Attempted immunization of swine against acute sarcocystosis using cystozoite-derived vaccines

Low-dose Sarcocystis miescheriana infections have recently been shown to protect pigs against acute sarcocystosis. Because this protective immunity was short-lasting, an alternative immunization strategy was examined. Four experimental vaccines were prepared from S. miescheriana cystozoites and tested in 13 pigs. Two vaccines were prepared from intact organisms (live and formalin-fixed cystozoites) and 2 from subcellular cystozoite fractions (pellicle and protoplasm extracts). The live vaccine was injected intraperitoneally and the remainder were suspended in Freund's incomplete adjuvant and injected intramuscularly. An additional 5 pigs were injected with adjuvant or saline placebos and used as controls. Serum samples were collected regularly and tested in enzyme immunoassays for specific IgM and IgG antibodies. Low levels of IgM antibodies were detected after 8 days and elevated levels of IgG antibodies were detected after 22 days. The success of vaccination was tested 40 days after vaccination by lethal homologous challenge of each pig with 3 X 10(6) sporocysts. Despite the presence of specific antibodies at the time of challenge, all pigs died from acute sarcocystosis 12 days later. The cystozoite vaccines were therefore antigenic but not immunogenic and did not induce any protective immunity.     

Class-specific antibody responses in pigs following immunization and challenge with sporocysts of Sarcocystis miescheriana

Sixteen pigs were each immunized by oral inoculation with 1000 sporocysts of Sarcocystis miescheriana at 8 weeks of age. Four equal groups were then challenged with 3 million sporocysts per animal at 40, 80, 120 or 160 days post-immunization (dpi). Host antibody responses were monitored using the enzyme-linked immunosorbent assay (ELISA) and the indirect fluorescent-antibody test (IFAT). When using class-specific ELISAs, dramatic increases in specific IgM-antibodies were observed at 21 dpi and increasing levels of IgG-antibodies were detected at 34 dpi. IgM-antibody titres dropped relatively quickly to insignificant levels, whereas IgG-antibody titres persisted at high levels until the end of the experiment. Following challenge, elevated levels of IgM-antibodies were detected, whereas IgG-antibody titres remained unchanged. The dynamics of the antibody titres detected by the IgG-IFAT closely corresponded to those detected by the IgG-ELISA. Despite the presence of specific antibodies at the time of challenge and the continued production of IgM-antibodies after challenge, the protective immunity decreased after 40 dpi and had disappeared by 120 dpi. Furthermore, none of the techniques used were suitable for the detection of specific antibodies during the early phase of acute sarcocystosis around 12 days after challenge.     

Persistence of acquired immunity to Sarcocystis miescheriana infection in growing pigs

Sixteen 8-week-old pigs were each experimentally immunized by subclinical infections with 10(3) Sarcocystis miescheriana (syn. S. suicanis) sporocysts and 8 other pigs served as non-immunized controls. Four groups of pigs (each consisting of 4 immunized plus 2 control pigs) were then challenged by infection with 3 X 10(6) sporocysts at either 40, 80, 120 or 160 days post-immunization (dpi) to determine the persistence of the protective immunity against acute sarcocystosis. Pigs challenged 40 dpi demonstrated a solid immunity to lethal challenge and disease. They survived challenge following a mild fever phase whereas both controls died from acute disease. This immunity however, did not prevent the further establishment of parasitic cysts within the host musculature following challenge. The protective immunity against acute disease persisted to 80 dpi, but was not evident thereafter. At necropsy, the clinical and pathological findings in all pigs which had been subjected to challenge were consistent with anamnestic responses of sensitized hosts to re-infection.     

Hemostatic alterations in pigs fed sublethal doses of Sarcocystis miescheriana

Effects of non-lethal Sarcocystis miescheriana infections on the blood coagulation system were investigated. Nine pigs were inoculated orally with 2 X 10(5) sporocysts (Group A) and nine pigs (Group B) served as non-infected controls. Blood samples were taken from the vena jugularis externa every 2 or 3 days until 19 days post-infection (dpi). The following parameters were investigated: partial thromboplastin time (PTT), prothrombin time (PT), thrombin time (TT), thrombin coagulase time (TCT), fibrinogen (FIB), factor (F) VIII, F XI, F XII, antithrombin III (AT III), alpha 2 macroglobulin (alpha 2 MG), alpha 2 antiplasmin (alpha 2 AP), pre-kallikrein (PK), and the number of circulating thrombocytes. All infected pigs suffered from acute sarcocystiosis between 12 and 19 dpi. Clinical illness was most severe from 14 to 17 dpi. At this time, PTT and FIB increased, and TT and TCT decreased slightly. The activities of the clotting factors increased at 17 and 19 dpi. However, only F VIII activity was significantly higher in the infected pigs than in the controls at 17 and 19 dpi. PK was significantly lower in the infected pigs at 12, 14, and 17 dpi. Thrombocyte counts were reduced with the onset of the acute phase of illness and some pigs had marked thrombocytopenia. These results indicate low-grade disseminated intravascular coagulation (DIC) in the course of mild S. miescheriana infections in pigs.     

Heart failure in a pig chronically infected with Sarcocystis miescheriana--a case report

A pig infected with 2 x 10(5) sporocysts of Sarcocystis miescheriana which had survived the acute phase of the disease from 12 dpi until 17 dpi retarded in growth and finally died at 60 dpi. From gross pathological examination heart failure was assumed as the cause of death. Histopathologically severe Myocarditis eosinophilica fibrosa was diagnosed. The sections through the heart muscle contained numerous degenerating and some intact sarcocysts.     

Antibody development and cellular immune responses in sheep immunized and challenged with Sarcocystis tenella sporocysts

Four specific-pathogen-free (SPF) sheep were experimentally infected with 10(3) or 10(4) Sarcocystis tenella (syn. S. ovicanis) sporocysts and another two sheep served as uninfected controls. All sheep were challenged 49 days later by infection with 2.5 X 10(5) sporocysts and their humoral and cellular responses to infection and challenge were assessed weekly by enzyme immunoassays and lymphocyte transformation assays. The control sheep died from acute sarcocystosis 29-30 days after challenge, whereas the immunized sheep survived and were protected against acute disease. Specific IgM and IgG antibodies were detected in the immunized sheep from 28 days after infection onwards. Lymphocytes collected before and after challenge did not exhibit any significant differences in their responses to stimulation with S. tenella cystozoite or sporozoite antigens. Furthermore, lymphocytes collected before challenge did not differ from the controls in their responses to stimulation with the mitogens lipopolysaccharide or phytohaemagglutinin. However, lymphocytes collected after challenge did exhibit increased blastogenic responses to stimulation with both mitogens from 21-28 days after challenge onwards. The infected sheep were necropsied 46 days after challenge, and histological and ultrastructural studies revealed numerous infiltrates of lymphocytes, histiocytes and plasma cells in the skeletal muscles, sometimes in association with degenerating parasitic cysts and macrophage myophagia. Parasites were not completely eliminated nor prevented from further establishment, therefore the protective immunity was not sterile but rather a state of premunition.     

In vitro and in vivo interaction between sporocysts of Sarcocystis muris and mouse peritoneal macrophages

The interaction between the sporocysts of Sarcocystis muris and mouse peritoneal macrophages was studied both in vitro and in vivo in an attempt to determine whether or not resident peritoneal macrophages might effect the excystation of S. muris sporozoites from sporocysts injected intraperitoneally. Sporocysts of S. muris were phagocytosed by peritoneal macrophages both in vitro and in vivo. The addition of either unheated mouse serum or fetal calf serum did not significantly alter the level of phagocytosis. The percentage of phagocytosis in vivo and by thioglycolate-, proteose peptone- and BCG-elicited macrophages in vitro was greater than that shown by unstimulated macrophages in vitro. After 8 h incubation in vivo and in vitro a small proportion of sporocysts (less than 5%) was seen to have collapsed walls and up to 5% to have stained sporozoites, suggesting increased permeability of the sporocyst wall. The significance of increased permeability of the cyst wall in the process of sporozoite excystation is discussed.     

Characterization of monoclonal antibodies against ovine Sarcocystis spp. antigens by immunoblotting and immuno-electron microscopy

Six monoclonal antibodies were raised in mice against purified cytozoite extracts of Sarcocystis gigantea and S. tenella from sheep. Each monoclonal antibody was evaluated for specificity by enzyme immunoassay, immunoblotting and immuno-electron microscopy using homologous and heterologous antigenic preparations. All six monoclonal antibodies exhibited good species-specificity when reacted against crude soluble cystozoite antigens in enzyme immunoassays. However, only two monoclonal antibodies (IgM and IgG2a) exhibited reactivity in Western blots against specific protein bands. Both reacted against S. gigantea antigens of 100,000, 43,000 and 39,000 molecular weight. Neither monoclonal antibody reacted against the heterologous species S. tenella. Ultrastructural studies performed with colloidal-gold conjugated antisera revealed that both monoclonal antibodies reacted against antigens located around micronemes and amylopectin granules in S. gigantea cystozoites. Another monoclonal antibody (IgGI) reacted only against microneme determinants in S. tenella cystozoites. In contrast, polyclonal sheep and rabbit immune sera cross-reacted against a wide range of cystozoite antigens.     

Lipid metabolism and Sarcocystis miescheriana infection in growing swine.

Sixteen 2-month-old pigs were divided into four equal groups and infected with either 500,000, 1,000,000 or 3,000,000 sporocysts of Sarcocystis miescheriana. Four pigs served as uninfected controls. Pigs were bled weekly and serum was collected beginning 14 days prior to infection and continuing until 63 days after infection. Body fat composition, as measured by the specific gravity of the carcass, was not affected by infection. There were no significant effects of infection on serum concentrations of glucose, insulin, triglycerides, and total, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol. A slight depression in HDL cholesterol occurred during the acute phase of infection. Tumor necrosis factor (TNF) was not detected in serum from infected swine when assayed by a cytotoxicity assay using TNF-sensitive WEHI 164 clone 13 cells. Attempts to stimulate TNF production in RAW 264.7 cells with parasitic lysates gave mixed results. This study suggests that the disruption of lipid metabolism is not the primary cause of growth retardation in growing swine infected with S. miescheriana.     

Variation in clinical and parasitological traits in Pietrain and Meishan pigs infected with Sarcocystis miescheriana

Future prophylaxis needs new concepts, including natural disease resistance of hosts against infectious agents. Genomic approaches to detect and improve disease resistance in farm animals and the molecular mechanisms involved in host-parasite interactions depend to a high degree on the trait differences between founder breeds, i.e. on the animal model. The present study evaluates differences in susceptibility/resistance against Sarcocystis miescheriana in the European Pietrain (PI) and the Chinese Meishan (ME) pig breeds, based on 25 individuals, infected orally with 5x10(4) sporocysts of S. miescheriana. Significant differences appeared in clinical, serological, haematological and parasitological findings. The major discriminating period post infection (p.i.) was between days 42 and 45. Severity of signs was negatively correlated with specific immunoglobulin titres during the first 3 weeks p.i. and positively with the load of bradyzoites in muscle tissues of the pigs. Loads of bradyzoites in muscle tissues were 20 times higher in PI than in ME. Sarcocystis-specific differences between the two breeds were in the range of 1-2 standard deviations. The study lays the foundation for further experiments to analyse chromosomal regions, candidate genes, and thus the molecular basis of Sarcocystis susceptibility/resistance as a model for host-parasite interaction in protozoan infectious disease.     

应用斑点酶联免疫吸附试验快速检测猪弓形虫抗体

将斑点酶联免疫吸附试验(Dot—ELISA)用于检测猪弓形虫抗体,并与常规ELISA和IHA法进行了比较。结果,对102份滴度下降的猪阳性血清检测,弓形虫抗体阳性检出率,Dot—ELISA为66.67％(68/102),常规ELISA为48.04％(49/102),IHA为27.45％(28/102);对675头商品猪血清检测,弓形虫抗体阳性检出率,Dot—ELISA为48.15％(325/675),常规ELISA为41.93％(283/675),IHA为33.80％(228/675);与3种寄生虫(猪囊虫、猪旋毛虫、住肉孢子虫)阳性血清无交叉反应;对123份弓形虫抗体阳性和158份阴性猪血清进行3次重复性试验,结果完全一致。结果证明,该法敏感性高,特异性强,操作简便快速(于接到病料后2h报告结果),便于在基层推广使用。     

Sarcocystis infection in pigs of Hissar, Haryana, India and its transmission to dogs

Sarcocystis zoites were found in pepsin digests of 68.8% of 157 pigs from Hissar, Haryana. Sarcocystis-infected meat was fed to 4 young dogs and 2 cats. The dogs shed Sarcocystis sporocysts in their faeces 12 days after eating infected meat whereas cats did not shed sporocysts.     

Clinical, haematological and plasma biochemical changes in specified-pathogen-free (sporozoa) lambs experimentally infected with low numbers of Sarcocystis tenella sporocysts

Two groups of lambs raised free of sporozoan infection were inoculated with Sarcocystis tenella sporocysts and compared with controls. Lambs from Group 1 were inoculated with 5000 sporocysts and those in Group 2 were given 20,000. Transient increases in rectal temperatures occurred between 23 and 39 days post-inoculation (dpi), although the lambs appeared normal and retained their appetites. Packed cell volumes (PCV) of lambs given 20,000 sporocysts decreased dramatically from 28 to 38 dpi after which they slowly returned to near pre-inoculation levels by 99 dpi. The anaemia was normocytic/normochromic. White cell counts (WCC) rose in infected lambs from 49 dpi, reflecting principally an increase in lymphocyte numbers. Plasma albumin of Group 2 decreased at 28 dpi and remained depressed until the experiment was terminated at 99 dpi. Plasma globulin of infected groups increased from 31 (Group 2) and 35 dpi (Group 1). Plasma alkaline phosphatase (ALP) of Group 2 decreased from 28 dpi and remained depressed to 99 dpi. Lactate dehydrogenase (LDH) of Group 2 was elevated at 24 and 28 dpi and from 42 to 78 dpi, while aspartate aminotransferase (AST) of the same group was elevated from 45 to 66 dpi. Creatine kinase (CK) of Group 2 was elevated from 52 to 71 dpi.     

Prevalence of Sarcocystis in domestic pigs in India.

Muscle samples from 890 slaughtered pigs were examined for the presence of sarcocysts. A high prevalence rate of 67.98% was observed. Two types of microsarcocysts were recorded. The sarcocyst wall of one type had redial striations and the other possessed hair-like villar protrusions. The species were identified as Sarcocystis miescheriana and Sarcocystis suihominis; there was a slightly higher incidence of the latter species (47.11%) than of the former (43.14%). S. suihominis has been identified for the first time from pigs in India.     

Comparison of Dot-ELISA, ELISA and IFAT for the detection of IgG antibodies to Sarcocystis muris in experimentally infected and immunized mice

The dot enzyme-linked immunosorbent assay (Dot-ELISA) was used for the detection of IgG antibodies to Sarcocystis muris and compared with the enzyme-linked immunosorbent assay (ELISA) and the indirect fluorescent antibody test (IFAT). In experimentally infected mice, first positive reactions occurred in the Dot-ELISA between 18 and 32 days after infection (dpi), in the ELISA between 18 and 49 dpi, and in the IFAT between 11 and 25 dpi. Maximum titers were 1:40 960 in the Dot-ELISA, 1:1280 in the ELISA and 1:2560 in the IFAT. High titers persisted until the end of the examination period (182 dpi) in all 3 tests. In immunized mice, all 3 tests detected antibodies 7 days after the first injection of protein antigen. The highest titers of 1:5120 and 1:10 240 were recorded in the Dot-ELISA after 35 days; titers of 1:1280 and 1:2560 were observed in the ELISA, and titers of 1:160 and 1:320 in the IFAT after 42 days. No false-positive reactions occurred in the Dot-ELISA and in the IFAT when 177 sera from non-infected mice were examined, but 1% (2/177) of the sera reacted positively in the ELISA. Sixty-three percent (94/150) of sera from mice infected experimentally with Toxoplasma gondii showed slight positive reactions up to 1:40 in the Dot-ELISA; 9% (13/150) of the sera reacted positively up to 1:40 in the IFAT and 4% (6/150) up to 1:20 in the ELISA. The Dot-ELISA appears to be a good alternative to the ELISA and the IFAT in the serodiagnosis of sarcosporidiosis and should be further evaluated for the serodiagnosis of other parasitic diseases.     

Prevalence of Sarcocystis cysts in pigs and sheep in Spain

Samples of serum and diaphragm muscle were collected from 100 pigs, and serum samples and oesophagi were collected from 100 sheep. The diaphragm muscle and oesophageal tissues were examined for the presence of macroscopic and microscopic Sarcocystis cysts by compression between trichinoscope plates as well as by tissue digestion with pepsin solution. The sera were examined by the indirect haemagglutination test (IHA), using antigens from Sarcocystis gigantea. With these methods, 95% of the sheep and 43% of the pigs were found to be infected with Sarcocystis sp.     

Sarcocystis infections in Georgia swine

Tissues from 168 mature sows obtained at slaughter in northern and southern Georgia were examined for infection with Sarcocystis spp. Digestion techniques revealed zoites in 28 (16.5%) sows. Infected meat was fed to laboratory-reared dogs, cats, raccoons (Procyon lotor), and opossums (Didelphis marsupialis). Dogs and raccoons shed sporocysts (8.3 mum X 11.2 mum) 12 to 14 days after infection. Cats and opossums were refractory to infection. Sarcocystis-free pigs were infected with 50,000 sporocysts of swine-dog origin. Tissues from laboratory-infected swine were fed to dogs and raccoons. Both species shed sporocysts 14 days later. This is the 1st time in which a definitive host has been demonstrated for species of Sarcocystis occurring in North American swine. The raccoon constitutes a new definitive host for S suicanis Erber 1977. In contrast to previously reported low prevalences of Sarcocystis infections in swine, the relatively high prevalence reported here indicates that S suicanis may be of importance to swine producers in Georgia.     

Effects of Sarcocystis miescheriana infection on carcass quality and on the water-binding capacity of the meat of halothane-tested fattening pigs

Ten halothane-positive pigs (stress sensitive, group A) and ten halothane-negative pigs (stress insensitive, Group C) with a mean body weight of 36 kg were each inoculated orally with 50,000 sporocysts of Sarcocystis miescheriana. Twelve halthane-positive pigs (Group B) and ten halothane-negative pigs (Group D) served as non-infected controls. Thirteen weeks post infection (p.i.) the lean: fat ratios of the pigs of the infected groups A and C were lower (A, 1:0.41 +/- 0.09; C, 1:0.50 +/- 0.10) than those of the pigs of the non-infected groups B and D (B, 1:0.50 +/- 0.08; D, 1:0.55 +/- 0.08). The back-fat thickness, the fat thickness 'A' and the fat thickness 'B' were thinner in infected pigs than in non-infected pigs. The difference in Lendenst?rkespeckquotient (Loin Fat Thickness Quotient) (LSQ) between infected and non-infected pigs was not statistically significant. The values of the water-holding capacity were lower in infected pigs than in non-infected pigs, the difference being statistically significant only in the halothane-negative groups (C, 0.45 +/- 0.02; D, 0.48 +/- 0.04). The water-absorbing capacity was significantly higher in the infected groups (A, 5.92 +/- 3.99%; B, 2.26 +/- 1.08%; C, 8.96 +/- 2.90%; D, 4.97 +/- 2.51%). In conclusion, it can be said that there was a slight tendency towards a better carcass quality and a better water-binding capacity in infected pigs, although this was combined with reduced growth rates.     

Unusual biphasic disease in domestic pigeons (Columba livia f. domestica) following experimental infection with Sarcocystis calchasi

A novel Sarcocystis species has recently been reported in the domestic pigeon (Columba livia f. domestica) as intermediate host, causing severe central nervous signs similar to Paramyxovirus-1 or Salmonella Typhimurium var. cop. infection. Transmission of the parasite via the northern goshawk (Accipiter gentilis) as definitive host has been established. Experimental infection of domestic pigeons with sporocysts excreted by experimentally infected northern goshawks reproduced the natural infection in the pigeon, proving the causative role of the parasite in the disease. Here, we describe in greater detail the course of the fulminant biphasic disease depending on the infectious dose. Pigeons infected with 10(3) or 10(4) sporocysts showed clinical signs of polyuria and apathy around 10-11 days postinfection (dpi) and sudden neurological signs 51-57 dpi as a second phase of disease. Pigeons infected with higher doses died within 7-12 dpi, also showing polyuria and apathy but without nervous signs. At necropsy, livers and spleens had multifocal necroses and infestations with parasitic stages, namely, schizonts. Moreover, lesions and schizonts were also found in the lung, bone marrow, and next to blood vessels in the connective tissue of various organs. Pigeons infected with 102 sporocysts remained symptomless until 58-65 dpi, when sudden central nervous signs occurred. Major histopathologic findings of pigeons with neurological signs were encephalitis and myositis of virtually every skeletal muscle with high infestations of sarcocysts. Only mild myocarditis and very few cysts were found in the heart muscles. Importantly, a sentinel pigeon developed identical lesions when compared to those of low-dose infected pigeons, suggesting a risk of mechanical transmission of sporocysts from freshly infected to uninfected pigeons in a flock. By contrast, chickens failed to develop any clinical signs or pathologic lesions in the same experiment. The findings further characterize the new highly pathogenic disease in domestic pigeons, which clinically mimics paramyxovirosis and salmonellosis in both phases of the disease and exclude chickens as further intermediate host species.     

Kinetics of circulating antigens in pigs experimentally infected with Taenia solium eggs

Three groups of four piglets were experimentally infected with different doses (10(3), 10(4) and 10(5)) of Taenia solium eggs whereas a fourth group of two pigs received gravid proglottids. At autopsy 6 months post infection, the two latter pigs were heavily infected with more than 3000 living cysts per kg of muscle. Ten of the 12 other pigs harboured light infections, i.e. between 2 and 107 cysticerci, 42.4% of which were degenerated. The two remaining pigs had no detectable cysts at post mortem examination. Circulating antigens (CA) were detected in the sera of all pigs harbouring living cysticerci using a monoclonal antibody based ELISA. CA were first detected between 2 and 6 weeks post infection and remained present generally throughout the entire observation period even in pigs carrying only five to eight living cysts, although strong fluctuations of the level of CA were observed in some pigs. In animals without living cysts at post mortem CA were only detected for a short period and disappeared presumably when the cysticerci became degenerated. The minimum number of living cysts, which could be detected using this ELISA, was 1.