Molecular Epidemiology, Species Distribution, and Zoonotic Importance of the Neglected Meat-Borne Pathogen Sarcocystis spp. in Cattle (Bos taurus): A Global Systematic Review and Meta-analysis.

BACKGROUND: Sarcocystis species are diverse apicomplexan parasites, though only two zoonotic species (S. hominis and S. heydorni) circulate between cattle and humans. Due to the importance of cattle in the human food chain and to prevent the consequences of parasitosis in humans, the first global systematic review and meta-analysis on molecular epidemiology, species distribution, and zoonotic significance of Sarcocystis infection in cattle was performed. METHODS: For this aim, four international English databases (PubMed, Scopus, Google Scholar, and Web of Science) were systematically searched till 20th September 2021, and random-effect models were drawn to calculate total estimates and their 95% confidence intervals (CIs). RESULTS: Finally, 44 papers from 21 countries were qualified for this review which examined 8526 cattle regarding Sarcocystis infection, rendering a total prevalence of 62.7% (95% CI 53-71.5%). Globally, 12 Sarcocystis spp. have been reported from cattle, including S. cruzi, S. hominis, S. hirsuta, S. rommeli, S. heydorni, S. bovifelis, S. bovini, S. sinensis, S. gigantea, S. fusiformis, S. hjorti and S. tenella. Among them, S. cruzi (37 studies), S. hominis (22 studies) and S. hirsuta (19 studies) were the 3 most common species, with 76.4% (95% CI 64.8-85%), 30.2% (95% CI 19.3-44%) and 8.7% (95% CI 3.8-18.6%), respectively. However, molecular identification was not performed in 48.4% (95% CI 27.3-70.1%) of the positive samples. CONCLUSION: Despite the zoonotic significance of Sarcocystis spp., particularly S. hominis, the epidemiology and distribution of Sarcocystis infection in cattle remains unclear and demands more extensive researches around the world.

Lesions caused by Sarcocystis spp., parasites of opossums (Didelphis aurita), in acute and chronic infections in birds (Melopsittacus undulatus).

Protozoa of the genus Sarcocystis are obligatory heterogenous parasites with both definitive and intermediate hosts. Opossums (Didelphis aurita) can shed multiple species of Sarcocystis with birds as the intermediate host. The pathologies of Sarcocystis species in birds have not been thoroughly elucidated. Therefore, the aim of the present study to determine the main lesions that can occur in acute and chronic infections in intermediate hosts, when they ingest infective sporocysts that are shed in the opossum's feces, using budgerigars as a model. To this end, 12 budgerigars, Melopsittacus undulatus, were divided into two groups that received an inoculum with 60 and 120 sporocysts. Birds that died or were euthanized were necropsied, and the lung, tongue, liver, brain, heart, and skeletal striated muscles were collected and fixed in 10% formalin for histopathological analysis. The infectivity varied according to the sample and infective dose. Acute histopathological lesions were characterized by evidence of slightly degenerated hepatocyte cords that permeated the region of the blood vessel and hepatic sinusoids. Pulmonary tissue lesions were also observed in the parabronchial region with the presence of inflammatory infiltrates associated with areas of edema and atelectasis. In chronic infections, few mature cysts were observed in the chest, and many mature cysts in the thigh and tongue muscles. Thus, it was possible to conclude that lesions are highly characteristic in acute infection and, in chronic infections, cysts were present but without major lesions. In this case, the preferred organs of parasitism were the thigh and the tongue.

Sarcocystosis in Ruminants of Iran, as Neglected Food-Borne Disease: A Systematic Review and Meta-analysis.

PURPOSE: Sarcocystis is a zoonotic parasitic pathogen which endangers the safety of meat and meat products. This systematic review and meta-analysis aimed to evaluate the prevalence rate and status of Sarcocystis spp. in ruminants as important food sources in Iran. METHODS: Data were collected from papers indexed in five English language electronic databases (PubMed, Scopus, Web of Science, Science Direct, and Google Scholar) and four Persian electronic databases (IranMedex, SID, IranDoc, and Magiran) from January to April 2019. Papers were selected based on inclusion criteria. Data analysis was performed in StatsDirect statistical software, version 2.7.2. RESULTS: The searching process resulted in the identification of 73 studies. Data analyses revealed that the total prevalence (95% confidence intervals) of Sarcocystis spp. in Iranian ruminants was 74.40% (64.01-83.56). In addition, a significant association was also observed between sarcocystosis infection in Iranian ruminants and year, host, location, and diagnostic technique (P < 0.001). CONCLUSIONS: According to our data, the prevalence of Sarcocystis infection in ruminants is relatively high. High pathogenicity of some Sarcocystis spp. and the negative impact that the spread of some parasites among ruminants can have on human and animal health necessitate the direction of more attention toward monitoring, controlling, and preventing sarcocystosis.

Sarcocystis neurona-Induced Myeloencephalitis Relapse Following Anticoccidial Treatment.

Sarcocystis neurona is a ubiquitous parasite in the eastern United States, which is the principal causative agent in the neurologic disorder equine protozoal myeloencephalitis (EPM). While much is known about this protozoa's life cycle in its natural host, the opossum (Didelphis virginiana), little is known of how it acts in the aberrant equine host, which displays a high incidence of exposure with a relatively low rate of morbidity. For this study, we employed the popular interferon gamma knockout mouse model to determine the potential for recrudescence of S. neurona infection after treatment with the anticoccidial drug diclazuril. Mice were infected with S. neurona merozoites, and 7-days post-infection (DPI) they were treated with diclazuril for 30 or 60 days or not treated at all. All infected non-treated mice developed neurologic signs consistent with S. neurona infection within 30 DPI. All diclazuril-treated infected mice remained clinically normal while on treatment but developed neurologic signs within 60 days of treatment cessation. Histological examination of cerebella from all infected mice demonstrated characteristic lesions of S. neurona infection, regardless of treatment status. Cerebellar samples collected from infected treated mice, displaying neurologic signs, produced viable S. neurona in culture. However, cerebellar samples collected from infected and neurologically normal mice at the end of a 30-day treatment period did not produce viable S. neurona in culture. Analysis of the humoral immune response in infected mice showed that during treatment IgM antibody production decreased, suggesting the organism was sequestered from immune surveillance. The cessation of treatment and subsequent development of neurologic disease resulted in increased IgM antibody production, suggesting recognition by the immune system at that time. Based on the study results the authors propose that diclazuril was able to inhibit the replication and migration of S. neurona but not fully eliminate the parasite, suggesting recrudescence of infection after treatment is possible.

High prevalence of Sarcocystis spp. infections in cattle (Bos taurus) from central China.

Myocardium and diaphragm samples of cattle (n = 521) from HeNan Province (China) were screened for Sarcocystis sarcocysts by histological examination, pepsin digestion, and molecular assays. Morphology and molecular assays were used for identification. The prevalence of Sarcocystis infection in cattle was 41.5% (216/521). Histological examination identified sarcocysts in the myocardium (49.4%, 200/405) and diaphragm (13.8%, 16/116) of cattle. Two species were identified, namely S. cruzi (41.3%, 215/521) and S. hominis (0.2%, 1/521). The findings of the present study indicate a high prevalence of S. cruzi infection in cattle from central China.

Sarcocystosis in South American camelids: The state of play revisited.

Members of the genus Sarcocystis (Apicomplexa: Sarcocystidae) are intracellular protozoan parasites that infect a wide range of domestic and wild animals, resulting in economic losses in production animals worldwide. Sarcocystis spp. have indirect life-cycles where canids and felids serve as main definitive hosts while a range of domestic and wild animals serve as intermediate hosts, including South American camelids (SACs) such as alpacas, llamas and guanacos. These animals primarily occur in South American countries on Andean, elevated plains but in recent years, alpacas and llamas have become emerging animal industries in other parts of the world such as Australia, Europe and the USA due to their high-quality fiber, meat and hides. For instance, alpaca meat is becoming popular in many parts of the world due to its lower cholesterol content than other red meat, thereby it has the potential of a valuable product for both local and international markets. However, SAC meat can be degraded and/or even condemned due to the presence of macroscopic sarcocysts in skeletal muscles, leading to significant economic losses to farmers. The infection is generally asymptomatic, though highly pathogenic or even fatal Sarcocystis infections have also been reported in alpacas and llamas. Despite the economic importance of sarcocystosis in SACs, little is known about the life-cycle of parasites involved, disease transmission, epidemiology, pathogenesis, diagnosis, control and public health significance. This review article provides an in-depth analysis of the existing knowledge on the taxonomy, epidemiology, clinicopathology and diagnosis of Sarcocystis in SACs, highlights knowledge gaps and proposes future areas of research that could contribute to our better understanding of sarcocystosis in these animals.

Sarcocystis myopathy in a patient with HIV-AIDS.

Sarcocystosis is a zoonotic infection that causes intestinal and muscular illnesses in humans. Sarcocystosis was until recently considered rare in humans. To complete their life cycle, Sarcocystis species require both a definitive and an intermediate host. Humans are the definitive host when infected by one of two species: Sarcocystis hominis (from eating undercooked beef) or Sarcocystis suihominis (from eating uncooked pork). Infection with either of these species results in intestinal sarcocystosis, causing a self-limited disease characterized by nausea, abdominal pain, and diarrhea. Humans act as the intermediate host when infected by Sarcocystis nesbitti, resulting in the markedly different clinical picture of muscular sarcocystosis. Most documented cases of muscular sarcocystosis were assumed to be acquired in Malaysia, in addition to other regions of Southeast Asia and India. Published cases of muscular sarcocystosis from the Middle East, Central and South America, and Africa are all rare. Although the clinical presentation of muscular sarcocystosis remains to be fully characterized, fever, myalgia, and headache are among the most common symptoms. Here, we report a patient from sub-Saharan Africa with chronic Sarcocystis myopathy and well-controlled HIV-AIDS.

Caracterização molecular de Sarcocystis spp. em amostras de carne / Molecular characterization of Sarcocystis spp. in samples of meat

A sarcocistose é uma doença distribuída mundialmente, podendo acometer aves, répteis e diversos mamíferos, incluindo o homem. O objetivo desse trabalho foi detectar a presença de Sarcocystis spp. e caracterizar as espécies encontradas em 375 amostras de produtos cárneos (filé mignon bovino, carne moída bovina e salame colonial). Para isso, foi realizada a detecção do parasita através da técnica de PCR para amplificação parcial do gene 18S rRNA e sua caracterização molecular utilizando o polimorfismo no comprimento do fragmento de restrição (RFLP) com as enzimas de restrição Bcl I, Rsa I e Alu I. A ocorrência de Sarcocystis spp. foi de 17% (64/375) do total de amostras testadas pelo PCR. Entre os produtos cárneos avaliados, 5,6% (7/125) das amostras de filé mignon, 12,8% (16/125) de carne moída e 32,8% (41/125) de embutido colonial, foram positivas para presença do DNA do Sarcocystis spp. Entre estas amostras positivas, as espécies caracterizadas foram Sarcocystis hirsuta e Sarcocystis hominis com prevalências de 93,7% (60/64) e 6,3% (4/64), respectivamente. Considerando à relevância da sarcocistose na área da saúde pública, a ocorrência de S. hominis encontrado neste estudo, pode ser um fator de risco para a contaminação humana. Porém, a presença do DNA deste protozoário não significa necessariamente potencial de infecção aos humanos, pois cuidados nos processos de fabricação podem reduzir a viabilidade dos cistos.(AU)

The sarcocystosis is a worldwide spread disease and can affect birds, reptiles and many mammals, including man. The aim of this study was to detect the presence of Sarcocystis spp. and characterize the species found in 375 samples of meat products (filet mignon, ground beef and colonial salami). For this, we carried out the detection of the parasite by PCR for the amplification of the partial 18S rRNA gene and molecular characterization using the restriction fragment length polymorphism (RFLP) with restriction enzymes Bcl I, Alu I and Rsa I. The occurrence of Sarcocystis spp. was 17% (64/375) of all samples. Among the meat products evaluated, the filet mignon samples were positive in 5.6% (7/125), the ground beef in 12.8% (16/125) and the colonial salami in 32.8% (41/125). Of the positive samples, Sarcocystis hirsuta and Sarcocystis hominis were detected, with prevalence of 93.7% (60/64) and 6.3% (4/64), respectively. Considering the relevance of sarcocystosis in public health, the occurrence of S. hominis found may be a risk factor to human contamination. However, the presence of DNA of this parasite does not necessarily mean potential of infection to humans, because good practices in the manufacturing processes can reduce the viability of the cysts.(AU)

Characterization of Sarcocystis fayeri's actin-depolymerizing factor as a toxin that causes diarrhea.

Raw horsemeat has the potential to induce food poisoning which often presents with diarrheal symptoms. A sample of horsemeat was found to be infected with Sarcocystis fayeri, and a 15-kDa protein isolated from the cysts of S. fayeri was found to clearly show its diarrhea-inducing activity. A nested polymerase chain reaction was used to clone the cDNA of the 15-kDa protein. The deduced amino acid sequence showed homology to actin-depolymerizing factor (ADF). A recombinant 15-kDa protein depolymerized prepolymerized actins in a test tube. The 15-kDa protein possessed conserved amino acid sequences of ADF of Toxoplasma gondii and Eimeria tenella. These characteristics indicate that the 15-kDa protein of S. fayeri belongs to the ADF/cofilin protein family. The recombinant 15-kDa protein evoked fluid accumulation in the looped ileum, resulting in diarrhea, but it did not kill the cultured fibroblast cells, macrophages or intestinal mucosal cells. In addition, the culture supernatant of the macrophages treated with the recombinant 15-kDa protein killed the fibroblast L929 cells. This fact indicates that ADF of S. fayeri induced cytotoxic substances, such as tumor necrosis factor-α, according to the published reports. Although further experiments are needed now to elucidate the enterotoxic mechanism of S. fayeri's ADF, our findings may offer new insight into research on parasites and parasite-instigated food poisoning.

Meningoencephalitis caused by pathogenic Sarcocystis species in a naturally infected sheep in Turkey.

A 3-year-old sheep was examined after an acute onset of hind limb paralysis and ataxia. At necropsy, central nervous system, pulmonary and intestinal hyperaemia and ecchymoses in the aortic arch were observed. Main microscopic lesions were confined to the heart, cerebrum and cerebellum. There were a multifocal mild myocarditis and nonsuppurative meningoencephalitis together with protozoal cysts in the heart and the brain. Protozoal cystic structures were observed within many of the myocardial fibers as well as in the cerebrum and cerebellum. Using light microscopy it could not be morphologically determined whether these organisms were Toxoplasma (T.) gondii or Neospora (N.) caninum. Additional diagnostic methods like immunohistochemistry and polymerase chain reaction provided differentiation of Sarcocystis from T. gondii and N. caninum. Transmission electron microscopy demonstrated characteristic features of Sarcocystis sp. as previously described. This is the first confirmed diagnosis of Sarcocystis sp. in the central nervous system of a sheep from Turkey.

Environmental Persistence Influences Infection Dynamics for a Butterfly Pathogen.

Many pathogens, including those infecting insects, are transmitted via dormant stages shed into the environment, where they must persist until encountering a susceptible host. Understanding how abiotic conditions influence environmental persistence and how these factors influence pathogen spread are crucial for predicting patterns of infection risk. Here, we explored the consequences of environmental transmission for infection dynamics of a debilitating protozoan parasite (Ophryocystis elektroscirrha) that infects monarch butterflies (Danaus plexippus). We first conducted an experiment to observe the persistence of protozoan spores exposed to natural conditions. Experimental results showed that, contrary to our expectations, pathogen doses maintained high infectivity even after 16 days in the environment, although pathogens did yield infections with lower parasite loads after environmental exposure. Because pathogen longevity exceeded the time span of our experiment, we developed a mechanistic model to better explore environmental persistence for this host-pathogen system. Model analysis showed that, in general, longer spore persistence led to higher infection prevalence and slightly smaller monarch population sizes. The model indicated that typical parasite doses shed onto milkweed plants must remain viable for a minimum of 3 weeks for prevalence to increase during the summer-breeding season, and for 11 weeks or longer to match levels of infection commonly reported from the wild, assuming moderate values for parasite shedding rate. Our findings showed that transmission stages of this butterfly pathogen are long-lived and indicated that this is a necessary condition for the protozoan to persist in local monarch populations. This study provides a modeling framework for future work examining the dynamics of an ecologically important pathogen in an iconic insect.

Detection and Molecular Identification of Sarcocystis rileyi (Apicomplexa: Sarcocystidae) From a Northern Shoveler (Anas clypeata) in Mexico.

We detected macroscopic Sarcocystis cysts in a Northern Shoveler ( Anas clypeata ) in the Lerma Marshes, State of Mexico, Mexico in February 2014. The 5.0×2.0-mm macrocysts in the breast muscle of the duck were ovoid and yellow. Using an optical microscope, we saw parasitic forms of a Sarcocystis sp. among muscular fibers; the cysts measured 3.5×1.1 mm. The external wall had a smooth surface and the internal wall had a spongy texture. We identified the macrocysts as Sarcocystis rileyi according to sequences of the 18S rRNA gene, 28S rRNA gene, and ITS-1 region. Sarcocystosis should be considered in similar assessments in wild waterfowl in Mexico. Awareness of S. rileyi among anatids in the Lerma Marshes will contribute to more-effective conservation and management actions.

Intra-uterine exposure of horses to Sarcocystis spp. antigens / Exposição intrauterina ao Sarcocystis spp. em equinos

The aim of this study was to examine the intra-uterine exposure to Sarcocystis spp. antigens, determining the number of foals with detectable concentrations of antibodies against these agents in the serum, before colostrum ingestion and collect data about exposure of horses to the parasite. Serum samples were collected from 195 thoroughbred mares and their newborns in two farms from southern Brazil. Parasite specific antibody responses to Sarcocystis antigens were detected using the indirect immunofluorescent antibody test (IFAT) and immunoblot analysis. In 84.1% (159/189) of the pregnant mares and in 7.4% (14/189) of foals we detected antibodies anti-Sarcocystis spp. by IFAT. All samples seropositive from foals were also positive in their respective mares. Serum samples of seropositive foals by IFAT, showed no reactivity on the immunoblot, having as antigens S. neurona merozoites. In conclusion, the intra-uterine exposure to Sarcocystis spp. antigens in horses was demonstrated, with occurrence not only in mares, but also in their foals, before colostrum ingestion these occurrences were reduced.

O objetivo deste estudo foi avaliar a exposição intrauterina ao Sarcocystis spp., para determinar o número de potros que possuem concentrações detectáveis de anticorpos contra esses agentes no soro, antes da ingestão do colostro, por meio da coleta de dados sobre a exposição a esses protozoários nos equinos. Amostras de soro foram coletadas de 195 éguas puro-sangue e seus respectivos potros recém-nascidos, em duas fazendas localizadas na região Sul do Brasil. Os testes utilizados na detecção de anticorpos específicos para o Sarcocystis foram a reação de imunofluorescência indireta (RIFI) e análise por meio de immunoblot. Pela RIFI, em 84,1% (159/189) das éguas e em 7,4% (14/189) dos potros foram detectados anticorpos anti-Sarcocystis spp. Todas as amostras soropositivas dos potros também foram positivas para suas respectivas mães. As amostras de soro dos potros soropositivos na RIFI, não apresentaram reatividade no immunoblot, tendo como antígenos merozoítos de S. neurona. Em conclusão, foi demonstrada a exposição intrauterina de Sarcocystis spp. em equinos, com ocorrência em éguas, porém, em seus respectivos potros, antes da ingestão de colostro a ocorrência foi reduzida.

Acute, fatal Sarcocystis calchasi-associated hepatitis in Roller pigeons (Columba livia f. dom.) at Philadelphia Zoo.

Four Roller pigeons (Columba livia f. dom.) at the Philadelphia Zoo died suddenly. Necropsy examination revealed macroscopic hepatitis. Microscopically, the predominant lesions were in liver, characterized with necrosis and mixed cell inflammatory response. Sarcocystis calchasi-like schizonts and free merozoites were identified in liver. Transmission electron microscopy confirmed that schizonts were in hepatocytes. A few schizonts were in spleen. PCR using S. calchasi-specific primers confirmed the diagnosis. Neither lesions nor protozoa were found in brain and muscles. This is the first report of acute visceral S. calchasi-associated sarcocystosis in naturally infected avian hosts.

A review of Sarcocystis spp. shed by opossums (Didelphis spp.) in Brazil / Revisão sobre Sarcocystis spp. excretados por gambás (Didelphis spp.) no Brasil

South American opossums are the definitive hosts of Sarcocystis neurona, Sarcocystis falcatula, Sarcocystis speeri and Sarcocystis lindsayi. The sporocysts of these species of Sarcocystis are morphologically similar and methods like infectivity and pathogenicity for intermediate hosts (immunodeficient mice and psittacine birds) and molecular tools are used for identification. Opossums are synanthropic wild animals, and widely distributed in Brazilian territory. Previous studies have shown high environmental contamination with S. neurona sporocysts in several Brazilian regions. This paper reviews information on Sarcocystis spp. shed by various opossum species and its occurrence in Brazil.(AU)

Os gambás Sul-americanos são os hospedeiros definitivos de Sarcocystis falcatula, Sarcocystis neurona, Sarcocystis speeri e Sarcocystis lindsayi. Estas espécies de Sarcocystis são morfologicamente similares, mas podem ser distinguidas por sua patogenicidade e infectividade em hospedeiros intermediários (aves e camundongos imunodeficientes) e técnicas moleculares. Os gambás são animais silvestres e sinantrópicos e amplamente distribuídos no território nacional. Estudos anteriores demonstraram uma alta contaminação ambiental com esporocistos de S. neurona em diversas regiões brasileiras. Este artigo revisa informações sobre Sarcocystis spp. excretados por gambás e sua ocorrência no Brasil.(AU)

Prevalence and Risk Factors of Intestinal Parasites in Cats from China.

The prevalence of intestinal parasites in cats from China was largely unknown prior to this study. The aim of the present study was to investigate the presence of intestinal parasites in cats from central China and also identify risk factors for parasitism. Fecal samples from 360 cats were examined using sugar flotation procedure and fecal smear test by microscope. Cats had mixed two or three kinds of parasites infections. Of the 360 cats feces, intestinal parasites positive feces were 149 (41.39%). 64 (17.78%) were infected with Toxocara cati, 61 (16.94%) with Isospora felis, 41 (11.39%) with Isospora rivolta, 33 (9.17%) with Paragonimus, 23 (6.39%) with hookworms, 11 (3.06%) with Toxoplasma-like oocysts, 10 (2.78%) with Trichuris, 4 (1.11%) with lungworm, 2 (0.56%) with Sarcocystis, and 1 (0.28%) with Trematode. The cats' living outdoor was identified as risk factor by statistical analysis. These results provide relevant basic data for assessing the infection of intestinal parasites in cats from central region of China. In conclusion, there was high prevalence of intestinal parasites in cats from China.

Selection of Reference Genes for RT-qPCR Analysis in the Monarch Butterfly, Danaus plexippus (L.), a Migrating Bio-Indicator.

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a powerful technique to quantify gene expression. To facilitate gene expression study and obtain accurate results, normalization relative to stably expressed reference genes is crucial. The monarch butterfly, Danaus plexippus (L.), is one of the most recognized insect species for its spectacular annual migration across North America. Besides its great voyages, D. plexippus has drawn attention to its role as a bio-indicator, ranging from genetically modified organisms (GMOs) to natural ecosystems. In this study, nine reference genes from D. plexippus genome were selected as the candidate reference genes. The expression profiles of these candidates under various biotic and abiotic conditions were evaluated using the four readily available computational programs, BestKeeper, Normfinder, geNorm, and ΔCt method, respectively. Moreover, RefFinder, a web-based computational platform integrating the four above mentioned algorisms, provided a comprehensive ranking of the stability of these reference genes. As a result, a suite of reference genes were recommended for each experimental condition. Specifically, elongation factor 1α (EF1A) and ribosomal protein 49 (RP49) were the most stable reference genes, respectively, under biotic (development, tissue, and sex) and abiotic (photoperiod, temperature, and dietary RNAi) conditions. With the recent release of a 273-million base pair draft genome, results from this study allow us to establish a standardized RT-qPCR analysis and lay a foundation for the subsequent genomic and functional genomic research in D. plexippus, a major bio-indicator and an emerging model for migratory animals.

[Control of toxicity of Sarcocystis fayeri in horsemeat by freezing treatment and prevention of food poisoning caused by raw consumption of horsemeat].

More than 27 outbreaks per year of food poisoning caused by consuming horse meat were reported in Kumamoto Prefecture (including Kumamoto City) from January 2009 to September 2011. It was found that the causative agent of the outbreaks was a protein with a molecular weight of 15 kDa that had originated from bradyzoites of Sarcocystis fayeri parasitizing the horse meat. Rabit ileal loop tests showed that pepsin treatment of homogenates of frozen horse meat containing the cysts of S. fayeri induced loss of toxicity, presumably by digestion of the proteinous causative agent(s). Slices of horse meat containing the cysts were frozen at below -20°C for various periods. The cysts were collected after thawing the slices, then treated in an artificial stomach juice containing pepsin. The bradyzoites of the cysts kept at -20°C for 48 hr or more completely disappeared. Simultaneously, the 15 kDa protein also disappeared in the frozen cysts. After notifying the public and recommending freezing treatment of horse meat, no subsequent cases of food poisoning were reported. This indicates that freezing of horse meat is effective to prevent the occurrence of food poisoning caused by consuming raw horse meat containing S. fayeri.

PAN-modular structure of microneme protein SML-2 from the parasite Sarcocystis muris at 1.95 Å resolution and its complex with 1-thio-ß-D-galactose.

The microneme protein SML-2 is a member of a small family of galactose-specific lectins that play a role during host-cell invasion by the apicomplexan parasite Sarcocystis muris. The structures of apo SML-2 and the 1-thio-ß-D-galactose-SML-2 complex were determined at 1.95 and 2.1 Å resolution, respectively, by sulfur-SAD phasing. Highly elongated dimers are formed by PAN-domain tandems in the protomer, bearing the galactose-binding cavities at the distal apple-like domains. The detailed structure of the binding site in SML-2 explains the high specificity of galactose-endgroup binding and the broader specificity of the related Toxoplasma gondii protein TgMIC4 towards galactose and glucose. A large buried surface of highly hydrophobic character and 24 intersubunit hydrogen bonds stabilize the dimers and half of the 12 disulfides per dimer are shielded from the solvent by the polypeptide chain, thereby enhancing the resistance of the parasite protein towards unfolding and proteolysis that allows it to survive within the intestinal tracts of the intermediate and final hosts.

Polyparasitism is associated with increased disease severity in Toxoplasma gondii-infected marine sentinel species.

In 1995, one of the largest outbreaks of human toxoplasmosis occurred in the Pacific Northwest region of North America. Genetic typing identified a novel Toxoplasma gondii strain linked to the outbreak, in which a wide spectrum of human disease was observed. For this globally-distributed, water-borne zoonosis, strain type is one variable influencing disease, but the inability of strain type to consistently explain variations in disease severity suggests that parasite genotype alone does not determine the outcome of infection. We investigated polyparasitism (infection with multiple parasite species) as a modulator of disease severity by examining the association of concomitant infection of T. gondii and the related parasite Sarcocystis neurona with protozoal disease in wild marine mammals from the Pacific Northwest. These hosts ostensibly serve as sentinels for the detection of terrestrial parasites implicated in water-borne epidemics of humans and wildlife in this endemic region. Marine mammals (151 stranded and 10 healthy individuals) sampled over 6 years were assessed for protozoal infection using multi-locus PCR-DNA sequencing directly from host tissues. Genetic analyses uncovered a high prevalence and diversity of protozoa, with 147/161 (91%) of our sampled population infected. From 2004 to 2009, the relative frequency of S. neurona infections increased dramatically, surpassing that of T. gondii. The majority of T. gondii infections were by genotypes bearing Type I lineage alleles, though strain genotype was not associated with disease severity. Significantly, polyparasitism with S. neurona and T. gondii was common (42%) and was associated with higher mortality and more severe protozoal encephalitis. Our finding of widespread polyparasitism among marine mammals indicates pervasive contamination of waterways by zoonotic agents. Furthermore, the significant association of concomitant infection with mortality and protozoal encephalitis identifies polyparasitism as an important factor contributing to disease severity in marine mammals.