The in vitro excystation of Sarcocystis gigantea sporocysts

Studies on the in vitro excystation of Sarcocystis gigantea sporocysts revealed that pretreatment before exposure to trypsin and bile was an essential prerequisite. However, in contrast to Sarcocystis tenella and Sarcocystis capracanis, incubation in cysteine hydrochloride under CO2 was largely unsuccessful for excysting Sarcocystis gigantea: of the pretreatments tested, only exposure to sodium hypochlorite proved effective. Excystation from sodium hypochlorite-pretreated S. gigantea sporocysts took place in trypsin and bile between temperatures of 30 and 43 degrees C and occurred rapidly at 39 degrees C. While the presence of bile or bile salts was essential for this process, that of trypsin was not, although more sporocysts excysted in its presence than in its absence. Excystation occurred in the presence of all bile types tested but not when Tween 80 was substituted for bile. The highest levels of excystation were recorded when cattle or sheep bile or sodium taurocholate were used and the lowest when chicken or pig bile were employed. Neither the concentration of sheep bile above 2.5%, nor hydrogen ion concentration (pH range 5.0-10.0) appeared to have any marked effect on the level of excystation obtained.     

The in vivo excystation of Sarcocystis gigantea and S. tenella sporocysts

In vivo studies on the excystation of Sarcocystis gigantea and S. tenella sporocysts indicated that this process was, as in vitro, a diphasic one involving both pretreatment and treatment phases. The studies also tended to support in vitro observations that the requirements for the excystation of these two species are quite different. The results suggested that for neither species was the pretreatment stimulus likely to be provided by conditions in the rumen alone. However, exposure to abomasal conditions only induced moderate levels of excystation in both when they were subsequently treated with trypsin and bile. For S. gigantea, 0.25 to 4 h abomasal exposure was most effective; for S. tenella, 24 hours. The stimuli necessary to complete the excystation process could, apparently, be provided by 1 h placement in the duodenum for S. gigantea but not for S. tenella.     

Recovery of Sarcocystis gigantea sporocysts from cat faeces

A method for the mass recovery of S. gigantea sporocysts from cat faeces involving homogenisation and centrifugation in water, passage through 250- and 53-microns sieves and floatation in 1.2 SG NaCl solution, is described. An examination of the various processes involved in this procedure showed that the greatest yields were obtained when a proportion of faeces to floatation medium of 1:20 and centrifugation at 6000 X g for at least 5 min was used. Ninety-six per cent of the sporocysts recovered were obtained from the first centrifugation in aqueous NaCl solution (SG 1.2). Although neither sieving nor additional washing of homogenised samples prior to floatation significantly affected sporocyst recovery, both reduced the amount of debris present. A considerable reduction in the amount of debris resulted from feeding infected cats on tinned fish rather than tinned meat. The addition of CCl4 to the NaCl solution also improved sporocyst purity but with a marked reduction in the numbers recovered.     

Evaluation of a concentration method for counting Sarcocystis gigantea sporocysts in cat faeces

A technique for determining the numbers of S. gigantea sporocysts in cat faeces using a concentration procedure and haemocytometer was evaluated. The results showed that it was more accurate than a modified McMaster method and had a mean recovery rate of 73% at four levels of infection ranging from about 2000 to over 20,000 sporocysts per gram of faeces.     

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.     

巨型住肉孢子虫烯醇酶基因克隆与表达

巨型住肉孢子虫(Sarcocystis gigantea)是羊体内一种常见寄生虫,多寄生于羊横纹肌内形成包囊,导致羊肉大量废弃,给畜牧业带来巨大经济损失。本研究尝试筛选能有效诊断住肉孢子虫感染的抗原。通过免疫印迹及质谱分析筛选到巨型住肉孢子虫候选诊断抗原烯醇酶,利用染色体步移技术扩增两侧翼未知序列并表达重组蛋白。应用生物信息学分析和免疫印迹对该蛋白诊断价值进行评价。结果筛选到的候选蛋白为巨型住肉孢子虫烯醇酶(SgENO),克隆得到1 181 bp的基因并获得重组蛋白rSgENO。对rSgENO应用进行初步评价,发现其与其他顶复亚门原虫的烯醇酶氨基酸相似性均较高(71%～92.1%),且能被弓形虫和新孢子虫阳性血清所识别,具有交叉反应性。本研究克隆并表达了巨型住肉孢子虫烯醇酶,后期评价发现该重组蛋白与新孢子虫和弓形虫有较强的交叉反应性,不能用于羊住肉孢子虫的血清学诊断,但有成为疫苗候选分子的潜力。     

Viability of Sarcocystis neurona sporocysts and dose titration in gamma-interferon knockout mice

Gamma-interferon knockout mice have become the model animal used for studies on Sarcocystis neurona. In order to determine the viability of S. neurona sporocysts and to evaluate the course of the disease in these mice, sporocysts were collected from opossums (Didelphis virginiana), processed, and stored for varying periods of time. Gamma-interferon knockout mice were then inoculated orally with different isolates at different doses. These animals were observed daily for clinical signs until they died or it appeared necessary to humanely euthanize them. 15 of 17 (88%) mice died or showed clinical signs consistent with neurologic disease. The clinical neurologic symptoms observed in these mice appeared to be similar to those observed in horses. 15 of 17 (88%) mice were euthanized or dead by day 35 and organisms were observed in the brains of 13 of 17 (77%) mice. Dose appeared not to effect clinical signs, but did effect the amount of time in which the course of disease was completed with some isolates. The minimum effective dose in this study was 500 orally inoculated sporocysts. Efforts to titrate to smaller doses were not attempted. Direct correlation can be made between molecularly characterized S. neurona sporocysts and their ability to cause neurologic disease in gamma-interferon knockout mice.     

Immunoconversion against Sarcocystis neurona in normal and dexamethasone-treated horses challenged with S. neurona sporocysts

Equine protozoal myeloencephalitis is a common neurologic disease of horses in the Americas usually caused by Sarcocystis neurona. To date, the disease has not been induced in horses using characterized sporocysts from Didelphis virginiana, the definitive host. S. neurona sporocysts from 15 naturally infected opossums were fed to horses seronegative for antibodies against S. neurona. Eight horses were given 5x10(5) sporocysts daily for 7 days. Horses were examined for abnormal clinical signs, and blood and cerebrospinal fluid were harvested at intervals for 90 days after the first day of challenge and analyzed both qualitatively (western blot) and quantitatively (anti-17kDa) for anti-S. neurona IgG. Four of the challenged horses were given dexamethasone (0.1mg/kg orally once daily) for the duration of the experiment. All challenged horses immunoconverted against S. neurona in blood within 32 days of challenge and in CSF within 61 days. There was a trend (P = 0.057) for horses given dexamethasone to immunoconvert earlier than horses that were not immunosuppressed. Anti-17kDa was detected in the CSF of all challenged horses by day 61. This response was statistically greater at day 32 in horses given dexamethasone. Control horses remained seronegative throughout the period in which all challenged horses converted. One control horse immunoconverted in blood at day 75 and in CSF at day 89. Signs of neurologic disease were mild to equivocal in challenged horses. Horses given dexamethasone had more severe signs of limb weakness than did horses not given dexamethasone; however, we could not determine whether these signs were due to spinal cord disease or to effects of systemic illness. At necropsy, mild-moderate multifocal gliosis and neurophagia were found histologically in the spinal cords of 7/8 challenged horses. No organisms were seen either in routinely processed sections or by immunohistochemistry. Although neurologic disease comparable to naturally occurring equine protozoal myeloencephalitis (EPM) was not produced, we had clear evidence of an immune response to challenge both systemically and in the CNS. Broad immunosuppression with dexamethasone did not increase the severity of histologic changes in the CNS of challenged horses. Future work must focus on defining the factors that govern progression of inapparent S. neurona infection to EPM.     

Canine coronavirus inactivation with physical and chemical agents

Canine coronavirus (CCoV) is responsible for mild or moderate enteritis in puppies. The virus is highly contagious and avoiding contact with infected dogs and their excretions is the only way to ensure disease prevention. Since no studies have yet focused on the sensitivity of CCoV to chemical biocides the present investigation examined the efficiency of physical and chemical methods of viral inactivation. CCoV infectivity was stable at +56 degrees C for up to 30 min, but tended to decrease rapidly at +65 degrees C and +75 degrees C. Germicidal ultra-violet (UV-C) light exposure demonstrated no significant effects on virus inactivation for up to 3 days. CCoV was observed to be more stable at pH 6.0-6.5 while extreme acidic conditions inactivated the virus. Two tested aldehydes inactivated the virus but their action was temperature- and time-dependent. The methods for CCoV inactivation could be applied as animal models to study human coronavirus infection, reducing the risk of accidental exposure of researchers to pathogens during routine laboratory procedures.     

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.     

Antibody index and specific antibody quotient in horses after intragastric administration of Sarcocystis neurona sporocysts

OBJECTIVE: To investigate the use of a specific antibody index (AI) that relates Sarcocystis neurona-specific IgG quotient (Q(SN)) to total IgG quotient (Q(IgG)) for the detection of the anti-S neurona antibody fraction of CNS origin in CSF samples obtained from horses after intragastric administration of S neurona sporocysts. ANIMALS: 18 adult horses. PROCEDURES: 14 horses underwent intragastric inoculation (day 0) with S neurona sporocysts, and 4 horses remained unchallenged; blood and CSF samples were collected on days - 1 and 84. For purposes of another study, some challenged horses received intermittent administration of ponazuril (20 mg/kg, PO). Sarcocystis neurona-specific IgG concentrations in CSF (SN(CSF)) and plasma (SN(plasma)) were measured via a direct ELISA involving merozoite lysate antigen and reported as ELISA units (EUs; arbitrary units based on a nominal titer for undiluted immune plasma of 100,000 EUs/mL). Total IgG concentrations in CSF (IgG(CSF)) and plasma (IgG(plasma)) were quantified via a sandwich ELISA and a radial immunodiffusion assay, respectively; Q(SN), Q(IgG), and AI were calculated. RESULTS: Following sporocyst challenge, mean +/- SEM SN(CSF) and SN(plasma) increased significantly (from 8.8 +/- 1.0 EUs/mL to 270.0 +/- 112.7 EUs/mL and from 1,737 +/- 245 EUs/mL to 43,169 +/- 13,770 EUs/mL, respectively). Challenge did not affect total IgG concentration, Q(SN), Q(IgG), or AI. CONCLUSIONS AND CLINICAL RELEVANCE: S neurona-specific IgG detected in CSF samples from sporocyst-challenged horses appeared to be extraneural in origin; thus, this experimental challenge may not reliably result in CNS infection. Calculation of a specific AI may have application to the diagnosis of S neurona-associated myeloencephalitis in horses.     

Susceptibility of an avian pneumovirus isolated from Minnesota turkeys to physical and chemical agents

Survival characteristics of a Minnesota avian pneumovirus (APV) isolated from a turkey nasoturbinate, propagated in tissue culture, and exposed to various physiochemical treatments were determined. These characteristics included survivability under various conditions. Specifically, APV was viable at temperatures of -70 C and -20 C for over 26 wk, 4 C for less than 12 wk, 20 C for less than 4 wk, 37 C for 48 hr, and 50 C for less than 6 hr. In addition, APV survived 12 freeze/thaw cycles with no loss of activity. With a variable pH for 1 hr, the titer was unaffected between the levels of pH 5 and 9. Several disinfectants, including quaternary ammonia, ethanol, iodophor, a phenol derivative, a biguanide, and bleach, were all effective in reducing the viability of the virus. After 7 days of drying at room temperature, the APV remained viable and was recovered on cell culture.     

Concurrent presence of Sarcocystis neurona sporocysts, Besnoitia darlingi tissue cysts, and Sarcocystis inghami sarcocysts in naturally infected opossums (Didelphis virginiana).

Opossums (Didelphis virginiana) are exposed to a wide range of coccidia through feeding on a variety of foods, including, but not limited to, carrion, insects, and nestling birds. Abundant D. virginiana populations in urban and suburban areas can be important reservoirs of parasitic infection because of their profuse and prolonged excretion of the sporocysts of several species of Sarcocystis, their omnivorous diet, and their relatively long life span. This report describes 2 adult female opossums found to be simultaneously infected with the tissue cysts of Besnoitia darlingi, sarcocysts of Sarcocystis inghami, as well as with the intestinal sporocysts of S. neurona. Cysts typical of B. darlingi based on gross, histological, and ultrastructural characteristics were disseminated throughout the visceral organs, musculature, ears, and skin. The S. neurona and B. darlingi infections were confirmed by comparative sequence analysis of polymerase chain reaction-amplified diagnostic genetic loci. Sarcocysts of S. inghami are also described. Such examples of multiple parasitic infections show that concurrent infections occur naturally. The propensity for species to coexist should be considered in the differential diagnosis of tissue cyst-forming coccidian protozoa and may have important epidemiological and evolutionary implications.     

Migration and development of Sarcocystis neurona in tissues of interferon gamma knockout mice fed sporocysts from a naturally infected opossum

Migration and development of Sarcocystis neurona was studied in 50 gamma interferon knockout mice fed graded doses of S. neurona sporocysts from the intestine of a naturally infected opossum. Mice were examined at necropsy 1-62 days after feeding sporocysts (DAFS). All tissue sections were reacted with anti-S. neurona-specific polyclonal rabbit serum in an immunohistochemical (IHC) test. Between 1 and 3 DAFS, organisms were seen mainly in intestines. Between 4 and 11 DAFS, organisms were seen in several visceral tissues. Beginning with 13 DAFS, schizonts and merozoites were present in sections of brains of all infected mice. All regions of the brain were parasitized but the hind brain was most severely affected. S. neurona was found in the spinal cord of all 10 mice examined 22-30 DAFS. Of the 28 infected mice examined 20-62 DAFS, S. neurona was found in the brains of all 28, lungs of 14, hearts of 8 and eyes of 3. More organisms were seen in IHC-stained sections than in sections stained with hematoxylin and eosin. Treatment of tissues with glutaraldehyde, Karnovsky fixative, and ethylene diamino tetra acetic acid (EDTA, used for decalcification) did not affect staining of organisms by IHC.     

Resistance of Berne virus to physical and chemical treatment

Thermal inactivation of Berne virus proceeded at a linear rate between 31 degrees and 43 degrees C. Storage at temperatures lower than -20 degrees C preserved the infectivity, while at 4 degrees C appreciable loss occurred between 92 and 185 days. Freeze-drying or desiccation at 22 degrees C caused only insignificant loss of infectivity. Virus preparations were not affected by pH values between 2.5 and 10.3. Inactivation by UV occurred more rapidly than with herpes, toga and rhabdoviruses. Berne virus infectivity was sensitive to pronase and B. subtilis proteinase. It was not inactivated by trypsin and chymotrypsin treatment, which resulted in enhancement of infectivity; low concentrations of pronase (less than 10 micrograms ml-1) had a similar effect on Berne virus. Neither phospholipase C or RNase, alone or in combination, nor sodium deoxycholate (0.1%) inactivated the virus; in contrast, Triton X-100 (0.1%; 1.0%) caused rapid inactivation with a constant level of residual infectivity.     

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.     

苜蓿皂甙的理化性质及其生物学功能

虽然苜蓿皂甙作为一种抗营养因子限制了苜蓿在畜牧生产中的广泛应用,但苜蓿皂甙能够降低单胃动物血浆胆固醇,为人类提供安全、保健的畜产品。为此本文对苜蓿皂甙的理化性质、制备及生物学功能进行了综述。