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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. [Display omitted] • 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. [ABSTRACT FROM AUTHOR]

[Display omitted] • The influence of in vitro maintenance on phenotypic traits of Toxoplasma gondii was studied. • Cell culture adaptation in the short term influences cyst formation and mouse virulence. • Toxoplasma gondii isolates experience a loss of cyst formation after 40 culture passages. • Changes in mouse virulence correlated with in vitro parasite growth and cyst formation. Most Toxoplasma gondii research has been carried out using strains maintained in the laboratory for long periods of time. Long-term passage in mice or cell culture influences T. gondii phenotypic traits such as the capability to produce oocysts in cats and virulence in mice. In this work, we investigated the effect of cell culture adaptation in the short term for recently obtained type II (Tg ShSp1 (Genotype ToxoDB#3), Tg ShSp2 (#1), Tg ShSp3 (#3) and Tg ShSp16 (#3)) and type III (#2) isolates (Tg ShSp24 and Tg PigSp1). With this purpose, spontaneous and alkaline stress-induced cyst formation in Vero cells during 40 passages, from passage 10 (p 10) to 50 (p 50), and isolate virulence at p 10 versus p 50 were studied using a harmonized bioassay method in Swiss/CD1 mice. T. gondii cell culture maintenance showed a drastic loss of spontaneous and induced production of mature cysts after ≈25–30 passages. The Tg ShSp1, Tg ShSp16 and Tg ShSp24 isolates failed to generate spontaneously formed mature cysts at p 50. Limited cyst formation was associated with an increase in parasite growth and a shorter lytic cycle. In vitro maintenance also modified T. gondii virulence in mice at p 50 with events of exacerbation, increasing cumulative morbidity for Tg ShSp2 and Tg ShSp3 isolates and mortality for Tg ShSp24 and Tg PigSp1 isolates, or attenuation, with absence of mortality and severe clinical signs for Tg ShSp16, and better control of the infection with the lowest parasite and cyst burdens in lungs and brain for the Tg ShSp1 isolate. The present findings show deep changes in relevant phenotypic traits in laboratory-adapted T. gondii isolates and open new discussion about their use for inferring keys to parasite biology and virulence. [ABSTRACT FROM AUTHOR]

• The first description of H. lusitanicum microbial community. • Francisella- LE dominates the microbial community of H. lusitanicum. • Francisella- LE is likely the primary symbiont of H. lusitanicum. • Future studies need to evaluate the role of Francisella -LE in the biology of H. lusitanicum and the nature of its interactions with other pathogenic and non-pathogenic microbes. 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. [ABSTRACT FROM AUTHOR]

The protozoon Neospora caninum is a major cause of abortion in cattle worldwide. Canids ( Canis familiaris , Canis latrans , Canis lupus ) are definitive hosts whereas many other animal species, including pigs, are intermediate hosts for the parasite. Between 2012 and 2014, serum samples from 1059 feral swine ( Sus scrofa ) from 29 states of the USA were tested for N. caninum antibodies, using the N. caninum agglutination test (NAT). Of these, 159 (15.0%) feral pigs from 21 states tested positive, with a range of titers of 1:25 (cut-off) (n = 153), 1:200 (1), 1:400 (1), 1:800 (3) and 1:3200 (1). Results indicate widespread exposure of feral swine to N. caninum infection across the USA. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is a major cause of reproductive failure in small ruminants. Genotypic diversity of T. gondii strains has been associated with variations in phenotypic traits in in vitro and murine models. However, whether such diversity could influence the outcome of infection in small ruminants remains mostly unexplored. Here, we investigate the outcome of oral challenge in sheep at mid-pregnancy with 10 sporulated oocysts from three different T. gondii isolates belonging to archetypal II and III and selected according to their genetic and phenotypic variations shown in previous studies. Seventy-three pregnant sheep were divided in four groups: G1 infected with TgShSp1 isolate (type II, ToxoDB#3), G2 with TgShSp16 isolate (type II, ToxoDB#3), G3 with TgShSp24 isolate (type III, ToxoDB#2) and G4 of uninfected control sheep. Two different approaches were carried out within this study: (i) the outcome for the pregnancy after infection (n = 33) and (ii) the lesions and parasite tropism and burden at 14 and 28 days post infection (dpi) (n = 40). The onset of hyperthermia and seroconversion occurred one and two days later, respectively in G1 when compared to G2 and G3. However, sheep that suffered from reproductive failure, either by abortion, foetal dead at the time of euthanasia or stillbirth were similar among infected groups (50%, 40% and 47%, respectively). Histological lesions in placentomes and foetal tissues from euthanized animals from the second approach were only detected at 28 dpi and mainly in G1. At 14 dpi, T. gondii -DNA was only detected in G1 in the 11% of the placentomes. However, at 28 dpi the frequency of detection in placentomes was higher in G1 (96%) than in G2 and G3 (7% and 47%, respectively) besides in foetuses was lower in G2 (20%) than in G1 and G3 (100% and 87%, respectively). Regarding late abortions, stillbirths, and lambs of G1, G2 and G3, the frequency of microscopic lesions was similar between groups (79%, 78% and 67%, respectively) whereas T. gondii -DNA was evidenced in 100%, 55% and 100%, respectively. These recently obtained T. gondii isolates led to similar reproductive losses but intra- and inter-genotype variations in the rise of hyperthermia, dynamics of antibodies, frequency of lesions and parasite detection and distribution. Thus, the different phenotypic traits of the isolates could influence the outcome of the infection and mechanisms responsible for it, and further investigations are warranted. [Display omitted] • In pregnant sheep, reproductive losses after infection with T. gondii oocysts were similar despite the isolate employed. • Clinical outcomes reveal inter- and intra-genotypic differences. • Histological severity of microscopic lesions does not correlate with reproductive failure. • Invasion of the placenta causes unacknowledged early vascular lesions at 28 dpi. • Previous in vitro phenotypic traits cannot be directly inferred to pregnant sheep model. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

There is a surprisingly scarce amount of epidemiological and molecular data on the prevalence, frequency, and diversity of the intestinal protozoan parasites Giardia duodenalis and Cryptosporidium spp. in wildlife in general and mesocarnivore species in particular. Consequently, the extent of the cyst/oocyst environmental contamination attributable to these wild host species and their potential implications for public veterinary health remain largely unknown. In this molecular epidemiological survey a total of 193 individual faecal samples from badgers ( Meles meles , n = 70), ferrets ( Mustela putorius furo , n = 2), genets ( Genetta genetta , n = 6), Iberian lynxes ( Lynx pardinus , n = 6), beech martens ( Martes foina , n = 8), mongooses ( Herpestes ichneumon , n = 2), otters ( Lutra lutra , n = 2), polecats ( Mustela putorius , n = 2), red foxes ( Vulpes vulpes , n = 87), wildcats ( Felis silvestris , n = 2), and wolves ( Canis lupus , n = 6) were obtained from road-killed, hunted, and accidentally found carcasses, and from camera-trap surveys or animals entering rescue shelters, during the period December 2003–April 2016. Investigated specimens were collected in five Spanish autonomous regions including Andalusia ( n = 1), Asturias ( n = 69), Basque Country ( n = 49), Castile-La Mancha ( n = 38), and Extremadura ( n = 36). The presence of cysts/oocysts was confirmed by PCR-based methods targeting the small subunit ( ssu ) ribosomal RNA gene of these parasite species. Genotyping of the obtained isolates were attempted at appropriate markers including the glutamate dehydrogenase ( G. duodenalis ) and the 60-kDa glycoprotein ( C. parvum and C. ubiquitum ) loci. Overall, G. duodenalis was detected in 8% (7/87) of red foxes, a single beech marten, and a single wolf, respectively. Cryptosporidium was identified in 3% (2/70) of badgers, 8% (7/87) of red foxes, a single genet, and a single mongoose, respectively. None of the nine G. duodenalis isolates generated could be genotyped at the assemblage/sub-assemblage level. Out of the nine Cryptosporidium isolates successfully characterized, three were identified as C. canis (one in a mongoose and two in red foxes), and three as C. parvum (one in a badger and three in red foxes). The remaining three isolates were assigned to C. felis (in a red fox), C. hominis (in a badger), and C. ubiquitum (in a red fox), respectively. Two additional Cryptosporidium isolates infecting a badger and a genet, respectively, were untypable. The red fox was confirmed as a suitable host of potentially zoonotic Cryptosporidium species, mainly C. parvum and C. ubiquitum . The high mobility and wide home range of red foxes, together with their increasing presence in urban and peri-urban settings, may led to the overlapping of sylvatic and domestic cycles of the parasite, and consequently, to an increased risk of cryptosporidiosis in production animals and humans. The detection of C. hominis oocysts in a badger raises the question of whether this finding represents a true infection or a sporadic event of mechanical passage of C. hominis oocyst of anthroponotic origin. [ABSTRACT FROM AUTHOR]

The protozoan Toxoplasma gondii and the metazoan Trichinella spp. infect virtually all warm-blooded animals, including birds, humans, livestock, and marine mammals. Both parasitic infections can cause serious illness in human beings and can be acquired by ingesting under-cooked meat harboring infective stages. Approximately 3500 black bears ( Ursus americanus ) are legally-harvested each year in Pennsylvania, USA during the November hunting season. Among animals found infected with T. gondii , the prevalence of T. gondii is the highest among black bears in the USA; however, little is currently known of epidemiology of toxoplasmosis in this host species. Serum samples were collected during the winters of 2015 and 2016 from adult female bears and their nursing cubs or yearlings while they were still in their dens. Additionally, archived sera from bear samples collected throughout the year, including hunter-harvested bears in November and trapped bears in the summer, were serologically tested. Antibodies to T. gondii were assayed by the modified agglutination test (MAT, cut-off 1:25) and antibodies to Trichinella spp. were assayed using a commercial enzyme-linked immunosorbent assay (ELISA). Overall, T. gondii antibodies were found in 87.6% (206/235) of adults, and 44.1% (30/68) of yearlings. In March 2015/2016 sampling, antibodies to T. gondii were found in 94% (30/32) adult female bears while in their den. Antibodies were detected in 5% (3/66) of the nursing cubs in the dens of these sows. One positive cub had a MAT titer of 1:160 and two were positive at the 1:25 dilution but not at 1:50. The adult females of these cubs had MAT titers ranging from 1:400 to 1:3200. Antibodies to Trichinella spp. were found in 3% (6/181) of adults and 3.6% (1/28) of yearlings; these 7 bears were also seropositive for T. gondii . No antibodies to Trichinella spp. were detected in the sera of 44 nursing cubs tested. The finding of T. gondii antibodies in only 3 of 66 cubs, and higher antibody titers in their respective sows indicates that the colostrally-acquired antibodies wane to undetectable levels by 8–10 weeks, while the cubs are still in the den. The results indicate that there is no transplacental transmission of T. gondii , that antibodies acquired from colostrum are largely undetectable by the time cubs emerge from the den, and nearly that 50% of bears acquire infection postnatally by 10 months of age. This is the first report of disappearance of transcolostral antibodies of any infection in bears. [ABSTRACT FROM AUTHOR]

Bovine besnoitiosis, a parasitic disease caused by Besnoitia besnoiti, has been reported mainly in beef cattle raised under extensive pastoral systems and is considered to be re-emerging in Western Europe. Horizontal transmission probably occurs either by means of blood sucking arthropods or as a consequence of direct contact between infected and non-infected cattle. However, the role that wild ruminants (e.g., red deer ( Cervus elaphus ) and roe deer ( Capreolus capreolus )) may play in the parasite life cycle as putative reservoirs remains elusive. Thus, we investigated the presence of Besnoitia spp. infection in 2608 wild ruminants located in areas where bovine besnoitiosis is present and identified the Besnoitia species detected. First, a serosurvey was conducted in red deer ( n = 309), roe deer ( n = 417), Pyrenean chamois ( Rupicapra p. pyrenaica, n = 383) and Iberian wild goat ( Capra pyrenaica hispanica , n = 288) from two areas of Aragon, northeastern Spain, where bovine besnoitiosis is endemic. Second, red deer ( n = 820), roe deer ( n = 37), fallow deer ( Dama dama , n = 166), Iberian wild goat ( n = 86) and European mouflon ( Ovis orientalis musimon , n = 102) from southwestern Spain, where new outbreaks have recently been reported, were also sampled. The presence of Besnoitia spp.-specific antibodies was confirmed by western blot in one red deer and one roe deer from the Pyrenees, and Besnoitia spp. DNA was detected by ITS1-PCR in the seropositive red deer. Besnoitia genotyping based on 6 microsatellite (MS) analyses was carried out in red deer samples and compared with B. besnoiti genotypes from 7 in vitro isolates and 3 infected bovines, B. tarandi (1 isolate) and B. bennetti (from tissues of an infected donkey) for Besnoitia spp. assignation. Multilocus MS analysis of B. besnoiti, B. tarandi and B. bennetti showed specific genotypes for each species. A restricted genetic diversity with two genotypes by variation in a unique MS marker was revealed among the 7 B. besnoiti isolates. Incomplete Besnoitia spp. genotype of 3 MS markers from red deer samples entirely matched the B. besnoiti genotypes. Accordingly, this work gives clues for the presence of B. besnoiti infection in red deer from Western Europe. Further molecular genotyping is needed to confirm that red deer may act as an intermediate host of B. besnoiti , although the low prevalences that were found indicate that wild ruminant species do not pose a significant risk of transmitting the infection to cattle. [ABSTRACT FROM AUTHOR]