Lack of Detection of Toxoplasma gondii in Pipistrellus spp. Bats from Densely Cat-Populated Areas of NE Spain.

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.

Apicomplexans in small mammals from Chile, with the first report of the Babesia microti group in South American rodents.

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.

SEROSURVEY FOR SELECTED PARASITIC AND BACTERIAL PATHOGENS IN DARWIN'S FOX (LYCALOPEX FULVIPES): NOT ONLY DOG DISEASES ARE A THREAT.

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.

Detection of Eumonospora henryae (Apicomplexa: Sarcocystidae) from Falco columbarius (Falconiformes: Aves): Comparison of host-parasite phylogram and comments on the family Sarcocystidae Poche, 1913.

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.

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.

Disseminated protozoal infection in a wild feathertail glider (Acrobates pygmaeus) in Australia.

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.

Resurrection of the genus Eumonospora (Apicomplexa: Sarcocystidae) for Caryospora species without Stieda body.

The coccidian genus Eumonospora Allen, 1933 is re-established. Despite morphological features and host preference among species, coccidian with octasporozoic and monosporocystic oocysts are traditionally consider to belonging in the genus Caryospora Léger, 1904 (Apicomplexa: Eimeriidae). Recently, the genus Avispora Schuster et al., 2016 was proposed for above caryosporoids parasitizing birds based on combined morphological and phylogenetic analyses. However, diagnostic morphological characters of the genus Avispora, the absence of Stieda and substieda bodies, has already been mentioned in the description of the genus Eumonospora Allen, 1933 (Apicomplexa: Sarcocystidae), and thus Avispora is considered to be a junior synonym of Eumonospora. In this study, caryosporoid coccidians were detected from five owl species; Bubo scandiacus, Ptilopsis leucotis, Athene noctua, Strix nebulosa, and Pulsatrix perspicillata (Strigiformes: Strigidae) and identified as Avispora henryae (Yakimoff & Matikaschwaili, 1932) described from Bubo bubo (Strigiformes: Strigidae). Eumonospora henryae (Yakimoff & Matikaschwili, 1932) comb. nov. is redescribed for this species based not only on morphological features but also on phylogenetical analyses. The key of the genus Eumonospora and a list to the species known at present are also provided.

Endemic, exotic and novel apicomplexan parasites detected during a national study of ticks from companion animals in Australia.

BACKGROUND: Apicomplexan tick-borne pathogens that cause disease in companion animals include species of Babesia Starcovici, 1893, Cytauxzoon Neitz & Thomas, 1948, Hepatozoon Miller, 1908 and Theileria Bettencourt, Franca & Borges, 1907. The only apicomplexan tick-borne disease of companion animals that is known to occur in Australia is babesiosis, caused by Babesia canis vogeli Reichenow, 1937 and Babesia gibsoni Patton, 1910. However, no molecular investigations have widely investigated members of Apicomplexa Levine, 1980 in Australian ticks that parasitise dogs, cats or horses, until this present investigation. RESULTS: Ticks (n = 711) removed from dogs (n = 498), cats (n = 139) and horses (n = 74) throughout Australia were screened for piroplasms and Hepatozoon spp. using conventional PCR and Sanger sequencing. The tick-borne pathogen B. vogeli was identified in two Rhipicephalus sanguineus Latreille ticks from dogs residing in the Northern Territory and Queensland (QLD). Theileria orientalis Yakimov & Sudachenkov, 1931 genotype Ikeda was detected in three Haemaphysalis longicornis Neumann ticks from dogs in New South Wales. Unexpectedly, the exotic tick-borne pathogen Hepatozoon canis James, 1905 was identified in an Ixodes holocyclus Neumann tick from a dog in QLD. Eight novel piroplasm and Hepatozoon species were identified and described in native ticks and named as follows: Babesia lohae n. sp., Babesia mackerrasorum n. sp., Hepatozoon banethi n. sp., Hepatozoon ewingi n. sp., Theileria apogeana n. sp., Theileria palmeri n. sp., Theileria paparinii n. sp. and Theileria worthingtonorum n. sp. Additionally, a novel cf. Sarcocystidae sp. sequence was obtained from Ixodes tasmani Neumann but could not be confidently identified at the genus level. CONCLUSIONS: Novel species of parasites in ticks represent an unknown threat to the health of companion animals that are bitten by these native tick species. The vector potential of Australian ticks for the newly discovered apicomplexans needs to be assessed, and further clinical and molecular investigations of these parasites, particularly in blood samples from dogs, cats and horses, is required to determine their potential for pathogenicity.

RETROSPECTIVE SURVEY FOR PATHOGENS IN STRANDED MARINE MAMMALS IN NORTHEASTERN BRAZIL: BRUCELLA SPP. INFECTION IN A CLYMENE DOLPHIN ( STENELLA CLYMENE).

: We surveyed 13 carcasses of marine mammals (12 Trichechus manatus and one Stenella clymene) that had stranded in northeastern Brazil during 1990-2013 for infectious diseases by screening tissues from the collection of the Brazilian National Center of Research and Conservation of Aquatic Mammal, Chico Mendes Institute for Biodiversity Conservation. Brucella spp. and Mycobacterium spp. were investigated by culturing and PCR of tissue samples, whereas Sarcocystidae parasites, Leptospira spp., and Morbillivirus were surveyed for using specific PCR assays. Brucella spp. and Mycobacterium spp. were not isolated through microbiologic culturing, and all animals were negative for detection of Sarcocystidae parasites, Leptospira spp., Mycobacterium spp., and Morbillivirus by PCR assays. All manatees were negative for Brucella spp. infection, but Brucella ceti was detected in the brain tissue of an S. clymene calf by using a PCR assay.

Detecção molecular de Neospora caninum em macaco-da-noite (Aotus azarae) de vida livre no estado do Mato Grosso: relato de caso / Molecular detection of Neospora caninum in azara's night monkey (Aotus azarae) of wildlife in the state of Mato Grosso: Case report

Estudos indicam, por meio de infecção experimental, que primatas não humanos são susceptíveis à infecção por Neospora caninum. Relata-se um caso de um macaco-da-noite (Aotus azarae infulatus), que apresentou sinais inespecíficos e não respondeu à terapêutica clínica de suporte, evoluindo a óbito, encaminhado em seguida para exame anatomopatológico. Amostras de tecidos foram coletadas e processadas rotineiramente para confecção de lâminas histológicas. Microscopicamente, a principal lesão foi observada no coração e consistia em miocardite necrótica multifocal por protozoário, com a presença de estruturas compatíveis com o estágio de taquizoítos de protozoários dos gêneros Neospora sp. ou Toxoplasma sp. No sistema nervoso central, predominantemente no tronco encefálico, havia estruturas semelhantes às descritas no coração. Os resultados da reação em cadeia pela polimerase (PCR) foram positivos para N. caninum e negativos para Toxoplasma gondii, usando DNA extraído do sangue e dos tecidos. Este relato de caso fornece evidências histológicas e moleculares de que o primata em questão foi susceptível a uma infecção natural, porém estudos devem ser realizados para investigar o real papel dos primatas no ciclo de vida de N. caninum.(AU)

Studies indicate through experimental infection that non-human primates are susceptible to infection by Neospora caninum. This report is of a case of a night monkey (Aotus azarae infulatus) that presented nonspecific signs and did not respond to supportive clinical therapy evolving to death, followed by a pathology examination. Tissue specimens were routinely collected and processed for the preparation of histological slides. Microscopically, the main lesion was observed in the heart and consisted of multifocal necrotic myocarditis by protozoa, with the presence of structures compatible with the stage of protozoan tachyzoites of the genus Neospora sp. or Toxoplasma sp. In the central nervous system, predominantly in the brainstem there were structures similar to those described in the heart. Polymerase chain reaction (PCR) results were positive for N. caninum and was negative for Toxoplasma gondii using DNA extracted from blood and tissues. This case report provides histological and molecular evidence that the primate in question was susceptible to a natural infection, but studies should be conducted to investigate the real role of primates in the life cycle of N. caninum.(AU)

Antibodies against Neospora caninum, Sarcocystis spp. and Toxoplasma gondii detected in buffaloes from Rio Grande do Sul, Brazil / Anticorpos contra Neospora caninum, Sarcocystis spp. e Toxoplasma gondii detectados em búfalos no Rio Grande do Sul

The presence of antibodies against Neospora caninum, Sarcocystis spp. and Toxoplasma gondii was evaluated in buffaloes (Bubalus bubalis) from Rio Grande do Sul state (RS), southern Brazil. Serum samples (n=220) were analyzed for antibodies by indirect fluorescent antibody test (IFAT). Antibody presence was considered when the titers were equal or higher than 100 for these protozoa. A total of 60.5% (133/220) buffalo serum samples were positive for at least one of the protozoa evaluated in this study. Antibodies for N. caninum, Sarcocystis spp. and T. gondii were found in 36.4% (80/220), 25.5% (56/220) and 16.8% (37/220) of the buffaloes respectively, indicating a higher frequency of N. caninum infection (p=0.0133). The IFAT is a suitable method to diagnose N. caninum, Sarcocystis spp. and T. gondii infection in buffaloes for detecting IgG antibodies. This study demonstrates the presence of these three protozoa in buffalo herds in RS, Brazil, which may be source of infection to other animals. The high frequency of animals positive for N. caninum is important and could be related to reproductive problems. Additionally, the presence of Sarcocystis spp. and T. gondii in buffaloes can be a possible public health issue.(AU)

A presença de anticorpos contra Neospora caninum, Sarcocystis spp. e Toxoplasma gondii foi avaliada em búfalos (Bubalus bubalis) no estado do Rio Grande do Sul (RS), Região Sul do Brasil. Amostras de soro de 220 bubalinos foi analisada para presença de anticorpos, através de reação de imunofluorescência indireta (RIFI). Foram consideradas positivas as amostras que apresentaram títulos de anticorpos maiores ou iguais a 100, para os protozoários estudados. Um total de 60,5% (133/220) das amostras sorológicas dos búfalos foram positivas para pelo menos um dos parasitas pesquisados. Anticorpos para N. caninum, Sarcocystis spp. e T. gondii foram encontrados em 36,4% (80/220); 25,5% (56/220) e 16,8% (37/220) dos búfalos respectivamente, indicando que houve uma maior frequência de infecção de N. caninum em relação aos demais protozoários (p=0.0133). A RIFI é um método adequado para o diagnóstico sorológico da infecção por N. caninum, Sarcocystis spp. e T. gondii em búfalos. Este estudo demonstrou a presença destes três protozoários em bubalinos no RS, Brasil, que pode ser fonte de infecção para outros animais. A elevada ocorrência de animais positivos para N. caninum é importante e pode estar relacionada a problemas reprodutivos. Adicionalmente, a presença de Sarcocystis spp. e T. gondii em búfalos, pode significar um possível problema de saúde pública.(AU)

Seroepidemiology of Sarcocystis neurona, Toxoplasma gondii and Neospora spp. among horses in the south of the state of Minas Gerais, Brazil / Soroepidemiologia de Sarcocystis neurona, Toxoplasma gondii e Neospora spp. em Equinos no Sul do Estado de Minas Gerais, Brasil

Abstract The present study used the indirect fluorescent antibody test (IFAT) to determine the seroprevalence of Sarcocystis neurona, Toxoplasma gondii and Neospora spp., and evaluated the variables associated with these infections among 506 apparently healthy horses, reared in the south of the state of Minas Gerais, Brazil. This study was conducted between April 2012 and October 2013. Among the horses, the true prevalence of S. neurona was 26% (95% CI: 22.0-30.4%), T. gondii 19.9% (95% CI: 15.5-24.8%) and Neospora spp. 23.9% (95% CI: 19.9-28.1%); and among the farms, 88.3% (95% CI: 74.4-91.6%), 71.6% (95% CI: 41-92.8%) and 85% (95% CI: 70.7-96.1%), respectively. Regarding mixed infection, 17 horses (3.4%) were seropositive for both S. neurona and T. gondii, 16 (3.2%) for T. gondii and Neospora spp. and 14 (2.8%) for S. neurona and Neospora spp. The associations between seropositivity and variables relating to the structure of the farm, management and health were analyzed using the logistic regression analysis, through the generalized estimating equations (GEE). The results suggest that the south of Minas Gerais is an enzootic area for S. neurona, T. gondii and Neospora spp. among horses, with prevalence of asymptomatic subclinical or chronic infections.

Resumo Este estudo determinou, pela reação de imunofluorescência indireta (RIFI), a soroprevalência de Sarcocystis neurona, Toxoplasma gondii e Neospora spp., e avaliou as variáveis associadas com a infecção, em 506 equinos, aparentemente sadios, criados no Sul de Minas, Brasil. O estudo foi realizado no período de abril de 2012 a outubro de 2013. Entre equinos, a prevalência verdadeira para S. neurona foi de 26% (IC 95%= 22,0-30,4%); para T. gondii de 19,9% (IC 95%= 15,5-24,8%); e para Neospora spp. de 23,9% (IC 95%= 19,9-28,1%); e entre propriedades, 88,3% (IC 95%= 74,4-91,6%), 71,6% (IC 95% = 41-92,8%), e 85% (IC 95%= 70,7-96,1%), respectivamente. Em relação à infecção mista, 17 (3,4%) equinos foram soropositivos para S. neurona e T. gondii, 16 (3,2%) para T. gondii e Neospora spp., e 14 (2,8%) para S. neurona e Neospora spp. A associação entre soropositividade e variáveis relacionadas à estrutura da propriedade, manejo e sanidade, foi analisada, utilizando-se a análise de regressão logística estimada por Generalized Estimating Equations (GEE). Os resultados sugerem que o Sul de Minas é área enzoótica para S. neurona, T. gondii e Neospora spp. em equinos, com predomínio de infecções subclínicas ou crônicas, assintomáticas.

Detection and characterization of diverse coccidian protozoa shed by California sea lions.

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.

Molecular phylogeny of Toxoplasmatinae: comparison between inferences based on mitochondrial and apicoplast genetic sequences / Filogenia molecular de Toxoplasmatinae: comparação entre inferências baseadas em sequências genéticas mitocondriais e de apicoplasto

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.

Resumo A filogenia da subfamília Toxoplasmatinae tem sido amplamente investigada com diversos marcadores moleculares. Neste estudo, a filogenia molecular da subfamília Toxoplasmatinae foi analisada através de genes de apicoplasto e mitocondriais. Foram analisadas sequências parciais de genes de apicoplasto codificadores da protease caseinolítica (clpC), e da subunidade beta da RNA polimerase (rpoB) e de gene mitocondrial codificador de citocromo B (cytB). Foram investigadas cepas adaptadas em laboratório de Sarcocystis neurona eSarcocystis falcatula, parasitos estreitamente relacionados, além de Neospora caninum, Neospora hughesi, Toxoplasma gondii (cepas RH, CTG e PTG),Besnoitia akodoni, Hammondia hammondi e duas linhagens geneticamente divergentes de Hammondia heydorni. A análise molecular, baseada em genes de organelas, não diferenciou claramenteN. caninum de N. hughesi, porém foi possível confirmar as duas linhagens de H. heydorni. Foram encontradas pequenas diferenças entre as cepas adaptadas em laboratório deS. falcatula e S. neurona em todos os marcadores moleculares avaliados. Concluindo, filogenias congruentes foram reconstruídas com os três diferentes genes que podem ser úteis em triagem de parasitos sarcocistídeos não identificados, para identificar sua relação com organismos da família Sarcocystidae. Os estudos evolutivos com genes organelares confirmam que o gênero Hammondia é parafilético. Osprimers utilizados para amplificação declpC e rpoB foram capazes de amplificar sequências genéticas de organismos do gênero Sarcocystis e da subfamília Toxoplasmatinae.

Diversity of Sarcocystis spp shed by opossums in Brazil inferred with phylogenetic analysis of DNA coding ITS1, cytochrome B, and surface antigens.

Although few species of Sarcocystis are known to use marsupials of the genus Didelphis as definitive host, an extensive diversity of alleles of surface antigen genes (sag2, sag3, and sag4) has been described in samples of didelphid opossums in Brazil. In this work, we studied 25 samples of Sarcocystis derived from gastrointestinal tract of opossums of the genus Didelphis by accessing the variability of sag2, sag3, sag4, gene encoding cytochrome b (cytB) and first internal transcribed spacer (ITS1). Reference samples of Sarcocystis neurona (SN138) and Sarcocystis falcatula (SF1) maintained in cell culture were also analyzed. We found four allele variants of cytB, seven allele variants of ITS1, 10 allele variants of sag2, 13 allele variants of sag3, and 6 allele variants of sag4. None of the sporocyst-derived sequences obtained from Brazilian opossums revealed 100% identity to SN138 at cytB gene, nor to SN138 or SF1 at ITS1 locus. In addition, none of the sag alleles were found identical to either SF1 or SN138 homologous sequences, and a high number of new sag allele types were found other than those previously described in Brazil. Out of ten sag2 alleles, four are novel, while eight out of 13 sag3 alleles are novel and one out of six sag4 alleles is novel. Further studies are needed to clarify if such a vast repertoire of allele variants of Sarcocystis is the consequence of re-assortments driven by sexual exchange, in order to form individuals with highly diverse characteristics, such as pathogenicity, host spectrum, among others or if it only represents allele variants of different species with different biological traits.