Antiplasmodial, antimalarial activities and toxicity of African medicinal plants: a systematic review of literature.

BACKGROUND: Malaria still constitutes a major public health menace, especially in tropical and subtropical countries. Close to half a million people mainly children in Africa, die every year from the disease. With the rising resistance to frontline drugs (artemisinin-based combinations), there is a need to accelerate the discovery and development of newer anti-malarial drugs. A systematic review was conducted to identify the African medicinal plants with significant antiplasmodial and/or anti-malarial activity, toxicity, as wells as assessing the variation in their activity between study designs (in vitro and in vivo). METHODS: Key health-related databases including Google Scholar, PubMed, PubMed Central, and Science Direct were searched for relevant literature on the antiplasmodial and anti-malarial activities of African medicinal plants. RESULTS: In total, 200 research articles were identified, a majority of which were studies conducted in Nigeria. The selected research articles constituted 722 independent experiments evaluating 502 plant species. Of the 722 studies, 81.9%, 12.4%, and 5.5% were in vitro, in vivo, and combined in vitro and in vivo, respectively. The most frequently investigated plant species were Azadirachta indica, Zanthoxylum chalybeum, Picrilima nitida, and Nauclea latifolia meanwhile Fabaceae, Euphorbiaceae, Annonaceae, Rubiaceae, Rutaceae, Meliaceae, and Lamiaceae were the most frequently investigated plant families. Overall, 248 (34.3%), 241 (33.4%), and 233 (32.3%) of the studies reported very good, good, and moderate activity, respectively. Alchornea cordifolia, Flueggea virosa, Cryptolepis sanguinolenta, Zanthoxylum chalybeum, and Maytenus senegalensis gave consistently very good activity across the different studies. In all, only 31 (4.3%) of studies involved pure compounds and these had significantly (p = 0.044) higher antiplasmodial activity relative to crude extracts. Out of the 198 plant species tested for toxicity, 52 (26.3%) demonstrated some degree of toxicity, with toxicity most frequently reported with Azadirachta indica and Vernonia amygdalina. These species were equally the most frequently inactive plants reported. The leaves were the most frequently reported toxic part of plants used. Furthermore, toxicity was observed to decrease with increasing antiplasmodial activity. CONCLUSIONS: Although there are many indigenous plants with considerable antiplasmodial and anti-malarial activity, the progress in the development of new anti-malarial drugs from African medicinal plants is still slothful, with only one clinical trial with Cochlospermum planchonii (Bixaceae) conducted to date. There is, therefore, the need to scale up anti-malarial drug discovery in the African region.

Prevalence of antibodies to Neospora caninum in the serum of camels (Camelus dromedarius) from central Punjab, Pakistan.

Infection with the Apicomplexan parasite Neospora caninum occurs in domestic and wild animals worldwide. This parasite causes significant economic losses in the dairy industry worldwide. The current study was designed to assess the prevalence of N. caninum antibodies in the sera of camels (Camelus dromedarius) from Rakh Mahni, Bhakkar, Pakistan, to better understand the epidemiology of this important cause of bovine abortion in Pakistan. Eighty-one sera samples collected from apparently healthy camels (male = 19, female = 62) were tested for antibodies against N. caninum by using a commercially available competitive Enzyme Linked Immunosorbent assay diagnostic kit. Nine (11.1%) of 81 sera samples were found positive for antibodies to N. caninum. No significant (P > 0.05) difference in seroprevalence was observed between male and female camels or between different breeds. An increasing trend of seropositivity to N. caninum was seen with the age of animals indicating postnatal exposure to N. caninum infection. However, this increase in prevalence was not significant (P > 0.05). However, a significant (P < 0.05) difference in seroprevalence was noted among feeding style of animals, i.e., stall feeding, grazing, and mixed (grazing and stall feeding) feeding style. The prevalence was significantly (P < 0.05) higher in female animals 26.1% (6/23) with abortion history that of animals have had no such history 5.1% (2/39). Presence of antibodies to N. caninum in the sera of camels indicates that further studies are required to evaluate the importance of N. caninum as a cause of abortion or neonatal disease in these animals.

Risk factors and sources of foodborne hepatitis E virus infection in the United States.

Hepatitis E virus (HEV) is an important human pathogen with pigs and other species serving as natural animal reservoirs. Ample evidence documents sporadic cases of hepatitis E acquired via consumption of undercooked meat. Chronic hepatitis E cases in immunosuppressed individuals are mostly caused by zoonotic HEV of swine origin. We report here the identification of genotype 3 HEV from non-liver commercial pork from local grocery stores in southwest Virginia, and association of HEV seropositivity to the consumption of undercooked meat in healthy young adults at a university in the United States. These results raise concerns about foodborne HEV transmission in the United States. J. Med. Virol. 88:1641-1645, 2016. © 2016 Wiley Periodicals, Inc.

Caspase-11 Modulates Inflammation and Attenuates Toxoplasma gondii Pathogenesis.

Toxoplasma gondii is an obligate intracellular parasite that is the etiologic agent responsible for toxoplasmosis. Infection with T. gondii results in activation of nucleotide binding domain and leucine rich repeat containing receptors (NLRs). NLR activation leads to inflammasome formation, the activation of caspase-1, and the subsequent cleavage of IL-1ß and IL-18. Recently, a noncanonical inflammasome has been characterized which functions through caspase-11 and appears to augment many biological functions previously considered to be dependent upon the canonical inflammasome. To better elucidate the function of this noncanonical inflammasome in toxoplasmosis, we utilized Asc (-/-) and Casp11 (-/-) mice and infected these animals with T. gondii. Our data indicates that caspase-11 modulates the innate immune response to T. gondii through a mechanism which is distinct from that currently described for the canonical inflammasome. Asc (-/-) mice demonstrated increased disease pathogenesis during the acute phase of T. gondii infection, whereas Casp11 (-/-) mice demonstrated significantly attenuated disease pathogenesis and reduced inflammation. This attenuated host response was associated with reduced local and systemic cytokine production, including diminished IL-1ß. During the chronic phase of infection, caspase-11 deficiency resulted in increased neuroinflammation and tissue cyst burden in the brain. Together, our data suggest that caspase-11 functions to protect the host by enhancing inflammation during the early phase of infection in an effort to minimize disease pathogenesis during later stages of toxoplasmosis.

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.

GAMOGONY OF SARCOCYSTIS STRIXI IN MAMMALIAN CELL CULTURES.

We are interested in the disease ecology of Sarcocystis species that infect birds of prey as definitive and intermediate hosts. The present study was done to test our hypothesis that a laboratory model can be developed for sarcocystis infection in mammals using gamma interferon gene knockout (KO) mice as a source of Sarcocystis strixi bradyzoites and mammalian cell cultures as a source of sporulated S. strixi oocysts. Sporocysts of S. strixi from a naturally infected barred owl (Strix varia) were fed to KO mice to produce sarcocysts, and the enclosed bradyzoites were obtained by acid-pepsin digestion of abdominal and thigh muscles. Bradyzoites, metrocytes, and an unusual spherical stage were seen in digest before the inoculation of host cells. The spherical stages stained dark with Giemsa stain, but no nucleus was observed, and they were seen free and associated with the concave portion of some bradyzoites. Examination of infected cell cultures demonstrated that macrogamonts and microgamonts were present at 24 hr post-inoculation. Since sporulated oocysts were not observed, we had to reject our current hypothesis.

Horses affected by EPM have increased sCD14 compared to healthy horses.

Equine protozoal myeloencephalitis (EPM) is a debilitating neurologic disease affecting horses across the Americas. Gaps in understanding the inflammatory immune response in EPM-affected horses create difficulties with diagnosis and treatment, subsequently negatively impacting the prognosis of affected horses. The purpose of the current study was to evaluate circulating levels of the inflammatory immune marker soluble CD14 (sCD14), in horses with EPM (n = 7) and determine if they differed from healthy neurologically normal horses (n = 6). Paired sera and cerebrospinal fluid (CSF) samples were analyzed for sCD14. Inclusion criteria for EPM horses consisted of the presence of neurologic signs consistent with EPM, Sarcocystis neurona surface antigens 2, 4/3 (SnSAG 2, 4/3) ELISA serum: CSF antibody ratio ≤ 100, and a postmortem diagnosis of EPM. Control horses were neurologically normal, healthy horses with SnSAG 2, 4/3 ELISA serum: CSF antibody ratios of > 100. Serum anti-Sarcocystis neurona antibodies indicate that healthy control horses were exposed to S. neurona but resistant to developing clinical EPM. EPM cases had significantly greater concentrations of sCD14 in CSF samples compared to control horses and increased serum sCD14 concentrations. A positive correlation between sCD14 serum and CSF concentrations was observed in EPM-affected horses but not healthy horses. Soluble CD14 is an inflammatory marker, and the study results suggest it is elevated in EPM patients. When performed in conjunction with clinical evaluation and standard antibody testing, there may be potential for sCD14 to be utilized as a correlate for EPM.

Neosporosis, Toxoplasmosis, and Sarcocystosis in Ruminants: An Update.

Neospora caninum, Toxoplasma gondii, and Sarcocystis spp. are related Apicomplexan parasites that have 2 hosts in their life cycles. The definitive hosts excrete unsporulated (Neospora caninum, T gondii) or sporulated (Sarcocystis spp.) oocysts in their feces after ingesting tissue cysts from the tissues of ruminant intermediate hosts. These coccidians can cause abortion and neonatal mortality in ruminants. T gondii and Sarcocystis hominis (from cattle) are zoonotic. This article reviews information on the etiology, life cycle, diagnosis, control and prevention of these parasites and the diseases they cause in ruminants.

Canine and bovine Neospora caninum control sera examined for cross-reactivity using Neospora caninum and Neospora hughesi indirect fluorescent antibody tests.

Neospora caninum is a well known protozoan parasite of domestic and wild animals. Neospora hughesi is a closely related protozoan with an unknown life cycle, host range, and infection prevalence. Many serologic surveys of N. caninum have been performed without consideration of potential cross-reactions with N. hughesi, which could confound results. The aim of this study was to investigate whether postexposure sera from animals experimentally infected with N. caninum exhibit significant reactivity differences when tested using N. caninum and N. hughesi Immunofluorescent Antibody Tests (IFAT). Pre- and postinfection serum samples from 10 dogs, 20 calves, and 17 cows were tested by dual IFATs. All pre-exposure samples for N. caninum tested seronegative for both organisms. All postexposure samples that were seropositive for N. caninum were also positive for N. hughesi, although N. hughesi antibody titers were usually 1 dilution lower (P < 0.02). Serologic surveys for N. caninum may be confounded by cross-reacting titers with N. hughesi, but true positive N. caninum antibody titers are greater than, or equal to, cross-reacting N. hughesi antibody titers.

Inhibition of Toxoplasma gondii and Plasmodium falciparum infections in vitro by NSC3852, a redox active antiproliferative and tumor cell differentiation agent.

We searched the National Cancer Institute (NCI) compound library for structures related to the antitumor quinoline NSC3852 (5-nitroso-8-quinolinol) and used a computer algorithm to predict the antiprotozoan activity for each of 13 structures. Half of these compounds inhibited Toxoplasma gondii tachyzoite propagation in human fibroblasts at < or =1 microM. The active compounds comprise a series of low-molecular-weight quinolines bearing nitrogen substituents in the ring-5 position. NSC3852 (EC(50) 80 nM) and NSC74949 (EC(50) 646 nM) were the most potent. NSC3852 also inhibited Plasmodium falciparum growth in human red blood cells (EC(50) 1.3 microM). To investigate the mechanism for NSC3852's anti-T. gondii activity, we used chemiluminescence assays to detect reactive oxygen species (ROS) formation in freshly isolated tachyzoites and in infected host cells; the absence of ROS generation by NSC3852 in these assays indicated NSC3852 does not redox cycle in T. gondii. Inhibitors of enzyme sources of free radicals such as superoxide anion, nitric oxide (NO), and their reaction product peroxynitrite did not interfere with the anti-T. gondii activity of NSC3852. However, inhibition of T. gondii tachyzoite propagation by NSC3852 involved redox reactions because tachyzoites were protected from NSC3852 by inclusion of the cell permeant superoxide dismutase mimetic, MnTMPyP, or N-acetylcysteine in the culture medium. We conclude that the Prediction of Activity Spectra for Substances (PASS) computer program is useful in finding new compounds that inhibit T. gondii tachyzoites in vitro and that NSC3852 is a potent T. gondii inhibitor that acts by indirect generation of oxidative stress in T. gondii.

Coccidiosis in dogs-100 years of progress.

Until 1970, coccidian parasites of dogs were considered to have a direct fecal-oral life cycle like Eimeria in poultry. They were thought to be non-host specific and infect both dogs and cats. Studies conducted in the 1970s revealed that dog coccidia were host-specific and had transport or paratenic hosts that were infected with an encysted stage containing a single organism, the monozoic tissue cyst. There are still considerable confusion and uncertainties concerning the life cycles and pathogenicity of coccidian parasites of dogs. The present paper reviews the history, taxonomy, life cycles, pathogenicity, epidemiology, diagnosis, and treatment of conventional coccidian parasites previously called Isospora spp., currently designated Cystoisospora spp. that infect canines.

Giardia duodenalis assemblages in cats from Virginia, USA.

Giardia duodenalis is considered a species complex that is divided into 8 genetically distinct but morphologically identical assemblages (A-H). Assemblages C-H are generally host adapted, while A and B infect both people and animals and are considered potentially zoonotic. Furthermore, within assemblage A there are four subtypes (AI, AII, AIII, and AIV) of varying zoonotic potential; human isolates belong to AI and AII, while animal isolates belong to AI, AIII and AIV. Assemblages A, B, C, D, and F have all been reported from cats. The objective of this study was to determine the assemblage(s) of G. duodenalis present in cats from Virginia using multilocus genotyping and to assess if there were any differences among the assemblage(s) found in the populations of cats surveyed (free-roaming, shelter, owned) or their geographic location within Virginia. Samples that were positive for G. duodenalis cysts by microscopy using centrifugal flotation with ZnSO4 solution and/or direct immunofluorescence assay were genotyped using PCR and sequencing targeting fragments of the SSU rRNA, gdh, bg, and tpi genes. In total, 54 cyst-positive samples were analyzed by PCR and sequencing: 43 produced amplicons, and 37 samples had interpretable sequence data at one or more loci. Assemblage F was detected in 21/37 samples, AI was detected in 12/37 samples, and in 4/37 samples both assemblages F and AI were detected. The potentially zoonotic assemblage AI was detected in cats from two widely separated animal shelters and from one free-roaming cat. These genotyping data demonstrate that potentially zoonotic G. duodenalis assemblages are present in cats in Virginia.

Prevalence of Sarcocysts in the Muscles of Raptors from a Rehabilitation Center in North Carolina.

The life cycle of Sarcocystis species is heteroxenous (2-host), with carnivores being the definitive host and herbivores serving as intermediate hosts in predator-prey relationships. Raptors (eagles, hawks, falcons, and owls) are apex predators and are not consumed routinely by other carnivores, making the occurrence of sarcocysts in their muscles unusual. Recent reports of sarcocysts in eagles and owls with Sarcocystis encephalitis suggests that this condition may be becoming more frequent, and Sarcocystis falcatula has been implicated as the agent of encephalitis in golden ( Aquila chrysaetos) and bald eagles ( Haliaeetus leucocephalus) as well as great horned owls ( Bubo virginianus). The present study was done to determine the prevalence of sarcocysts of Sarcocystis species in the muscles of raptors from the southeastern United States. Pectoral and heart muscle from 204 raptor patients from the Carolina Raptor Center, Huntersville, North Carolina were tested for the presence of Sarcocystis species using histology. Only a few sarcocysts were seen in sections of pectoral muscle from 39 of 204 raptors (19.1%) and heart muscle from 9 that also had sarcocysts in their pectoral muscle. Two structural types of sarcocysts, thin-walled (1 µm; 62%) or thick-walled (>2 µm, 38%), were seen. Statistical analysis of raptor age and gender was done by Fisher's exact test on samples from raptors with 20 or more samples per group. The prevalence of sarcocysts by age (2 yr or more) was significant for red-shouldered hawks ( Buteo lineatus) ( P = 0.022) and Cooper's hawks ( Accipiter cooperii) ( P = 0.028). Sarcocyst prevalence in male raptors from these groups evaluated statistically were always less than in females. Prevalence in female red-tailed hawks ( Buteo jamaicensis) (42.1%) was significantly greater than in males (6.7%) using Fisher's exact test ( P = 0.047). Examination of case histories from the 39 sarcocyst-positive raptors did not reveal an association with sarcocysts in raptor pectoral or heart muscle and in a diagnosis of encephalitis. Additional studies are needed to determine the epidemiology and relationships of Sarcocystis spp. that use raptors as intermediate hosts and the importance of Sarcocystis spp. in the overall wellbeing of raptors in their natural environments.

Prevalence of Dirofilaria immitis Infections In Dogs and Cats In Hainan Island/Province and Three Other Coastal Cities of China Based On Antigen Testing and PCR.

Canine and feline heartworm disease caused by Dirofilaria immitis is a serious and sometimes fatal infection transmitted by mosquitos. Little is known about the prevalence or distribution of D. immitis infection in dogs and cats on the island of Hainan island/province or coastal cities of China. The present study examined the occurrence of D. immitis infections in dogs (n = 869) and cats (n = 51) in Hainan island/province and prevalence in dogs from 3 coastal cities (Shenzhen [n = 55], Shanghai [n = 69], and Hangzhou [n = 45]) in southern and eastern China. A commercial antigen detection (AD) test and 2 PCR methods (16S ribosomal RNA and Wolbachia surface protein [ WSP] genes) were used to determine the prevalence of D. immitis from animals >6 mo old with no previous history of D. immitis preventive treatment or heartworm infection. Gene sequencing was used to confirm positive PCR samples. The AD test was not used on cat samples. Using the AD test, the prevalence in dogs was 0.5% (4/869) in Hainan island/province, 0% (0/55) in Shenzhen, 1.5% (1/69) in Shanghai, and 0% (0/45) in Hangzhou. Prevalence by 16S rRNA gene PCR was 7.4% (64/869) of dogs from Hainan island/province, 0% (0/55) in dogs from Shenzhen, 1.5% (1/69) in dogs from Shanghai, and 0% (0/45) in dogs from Hangzhou. Prevalence by WSP gene PCR in dogs was 5.3% (46/869) in Hainan island/province, 0% (0/55) in Shenzhen, 1.5% (1/69) in Shanghai, and 0% (0/45) in Hangzhou. Prevalence in the 51 cats from Hainan island/province was 9.8% and 5.9% by 16S rRNA and WSP gene PCR, respectively. The present study demonstrates that canine heartworm exposure occurs in dogs and cats in Hainan island/province and that PCR methods detected a higher prevalence than did the AD method. The 16S rRNA gene PCR detected more positive samples than did the WSP gene PCR in both dogs and cats. The 3 coastal cities had very few dogs that had evidence of D. immitis exposure.

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.

Comparison of diagnostic techniques for detection of Giardia duodenalis in dogs and cats.

BACKGROUND: An evaluation of currently available in-clinic diagnostic tests for Giardia duodenalis infection of dogs and cats has not been performed. In addition, there is discordance among published diagnostic comparisons. The absence of a true gold standard for detecting Giardia duodenalis also complicates diagnostic evaluations. OBJECTIVES: To evaluate diagnostic tests commercially available in the United States for detecting Giardia duodenalis in dogs and cats, in comparison to a widely used reference test, the direct immunofluorescent assay (IFA), and also to compare the results of 2 methods of analysis: comparison of diagnostic tests to a reference test (IFA) and Bayesian analysis. ANIMALS: Fecal samples from a convenience sample of 388 cats and dogs located in Colorado, Oklahoma, and Virginia. METHODS: Fecal samples were tested for Giardia duodenalis by zinc sulfate centrifugal fecal flotation and 4 different commercial diagnostic immunoassays. Results were analyzed via Bayesian analysis and by comparison to the IFA as the reference test. RESULTS: Sensitivity and specificity by comparison to IFA was ≥82% and ≥90%, respectively, for all diagnostic tests in dogs and cats. When analyzed via Bayesian analysis, sensitivity and specificity were ≥83% and ≥95%, respectively. When ZnSO4 centrifugal fecal flotation results were combined with immunoassay results, there was no longer a significant difference between the sensitivities of the commercial in-clinic immunoassays. CONCLUSION AND CLINICAL RELEVANCE: The Bayesian analysis validates using IFA as the reference test. Differences in commercial in-clinic immunoassay sensitivities can be mitigated when the results are combined with ZnSO4 centrifugal fecal flotation results.

Confirmation of Sarcocystis jamaicensis Sarcocysts in IFN-γ Gene Knockout Mice Orally Inoculated with Sporocysts from a Red-Tailed Hawk ( Buteo jamaicensis).

Here, we report confirmation of sarcocysts of Sarcocystis jamaicensis in an experimental intermediate host, IFN-γ gene knockout (KO) mice orally inoculated sporocysts from its natural definitive host, a red-tailed hawk ( Buteo jamaicensis) (RTH). A RTH submitted to the Carolina Raptor Center, Huntersville, North Carolina, was euthanized because it could not be rehabilitated and released. Fully sporulated sporocysts from intestinal scrapings of the RTH were orally fed to 2 laboratory-reared outbred Swiss Webster mice (SW; Mus musculus) and to 2 KO mice. The sporocysts were infective for KO mice but not to SW mice. Both SW mice remained asymptomatic, and neither schizonts nor sarcocysts were found in their tissues when euthanized on day 54 post-inoculation (PI). The KO mice developed neurological signs and were necropsied 38-54 days PI. Schizonts/merozoites were found in both KO mice euthanized and they were confined to the brain. The predominant lesion was meningoencephalitis. Microscopic sarcocysts were found in muscles of both KO mice. When viewed with light microscopy, the sarcocyst wall appeared thin (<1 µm thick) and smooth. Ultrastructural details of sarcocysts are described.

Strains of Sarcocystis neurona exhibit differences in their surface antigens, including the absence of the major surface antigen SnSAG1.

A gene family of surface antigens is expressed by merozoites of Sarcocystis neurona, the primary cause of equine protozoal myeloencephalitis (EPM). These surface proteins, designated SnSAGs, are immunodominant and therefore excellent candidates for development of EPM diagnostics or vaccines. Prior work had identified an EPM isolate lacking the major surface antigen SnSAG1, thus suggesting there may be some diversity in the SnSAGs expressed by different S. neurona isolates. Therefore, a bioinformatic, molecular and immunological study was conducted to assess conservation of the SnSAGs. Examination of an expressed sequence tag (EST) database revealed several notable SnSAG polymorphisms. In particular, the EST information implied that the EPM strain SN4 lacked the major surface antigen SnSAG1. The absence of this surface antigen from the SN4 strain was confirmed by both Western blot and Southern blot. To evaluate SnSAG polymorphisms in the S. neurona population, 14 strains were examined by Western blots using monospecific polyclonal antibodies against the four described SnSAGs. The results of these analyses demonstrated that SnSAG2, SnSAG3, and SnSAG4 are present in all 14 S. neurona strains tested, although some variance in SnSAG4 was observed. Importantly, SnSAG1 was not detected in seven of the strains, which included isolates from four cases of EPM and a case of fatal meningoencephalitis in a sea otter. Genetic analyses by PCR using gene-specific primers confirmed the absence of the SnSAG1 locus in six of these seven strains. Collectively, the data indicated that there is heterogeneity in the surface antigen composition of different S. neurona isolates, which is an important consideration for development of serological tests and prospective vaccines for EPM. Furthermore, the diversity reported herein likely extends to other phenotypes, such as strain virulence, and may have implications for the phylogeny of the various Sarcocystis spp. that undergo sexual stages of their life cycle in opossums.

SnSAG5 is an alternative surface antigen of Sarcocystis neurona strains that is mutually exclusive to SnSAG1.

Sarcocystis neurona is an obligate intracellular parasite that causes equine protozoal myeloencephalitis (EPM). Previous work has identified a gene family of paralogous surface antigens in S. neurona called SnSAGs. These surface proteins are immunogenic in their host animals, and are therefore candidate molecules for development of diagnostics and vaccines. However, SnSAG diversity exists in strains of S. neurona, including the absence of the major surface antigen gene SnSAG1. Instead, sequence for an alternative SnSAG has been revealed in two of the SnSAG1-deficient strains. Herein, we present data characterizing this new surface protein, which we have designated SnSAG5. The results indicated that the protein encoded by the SnSAG5 sequence is indeed a surface-associated molecule that has characteristics consistent with the other SAGs identified in S. neurona and related parasites. Importantly, Western blot analyses of a collection of S. neurona strains demonstrated that 6 of 13 parasite isolates express SnSAG5 as a dominant surface protein instead of SnSAG1. Conversely, SnSAG5 was not detected in SnSAG1-positive strains. One strain, which was isolated from the brain of a sea otter, did not express either SnSAG1 or SnSAG5. Genetic analysis with SnSAG5-specific primers confirmed the presence of the SnSAG5 gene in Western blot-positive strains, while also suggesting the presence of a novel SnSAG sequence in the SnSAG1-deficient, SnSAG5-deficient otter isolate. The findings provide further indication of S. neurona strain diversity, which has implications for diagnostic testing and development of vaccines against EPM as well as the population biology of Sarcocystis cycling in the opossum definitive host.

Experimental infection of cats (Felis catus) with Tritrichomonas foetus isolated from cattle.

Tritrichomonas foetus is recognized as the causative agent of venereal trichomoniasis in cattle. It is characterized by embryonic and early fetal death and post-coital pyometra, and feline trichomoniasis, manifest as chronic, large bowel diarrhea. Many of the infected cats are less than 2 years old and specific routes of transmission remain unknown. We recently demonstrated that feline isolates of T. foetus can successfully infect heifers, resulting in pathologic changes similar, but not identical to those previously reported as representative of bovine trichomoniasis. In this study, we experimentally infected six cats less than 1 year of age with a bovine (D-1) isolate of T. foetus and one cat with a feline (AUTf-1) isolate of T. foetus. Within 2 weeks, the cat infected with the feline (AUTf-1) isolate was culture positive for trichomonads in weekly fecal samples. At the end of 5 weeks, only one cat infected with the bovine (D-1) isolate was fecal culture positive for trichomonads. At necropsy, the intestine of each cat was removed and divided into five sections (ileum, cecum, anterior, medial and posterior colon). Contents from each section were collected and cultured. The cat infected with the feline (AUTf-1) isolate was culture positive in the ileum, cecum, medial and posterior colon. Two cats infected with the bovine (D-1) isolate were culture positive in the cecum only. Additionally, each intestinal section was submitted to a pathologist for histopathological examination. The combined results indicate that there are demonstrable differences between the feline (AUTf-1) and bovine (D-1) isolates regarding their infectivity in cats.