burgdorferi s.l. (B. afzelii and B. garinii; 2 sites) and Anaplasma phagocytophilum (3 sites) were detected in I. ricinus. Anaplasma ovis was detected at one site in I. ricinus and Rh. sanguineus s.l. SFG Rickettsia were detected at all the study sites: R. monacensis and R. helvetica in I. ricinus at the 3 sites where this tick is present; R. massiliae in Rh. sanguineus s.l. (1 site); and R. hoogstraalii and Candidatus R. barbariae in Rh. bursa in Corsica. These results show that there is a risk of tick-borne diseases for humans and domestic and wild animals frequenting these mountain areas.

Title: Prévalence d'agents pathogènes vectorisés chez des tiques collectées chez des ongulés sauvages (mouflons, chamois) dans 4 zones montagneuses en France. Abstract: Les tiques sont des vecteurs majeurs de différents agents pathogènes d'importance sanitaire, tels que des bactéries, des virus et des parasites. Les problématiques liées aux tiques et aux pathogènes vectorisés augmentent en zones de montagne, en lien notamment avec le réchauffement climatique. Nous avons collecté des tiques (n = 2 081) sur des chamois et des mouflons dans 4 zones montagneuses en France. Six espèces ont été identifiées : Ixodes ricinus, Rhipicephalus bursa, Rh. sanguineus s.l., Haemaphysalis sulcata, H. punctata et Dermacentor marginatus. Nous avons observé une forte variation de la composition en espèces de tiques entre les sites d'étude, en lien notamment avec le climat des sites. Nous avons ensuite recherché les ADN d'agents pathogènes vectorisés sur 791 tiques : Babesia/Theileria spp, Borrelia burgdorferi s.l., Anaplasma phagocytophilum, A. marginale, A. ovis, et de Rickettsia du groupe des fièvres boutonneuses (SFG). Theileria ovis a été détecté uniquement en Corse chez Rh. bursa. Babesia venatorum (2 sites), Borrelia burgdorferi s.l. (B. afzelii and B. garinii; 2 sites) et Anaplasma phagocytophilum (3 sites) ont été détectés chez I. ricinus. Anaplasma ovis a été détecté dans un site chez I. ricinus et Rh. sanguineus s.l.. Les Rickettsia SFG ont été détectées dans tous les sites d'étude : Rickettsia monacensis et R. helvetica chez I. ricinus dans les 3 sites où cette tique est présente; R. massiliae chez Rh. sanguineus s.l. (1 site); et R. hoogstraalii et Candidatus R. barbariae chez Rh. bursa en Corse. Ces résultats montrent un risque de transmission de maladies par les tiques pour les personnes et les animaux domestiques et sauvages fréquentant ces zones de montagne.

Lack of efficacy of fenbendazole against Giardia duodenalis in a naturally infected population of dogs in France.

Giardiosis is a worldwide intestinal parasitosis, affecting both humans and animals. Treatment in dogs remains limited and the lack of efficacy of the few approved medications is a rising concern. In this study, 23 dogs raised by veterinary students and naturally infected with Giardia duodenalis were treated in home conditions with fenbendazole (50 mg/kg orally for 5 consecutive days). Fecal samples were collected immediately before treatment (FS1), 2-4 days after treatment (FS2) and 8-10 days after treatment (FS3). Giardia duodenalis cyst excretion was measured quantitatively by direct immunofluorescence assay (DFA) at FS1, FS2 and FS3. Molecular typing with a nested PCR targeting the SSU rDNA locus was also performed at FS1 and FS2. Fecal consistency improved in 16/21 dogs (76%) and mean cyst shedding was reduced by 84% after treatment. However, only 8/23 dogs (35%) achieved therapeutic success (≥90% reduction of cysts) and only 4/23 dogs (17%) had complete elimination of G. duodenalis. Molecular typing showed that dogs harbored only canine-specific assemblages, with a high prevalence of assemblage C in analyzed samples (30/39). We also detected different assemblages after treatment and nucleotide substitutions in assemblage C sequences that have not been described previously. Eight to ten days after treatment, high Giardia cyst excretion was measured, suggesting possible reinfection despite hygiene measures and/or multiplication. These data suggest that fenbendazole treatment may improve fecal consistency but has limited therapeutic efficacy against giardiosis in this population of dogs. Further research is still needed to assess the efficacy of fenbendazole against canine giardiosis.

Title: Absence d'efficacité du fenbendazole contre Giardia duodenalis dans une population de chiens naturellement infectés en France. Abstract: La giardiose est une parasitose intestinale mondiale, touchant à la fois l'homme et les animaux. Chez le chien, le traitement reste limité et le manque d'efficacité des quelques médicaments autorisés inquiète de plus en plus. Dans cette étude, 23 chiens d'étudiants vétérinaires et infectés naturellement par Giardia duodenalis ont été traités en conditions réelles avec du fenbendazole (50 mg/kg par voie orale pendant 5 jours consécutifs). Des échantillons de selles ont été collectés juste avant le traitement (FS1), 2­4 jours après traitement (FS2) et 8­10 jours après traitement (FS3). L'excrétion de kystes de G. duodenalis a été mesurée quantitativement par immunofluorescence directe (IFD) à FS1, FS2 et FS3. Un génotypage par PCR nichée ciblant le locus SSU ADNr a également été réalisé à FS1 et FS2. La consistance des selles a été améliorée chez 16/21 (76 %) chiens et la moyenne d'excrétion des kystes a été réduite de 84 % juste après le traitement. Seulement 8/23 (35 %) chiens ont atteint un succès thérapeutique (≥ 90 % de réduction d'excrétion de kystes) et 4/23 (17 %) chiens ont eu une élimination complète de G. duodenalis. L'analyse des séquences a montré que les chiens présentaient seulement des assemblages génotypiques spécifiques de l'espèce canine, avec une forte prévalence de l'assemblage C dans les échantillons analysés (30/39). Des changements d'assemblage après traitement et des substitutions nucléotidiques jamais décrites au sein de l'assemblage C ont également été observés. Huit à dix jours après traitement, une forte excrétion de kystes de G. duodenalis a été mesurée : malgré les mesures hygiéniques, une réinfection et/ou une multiplication semblent probables. Ces données suggèrent que le traitement au fenbendazole peut améliorer la consistance des selles mais a une efficacité thérapeutique limitée contre la giardiose dans cette population de chiens. Des recherches supplémentaires sont encore nécessaires pour évaluer l'efficacité du fenbendazole contre la giardiose canine.

Diversity, geographic distribution, and habitat-specific variations of microbiota in natural populations of the chicken mite, Dermanyssus gallinae.

Dermanyssus gallinae is considered to be the most economically significant ectoparasite to affect egg-laying poultry in Europe. This mite can also act as a vector for a number of pathogens. The array of bacteria associated with D. gallinae mites could provide insight into the biology and population dynamics of arthropods, but at the present time little information is available. To understand the intra- and interpopulation diversity of its associated microbiota, we analyzed the whole internal bacterial community of natural populations of D. gallinae originating from two types of poultry farm habitats (standard and free-range) in two regions of France (Brittany and the Rhone-Alpes). Total DNA was extracted from individual or pooled mites, and polymerase chain reaction temporal temperature gradient gel electrophoresis of 16S rRNA was then done to separate bacterial DNA fragments associated with the host arthropod. A large diversity of bacteria was detected, but principally firmicutes and gamma-Proteobacteria. Between-group analyses of temporal temperature gradient gel electrophoresis-banding patterns revealed that bacterial populations clustered into categories according to their geographic origin and the habitat specifics of the farms. Some degree of stability of bacterial populations was observed within a specific time scale. These results suggest that environmental factors either recent (e.g., poultry farming practices) or long-standing (e.g., geographic isolation) may affect the bacterial communities present in D. gallinae. Further knowledge of the microbiota associated with D. gallinae and its variation would indeed offer new perspectives for biological control methods to prevent the establishment, proliferation, and transmission of pathogenic bacteria.

Bacterial taxa associated with the hematophagous mite Dermanyssus gallinae detected by 16S rRNA PCR amplification and TTGE fingerprinting.

Dermanyssus gallinae (Arthropoda, Mesostigmata) is suspected to be involved in the transmission of a wide variety of pathogens, but nothing is known about its associated non-pathogenic bacterial community. To address this question, we examined the composition of bacterial communities in D. gallinae collected from standard poultry farms in Brittany, France. Genetic fingerprints of bacterial communities were generated by temporal temperature gradient gel electrophoresis (TTGE) separation of individual polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments, followed by DNA sequence analysis. Most of the sequences belonged to the Proteobacteria and Firmicute phyla, with a majority of sequences corresponding to the Enterobacteriales order and the Staphylococcus genus. By using statistical analysis, we showed differences in biodiversity between poultry farms. We also determined the major phylotypes that compose the characteristic microbiota associated with D. gallinae. Saprophytes, opportunistic pathogens and pathogenic agents such as Pasteurella multocida, Erysipelothrix rhusiopathiae and sequences close to the genus Aerococcus were identified. Endosymbionts such as Schineria sp., Spiroplasma sp. Anistosticta, "Candidatus Cardinium hertigii" and Rickettsiella sp. were also present in the subdominant bacterial community. Identification of potential targets within the symbiont community may be considered in the future as a means of ectoparasite control.

The poultry red mite (Dermanyssus gallinae): a potential vector of pathogenic agents.

The poultry red mite, D. gallinae has been involved in the transmission of many pathogenic agents, responsible for serious diseases both in animals and humans. Nowadays, few effective methods are available to control the ectoparasite in poultry farms. Consequently, this is an emerging problem which must be taken into account to maintain good health in commercial egg production. This paper addresses the vector capacity of the ectoparasite with special emphasis on salmonellae, pathogenic agents responsible for many of the most important outbreaks of food-borne diseases worlwide. It has been experimentally shown that D. gallinae could act as a biological vector of S. enteritidis and natural carriage of these bacteria by the mite on poultry premises has also been reported. It was also found that D. gallinae carried other pathogens such as E. coli, Shigella sp., and Staphylococcus, thus increasing the list of pathogenic agents potentially transmitted by the mite.

Endosymbiotic bacteria living inside the poultry red mite (Dermanyssus gallinae).

This study investigated the endosymbiotic bacteria living inside the poultry red mite collected from five samples of one commercial farm from the UK and 16 farms from France using genus-specific PCR, PCR-TTGE and DNA sequencing. Endosymbiotic bacteria are intracellular obligate organisms that can cause several phenotypic and reproductive anomalies to their host and they are found widespread living inside arthropods. The farm sampled from the UK was positive for bacteria of the genera Cardinium sp. and Spiroplasma sp. From France, 7 farms were positive for Cardinium sp., 1 farm was positive for Spiroplasma sp., 1 farm was positive for Rickettsiella sp. and 2 farms were positive for Schineria sp. However, it was not possible to detect the presence of the genus Wolbachia sp. which has been observed in other ectoparasites. This study is the first report of the presence of endosymbionts living inside the poultry red mite. The results obtained suggest that it may be possible that these bacterial endosymbionts cause biological modifications to the poultry red mite.

Detection of tick-borne pathogens in questing Ixodes ricinus in the French Pyrenees and first identification of Rickettsia monacensis in France.

Ticks are important vectors of several human and animal pathogens. In this study, we estimated the prevalence of important tick-borne infections in questing ticks from an area in Southwestern France (Hautes-Pyrénées) inhabited by Pyrenean chamois (Rupicapra pyrenaica pyrenaica) experiencing high tick burden. We examined adult and nymph ticks collected by the flag dragging method from 8 to 15 sites in the Pic de Bazès during the years 2009, 2011, 2013 and 2015. PCR assays were conducted on selected ticks for the detection of Borrelia burgdorferi s.l., Babesia spp., Rickettsia spp., spotted fever group (SFG) Rickettsia and Anaplasma phagocytophilum. Randomly selected positive samples were submitted for sequence analysis. A total of 1971 questing ticks were collected including 95 males, 101 females and 1775 nymphs. All collected ticks were identified as Ixodes ricinus. Among them, 696 ticks were selected for pathogen detection and overall prevalence was 8.4% for B. burgdorferi s.l.; 0.4% for Babesia spp.; 6.1% for A. phagocytophilum; 17.6% for Rickettsia spp.; and 8.1% for SFG Rickettsia. Among the sequenced pathogens, we detected in this population of ticks the presence of Babesia sp. EU1 and Rickettsia helvetica, as well as Rickettsia monacensis for the first time in France. The detection of these pathogens in the Pic de Bazès highlights the potential infection risks for visitors to this area and the Pyrenean chamois population.

TITLE: Détection d'agents pathogènes présents chez les tiques errantes, Ixodes ricinus, dans les Pyrénées françaises et première identification de Rickettsia monacensis en France. ABSTRACT: Les tiques sont des vecteurs importants de plusieurs maladies animales et humaines. Dans cette étude, nous avons estimé la prévalence de maladies vectorisées chez les tiques errantes dans une région du sud-ouest de la France (Hautes-Pyrénées) occupée par des chamois des Pyrénées (Rupicapra pyrenaica pyrenaica) fortement infestés par les tiques. Nous avons utilisé des tiques adultes et des larves collectées par la technique du drapeau sur 8­15 sites du Pic de Bazès au cours des années 2009, 2011, 2013 et 2015. Des analyses PCR ont été menées sur des tiques sélectionnées pour la détection de Borrelia burgdorferi s.l., Babesia spp., Rickettsia spp., Rickettsia du groupe de la fièvre pourprée (GFP) et Anaplasma phagocytophilum. Des échantillons positifs sélectionnés au hasard ont été soumis à une analyse de séquences. Un total de 1971 tiques a été collecté dont 95 mâles, 101 femelles et 1775 larves. Toutes les tiques recueillies ont été identifiées comme étant Ixodes ricinus. Parmi celles-ci, 696 tiques ont été sélectionnées pour la détection des agents pathogènes et la prévalence globale était de 8,4 % pour B. burgdorferi s.l., 0,4 % pour Babesia spp., 6,1 % pour A. phagocytophilum, 17,6 % pour Rickettsia spp. et 8,1 % pour Rickettsia GFP. Parmi les agents pathogènes séquencés, nous avons détecté dans cette population de tiques la présence de Babesia sp. EU1, Rickettsia helvetica et, pour la première fois en France, Rickettsia monacensis. La détection de ces agents pathogènes dans le Pic de Bazès souligne les risques potentiels d'infection pour les visiteurs de cette région et pour la population de chamois des Pyrénées.

Prevalence and genetic diversity of the intestinal parasites Blastocystis sp. and Cryptosporidium spp. in household dogs in France and evaluation of zoonotic transmission risk.

Several parasites including the protozoa Blastocystis sp. and Cryptosporidium spp. may be causative agents of gastrointestinal symptoms in domestic dogs, and there may be a potential risk of transmission to owners. While France is one of the largest European countries in terms of its canine population, little data is available about the molecular epidemiology of these two parasites. The purpose of this study was to determine the prevalence of intestinal parasites in household dogs in France, and to evaluate the zoonotic risk of Blastocystis sp. and Cryptosporidium spp. by genotyping the corresponding isolates. To this end, 116 faecal samples were collected from household dogs regardless of breed, age or gender, living in the Lyons area, France. Various intestinal protozoa and helminths were identified by light microscopy. Screening for Blastocystis sp. and Cryptosporidium spp. were subsequently performed by PCR targeting the small subunit (SSU) rDNA coding region, followed by direct sequencing of the PCR products and analysis of the sequences obtained for genotyping. The overall prevalence of dogs infected with at least one gastrointestinal parasite was 42.2% (49/116). After light microscopy examination of faecal samples, the most common parasites found were the protozoa Giardia sp. (25.0%) and Cystoisospora sp. (19.8%). Using molecular methods, four dogs (3.4%) were shown to be infected by Blastocystis sp. and carried either subtype (ST) 2, commonly identified in various animal groups, or ST10, frequently found in bovids. Three dogs (2.6%) were positive for C. canis, infecting humans episodically. The low prevalence of both parasites, combined with the identification of C. canis and Blastocystis sp. ST2 and ST10 in the canine population, strongly suggests that dogs play a negligible role as zoonotic reservoirs for both parasites and do not seem to be natural hosts of Blastocystis sp.

Prevalence of Tritrichomonas foetus infections in French catteries.

Recently, Tritrichomonas foetus, the known etiologic agent of bovine trichomonosis was identified in domestic cats in many countries around the world. In felids, this parasite would be a significant cause of large-bowel diarrhoea. Therefore the aim of the present study was to determine for the first time the prevalence of T. foetus infection in French catteries. In this epidemiological survey, rectal swabs from 140 cats participating in three international shows were tested for the presence of motile parasites by microscopy after culturing. The prevalence of T. foetus infection was 14.3% among cats (20/140) and 15.9% among catteries (18/117). These values were similar to those previously obtained in other European countries. Except for the age, no significant associations were found between the presence of T. foetus and various risk factors of infection such as the size of the cattery, the type of food, or the vicinity of a dog. Internal transcribed region of the ribosomal DNA unit was sequenced from the 20 T. foetus isolates identified in this study. They exhibited 100% identity and are homologous with other sequences of strains isolated from domestic cats in other countries.

A preliminary study of natural and experimental infection of the lesser mealworm Alphitobius diaperinus (Coleoptera: Tenebrionidae) with Histomonas meleagridis (Protozoa: Sarcomastigophora).

Histomonas meleagridis is a protozoan parasite causing histomoniasis (histomonosis), a disease of gallinaceous fowl. In order to determine whether the lesser mealworm, Alphitobius diaperinus, is capable of harbouring H. meleagridis, the presence of the parasite was tested by polymerase chain reaction in lesser mealworms collected in the field. Parasite DNA was detected in two larvae from two farms undergoing an outbreak of histomoniasis. Insects were also artificially infected, killed after incubation and analysed by polymerase chain reaction for the presence of parasite DNA. After 4 days, two larvae (out of 20) remained positive. In another experimental infection to investigate the viability of histomonads in the larvae, living parasites were detected in five of 20 larvae 4 days after infection. These results indicate that although A. diaperinus can become infected with H. meleagridis it appears to have a low susceptibility to infection and would probably not be a major route of contamination between flocks.

Colonization and organ invasion in chicks experimentally infected with Dermanyssus gallinae contaminated by Salmonella Enteritidis.

The poultry red mite (Dermanyssus gallinae) is the most important and common ectoparasite of laying hens in Europe. This haematophagous mite has been experimentally demonstrated to be a vector of Salmonella Enteritidis by acquiring bacteria through the blood meal or cuticular contact. We have evaluated another route of infection by orally inoculating chicks with mites previously infected by S. Enteritidis. Two methods of infecting the mites were tested: mites contaminated by cuticular contact or during the blood meal. After the washing of mites with paraformaldehyde, groups of 10 Salmonella-contaminated mites were inoculated individually into 1-day-old chicks. The titre of the inoculum suspension was evaluated by crushing mites and followed by bacteriological counting. It was 3x10(4) colony-forming units/chick and 2.7x10(6) colony-forming units/chick, respectively, for cuticular contact and orally mediated contamination of mites. Each bird was found to be positive 12 days post-inoculation. Salmonella colonized the intestinal tracts and invaded the livers and spleens. The caecal content concentration reached a mean level of S. Enteritidis of 8.5x10(4) most probable number (MPN) Salmonella/g. This experiment demonstrated the ability of mites to orally infect 1-day-old chicks with subsequent colonization and multiplication of Salmonella. Consequently, mites infected by S. Enteritidis constitute potential reservoir hosts of this bacterium, allowing it to persist in the poultry house as a source of infection for newly introduced animals. If contaminated mites are found in poultry facilities, effective red mite control should be performed before new batches are introduced into the facility.

Comparison of four methods of extracting DNA from D. gallinae (Acari: Dermanyssidae).

Dermanyssus gallinae is one of the most serious ectoparasites of poultry and it has been implicated as a vector of several major pathogenic diseases. Molecular detection of such pathogens in mites is crucial and therefore, an important step is the extraction of their DNA from mites. So, we compared four DNA extraction protocols from engorged and unfed individual mites: a conventional method using a Cethyl Trimethyl Ammonium Bromide buffer (CTAB), a Chelex resin, a Qiamp DNA extraction kit and a more recent one filter-based technology (FTA). The DNA samples have been tested for their ability to be amplified by an amplification of a D. gallinae 16S rRNA gene region. The best results were obtained using CTAB and Qiagen methods at the same time with unfed and engorged mites (96% and 100% of amplified samples). FTA produced similar results when using unfed mites but not when processing engorged ones (96% and 70%). Finally, the Chelex method was the least efficient in terms of DNA amplification, especially when applied on engorged individuals (50%). The possible inhibitor role of these Chelex extracted DNA was demonstrated by the means of a PCR control on PUC plasmid. No difference was observed with CTAB, Qiamp DNA extraction kit or FTA methods using DNA extracted one year before.

Molecular phylogenies of Blastocystis isolates from different hosts: implications for genetic diversity, identification of species, and zoonosis.

Small-subunit (SSU) rRNA gene sequences were obtained by PCR from 12 Blastocystis isolates from humans, rats, and reptiles for which elongation factor 1alpha (EF-1alpha) gene sequences are already available. These new sequences were analyzed by the Bayesian method in a broad phylogeny including, for the first time, all Blastocystis sequences available in the databases. Phylogenetic trees identified seven well-resolved groups plus several discrete lineages that could represent newly defined clades. Comparative analysis of SSU rRNA- and EF-1alpha-based trees obtained by maximum-likelihood methods from a restricted sampling (13 isolates) revealed overall agreement between the two phylogenies. In spite of their morphological similarity, sequence divergence among Blastocystis isolates reflected considerable genetic diversity that could be correlated with the existence of potentially >/=12 different species within the genus. Based on this analysis and previous PCR-based genotype classification data, six of these major groups might consist of Blastocystis isolates from both humans and other animal hosts, confirming the low host specificity of Blastocystis. Our results also strongly suggest the existence of numerous zoonotic isolates with frequent animal-to-human and human-to-animal transmissions and of a large potential reservoir in animals for infections in humans.