Survival of Isospora suis oocysts under controlled environmental conditions

Isospora suis is a coccidian parasite infecting piglets soon after birth. While the gross epidemiology of I. suis is well known, little knowledge exists on the ecology of the oocysts. To study the development and survival of oocysts of I. suis under controlled laboratory conditions, known numbers of oocysts ( approximately 200 in each of 4 replicates) were exposed to all combinations of 4 relative humidities (53-100% RH) and 3 temperatures (20 degrees , 25 degrees , 30 degrees C). Determination of viability was based on morphological and fluorescent properties of the oocyst as well as on the permeability of the oocyst wall characterized by inclusion/exclusion of the fluorescent dye propidium iodide. The viability of the oocysts was studied over time by fluorescence and light microscopy until <5% of the oocysts were considered to be viable. The sporulation rate increased with temperature, however, the infective sporocyst stage was reached within 24h at all temperatures, while RH did not seem to affect sporulation. Results show a rapid reduction in viable oocysts exposed to high temperatures (25 degrees C and 30 degrees C) in combination with low relative humidities (53% RH and 62% RH), at which conditions oocysts died within 24h. Viability was higher when oocysts were exposed to higher relative humidities (75% RH and 100% RH) as well as a lower temperature (20 degrees C). However, even at 75% RH the oocysts died within 24-60 h at 30 degrees C to 20 degrees C, respectively, while the most favourable condition appeared to be 100% RH and 25 degrees C at which condition the percentage of viable oocysts decreased from 100% to 17% in 96 h. The results indicate that it may be possible to reduce the infection pressure of I. suis in modern sow herds by changing the environmental conditions and/or the management within the farrowing pens, and thereby increase animal welfare without relying on the use of routine medication.     

Prevalence of oocysts of Isospora suis and Eimeria spp from sows on farms with and without a history of neonatal coccidiosis

Oocysts of Isospora suis were not found in any of 77 fecal samples from sows on farms with a history of neonatal coccidiosis. Oocysts of Isospora suis were found in 1 of 172 fecal samples from farms without a history of neonatal coccidiosis. Oocysts of Eimeria spp were found in 81.8% of the sows from farms with a history of neonatal coccidiosis and in 94.8% of the sows from farms without a history of neonatal coccidiosis. Oocysts of Isospora spp from birds were encountered as a pseudoparasite in several fecal samples.     

东北虎源等孢球虫在猫体内的卵囊排出规律观察

为研究猫体内的卵囊排出规律,本研究分离的东北虎源等孢球虫纯种卵囊,在猫体内多次传代后获得充足的卵囊.对猫进行人工感染30、10~3和10~4个孢子化卵囊,观察猫的卵囊排出规律,包括潜隐期、显露期、排卵囊高峰期及每克粪便中卵囊数(OPG 值).通过对比,验证了潜隐期、显露期、OPG值与感染剂量存在相关关系,即感染剂量越小,潜隐期越长,显露期越长,OPG值越小,反之亦然.     

Modified flotation method with the use of Percoll for the detection of Isospora suis oocysts in suckling piglet faeces

The modification of flotation method for the examination of diarrhoeic piglet faeces for the detection of Isospora suis oocysts was elaborated. The method was based on removing fractions of fat from the sample of faeces by centrifugation with a 25% Percoll solution. The investigations were carried out in comparison to the McMaster method. From five variants of the Percoll flotation method, the best results were obtained when 2ml of flotation liquid per 1g of faeces were used. The limit of detection in the Percoll flotation method was 160 oocysts per 1g, and was better than with the McMaster method. The efficacy of the modified method was confirmed by results obtained in the examination of the I. suis infected piglets. From all faecal samples, positive samples in the Percoll flotation method were double the results than that of the routine method. Oocysts were first detected by the Percoll flotation method on day 4 post-invasion, i.e. one-day earlier than with the McMaster method. During the experiment (except for 3 days), the extensity of I. suis invasion in the litter examined by the Percoll flotation method was higher than that with the McMaster method. The obtained results show that the modified flotation method with the use of Percoll could be applied in the diagnostics of suckling piglet isosporosis.     

Ultrastructure of developing Isospora suis in cultured cells

The ultrastructure of Isospora suis sporozoites, type-1 meronts, and type-1 merozoites was examined, using transmission electron microscopy of infected cultured cells. The ultrastructure of sporozoites and type-1 merozoites was similar. Each possessed trimembranous pellicles, subpellicular microtubules, a conoid, anterior and posterior polar rings, rhoptries, micronemes, a single vesicular nucleus, tubular mitochondria, Golgi complexes, ribosomes endoplasmic reticula, inactive micropores, amylopectin bodies, lipid bodies, dense bodies, and crystalloid bodies. Merozoites were produced by endodyogeny. Ultrastructural events associated with merozoite production by type-1 meronts are described.     

Comparison of different methods for examining the feces of suckling piglets for Isospora suis

A highly sensitive diagnostic method is of great importance for detection of I. suis (Isospora suis). In the present study KSFV (combined sedimentation-flotation method) using different flotation solutions (NaCl, ZnSO4, NaCl-sucrose) was investigated regarding sensitivity, costs and expenditure of human labour in comparison to FV (flotation method) using NaCl-sucrose solution and to AM (autofluorescence microscopy). Ninety faecal samples were examined. Using AM, oocysts were detected in 83.3% of the samples. In cases of KSFV and FV, respectively, 41.1% to 53.3% of samples were tested positive. The quantity of detected oocysts was markedly higher in AM than in KSFV or FV. Therefore AM is the most sensitive of the comprised methods. The expenditure of labour is lowest in AM, time exposure is lower in FV than in KSFV. For performance of AM a fluorescence microscope is required contrary to KSFV and FV.     

Development of Isospora suis from pigs in primary porcine and bovine cell cultures

Sporozoites of Isospora suis penetrated and developed by endodyogeny in primary porcine kidney (PPK) and primary fetal bovine kidney (PFBK) cell cultures. Motile merozoites and binucleate Type I meronts were observed in both types of cultured cells. Multinucleate Type II meronts developed in PPK cell cultures only. These multinucleate meronts were always found singly, were nonmotile and did not form merozoites.     

Pathogenicity of Isospora suis in gnotobiotic and conventionalised piglets

Isospora suis is unequivocally a primary pathogen of swine. Inoculation of I suis in conventionalised and germ-free piglets caused a biphasic disease course with marked diarrhoea, villous atrophy and necrosis of the intestinal epithelium at four to six and eight to 10 days after inoculation. The presence of a normal bacterial flora markedly (P less than 0.05) influenced the survival rate of piglets but did not appear to affect the histopathological changes observed. Mild limited focal necrosis and bile stasis were present in the liver at eight to 10 days after inoculation. In this period there was also ectasia of lymph vessels in the intestinal lymph nodes.     

PCR-based differentiation of three porcine Eimeria species and Isospora suis

Isospora suis and Eimeria are frequent coccidian parasites of pigs. The unsporulated oocysts of Eimeria species and of I. suis are difficult to differentiate. Therefore, a species-specific PCR was developed. PCR products were amplified from Eimeria polita, Eimeria porci, and Eimeria scabra using primers from the conserved 18S rRNA regions and were subsequently sequenced. Based on variable sequence regions, primers were constructed for the differentiation of the three Eimeria species and I. suis. Using a combination of PCRs detecting one or two species, all four coccidian species were detected (theoretical lower detection level: DNA content of 250 oocysts of each Eimeria species or 25 oocysts of Isospora in 1microl) and differentiated. The PCR-based differentiation of the above mentioned species provides a useful alternative to microscopy.     

An observational study on the prevalence and impact of Isospora suis in suckling piglets in southwestern Ontario, and risk factors for shedding oocysts

An observational study was conducted to determine the prevalence of Isospora suis oocysts in fecal samples from suckling piglets in Ontario, and to evaluate the relationship between the presence of I. suis oocysts and diarrhea. Fifty farms and 709 litters of piglets were included in the study. Oocysts were detected on 70% of farms, with 187 litters infected. A litter of pigs that was positive for oocysts was significantly more likely to exhibit diarrhea than a litter that was negative [odds ratio (OR) = 4.0; 95% confidence interval (CI) = 2.8 to 5.8; P < 0.001]. Management and housing factors were examined with respect to risk factors for the presence of I. suis. Farms that did not use a detergent when cleaning farrowing crates were 10-times more likely to be positive for I. suis than those that used a detergent (P = 0.007). It was concluded that coccidiosis is a common problem on Ontario swine farms.     

Shedding of Oocysts in Piglets Experimentally Infected with Isospora suis

Christensen, S. AA. Henriksen: Shedding of oocysts in piglets experimentally infected with Isospora suis. Acta vet scand., 1994, 35, 165-172.–Forty-seven piglets were inoculated with doses of 100 to 50,000 sporulated oocysts of Isospora suis. After 5-7 days oocysts were found in faeces. The patent period extended from 8 to 16 days. The shedding of oocysts showed a cyclic pattern with 2-3 peaks separated by intervals of approximately 5 days. Subpatent periods were often seen between the peaks.The level of oocyst shedding during the initial days of the patent period reflected, to some extent, the inoculation dose. However, a maximum of OPG at the 100,000 level was observed among one or more piglets from all groups, regardless of the inoculation dose. Among the majority of piglets inoculated with more than 100 oocysts, the highest OPG-figures were observed in the first peak of the cyclic pattern. Unlike this, the maximum of OPG was observed in the second peak of the cycle among 6 of the 7 piglets inoculated with 100 oocysts only. The triphasic pattern was most pronounced in the low dosed group.The marked upscaling of oocyst production, as particularly registered in the low dosed groups, seams to explain at least part of the problems met under practical conditions, when trying to eliminate the transmission of oocysts between successive litters in the farrowing boxes.The cyclic excretion pattern and an apparent absence of autoinfections may indicate that the development of I. suis in the host includes several oocyst producing generations descending from the same initial infection.The presence of subpatent periods can probably explain the marked variation in OPG, as they are often recorded when examining faecal samples from piglets, even when the samples are originating from the same litter.     

The effects of nitric oxide donors on the sporulation of Eimeria tenella oocysts

Nitric oxide (NO) is involved in many physiological processes including regulation of blood pressure, immune response, and neural communication. The effects of NO donors on the sporogony of Eimeria tenella oocysts and the blocking effects of NO scavenger, hemoglobin (Hb), against NO donors were investigated in this study. The results showed that the sporulation of oocysts could be completely inhibited by acidic sodium nitrite (NaNO2), S-nitroso-glutathione (GSNO) or S-nitroso-N-acetyl-dl-penicillamine (SNAP) in profound dose-dependent and time-dependent manners, and the inhibiting process was irreversible and not affected by feces of chickens. However, not all NO donors in this study, such as sodium nitroprusside (SNP), demonstrated above-mentioned effect. The NO scavenger Hb could significantly prevent the detrimental effects of NO donors on the sporogony of oocysts in a dose-dependent manner. At present, the inhibiting mechanism of NO donors on the sporulation of oocysts was still unclear.     

The role of Isospora suis as a pathogen in conventional piglet production in Germany

In order to evaluate the prevalence of Isospora suis in conventional piglet production in Germany, pooled faecal samples from 327 pig litters from 18 pig production units (20-320 sows each) were examined. At least 10 litters from each farm were investigated. I. suis was present on 83% of the farms and 42.5% of the litters, the infection rate being highest in the third week of age (48.2%). I. suis was found more frequently in samples of diarrhoea than in firm faeces (49.2% compared to 22.2%). Twenty naturally infected piglets from six of these farms underwent examination post mortem, including histology, virology and bacteriology. Histological examination revealed atrophy of the villi in various degrees, mild crypt hyperplasia, fusion of the villi, metaplastic epithelium, erosions and necrosis, especially in the medium and the posterior jejunum and in the ileum. Asexual and sexual developmental stages of the parasite were found in varying numbers in the epithelium of the whole of the small intestine. Bacteria and viruses were mostly excluded as the cause of diarrhoea, and it was concluded that I. suis was the primary pathogen inducing distinct changes and clinical symptoms of diarrhoea.     

Occurrence of Isospora suis in Germany, Switzerland and Austria

Nationwide surveys for the occurrence of Isospora suis were carried out in Germany, Austria and Switzerland including a questionnaire regarding herd size, health status and management practices and a coccidiosis sampling kit for pooled faecal samples from litters of suckling piglets. A total of 184 veterinary practices participated in the survey and returned 1745 samples (331 kits) from 324 farms in the north (n = 98), south (n = 84), centre/east (n = 42) and west (n = 10) of Germany, Austria (n = 61) and Switzerland (n = 29) with larger farms in north and centre/east (average number of sows: 270 and 500) and smaller ones in the south (95), Austria (60) and Switzerland (43). Larger farms tended to have better hygienic standards (slatted floors, disinfection of the farrowing units). The majority of the participating farms (93.5%) reported problems with diarrhoea in piglets at 2-3 weeks of age, significantly associated (P < 0.001) with uneven weaning weights (94.9%). Toltrazuril (5%; Baycox) was used only rarely; however, in these farms unevenness of weaning weights was less frequently observed (P = 0.011). A 76.2% of the farms were positive for I. suis (samples contained mostly low or moderate oocyst numbers), especially in the south (P < 0.001). Oocysts were more frequently found in samples from farms with reported diarrhoea (P = 0.011), uneven weight gain (P = 0.019) or in herds of small size (P < 0.001). Disinfection, floor type or treatment with toltrazuril did not affect the frequency of observation of oocysts.     

一氧化氮对球虫(E.tenella)卵囊孢子生殖的抑制特性

本研究利用NO供体以及几种抗氧化剂和NO清除剂来分析外源性NO对E.tenella卵囊孢子生殖的抑制特性.试验结果表明:S-亚硝基硫醇类NO供体(SNAP和GSNO)对卵囊的抑制作用具有明显的剂量-效应荚系,8 mmol/L的SNAP和GSNO均可完全抑制新鲜卵囊的孢子生殖;KCN对卵囊的抑制呈现为剂量-效应关系,但NaN3没有抑制作用.维生素C、甘露醇和水杨酸钠等抗氧化物质以及FeSO4和DTT均不能消除GSNO对卵囊孢子生殖的抑制作用.K2Cr2O7和KMnO4均会明显抑制GSNO对卵囊的作用.NO清除剂血红蛋白(Hb)能够消除NO供体对卵囊的抑制作用,并呈现剂量-效应关系.     

Effects of clopidol on sporulation and infectivity of Eimeria tenella oocysts

Feed additive anticoccidials currently used in Japan were examined for possible effects on oocyst sporulation of Eimeria tenella. Monensin, salinomycin, lasalocid, amprolium plus ethpabate, amporolium plus ethopabate plus sulfaquinoxaline, clopidol, or nicarbazin were given to chickens continuously via the feed at the recommended use level or one-half of that level. Oocysts discharged in feces 7-8 days post inoculation (PI) were collected and aerated for sporulation. Low sporulation rate was noted, when clopidol at 62.5 mg kg-1 was given from 4 to 7 days PI. These oocysts were as infective as oocysts from controls, based on weight gain, feed efficiency, gross lesion score of cecae, and oocyst count 7 days PI. The results of the study indicated that the second schizogony and gametogony are vulnerable to clopidol, as evidenced by oocyst sporulation, but infectivity of these sporulated oocysts was not affected.     

猪等孢球虫检测及防控研究进展

猪等孢球虫是一种危害仔猪生长的专性寄生性原虫,感染该寄生虫的仔猪生长缓慢,且常表现出腹泻症状,甚至死亡,对养猪业造成极大危害。本文综合了国内外研究成果,从流行病学特征、形态特征、生活史、检测方法及防控措施等方面对仔猪等孢球虫进行了综述,并对猪等孢球虫的防控手段、内生发育史、基因组学等生物学特性以及与肠道菌群的相互作用等问题进行了展望,以期为猪等孢球虫的病原鉴定及有效防控提供科学依据。     

Pathogenesis of experimental combined infections with Isospora suis and rotavirus in conventional and gnotobiotic piglets

51 gnotobiotic and 63 conventional, one-, or two-days-old piglets were divided into five groups and infected orally either with Isospora suis or rotavirus alone, or with both agents simultaneously or successively with alternative sequences and various intervals. 15 gnotobiotic and 10 conventional piglets served as controls. The development of small intestinal lesions after infection with I. suis was biphasic. The dominant alteration resulting from rotavirus infection was villus atrophy, considerably more pronounced and extensive in gnotobiotic than in conventional piglets. Synergistic action of I. suis and rotavirus was manifested both clinically, and morphologically. This action culminated at the time of the actual, or presumed development of merogony of I. suis, i.e. on DPI 3 to 5. The action develops only if the intestinal epithelium is damaged functionally and morphologically by a preceding rotavirus infection. It is concluded that the synergistic action is based on a competition of rotavirus and I. suis for mature, enzymatically active absorptive cells.