黄牛、水牛体内人肉孢子虫18S rRNA基因研究及虫种鉴定

本文对自然感染的水牛源的人肉孢子虫以及黄牛源人孢子虫ＤＮＡ的１８Ｓ ｒＲＮＡ基因的ＰＣＲ扩增产物进行了测序。对年获的８６３ｂｐ的１８Ｓ ｒＲＮＡ基因分析比较表明,二者有较高的同源性,因此认为二者可能同是一种肉孢子虫－－人肉孢子虫（Ｓａｒｃｏｃｙｓｔｉｓ ｈｏｍｉｎｉｓ Ｒａｉｌｌｉｅｔ ａｎｄ Ｌｕｃｅｔ,１８９１）。由此推断,不仅黄牛可作为人肉孢子虫的中间宿主,水牛也可作为人肉孢子虫的中间宿主。     

Characterization of Sarcocystis species in domestic animals using a PCR-RFLP analysis of variation in the 18S rRNA gene: a cost-effective and simple technique for routine species identification

Thirteen restriction endonucleases were used to investigate nuclotide sequence variation in the 18S rRNA DNA of 88 individuals from ten Sarcocystis taxa collected as cysts from their intermediate hosts, swine, cattle and water buffalo. A DNA sequence of approximately 900 bp was used. A total of 26 electromorphs were detected. The electromorphs were sorted into seven different haplotypes that coincided with the six named species and an unidentified species from cattle. These findings support those of our morphological examinations, which suggested that the taxa resembling Sarcocystis hirsuta, S. hominis, both found in water buffalo, and S. sinensis found in cattle, are not new species but are in fact S. hirsuta and S. hominis as found in cattle, and S. sinensis as found in water buffalo; this finding supports the idea that these species can utilize both cattle and water buffalo as intermediate hosts and are not restricted to one or the other host group as previously thought. PCR-RFLP resolved by agarose gel electrophoresis is shown to be an easy and rapid method of discriminating between these species.     

Experimental Sarcocystis hominis infection in a water buffalo (Bubalus bubalis)

A water buffalo (Bubalus bubalis) was fed 5.0 x 10(5) Sarcocystis hominis sporocysts from a human volunteer who had ingested S. hominis cysts from naturally infected cattle. A necropsy was performed on the buffalo 119 days after inoculation, and a large number of microscopic sarcocysts (approximately 5,000/g) were found in skeletal muscles. Ultrastructurally, the sarcocyst wall from buffalo muscles has upright villar protrusions measuring about 5.6 x 0.8 microm with numerous microtubules that run from the base to the apex. Sarcocysts from this buffalo were infective to 2 human volunteers, confirming their identity as S. hominis. Therefore, we believe that buffaloes can act experimentally as the intermediate host for S. hominis.     

A PCR-based RFLP analysis of Sarcocystis cruzi (Protozoa: Sarcocystidae) in Yunnan Province,PR China,reveals the water buffalo (Bubalus bubalis) as a natural intermediate host

A polymerase chain reaction-based restriction fragment length polymorphism (RFLP) approach is used to examine Sarcocystis cruzi-like taxa from the atypical intermediate host, water buffalo, in Yunnan, People's Republic of China. The loci examined lie within the 18S rRNA gene. A total of 15 water buffalo isolates are compared with those of 10 S. cruzi from cattle. RFLP patterns for the S. cruzi isolates from cattle and the S. cruzi-like taxon from water buffalo are found to be identical with all the 12 restriction enzymes used. Interpopulation variation between samples from Kunming and Gengma (Yunnan) is found to be undetectable at these loci for both S. cruzi and the S. cruzi-like taxon. But RFLPs are found between the S. cruzi taxa and S. suihominis from pigs at the same study sites. These findings support the hypothesis that S. cruzi is able to use the water buffalo as an intermediate host and is not restricted to cattle as was previously supposed.     

An electron microscopic study of the macro- and microcysts of coccidia in the genus Sarcocystis from buffalo

Two sarcosporidian species from muscles of the water buffalo examined in Azerbaijan were studied. The one making macrocysts was identified as S. fusiformis, whereas the other producing microcysts appeared to be not S. levinei as it might have been expected. Indeed, the microcysts found in the muscles of the buffalo in the slaughterhouse [correction of slaughtery] of Baku differed essentially in morphology of the cyst wall from that of S. levinei, sooner resembling that of S. cruzi from cattle. The cyst wall of these microcysts was seen to make deep invaginations towards the ground substance of cysts. From these invaginations some curved channels were grown outlined with a filamentous material. The described pattern of cyst wall is not characteristic of the S. cruzi sarcocyst. No specification was achieved, and the sarcocysts found in the bubaline muscles are designated as Sarcocystis sp. from the buffalo.     

Structural and functional characterization of cyst cells in Sarcocystis sp. (Sporozoa, Apicomplexa)

By means of light and electron microscopy, the structural pattern of muscle cysts (sarcocysts) was examined for the four species of the genus Sarcocystis: S. muris (from murine skeletal muscles), Sarcocystis sp. and S. fusiformis (from, respectively, heart and skeletal muscles of buffalo), and S. ovifelis (from ovine tong muscles). The orderly fashion of the interior of the cyst is attained by partitition of its space into numerous compartments with the involvement of the intermediate filaments. These, in their turn, are bound to each other by thin filaments to make eventually a common filamentous net. The net limits separate groups of cells referred to as cyst zoites. The common net of filaments and microtubules (when present) may be regarded not only as the organizer of the cyst interior cytoskeleton, but also as the main mechanism of substance transportation in various directions: from the host cell to the sarcocyst, and within or outside the cyst. The role of dedifferentiation, proliferation and differentiation processes is suggested in the establishment of the fixed sequence of events throughout the unidirectional development of cyst cells and their interaction, from precystic meronts to cyst merozoites (gamonts). Special attention is paid to metrocyte morphogenesis and functioning. In the present work, metrocytes subjected to apoptosis were recognized. It is suggested that phenomenon of programmed cell death in metrocytes may be associated with the control of cell number in mature and ageing sarcocysts.     

Sarcocystis cruzi: comparative studies confirm natural infections of buffaloes

Controversy exists concerning whether cattle and water buffalo sustain infections with cysts of distinct arrays of species in the genus Sarcocystis. In particular, morphologically similar parasites have been alternately ascribed to Sarcocystis cruzi or to Sarcocystis levinei, depending on their occurrence in cattle or water buffalo. We used light and transmission electron microscopy, genetic analysis, and experimental infections of definitive canine hosts to determine whether consistent differences could be identified from parasites derived from several natural infections of each host, examining several tissue types (esophagus, skeletal muscles, and heart). Cysts derived from cattle and water buffalo shared similar structure; variation among 18S rRNA sequences did not segregate consistently according to intermediate host type; parasites derived from cattle and water buffalo induced similar outcomes in the canine definitive host. One cattle specimen harbored unusually large (macroscopic) sarcocysts which nonetheless conformed to previously reported ultrastructural and genetic features of S. cruzi. Finding no consistent basis to differentiate between them, we conclude that the parasites infecting each host and tissue type correspond to S. cruzi. In our sample, no phylogenetically distinct taxon was sampled which might correspond to a distinct taxon previously described as S. levinei. Either that taxon was missed by our sampling effort, or it may represent a junior synonym to S. cruzi, which would then cycle between dogs and a broader range of intermediate bovine hosts than was previously considered.     

试验感染水牛体内枯氏住肉孢子虫内生发育的超微结构

通过人工感染试验在电镜下对一种住肉孢子虫在水牛体内的发育进行了研究。结果揭示在血管内皮细胞中的裂殖生殖和在肌细胞内的包囊发育过程与枯氏住肉孢子虫的一致,裂殖体、裂殖子、包囊壁和囊内母细胞、缓殖子的超微结构亦与枯氏住肉孢子虫的相同。首次通过试验感染的发育史研究证明,流行于我国湖南水牛的一种小包囊属于枯氏住肉孢子虫,水牛是黄牛枯氏住肉孢子虫的新宿主。     

Prevalence of Sarcocystis spp. cysts in Japanese and imported beef (Loin: Musculus longissimus)

A survey was carried out to investigate the occurrence of Sarcocystis infection in the loin (Musculus longissimus) of Japanese and imported beef. In all, the muscle tissue of 482 samples were examined by histological method. The prevalence of Sarcocystis unspecified species cysts was lower in Japanese beef (total 6.31%: 0% in Holstein castrated, 12.96% in Holstein milk cow, 3.33% in Japanese shorthorn and 11.58% in Japanese black cattle) than in beef imported from America (36.78%) or Australia (29.49%). The infection density of imported beef, especially in American, was higher than in Japanese beef. All detected cysts except one were identified as Sarcocystis cruzi. One thick walled cyst was found in Australian beef but it could not be distinguished as either Sarcocystis hirsuta or Sarcocystis hominis.     

Morphological and biochemical correlates in the characterization of Sarcocystis spp

Isoenzyme electrophoretic techniques were applied to the characterization of seven Sarcocystis spp. that had been identified by conventional morphological studies. Cystozoites were harvested from macroscopic cysts from sheep, cattle, and mice and from microscopic cysts from sheep, cattle, and goats. Soluble cystozoite extracts were subjected to cellulose acetate gel electrophoresis and characterized at 15 of the 39 enzyme loci examined. Genetic relationships among isolates were examined by simple phenetic clustering. Two different morphological types of macroscopic cysts from sheep, identified as S. gigantea (syn. S. ovifelis) and S. medusiformis, consistently differed at 40% of the loci examined. Such genetic divergence confirms their separate morphotypic classification. Both differed from microscopic cyst isolates from sheep at 87% of the loci examined; however, two different morphotypes of microscopic cysts were found in the sheep sampled (thick-walled and thin-walled cysts). Until sufficient numbers of each type can be isolated and examined separately, both were regarded as belonging to the species S. tenella (syn. S. ovicanis). Macroscopic and microscopic cysts from cattle consistently differed at 80% of the loci thereby supporting their separate classification as S. hirsuta (syn. S. bovifelis) and S. cruzi (syn. S. bovicanis), respectively. Isolates from goats (microscopic cysts identified as S. capracanis) differed from S. tenella and S. cruzi at 20% and 47% of the loci, respectively. All macroscopic cyst isolates from the various host animal species (including S. muris from mice) differed from each other at nearly all loci. Isoenzyme electrophoretic techniques therefore provided genetic evidence supporting the classification of these various Sarcocystis spp. by their morphological characteristics.     

Light and electron microscopical study on sarcocysts from muscles of the rhesus monkey (Macaca mulatta), Baboon (Papio cynocephalus) and Tamarin (Saguinus(=Oedipomidas) oedipus).

Sarcocystis cysts from muscles of monkeys (baboon, tamarin and rhesus monkey) were studied by means of light and electron microscopy. The differences in the morphology of the cyst wall and parasites clearly indicate that the three monkey species examined were each parasitized by at least a specific Sarcocystis species being not identical with S. nesbitti or S. kortei. The large numbers of cysts found within the muscle fibres point out the important role that have these monkeys as intermediate hosts in the life cycle of Sarcocystis species, where the final hosts are still unknown.     

Non-invasive methods for identifying oocysts of Sarcocystis spp. from definitive hosts

Because the excreted sporocysts and/or oocysts of various species of Sarcocystis may not be discriminated morphologically, we sought to validate a diagnostic technique based on variation in the 18S rDNA sequence. Oocysts and/or sporocysts from three taxa of Sarcocystis were collected from human, feline, and canine definitive hosts that had fed upon meats infected with the muscle cysts of Sarcocystis hominis, Sarcocystis fusiformis and a species of Sarcocystis from water buffalo that could not be distinguished from Sarcocystis cruzi. Using a new collection method employing filter paper, these excreted oocysts and sporocysts were subjected to DNA extraction, as were the corresponding muscle cysts. Methods employing PCR–RFLP and DNA sequencing of a partial 18S rDNA gene (ssrRNA) sequence were then used to successfully distinguish among the three taxa. The same, unique restriction digestion pattern characterizes the tissue cysts and oocysts and/or sporocysts of each parasite taxon. The technique makes possible amplification and identification of species specific gene sequences based on DNA extracted from as few as 7 excreted sporocysts (the equivalent of 3 and 1/2 oocysts) from freshly prepared material, or as few as 50 sporocysts from feces samples that had been stored in potassium dichromate (K₂Cr₂O₇) for as long as 6 years. This represents the first report using molecular diagnostic procedures to diagnose oocysts of Sarcocystis in faecal samples, describing a valuable new tool for studying the epidemiology of various Sarcocystis species.     

Evidence supporting zoonotic transmission of Cryptosporidium in rural New South Wales

Cryptosporidium hominis, which has an anthroponotic transmission cycle and Cryptosporidium parvum, which is zoonotic, are the primary species of Cryptosporidium that infect humans. The present study identified the species/genotypes and subgenotypes of Cryptosporidium in 7 human and 15 cattle cases of sporadic cryptosporidiosis in rural western NSW during the period from November 2005 to January 2006. The species/genotype of isolates was determined by PCR sequence analysis of the 18S rRNA and C. parvum and C. hominis isolates were subgenotyped by sequence analysis of the GP60 gene. Fourteen of 15 cattle-derived isolates were identified as C. parvum and 1 as a C. bovis/C. parvum mixture. Of the human isolates, 4 were C. parvum and 3 were C. hominis. Two different subgenotypes were identified with the human C. hominis isolates and six different subgenotypes were identified within the C. parvum species from humans and cattle. All four of the C. parvum subtypes found in humans were also found in the cattle, indicating that zoonotic transmission may be an important contributor to sporadic human cases cryptosporidiosis in rural NSW.     

Ultrastructure of Sarcocystis spp. (Protozoa: Apicomplexa) in rodents from North Sulawesi and West Java, Indonesia

Tissue cysts of the protozoan genus Sarcocystis were detected in the skeletal muscles of 16 (40%) of 40 wild rodents captured in North Sulawesi and West Java, Indonesia. Two types of cysts were found to differ in their morphological characteristics. Macroscopic and microscopic cysts bounded by thick radially-striated cyst walls were detected at both locations in a total of 13 rodents belonging to seven different species (Bunomys chrysocomus, B. fratrorum, Maxomys bartelsii, M. musschenbroekii, Paruromys dominator, Rattus xanthurus and R. exulans). The primary cyst walls contained numerous broad spatula-like protrusions and the cysts were identified as S. singaporensis Zaman and Colley, 1976. In contrast, microscopic cysts bounded by thin smooth cyst walls were detected in seven rodents belonging to three different species captured at Toraut in North Sulawesi (B. chrysocomus, B. fratrorum and P. dominator). Ultrastructural examination revealed numerous slender hair-like protrusions of their primary cyst walls. It is proposed that these cysts be named S. sulawesiensis sp. n. on the basis of their unique morphological characteristics, their intermediate host range and their limited geographic distribution. Mixed infections by both species were found in three rodent species (B. chrysocomus, B. fratrorum and P. dominator).     

A Light Microscopic Comparison of The Cysts of Four Species of Sarcocystis Infecting The Domestic Reindeer (Rangifer Tarandus) in Northern Norway

Fresh preparations of micro-isolated sarcocysts from skeletal and cardiac muscle of 12 reindeer were examined by light microscopy. On the basis of cyst structure and cyst wall structure 4 Sarcocystis spp. could be differentiated. New names have been proposed for 2 previously unnamed Sarcocystis spp. of reindeer, and S. grueneri has been redefined. S. rangiferi n. sp. had macroscopic cysts in skeletal muscle measuring 2106×403 µm. The cyst wall protrusions were finger-like and measured 13.2×6.7 µm. The cysts were surrounded by a layer of fibrillar material. S. tarandi n. sp. had micro- to macroscopic cysts primarily in skeletal muscle, but a few cysts were found in the heart of one animal. In skeletal muscle the cysts measured 999×75µm; in the heart the cysts were shorter and wider. The cyst wall protrusions were fingerlike and measured 9.2×2.2 µm. S. grueneri had micro- to macroscopic cysts in cardiac muscle measuring 581×137 µm. The cyst wall was thin and relatively smooth with no visible protrusions. Sarcocystis sp. had micro- to macroscopic, slender cysts in skeletal muscle measuring 916×64 µm. The cyst wall had tightly packed, short, knob-like protrusions. The cysts of this species were previously classified as cysts of S. grueneri.     

Isolation and sequence characterization of mammary derived growth inhibitor gene of riverine buffalo (Bubalus bubalis)

In this study, attempts have been made to identify and characterize water buffalo (Bubalus bubalis) mammary derived growth inhibitor (MDGI) gene, isolated from a mammary gland cDNA library of lactating buffalo. The complete MDGI cDNA was of 698 nucleotides, consisting 61 nucleotides in 5' UTR, coding region of 402 nucleotides, and 235 nucleotides representing the 3' UTR. Comparison of nucleotide and deduced amino acid sequence data with that of MDGI/fatty acid binding protein (FABP) of other species shows three buffalo specific nucleotide changes while seven nucleotide changes were common to cattle and buffalo. Buffalo and cattle MDGI had 100% amino acid sequence similarity, which also shared three amino acid changes: 34 (Ala-Gly), 109 (Leu-Met), and 132 (Glu-Gln) as compared to other species. Comparison with FABPs reported from other cattle tissues revealed highest amino acid sequence similarity with FABP-heart (100%) and least with FABP-liver (20.5%). Phylogenetic analysis revealed cattle MDGI to be closest to buffalo, while mouse MDGI was distantly placed, whereas different tissue derived FABPs of cattle showed FABP-heart closest and FABP-epidermis most distantly placed from buffalo MDGI. This report also differs from the earlier findings that MDGI is intermediate of FABP-heart and adipose.     

Infectivity of sarcocystis spp. from bison, elk, moose, and cattle for cattle via sporocysts from coyotes

Bison bison (bison), Cervus canadensis (elk), Alces alces (moose), and Bos taurus (cattle) musculature containing Sarcocystis spp. cysts was fed to laboratory raised Canis latrans (coyotes), Sporocysts collected from the feces of coyotes fed musculature of each of the ruminant species were fed to four groups of three laboratory-raised domestic calves, respectively, to determine if Sarcocystis spp. was transmissible from wild to domestic ruminants and if so, to compare clinical signs of infection and morphologic features of cysts with those resulting from infection with Sarcocystis bovicanis. All calves fed sporocysts of Sarcocystis from coyotes that ate bison or cattle muscle had similar clinical signs and harbored morphologically similar parasites, suggesting that both bison and cattle are intermediate hosts for S. bovicanis and that this species is transmissible between the two ruminant species. All calves fed sporocysts from coyotes that ate elk muscle or moose muscle remained asymptomatic but one calf in each group had intramuscular cysts. The finding of relatively large numbers of intramuscular cysts in one calf fed sporocysts of elk origin and smaller numbers in one calf fed sporocysts of moose origin could represent either spurious natural infections or indicate low infectivity of Sarcocystis spp. from elk and moose to cattle.     

Ultrastructural development of the sarcocyst of Sarcocystis rauschorum (Apicomplexa: Sarcocystidae) in the varying lemming Dicrostonyx richardsoni

The development of the sarcocyst of Sarcocystis rauschorum in its intermediate host was studied. Lemmings were orally administered sporocysts of S. rauschorum obtained from snowy owls (Nyctea scandiaca). Beginning at 9 days postinoculation (DPI) and at various intervals to 84 DPI, skeletal muscle tissue taken from the infected lemmings was examined by electron microscopy. At 9 DPI the sarcocysts contained few metrocytes and the cyst wall was flat. The metrocytes underwent endodyogeny, and within a few days the cyst wall of the rapidly growing sarcocyst developed numerous tubulovesicular invaginations into the electron-dense layer, and the wall had a few irregular infoldings. By 21 DPI, banana-shaped bradyzoites appeared, and by 84 DPI the mature cysts were filled with bradyzoites in groups subdivided by septa and by deep infoldings of the cyst wall. The fine structure of the wall remained simple throughout maturation, with no conspicuous invagination or protrusion. The sarcocyst produced in response to S. rauschorum is unlike those from many species of Sarcocystis, which have complex walls that change markedly as the sarcocysts mature; however, its simple appearance is similar to other species that have rodents as intermediate hosts and raptorial birds as definitive hosts.