Low prevalence of viable Toxoplasma gondii in fresh, unfrozen American pasture-raised pork and lamb from retail meat stores in the United States.

In a national survey of fresh, unfrozen, American pasture-raised lamb and pork, the prevalence of viable Toxoplasma gondii was determined in 1500 samples selected by random multistage sampling (750 pork, 750 lamb) obtained from 250 retail meat stores from 10 major geographic areas in the USA. Each sample consisted of a minimum of 500g of meat purchased from the retail meat case. To detect viable T. gondii, 50g meat samples of each of 1500 samples were bioassayed in mice. Viable T. gondii was isolated from 2 of 750 lamb samples (unweighted: 0.19%, 0.00-0.46%; weighted: 0.04%, 0.00-0.11%) and 1 of 750 pork samples (unweighted: 0.12%, 0.00-0.37%; weighted: 0.18%, 0.00-0.53%) samples. Overall, the prevalence of viable T. gondii in these retail meats was very low. Nevertheless, consumers, especially pregnant women, should be aware that they can acquire T. gondii infection from ingestion of undercooked meat. Cooking meat to an internal temperature of 66°C kills T. gondii.

The aromatic amino acid hydroxylase genes AAH1 and AAH2 in Toxoplasma gondii contribute to transmission in the cat.

The Toxoplasma gondii genome contains two aromatic amino acid hydroxylase genes, AAH1 and AAH2 encode proteins that produce L-DOPA, which can serve as a precursor of catecholamine neurotransmitters. It has been suggested that this pathway elevates host dopamine levels thus making infected rodents less fearful of their definitive Felidae hosts. However, L-DOPA is also a structural precursor of melanins, secondary quinones, and dityrosine protein crosslinks, which are produced by many species. For example, dityrosine crosslinks are abundant in the oocyst walls of Eimeria and T. gondii, although their structural role has not been demonstrated, Here, we investigated the biology of AAH knockout parasites in the sexual reproductive cycle within cats. We found that ablation of the AAH genes resulted in reduced infection in the cat, lower oocyst yields, and decreased rates of sporulation. Our findings suggest that the AAH genes play a predominant role during infection in the gut of the definitive feline host.

Sarcocystis cymruensis: discovery in Western Hemisphere in the Brown rat (Rattus norvegicus) from Grenada, West Indies: redescription, molecular characterization, and transmission to IFN-γ gene knockout mice via sporocysts from experimentally infected domestic cat (Felis catus).

Rodents are intermediate hosts for many species of Sarcocystis. Little is known of Sarcocystis cymruensis that uses the Brown rat (Rattus norvegicus) as intermediate hosts and the domestic cat (Felis catus) as experimental definitive host. Here, we identified and described Sarcocystis cymruensis in naturally infected R. norvegicus from Grenada, West Indies. Rats (n = 167) were trapped in various locations in two parishes (St. George and St. David). Microscopic, thin (< 1 µm) walled, slender sarcocysts were found in 11 of 156 (7.0%) rats skeletal muscles by squash examination. A laboratory-raised cat fed naturally infected rat tissues excreted sporocysts that were infectious for interferon gamma gene knockout (KO) mice, but not to Swiss Webster outbred albino mice. All inoculated mice remained asymptomatic, and microscopic S. cymruensis-like sarcocysts were found in the muscles of KO mice euthanized on day 70, 116, and 189 post inoculation (p.i.). Sarcocysts from infected KO mice were infective for cats at day 116 but not at 70 days p.i. By transmission electron microscopy, the sarcocyst wall was "type 1a." Detailed morphological description of the cyst wall, metrocytes, and bradyzoites is given for the first time. Additionally, molecular data on S. cymruensis are presented also for the first time. Molecular characterization of sarcocysts 18S rDNA and 28S rDNA, ITS-1, and cox1 loci showed the highest similarity with S. rodentifelis and S. muris. In conclusion, the present study described the natural infection of S. cymruensis in Brown rat for the first time in a Caribbean country and provided its molecular characteristics.

Prevalence and genetic characterization of Toxoplasma gondii in free-range chickens from grocery stores and farms in Maryland, Ohio and Massachusetts, USA.

Chickens are considered important in the epidemiology of Toxoplasma gondii. Chicken hearts (n = 1185) obtained from grocery stores were tested for T. gondii infection. Antibodies to T. gondii were assayed in fluid removed from the heart cavity using the modified agglutination test (MAT) at 1:5, 1:25, and 1:100 dilutions. MAT antibodies were detected in 222 hearts at 1:5 dilution and 8 hearts at 1:25 dilution, but none were positive at 1:100 dilution. Seropositive (n = 230, 19.4%) chicken hearts were bioassayed in mice and seronegative (n = 157) chickens were bioassayed in cats. Viable T. gondii was not isolated from any hearts by bioassays in mice. The 2 cats fed 60 and 97 hearts did not excrete T. gondii oocysts. The results indicate a low prevalence of viable T. gondii in chickens from grocery stores. Molecular typing of 23 archived T. gondii strains isolated from free-range chickens from Ohio and Massachusetts using the 10 PCR-RFLP markers including SAG1, SAG2 (5'-3'SAG2 and altSAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed that seven were ToxoDB PCR-RFLP genotype #1, 11 were genotype #2, one was genotype #3, three were genotype #170, and one was mixed genotype. These results indicate that the clonal genotypes #1 (type II), #2 (type III), and #3 (type II variant) are common in free-range chickens.

Sarcocystis rommeli, n. sp. (Apicomplexa: Sarcocystidae) from Cattle (Bos taurus) and its Differentiation from Sarcocystis hominis.

Cattle (Bos taurus) are intermediate hosts for three named species of Sarcocystis, S. cruzi, S. hirsuta, and S. hominis. Recently, a fourth species was identified and named S. sinensis. However, S. sinensis originally named a species of Sarcocystis in water buffalo (Bubalus bubalis) in China. Based on unverifiable evidence, it was suggested that the same parasite infects cattle. In addition, S. sinensis was recently declared as nomen nudum because its naming violated the rules of International Code of Zoological Nomenclature. Thus, the fourth species using cattle as an intermediate host does not have a valid name. Here, we propose a new name, Sarcocystis rommeli for the S. sinensis-like parasite from cattle in Argentina, and differentiate it ultrastructurally from S. hominis sarcocysts from experimentally infected cattle. Sarcocystis rommeli sarcocysts were microscopic with a 5-µm-thick wall with slender villar protrusions (Vp); the Vp were up to 5 µm long, up to 0.5 µm wide, and of uneven thickness, often bent at an angle. The ground substance layer (Gs) was up to 0.8 µm thick and smooth. Vesicular structures were seen at the base of the Vp. The bradyzoites were 10-12 µm long. Sarcocystis hominis sarcocysts had Vp that were often upright, up to 7.5 µm long, and up to 1.8 µm wide; the Gs was up to 2 µm thick and without vesicles. Its sarcocyst wall was up to 5.6 µm thick, the vp were bent at an angle, up to 5.8 µm long, the Gs was up to 2 µm thick, but without vesicles seen in S. rommeli. Beef containing sarcocysts of S. rommeli was not orally infectious for two human volunteers and a red fox (Vulpes vulpes). The Sarcocystis described here is molecularly different from S. cruzi, S. hirsuta, and S. hominis based on 18S rRNA and cox1 gene sequences.

Antibody Detection and Molecular Characterization of Toxoplasma gondii from Bobcats (Lynx rufus), Domestic Cats (Felis catus), and Wildlife from Minnesota, USA.

Little is known of the epidemiology of toxoplasmosis in Minnesota. Here, we evaluated Toxoplasma gondii infection in 50 wild bobcats (Lynx rufus) and 75 other animals on/near 10 cattle farms. Antibodies to T. gondii were assayed in serum samples or tissue fluids by the modified agglutination test (MAT, cut-off 1:25). Twenty nine of 50 bobcats and 15 of 41 wildlife trapped on the vicinity of 10 farms and nine of 16 adult domestic cats (Felis catus) and six of 14 domestic dogs resident on farms were seropositive. Toxoplasma gondii oocysts were not found in feces of any felid. Tissues of all seropositive wild animals trapped on the farm were bioassayed in mice and viable T. gondii was isolated from two badgers (Taxidea taxus), two raccoons (Procyon lotor), one coyote (Canis latrans), and one opossum (Didelphis virginiana). All six T. gondii isolates were further propagated in cell culture. Multi-locus PCR-RFLP genotyping using 10 markers (SAG1, SAG2 (5'-3'SAG2, and alt.SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico), and DNA from cell culture derived tachyzoites revealed three genotypes; #5 ToxoDataBase (1 coyote, 1 raccoon), #1 (1 badger, 1 raccoon, 1 opossum), and #2 (1 badger). This is the first report of T. gondii prevalence in domestic cats and in bobcats from Minnesota, and the first isolation of viable T. gondii from badger.

Sarcocystis masoni, n. sp. (Apicomplexa: Sarcocystidae), and redescription of Sarcocystis aucheniae from llama (Lama glama), guanaco (Lama guanicoe) and alpaca (Vicugna pacos).

There is considerable confusion concerning the species of Sarcocystis in South American camelids (SAC). Several species names have been used; however, proper descriptions are lacking. In the present paper, we redescribe the macroscopic sarcocyst forming Sarcocystis aucheniae and describe and propose a new name, Sarcocystis masoni for the microscopic sarcocyst forming species. Muscles samples were obtained from llamas (Lama glama) and guanacos (Lama guanicoe) from Argentina and from alpacas (Vicugna pacos) and llamas from Peru. Individual sarcocysts were processed by optical and electron microscopy, and molecular studies. Microscopic sarcocysts of S. masoni were up to 800 µm long and 35-95 µm wide, the sarcocyst wall was 2·5-3·5 µm thick, and had conical to cylindrical villar protrusions (vp) with several microtubules. Each vp had 11 or more rows of knob-like projections. Seven 18S rRNA gene sequences obtained from sarcocysts revealed 95-96% identity with other Sarcocystis spp. sequences reported in the GenBank. Sarcocysts of S. aucheniae were macroscopic, up to 1·2 cm long and surrounded by a dense and laminar 50 µm thick secondary cyst wall. The sarcocyst wall was up to 10 µm thick, and had branched vp, appearing like cauliflower. Comparison of the 11 sequences obtained from individual macroscopic cysts evidenced a 98-99% of sequence homology with other S. aucheniae sequences. In conclusion, 2 morphologically and molecularly different Sarcocystis species, S. masoni (microscopic cysts) and S. aucheniae (macroscopic cysts), were identified affecting different SAC from Argentina and Peru.

Ancient, globally distributed lineage of Sarcocystis from sporocysts of the Eastern rat snake (Pantherophis alleghaniensis) and its relation to neurological sequalae in intermediate hosts.

There is an emerging concern that snakes are definitive hosts of certain species of Sarcocystis that cause muscular sarcocystosis in human and non-human primates. Other species of Sarcocystis are known to cycle among snakes and rodents, but have been poorly characterized in the USA and elsewhere. Although neurological sequalae are known for certain species of Sarcocystis, no such neurological symptoms are known to typify parasites that naturally cycle in rodents. Here, sporocysts of a species of Sarcocystis were found in the intestinal contents of a rat snake (Pantherophis alleghaniensis) from Maryland, USA. The sporocysts were orally infective for interferon gamma gene knockout (KO) mice, but not to Swiss Webster outbred mice. The KO mice developed neurological signs, and were necropsied between 33 and 52 days post-inoculation. Only schizonts/merozoites were found, and they were confined to the brain. The predominant lesion was meningoencephalitis characterized by perivascular cuffs, granulomas, and necrosis of the neuropil. The schizonts and merozoites were located in neuropil, and apparently extravascular. Brain homogenates from infected KO mice were infective to KO mice and CV-1 cell line. DNA extracted from the infected mouse brain, and infected cell cultures revealed the highest identity with Sarcocystis species that employ snakes as definitive hosts. This is the first report of Sarcocystis infection in the endangered rat snake (P. alleghaniensis) and the first report of neurological sarcocystosis in mice induced by feeding sporocysts from a snake. These data underscore the likelihood that parasites in this genus that employ snakes as their definitive hosts constitute an ancient, globally distributed monophyletic group. These data also raise the possibility that neurological sequalae may be more common in intermediate hosts of Sarcocystis spp. than has previously been appreciated.

Seroprevalence, isolation, first genetic characterization of Toxoplasma gondii, and possible congenital transmission in wild moose from Minnesota, USA.

Toxoplasma gondii infections are widespread in white-tailed deer (Odocoileus virginianus) but little is known of its prevalence in other cervids in the USA. Moose (Alces alces) is a popular large game animal, hunted for its meat and trophy antlers. Here, we report seroprevalence, isolation, and genetic characterization of T. gondii from moose from Minnesota. Antibodies against T. gondii were detected in 8 of 79 (10%) moose tested by the modified agglutination test (MAT 1:25 or higher). The myocardium of 68 moose was bioassayed individually in mice, irrespective of serological status. T. gondii was detected in three moose (2 adults, 1 3 weeks old). The parasite from 2 adults was further propagated in cell culture. PCR-RFLP genotyping of cell culture derived tachyzoites using 10 genetic markers, SAG1, SAG2 (5' and 3' SAG2, and alt.SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed two different ToxoDB PCR-RFLP genotypes (#5, designated TgMooseUS1, and #7, TgMooseUS2). The mice inoculated with myocardium of the juvenile moose developed antibodies against T. gondii, and DNA extracted from infected mouse brain was only partially characterized by PCR-RFLP genotyping, which suggests a potential new genotype. Result documented prevalence of T. gondii in moose, and its possible transplacental/transmammary transmission of T. gondii in moose.

Toxoplasma gondii isolates from mouflon sheep (Ovis ammon) from Hawaii, USA.

Little is known of Toxoplasma gondii isolates circulating in wildlife. The mouflon (Ovis ammon) is very popular game animal, hunted for its trophy horns. Here, we report the isolation and genetic characterization of T. gondii from two mouflons from Hawaii, USA. Both sheep had antibodies titers of 1:800 or higher. Viable T. gondii were isolated and nested PCR-RFLP genotyping revealed two genotypes, a clonal Type III (designated TgMouflonUS1), and a new genotype (designated TgMouflonUS2, and ToxoDB PCR-RFLP genotypes #249). This is the first report of T. gondii infection, isolation and genetic characterization in mouflons from the USA.

Sarcocystis caninum and Sarcocystis svanai n. spp. (Apicomplexa: Sarcocystidae) Associated with Severe Myositis and Hepatitis in the Domestic Dog (Canis familiaris).

There are several reports of Sarcocystis sarcocysts in muscles of dogs, but these species have not been named. Additionally, there are two reports of Sarcocystis neurona in dogs. Here, we propose two new names, Sarcocystis caninum, and Sarcocystis svanai for sarcocysts associated with clinical muscular sarcocystosis in four domestic dogs (Canis familiaris), one each from Montana and Colorado in the USA, and two from British Columbia, Canada. Only the sarcocyst stage was identified. Most of the sarcocysts identified were S. caninum. Sarcocysts were studied using light microscopy, transmission electron microscopy (TEM), and polymerase chain reaction. Based on collective results two new species, S. caninum and S. svanai were designated. Sarcocystis caninum and S. svanai were structurally distinct. Sarcocystis caninum sarcocysts were up to 1.2 mm long and up to 75 µm wide. By light microscopy, the sarcocyst wall was relatively thin and smooth. By TEM, the sarcocyst wall was "type 9", 1-2 µm thick, and contained villar protrusions that lacked microtubules. Bradyzoites in sections were 7-9 µm long. Sarcocysts of S. svanai were few and were identified by TEM. Sarcocystis svanai sarcocysts were "type 1", thin walled (< 0.5 µm), and the wall lacked villar protrusions but had tiny blebs that did not invaginate. DNA was extracted either from infected frozen muscle biopsies or formalin-fixed paraffin-embedded sections. Dogs were either singly infected with S. caninum or multiply co-infected with S. caninum and S. svanai (the result of a mixed infection) based on multilocus DNA sequencing and morphology. BLASTn analysis established that the sarcocysts identified in these dogs were similar to, but not identical to Sarcocystis canis or Sarcocystis arctosi, parasites found to infect polar bears (Ursus maritimus) or brown bears (Ursus arctosi), respectively. However, the S. caninum sequence showed 100% identify over the 18S rRNA region sequenced to that of S. arctica, a parasite known to infect Arctic foxes (Vulpes lagopus).

Isolation and Genetic Characterization of Toxoplasma gondii from Black Bears (Ursus americanus), Bobcats (Lynx rufus), and Feral Cats (Felis catus) from Pennsylvania.

Toxoplasma gondii infects virtually all warm-blooded hosts worldwide. Recently, attention has been focused on the genetic diversity of the parasite to explain its pathogenicity in different hosts. It has been hypothesized that interaction between feral and domestic cycles of T. gondii may increase unusual genotypes in domestic cats and facilitate transmission of potentially more pathogenic genotypes to humans, domestic animals, and wildlife. In the present study, we tested black bear (Ursus americanus), bobcat (Lynx rufus), and feral cat (Felis catus) from the state of Pennsylvania for T. gondii infection. Antibodies to T. gondii were found in 32 (84.2%) of 38 bears, both bobcats, and 2 of 3 feral cats tested by the modified agglutination test (cut off titer 1:25). Hearts from seropositive animals were bioassayed in mice, and viable T. gondii was isolated from 3 of 32 bears, 2 of 2 bobcats, and 2 of 3 feral cats. DNA isolated from culture-derived tachyzoites of these isolates was characterized using multilocus PCR-RFLP markers. Three genotypes were revealed, including ToxoDB PCR-RFLP genotype #1 or #3 (Type II, 1 isolate), #5 (Type 12, 3 isolates), and #216 (3 isolates), adding to the evidence of genetic diversity of T. gondii in wildlife in Pennsylvania. Pathogenicity of 3 T. gondii isolates (all #216, 1 from bear, and 2 from feral cat) was determined in outbred Swiss Webster mice; all three were virulent causing 100% mortality. Results indicated that highly mouse pathogenic strains of T. gondii are circulating in wildlife, and these strains may pose risk to infect human through consuming of game meat.

Sarcocystis oreamni, n. sp. (Apicomplexa: Sarcocystidae) from the mountain goat (Oreamnos americanus).

Numerous species of Sarcocystis have been reported from wild ruminants, but none has been named from the Rocky Mountain goat (Oreamnos americanus). Mature sarcocysts were found in frozen muscle samples of three of seven mountain goats from Alaska, USA. Two morphological types of sarcocysts were found; one had Sarcocystis cornagliai-like sarcocysts, previously named from the Alpine ibex (Capra ibex) from Europe. Two other goats were infected with a new species, Sarcocystis oreamni. Sarcocystis oreamni sarcocysts were microscopic with 2 µm-thick sarcocyst wall. By transmission electron microscopy, the sarcocyst wall had 1.7 µm-thick with unusual molar tooth-like villar protrusions (vp), type 29. The vp had an electron dense core and two disc-shaped plaques at the tip with fine microtubules. Bradyzoites were 8.6-9.1 µm long. Single nucleotide polymorphism (SNP) identified in 18S rRNA, and 28S rRNA loci of rDNA regions that suggested S. oreamni molecularly apart from related species. The phylogenetic analysis based on 18S rRNA and 28S rRNA sequences suggested S. oreamni is related with Sarcocystis species that employ members of the Canidae family as their definitive host.

Sarcocystis mehlhorni, n. sp. (Apicomplexa: Sarcocystidae) from the black-tailed deer (Odocoileus hemionus columbianus).

Infection with Sarcocystis is common in many species of wild cervids but none is reported from the black-tailed deer (Odocoileus hemionus columbianus). Here, we report Sarcocystis infection in two black-tailed deer from northwest USA for the first time. Sarcocysts were microscopic, up to 556 µm long and mature. The sarcocyst wall was up to 1.39 µm thick and had rectangular 1.17-µm-long villar protrusions, type 17, with thin (230 nm) electron dense ground substance layer. Molecular characterization and phylogenetic analysis indicated that Sarcocystis in the black-tailed deer is related to structurally distinct Sarcocystis species in cervids. A new name, Sarcocystis mehlhorni, is proposed for the Sarcocystis species in black-tailed deer.

An update on Toxoplasma gondii infections in northern sea otters (Enhydra lutris kenyoni) from Washington State, USA.

Toxoplasmosis in marine mammals is epidemiologically and clinically important. Toxoplasma gondii antibodies (by modified agglutination test, cut-off ≥1:25) were detected in serum of 65 of 70 (92.9%) northern sea otters (Enhydra lutris kenyoni) from Washington State, USA. Brains and/or muscles of 44 sea otters were bioassayed in mice (INF-γ knock-out [KO], Swiss Webster outbred [SW]) and viable T. gondii was isolated from 22 of 44 (50%); T. gondii strains were lethal to KO mice but not SW mice. These T. gondii isolates were further propagated in cell culture. Multi-locus PCR-RFLP genotyping of cell culture-derived tachyzoites revealed four different genotypes among 22 isolates including ToxoDB PCR-RFLP genotype #5 (14 isolates), #1 (three isolates), #3 (four isolates), and #167 (one isolate). PCR-DNA sequencing based genotyping using polymorphic gene GRA6 revealed one of four different alleles. Among the 14 RFLP genotype #5 strains, 10 have GRA6 sequences that match with the Type A, one match with the Type X, two strains did not generate sequence data, and one strain had double peaks at known polymorphic sites indicating a mixed infection. The seven strains belong to genotypes #1 and #3, all have identical sequences to T. gondii Type II reference isolate ME49. Genotype #167 strain has identical sequence to Type I reference strain. In summary, we observed high seroprevalence, and high rate of isolation of T. gondii from northern sea otters and predominant genotype #5 that has been previously reported a dominant and widespread strain among terrestrial wildlife in North America. GRA6 sequence analysis of the genotype #5 isolates indicated the dominance of Type A lineage in sea otters in Washington State.

Dissection of the in vitro developmental program of Hammondia hammondi reveals a link between stress sensitivity and life cycle flexibility in Toxoplasma gondii.

Most eukaryotic parasites are obligately heteroxenous, requiring sequential infection of different host species in order to survive. Toxoplasma gondii is a rare exception to this rule, having a uniquely facultative heteroxenous life cycle. To understand the origins of this phenomenon, we compared development and stress responses in T. gondii to those of its its obligately heteroxenous relative, Hammondia hammondi and have identified multiple H. hammondi growth states that are distinct from those in T. gondii. Of these, the most dramatic difference was that H. hammondi was refractory to stressors that robustly induce cyst formation in T. gondii, and this was reflected most dramatically in its unchanging transcriptome after stress exposure. We also found that H. hammondi could be propagated in vitro for up to 8 days post-excystation, and we exploited this to generate the first ever transgenic H. hammondi line. Overall our data show that H. hammondi zoites grow as stringently regulated, unique life stages that are distinct from T. gondii tachyzoites, and implicate stress sensitivity as a potential developmental innovation that increased the flexibility of the T. gondii life cycle.

Recombinant Calponin of human filariid Brugia malayi: Secondary structure and immunoprophylactic potential.

In the search for potential vaccine candidates for the control of human lymphatic filariasis, we recently identified calponin-like protein, that regulates actin/myosin interactions, in a proinflammatory fraction F8 (45.24-48.64kDa) of Brugia malayi adult worms. In the present study, the gene was cloned, expressed, and the recombinant Calponin of B. malayi (r-ClpBm) was prepared and characterized. r-ClpBm bears homology with OV9M of Onchocerca volvulus, a non-lymphatic filariid that causes loss of vision and cutaneous pathology. r-ClpBm was found to be a ∼45kDa protein that folds into a predominantly α-helix conformation. The protective efficacy of r-ClpBm against B. malayi infection in Mastomys coucha was investigated by assessing the course of microfilaraemia and adult worm burden in the host immunized with r-ClpBm and subsequently infected with infective third stage larvae (L3). Expression of the Calponin was detected in all life stages (microfilariae, L3, L4, L5 and adults) of the parasite and immunization with r-ClpBm partially protected M. coucha against establishment of infection as inferred by ∼42% inhibition in parasite burden. Upregulated cellular proliferation, TNF-α, IFN-γ, IL-1ß, IL-4, nitric oxide (NO) release, expression of iNOS, and specific IgG, IgG1 and IgG2b in immunized animals correlated with parasitological findings. r-ClpBm immunization caused degranulation in majority of mast cells indicating possible involvement of mast cell products in reducing the parasite survival. It appears that complex mechanisms including Th1, Th2, NO and mast cells are involved in the clearance of infection. To the best of our knowledge this is the first report on cloning, expression of the gene and purification of r-ClpBm, determination of its secondary structure and its ability to partially prevent establishment of B. malayi infection. Thus, r-ClpBm may further be studied and developed in combination with other protective molecules of B. malayi as a component of potential filarial cocktail vaccine candidate.

Isolation, molecular characterization, and in vitro schizogonic development of Sarcocystis sp. ex Accipiter cooperii from a naturally infected Cooper's hawk (Accipiter cooperii).

Raptors serve as the definitive host for several Sarcocystis species. The complete life cycles of only a few of these Sarcocystis species that use birds of prey as definitive hosts have been described. In the present study, Sarcocystis species sporocysts were obtained from the intestine of a Cooper's hawk (Accipiter cooperii) and were used to infect cell cultures of African green monkey kidney cells to isolate a continuous culture and describe asexual stages of the parasite. Two clones of the parasite were obtained by limiting dilution. Asexual stages were used to obtain DNA for molecular classification and identification. PCR amplification and sequencing were done at three nuclear ribosomal DNA loci; 18S rRNA, 28S rRNA, and ITS-1, and the mitochondrial cytochrome c oxidase subunit 1 (cox1) locus. Examination of clonal isolates of the parasite indicated a single species related to S. columbae (termed Sarcocystis sp. ex Accipiter cooperii) was present in the Cooper's hawk. Our results document for the first time Sarcocystis sp. ex A. cooperii occurs naturally in an unknown intermediate host in North America and that Cooper's hawks (A. cooperii) are a natural definitive host.

Seroprevalence, isolation and co-infection of multiple Toxoplasma gondii strains in individual bobcats (Lynx rufus) from Mississippi, USA.

Toxoplasma gondii causes lifelong chronic infection in both feline definitive hosts and intermediate hosts. Multiple exposures to the parasite are likely to occur in nature due to high environmental contamination. Here, we present data of high seroprevalence and multiple T. gondii strain co-infections in individual bobcats (Lynx rufus). Unfrozen samples (blood, heart, tongue and faeces) were collected from 35 free ranging wild bobcats from Mississippi, USA. Toxoplasma gondii antibodies were detected in serum by the modified agglutination test (1:≥200) in all 35 bobcats. Hearts from all bobcats were bioassayed in mice and viable T. gondii was isolated from 21; these strains were further propagated in cell culture. Additionally, DNA was extracted from digests of tongues and hearts of all 35 bobcats; T. gondii DNA was detected in tissues of all 35 bobcats. Genetic characterisation of DNA from cell culture-derived isolates was performed by multiplex PCR using 10 PCR-RFLP markers. Results showed that ToxoDB genotype #5 predominated (in 18 isolates) with a few other types (#24 in two isolates, and #2 in one isolate). PCR-DNA sequencing at two polymorphic markers, GRA6 and GRA7, detected multiple recombinant strains co-infecting the tissues of bobcats; most possessing Type II alleles at GRA7 versus Type X (HG-12) alleles at GRA6. Our results suggest that individual bobcats have been exposed to more than one parasite strain during their life time.