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Domestic pigs are considered as one of the main intermediate hosts in the zoonotic transmission of Toxoplasma gondii in many countries. Serological and molecular studies are warranted to better understand the epidemiology and transmission patterns of this parasite worldwide. To date, seroepidemiological information on T. gondii in domestic pigs in Cuba is very scarce and there are no reports of T. gondii genotypes circulating in this country. Here, we aimed to estimate the seroprevalence of T. gondii and provide genetic characterization of the strains circulating in slaughtered pigs intended for human consumption in Central Cuba. Seroprevalence was determined in 450 serum samples from slaughtered pigs in Villa Clara province using ELISA. Anti-Toxoplasma gondii IgG antibodies were detected in 100 animals (22.2%, 95% CI: 18.5-26.2). Conventional PCR of the 529-bp marker of T. gondii was performed in hearts and diaphragm tissues of all ELISA-seropositive pigs. Toxoplasma gondii DNA was detected in four animals. Further genetic characterization of the positive DNA samples was performed by multilocus PCR-RFLP and PCR-sequencing typing tools. Molecular analysis revealed four different genetic profiles that were combinations of type I, II, III and u-1 alleles, suggesting the circulation of non-clonal genotypes of T. gondii in domestic pigs in Cuba. Our results indicate that T. gondii is widely distributed in slaughtered pigs in this country, which might have important implications for public health. To the best of our knowledge, this is the first report on genetic characterization of T. gondii in Cuba. Although preliminary, the results suggest a high genetic diversity of T. gondii in the study region. Further studies based on parasite isolation are needed to definitively identify the genotypes circulating and characterize the virulence of strains detected in pigs in Cuba, and to assess the risk of zoonotic transmission from pork products in this country.

Little information is currently available on the occurrence and genetic diversity of pathogenic and commensal protist species in captive and semi-captive non-human primates (NHP) resident in zoological gardens or sanctuaries in low- and medium-income countries. In this molecular-based study, we prospectively collected individual faecal samples from apparently healthy NHP at the Abidjan Zoological Garden (AZG) in Côte d’Ivoire, the Tacugama Sanctuary (TS) in Sierra Leone, and the Quistococha Zoological Garden (QZG) in Peru between November 2018 and February 2020. We evaluated for the presence of pathogenic (Cryptosporidium spp., Entamoeba histolytica, Giardia duodenalis, Blastocystis sp., Enterocytozoon bieneusi, Balantioides coli) and commensal (Entamoeba dispar, Troglodytella abrassarti) protist species using PCR methods and Sanger sequencing. Giardia duodenalis was the most prevalent species found (25.9%, 30/116), followed by Blastocystis sp. (22.4%, 26/116), and E. dispar (18.1%, 21/116). We detected E. bieneusi (4.2%, 1/24) and T. abrassarti (12.5%, 3/24) only on NHP from AZG. Cryptosporidium spp., E. histolytica, and B. coli were undetected at the three sampling sites investigated here. Sequence analyses revealed the presence of zoonotic sub-assemblages BIII (n = 1) in AZG and BIV (n = 1) in TS within G. duodenalis. We identified Blastocystis subtype ST3 (100%, 6/6) in AZG, ST1 (80.0%, 12/15), ST2 (6.7%, 1/15), and ST3 (13.3%, 2/15) in TS, and ST2 (80.0%, 4/5) and ST3 (20.0%, 1/5) in QZG. The only E. bieneusi isolate detected here was identified as zoonotic genotype CAF4. Our PCR-based data indicate that potentially pathogenic protist species including G. duodenalis, Blastocystis sp., E. bieneusi, and B. coli are present at variable rates in the three NHP populations investigated here. The identification of zoonotic genotypes within these species indicates that human-NHP transmission is possible, although the extent and directionality of these events need to be elucidated in future molecular surveys., Graphical abstract Image 1, Highlights • Giardia and Blastocystis are highly prevalent in confined non-human primates. • Diarrhoea-causing Cryptosporidium and Entamoeba histolytica were undetected. • First description of Enterocytozoon bieneusi genotype CAF4 in non-human primates. • Confined non-human primates harbour protist species with zoonotic potential. • Cross-species (including human) transmission is possible in zoos and sanctuaries.

The phylum Microsporidia encompasses a diverse group of obligate, intracellular, and spore-forming organisms able to infect a wide range of animal hosts. Among them, Enterocytozoon bieneusi is the most frequently reported species in humans and animals. Little is known about the presence and epidemiology of E. bieneusi in wildlife. We investigated E. bieneusi occurrence and genetic diversity in wild and domestic mammals, through molecular-detection methods, from different regions across Portugal. A total of 756 samples were collected from 288, 242, and 226 wild carnivores, wild ungulates, and domestic animals, respectively. Overall, eight specimens were E. bieneusi-positive (1.1%, 8/756) obtained from five wild (Iberian lynx, Iberian wolf, red fox, stone marten, and wild boar) and one domestic (sheep) host. Nucleotide sequence analysis identified four genotypes of E. bieneusi, Type IV, Wildboar3, BEB6, and PtEbIX. Three of those genotypes belong to Groups 1 (Type IV and Wildboar3) and 2 (BEB6), which are known to contain genotypes capable of infecting a variety of hosts, including humans, highlighting their public health importance. PtEbIX belongs to the dog-specific Group 11. This study represents the first, largest, and most comprehensive molecular-based epidemiology survey carried out in Portugal in wild and domestic animals to date and the first worldwide identification of E. bieneusi in wolf species. Our study showed that wild carnivores and ungulates may act as reservoirs of zoonotic genotypes of E. bieneusi, establishing their role in maintaining the sylvatic cycle of this parasite while representing a potential source of infection for humans and domestic animals.

Rodents and other micromammals constitute important reservoirs of infectious diseases; their role in the life cycle of apicomplexan parasites such as Toxoplasma gondii, Neospora caninum, and Sarcocystis spp. still needs clarification. In the present study, we analyzed by PCR and Sanger sequencing methods the presence of specific parasite DNA within brain and heart tissues of 313 individuals of five synanthropic small mammal species (Apodemus sylvaticus, Mus spretus, M. musculus, Rattus rattus, and Crocidura russula) collected in Barcelona metropolitan area (NE Spain). In addition, PCR-RFLP and microsatellites were also used as tools for genotypic characterization of T. gondii and N. caninum, respectively. Specific DNA of T. gondii, N. caninum, and Sarcocystis spp. was detected in 0.3% (n = 1), 1.3% (n = 4), and 3.8% (n = 12) of the animals, respectively. No mixed infections were observed. Crocidura russula stood out as the main host for Sarcocystis spp. Toxoplasma gondii-specific DNA detected in a house rat was genetically characterized by PCR-RFLP, presenting type II and III alleles (SAG1 [II], SAG3 [II], GRA6 [II], c22-8 [III], Apico [III]). Also, unsuccessful DNA sequencing and microsatellite typing were attempted in N. caninum-positive samples, which suggested a lack of PCR specificity and open avenues to speculate the host competence of rodents for N. caninum. Likewise, Sarcocystis spp. identity was studied by alignment and phylogenetic analyses of cox1 and 28S rRNA sequences from the 14 positive samples. It resulted in at least three unknown organisms closely similar (95.7-100% cox1-sequence homology) to Sarcocystis pantherophisi from the Eastern rat snake (Pantherophis alleghaniensis) (KU891603), suggesting together with 28S rRNA sequences analyses, three Sarcocystis sp. with a life cycle conformed by rodents as intermediate host (IH) and snakes as definitive hosts (DH) infecting the periurban micromammals surveyed. Prevalence figures found in this first survey carried out in Spain agree with other international studies focused on periurban areas. Further surveys should be conducted in farms and their surroundings in order to unravel the role of wild micromammals in the epidemiology of such protozoan parasites affecting our livestock, and therefore human population.

During 2011–2015, we conducted a Crimean-Congo hemorrhagic fever virus (CCHFV) survey in captured ticks that were feeding mainly on wild and domestic ungulates in Spain, where presence of this virus had been reported previously. We detected CCHFV RNA in Hyalomma lusitanicum and H. marginatum ticks for 3 of the 5 years. The rate of infected ticks was 2.78% (44/1,579), which was similar to those for other countries in Europe with endemic foci for CCHFV (Kosovo, Bulgaria, and Albania). These data confirm the established spread of CCHFV into western Europe. Phylogenetic study of the small RNA segment showed Africa-3 clade as the only genotype identified, although we observed cocirculation of genetic variants during 2011 and 2015. We could not rule out genetic reassortments because of lack of sequence data for the medium and large RNA segments of the virus genome.

Various muscle tissue samples from 60 moose (Alces alces) in the Baltic region were examined for Sarcocystis species. Sarcocysts were detected in 49 out of 60 (81.7%) moose investigated. Six species, Sarcocystis alces, Sarcocystis hjorti, Sarcocystis linearis, Sarcocystis silva, Sarcocystis ovalis, and Sarcocystis sp., were identified using light microscopy (LM), and DNA sequence analysis (cox1 and 18S rDNA). Sarcocysts of S. alces, S. ovalis, and S. hjorti were studied using transmission electron microscopy (TEM); sarcocyst walls of S. alces, S. ovalis, and S. hjorti were type 25, type 24, and type 7a, respectively. Sarcocystis linearis previously found in roe deer and red deer was also shown to use moose as an intermediate host for the first time. The unknown Sarcocystis sp. was rare and might employ another main intermediate host. Phylogenetic results demonstrated that Sarcocystis sp. was most closely related to Sarcocystis tarandivulpes, using canids as definitive hosts.

Barbary sheep (Ammotragus lervia) is a North African native wild Caprinae, introduced in the 1970s in new territories such as Spain, the USA, and Mexico. Here, we describe Sarcocystis species in Barbary sheep. Sarcocysts were found in 19 out of 56 adult A. lervia in Southern Spain and characterized morphologically and molecularly. By light microscopy, sarcocysts had thin ( 2 μm) walls. By transmission electron microscopy, sarcocysts with thick walls had Type 14 villar protrusions corresponding to S. tenella/S. capracanis of domestic sheep (Ovis aries) or goats (Capra hircus). Sarcocysts with thin walls had Type 7b villar protrusions that corresponded to S. arieticanis/S. hircicanis of domestic sheep or goats. Molecular analyses allowed the identification of only thick-walled Sarcocystis species. Six sarcocysts were assigned to S. tenella (99.2–100% and 95.6–100% sequence similarity within 18S rRNA and COI, respectively) and 19 sarcocysts were assigned to S. capracanis (98.5–99.8% and 97.9–99.0% sequence similarity within 18S rRNA and COI, respectively). Further studies are needed for taxonomic identification of sarcocysts in Barbary sheep because Sarcocystis species in sheep and goats are not cross transmissible despite morphological similarities.

Exploring tick associations with complex microbial communities and single-microbial partners, especially intracellular symbionts, has become crucial to understand tick biology. Of particular interest are the underlying interactions with biological consequences i.e. tick fitness, vector competence. In this study, we first sequenced the 16S rRNA bacterial phylogenetic marker in adult male ticks of Hyalomma lusitanicum collected from 5 locations in the province of Cáceres to explore the composition of its microbial community. Overall, 16S rRNA sequencing results demonstrated that the microbial community of H. lusitanicum is mostly dominated by Francisella-like endosymbionts (FLEs) (ranging from 52% to 99% of relative abundance) suggesting it is a key taxon within the microbial community and likely a primary endosymbiont. However, further research is required to explore the mechanisms underlying the interaction between FLEs and H. lusitanicum., This study and S.D-S were financially supported by the Consejería de Educación, Cultura y Deportes, JCCM, Spain co-financed by European Regional Development Fund (FEDER), project IMpROVE (SBPLY/17/180501/000342).

Little information is available on the occurrence and genetic variability of the diarrhoea-causing enteric protozoan parasite Giardia duodenalis in indigenous communities in Brazil. This cross-sectional epidemiological survey describes the frequency, genotypes, and risk associations for this pathogen in Tapirapé people (Brazilian Amazon) at four sampling campaigns during 2008–2009. Microscopy was used as a screening test, and molecular (PCR and Sanger sequencing) assays targeting the small subunit ribosomal RNA, the glutamate dehydrogenase, the beta-giardin, and the triosephosphate isomerase genes as confirmatory/genotyping methods. Associations between G. duodenalis and sociodemographic and clinical variables were investigated using Chi-squared test and univariable/multivariable logistic regression models. Overall, 574 individuals belonging to six tribes participated in the study, with G. duodenalis prevalence rates varying from 13.5–21.7%. The infection was positively linked to younger age and tribe. Infected children &lt, 15 years old reported more frequent gastrointestinal symptoms compared to adults. Assemblage B accounted for three out of four G. duodenalis infections and showed a high genetic diversity. No association between assemblage and age or occurrence of diarrhoea was demonstrated. These data indicate that the most likely source of infection was anthropic and that different pathways (e.g., drinking water) may be involved in the transmission of the parasite.

The Iberian lynx (Lynx pardinus) is one of the most endangered felid species in the world. Conservation efforts have increased its population size and distribution and reinforced their genetic diversity through captive breeding and reintroduction programmes. Among several threats that the Iberian lynx faces, infectious and parasitic diseases have underlined effects on the health of their newly reintroduced populations, being essential to identify the primary sources of these agents and assess populations health status. To achieve this, 79 fresh faecal samples from Iberian lynx and sympatric mesocarnivores were collected in the reintroduction area of Extremadura, Spain. Samples were submitted to copromicroscopic analyses to assess parasite diversity, prevalence, and mean intensity of parasite burden. Overall, 19 (24.1%, ±15.1-35.0) samples were positive for at least one enteric parasite species. Parasite diversity and prevalence were higher in the Iberian lynx (43.8%) compared with the others mesocarnivores under study (e.g., the red fox Vulpes vulpes and the Egyptian mongoose Herpestes ichneumon). Ancylostomatidae and Toxocara cati were the most prevalent (15.6%) parasites. Obtained results revealed that Iberian lynx role as predator control might have reduced parasite cross-transmission between this felid and mesocarnivores due to their decreasing abundances. Surveillance programs must include regular monitoring of this endangered felid, comprising mesocarnivores, but also domestic/feral and wild cat communities. This research was funded by the European Union through its LIFE project Life + IBERLINCE (LIFE + 10NAT/ES/570) “Recuperación de la distribución histórica del lince ibérico (Lynx pardinus) en España y Portugal”. R. T. Torres is funded by national funds (OE), through FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the framework contract foreseen in the numbers 4, 5, and 6 of the article 23, of the Decree-Law 57/2016, of 29 August, changed by Law 57/2017, of 19 July. Thanks are due to FCT/MCTES for the financial support to CESAM (UIDP/50017/2020 + UIDB/50017/2020) and CIISA Project UIDB/00276/2020 through national funds. Sí

Background Several studies have independently evaluated the occurrence of hepatitis E virus (HEV) and enteroparasites in swine, but no surveys have been conducted to jointly assess the prevalence and genetic diversity of enteroparasites in pigs and wild boars, their sympatric transmission between hosts, and their potential interaction with HEV. Methods We prospectively collected serum and faecal samples from black Iberian domestic pigs and wild boars from southern Spain between 2015‒2016. We evaluated for HEV in serum and faeces, and for the presence of enteroparasites (Giardia duodenalis, Cryptosporidium spp., Blastocystis sp., Neobalantidium coli and Strongyloides spp.) in the same faecal samples. The prevalence of each intestinal parasite species was calculated. Results A total of 328 animals (56.7% black Iberian pigs and 43.3% wild boars) were included in the study. The overall global prevalence of HEV in serum was 16.8%. The overall global prevalence of each enteroparasite species was 19.5% for G. duodenalis, 8.2% for Cryptosporidium spp., 41.8% for Blastocystis sp., 31.4% for N. coli, and 8.8% for Strongyloides spp. HEV-infected animals showed a significantly lower prevalence of G. duodenalis (3.2 vs 20%; P = 0.002) and Blastocystis sp. (38.7 vs 80%; P G. duodenalis and Blastocystis sp. infections showed a significantly lower rate of HEV infection than those not harbouring these enteroparasites (P Conclusions Our study found a high prevalence of enteroparasites in black Iberian pigs and wild boars in southern Spain, suggesting a sympatric co-transmission of some of the species investigated. It is suggested that extracellular G. duodenalis and Blastocystis sp. might have a protective effect on HEV acquisition in swine.

Enterocytozoon bieneusi is a human pathogen with a broad range of animal hosts. Initially, E. bieneusi was considered an emerging opportunistic pathogen in immunocompromised, mainly HIV-infected patients, but it has been increasingly reported in apparently healthy individuals globally. As in other African countries, the molecular epidemiology of E. bieneusi in Mozambique remains completely unknown. Therefore, we undertook a study to investigate the occurrence and genetic diversity of E. bieneusi infections in children with gastrointestinal symptoms as well as in asymptomatic children in Mozambique. Individual stool specimens were collected from 1,247 children aged between 0 and 14 years-old living in urban and rural settings in Zambézia (n = 1,097) and Maputo (n = 150) provinces between 2016 and 2019. Samples were analysed for E. bieneusi by nested-PCR targeting the internal transcribed spacer (ITS) region of the rRNA gene. All positive amplicons were confirmed and genotyped. Penalised logistic regression (Firth) was used to evaluate risk associations. The overall prevalence of E. bieneusi in this children population was 0.7% (9/1,247). A 10-fold higher prevalence was found in Maputo (4.0%; 6/150) than in Zambézia (0.3%; 3/1,097). All E. bieneusi-positive samples were from children older than 1-year of age, and most (8/9) from asymptomatic children. Nucleotide sequence analysis of the ITS region revealed the presence of four genotypes, three previously reported (Peru11, n = 1; Type IV, n = 2, and S2, n = 2) and a novel genotype (named HhMzEb1, n = 4). Novel genotype HhMzEb1 was identified in both asymptomatic (75%, 3/4) and symptomatic (25%, 1/4) children from a rural area in Maputo province in southern Mozambique. Genotypes HhMzEb1, Peru11, S2, and Type IV belonged to the Group 1 that includes genotypes with low host specificity and the potential for zoonotic and cross-species transmission. Being infected by enteric protozoan parasites and no handwashing were identified as risk associations for E. bieneusi infection. This study reports the first investigation of E. bieneusi genotypes in Mozambique with the identification of three previously reported genotypes in humans as well as a novel genotype (HhMzEb1). Findings highlight the need to conduct additional research to elucidate the epidemiology of E. bieneusi in the country, especially in rural areas where poor hygiene conditions still prevail. Special attention should be paid to the identification of suitable animal and environmental reservoirs of this parasite and to the characterization of transmission pathways., Author summary Enterocytozoon bieneusi is an obligate intracellular parasite that infects a wide range of vertebrate hosts. It is the most important etiological agent of human microsporidiasis. Most clinical and epidemiological studies conducted to date have focused on immunodeficient or immunosuppressed individuals including HIV+ patients and solid organ transplant recipients, as in those E. bieneusi infection causes life-threatening chronic diarrhoea. In contrast, latent microsporidia infections in immunocompetent individuals have received far less attention. Molecular epidemiological studies in humans and animals have revealed that E. bieneusi encompasses a very large diversity of genetic variants (genotypes) with marked differences in host specificity and even geographical distribution. In Mozambique, as in many other African countries, the epidemiology of E. bieneusi is completely unknown. Therefore, to identify the occurrence and genetic diversity of this pathogen in Mozambique stool samples were obtained from children, including apparently healthy and symptomatic, in Zambézia and Maputo provinces and tested for E. bieneusi by molecular methods. Results demonstrated the presence of E. bieneusi genotypes for the first time in Mozambique. Four genotypes were identified, three genotypes that have previously been reported in humans (Peru11, Type IV, and S2) and a novel genotype (HhMzEb1). Two of the genotypes Peru11, Type IV have also been frequently identified in animals indicating that potentially zoonotic E. bieneusi genotypes are inadvertently circulating in the surveyed populations. Additional population genetic studies are needed to elucidate the actual extent of the epidemiology and transmission dynamics of E. bieneusi in Mozambique.

© The Author(s) 2020., [Background]: Toxoplasma gondii is a major cause of abortion in small ruminants and presents a zoonotic risk when undercooked meat containing cysts is consumed. The aim of the present study was to investigate the genetic diversity among the T. gondii strains circulating in ovine livestock in Spain., [Methods]: Selected samples collected from abortion outbreaks due to toxoplasmosis (n = 31) and from chronically infected adult sheep at slaughterhouses (n = 50) in different Spanish regions were bioassayed in mice, aiming at parasite isolation. In addition, all original clinical samples and the resulting isolates were genotyped by multi-nested PCR-RFLP analysis of 11 molecular markers and by PCR-DNA sequencing of portions of the SAG3, GRA6 and GRA7 genes., [Results]: As a result, 30 isolates were obtained from 9 Spanish regions: 10 isolates from abortion-derived samples and 20 isolates from adult myocardial tissues. Overall, 3 genotypes were found: ToxoDB#3 (type II PRU variant) in 90% (27/30) of isolates, ToxoDB#2 (clonal type III) in 6.7% (2/30), and ToxoDB#1 (clonal type II) in 3.3% (1/30). When T. gondii-positive tissue samples (n = 151) were directly subjected to RFLP genotyping, complete restriction profiles were obtained for 33% of samples, and up to 98% of the specimens belonged to the type II PRU variant. A foetal brain showed a clonal type II pattern, and four specimens showed unexpected type I alleles at the SAG3 marker, including two foetal brains that showed I + II alleles as co-infection events. Amplicons of SAG3, GRA6 and GRA7 obtained from isolates and clinical samples were subjected to sequencing, allowing us to confirm RFLP results and to detect different single-nucleotide polymorphisms., [Conclusions]: The present study informed the existence of a predominant type II PRU variant genotype (ToxoDB#3) infecting domestic sheep in Spain, in both abortion cases and chronic infections in adults, coexisting with other clonal (ToxoDB#1 and ToxoDB#2), much less frequent genotypes, as well as polymorphic strains as revealed by clinical sample genotyping. The use of multilocus sequence typing aided in accurately estimating T. gondii intragenotype diversity., This research was supported by projects funded by the Spanish Ministry of Science and Innovation (AGL2016-75935-C2-R) and the Community of Madrid (PLATESA2-CM-P2018/BAA-4370). MF and RC were funded by UCM-Santander/2017 pre-doctoral grants, and PLATESA2 post-doctoral grants, respectively. CG was funded by DGAPA, National Autonomous University of Mexico (UNAM). RC, EC and LO are part of the TOXOSOURCES consortium, supported by funding from the European Union’s Horizon 2020 Research and Innovation programme under grant agreement No. 773830: One Health European Joint Programme.

Thelazia callipaeda (Spirurida, Thelaziidae) is a vector-borne zoonotic eyeworm able to infect a broad spectrum of carnivores. Here, we describe the first case of bilateral infection by T. callipaeda in the eyes of an adult female Iberian wolf (Canis lupus signatus) in central Spain. Nematodes collected were morphologically identified (n = 42), and two specimens were molecularly characterized. At the sequence analysis of the partial mitochondrial cytochrome c oxidase subunit 1 gene, T. callipaeda haplotype 1 (the only haplotype circulating in Europe) was detected. The role of the Iberian wolf as a natural reservoir for T. callipaeda in the life cycle of this emerging zoonosis and the implications in conservation are discussed.

Recurrent self-mating can result in nearly clonal propagation of biological lineages, but even occasional outcrossing can serve to redistribute variation in future generations, providing cohesion among regional populations. The zoonotic parasite Trichinella spiralis has been suspected to undergo frequent inbreeding, resulting in genetically uniform larval cohorts which differ markedly from one another. Here, we explored the extent of inbreeding for this parasite by determining how genetic variation (at variable microsatellite markers) is distributed among 1379 larvae derived from 41 wild boars in Extremadura, Spain. In particular, we sought to determine how much of the genetic variation in this region's parasites occurs among the larvae of any given wild boar, and whether each derives from one, or more, parental lineages. We found strong evidence for inbreeding, resulting in genetically distinct parasite subpopulations among the parasites derived from many pairs of wild boar. Fully two-thirds of these parasite cohorts appear to derive from inbred parents; in 10% of the wild boars, parasites were so inbred as to become absolutely fixed in all of the assayed genetic loci. In spite of this, more than one pair of parents appear to have given rise to the infections in one-third of the sampled wild boars, resulting in mixed infections. These mixed infections should slow losses of heterozygosity and multi-locus polymorphism in any given parasite lineage. Such outcrossing should limit distinctions that would otherwise accumulate among transmission chains, thereby enforcing cohesion through the region's population in spite of its marked departure from panmixia. Conditions of transmission may differ in other regions, where such epidemiological features may engender different evolutionary outcomes.

Toxoplasma gondii causes lifelong chronic infection in both feline definitive hosts and intermediate hosts. Multiple exposures to the parasite are likely to occur in nature due to high environmental contamination. Here, we present data of high seroprevalence and multiple T. gondii strain co-infections in individual bobcats (Lynx rufus). Unfrozen samples (blood, heart, tongue and faeces) were collected from 35 free ranging wild bobcats from Mississippi, USA. Toxoplasma gondii antibodies were detected in serum by the modified agglutination test (1:≥200) in all 35 bobcats. Hearts from all bobcats were bioassayed in mice and viable T. gondii was isolated from 21; these strains were further propagated in cell culture. Additionally, DNA was extracted from digests of tongues and hearts of all 35 bobcats; T. gondii DNA was detected in tissues of all 35 bobcats. Genetic characterisation of DNA from cell culture-derived isolates was performed by multiplex PCR using 10 PCR-RFLP markers. Results showed that ToxoDB genotype #5 predominated (in 18 isolates) with a few other types (#24 in two isolates, and #2 in one isolate). PCR-DNA sequencing at two polymorphic markers, GRA6 and GRA7, detected multiple recombinant strains co-infecting the tissues of bobcats; most possessing Type II alleles at GRA7 versus Type X (HG-12) alleles at GRA6. Our results suggest that individual bobcats have been exposed to more than one parasite strain during their life time.

The roe deer (Capreolus capreolus) has been identified as an intermediate host for six known Sarcocystis species, S. capreolicanis, S. entzerothi, S. gracilis, S. linearis, S. oviformis, and S. silva. In this study, we identified Sarcocystis species in the diaphragm and tongue muscles from the Lithuanian and Spanish roe deer, respectively, on the basis of a microscopic examination and DNA analysis. A total of 43 and 27 sarcocysts were isolated and characterized from the Lithuanian and Spanish roe deer, respectively. Overall six Sarcocystis species were identified in roe deer from Lithuania, and only three of them, S. gracilis, S. linearis, and S. silva were found to have infecting animals from Spain. The current paper represents first molecular results of Sarcocystis species in the Spanish roe deer. Furthermore, transmission electron microscopy examination revealed specific wall structure of sarcocysts studied, S. linearis was characterized by ribbon-like villar protrusions (vp) (type 8a), and S. oviformis was distinguished by elongated vp resembling spades or mushroom-like structures (type 39). Based on 18S rDNA and cox1 sequences, Sarcocystis species from the roe deer showed considerable intraspecific genetic variability. However, similar values of intraspecific genetic variation were estimated at both genes analysed. The highest variability was observed for S. capreolicanis and S. linearis in both genes and for S. silva at cox1. Consequently, the level of genetic variability of Sarcocystis from the roe deer varied depending on species rather than on gene analysed or geographical area.

Background Typically, carnivores serve as definitive hosts for Sarcocystis spp. parasites; currently, their role as intermediate hosts is being elucidated. The present study aimed to identify and molecularly characterize Sarcocystis cysts detected in striated muscle of red foxes from different populations in Latvia, Lithuania and Spain. Methods Muscle samples from 411 red foxes (Vulpes vulpes) and 269 racoon dogs (Nyctereutes procyonoides) from Latvia, 41 red foxes from Lithuania and 22 red foxes from Spain were examined for the presence of Sarcocystis sarcocysts by light microscopy (LM). Sarcocystis spp. were identified by transmission electron microscopy (TEM) and molecular biology techniques. Results Sarcocystis cysts were detected in 11/411 (2.7%) Latvian, 3/41 (7.3%) Lithuanian, and 6/22 (27.3%) Spanish red foxes, however, cysts were not observed in the muscles of racoon dogs. Based on LM, TEM, 18S rDNA, 28S rDNA, ITS1, cox1 and rpoB sequences, Sarcocystis arctica and Sarcocystis lutrae cysts were identified in red fox muscles from Latvia and Lithuania, whereas only S. arctica was detected in Spain. The 18S rDNA, 28S rDNA and ITS1 sequences from the 21 isolates of S. arctica from Latvia, Lithuania and Spain were identical. By contrast, two and four haplotypes were determined based on mtDNA cox1 and apicoplast rpoB sequences, respectively. Polymorphisms were not detected between the two isolates of S. lutrae from Latvia and Lithuania. Based on phylogenetic results, S. arctica and S. lutrae were most closely related to Sarcocystis spp. using predatory mammals as intermediate hosts and to Sarcocystis species with a bird-bird life-cycle. Conclusions Based on current knowledge, the red fox and Arctic fox (Vulpes lagopus) could act as intermediate host for the same two Sarcocystis species. Molecular results suggest the existence of two genetic lineages of S. arctica, and such divergence relies on its geographical distribution but not on their intermediate host species.

Low antibody titers to Neospora caninum have been reported in humans, but infection has not been confirmed. We used N. caninum-specific PCR to test 600 clinical samples from patients with toxoplasmosis signs but Toxoplasma gondii-negative PCR results. We did not detect N. caninum DNA, demonstrating it is an unlikely opportunistic zoonotic agent.

Wild birds are important in the epidemiology of toxoplasmosis because they can serve as reservoir hosts, and vectors of zoonotic pathogens including Toxoplasma gondii. Canada goose (Branta canadensis) is the most widespread geese in North America. Little is known concerning T. gondii infection in both migratory, and local resident populations of Canada geese. Here, we evaluated the seroprevalence, isolation, and genetic characterization of viable T. gondii isolates from a migratory population of Canada geese. Antibodies against T. gondii were detected in 12 of 169 Canada geese using the modified agglutination test (MAT, cutoff 1:25). The hearts of 12 seropositive geese were bioassayed in mice for isolation of T. gondii. Viable parasites were isolated from eight. One isolate was obtained from a seropositive goose by both bioassays in mice, and in a cat; the cat fed infected heart excreted T. gondii oocysts. Additionally, one isolate was obtained from a pool of four seronegative (

Infection with Sarcocystis is common in many species of wild cervids but none is reported from the black-tailed deer (Odocoileus hemionus columbianus). Here, we report Sarcocystis infection in two black-tailed deer from northwest USA for the first time. Sarcocysts were microscopic, up to 556 μm long and mature. The sarcocyst wall was up to 1.39 μm thick and had rectangular 1.17-μm-long villar protrusions, type 17, with thin (230 nm) electron dense ground substance layer. Molecular characterization and phylogenetic analysis indicated that Sarcocystis in the black-tailed deer is related to structurally distinct Sarcocystis species in cervids. A new name, Sarcocystis mehlhorni, is proposed for the Sarcocystis species in black-tailed deer.

The muscles of herbivores commonly harbor sarcocysts of parasites belonging to species in the genus Sarcocystis, but such muscle parasites are rare in carnivores. Here, we report Sarcocystis arctica-like sarcocysts in muscles of Arctic foxes (Vulpes lagopus) from Alaska, USA, for the first time. The tongues of 56 foxes were examined for Sarcocystis infection using several methods. Sarcocystis bradyzoites were detected in pepsin digests of 13 (23.2%), and sarcocysts were found in histological sections stained with hematoxylin and eosin (HE) of 9 (16.0%). By light microscopy, sarcocysts were up to 4 mm long and up to 245 μm wide. In HE-stained sections, the sarcocyst wall appeared smooth and up to 1.5 μm thick without visible protrusions. By transmission electron microscopy, the sarcocyst wall had a wavy parasitophorous vacuolar membrane (pvm) folded as pleomorphic villar protrusions (vp), sometimes with anastomoses of villar tips. The vp and the ground substance (gs) layer were smooth and without microtubules. The gs was up to 2.0 μm thick. The total width of the wall including vp and the gs was up to 4.0 μm. The vp were up to 3.0 μm long and most closely resembled “type 9c.” All sarcocysts were mature and contained numerous 8.1 × 2.1 μm sized bradyzoites. Molecular characterization (at 18S rDNA, 28S rDNA, ITS-1, and cox1) showed the highest affinity for S. arctica of the Arctic fox (V. lagopus) from Norway. In the present investigation, we provide evidence that sarcocysts are common in tongues of Alaskan Arctic foxes suggesting that these carnivores are serving as intermediate hosts, and we also provide ultrastructure of S. arctica from the Arctic fox for the first time.

Bovine besnoitiosis is an emerging disease in Europe, presenting quick spread toward central and southern Spain. Characterization of an outbreak in a free-ranging Limousin and Avilena beef cattle herd from southwestern Spain territories is attempted. Serological survey in the herd revealed increase of number of infected animals, from 34.3 % on first diagnoses/exams on December 2013 to 42.5 % in the second on April 2014. Blood analysis and serum biochemistry showed important alterations like leukocytosis (+33.2 % of mean value), with lymphocytosis (+205.3 %) and increase of LDH (+25.1 %), associated with tissue damage. Clinical cases were only observed in Limousin animals. Along with typical lesions of acute and chronic besnoitiosis, inflammatory and degenerative processes and parasitic cysts were present in the corpus cavernosum and the corpus spongiosum of penis. By using polymerase chain reaction (PCR) sequencing of 18S rDNA, Besnoitia besnoiti was confirmed as causative agent; microsatellite sequence analyses showed the homology of isolates with previously studied strains.

Sarcocystis sarcocysts are common in muscles of herbivores but are rare in muscles of carnivores. Here, we report sarcocysts in the muscles of a gray wolf (Canis lupus) from Alaska, USA, for the first time. Sarcocysts extracted from the tongue of the wolf were up to 900 μm long and slender and appeared to have a relatively thin wall by light microscope. By transmission electron microscopy, the sarcocyst wall most closely resembled "type 9c," and had a wavy parasitophorous vacuolar membrane folded as pleomorphic villar protrusions (vp), with anastomoses of tips. The vp and the ground substance (gs) layer were smooth without tubules or granules. The gs was up to 2.0 μm thick. The total width of the wall including vp and the gs was 3.5 μm. The vp were up to 1.5 μm long. Mature sarcocysts contained numerous bradyzoites and few metrocytes. The bradyzoites were 9.5 μm long and 1.5 μm wide, and contained all organelles found in Sarcocystis bradyzoites with at least two rhoptries. Molecular characterization showed the highest identity for 18S rRNA, 28S rRNA, ITS-1, and cox1 sequences of Sarcocystis arctica of the Arctic fox (Vulpes lagopus) from Norway. The ultrastructure of S. arctica from the fox is unknown. Here, we provide ultrastructure of S. arctica from the Alaskan wolf for the first time. The definitive host of S. arctica remains unknown.

SUMMARYThere is considerable confusion concerning Sarcocystis species in camels. Five species: Sarcocystis cameli, Sarcocystis ippeni, Sarcocystis camelicanis, Sarcocystis camelocanis and Sarcocystis miescheri were named with inadequate descriptions and no type specimens. Here, we review literature on sarcocystosis in camels worldwide and redescribe structure of S. cameli and S. ippeni sarcocysts by light- and transmission electron microscopy (LM and TEM). Eight sarcocysts from the oesophagi of two camels (Camelus dromedarius) from Egypt were studied. By LM, all sarcocysts were thin-walled with barely visible projections on the cyst walls. By TEM, two structurally distinct sarcocysts were recognized by unique villar protrusions (vp) not found in sarcocysts from any other host. Sarcocysts of S. cameli had vp of type 9j. The sarcocyst wall had upright slender vp, up to 3·0 µm long and 0·5 µm wide; the total thickness of the sarcocyst wall with ground substance (gs) layer was 3·5 µm. On each vp, there were rows of knob-like protrusions that appeared to be interconnected. The vp had microtubules that originated at midpoint of the gs and continued up to the tip; microtubules were smooth, without any granules or dense areas. Bradyzoites were approximately 14–15 × 3–4 µm in size with typical organelles. Sarcocystis ippeni sarcocysts had type 32 sarcocyst wall characterized by conical vp with an electron dense knob. The total thickness of the sarcocyst wall (from the base of gs to vp tip) was 2·3–3·0 µm. The vp were up to 1·2 µm wide at the base and 0·25 µm at the tip. Microtubules in vp originated at midpoint of gs and continued up to tip; microtubules were criss-crossed, smooth and without granules or dense areas. Bradyzoites were 12·0–13·5 × 2·0–3·0 µm in size. Sarcocystis camelicanis, S. camelocanis and S. miescheri are considered invalid.

Toxoplasma gondii infections are widespread in white-tailed deer (Odocoileus virginianus) but little is known of its prevalence in other cervids in the USA. Moose (Alces alces) is a popular large game animal, hunted for its meat and trophy antlers. Here, we report seroprevalence, isolation, and genetic characterization of T. gondii from moose from Minnesota. Antibodies against T. gondii were detected in 8 of 79 (10%) moose tested by the modified agglutination test (MAT 1:25 or higher). The myocardium of 68 moose was bioassayed individually in mice, irrespective of serological status. T. gondii was detected in three moose (2 adults, 1 3 weeks old). The parasite from 2 adults was further propagated in cell culture. PCR-RFLP genotyping of cell culture derived tachyzoites using 10 genetic markers, SAG1, SAG2 (5′ and 3′ SAG2, and alt.SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed two different ToxoDB PCR-RFLP genotypes (#5, designated TgMooseUS1, and #7, TgMooseUS2). The mice inoculated with myocardium of the juvenile moose developed antibodies against T. gondii, and DNA extracted from infected mouse brain was only partially characterized by PCR-RFLP genotyping, which suggests a potential new genotype. Result documented prevalence of T. gondii in moose, and its possible transplacental/transmammary transmission of T. gondii in moose.

SUMMARYThe North American opossum (Didelphis virginiana) is the definitive host for at least three named species ofSarcocystis: Sarcocystis falcatula, Sarcocystis neuronaandSarcocystis speeri.The South American opossums (Didelphis albiventris, Didelphis marsupialisandDidelphis aurita) are definitive hosts forS. falcatulaandS. lindsayi. The sporocysts of theseSarcocystisspecies are similar morphologically. They are also not easily distinguished genetically because of the difficulties of DNA extraction from sporocysts and availability of distinguishing genetic markers. Some of these species can be distinguished by bioassay;S. neuronaandS. speeriare infective to gamma interferon gene knockout (KO) mice, but not to budgerigars (Melopsittacus undulatus); whereasS. falcatulaandS. lindsayiare infective to budgerigars but not to KO mice. The natural intermediate host ofS. speeriis unknown. In the present study, development of sarcocysts ofS. speeriin the KO mice is described. Sarcocysts were first seen at 12 days post-inoculation (p.i.), and they became macroscopic (up to 4 mm long) by 25 days p.i. The structure of the sarcocyst wall did not change from the time bradyzoites had formed at 50–220 days p.i. Sarcocysts contained unique villar protrusions, ‘type 38’. The polymerase chain reaction amplifications and sequences analysis of three nuclear loci (18S rRNA, 28S rRNAandITS1) and two mitochondrial loci (cox1andcytb) ofS. speeriisolate from an Argentinean opossum (D. albiventris) confirmed its membership among species ofSarcocystisand indicated an especially close relationship to another parasite in this genus that employs opossums as its definitive host,S. neurona.These results should be useful in finding natural intermediate host ofS. speeri.

Food safety regulations require the control of the presence of protozoa in meats destined for human consumption. Wild boar (Sus scrofa) meat may constitute a source of zoonoses. A 23.8% (688/2881) seroprevalence of anti-Toxoplasma gondii antibodies and 72.2% (662/910) Sarcocystis sarcocysts prevalence were detected among wild boars hunted in Southwestern areas of Spain. Identity of Sarcocystis spp. was performed by RFLP-PCR and sequencing, detecting S. miescheriana (7/8) and the zoonotic S. suihominis (1/8). Risk assessment studies of these coccidian in meats destined to human consumption are needed.

Numerous species of Sarcocystis have been reported from wild ruminants, but none has been named from the Rocky Mountain goat (Oreamnos americanus). Mature sarcocysts were found in frozen muscle samples of three of seven mountain goats from Alaska, USA. Two morphological types of sarcocysts were found; one had Sarcocystis cornagliai-like sarcocysts, previously named from the Alpine ibex (Capra ibex) from Europe. Two other goats were infected with a new species, Sarcocystis oreamni. Sarcocystis oreamni sarcocysts were microscopic with 2 μm-thick sarcocyst wall. By transmission electron microscopy, the sarcocyst wall had 1.7 μm-thick with unusual molar tooth-like villar protrusions (vp), type 29. The vp had an electron dense core and two disc-shaped plaques at the tip with fine microtubules. Bradyzoites were 8.6–9.1 μm long. Single nucleotide polymorphism (SNP) identified in 18S rRNA, and 28S rRNA loci of rDNA regions that suggested S. oreamni molecularly apart from related species. The phylogenetic analysis based on 18S rRNA and 28S rRNA sequences suggested S. oreamni is related with Sarcocystis species that employ members of the Canidae family as their definitive host.

Cattle (Bos taurus) are intermediate hosts for four species of Sarcocystis, namely Sarcocystis cruzi, Sarcocystis hirsuta, Sarcocystis hominis, and Sarcocystis rommeli. Of these four species, mature sarcocysts of S. cruzi are thin-walled (

Transmission of pathogens between domestic and wild life animals plays an important role in epidemiology. Feral pig populations are increasing and expanding in the USA, and may constitute a risk to non-biosecure domestic pig facilities by serving as reservoirs for pathogens. We surveyed, for Sarcocystis infection, the myocardium of 1006 feral pigs (Sus scrofa) trapped or hunted in 29 states during the Comprehensive Feral Swine Disease Surveillance Program of the USDA's Animal and Plant Health Inspection Service, Wildlife Services unit during 2012–2014. Sarcocysts were detected in histological sections of 25% (251/1006) of myocardium with an average parasitic load/intensity of infection of 3·03 sarcocysts/section (1·5×0·7 cm), and higher prevalence of myocarditis in severe infections. Microscopic examination of pepsin digests of 147 hearts revealed a higher prevalence of Sarcocystis bradyzoites (49%, 72/147) than when diagnosed by histology. A fragment of Sarcocystis 18S rRNA was amplified and digested with a restriction endonuclease, revealing a pattern consistent with Sarcocystis miescheriana in all 44 selected samples. Sequencing 31 of these 44 isolates confirmed their correspondence to S. miescheriana. Thus, S. miescheriana infection, but not the zoonotic parasite Sarcocystis suihominis, appears to be prevalent and widespread in feral pigs in the USA.

SUMMARYFour valid species of Sarcocystis have been reported from the water buffalo (Bubalus bubalis): Sarcocystis fusiformis, Sarcocystis buffalonis, Sarcocystis levinei and Sarcocystis dubeyi. Here, we redescribe structure of S. fusiformis sarcocysts by scanning and transmission electron microscopy (SEM, TEM). Twenty-one macroscopic sarcocysts from oesophagus of the water buffalo in Egypt were examined by light microscopy, SEM and TEM. The sarcocyst wall was up to 9 μm thick, depending on the section and the technique. In 5 μm paraffin-embedded sections, the sarcocyst wall was indistinct, 2–5 μm thick and appeared smooth. In 1 μm plastic-embedded sections stained with toluidine blue, the sarcocyst wall was 2·5–5·2 μm thick and had branched villar protrusions (vp)-like branches of a dead tree. By SEM, the sarcocyst wall had a mesh-like structure with irregularly shaped vp that were folded over the sarcocyst wall. On each vp there were uniform papillomatous structures that were 100 nm wide. By TEM, vp were up to 6 μm long and contained filamentous tubular structures, most of which were parallel to the long axis of the projections; granules were absent from these tubules. By TEM, bradyzoites within the same cyst varied from 11·2 to 16·8 μm in length. By TEM, bradyzoites had a very long (10 μm) convoluted mitochondrion, up to 12 dense granules, but only 2 rhoptries. This redescription should help to differentiate the sarcocysts of S. fusiformis from similar sarcocysts in domestic and wild ruminants.

1 United States Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Animal Parasitic Diseases Laboratory, Beltsville, MD 20705-2350, USA 2 Department of Anatomy and Physiology, Faculty of Veterinary Science, University of Pretoria, Onderstepoort 0110, South Africa 3 Alaska Department of Fish and Game, Division of Wildlife Conservation, Juneau, AK 99811, USA Parasitol Res (2016) 115:457 DOI 10.1007/s00436-015-4804-5

Toxoplasma gondii is a major zoonotic agent which may cause harmful effects mainly in pregnant and immunocompromised hosts. Despite many efforts on its genetic characterization, an entirely clear picture of the population structure in Europe has not been achieved yet. The present study aimed to summarize the available genotyping information and to map the distribution of circulating strains. There is consensus on type II T. gondii genotypes prevailing in Europe, but the absence of harmonization in the use of typing methods limits detailed knowledge. Standardized, high-end typing tools and integrative strategies are needed to fill the gaps and complete an accurate image of the T. gondii genetic population in Europe.

Toxoplasma gondii, a major zoonotic pathogen, possess a significant genetic and phenotypic diversity that have been proposed to be responsible for the variation in clinical outcomes, mainly related to reproductive failure and ocular and neurological signs. Different T. gondii haplogroups showed strong phenotypic differences in laboratory mouse infections, which provide a suitable model for mimicking acute and chronic infections. In addition, it has been observed that degrees of virulence might be related to the physiological status of the host and its genetic background. Currently, mortality rate (lethality) in outbred laboratory mice is the most significant phenotypic marker, which has been well defined for the three archetypal clonal types (I, II and III) of T. gondii; nevertheless, such a trait seems to be insufficient to discriminate between different degrees of virulence of field isolates. Many other non-lethal parameters, observed both in in vivo and in vitro experimental models, have been suggested as highly informative, yielding promising discriminatory power. Although intra-genotype variations have been observed in phenotypic characteristics, there is no clear picture of the phenotypes circulating worldwide; therefore, a global overview of T. gondii strain mortality in mice is presented here. Molecular characterization has been normalized to some extent, but this is not the case for the phenotypic characterization and definition of virulence. The present paper proposes a baseline (minimum required information) for the phenotypic characterization of T. gondii virulence and intends to highlight the needs for consistent methods when a panel of T. gondii isolates is evaluated for virulence.

Toxoplasma gondii constitutes a major zoonotic agent but also has been frequently identified as an important cause of clinical disease (e.g., abortion, pneumonia, encephalitis) in wildlife; specifically, T. gondii has been associated with neurological disease in cetaceans. This study investigated the genetic diversity of T. gondii strains involved in infections in dolphins found stranded in the Mediterranean coastlines of Italy. Tissue samples from 16 dolphins (Stenella coeruleoalba and Tursiops truncatus species) positive for T. gondii-DNA presence by PCR were examined by histology and subjected to further genetic characterization of strains detected by PCR-RFLP and multilocus PCR-sequencing assays. According to fully genotyped samples, the genotypes ToxoDB#3 (67%) and #2 (22%) were detected, the latter being reported for the first time in cetaceans, along with a mixed infection (11%). Subtyping by PCR-seq procedures provided evidence of common point mutations in strains from southwestern Europe. Despite evidence of T. gondii as a cause of neurological disease in dolphins, sources of infections are difficult to identify since they are long-living animals and some species have vast migration areas with multiple chances of infection. Finally, the genetic diversity of T. gondii found in the dolphins studied in the Mediterranean coastlines of Italy reflects the main genotypes circulating inland in the European continent.