Detection of Sarcocystis hominis, Sarcocystis bovifelis, Sarcocystis cruzi, Sarcocystis hirsuta and Sarcocystis sigmoideus sp. nov. in carcasses affected by bovine eosinophilic myositis.

Bovine eosinophilic myositis is an inflammatory myopathy characterized by multiple focal or diffuse grey to green patches leading to condemnation of affected carcasses. Although its etiology is still uncertain, there is evidence that Sarcocystis species may play a role in the development of eosinophilic myositis. The goal of the present study was to identify Sarcocystis spp. in intralesional and extralesional tissues of condemned cattle carcasses, in order to evaluate the possible role of different bovine Sarcocystis spp. in the etiology of bovine eosinophilic myositis. Muscle samples (n = 100) of 26 affected carcasses were collected in Northern Italy. One to five samples with lesions and two aliquots of tissue without lesions were collected from each carcass; lesions were grossly categorized in green focal lesions and green diffuse patches. Genomic DNA was extracted and analyzed by multiplex-PCR targeting different Sarcocystis spp. Unidentified species were characterized morphologically (light microscopy, histology), ultrastructurally (scanning and transmission electron microscopy) and on the molecular level (complete 18S rRNA gene and partial cox1 gene sequencing). A bovine eosinophilic myositis prevalence of 0.017% was visually assessed by routine carcass inspection between 2014 and 2019 in Italy (184/1,108,150 slaughtered cattle). Out of 26 carcasses, 25 revealed the presence of at least one Sarcocystis species (96.2%). The presence of Sarcocystis spp. DNA was significantly more frequent in intralesional than in extralesional samples. Considering the different species, Sarcocystis bovifelis and Sarcocystis hominis were significantly more frequent in intralesional (41.7% and 50%, respectively) than in extralesional samples (1.9% and 15.4%, respectively), while there was no significant difference between the presence of Sarcocystis cruzi and Sarcocystis hirsuta in intralesional (27.1% and 2.1%, respectively) and extralesional (30.8% and 1.9%, respectively) samples. The presence of an unnamed Sarcocystis sp. showing thick-walled (3.7-5.4 µm) cysts with densely packed, flattened, undulating and narrow protrusions, which showed an S-shape in side view, was recorded in the diaphragm of two carcasses. Genomic DNA from individual sarcocysts isolated from the diaphragm was successfully amplified and further sequenced. Sequence comparison revealed <94.6% and 83.4% identity at 18S rRNA and cox1 genes, respectively, with other named Sarcocystis spp., while the phylogenetic analysis clearly separated the unnamed Sarcocystis sp. from the other Sarcocystis spp. using cattle as intermediate hosts. The present study contributes to the understanding of the importance of different Sarcocystis spp. in the pathogenesis of bovine eosinophilic myositis. The results emphasize the association of Sarcocystis hominis and Sarcocystis bovifelis with bovine eosinophilic myositis and highlight the presence of a new Sarcocystis sp. using cattle as intermediate hosts. The name Sarcocystis sigmoideus sp. nov. is proposed for the newly described Sarcocystis species.

Zoonotic nematode in the city of La Plata, Argentina: Report of a case of Calodium hepaticum in Rattus rattus.

This study describes a case of Calodium hepaticum (Trichinellida: Capillariidae) infection in an adult rat (Rattus rattus) from the periurban area of the city of La Plata in the province of Buenos Aires, Argentina. The rat was found with neurological signs (ataxia, lethargy, and episodes of unresponsiveness) in the food storage of a goat production facility. The liver was observed with hepatomegaly and diffuse and irregular yellowish-white spots appearing in striae or small nodules on the external surface and inside the liver. Subsequent microscopic and histopathological studies were performed. Eggs were observed by direct microscopy of the impression smear of liver tissue. A multifocal granulomatous tissue reaction with different stages of fibrocellular tissue was observed in the liver parenchyma. The granulomas contained adults and degenerated eggs delimited by an intense infiltrate of mononuclear cells. Macro and microscopic observations and histopathological liver lesions were compatible with C. hepaticum infection. To our knowledge, this is the first confirmation of C. hepaticum infection in R. rattus in Argentina, increasing the host record of this parasite and a new record of distribution in goat production systems in the country.

Morphological and molecular characterization of Sarcocystis spp. in pigs (Sus scrofa domestica) from Argentina.

Sarcocystis spp. are intracellular protozoan parasites with an obligatory heteroxenous life cycle. The objective of this study was to identify Sarcocystis spp. in pig muscles from Argentina, by light and transmission electron microscopy (TEM), and molecular studies. Muscles samples from 561 pigs (Sus scrofa domestica) were classified according to the breeding system in: intensive farming (IF, n = 295; animals kept in confinement during most of their productive cycle), or semi-extensive farming (SEF, n = 266; animals bred outdoors, generally family or backyard production). Results showed that 24.8% (139/561) were positive by light microscopy, with a significantly higher prevalence in the SEF (34.6%; 92/266) than the IF pigs (15.9%; 47/295) (p < 0.05). Of the 202 samples analyzed by PCR, 96 were positive (47.5%) for the 18S rRNA (18S ribosomal RNA) fragment. All samples analyzed by the S. suihominis specific coxI (mitochondrial cytochrome c oxidase subunit I) PCR (n = 235; 96 positives by 18S rRNA PCR and 139 positives by light microscopy) were negative. Fourteen individual cysts were positive for the 18S rRNA PCR and sequenced. Consensus sequences obtained from the 18S rRNA fragment PCR ranged from 613 to 880 bp and showed 100% of identity between them and with previously reported S. miescheriana sequences. In all the pig samples analyzed by TEM, cyst wall ultrastructure was compatible with S. miescheriana. This is the first study that provides infection rates and describes and identifies morphological and molecular features of Sarcocystis spp. cysts in pigs from Argentina.

Angiostrongylus dujardini infection in a coconut lorikeet (Trichoglossus haematodus) from a zoological garden in Switzerland.

Angiostrongylus spp. (Metastrongyloidea) can cause severe disease in several animal species and humans. This report describes an infection with Angiostrongylus dujardini in a captive coconut lorikeet (Trichoglossus haematodus) from a zoo in Switzerland. The bird was reported being attacked by conspecifics, removed from the flock, and hospitalized. It showed lethargy, moderately reduced body condition, and lack of reaction to visual stimuli. Analgesic and antibiotic treatment were initiated but because of worsening of its general condition, the bird was euthanized the following day. Necropsy revealed multifocal, subcutaneous hemorrhages, diffusely reddened lungs and a moderately dilated right heart with several intraluminal nematodes embedded in a coagulum. Four worms were collected and microscopically examined. They were identified as adult females, measuring 19-21 mm long x 0.4-0.5 mm wide, with general morphological and morphometric characteristics consistent with angiostrongylid nematodes. In lung sections, multifocal collection of thin-walled embryonated eggs in variable stages of development was observed along with fully developed nematode larvae within the lumina of alveoli and lung vessels. Associated granulomatous infiltrates indicated a severe, multifocal, chronic, granulomatous pneumonia. The diagnosis of A. dujardini infection was formulated by morphological examination of adult and larval stages, supported by molecular analysis (PCR-amplification and sequencing of the ITS2, 5.8S and 28S rDNA flanking regions). This is the first report of A. dujardini infection in an avian species, providing evidence that birds can serve as accidental hosts of this parasite in addition to mammals, and that the parasite can reach maturity and multiply in the avian cardiorespiratory system.

What is the role of Swiss domestic cats in environmental contamination with Echinococcus multilocularis eggs?

BACKGROUND: The role of the domestic cat as definitive host for Echinococcus multilocularis and thus in environmental contamination with eggs has not yet been entirely resolved. This study aimed to assess the prevalence of E. multilocularis and other gastrointestinal parasites in Swiss domestic cats and to compare the diagnostic sensitivity of different methods for the detection of intestinal taeniid infection. METHODS: Faecal samples from 146 cats were included in the study. Faecal samples only were available from 55 cats; for the other 91 cats, necropsy was performed in addition to faecal sample testing. All (n = 146) faecal samples were analysed by a combined sedimentation/flotation technique (44% ZnCl2) and by the sodium acetate-acetic acid-formalin (SAF) sedimentation technique; when sufficient material was available (n = 121 samples) the Baermann-Wetzel technique was also used. Additionally, all samples were analysed by two coproantigen (copro)-quantitative PCRs (qPCR): (i) a multiplex qPCR able to detect and differentiate between E. multilocularis, Echinococcus granulosus sensu lato and Taenia spp./other cestodes (CEST-qPCR) and (ii) an E. multilocularis-specific qPCR (EM-qPCR). Finally, the intestines were examined macroscopically and microscopically for parasite stages at necropsy (n = 91) and using an intestinal scraping technique (IST) (n = 64). RESULTS: Of the 146 cats examined, 24 (17.1%) were infected by intestinal parasites, namely Hydatigera (syn. Taenia) taeniaeformis (8.9%), Toxocara cati (6.1%), Capillaria sp. (3.4%), hookworms (3.4%), Mesocestoides litteratus (1.4%), Giardia sp. (1.4%), Cystoisospora rivolta (1.4%), Cystoisospora felis (0.7%), Toxoplasma gondii (0.7%), Hammondia hammondi (0.7%) and Strongyloides sp. (0.7%). Necropsy and the IST revealed adult H. taeniaeformis in 12 animals, of which eight faecal samples were positive by the CEST-qPCR (sensitivity = 67%) and six samples by the sedimentation/flotation technique (sensitivity = 50%). No E. multilocularis infection was detected in the sampled cats. Using Bayesian latent class analysis, the mean posterior prevalence probability was 0.0% (95% confidence interval 0-0.83%) for E. multilocularis. CONCLUSIONS: There was no evidence of E. multilocularis infection among the 146 cats examined, suggesting that the prevalence of this parasite is low (< 1%) in the Swiss domestic cat population. Nonetheless, some of the sampled cats were infected by parasites that have rodents as intermediate hosts, demonstrating successful predation by these cats, and some were infected with zoonotic parasites. Cats therefore should not be disregarded as potential hosts for E. multilocularis and other zoonotic parasites.

First report of Sarcocystis spp. (Apicomplexa, Sarcocystidae) in Lagostomus maximus (Desmarest, 1917) (Rodentia, Chinchillidae) in Argentina.

Sarcocystis is a genus of intracellular parasitic protozoa that infects various species of mammals, birds, and reptiles worldwide. At least 46 Sarcocystis species naturally infect rodents as intermediate hosts producing tissue cysts. This study aimed to provide the first report and molecular characterisation of Sarcocystis spp. in muscles from plains viscacha (Lagostomus maximus) in Argentina. Muscle samples of 53 plains viscachas from three provinces of Argentina were processed by homogenisation and optical microscopy to detect tissue cysts. Positive samples were analysed by PCR-sequencing, using the following markers: 18S rRNA, ITS1, and coxI. The 18S rRNA and coxI consensus sequences were aligned with other sequences from Sarcocystis spp., and phylogenetic trees were constructed. Of all animals processed, 13.2% (7/53) harboured Sarcocystis sp. cysts. 18S rRNA consensus sequences were obtained from four muscle samples and one individual cyst, and they showed 99.88-100% similarity, except for the cyst sequence, which showed 97.11% homology. Similarities of only 96-97% were recorded in the 18S rRNA fragment with other Sarcocystis spp. whose sequences are available in the GenBank. The five coxI fragment sequences obtained were 100% identical and showed an identity of 99.41-99.48% with S. canis. For ITS1 only short and low-quality sequences were obtained. In the phylogenetic trees, all the sequences from plains viscachas were positioned together in a branch separated from other Sarcocystis spp. These results could be related to new Sarcocystis spp. producing sarcocysts in plains viscachas. Besides, comprehensive cyst morphological analysis using TEM from the new Sarcocystis species will allow a description of the cyst wall ultrastructure. In this sense, further studies are needed to deepen these findings and elucidate other potential intermediate and possible definitive hosts.

Identification of a new Sarcocystis sp. in marsh deer (Blastocerus dichotomus) from wetlands of Argentina.

The marsh deer (Blastocerus dichotomus) is the largest South American native deer species and is listed as "Vulnerable" by IUCN due to the population reduction. As part of a conservation and disease surveillance program, muscle samples from 14 marsh deer found dead in 2016 and 2017 in northeast Argentina were obtained at necropsy. Samples from each animal were processed as pooled muscles (heart, diaphragm, tongue and hindlimb) by homogenization and direct microscopical observation to detect intracellular Sarcocystis spp. cysts. Sarcocysts were observed in six samples, and several cysts recovered from two samples were processed by transmission electron microscopy. The cysts were thin-walled and showed a cyst-wall ultrastructure with ribbon-like protrusions similar to other species using cervids as intermediate host and canids as definitive hosts. Genomic DNA from individual sarcocysts from three marsh deer were successfully amplified by PCR of 18S rRNA and COI gene fragments and further sequenced. Sequence comparison revealed a 99.3-100% identity among them and only 93.7-96.6% and 88.8-89.7% identity at 18S rRNA and COI markers, respectively, with other Sarcocystis spp. Despite morphological similarities, the high sequence divergence at 18S rRNA and COI fragments allowed the assumption that Sarcocystis sp. from marsh deer is a different species from others using cervids as intermediate hosts. Therefore, we propose the name Sarcocystis blastoceris n. sp. for the species infecting marsh deer.

Identification of Sarcocystis spp. in wild boars (Sus scrofa) from Argentina.

Sarcocystis spp. are intracellular protozoan parasites with an obligatory heteroxenous life cycle. The objective of this study is to identify Sarcocystis spp. in wild boar muscles from Argentina by light and transmission electron microscopy and molecular characterization. Muscle samples from diaphragm, tongue, masseter, intercostals, heart, and forelimbs of 240 wild boars were analyzed. Of the animals, 48.3% (116/240) were positive for sarcocysts by light microscopy, whereas 45.8% (110/240) were positive for Sarcocystis spp. by PCR targeting 18S rRNA fragment. These samples were subjected to a specific PCR for S. suihominis coxI gene, 3.6% (4/110) of which were weak positives. Unfortunately, sequence analysis was inconclusive. This could be related to a potentially low S. suihominis cyst load in the samples, or to an incomplete primer matching with the South American S. suihominis sequences. Seventeen individual sarcocysts were positive by PCR for the 18S rRNA fragment, whose sequences showed 99.75-100% identity with each other and with previously reported S. miescheriana sequences. A total of 21 cysts collected from 11 muscle samples and analyzed by TEM presented a cyst wall type compatible with S. miescheriana, and one cyst presented an ultrastructure compatible with S. suihominis. The latter came from a sample that also contained S. miescheriana cysts, indicating that the animal was co-infected. This is the first study that provides infection rates and describes and identifies morphological and molecular features of Sarcocystis spp. cysts in wild boars from South America.

Association of bovine viral diarrhea virus, bovine herpesvirus 1, and Neospora caninum with late embryonic losses in highly supplemented grazing dairy cows.

The objectives of this study were: 1- to evaluate the association of Bovine Viral Diarrhea Virus (BVDV), Bovine Herpes Virus 1 (BoHV-1), and Neospora caninum (N. caninum) with the risk for Late Embryonic Loss (LEL) in grazing dairy cows, 2- to evaluate blood progesterone concentration at the time of LEL occurrence, and 3- to describe a novel ultrasound-guided technique for conceptus sampling. We run a prospective cohort study involving 92 cows (46 LEL and 46 NLEL). An LEL cow was that having an embryo with no heartbeat, detached membranes, or floating structures, including embryo remnants detected at pregnancy check by ultrasonography (US) 28-42 days post-AI, whereas an NLEL cow was that with embryo heartbeats detectable by US at pregnancy check 28-42 d post-IA. We took two blood samples from every cow at pregnancy check by US (the day of LEL detection) and 28 d later to perform serological diagnosis of BVDV, BoHV-1, and N. caninum; and to measure blood progesterone concentration at pregnancy check (28-42 d post-AI). We also sampled the conceptus from all the LEL cows. We performed PCR to detect BVDV, BoHV-1, and N. caninum in sampled conceptuses from LEL cows. Finally, we evaluated the associations of risk factors (serological titers, seroconversion, and progesterone) with LEL odds with logistic models. The risk for LEL was associated with serological titers to BVDV (P = 0.03) and tended to be associated with seroconversion to BVDV, given that 19.6% (9/46) in LEL and 6.5% (3/46) in NLEL cows seroconverted to BVDV (P = 0.09). In addition, BVDV was detected in conceptuses from LEL cows that seroconverted to BVDV but not in LEL cows that did not seroconvert. Conversely, the risk for LEL was not associated with the titers or seroconversion to BoHV-1 and N. caninum. BoHV-1 and N. caninum were not identified in any of the conceptuses. Finally, blood progesterone concentration was similar in LEL and NLEL cows, and it was not associated with the risk for LEL (P = 0.54). In conclusion, BVDV infection is a risk factor for LEL in dairy cows.

Pampas fox (Lycalopex gymnocercus) of the Argentine Pampas as intermediate host for Neospora caninum.

The Pampas fox (Lycalopex gymnocercus) is the most abundant wild canid from South America. This wild canid inhabits grasslands, open woodlands, and areas highly modified by extensive ranching and agricultural activities. We aimed to evaluate Neospora caninum infection in tissues from the Pampas fox from Argentina. A total of 41 free-living Pampas foxes were sampled in rural areas located in the Humid Pampas region, Argentina. Brain tissue and different muscles were assessed by histologic and molecular methods. No N. caninum cysts were observed in brain and muscle tissue samples analyzed by histology and immunohistochemistry. Molecular N. caninum identification from brain tissue was based on amplification by PCR of Nc-5 gene and ITS1 rRNA fragments and subsequent sequencing. The presence of N. caninum DNA was 74% (23/31) for the Nc-5 gene and was confirmed by a second ITS1 PCR in 55% (17/31) of the brain tested. Thirteen ITS1 consensus sequences were obtained, and all have a 99.58-100% similarity with N. caninum reference sequences. Only 4% (1/23) of muscles samples analyzed were positive for the Nc-5 gene of N. caninum. This study demonstrated a high prevalence of N. caninum DNA in brain from free-ranging Pampas fox of the Pampa Argentine, thus confirming that this wild canid is a wide distributed intermediate host.

Sarcocystis neurona and related Sarcocystis spp. shed by opossums (Didelphis spp.) in South America.

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.

Sarcocystis neurona and related Sarcocystis spp. shed by opossums (Didelphis spp.) in South America / Sarcocystis neurona e Sarcocystis spp. relacionados, excretados por gambás (Didelphis spp.) na América do Sul

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.

Resumo Parasitos protozoários do gênero Sarcocystis são coccídios heteroxenos formadores de cistos, que infectam variadas espécies animais e compreendem cerca de 200 espécies descritas. Pelo menos quatro Sarcocystis spp. (S. falcatula, S. neurona, S. lindsayi e S. speeri) utilizam gambás (Didelphis spp.) como hospedeiros definitivos; e duas delas, S. neurona and S. falcatula são conhecidas por causarem doença em equinos e aves, respectivamente. Gambás didelfídeos são restritos ao continente americano, contudo são distribuídos de forma heterogênea nas Américas. Cinco Didelphis spp. são distribuídos na América do Sul (D. aurita, D. albiventris, D. marsupialis, D. imperfecta e D. pernigra), enquanto somente uma espécie (D. virginiana) é encontrada na América do Norte. Trabalhos conduzidos, nas últimas décadas, mostram que Sarcocystis spp. derivados de Didelphis spp. sul-americanos possuem diferenças biológicas e genéticas, quando comparados a Sarcocystis spp. excretados pelo gambá norte-americano D. virginiana. O objetivo desta revisão é discutir a situação peculiar das espécies de Sarcocystis na América do Sul com um foco especial em diagnóstico, epidemiologia e infecções animais, assim como nas características genéticas desses parasitos.

Sarcocystis spp. infection in South American deer huemul (Hippocamelus bisulcus) and pudu (Pudu puda) from Patagonian National Parks, Argentina.

Sarcocystis spp. are intracellular protozoan parasites with heteroxenous life cycles. This study described Sarcocystis spp. infection in adult South American native deer huemul (Hippocamelus bisulcus) and pudu (Pudu puda). Heart, diaphragm, tongue, and skeletal muscle samples were collected from 5 huemuls and 2 pudus, found dead in National Parks. Direct microscopic examination, transmission electron microscopy, PCR, and sequencing were performed. Sarcocystis spp. microscopic thin-walled cysts were identified in 3 huemuls and 1 pudu. Several cysts from 1 huemul and 1 pudu were observed by TEM; ultrastructure was similar to previously reported as cyst wall type 17 and types 2 and 8, respectively. Fragments of the 18S rRNA and cytochrome c oxidase subunit I (cox1) genes were amplified and sequenced from 3 individual cysts from 2 huemuls and 2 cysts from the pudu. The sequences from huemuls showed a high identity among them (> 99%) at both amplified targets. The highest identities were > 99.7% at 18S rRNA and 93% at cox1 with S. tarandivulpes sequences. The 18S rRNA gene sequences from pudus showed an identity > 99.7% with Sarcocystis sp., S. taeniata, and S. linearis sequences, while the cox1 sequences were different, one showing 99.42% identity with S. venatoria and the other 98.22% with S. linearis. A single species, similar to S. tarandivulpes, was identified in all huemul samples while 2 molecularly different Sarcocystis spp. were found in 1 pudu with high similarities to either S. venatoria or to S. linearis, S. taeniata-like, and S. morae. Based on the cox1 sequence identities, at least the Sarcocystis sp. in huemuls might represent a new species, primarily occurring in this host. Additional sarcocyst isolates from both hosts need to be examined molecularly in order to firmly establish whether these species are indeed native to huemuls and/or pudus or are derived from introduced deer species.

Evaluation of biological behavior of Toxoplasma gondii atypical isolates # 14 and # 163.

Toxoplasma gondii is an obligate intracellular protozoan parasite capable of infecting warm-blooded animals, including humans. A highly diverse genetic population has been reported in Central and South America, predominating mainly atypical genotypes. Different genotypes showed different biological behavior in mice. The aim of this study was to evaluate the biological behavior of T. gondii isolates obtained from Macropus rufogriseus (TgMr) and Saimiri boliviensis (TgSb) identified as atypical genotypes # 14 and # 163, respectively. Strains RH, ME49 and VEG were used as reference for clonal types I, II and III, respectively. In vitro invasion and replication capacity assays were analyzed at 6 and 18 hpi, respectively. In vivo assay was performed in Swiss mice (n = 30) using 1 × 102 and 1 × 103 parasites/mouse as infective doses (ME49, VEG, TgMr, TgSb and negative control). Morbi-mortality and tissues PCR were assessed. Lymphoproliferation assays were performed and gamma interferon was measured by ELISA. The ME49 strain showed the highest invasion, followed by TgSb and VEG, while RH and TgMr presented the lowest invasions. The RH strain and the TgSb isolate showed more endodyogeny events (fastest doubling times) than VEG and ME49 strains and the TgMr isolate. Both atypical isolates showed high virulence (100% morbi-mortality, at 8-10 dpi) and parasite DNA was detected in all tissue samples. Splenocytes from mice inoculated with TgMr and TgSb registered the highest values of gamma interferon. An in vitro invasion-replication index was established which correlates inversely with virulence in mice. In conclusion, T. gondii atypical isolates # 14 and # 163 showed a different in vitro behavior than clonal strains, with low invasion-replication indexes but being highly virulent in mouse model.

Fatal Sarcocystis cruzi-induced eosinophilic myocarditis in a heifer in Uruguay.

Sarcocystis spp. are causative agents of bovine eosinophilic myositis and/or myocarditis, which are chronic subclinical myopathies that are occasionally responsible for condemnation at slaughterhouses. Sarcocystis cruzi is a protozoan parasite of worldwide distribution transmitted by canids, most commonly associated with subclinical infection in cattle. Although S. cruzi infections can rarely lead to fatal systemic disease, fatal cardiac cases with confirmation of the etiologic diagnosis have not been reported, to our knowledge. We describe herein an unusual case of S. cruzi-induced fatal bovine eosinophilic myocarditis. A 22-mo-old, Holstein-Hereford heifer, in a group of 110 cattle on pasture, manifested growth retardation and died in February 2017. Autopsy revealed myriad yellow-green 1-3-mm coalescing foci, surrounded by fibrosis, affecting ~75% of the ventricular myocardium. Pulmonary edema, ascites, and hydrothorax were consistent with chronic congestive heart failure. Histology revealed severe eosinophilic, granulomatous, necrotizing myocarditis, with multinucleate giant cells, fibrosis, and mineralization. Numerous thin-walled protozoan cysts resembling Sarcocystis spp. were present in the necrotic foci and within the sarcoplasm of adjacent cardiomyocytes. PCR and sequencing of the 18S rRNA gene revealed 99.9-100% homology with S. cruzi. Sarcocystosis can be a rare cause of fatal myocarditis in cattle.

Low prevalence of infection by Sarcocystis neurona in horses from the State of Alagoas, Brazil / Baixa prevalência da infecção por Sarcocystis neurona em cavalos do estado de Alagoas, Brasil

Abstract The aim of this study was to determine the prevalence of infection by Sarcocystis neurona in horses and identify potential risk factors. Were analyzed 427 samples from 36 farms in 21 municipalities in the Alagoas State, Brazil. Presence of anti-S. neurona antibodies was diagnosed by indirect immunofluorescence antibody test (IFAT) and was confirmed using the immunoblot test. Risk factors were assessed through investigative questionnaires on animal management on the farms. The prevalence of anti-S.neurona antibodies was 2.8% (confidence interval, CI: 1.5-4.9%) from IFAT and 1.6% (CI:0.8-3.34%) from immunoblot, and there were positive horses on 16.6% of the studied farms. None of the variables studied presented associations with serological status for S. neurona. This is the first report on infection by S. neurona in horses reared in Alagoas, Brazil showing a low exposure to S. neurona in this region, but with significant numbers of foci.

Resumo Objetivou-se neste estudo determinar a prevalência e os fatores de risco associados à infecção por Sarcocystis neurona em equinos. Foram analisadas 427 amostras de 36 propriedades localizadas em 21 municípios do estado de Alagoas. O diagnóstico de anticorpos anti-S. neurona foi realizado pela técnica de Imunofluorescência Indireta (IFI) e confirmada por immunoblot. O estudo dos fatores de risco foi realizado a partir de questionários investigativos sobre o manejo dos animais nas propriedades. A prevalência de anticorpos anti-S. neurona foi de 2,8% (I.C. 1,5-4,9%) na IFI e de 1,6% (I.C. 0,8-3,34%) no immunoblot com equinos positivos em 16,6% das propriedades estudadas. Nenhuma variável estudada apresentou associação com o status sorológico para S. neurona. Este é o primeiro relato da infecção por S. neurona em equinos criados no Estado de Alagoas, Brasil, confirmando que os animais desta região têm baixa exposição a S. neurona, mas com significativo número de focos.

Toxoplasma gondii and Neospora caninum infections in synanthropic rodents from Argentina / Infecções por Toxoplasma gondii e Neospora caninum em roedores sinantrópicos da Argentina

Abstract Toxoplasma gondii and Neospora caninum are closely related coccidian parasites (phylum Apicomplexa). This is the first study from urban synanthropic rodent species that involved serological and molecular diagnosis of T. gondii and N. caninum infection, and genotyping of T. gondii in Argentina. A total of 127 rodent samples were trap captured: Mus musculus (n = 78), Rattus norvegicus (n = 26) and Rattus rattus (n = 23). Antibodies against T. gondii and N. caninum were detected by IFAT in 32.8% (40/122) and 0.8% (1/122) of rodent samples, respectively, demonstrating contact with these protozoans. Additionally, T. gondii DNA was detected in 3.3% (4/123) of rodent central nervous system samples and 2 samples were genotyped by multilocus nPCR-RFLP. Neospora caninum DNA was not detected by PCR. The 2 genotyped samples were type III allele for all markers except for SAG-1 (type I for Rat1Arg and type II/III for Rat2Arg) and were identified as #48 and #2 (likely) according to the allele combinations reported on Toxo DB (Toxo-DB). The results of the present study revealed a wide distribution of T. gondii and less for N. caninum, in synanthropic rats and mice in the studied area.

Resumo Toxoplasma gondii e Neospora caninum são parasitas coccídeos intimamente relacionados (filo Apicomplexa). Este é o primeiro estudo de espécies de roedores sinantrópicos urbanos, o qual envolveu diagnósticos sorológicos e moleculares da infecção por T. gondii e N. caninum e genotipagem de T. gondii na Argentina. Um total de 127 amostras de roedores foram obtidas: Mus musculus (n = 78), Rattus norvegicus (n = 26) e Rattus rattus (n = 23). Anticorpos contra T. gondii e N. caninum foram detectados pela IFAT em 32,8% (40/122) e 0,8% (1/122) das amostras de roedores, respectivamente, demonstrando contato com esses protozoários. Adicionalmente, o DNA de T. gondii foi detectado em 3,3% (4/123) das amostras do sistema nervoso central de roedores e duas amostras foram genotipadas por nPCR-RFLP multilocus. O DNA de N. caninum não foi detectado por PCR. As 2 amostras genotipadas eram do tipo III para todos os marcadores, exceto para SAG-1 (tipo I para Rat1Arg e tipo II / III para Rat2Arg) e foram identificadas como # 48 e # 2 (provavelmente) de acordo com as combinações de alelos relatadas no Toxo DB (Toxo-DB). Os resultados do presente estudo indicam uma ampla distribuição de T. gondii e menor para N. caninum , em ratos e camundongos sinantrópicos na área estudada.

Toxoplasmosis congénita: Diagnóstico serológico, RPC, aislamiento y caracterización molecular de Toxoplasma gondii / Congenital toxoplasmosis: Serology, PCR, parasite isolation and molecular characterization of Toxoplasma gondii

Resumen Introducción El diagnóstico de toxoplasmosis congénita (TC) en el recién nacido es muy importante porque debe recibir tratamiento siempre, sintomático o no, para evitar o aminorar las secuelas de la enfermedad. Objetivo Evaluación comparativa de los métodos disponibles en la institución para el diagnóstico de TC. Materiales y Métodos Se evaluaron métodos diagnósticos en 67 niños cuyas madres cursaron toxoplasmosis aguda durante el embarazo. Se utilizó la técnica de Sabin Feldman para IgG al nacimiento y durante el seguimiento serológico hasta el año de vida. Para determinar IgM, IgA e IgE se utilizó la técnica immunosorbent agglutination assay (ISAGA). El diagnóstico directo se realizó por reacción de polimerasa en cadena (RPC), aislamiento y caracterización molecular del parásito. Resultados La sensibilidad (S) de ISAGA IgM fue 87%, ISAGA IgA 91% y la especificidad (E) fue 100% para ambas; cuando se realizaron en conjunto, la S aumentó a 98%. La detección de IgE contribuyó al diagnóstico cuando se la detectó sólo en la sangre del neonato y no en sangre materna. Se aisló el parásito en cuatro casos de TC, uno fue genotipo II y los otros tres, genotipos "atípicos". La S del aislamiento fue 80% y la E 100%. Conclusión Los métodos serológicos utilizados mostraron una buena eficacia diagnóstica. Un caso fue detectado sólo por el aislamiento y la caracterización molecular tiene gran valor epidemiológico.

Background. Congenital toxoplasmosis diagnosis in the newborn is a very important issue due to the need for early treatment to prevent future sequels. Aim To compare available methods at the institution for the diagnosis of congenital toxoplasmosis. Material and Methods In this study we have evaluated the different diagnostic tests used in 67 congenital exposed newborns, including serological tests, PCR, parasite isolation and molecular characterization. Results The ISAGA IgM and IgA tests showed sensitivity (Se) of 87 and 91%, respectively, and specificity (Sp) of 100%. When ISAGA IgM and IgA were performed simultaneously, the Se increased to 98% and the Sp was 100%. The presence of IgE contributed to the diagnosis when it was detected in the child's serum but not in maternal blood. In four congenital infected children the parasite was isolated and genotyped: one was genotype II and the other three were "atypical" genotypes. No parasite was isolated in children without congenital toxoplasmosis. Discussion Overall, serological tests showed a good diagnostic performance although in one case they were all negative and isolation was the only tool to identify the infection. We conclude that it is essential to use all diagnostic tests in every single exposed child, including if possible, molecular characterization due to its epidemiological implication.

Molecular characterization of Sarcocystis spp. in intestine mucosal scrapings and fecal samples of Pampas fox (Lycalopex gymnocercus).

Sarcocystis spp. are obligatory intracellular protozoan parasites which can infect humans and animals. Most of Sarcocystis species were identified based on the detection of muscle cysts in different intermediate hosts (IH). Regarding to natural infection in definitive host, there are few reports which have reached to determining species of Sarcocystis. The present work was aimed to studying the occurrence of Sarcocystis spp. (oocysts and sporocysts) in mucosal scrapings of small intestine and fecal samples of one the most abundant wild canids from South America, Lycalopex gymnocercus (Pampas fox), and to identify the Sarcocystis spp. using molecular tools. A total of 131 free-living L. gymnocercus were sampled in rural areas located in several departments from Buenos Aires province, Argentina. Fecal samples from all the animals and 33 small intestines were analyzed. Fecal and mucosal scrapings samples were analyzed by sugar flotation method and once oocysts or sporocysts were detected, sedimentation was performed and DNA extracted with a commercial kit. A PCR was conducted using primers targeting a fragment of the 18S rRNA gene and the amplicons were purified and sequenced. Of the total Pampas foxes analyzed, 23 (17.6%) had Sarcocystis spp. oocysts/sporocysts in fecal and/or mucosal samples. Sarcocystis spp. sporocysts were detected in 13.0% (17/131) of fecal samples and in 39.4% (13/33) of mucosal samples by the initial sugar flotation. Twenty one L. gymnocercus samples were processed by DNA extraction and PCR. Molecular identification of Sarcocystis spp. infection was successfully achieved in 14 foxes and was distributed as follows: 4.6% S. cruzi (6/131), 3.8% Sarcocystis spp. using birds as IH (S. albifronsi and S. anasi among others, 5/131), 0.8% S. tenella (1/131) and 1.5% (2/131) with low homology (97%) with S. miescheriana. In one fecal sample with spherical oocysts, the sequencing results showed a 100% sequence identity with Hammondia heydorni. The results show that the mucosal scrapings are the eligible sample to identify prevalence and to proceed with species identification. Lycalopex gymnocercus is suggested as definitive host for S. cruzi, S. tenella and probably various Sarcocystis spp. using birds as intermediate hosts as well as for H. heydorni.

Pampas fox (Lycalopex gymnocercus) new intermediate host of Sarcocystis svanai (Apicomplexa: Sarcocystidae).

Several Sarcocystis spp. have carnivores as definitive host and sarcocysts are common in muscles of herbivores (intermediate host). However, sarcocysts have been found in muscles of wild and domestic carnivores suggesting they are intermediate host for some Sarcocystis spp. Here, we report mature sarcocysts in the muscles of Pampas fox (Lycalopex gymnocercus). A total of 36 free-living foxes were analyzed. Different skeletal muscles were assessed by microscopic and molecular methods. Cysts and/or DNA of Sarcocystis sp. were detected in 61.1% (22/36) foxes. Histopathology revealed the presence of sarcocysts in 52.8% (19/36) foxes. The tongue and masseter were the muscles more frequently infected. Of all the samples processed by homogenization of pooled muscles of each animal, 45.4% (10/22) evidenced muscle cysts and 68.2% (15/22) resulted positives by PCR. Individual cysts obtained from the ten positive samples in direct microscopic examination were all positive by PCR. Five amplicons from individual cysts from different samples were selected for sequencing together with four PCR products obtained from the pooled muscles. All nine sequences shared a high identity among them (99.8-100%) and showed the highest identity by BLAST (99%) with a S. svanai sequence (KM362428) from a North American dog. By transmission electron microscopy, the sarcocyst wall was thin (<1µm), had minute undulations, with tiny evaginations and without evident villar protrusions. The cyst wall type is referred as "type 1". Sarcocystis svanai infects L. gymnocercus with a high prevalence and the presence of mature sarcocysts suggests the role of the Pampas fox as natural intermediate host. The definitive host of S. svanai remains unknown.