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The wild boar, an impactful invasive species in Brazil, is subject to population control activities, which often include the use of hunting dogs. Hunters commonly consume wild boar meat, which is also used to feed their dogs, posing a risk of Toxoplasma gondii infection for humans and both T. gondii and Neospora caninum for dogs. The study aimed to investigate the prevalence of infection in wild boars (n = 127) and hunting dogs (n = 73) from São Paulo, Rio Grande do Sul, and Paraná states. We employed histopathological, serological (indirect fluorescent antibody test), and molecular techniques (endpoint polymerase chain reaction). Histopathology slides of wild boar tissue (central nervous system, heart, skeletal muscle, liver, spleen, kidney, gastrointestinal tract, pancreas, lymph nodes, and thyroid) sections revealed no T. gondii or N. caninum cysts (0/47). Antibodies anti-T. gondii were detected in 35/108 (32.4%) and anti-N. caninum in 45/108 (41.7%) wild boars. Only 2/18 (11.1%) wild boar tissue homogenate samples tested positive for T. gondii on endpoint PCR. Hunting dogs showed antibodies against T. gondii in 62/73 (85%) and against N. caninum in 31/73 (42%). The presence of antibodies against T. gondii and N. caninum in wild boars and hunting dogs, along with T. gondii DNA detection in wild boars, indicates the circulation of these parasites. Educating hunters on preventing these foodborne diseases, including zoonotic risks, is crucial.

The genus Sarcocystis has approximately 200 species that are able to infect homeothermic and poikilothermic animals. So far, 23 Sarcocystis spp. have snakes as definitive hosts (DHs) and mammals and/or reptiles as intermediate hosts (IHs). Some of these species cause disease or even death in their IH. At least two Sarcocystis spp. from snakes have relevance in public health. Sarcocystis nesbitti causes serious myopathy in humans, and S. singaporensis is lethal for rats and was successfully tested in the biological control of these rodents. Molecular identification was not reported for half of Sarcocystis spp. shed by snakes. For some snake species, their role as DHs for Sarcocystis spp. is totally unknown, including certain snakes which are bred as companion animals. The aim of this review is to discuss the role of snakes as DHs or IHs of Sarcocystis spp. and the future directions for the identification of the parasites and the elucidation of their life cycles. [ABSTRACT FROM AUTHOR]

The genus Sarcocystis has approximately 200 species that are able to infect homeothermic and poikilothermic animals. So far, 23 Sarcocystis spp. have snakes as definitive hosts (DHs) and mammals and/or reptiles as intermediate hosts (IHs). Some of these species cause disease or even death in their IH. At least two Sarcocystis spp. from snakes have relevance in public health. Sarcocystis nesbitti causes serious myopathy in humans, and S. singaporensis is lethal for rats and was successfully tested in the biological control of these rodents. Molecular identification was not reported for half of Sarcocystis spp. shed by snakes. For some snake species, their role as DHs for Sarcocystis spp. is totally unknown, including certain snakes which are bred as companion animals. The aim of this review is to discuss the role of snakes as DHs or IHs of Sarcocystis spp. and the future directions for the identification of the parasites and the elucidation of their life cycles.

Simple Summary: Sarcocystidae is a family of protozoa whose life cycles usually include two hosts: an intermediate or paratenic host, where protozoa form tissular cysts, and a definitive host, whose intestines usually become infected after consuming tissular cysts. In Chile, studies of sarcocystids have been performed mostly on domestic animals, and scarce data are available from wildlife. A total of 207 rodents, encompassing six native species, from 13 localities in Central and Southern Chile, were studied to assess the presence of Sarcocystidae. DNA was isolated from several tissues and phylogenetic analyses were performed. Blood smear examinations and histological studies of several organs were performed. Overall, three individuals of rodents were PCR-positive and three different genotypes were retrieved belonging to Sarcocystis, Besnoitia, and Toxoplasmatinae protozoa. No protozoa were found during microscopic examinations. Although it was not possible to confirm whether the findings corresponded to parasitism or accidental encounter, the phylogenetic positions of the genotypes support the hypothesis that they are parasites of rodents. All three genotypes are suggested as potential new taxa in the Sarcocystidae family. In Chile, studies of parasites from the family Sarcocystidae (Apicomplexa) have mostly been related to domestic animals. We aimed to assess the presence of Sarcocystidae taxa in cricetid rodents from Central and Southern Chile. We studied 207 rodents, encompassing six species, from 13 localities. We isolated DNA from tissue samples, amplified the Sarcocystidae 18S rRNA gene with polymerase chain reaction, and performed phylogenetic analyses using maximum likelihood and Bayesian inferences. In addition, we examined blood smears and performed histological studies in organs from Sarcocystidae DNA-positive animals. Three specimens were DNA-positive and three genotypes were retrieved and named: Sarcocystis sp. P61, related to Sarcocystis strixi, was detected in two Abrothrix olivacea. Toxoplasmatinae gen. sp. P99 was retrieved from those same two specimens, and was related to Toxoplasma and other genera, although it branched independently. Besnoitia sp. R34 was detected in one Abrothrix hirta, and was clustered with congeneric species associated with rodents. No protozoa were found during microscopic studies; thus, it was not possible to confirm parasitic interactions rather than accidental encounters. However, the close relatedness of the retrieved genotypes to parasites of rodents supports the hypothesis of host–parasite associations. All three genotypes are suggested as potential new taxa, including a putative new genus. [ABSTRACT FROM AUTHOR]

Background: Limited data are currently available on protozoan parasites of the genus Sarcocystis that infect their avian hosts within the order Anseriformes (waterfowl). To date, no Sarcocystis species has been recorded in ducks in China. Methods: Leg muscles were sampled from 26 domestic ducks (Anas platyrhynchos) in China in 2021. Morphological characteristics of sarcocysts detected in the muscle tissue were described using light microscopy (LM) and transmission electron microscopy (TEM). Genomic DNA was extracted from single sarcocysts obtained from different ducks, and three genetic markers, 18S ribosomal DNA (18S rDNA), 28S ribosomal DNA (28S rDNA) and mitochondrial (mt) cytochrome oxidase subunit 1 (cox1), were amplified and cloned for sequence analyses. Results: Sarcocysts were observed by LM in only three of the 28 samples (10.7%). These sarcocysts had a thick cyst wall with numerous brush-like villar protrusions (vps) of 3.8–4.3 μm in length (n = 30) on the cyst surface. TEM observation showed that the sarcocysts had lanceolated vps. Each vps narrowed in the stalk and contained a bundle of microtubules that extended into the ground substance. Comparisons of the new sequences with those deposited in GenBank showed that the most similar sequences were those of Sarcocystishalieti in the great cormorant Phalacrocorax carbo and European starling Sturnus vulgaris, and Sarcocystis calchasi in the domestic pigeon (Columba livia) at the 18S rDNA (99.1% identity); Sarcocystiswenzeli from the domestic chicken Gallus gallus at the 28S rDNA (95.9–96.0% identity); and Sarcocystis speeri from the opossum at the mtcox1 (98.2% identity). The new 18S rDNA, 28S rDNA and mitochondrial cox1 sequences shared up to 99.0%, 95.6% and 97.7% identity, respectively, with those of Sarcocystis spp. obtained from Anseriformes avian hosts. Phylogenetic analysis inferred from the sequences of the three genetic markers placed the organism within a group of Sarcocystis spp. obtained from avian or carnivorous intermediate hosts and avian, marsupial or carnivorous definitive hosts. Based on the morphological observation and molecular analyses, the organism found in the Chinese domestic ducks was regarded as a new species and named Sarcocystis platyrhynchosi n. sp. Conclusions: Based on morphology and sequence analyses, the microcysts diagnosed in the domestic ducks examined in this study were named as a new species. This is the first record of Sarcocystis spp. from waterfowl in China. Sarcocysts of similar morphology occur frequently in different Anseriformes birds, and the relationships among these species need to be further clarified in future studies using more molecular markers. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii infection in healthy animals is often asymptomatic. However, some species with little history of contact with the parasite, such as marsupials and New World primates, present high mortality rates after infection. Despite its potential conservation concern, T. gondii infection in insectivorous bats has received little attention, and its impact on bat populations' health is unknown. To assess the putative role of insectivorous bats in the cycle of T. gondii, samples of three species of bats (Pipistrellus pipistrellus, P. pygmaeus and P. kuhlii) collected between 2019 and 2021 in NE Spain were tested for the presence of the parasite using a qPCR. All tissues resulted negative (0.0% prevalence with 95% CI: [0.0–2.6]) for the presence of T. gondii. Unlike previous studies on insectivorous bats from Europe, Asia and America, the present study suggests that Pipistrellus spp. bats do not play a significant role in the epidemiology of T. gondii in NE Spain. Further studies are encouraged to elucidate both the epidemiology of T. gondii and its potential impact on the health of microchiropteran species in Europe. [ABSTRACT FROM AUTHOR]

Sarcocystis bertrami (synonym: Sarcocystis fayeri) is a coccidian parasite that infects horses and donkeys in several countries. Dogs are known as definitive hosts of the parasite, however, the patent period is not well defined, and S. bertrami shed by dogs has never been confirmed by molecular methods. Here we investigated the shedding of S. bertrami by experimentally infected dogs and examined the excreted parasites by morphological and molecular tools. Three dogs of small breeds (one Yorkshire terrier and two miniature Pinschers) were acquired with ages of 30 and 60 days and were exclusively fed commercial dog food. Two dogs consumed equine muscle tissues containing cysts of S. bertrami. The third dog served as negative control and was simultaneously fed commercial dog food. The two animals that received equine tissues shed sporocysts and/or oocysts in their feces after prepatent periods of 13 and 23 days. The patent periods were 47 and 14 days. Sporocysts showed average dimensions of 14.19 µm (± 0.53) x 10.06 µm (± 0.44). The control dog did not shed sporocysts or oocysts of the parasite. Interestingly, patent periods had never been reported, and for one dog, the patent period (47 days) was longer than that reported for other Sarcocystidae parasites. PCRs to the gene 18S and to the internal transcribed spacer 1 (ITS1) of the rDNA were successfully performed with DNA extracted from sporocysts. ITS1 sequences were also obtained from the equine tissue cysts used to infect the dogs. Nucleotide sequences of cloned fragments of 18S from sporocysts, and ITS1 from both stages (tissue cysts and sporocysts) matched with S. bertrami (18S : 97.50–99.88 %; ITS1: 88.76–95.21 %), although high molecular diversity was observed with data from these loci. PCR to cox1 using sporocysts' DNA failed to amplify any product. The possibility of the existence of an additional and undescribed Sarcocystis species in the excreted sporocysts, besides S. bertrami , cannot be excluded from this experiment. To our knowledge, this is the first molecular confirmation of S. bertrami in canine feces. Sporocyst dimensions and prepatent periods observed in this study were similar to those previously described for S. bertrami and S. fayeri. In conclusion, the molecular, morphological and biological data generated here fit in previous descriptions for both S. bertrami and S. fayeri. [Display omitted] • Two dogs were fed Brazilian horse meat containing Sarcocystis bertrami cysts. • The dogs shed sporocysts, which were confirmed to be S. bertrami by molecular tools. • The minimum prepatent period was 13 days and the maximum patent period was 47 days. • ITS1 sequences from sporocysts had identities of 88.53–94.49 % with S. bertrami from Chinese horses. • High molecular diversity was observed for 18S and ITS1 from S. bertrami sporocysts. [ABSTRACT FROM AUTHOR]

Background: Sarcocystis is a genus of coccidian protozoa that at least seven species of it can parasitize cattle. The global prevalence of Sarcocystis is close to 100% in adult cattle. The main aim of this study was to identify the infection rate of Sarcocystis spp. in heart of cattle in Tehran, Iran by microscopy and PCR-RFLP methods. Methods: Totally, 100 bovine heart samples were collected from the main slaughterhouse of Shahriar, Meysam slaughterhouse, west of Tehran in 2016. At first, heart samples were completely examined for the presence of sarcocystic macrocysts. Then, for microscopic examination, 50 g of each heart was digested in sterile condition using pepsin acid digestion method. Then, the species of the parasite were detected by PCR-RFLP technique and sequencing. Results: Overall, 97 of 100 of the heart muscle samples were infected with Sarcocystis. All the samples were detected as S. cruzi through similar patterns in PCRRFLP. Conclusion: S. cruzi is the most common species in the heart of cattle slaughtered in Shahriar. [ABSTRACT FROM AUTHOR]

Neospora caninum is an apicomplexan (family: Sarcocystidae) protozoan parasite with a global distribution. In the N. caninum life cycle, dogs and other related canids are the definitive hosts, while other animals such as water buffaloes (Bubalus bubalis) constitute the intermediate host for this parasite. In many countries, the water buffalo is of high economic importance, providing valuable high-quality products for human needs. Although knowledge concerning the prevalence of this parasite in intermediate animal host populations is crucial, data from water buffalo are scarce. Keeping this in mind, the aim of this study was to examine the presence and assess the prevalence rates of N. caninum infection in water buffaloes in Northern Greece, where this animal husbandry sector started to raise, as well as to determine associated risk factors, with the application of a commercially available Neospora ISCOM ELISA test kit, developed for the detection of antibodies against N. caninum in milk samples The study was conducted during January-June 2023 in a total of 172 individual raw milk samples, collected from dairy buffaloes, reared under a semi extensive system, in three different farms. Information on the status of N. caninum infection in buffaloes from Greece was so far unknown with a lack of epidemiological or risk factors associated. For the detection of N. caninum, the commercially available Neospora ISCOM ELISA test kit (SANOVIR ®, Svanova Biotech AB, Uppsala, Sweden) was utilized. Results demonstrated the presence of N. caninum in water buffaloes from Greece for the first time. All positive N. caninum animal were asymptomatic, with absence of any disease signs. The overall prevalence of infection was 22.10%, whereas the main risk factors include the presence of dogs as well as the low biosecurity measures., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this study., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The genus Eumonospora Allen, 1933 (Apicomplexa: Sarcocystidae), an avian coccidia, is characterized by monosporocystic and octasporozoic oocysts without Stieda and substieda bodies. Some members of Eumonospora, which infect several raptor species, exhibit high levels of pathogenicity, making eumonosporiosis the leading cause of death in captive-bred raptors. The host specificity of these species appears to be mesostenoxenous, as evidenced by unsuccessful transmission between different orders of avian hosts. However, several studies have detected Eumonospora spp. in taxonomically distant avian hosts, indicating that some of these species may be euryxenous. In the current study, diarrheic fecal examination of a captive-bred juvenile merlin (Falconiformes: Aves) in Tokyo, Japan, was conducted, and a large number of oocysts were morphologically and molecularly identified as E. henryae (Yakimoff and Matschulsky, 1932), a coccidia species reported only in Strigiformes. This is a new recorded host for this coccidia. Phylogenetic analyses via Bayesian inference and maximum likelihood methods using concatenated genomic datasets consisting of nuclear 18S rDNA, nuclear 28S rDNA and mitochondrial cytochrome C oxidase subunit 1 gene, revealed a well-supported monophyletic clade of Eumonospora spp. belonging to the family Sarcocystidae Poche 1913, which largely corresponded to the avian host phylogram. Therefore, based on distinguishable oocyst morphology, a new subfamily, Eumonosporinae, within the family Sarcocystidae, is proposed, and a reconsideration of the definition of Sarcocystidae is suggested. Further molecular characterization of this emerging pathogen, as well as clarification of its complete life cycle, including cyst-forming ability, is required for more appropriate generic assessment.

The wild boar, an impactful invasive species in Brazil, is subject to population control activities, which often include the use of hunting dogs. Hunters commonly consume wild boar meat, which is also used to feed their dogs, posing a risk of Toxoplasma gondii infection for humans and both T. gondii and Neospora caninum for dogs. The study aimed to investigate the prevalence of infection in wild boars (n = 127) and hunting dogs (n = 73) from São Paulo, Rio Grande do Sul, and Paraná states. We employed histopathological, serological (indirect fluorescent antibody test), and molecular techniques (endpoint polymerase chain reaction). Histopathology slides of wild boar tissue (central nervous system, heart, skeletal muscle, liver, spleen, kidney, gastrointestinal tract, pancreas, lymph nodes, and thyroid) sections revealed no T. gondii or N. caninum cysts (0/47). Antibodies anti- T. gondii were detected in 35/108 (32.4%) and anti- N. caninum in 45/108 (41.7%) wild boars. Only 2/18 (11.1%) wild boar tissue homogenate samples tested positive for T. gondii on endpoint PCR. Hunting dogs showed antibodies against T. gondii in 62/73 (85%) and against N. caninum in 31/73 (42%). The presence of antibodies against T. gondii and N. caninum in wild boars and hunting dogs, along with T. gondii DNA detection in wild boars, indicates the circulation of these parasites. Educating hunters on preventing these foodborne diseases, including zoonotic risks, is crucial. [Display omitted] • Toxoplasma gondii 's overall prevalence in wild boars from Brazil was 27.6%. • Neospora caninum 's seroprevalence in wild boars from Brazil was 41.7%. • T. gondii 's seroprevalence in hunting dogs from Brazil was 85%. • N. caninum 's seroprevalence in hunting dogs from Brazil was 42%. • N. caninum infection was more frequent in female wild boars. [ABSTRACT FROM AUTHOR]

Lagomorpha coccidiosis, caused by coccidia, is a prevalent disease affecting rabbits, hares and pikas. This meta-analysis aimed to estimate the pooled prevalence of coccidia infection in lagomorphs and identify potential risk factors. A systematic search of six databases yielded 102 studies published between 1981 and 2023. The pooled prevalence of Eimeriidae, Sarcocystidae and Cryptosporidiidae in lagomorphs was 76.4%, 6.2% and 3.9%, respectively. Rabbits had the highest prevalence of Eimeriidae (76.8%) and Sarcocystidae (7.4%), while pikas had the highest prevalence of Cryptosporidiidae (6.2%). Juvenile rabbits exhibited the highest prevalence of Eimeriidae (84.6%) and Cryptosporidiidae (9.9%). Northwest China had the highest prevalence of Eimeriidae (87.8%). Over time, the prevalence of Eimeriidae declined (Coefficient: −0.0062; P<0.05), but remained high (65.0%) in the past five years. Our findings highlight the prevalence of Eimeriidae infection in lagomorphs and the need for further research on Sarcocystidae and Cryptosporidiidae infections. We emphasize the importance of developing lagomorpha coccidia vaccines and implementing vaccination schedules for juvenile rabbits to mitigate coccidia infections. • A systematic search of six databases identified 102 studies from 1981 to 2023. • Rabbits have highest Eimeriidae (76.8%) and Sarcocystidae (7.4%) prevalence. • Northwest China has highest Eimeriidae prevalence (87.8%). • Declining trend in Eimeriidae prevalence, yet high (65.0%) in the past five years. [ABSTRACT FROM AUTHOR]

Small mammals play an essential role as disseminators of pathogens because they reach high population densities and have ubiquitous distributions. In the Northern Hemisphere rodents are well recognized as reservoirs for tick-borne bacteria of the Anaplasmataceae family and also apicomplexan protozoans. In contrast, South American rodents hosting these microorganisms have been rarely identified. In this study, we collected blood from rodents and marsupials in northern Chile and screened for Anaplasmataceae bacteria and apicomplexan protozoa. Overall, 14.7% of the samples were positive for Babesia, Hepatozoon, and Sarcocystidae using conventional PCR assays targeting the structural 18S rRNA locus (18S). Phylogenetic analyses performed with amplicons derived from 18S and cytochrome c oxidase (COI) gene provided evidence of a Babesia sp. belonging to the Babesia microti group in Phyllotis darwini, and a novel Babesia genotype in P. darwini and Abrothrix jelskii. Furthermore, four novel genotypes of Hepatozoon retrieved from Abrothrix olivacea, P. darwini, and Oligoryzomys longicaudatus, formed independent lineages within a clade that includes additional Hepatozoon spp. detected in South American rodents. Moreover, an incidental finding of a previously detected apicomplexan, herein designated as Sarcocystidae sp., was recorded in Thylamys opossums with a high prevalence, indicating a possible specific association with these mammals. Phylogenetic analysis of Sarcoystidae sp. clearly demonstrated its relatedness to apicomplexans detected in Australian marsupials. Our results expand the range of mammals hosting tick-borne apicomplexans in South America, highlight a novel clade consisting of South American babesias, and report for the first time the B. microti group infecting rodents in the region. [ABSTRACT FROM AUTHOR]

This is the first report of a disseminated protozoal infection in a wild feathertail glider (Acrobates pygmaeus) from south-eastern Australia. The glider was found dead in poor body condition. Histologically, large numbers of zoites were seen predominantly in macrophages in the liver, spleen and lung, with protozoal cysts present in the liver. Molecular and phylogenetic analyses inferred that the protozoan parasite belongs to the family Sarcocystidae and is closely related to previously identified apicomplexans found in yellow-bellied gliders (Petaurus australis) in Australia and southern mouse opossums (Thylamys elegans) in Chile.

Tissue-cyst forming coccidia in the family Sarcocystidae are etiologic agents of protozoal encephalitis in marine mammals including the federally listed Southern sea otter (Enhydra lutris). California sea lions (Zalophus californianus), whose coastal habitat overlaps with sea otters, are definitive hosts for coccidian protozoa provisionally named Coccidia A, B and C. While Coccidia A and B have unknown clinical effects on aquatic wildlife hosts, Coccidia C is associated with severe protozoal disease in harbor seals (Phoca vitulina). In this study, we conducted surveillance for protozoal infection and fecal shedding in hospitalized and free-ranging California sea lions on the Pacific Coast and examined oocyst morphology and phenotypic characteristics of isolates via mouse bioassay and cell culture. Coccidia A and B were shed in similar frequency, particularly by yearlings. Oocysts shed by one free-ranging sea lion sampled at Año Nuevo State Park in California were previously unidentified in sea lions and were most similar to coccidia infecting Guadalupe fur seals (Arctocephalus townsendi) diagnosed with protozoal disease in Oregon (USA). Sporulated Coccidia A and B oocysts did not replicate in three strains of mice or in African green monkey kidney cells. However, cultivation experiments revealed that the inoculum of fecally-derived Coccidia A and B oocysts additionally contained organisms with genetic and antigenic similarity to Sarcocystis neurona; despite the absence of detectable free sporocysts in fecal samples by microscopic examination. In addition to the further characterization of Coccidia A and B in free-ranging and hospitalized sea lions, these results provide evidence of a new role for sea lions as putative mechanical vectors of S. neurona, or S. neurona-like species. Future work is needed to clarify the distribution, taxonomical status, and pathogenesis of these parasites in sea lions and other marine mammals that share their the near-shore marine environment.

Background: Data on the genus Sarcocystis in insectivores are limited. The Asian gray shrew Crocidura attenuata is one of the most common species of the insectivore family Soricidae in South Asia and Southeast Asia. To our knowledge, species of Sarcocystis have never been recorded previously in this host. Methods: Tissues were obtained from 42 Asian gray shrews caught in 2017 and 2018 in China. Sarcocysts were observed using light microscopy (LM) and transmission electron microscopy (TEM). To describe the parasite life cycle, muscle tissues of the host infected with sarcocysts were force-fed to two beauty rat snakes Elaphe taeniura. Individual sarcocysts from different Asian gray shrews, and oocysts/sporocysts isolated from the small intestines and feces of the experimental snakes, were selected for DNA extraction, and seven genetic markers, namely, two nuclear loci [18S ribosomal DNA (18S rDNA) and internal transcribed spacer region 1 (ITS1)], three mitochondrial genes [cytochrome oxidase subunit 1 (cox1), cox3 and cytochrome b], and two apicoplast genes (RNA polymerase beta subunit and caseinolytic protease C), were amplified, sequenced and analyzed. Results: Sarcocysts were found in 17 of the 42 (40.5%) Asian gray shrews. Under LM, the microscopic sarcocysts showed saw- or tooth-like protrusions measuring 3.3–4.5 μm. Ultrastructurally, the sarcocyst wall contained numerous lancet- or leaf-like villous protrusions, similar to those described for type 9h of the common cyst wall classification. The experimental beauty rat snakes shed oocysts/sporocysts measuring 11.9–16.7 × 9.2–10.6 μm with a prepatent period of 10–11 days. Comparison of the newly obtained sequences with those previously deposited in GenBank revealed that those of 18S rDNA and cox1 were most similar to those of Sarcocystis scandentiborneensis recorded in the tree shrews Tupaia minor and Tupaiatana (i.e., 97.6–98.3% and 100% identity, respectively). Phylogenetic analysis based on 18S rDNA or ITS1 sequences placed this parasite close to Sarcocystis spp. that utilize small animals as intermediate hosts and snakes as the known or presumed definitive host. On the basis of morphological and molecular characteristics and host specificity, the parasite was proposed as a new species, named Sarcocystis attenuati. Conclusions: Sarcocysts were recorded in Asian gray shrews, to our knowledge for the first time. Based on morphological and molecular characterization, a new species of parasite is proposed: Sarcocystisattenuati. According to the LM and TEM results, S. attenuati sarcocysts are distinct from those of Sarcocystis spp. in other insectivores and those of S. scandentiborneensis in tree shrews. The 18S rDNA or cox1 sequences of Sarcocystis attenuati shared high similarity with those of Sarcocystisscandentiborneensis, Sarcocystis zuoi, Sarcocystis cf. zuoi in the Malayan field rat, and Sarcocystis sp. in the greater white-toothed shrew. Therefore, we suggest that more research on the relationships of these closely related taxa should be undertaken in the future. [ABSTRACT FROM AUTHOR]

The Darwin's fox (Lycalopex fulvipes) is one of the most endangered carnivores worldwide, with the risk of disease spillover from domestic dogs being a major conservation threat. However, lack of epidemiologic information about generalist, non–dog-transmission-dependent protozoal and bacterial pathogens may be a barrier for disease prevention and management. To determine the exposure of some of these agents in Darwin's fox populations, 54 serum samples were collected from 47 Darwin's foxes in Southern Chile during 2013–18 and assessed for the presence of antibodies against Brucella abortus, Brucella canis, Coxiella burnetii, pathogenic Leptospira (serovars Grippotyphosa, Pomona, Canicola, Hardjo, and Copehageni), Toxoplasma gondii, and Neospora caninum. The highest seroprevalence was detected for T. gondii (78%), followed by pathogenic Leptospira (14%). All the studied Leptospira serovars were confirmed in at least one animal. Two foxes seroconverted to Leptospira and one to T. gondii during the study period. No seroconversions were observed for the other pathogens. No risk factors, either intrinsic (sex, age) or extrinsic (season, year, and degree of landscape anthropization), were associated with the probability of being exposed to T. gondii. Our results indicate that T. gondii exposure is widespread in the Darwin's fox population, including in areas with minimal anthropization, and that T. gondii and pathogenic Leptospira might be neglected threats to the species. Further studies identifying the causes of morbidity and mortality in Darwin's fox are needed to determine if these or other pathogens are having individual or population-wide effects in this species. [ABSTRACT FROM AUTHOR]

Background: Species of Sarcocystis are parasitic protozoa in poikilothermic and homeothermic animals. Out of the 26 valid species in birds as intermediate hosts, none has been reported in those of the order Musophagiformes, such as the great blue turaco Corythaeola cristata (Vieillot, 1816), which is a bird endemic to Central and Western Africa. The examination of great blue turacos imported from the Central Africa Republic to Czech Republic allowed the morphological and molecular characterization of a new species of Sarcocystis. Methods: Four turacos imported from the Central Africa Republic to a private breeder (Czech Republic) underwent parasitological examination for the presence of sarcocysts through wet mounts of breast, heart and leg muscles. Found parasites were molecularly and histologically studied by four loci (18S rRNA, 28S rRNA, ITS1 and cox1) and haematoxylin and eosin staining, respectively. Results: Three out of four examined birds harboured numerous sarcocysts in the breast and leg muscles. No macroscopic lesions where observed. Sarcocysts were microscopic, elongate and ribbon-shaped with a wall characterised by the presence of finger-shaped villar protrusions and filled with numerous elongate, banana-shaped bradyzoites, 11.87–14.84 × 2.05–2.92 µm in size. The new species was most closely related to Sarcocystisalbifronsi, Sarcocystisanasi, Sarcocystisatraii, Sarcocystischloropusae, Sarcocystisrileyi, Sarcocystiswenzeli and Sarcocystis sp. isolate from chicken in the four loci. Conclusions: To our knowledge, this is the first species of Sarcocystis found in a musophagiform bird worldwide. Genetically, S. cristata sp. nov. represents a distinct species. Phylogenetic analyses are useful for predicting potential definitive hosts of the new Sarcocystis species. [ABSTRACT FROM AUTHOR]

Abstract Protozoan parasites of the genus Sarcocystis are obligatory heteroxenous cyst-forming coccidia that infect a wide variety of animals and encompass approximately 200 described species. At least four Sarcocystis spp. (S. falcatula, S. neurona, S. lindsayi and S. speeri) use opossums (Didelphis spp.) as definitive hosts, and two of them, S. neurona and S. falcatula, are known to cause disease in horses and birds, respectively. Opossums are restricted to the Americas, but their distribution in the Americas is heterogeneous. Five Didelphis spp. are distributed in South America (D. aurita, D. albiventris, D. marsupialis, D. imperfecta and D. pernigra) whereas just one opossum species (D. virginiana) is found in North America. Studies conducted in the last decades show that Sarcocystis spp., derived from South American Didelphis spp., have biological and genetic differences in relation to Sarcocystis spp. shed by the North American opossum D. virginiana. The aim of this review was to address the peculiar scenario of Sarcocystis species shed by South American opossums, with a special focus on diagnosis, epidemiology, and animal infections, as well as the genetic characteristics of these parasites.

Neospora caninum (Family: Sarcocystidae) is an obligate intracellular protozoan. It is one of the most critical abortifacients in ruminants. The seroprevalence of antibodies against N. caninum and its risk factors was investigated among 430 sheep from four North Egyptian governorates, Alexandria, Gharbia, Menofia, and Qalyubia, during the period from 2017 to 2018. Generally, the overall prevalence rate of N. caninum among sheep was 8.6%. The logistic regression analysis for the obtained data revealed that N. caninum increased significantly with age (OR = 2.4, 95% CI: 8.4–18.7) of the ewe (OR = 3.3, 95% CI: 7.6–14.9), particularly among sheep in contact with dogs (OR = 4.9, 95% CI: 7.5–14.3). Besides, locality, season, and pregnancy status of examined sheep had no significant effect on the appearance of N. caninum infection. the present findings confirm the presence of N. caninum among sheep in Egypt which probably play a role in reproductive failure in sheep. Therefore, sanitary measures and monitoring of the infection should be implemented to reduce the spreading of the infection. [ABSTRACT FROM AUTHOR]

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In this study, faecal samples of four American Staffordshire terrier dogs (used for illegal fighting) were analysed by DNA extraction, molecular-phylogenetic and parasitological methods, in order to examine the occurrence of protozoan, apicomplexan parasites. In one sample, the DNA of Sarcocystis morae was shown to be present. This species was identified based on 100% identity with already reported sequences of S. morae from cervids in Lithuania and Spain. The result was also confirmed by phylogenetic analysis. The sporocysts of the canine S. morae isolate measured 14.95 × 9.75 μm on average. This is the first molecular evidence in support of the final host role of domestic dogs in the life cycle of S. morae. The most likely source of the infection was raw meat given to the examined dog to increase its physical achievement. In conclusion, under similar circumstances dogs may participate in the life cycle of S. morae in a 'natural way', shedding sporocysts/oocysts when used for hunting or taken to walks in forested areas. [ABSTRACT FROM AUTHOR]

The genus Eumonospora Allen, 1933 (Apicomplexa: Sarcocystidae), an avian coccidia, is characterized by monosporocystic and octasporozoic oocysts without Stieda and substieda bodies. Some members of Eumonospora , which infect several raptor species, exhibit high levels of pathogenicity, making eumonosporiosis the leading cause of death in captive-bred raptors. The host specificity of these species appears to be mesostenoxenous, as evidenced by unsuccessful transmission between different orders of avian hosts. However, several studies have detected Eumonospora spp. in taxonomically distant avian hosts, indicating that some of these species may be euryxenous. In the current study, diarrheic fecal examination of a captive-bred juvenile merlin (Falconiformes: Aves) in Tokyo, Japan, was conducted, and a large number of oocysts were morphologically and molecularly identified as E. henryae (Yakimoff and Matschulsky, 1932), a coccidia species reported only in Strigiformes. This is a new recorded host for this coccidia. Phylogenetic analyses via Bayesian inference and maximum likelihood methods using concatenated genomic datasets consisting of nuclear 18S rDNA, nuclear 28S rDNA and mitochondrial cytochrome C oxidase subunit 1 gene, revealed a well-supported monophyletic clade of Eumonospora spp. belonging to the family Sarcocystidae Poche 1913, which largely corresponded to the avian host phylogram. Therefore, based on distinguishable oocyst morphology, a new subfamily, Eumonosporinae, within the family Sarcocystidae, is proposed, and a reconsideration of the definition of Sarcocystidae is suggested. Further molecular characterization of this emerging pathogen, as well as clarification of its complete life cycle, including cyst-forming ability, is required for more appropriate generic assessment. [Display omitted] • Eumonospora henryae , reported only from owls, is detected from Falco columbarius. • New host record for this monosporocystic and octasporozoic species. • Indicates broad host spectrum which across family boundary. • Monophyletic Eumonospora spp. (Sarcocystidae) clade was found in phylogenetic study. • A new sub-family, Eumonosporinae, is proposed based on oocyst morphology. [ABSTRACT FROM AUTHOR]

This is the first report of a disseminated protozoal infection in a wild feathertail glider (Acrobates pygmaeus) from south-eastern Australia. The glider was found dead in poor body condition. Histologically, large numbers of zoites were seen predominantly in macrophages in the liver, spleen and lung, with protozoal cysts present in the liver. Molecular and phylogenetic analyses inferred that the protozoan parasite belongs to the family Sarcocystidae and is closely related to previously identified apicomplexans found in yellow-bellied gliders (Petaurus australis) in Australia and southern mouse opossums (Thylamys elegans) in Chile. Image 1 • A novel apicomplexan parasite caused a disseminated fatal disease in a wild feathertail glider. • The apicomplexan parasite is a member of the family Sarcocystidae. • It is most closely related to parasites previously identified in yellow-bellied gliders in Australia and southern mouseopossums in Chile. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is known to cause sporadic clinical disease and fatality in marine mammals worldwide. It has been recognized primarily in odontocetes, pinnipeds, and mustelids; however, there is very limited information available in mysticetes. We report the results of pathological, immunohistochemical, and molecular genotyping analyses in a Bryde’s whale (Balaenoptera edeni) with disseminated toxoplasmosis. A 10.7-m-long, adult, male Bryde’s whale in poor body condition was stranded alive on August 21, 2018 in Pontal do Ipiranga, Linhares, Espirito Santo state (Brazil). The animal died shortly after stranding and was promptly autopsied. The main gross findings were diffuse axial skeletal muscle atrophy, generalized congestion, petechiation and ecchymoses, necrotizing splenitis, hepatitis, myocarditis, pneumonia, and lymphadenitis (prescapular, pulmonary, mediastinal, and mesenteric), bilateral scapulohumeral hemarthros, and severe pulmonary edema. A microscopic examination confirmed the aforementioned diagnoses, featuring a histopathologic signature characterized by multisystemic necrotizing inflammation with vasculitis and disseminated intravascular coagulation, thrombosis, and numerous intralesional protozoal cysts and extracellular tachyzoites morphologically compatible with T. gondii. The immunohistochemical and polymerase chain reaction (PCR) analysis targeting a repetitive 529-bp DNA fragment of T. gondii confirmed toxoplasmosis in the liver, spleen, lung, and lymph nodes. PCR-restriction fragment length polymorphism analysis using 11 markers identified a new non-archetypal genotype, ToxoDB-RFLP genotype #300. Further, the genotyping by microsatellite technique employed 15 markers and confirmed a unique non-archetypal T. gondii strain, designated as PS-TgBaledBrES1. These novel results add to the diversity of this parasite in the world and to the scarce data on T. gondii genotype distribution in cetaceans, representing the first record of toxoplasmosis in a Bryde’s whale and setting the baseline knowledge for future research on T. gondii genotyping research in marine mammals from South America.

Sarcocystosis is a parasitic disease caused by an intracellular protozoan parasite Sarcocystis belonging to the phylum Apicomplexa. These parasites have a requisite two‐host life cycle. Recently, there are many Sarcocystis species that identified morphologically. In the present study, diaphragmatic muscle samples from the domestic horse (Equus caballus) were examined for Sarcocystis infection. The natural infection with sarcocysts was recorded to be 62·5% for only microcysts in the infected muscles. Molecular analysis using the 18S rRNA gene was conducted to swiftly and accurately identify the recovered species. Studies on the expression of the 18S rRNA gene have confirmed that the present parasite isolates belong to the Sarcocystis genus. The sequence data showed significant identities (>80%) with archived gene sequences from species within the Sarcocystidae family, and a dendrogram showing the phylogenetic relationship was constructed. The most closely related species were the previously described Sarcocystis fayeri and Sarcocystis bertrami. The current data showed that the present species was identified as S. fayeri and deposited in GenBank (accession number MF614956.1). This study highlights the importance of the genetic data in the exact taxonomy within sarcocystid species. [ABSTRACT FROM AUTHOR]

A total of 57 tissue samples of domestic pigs (Sus scrofa) were collected from the meat outlets of five north Indian states and examined for sarcocystosis by histological and molecular methods. The genomic DNA extracted from five representative positive isolates was subjected to PCR amplification of the partial 18S rRNA gene followed by cloning and sequencing. Sequence analysis of the newly generated Indian isolates recorded 96.9–100.0% identity with published sequences of Sarcocystis suihominis. Two new haplotypes that have not been previously described manifested 99.5–100.0% nucleotide homology within themselves. In the phylogenetic analysis, Indian isolates of S. suihominis grouped together with S. suihominis originating from Italy, and they collectively formed a sister clade with Sarcocystis miescheriana within a clade containing various Sarcocystis spp. of ruminants having felids as final hosts. At the same time, this clade separated from a sister clade containing Sarcocystis spp. of bovid or cervid ruminants using canids as known or surmised definitive host. The current study established the phylogenetic relationship of Indian isolates of S. suihominis with various Sarcocystis spp. as well as with other taxa of Sarcocystidae family based on 18S rRNA gene for the first time. [ABSTRACT FROM AUTHOR]

DNA sequencing, using traditional Sanger sequencing methods, has revolutionized parasitology research, with advancements in both parasite detection and phylogenetic reconstruction. Currently, high-throughput sequencing (HTS) technologies are available and can be used to screen simultaneously for multiple parasites via a metabarcoding approach, in a cheaper, faster and still accurate manner. However, the effectiveness of this new tool still needs to be further assessed to determine the full extent of both its potential and limitations. We employed this approach to detect the presence of blood parasites in fossorial reptiles. In this way we intended to both validate a recent hypothesis that fossorial reptiles may not be parasitized and to examine the potency of this technique. We indicate the presence of species of Hepatozoon in Trogonophis wiegmanni, thereby refuting the hypothesis that these parasites are not present due to the fossorial lifestyle of the host. We also detected a species of Sarcocystis for the first time in a species of Blanus, further demonstrating the value of the HTS approach. [ABSTRACT FROM AUTHOR]

Background: Apicomplexan parasites of the genus Sarcocystis have an obligate two-host life-cycle and comprise about 200 species, which infect different cold- and warm-blooded hosts, including humans. Recently, morphological and molecular studies of sarcocysts in broadly spread carnivore hosts have been on the rise. The description of muscular tissues infection by Sarcocystis in the raccoon dog and the common raccoon from the Czech Republic is herein presented. Methods: During January-August 2019, 15 raccoon dogs and 1 common raccoon were examined from 5 districts (Česká Lípa, Liberec, Mladá Boleslav, Trutnov and Ústí nad Labem) of the Czech Republic. Muscle parts (diaphragm, forearm, hind-limb, tongue and heart) were examined in wet preparations under compression by light microscopy. After finding Sarcocystis sp., morphological characteristics and molecular analyses of 18S rRNA, 28S rRNA, ITS1 and cox1 loci were used to identify the species. Results: Sarcocysts were detected and identified in 1 out of 15 raccoon dogs and in the single common raccoon. Preferential infection sites were diaphragm and tongue, followed by forearm and hind limb. To our knowledge, this is the first identification of microscopic sarcocysts by multi-locus genetic analysis from both host species. Molecular analyses revealed 100% similarity at 18S rRNA, 28S rRNA, and cox1 genes with S. lutrae for both hosts and 98–100% identity at the ITS1 region of the isolate from the common raccoon. Conclusions: Both widely distributed non-indigenous wild carnivores represent new intermediate host records for S. lutrae and the first report of this parasite in a member of the family Procyonidae, but still with an unknown natural definitive host. Molecular data revealed that this parasite species appears more closely related to the Sarcocystis spp. using raptorial birds as definitive hosts. Therefore, further studies aimed at its identification, including the complete life-cycle, remain necessary. [ABSTRACT FROM AUTHOR]

Background: Broad spectrums of pharmacological properties, including antimicrobial activity have been attributed to Zataria multiflora Boiss (Laminaceae). The in vivo efficacy and safety of Z. multiflora essential oil (ZM-EO) were evaluated against acute toxoplasmosis caused by Toxoplasma gondii (Sarcocystidae) in mice. Methods: Z. multiflora (aerial parts) was obtained from the rural districts of Kerman city (Kerman Province) Southwestern Iran, in May of 2016. Male NMRI mice were orally treated with normal saline (control group) and ZM-EO at the doses of 0.2 and 0.4 mL/kg once a day for 14 d (8 mice in each group). On the 15th day, the mice were infected with 104 tachyzoites of T. gondii RH strain by intraperitoneal route. The mortality rate and parasite load were determined in the infected mice. Additionally, 24 mice were applied to examine the sub-acute toxicity of ZM-EO at the above doses after treatment during 14 d. Results: GC/MS analysis displayed that the key constituents were thymol (45.4%), carvacrol (23%) and p-cymene (10.6%), respectively. Overall, 100% mortality was observed on the 8th and 9th days in treated mice with the concentrations of 0.2 and 0.4 mL/kg, respectively. The mean number of tachyzoites in the mice treated with 0.2 and 0.4 mL/kg of ZM-EO were 189×104 and 76×104 cell/mL, respectively, meaningfully (P<0.05) reduced compared with the control mice. Results also demonstrated that ZMEO had no important toxicity on mice. Conclusion: The results demonstrated the efficacy of ZM-EO against acute toxoplasmosis. Nevertheless, supplementary surveys are mandatory to examine its precise effects, mainly immunomodulatory effect on toxoplasmosis. [ABSTRACT FROM AUTHOR]

Canine coccidiosis caused by Cystoisospora canis and Cystoisospora ohioensis-complex is common in kennels. While often underestimated, coccidiosis may cause severe clinical signs in puppies and sometimes even lead to death, so preventative measures are important. This study examines Cystoisospora spp. infection at a Labrador retriever breeding facility in Madrid, Spain. To identify environmental factors associated with infection, dams were examined throughout a reproductive cycle (from oestrus to 60 days postpartum) and their puppies during their first 60 days of life. Also assessed was the efficacy of combined treatment with emodepside (0.9 mg/ml) and toltrazuril (18 mg/ml) at a dose of 0.5 ml/kg of weight, equivalent to 0.45 mg/kg and 9 mg/kg, respectively, in puppies on day 35 of life. Oocyst shedding was detected in 4.6-18.6% of 45 dams examined and in 2.2-9.1% of their litters (315 puppies). In both cases, peak opg elimination was recorded on day 30 postpartum/of life. The species of Cystoisospora detected were C. canis (91.3%) and C. ohioensis-complex (8.7%). While in both dams and puppies opg counts were higher in autumn when rainfall was at its highest, correlation between opg and rainfall emerged as significant only in puppies (p = 0.031). The treatment of 35 day-old puppies with toltrazuril was 100% effective in controlling this infection in the kennel. Our findings therefore suggest the need for a strict hygiene regime and the use of toltrazuril as blanket treatment to reduce Cystoisospora transmission in dog breeding facilities., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Guadalupe Miró reports equipment, drugs, or supplies and travel were provided by Complutense University of Madrid. Guadalupe Miró reports a relationship with Complutense University of Madrid that includes: board membership, employment, non-financial support, and travel reimbursement., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

This study investigated the presence and abundance of Cystoisospora suis oocysts in faecal samples from 131 one- to three-week-old pig litters belonging to eight intensively raised, indoor herds in Spain. Seven herds used preventive anticoccidial toltrazuril treatments administered orally or by intramuscular injection, and one did not use preventive anticoccidial treatments. The diagnosis was performed using two oocyst flotation-concentration methods, Bailenger's method in every herd and the more recent Joachim's method in four herds. Oocysts were detected in every farm, the proportion of oocyst-positive samples was higher with Bailenger's technique, and the estimated overall prevalence (95% confidence interval) was 40 (32-49)%, including 47 (29-65)% in non-medicated litters, 52 (38-67)% in orally medicated litters and 28 (16-40)% in intramuscularly medicated litters (p < 0.05). However, mixed logistic regression models indicated that the risk of infection was not significantly associated with preventive anticoccidial treatments (p > 0.05), and increased with age, was higher in herds with partially compared to fully slatted dung floors in farrowing pens and in litters with pigs with diarrhoea (p < 0.05). The median (range) oocysts per gram of faeces (OpG) in infected litters by Bailenger's method was 623 (35-49048) and mixed negative binomial models revealed no significant association between infection intensity in positive litters and pen's floor type and piglets age, faecal consistency and treatment status (p > 0.05). The apparent low efficacy of Toltrazuril suggests treatment administration failures, reduced residual efficacy or low susceptibility of C. suis strains in study farms and needs further investigation., (© 2024. The Author(s).)

Background: In September 2014, there was sudden upsurge in the number of Eurasian red squirrels (Sciurus vulgaris) found dead in the Netherlands. High infection levels with the parasite Toxoplasma gondii were demonstrated, but it was unclear what had caused this increase in cases of fatal toxoplasmosis. In the present study, we aimed to gain more knowledge on the pathology and prevalence of T. gondii infections in Eurasian red squirrels in the Netherlands, on the T. gondii genotypes present, and on the determinants of the spatiotemporal variability in these T. gondii infections. The presence of the closely related parasite Hammondia hammondi was also determined., Methods: Eurasian red squirrels that were found dead in the wild or that had died in wildlife rescue centres in the Netherlands over a period of seven years (2014-2020) were examined. Quantitative real-time polymerase chain reaction was conducted to analyse tissue samples for the presence of T. gondii and H. hammondi DNA. Toxoplasma gondii-positive samples were subjected to microsatellite typing and cluster analysis. A mixed logistic regression was used to identify climatic and other environmental predictors of T. gondii infection in the squirrels., Results: A total of 178 squirrels were examined (49/178 T. gondii positive, 5/178 H. hammondi positive). Inflammation of multiple organs was the cause of death in 29 squirrels, of which 24 were also T. gondii polymerase chain reaction positive. Toxoplasma gondii infection was positively associated with pneumonia and hepatitis. Microsatellite typing revealed only T. gondii type II alleles. Toxoplasma gondii infection rates showed a positive correlation with the number of days of heavy rainfall in the previous 12 months. Conversely, they showed a negative association with the number of hot days within the 2-week period preceding the sampling date, as well as with the percentage of deciduous forest cover at the sampling site., Conclusions: Toxoplasma gondii infection in the squirrels appeared to pose a significant risk of acute mortality. The T. gondii genotype detected in this study is commonly found across Europe. The reasons for the unusually high infection rates and severe symptoms of these squirrels from the Netherlands remain unclear. The prevalence of T. gondii in the deceased squirrels was linked to specific environmental factors. However, whether the increase in the number of dead squirrels indicated a higher environmental contamination with T. gondii oocysts has yet to be established., (© 2024. The Author(s).)

The Tioman Island of Malaysia experienced acute muscular sarcocystosis outbreaks from 2011 to 2014. So far, a previous study based on the 18S rRNA gene sequencing has reported S. singaporensis, S. nesbitti and Sarcocystis sp. YLL-2013 in water samples acquired from the island, thus confirming the waterborne nature of this emerging parasitic disease. This study aimed to improve the detection methods for Sarcocystis, in order to have a clearer picture of the true diversity of Sarcocystis species in Tioman. A new primer set (28S R7F-28S R8 Deg R) was designed to amplify the 28S rRNA gene of Sarcocystis. Subsequently, Sarcocystidae was detected in 65.6% (21/32) of water samples and 28% (7/25) of soil samples acquired between 2014 and 2015 from Tioman. Next-generation sequencing (NGS) on 18 of the positive samples was then performed using amplicons generated from the same primer set. This yielded 53 potentially unique Sarcocystidae sequences (290 bp), of which nine of the most abundant, prevalent and unique sequences were named herein. In contrast, NGS of the 18S rRNA gene V9 hypervariable region of 10 selected samples detected only two Sarcocystis species (160 bp). S. mantioni was the most ubiquitous sequence found in this study. [ABSTRACT FROM AUTHOR]

• Neosporosis is a common cause of abortion in cattle but it is rarely reported in horses. • Confirmed disseminated neosporosis is reported in an aborted equine fetus. • Description of lesions and immunohistochemistry in placenta, heart and lungs should help diagnosis. Neosporosis is a common cause of abortion in cattle worldwide but is rare in horses. Here, the first case of histologically, ultrastructurally, immunohistochemically, and molecularly confirmed equine abortion caused by neosporosis is reported. Samples of lung, heart, liver, skeletal muscle, tongue, brain, and the placenta from a female fetus aborted at 280 days of gestation were fixed in formalin and submitted for diagnosis. Histologically, there was disseminated neosporosis with severe lesions in lungs, liver and the heart. Protozoal tachyzoites in all tissues reacted with polyclonal anti- Neospora caninum rabbit antibodies. Transmission electron microscopic observation on lung tissue revealed tachyzoites consistent with Neospora , including many rhoptries. Polymerase-chain reaction (PCR) using primers designed to amplify the rRNA gene internal transcribed spacer 1 (ITS1) of the Sarcocystidae was performed on DNA extracted from fetal tissues. Comparison of the ITS1 amplified from the foal tissue to sequences available in GenBank revealed 100% sequence identity to the ITS1 from three isolates of Neospora hughesi. [ABSTRACT FROM AUTHOR]

Human toxoplasmosis, a protozoonosis caused by Toxoplasma gondii, has been described as a worldwide foodborne disease with important public health impact. Despite infection has reportedly varied due to differences in alimentary, cultural and hygienic habits and geographic region, social vulnerability influence on toxoplasmosis distribution remains to be fully established. Accordingly, the present study has aimed to assess T. gondii seroprevalence and factors associated to social vulnerability for infection in households of Ivaiporã, southern Brazil, with 33.6% population making half minimum wage or less, ranked 1,055th in population (31,816 habitants), 1,406th in per capita income (U$ 211.80 per month) and 1,021st in HDI (0.764) out of 5,570 Brazilian cities. Serum samples and epidemiological questionnaires were obtained from citizen volunteers with official City Secretary of Health assistance in 2015 and 2016. In overall, serosurvey has revealed 526/715 (73.57%) positive samples for anti-T. gondii antibodies by Indirect Fluorescent Antibody Test. Logistic regression has shown a significant increase associated to adults (p = 0.021) and elderly (p = 0.014) people, illiterates (p = 0.025), unemployment (p <0.001) and lack of household water tank (p = 0.039). On the other hand, sex (male or female), living area (urban or rural), yard hygiene, meat ingestion, sand or land contact, owning pets (dog, cat or both) were not significant variables of positivity for anti-T. gondii antibodies in the surveyed population. Although no significant spatial cluster was found, high intensity areas of seropositive individuals were located in the Kernel map where the suburban neighborhoods are located. In conclusion, socioeconomic vulnerability determinants may be associated to Toxoplasma gondii exposure. The increased risk due to illiteracy, adult or elderly age, unemployment and lack of household water tank were confirmed by multivariate analysis and the influence of low family income for seropositivity by the spatial analysis. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is an apicomplexan protozoan parasite with a complex life cycle composed of multiple stages that infect mammals and birds. Tachyzoites rapidly replicate within host cells to produce acute infection during which the parasite disseminates to tissues and organs. Highly replicative cells are subject to Double Strand Breaks (DSBs) by replication fork collapse and ATM, a member of the phosphatidylinositol 3-kinase (PI3K) family, is a key factor that initiates DNA repair and activates cell cycle checkpoints. Here we demonstrate that the treatment of intracellular tachyzoites with the PI3K inhibitor caffeine or ATM kinase-inhibitor KU-55933 affects parasite replication rate in a dose-dependent manner. KU-55933 affects intracellular tachyzoite growth and induces G1-phase arrest. Addition of KU-55933 to extracellular tachyzoites also leads to a significant reduction of tachyzoite replication upon infection of host cells. ATM kinase phosphorylates H2A.X (γH2AX) to promote DSB damage repair. The level of γH2AX increases in tachyzoites treated with camptothecin (CPT), a drug that generates fork collapse, but this increase was not observed when co-administered with KU-55933. These findings support that KU-55933 is affecting the Toxoplasma ATM-like kinase (TgATM). The combination of KU-55933 and other DNA damaging agents such as methyl methane sulfonate (MMS) and CPT produce a synergic effect, suggesting that TgATM kinase inhibition sensitizes the parasite to damaged DNA. By contrast, hydroxyurea (HU) did not further inhibit tachyzoite replication when combined with KU-55933. [ABSTRACT FROM AUTHOR]

Abstract Neospora caninum infection of cattle can be vertically transmitted, resulting in abortion or birth of infected calves. Vertical transmission occurs both in acutely or chronically infected cattle. There is little information on the immune response needed to prevent endogenous transplacental transmission, particularly from chronically infected cattle to their offspring in a natural environment. In this study, N. caninum seropositive pregnant cattle from three different farms with high avidity antibodies and low IgM titers were selected and their newborn colostrum-deprived calves were tested for anti- N. caninum antibodies. Based on these results, dams were grouped according to their congenital transmission status. The analysis of the immune profile of the chronically-infected pregnant cattle revealed that higher ratio between IgG1 and IgG2 anti- N. caninum serum titers and higher levels of systemic IFN-γ were associated with diminished vertical transmission rates, compared to dams with the opposite profile. Our results evidenced an association between the immune profile and vertical transmission in non-aborting chronically infected dams, and confirm that vertical transmission, even when not leading to abortion, is related to a defined immune profile. This is important information to accomplish successful vaccine development efforts. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is a ubiquitous, obligate intracellular eukaryotic parasite that causes congenital birth defects, disease inimmunocompromisedindividuals,andblindness.Proteinglycosylation plays an important role in the infectivity and evasion of immune responses of many eukaryotic parasites and is also of great relevance to vaccine design. Here we demonstrate that micronemal protein 2 (MIC2), a motility-associated adhesin of T. gondii, has highly glycosylated thrombospondin repeat (TSR) domains. Using affinity-purified MIC2 and MS/MS analysis along with enzymatic digestion assays, we observed that at least seven C-linked and three O-linked glycosylation sites exist within MIC2, with >95% occupancy at these O-glycosylation sites. We found that addition of O-glycans to MIC2 is mediated by a protein O-fucosyltransferase 2 homolog (TgPOFUT2) encoded by the TGGT1_273550 gene. Even though POFUT2 homologs are important for stabilizing motility-associated adhesins and for host infection in other apicomplexan parasites, loss of TgPOFUT2 in T. gondii had only a modest impact on MIC2 levels and the wider parasite proteome. Consistent with this, both plaque formation and tachyzoite invasion were broadly similar in the presence or absence of TgPOFUT2. These findings indicate that TgPOFUT2 O-glycosylates MIC2 and that this glycan, in contrast to previous findings in another study, is dispensable in T. gondii tachyzoites and for T. gondii infectivity. [ABSTRACT FROM AUTHOR]

The coccidian parasite Cystoisospora canis (syn. Isospora canis) can cause clinical disease in dogs. Three generation of schizonts have been reported in the small intestine of dogs before oocysts are excreted 9–11 days post inoculation (PI). Here, we re-evaluated asexual development of C. canis in 2 dogs necropsied 10 days after oral inoculation with 100,000 C. canis oocysts; both dogs had excreted oocysts 9 days PI. Asexual and sexual stages were seen in the lamina propria throughout the small intestine. Merozoites of different sizes were present, often in the same vacuole. They were arranged singly, in pairs, and many within a single parasitophorous vacuole. The maximum number of nuclei within developing merozoites in a group was 8, but it could not be discerned if they were individual nuclei or parts of merozoites. Findings of abundant asexual stages 1 day after dogs had started excreting oocysts indicated continued asexual multiplication beyond the prepatent period. The stages found resemble the 3 generations reported previously. The mode of division of the asexual generations remains unclear. The results of the present study indicate that there are many generations that are difficult to determine because of the multiplication of merozoites in the original host cell without leaving it to enter new host cells. From the literature, it is evident that cat and dog coccidia (Cystoisospora spp.) divide by more than 1 type of division, including endodyogeny. In the past, the schizont/meront groups containing more than 1 generation have been called "cysts." However, cyst is not an accurate term because it is best used for an orally infective stage of coccidia; monozoic tissue cysts of C. canis can occur in paratenic hosts in extraintestinal organs. We recommend the term "types" as originally proposed for intestinal stages of Toxoplasma gondii and used for the original description of the life cycle of C. suis of swine when describing endogenous stages of the Sarcocystidae. Ultrastructural studies are needed to determine the precise form of multiplication of canine Cystoisospora species. [ABSTRACT FROM AUTHOR]

Three-dimensional (3D) cell culture models bridge the gap between two-dimensional (2D) monolayer cultures and animal models. Physiologically relevant, 3D culture models have significantly advanced basic cell science and provide unique insights into host-pathogen interactions intrinsically linked to cell morphology. Toxoplasma gondii is an obligate intravacuolar parasite that chronically infects a large portion of the global human population. Our current understanding of Toxoplasma infection is largely based on 2D cell cultures, in which mammalian cells are grown on flat surfaces. However, 2D cell cultures may not recapitulate key conditions of in vivo infections as they introduce artificial pressures and tensions, which may subsequently alter infectious processes that are dependent on spatiality, e.g., invasion, replication and egress. In this study, we adapted a collagen-based 3D cell culture system to reproduce the 3D environment of T. gondii natural infections for investigation of the replication and egress of the parasite from the parasitophorous vacuole. Suspended in the 3D matrix, Toxoplasma-infected VERO cells have round morphology, as opposed to infected VERO cells in 2D monolayers. The doubling time of Toxoplasma in VERO cells within the matrix is comparable to that of parasites cultivated in VERO cell monolayers. In the absence of the pressure of flattened host cells grown in 2D cultures, the parasitophorous vacuole of T. gondii has a globular shape, with intravacuolar parasites distributed radially, forming 3D spherical ‘rosette’ structures. Parasites egress radially away from the ruptured host cell in 3D matrices, in contrast to Toxoplasma cultivated in 2D monolayer cultures, where the parasites escape perpendicularly from the flat surface below the host cells. These observations demonstrate the utility of collagen matrices for studying parasite modes of infection as these 3D assays more closely mimic in vivo conditions. [ABSTRACT FROM AUTHOR]

Neospora caninum is an intracellular protozoan parasite from the phylum Apicomplexa, mainly associated with abortions and causing enormous economic losses. We aimed, by the present study, to estimate the molecular prevalence and phylogenetic analyses of natural infection with N. caninum in Tunisian goats. A total number of 121 meat samples were collected from slaughtered goats in the regional slaughterhouse of Béja (Northwest Tunisia) and tested from N. caninum ITS1 gene using PCR followed by sequencing of PCR products. Phylogenetic analyses were used to identify this parasite infecting goats in Nortwest Tunisia. The overall molecular prevalence was 19% (23/121). The highest molecular prevalence of N. caninum was observed in goats aged between 2 and 4 years (31.9 ± 13.27%) (P = 0.004). There was no difference in the overall molecular prevalence of N. caninum according to both localities and animal breeds. Comparison of the partial sequences of the ITS1 gene revealed 99-100% similarity with GenBank sequences. A high similarity with all the blasted genotypes was reported for N. caninum sequences. This is the first molecular study and genetic characterisation of N. caninum in North African goats. [ABSTRACT FROM AUTHOR]

Tissue-cyst forming coccidia in the family Sarcocystidae are etiologic agents of protozoal encephalitis in marine mammals including the federally listed Southern sea otter (Enhydra lutris). California sea lions (Zalophus californianus), whose coastal habitat overlaps with sea otters, are definitive hosts for coccidian protozoa provisionally named Coccidia A, B and C. While Coccidia A and B have unknown clinical effects on aquatic wildlife hosts, Coccidia C is associated with severe protozoal disease in harbor seals (Phoca vitulina). In this study, we conducted surveillance for protozoal infection and fecal shedding in hospitalized and free-ranging California sea lions on the Pacific Coast and examined oocyst morphology and phenotypic characteristics of isolates via mouse bioassay and cell culture. Coccidia A and B were shed in similar frequency, particularly by yearlings. Oocysts shed by one free-ranging sea lion sampled at Año Nuevo State Park in California were previously unidentified in sea lions and were most similar to coccidia infecting Guadalupe fur seals (Arctocephalus townsendi) diagnosed with protozoal disease in Oregon (USA). Sporulated Coccidia A and B oocysts did not replicate in three strains of mice or in African green monkey kidney cells. However, cultivation experiments revealed that the inoculum of fecally-derived Coccidia A and B oocysts additionally contained organisms with genetic and antigenic similarity to Sarcocystis neurona; despite the absence of detectable free sporocysts in fecal samples by microscopic examination. In addition to the further characterization of Coccidia A and B in free-ranging and hospitalized sea lions, these results provide evidence of a new role for sea lions as putative mechanical vectors of S. neurona, or S. neurona-like species. Future work is needed to clarify the distribution, taxonomical status, and pathogenesis of these parasites in sea lions and other marine mammals that share their the near-shore marine environment.

Abstract Phylogenies within Toxoplasmatinae have been widely investigated with different molecular markers. Here, we studied molecular phylogenies of the Toxoplasmatinae subfamily based on apicoplast and mitochondrial genes. Partial sequences of apicoplast genes coding for caseinolytic protease (clpC) and beta subunit of RNA polymerase (rpoB), and mitochondrial gene coding for cytochrome B (cytB) were analyzed. Laboratory-adapted strains of the closely related parasites Sarcocystis falcatula and Sarcocystis neurona were investigated, along with Neospora caninum, Neospora hughesi, Toxoplasma gondii (strains RH, CTG and PTG), Besnoitia akodoni, Hammondia hammondiand two genetically divergent lineages of Hammondia heydorni. The molecular analysis based on organellar genes did not clearly differentiate between N. caninum and N. hughesi, but the two lineages of H. heydorni were confirmed. Slight differences between the strains of S. falcatula and S. neurona were encountered in all markers. In conclusion, congruent phylogenies were inferred from the three different genes and they might be used for screening undescribed sarcocystid parasites in order to ascertain their phylogenetic relationships with organisms of the family Sarcocystidae. The evolutionary studies based on organelar genes confirm that the genusHammondia is paraphyletic. The primers used for amplification of clpC and rpoB were able to amplify genetic sequences of organisms of the genus Sarcocystisand organisms of the subfamily Toxoplasmatinae as well.

Abstract: Two events have helped to shape protozoology/protistology as a specific scientific discipline. The first such event was the creation of the Society of Protozoologists in the U.S. in 1947 (and of its Journal of Protozoology, first published in 1954), the second event was the First International Conference on Protozoology, held in 1961 in Prague. The history of the Society of Protozoologists was comprehensively treated by Corliss (1998); the history of the Prague Conference is presented here as reminiscences and personal interpretation of events of the author, who was one of the conference organizers and a member of the organization committee. Special attention is given to the personality and scientific accomplishments of Otto Jírovec, the 1961 conference spiritual father and president. It is concluded that the Prague Conference, while establishing the tradition of protistology meetings, helped protistology to attain its present status as a fundamental science discipline, which discovers and interprets the web of life at one of its, basic, “microbial” levels. Protists literally permeate the earth biosphere and in a way represent the “dark matter” of the living world, still awaiting many discoveries. [ABSTRACT FROM AUTHOR]

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Carnivores usually act as definitive hosts of Sarcocystis species. However, the number of reports on sarcocyst formation in musculature of predators is on the increase. In the present study, muscle samples of 68 mustelids collected in Lithuania were examined for sarcocysts of Sarcocystis species. Sarcocysts were detected in diaphragm, tongue and limb muscles of ten animals (14.7%) but were not discovered in the heart. Based on 18S rDNA, 28S rDNA, cox1 and ITS1 sequence analysis, Sarcocystis lutrae was identified in three American minks (Neovison vison), two beech martens (Martes foina), three Eurasian badgers (Meles meles), one Eurasian otter (Lutra lutra) and one European polecat (Mustela putorius). The intraspecific variability of this Sarcocystis species was determined only in ITS1 region. Based on the phylogenetic analysis, no clear separation of S. lutrae by intermediate hosts or geographical locations was established. This paper represents the first identification of S. lutrae in the American mink, the beech marten and the European polecat. Current results indicate that S. lutrae is a common species in the muscles of various European mustelids. [ABSTRACT FROM AUTHOR]