Effects of temperature, pH and curing on the viability of Sarcocystis, a Japanese sika deer (Cervus Nippon centralis) parasite, and the inactivation of their diarrheal toxin.

Recently, the Sarcocystis parasite in horse and deer meat has been reported to be a causative agent of acute food poisoning, inducing nausea, vomiting and diarrhea. Compared with other causative agents, such as bacteria, viruses and other parasites, in deer meat, the Sarcocystis species parasite, including its stability under various conditions, is poorly understood. In this study, we assessed the viability of Sarcocystis spp. and the activity of their diarrhea toxin (a 15-kDa protein) in deer meat under conditions of freezing, cold storage, pH change and curing. In addition, the heat tolerance was assayed using purified bradyzoites. The results showed that the species lost viability by freezing at -20, -30 and -80°C for <1 hr, heating at 70°C for 1 min, alkaline treatment (pH 10.0) for 4 days and addition of salt at 2.0% for <1 day. Immunoblot assays showed that the diarrhea toxin disappeared together with the loss of viability. However, the parasite survived cooling at 0 and 4°C and acidification (pH 3.0 and 5.0) for more than 7 days with the diarrhea toxin intact. These results provide useful information for developing practical applications for the prevention of food poisoning induced by diarrheal toxin of Sarcocystis spp. in deer meat during cooking and preservation.

Immunoprotective responses against murine sarcocystosis by ß - Irradiated sporocysts.

This study aimed to induce protective immunity against infection with Sarcocystis muris in experimental mice using ß-irradiated sporocysts. Mice were vaccinated with 50 sporocysts of S. muris which were exposed to 1.84 µSv ß-irradiation for 2, 4 and 8 h. After challenge infection, different samples were taken for evaluation. Serum and intestinal wash were assayed for IFN-γ and IgA, respectively. Mesenteric lymph nodes (MLNs) and spleen were investigated for CD4+ and CD8+ T cells using immunohistochemistry. For liver, the morphological changes in parasitic stages and the count of infiltrated CD8+ T, NK1.1+ and FasL+ cells were also investigated. Real time (RT) - PCR was used for detection of liver MHC I, CD1d, IFN-γ, perforin and FasL as well as the parasite 18S ribosomal(r) RNA in liver and muscle tissues. Alterations of liver parasitic stages as well as a decrease in the infection with the parasite in both of liver and muscle tissues were dependent on radiation exposure time. An investigation for the mechanism of immunoprotection showed an increase in liver NK1.1+ & FasL+ cells, serum IFN-γ and intestinal IgA, while CD4+ and CD8+ T showed a remarkable increase in MLNs and spleen. FasL expression increased in the liver dependently on radiation exposure time, while perforin, MHC I and CD1d were not. ß-irradiated sporocysts with 1.84 µSv for 8 h s could induce the highest protection against infection with Sarcocystis. This could be largely relied on the increased infiltration of NK cells and associated higher expression of FasL in the liver.

Testing the Sarcocystis neurona vaccine using an equine protozoal myeloencephalitis challenge model.

Equine protozoal myeloencephalitis (EPM) is an important equine neurologic disorder, and treatments for the disease are often unrewarding. Prevention of the disease is the most important aspect for EPM, and a killed vaccine was previously developed for just that purpose. Evaluation of the vaccine had been hampered by lack of post vaccination challenge. The purpose of this study was to determine if the vaccine could prevent development of clinical signs after challenge with Sarcocystis neurona sporocysts in an equine challenge model. Seventy horses that were negative for antibodies to S. neurona and were neurologically normal were randomly assigned to vaccine or placebo groups and divided into short-term duration of immunity (study #1) and long-term duration of immunity (study #2) studies. S. neurona sporocysts used for the challenge were generated in the opossum/raccoon cycle isolate SN 37-R. Study #1 horses received an initial vaccination and a booster, and were challenged 34days post second vaccination. Study #2 horses received a vaccination and two boosters and were challenged 139days post third vaccination. All horses in study #1 developed neurologic signs (n=30) and there was no difference between the vaccinates and controls (P=0.7683). All but four horses in study #2 developed detectable neurologic deficits. The neurologic signs, although not statistically significant, were worse in the vaccinated horses (P=0.1559). In these two studies, vaccination with the S. neurona vaccine failed to prevent development of clinical neurologic deficits.

Daily feeding of diclazuril top dress pellets in foals reduces seroconversion to Sarcocystis neurona.

Thirty-three foals from a farm with a high exposure rate to Sarcocystis neurona were assigned to either an untreated or a diclazuril-treated group. Treated foals received daily 0.5 mg/kg of diclazuril pellets from 1 to 12 months of age. Monthly blood was tested for IgG against S. neurona using the indirect fluorescent antibody test. Following ingestion of colostral antibodies to S. neurona, there was a steady and continuous decline in seroprevalence to S. neurona until foals from both groups reached weaning age. Thereafter, the untreated foal group showed a significant increase in monthly seroprevalence compared to the diclazuril-treated foal group. The difference in temporal seroprevalence could be explained by the successful reduction of S. neurona infection in foals receiving a daily low-dose diclazuril.

[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.

[Research progress on human sarcocystosis].

Human sarcocystosis (both the intestinal and muscular forms) may be emerging as a significant, foodborne zoonotic infection in southeast Asia and southwest of China. This review summarizes recent findings in classification of Sarcocystis spp, epidemiologic features, clinical symptoms, diagnosis, treatment and prevention in human infections.

Evidence that antibodies against recombinant SnSAG1 of Sarcocystis neurona merozoites are involved in infection and immunity in equine protozoal myeloencephalitis.

Sarcocystis neurona is the principal etiologic agent of equine protozoal myeloencephalitis (EPM). An immunodominant protein of S. neurona, SnSAG-1, is expressed by the majority of S. neurona merozoites isolated from spinal tissues of horses diagnosed with EPM and may be a candidate for diagnostic tests and prophylaxis for EPM. Five horses were vaccinated with adjuvanted recombinant SnSAG1 (rSnSAG1) and 5 control (sham vaccinated) horses were vaccinated with adjuvant only. Serum was evaluated pre- and post-vaccination, prior to challenge, for antibodies against rSnSAG1 and inhibitory effects on the infectivity of S. neurona by an in vitro serum neutralization assay. The effect of vaccination with rSnSAG1 on in vivo infection by S. neurona was evaluated by challenging all the horses with S. neurona merozoites. Blinded daily examinations and 4 blinded neurological examinations were used to evaluate the presence of clinical signs of EPM. The 5 vaccinated horses developed serum and cerebrospinal fluid (CSF) titers of SnSAG1, detected by enzyme-linked immunosorbent assay (ELISA), post-vaccination. Post-vaccination serum from vaccinated horses was found to have an inhibitory effect on merozoites, demonstrated by in vitro bioassay. Following the challenge, the 5 control horses displayed clinical signs of EPM, including ataxia. While 4 of the 5 vaccinated horses did not become ataxic. One rSnSAG-1 vaccinated horse showed paresis in 1 limb with muscle atrophy. All horses showed mild, transient, cranial nerve deficits; however, disease did not progress to ataxia in rSnSAG-1 vaccinated horses. The study showed that vaccination with rSnSAG-1 produced antibodies in horses that neutralized merozoites when tested by in vitro culture and significantly reduced clinical signs demonstrated by in vivo challenge.

Lambs infected with UV-attenuated sporocysts of Sarcocystis ovicanis produced abnormal sarcocysts and induced protective immunity against a challenge infection.

The present study surveyed the prevalence of natural infection of the sheep esophagus muscle with sarcocysts of Sarcocystis ovicanis and examined induction of protective immunity using UV-attenuated sporocysts. The overall prevalence of natural infection of the sheep was 95%. Infectivity of the collected sarcocysts was confirmed by shedding of sporulated oocysts after feeding infected esophageal tissues to dogs. To induce protective immunity, lambs were immunized 3 times (once a week) with 1.5 x 10(4) sporocysts exposed to UV-light for 30 min (UV-30 group) or 60 (UV-60 group) min and then challenged with 1.5 x 10(4) normal sporocysts at the 3rd week post the 1st vaccination. These lambs showed high survival and less clinical signs of sarcocystosis than normal infected lambs. The attenuated sporocysts produced abnormal cysts; small in size and detached from the muscle fiber. These abnormalities were more obvious in UV-60 group than UV-30 group. Also, the IFN-gamma level and lymphocyte percentage were increased while the total leukocyte count was decreased in the UV-60 group compared with other groups. The high level of IFN-gamma may be an evidence for the induction of Th1 responses which may have protective effect against a challenge infection.

Effect of intermittent oral administration of ponazuril on experimental Sarcocystis neurona infection of horses.

OBJECTIVE: To evaluate the effect of intermittent oral administration of ponazuril on immunoconversion against Sarcocystis neurona in horses inoculated intragastrically with S neurona sporocysts. ANIMALS: 20 healthy horses that were seronegative for S neurona-specific IgG. PROCEDURES: 5 control horses were neither inoculated with sporocysts nor treated. Other horses (5 horses/group) each received 612,500 S neurona sporocysts via nasogastric tube (day 0) and were not treated or were administered ponazuril (20 mg/kg, PO) every 7 days (beginning on day 5) or every 14 days (beginning on day 12) for 12 weeks. Blood and CSF samples were collected on day - 1 and then every 14 days after challenge for western blot assessment of immunoconversion. Clinical signs of equine protozoal myeloencephalitis (EPM) were monitored, and tissues were examined histologically after euthanasia. RESULTS: Sera from all challenged horses yielded positive western blot results within 56 days. Immunoconversion in CSF was detected in only 2 of 5 horses that were treated weekly; all other challenged horses immunoconverted within 84 days. Weekly administration of ponazuril significantly reduced the antibody response against the S neurona 17-kd antigen in CSF. Neurologic signs consistent with EPM did not develop in any group; likewise, histologic examination of CNS tissue did not reveal protozoa or consistent degenerative or inflammatory changes. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of ponazuril every 7 days, but not every 14 days, significantly decreased intrathecal anti-S neurona antibody responses in horses inoculated with S neurona sporocysts. Protocols involving intermittent administration of ponazuril may have application in prevention of EPM.

Neosporosis, toxoplasmosis, and sarcocystosis in ruminants.

Neospora caninum, Sarcocystis spp, and Toxoplasma gondii are related coccidian parasites that can cause abortion and neonatal mortality in animals. In addition, T gondii and certain species of Sarcocystis are zoonotic. This article reviews information on the etiology, diagnosis, control, and prevention of these diseases.

Prophylactic administration of ponazuril reduces clinical signs and delays seroconversion in horses challenged with Sarcocystis neurona.

The ability of ponazuril to prevent or limit clinical signs of equine protozoal myeloencephalitis (EPM) after infection with Sarcocystis neurona was evaluated. Eighteen horses were assigned to 1 of 3 groups: no treatment, 2.5 mg/kg ponazuril, or 5.0 mg/kg ponazuril. Horses were administered ponazuril, once per day, beginning 7 days before infection (study day 0) and continuing for 28 days postinfection. On day 0, horses were stressed by transport and challenged with 1 million S. neurona sporocysts per horse. Sequential neurologic examinations were performed, and serum and cerebrospinal fluid were collected and assayed for antibodies to S. neurona. All horses in the control group developed neurologic signs, whereas only 71 and 40% of horses in the 2.5 and 5.0 mg/kg ponazuril groups, respectively, developed neurologic abnormalities. This was significant at P = 0.034 by using Fisher exact test. In addition, seroconversion was decreased in the 5.0 mg/kg group compared with the control horses (100 vs. 40%; P = 0.028). Horses with neurologic signs were killed, and a post-mortem examination was performed. Mild-to-moderate, multifocal signs of neuroinflammation were observed. These results confirm that treatment with ponazuril at 5.0 mg/kg minimizes, but does not eliminate, infection and clinical signs of EPM in horses.

Effect of daily administration of pyrantel tartrate in preventing infection in horses experimentally challenged with Sarcocystis neurona.

OBJECTIVE: To determine whether daily administration of pyrantel tartrate can prevent infection in horses experimentally challenged with Sarcocystis neurona. ANIMALS: 24 mixed-breed specific-pathogen-free weanling horses, 10 adult horses, 1 opossum, and 6 mice. PROCEDURE: Sarcocystis neurona-naïve weanling horses were randomly allocated to 2 groups. Group A received pyrantel tartrate at the labeled dose, and group B received a nonmedicated pellet. Both groups were orally inoculated with 100 sporocysts/d for 28 days, 500 sporocysts/d for 28 days, and 1000 sporocysts/d for 56 days. Blood samples were collected weekly, and CSF was collected monthly. Ten seronegative adult horses were monitored as untreated, uninfected control animals. All serum and CSF samples were tested by use of western blot tests to detect antibodies against S. neurona. At the end of the study, the number of seropositive and CSF-positive horses in groups A and B were compared by use of the Fisher exact test. Time to seroconversion on the basis of treatment groups and sex of horses was compared in 2 univariable Cox proportional hazards models. RESULTS: After 134 days of sporocyst inoculation, no significant differences were found between groups A and B for results of western blot tests of serum or CSF There were no significant differences in number of days to seroconversion on the basis of treatment groups or sex of horses. The control horses remained seronegative. CONCLUSIONS AND CLINICAL RELEVANCE: Daily administration of pyrantel tartrate at the current labeled dose does not prevent S. neurona infection in horses.

Prevention of meningo/encephalomyelitis due to Sarcocystis neurona infection in mice is mediated by CD8 cells.

Immunodeficient CD8 knockout mice were infected with Sarcocystis neurona merozoites, in order to determine the role of CD8 cells in protective immunity. Using a direct agglutination test, all infected mice seroconverted by selected time points. Infected mice developed splenomegaly and bilateral lymphadenopathy. Histological changes included marked follicular development in the spleen, endothelitis and moderate perivascular inflammation in the liver, and meningoencephalitis in the brain. Infected brains were positive for S. neurona by polymerase chain reaction. Corresponding to histopathological changes, there were decreased numbers of B-cells in the spleen. The mice did not have significant memory (CD44hi/CD4) or effector (CD45RBhi/CD4) populations present at the time of euthanasia. Flow cytometry confirmed the lack of CD8 cells. Taken together, these data support previous studies suggesting a critical role for CD8 cells in the prevention of menigoencephalitis in S. neurona-infected mice.

Sarcocystis spp. in human infections.

Sarcocystis species are intracellular protozoan parasites with an intermediate-definitive host life cycle based on a prey-predator relationship. Asexual stages develop in intermediate hosts after they ingest the oocyst stage from definitive-host feces and terminate with the formation of intramuscular cysts (sarcocysts). Sarcocysts in meat eaten by a definitive host initiate sexual stages in the intestine that terminate in oocysts excreted in the feces. Most Sarcocystis species infect specific hosts or closely related host species. For example, humans and some primates are definitive hosts for Sarcocystis hominis and S. suihominis after eating raw meat from cattle and pigs, respectively. The prevalence of intestinal sarcocystosis in humans is low and is only rarely associated with illness, except in volunteers who ingest large numbers of sarcocysts. Cases of infection of humans as intermediate hosts, with intramuscular cysts, number less than 100 and are of unknown origin. The asexual stages, including sarcocysts, can stimulate a strong inflammatory response. Livestock have suffered acute debilitating infections, resulting in abortion and death or chronic infections with failure to grow or thrive. This review provides a summary of Sarcocystis biology, including its morphology, life cycle, host specificity, prevalence, diagnosis, treatment, and prevention strategies, for human and food animal infections.

[Parasites in meat: a challenge for veterinarians in meat hygiene]. / Parasiten im Fleisch: Eine Herausforderung für die tierärztliche Fleischhygiene.

Meat hygiene is an important instrument of preventive public health since the end of the nineteenth century. The methods used during ante-mortem and post-mortem inspection are partly quite traditional. This is in particular true for the identification of parasites in meat. The aim of this review was to present facts on aetiology, prevalence and importance of meat-borne parasitic hazards. The capacity and the limits of the ante-mortem and post-mortem inspection for the identification of parasitic hazards are considered. Further, suggestions for the improvement of the meat hygiene are given. Cysticercosis, Toxoplasmosis and Sarcocystosis are exposed in detail. With regard to Cysticercosis, the integration of serological methods in the inspection procedure could result in a tenfold improvement of diagnostical sensitivity. With regard to Toxoplasmosis, it is obvious that meat containing Toxoplasma-cysts may reach the consumer, as animals infected with Toxoplasma gondii can neither be recognised in the ante-mortem inspection nor in the meat inspection. Systematical serological investigations on farm level would allow an appropriate judgement during meat inspection and minimize the consumer exposure to this parasite. Further, reliable methods for the detection of Toxoplasma-cysts have to be developed. With regard to Sarcocystosis, the compulsory inspection of the muscle surfaces to recognise a "massive infection with Sarcocystis" is very doubtful in its interpretation. There is a need for suitable microscopic, serological and molecular biological methods for the detection of Sarcocystis-species and reliable informations on the seroprevalence of the parasite in slaughtering animals.

Evaluation of immune responses in horses immunized using a killed Sarcocystis neurona vaccine.

Clinically normal horses developed cellular immunity to Sarcocystis neurona following IM vaccination with a commercial killed S. neurona vaccine, as indicated by the development of measurable anti-S. neurona IgG antibodies and additional intradermal skin testing. Large-scale independent assessments of the vaccine's performance and safety are in progress under field conditions. The next step in the evaluation of this vaccine would be to attempt experimental challenge after a reproducible reliable equine model of S. neurona encephalitis has been established that allows for reisolation of the pathogen after challenge.

Sarcocystis neurona-specific immunoglobulin G in the serum and cerebrospinal fluid of horses administered S neurona vaccine.

A vaccine against Sarcocystis neurona, which induces equine protozoal myeloencephalitis (EPM), has received conditional licensure in the United States. A major concern is whether the immunoglobulin G (IgG) response elicited by the vaccine will compromise the use of Western blotting (WB) as a diagnostic tool in vaccinated horses with neurologic disease. Our goals were to determine if vaccination (1) causes seroconversion: (2) causes at least a transient increase in S neurona-specific IgG in the cerebrospinal fluid (CSF); and (3) induces an IgG response that can be differentiated from that induced by natural exposure. Horses included in the study (n = 29) were older than 6 months with no evidence of neurologic disease. The presence or absence of anti-S neurona antibodies in the serum of each horse was determined by WB analysis. Seropositive horses had CSF collected and submitted for cytology, CSF index, and WB analysis. The vaccine was administered to all the horses and boostered 3-4 weeks later. On day 14 after the 2nd administration, serum and CSF were collected and analyzed. Eighty-nine percent (8 of 9) of the initial seronegative horses seroconverted after vaccination, of which 57% (4 of 7) had anti-S neurona IgG in their CSE Eighty percent (16 of 20) of the seropositive horses had an increase in serum S neurona IgG after vaccination. Of the 6 of 20 horses that were initially seropositive/CSF negative, 2 were borderline positive for anti-S neurona IgG in the CSF, 2 tested positive, and 2 were excluded because the CSF sample had been contaminated by blood. There were no WB banding patterns that distinguished samples from horses that seroconverted due to vaccination versus natural exposure. Caution must be used in interpreting WB analysis from neurologic horses that have been recently vaccinated for EPM.

Effect of a single dose of ponazuril on neural infection and clinical disease in Sarcocystis neurona-challenged interferon-gamma knockout mice.

Interferon gamma-knockout mice were challenged with 5000 Sarcocystis neurona sporocysts acquired from a naturally infected opossum. Ponazuril was administered once, by gavage, at day 1, 3, 7, 10, or 14 post-infection (pi). Ponazuril was given at either 20 or 200mg/kg. Mice that survived to day 30 pi were euthanized. Severity of CNS infection was quantified as schizont density in the cerebellum. Unchallenged mice in treatment and non-treatment groups remained free of disease and gained weight throughout the experiment. All challenged mice, regardless of treatment, developed histologic evidence of CNS infection even though clinical signs were prevented in some groups. The greatest treatment benefits were seen in mice given 200mg/kg ponazuril between days 4 and 14 pi. Weight gain over the course of the experiment occurred only in mice that were given 200mg/kg ponazuril on day 7 or 10 pi. With the exception of groups given 200mg/kg ponazuril on day 7 or 14 pi, mice in groups that got sporocysts developed abnormal neurologic signs. No deaths before day 30 pi occurred in mice given ponazuril at 20mg/kg on day 7 pi or 200mg/kg on day 1, 7, 10, or 14 pi. This effect was not significant. Mice given 200mg/kg on day 7 pi had significantly fewer cerebellar schizonts than did those of the control group that was not given ponazuril. These results indicate that single-dose administration of ponazuril for prevention of CNS infection is partially protective when given between days 4 and 14 pi.

Prevalence of Sarcocystis infection in meat-producing animals in Mongolia.

A survey of Sarcocystis infection was conducted in Mongolia between June 1998 and July 1999. Samples of muscle were taken from the diaphragm, heart, tongue, esophagus,and intercostal region of cattle, yak, hainag, sheep, horses, and camels. A muscle compress method was used to determine the prevalence of infection: cattle 90.0% (27/30), yak 93.3% (28/30), hainag 100% (30/30), sheep 96.9% (753/777), horses 75% (3/4) and camels 100% (5/5). Of the various muscles, heart was the most commonly infected in cattle (100%), yak (86.7%), and hainag (100%); tongue was most likly to be infected in sheep (100%) and horses (100%).

Effects of high temperature and disinfectants on the viability of Sarcocystis neurona sporocysts.

The effect of moist heat and several disinfectants on Sarcocystis neurona sporocysts was investigated. Sporocysts (4 million) were suspended in water and heated to 50, 55, 60, 65, and 70 C for various times and were then bioassayed in interferon gamma gene knockout (KO) mice. Sporocysts heated to 50 C for 60 min and 55 C for 5 min were infective to KO mice, whereas sporocysts heated to 55 C for 15 min and 60 C or more for 1 min were rendered noninfective to mice. Treatment with bleach (10, 20, and 100%), 2% chlorhexidine, 1% betadine, 5% o-benzyl-p-chlorophenol, 12.56% phenol, 6% benzyl ammonium chloride, and 10% formalin was not effective in killing sporocysts. Treatment with undiluted ammonium hydroxide (29.5% ammonia) for 1 hr killed sporocysts, but treatment with a 10-fold dilution (2.95% ammonia) for 6 hr did not kill sporocysts. These data indicate that heat treatment is the most effective means of killing S. neurona sporocysts in the horse feed or in the environment.