Morphological and molecular identification of three species of Sarcocystis in reindeer (Rangifer tarandus tarandus) in Iceland

Six Sarcocystis species have previously been described from reindeer in Norway based on sarcocyst morphology and DNA sequencing. The aim of this study was to determine whether reindeer in Iceland, which descend from reindeer imported from Norway in 1787, also were infected with Sarcocystis, and to identify and genetically characterise any species present. Muscle tissue from the heart, diaphragm and/or oesophagus was collected from 36 reindeer in Iceland. Pieces of all tissue samples were examined histologically. Frozen/thawed samples of cardiac muscle, oesophagus and/or diaphragm from 11 of the 36 reindeer were also examined under a stereoscopic microscope and sarcocysts present were identified to species either in situ or under a light microscope. Two cysts of each species, originating from two different reindeer were randomly selected for DNA analyses. The complete ssu rRNA gene was amplified by the polymerase chain reaction (PCR) and sequenced. In addition, two sarcocysts that could not be classified by microscopic examination were selected for partial ssu rRNA gene sequence analysis. By histology, sarcocysts were found in the diaphragm and/or oesophagus of 8 of 36 (22.2%) animals. By examination of fresh tissue, sarcocysts of Sarcocystis rangi, S. tarandivulpes and S. hardangeri were found in the oesophagus of seven of nine (77.8%) animals, suggesting a high prevalence of Sarcocystis in the Icelandic reindeer population. Cyst morphology and the ssu rRNA gene sequence of each of the three species were identical to isolates of the same species from Norwegian reindeer. DNA sequencing was useful in order to identify cysts with an ambiguous morphology. This is the first record of these Sarcocystis species in reindeer outside Norway.     

Phylogenetic relationships between Sarcocystis species from reindeer and other Sarcocystidae deduced from ssu rRNA gene sequences

Six Sarcocystis species from reindeer (S. grueneri, S. rangi, S. tarandivulpes, S. hardangeri, S. rangiferi and S. tarandi) have previously been genetically characterised. The aim of this study was to identify possible definitive hosts for S. hardangeri, S. rangiferi and S. tarandi by including the six species in phylogenetic analyses of the Sarcocystidae, and also to investigate the phylogenetic relationships between the species from reindeer and those from other hosts. The study also aimed at revealing whether the inclusion of six Sarcocystis species from the same intermediate host would have any effect on previously inferred phylogenetic relationships within the Sarcocystidae. The complete small subunit (ssu) rRNA gene sequences of all six Sarcocystis species from reindeer were used in the phylogenetic analyses along with ssu rRNA gene sequences of 85 other members of the Coccidea. Trees were constructed using Bayesian analysis and maximum likelihood estimations. All six Sarcocystis species from reindeer were placed together with other Sarcocystis species using an even-toed ungulate as their intermediate host. The three canine transmitted species, S. grueneri, S. rangi, S. tarandivulpes, formed a sister group to other Sarcocystis species with a canine definitive host. The position of S. hardangeri on the tree suggested that it uses another type of definitive host than the other Sarcocystis species in this clade. Considering the geographical distribution and infection intensity of S. hardangeri, corvid birds are perhaps its most likely definitive hosts. The phylogenetic position, geographical distribution, prevalence and morphological similarity to feline transmitted Sarcocystis species in closely related Cervidae suggest that the most likely definitive hosts of S. rangiferi and S. tarandi are felines, and in Norway notably the lynx. The overall phylogeny of the Sarcocystidae did not change by the inclusion of the six Sarcocystis species from reindeer. This study suggests that phylogentic analysis can be a useful tool in the search for possible definitive hosts for those Sarcocystis species for which they are unknown and difficult to find solely by other methods.     

Sarcocystis hardangeri and Sarcocystis rangi n. sp. from the domestic reindeer (Rangifer tarandus) in northern Norway

Fresh preparations of microisolated sarcocysts from striated muscle of several domestic reindeer from northern Norway were examined by light microscopy. In cardiac muscle, cysts of S. grueneri were found. In skeletal muscle, cysts of S. rangiferi, S. tarandi and S. tarandivulpes were found in all samples examined. In the abdominal muscles of some reindeer, one or two other types of cysts were found.Cysts of one type were macroscopic in size, and ovoid to cylindrical in shape. The cysts were surrounded by a 8–12 µm thick layer of fibrous material, and measured 1682×910 µm. The cysts had relatively few and irregularly distributed, 20–35 µm long, and 3–5 µm wide, linguiform cyst wall protrusions, which could only be seen after removal of the fibrous layer. These cysts were classified as cysts of S. hardangeri, a species previously described from wild reindeer in southern Norway.Cysts of the other type were long and slender, measuring 5460–12700 (8994 ± 2575) × 95–280 (180 ± 50) µm. The cysts had numerous very fine, flexible, hair-like cyst wall protrusions, which were 8–10 [xm long and less than 0.5 µm thick. These cysts are considered to belong to a new Sarcocystis species of reindeer, for which the name Sarcocystis rangi n, sp. is proposed. The reindeer is recorded as the intermediate host for 6 different species of Sarcocystis.     

Relationships among Sarcocystis species transmitted by New World opossums (Didelphis spp.)

At least three species of Sarcocystis (S. neurona, S. falcatula, S. speeri) have recently been shown to use opossums of the genus Didelphis as their definitive host. In order to evaluate the evolutionary relationships among Sarcocystis spp. isolates from the Americas, and to determine whether organisms representing the same parasite lineages are transmitted north and south of the Panamanian isthmus, we inferred the phylogenetic relationships from nucleotide sequence variation in parasites isolated from three opossum species (D. virginiana, D. albiventris, D. marsupialis). In particular, we used variation in the 25/396 marker to compare several isolates from Brazil, Argentina, and the United States to each other and to cloned S. neurona and S. falcatula whose morphology and host affinities have been defined in the laboratory. S. neurona was identified from a Brazilian D. albiventris, as well as from North American D. virginiana. Parasites resembling the Cornell isolate of S. falcatula are transmitted both south and north of the Panamanian isthmus by D. albiventris and D. virginiana, respectively. Distinct attributes at two genetic loci differentiated a Brazilian isolate of S. falcatula from all other known parasite lineages. We confirm S. neurona as the causative agent of recently reported neurologic disease in Southern sea otters, Enhydra lutris nereis. And we found that S. speeri could not be compared to the other opossum-derived Sarcocystis isolates on the basis of nucleotide variation at the 25/396 locus. The widespread distribution of certain species of Sarcocystis may derive from their ability to parasitize migratory bird hosts in their intermediate stage.     

Phylogenetic analysis of Sarcocystis nesbitti (Coccidia: Sarcocystidae) suggests a snake as its probable definitive host

Sarcocystis nesbitti was first described by Mandour in 1969 from rhesus monkey muscle. Its definitive host remains unknown. 18S rRNA gene of S. nesbitti was amplified, sequenced, and subjected to phylogenetic analysis. Among those congeners available for comparison, it shares closest affinity with those species of Sarcocystis which use snakes as definitive hosts. We therefore hypothesize that a snake may serve as the definitive host for S. nesbitti.     

The fox as definitive host for Sarcocystis sp. Gjerde, 1984 from skeletal muscle of reindeer (Rangifer tarandus). With a proposal for Sarcocystis tarandivulpes n. sp. as replacement name

Skeletal muscle of 5 wild reindeer was examined for sarcocysts and used for experimental infection of 6 foxes. Skeletal and cardiac muscle of another reindeer were only examined for sarcocysts. The skeletal muscle of all animals was infected with Sarcocystis sp.. In 2 of the animals cysts of S. hardangeri were also present. The single heart examined contained only cysts of S. grueneri.Four foxes given skeletal muscle containing apparently only cysts of Sarcocystis sp., started shedding Sarcocystis sporocysts, measuring on average 13.6×9.8 µm, after a prepatent period of 10–12 days. Two foxes given skeletal muscle containing cysts of both Sarcocystis sp. and S. hardangeri shed similar sporocysts, measuring on average 13.5×9.7 µm, after a prepatent period of 10–12 days.Based on the results from the present and previous investigations, Sarcocystis sp. is considered to have foxes (Vulpes vulpes and Alopex lagopus) and dogs (Ganis familiaris) as definitive hosts, becoming the second species of Sarcocystis with a known reindeer/Canidae life cycle. The name Sarcocystis tarandivulpes n. sp. is proposed as a replacement name for Sarcocystis sp. Gjerde, 1984 from skeletal muscle of reindeer.     

The raccoon dog (Nyctereutes procyonoides) as definitive host for Sarcocystis spp. of reindeer (Rangifer tarandus)

A raccoon dog (Nyctereutes procyonoides; Family: Canidae), was given cardiac muscle of reindeer infected with S. grueneri, and started shedding Sarcocystis sporocysts 10 days post feeding. The sporocysts measured 13.9 (12.4–15.7) × 10.1 (9.2–11.2) µm, and were excreted for at least 16 days. The raccoon dog is thus an additional definitive host for S. grueneri (Yakimoff & Sokoloff, 1934) Gjerde, 1984.Another raccoon dog was given skeletal muscle infected with 4 species of Sarcocystis, none of which was S. grueneri. The raccoon dog started shedding Sarcocystis sporocysts on day 10 post feeding, and excreted sporocysts for at least 16 days. The sporocysts measured 14.0 (12.3–15.6) × 10.1 (9.2–11.2) µm, and are considered to be sporocysts of S. tarandivulpes Gjerde, 1984.This is the first record of the raccoon dog as an experimental definitive host for Sarcocystis.     

Experimental infection of the deer ked (Lipoptena cervi) has no negative effects on the physiology of the captive reindeer (Rangifer tarandus tarandus)

The deer ked (Lipoptena cervi) is a haematophagous parasitic fly of cervids that spread to Finland in the early 1960's. Presently its northern distribution limit lies at approximately 65°N and it is gradually spreading northwards. In Finland the principal host species has been the moose (Alces alces), but the deer ked is about to establish contact with another potential host, the semi-domesticated reindeer (Rangifer tarandus tarandus) causing possible threats to reindeer health and management. The aim of this study was to investigate if the deer ked would have an influence on the welfare of the reindeer. Eighteen adult reindeer were divided into three experimental groups: the control group and two infected groups with 300 deer keds per reindeer introduced in August-September. One of the infected groups was treated with subcutaneous ivermectin in November. To gather comprehensive data on potential health hazards caused by the deer ked a wide array of physiological variables was measured during and at the end of the experiment in December. The keds caused no clear changes in the complete blood count, plasma clinical chemistry, amino acids, endocrinology, energy stores, enzyme activities or tissue fatty acid profiles of the host. The haematological, clinical chemical and endocrinological values displayed changes that could be related to the seasonal physiological adaptations of the species. In conclusion, at the duration and intensity of infection that were employed, the effects of the deer ked on the measured physiological variables of the reindeer were insignificant.     

Theileriosis in a reindeer (Rangifer tarandus tarandus) associated with a potentially novel Theileria sp

A 5‐year‐old male neutered reindeer (Rangifer tarandus tarandus) from Missouri was presented with a 3‐week history of anorexia, respiratory distress, lethargy, and weight loss. Blood smear review revealed that a small percentage of RBCs contained small (1–2 μm in length) pleomorphic piroplasms (signet ring, rod‐ or pear‐shaped, and elongate forms) with an eccentric magenta nucleus and basophilic cytoplasm. Nested PCR to specifically amplify a portion of the piroplasm small subunit ribosomal RNA (SSU rRNA) gene was performed on DNA extracted from an EDTA specimen of whole blood. Subsequent sequence analyses showed similarity between the reindeer hemoparasite and Theileria spp SSU rRNA gene sequences in the GenBank database, with highest similarity to those of a Theileria sp in a White‐tailed deer from North Texas (AY735132, AY735133). The reindeer and North Texas Theileria sp are genetically distinct from, albeit closely related to, the White‐tailed deer Theileria sp (subsequently referred to as T cervi). To the authors' knowledge, this is the first identification of Theileria of this genotype in a reindeer. Historically, T tarandirangiferis infection was found with associated mortality in reindeer in Russia, but reports predate molecular characterization. Hence, the relationship of T tarandirangiferis with either T cervi or this agent remains unknown. T cervi is not typically pathogenic in White‐tailed deer in the US unless the animal is immune‐compromised by stress or disease; however, mortality from T cervi infection in reindeer has been reported.     

Differentiation of Setaria digitata and Setaria labiatopapillosa using molecular markers

5S rRNA intergenic regions of Setaria digitata and Setaria labiatopapillosa were PCR amplified with primers designed from the 5S rRNA gene of Brugia malayi. The ladder-like banding patterns obtained for the amplifications were distinctly different for the two species. Four amplified products were cloned into the pBS vector and completely sequenced. DNA clones from two individual samples of S. digitata, Sd4 and Sd6, showed 97% sequence homology to each other. All sequenced clones showed the presence of the spliced leader (SL) RNA gene with a 22 nucleotide spliced leader sequence. The phylogenetic tree constructed using these data and the 5S rRNA intergenic regions of several other filarial nematodes showed the Setaria species sharing a branch with Dirofilaria. RAPD-PCR analyses identified 107 bands of which 86 were polymorphic (80%). A dendrogram constructed for S. digitata and S. labiatopapillosa separated the two species into two distinct clusters. The polymorphic loci identified by the RAPD-PCR analyses can be studied further to develop species-specific probes/PCR primers for the identification of each species.     

Sarcocystis infection in wild reindeer (Rangifer tarandus) from Hardangervidda in southern Norway: with a description of the cysts of Sarcocystis hardangeri n. sp

Fresh preparations of micro-isolated sarcosysts from skeletal muscle of 5 wild reindeer were examined by light microscopy. Slender, spindelshaped cysts measuring 821 × 60 µm, and having short knob-like cyst wall protrusions were found in all animals. In 1 animal cysts different in structure from the cysts of the 4 previously known Sarcocystis spp. of reindeer were found, These cysts are considered to be cysts of a new Sarcocystis sp. of reindeer, for which the name Sarcocystis hardangeri has been proposed.S. hardangeri n. sp. had macroscopic, ovoid to cylindrical cysts measuring 1667 (900–2570) × 819 (450–1575) µm. The cysts were surrounded by a 8–10 µm thick layer of fibrillar material. After removal of this layer, relatively few and irregularly spaced, slanting protrusions became visible. The 20–30 µm long protrusions were tongue-like, and were lying close to the surface of the cyst.Cysts of S. grueneri, S. rangiferi and S. tarandi were not demonstrated in the 5 wild reindeer examined.     

An SEM Study of the Morphology of the Lower Respiratory-tract Surface of the Reindeer (Rangifer tarandus tarandus L.)

The morphological features of the surface of the lower respiratory tract (trachea, bronchus, bronchiolus, distal airways, and alveoli) from 10 reindeer (Rangifer tarandus tarandus L.), differing in age and sex, were studied using scanning-electron microscopy. The respiratory surface of the reindeer generally resembles that reported previously in similar studies for other mammalian species. Ciliated epithelial cells, goblet cells, microvillous cells, Clara cells, alveolar epithelial cells of type 1 and type 2, and alveolar macrophages could be distinguished by their universally characteristic surface morphologies. The rarity of Kohn pores in the alveolar walls of reindeer was considered to be the most striking difference in comparison to most other species.     

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.     

Papillar morphology of the rumen of forest reindeer (Rangifer tarandus fennicus) and semidomesticated reindeer (R. t. tarandus)

The papillar morphology of the ventral and dorsal rumen of the wild forest reindeer (Rangifer tarandus fennicus Lönn.) and semidomesticated reindeer (R. t. tarandus L.) was studied in October and November 1996. The morphological measurements which were carried out were: the lengths of the papillae, the number of the papillae per square centimetre, the cross‐sectional area and perimeter of sections cut from the middle of papillae. From these measurements mean papillar volume, areal papillar volume, mean papillar (epithelial) surface area, areal papillar surface and surface enlargement factor were calculated. No differences in these measurements between ventral and dorsal walls of the rumen were evident. The semidomesticated reindeer had longer papillar perimeters, larger mean and areal papillar surface areas, larger mean papillar volumes, and a larger surface enlargement factor in the ventral rumen than did forest reindeer. This may be a result of differences between feeding habits, the semidomesticated reindeer preferring a diet including more plants rich in carbohydrates e.g. lichens, which has resulted in a high production of volatile fatty acids and thus stimulation of papillar growth.     

PCR studies of feline leprosy cases

16S rRNA gene sequence analysis provided evidence for two different mycobacterial species, Mycobacterium lepraemurium and a potentially novel species, as causative agents of 'feline leprosy'. Comparison of 16S rRNA gene sequence data obtained for M. lepraemurium and the potentially novel species indicated 12 nucleotide differences over a 446 bp region encompassing the V2 and V3 hypervariable regions. From available 16S rRNA gene sequence data, M. lepraemurium shared greatest nucleotide identity with M. avium subsp paratuberculosis and M. avium. The novel species had a long helix 18 in the V3 region and shared greatest nucleotide identity with M. leprae, M. haemophilum and M. malmoense. The novel species had an additional 'A' nucleotide at position 105 of the aligned 16S rRNA gene sequence, the only other mycobacterial database sequence having this same extra nucleotide being M. leprae. This nucleotide variation was exploited to develop specific PCR assays for the two species. These were found to be effective and specific when tested against a panel of mycobacteria including species found in feline leprosy lesions and closely related mycobacteria and also when applied directly to formalin-fixed, paraffin-embedded tissues from feline leprosy cases.     

驯鹿干扰素β1基因的克隆及遗传进化分析

试验旨在从驯鹿鹿茸顶端组织中克隆干扰素β1（interferon beta 1,IFNβ1）基因全长序列,分析其分子特性,为研究其生物学活性及实际应用奠定基础。根据GenBank数据库中牛、羊等近缘物种IFNβ1基因的保守序列设计1对引物,以驯鹿鹿茸顶端间充质层组织基因组DNA为模板,采用PCR技术扩增得到驯鹿IFNβ1基因并克隆、测序,利用相关生物信息学软件对该序列进行分析。结果发现,驯鹿IFNβ1基因全长561 bp,共编码186个氨基酸,含有3个糖基化位点;其编码蛋白含有4个α螺旋、4个β折叠区、7个β转角。将驯鹿IFNβ1基因推导的氨基酸序列与其他14种哺乳动物进行遗传进化分析发现,其同源性为45.1%~92.0%;驯鹿与其他动物IFNβ1基因序列分析和系统进化树分析表明,IFNβ1基因存在种属差异性,亲缘关系越近,同源性越高。本研究结果为驯鹿IFNβ1基因的进一步研究和应用提供了基础试验数据。     

Pestivirus and alphaherpesvirus infections in Swedish reindeer (Rangifer tarandus tarandus L.)

Herding semi-domesticated reindeer has economic and social value for Sami people in the northern territories of Fennoscandia. However, with the intensification of reindeer husbandry, interspecies transmission of pathogens between reindeer and domestic animals may become a problem, especially for countries such as Sweden, Norway, and Finland where pestivirus and alphaherpesvirus have been eradicated in domestic ruminants. This study, which included 1158 Swedish reindeer, showed relatively high prevalence of antibodies against bovine viral diarrhoea virus (BVDV) (32%) and bovine herpesvirus-1 (BoHV-1) (53%). Adult animals were more often seropositive for BVDV and BoHV-1 (50% and 78%, respectively) than were calves (18 and 11%, respectively). While the seroprevalence of alphaherpesvirus was similar in different herding districts, pestivirus seropositivity was highest in the South and diminished towards the North of the Swedish reindeer herding area. High correlation of the seropositivity against both pathogens at both individual and herd levels may indicate possible mutual synergetic effects and may be explained by the immunosuppressive nature of the viruses. While alphaherpesvirus seroprevalence was probably related to putative cervid herpesvirus 2 (CvHV-2), the pestivirus infecting reindeer remains undefined. The virus neutralisation test of reindeer sera using different pestivirus strains, revealed higher titres against Border disease virus strains like 137/4 (BDV-1) and Reindeer-1 (BDV-2) than against BVDV-1. However, the virus was not identified by real time RT-PCR in any of the samples (n=276) from seronegative reindeers. The study showed that pestivirus and alphaherpesvirus infections are endemic in the Swedish reindeer population.     

3株柔嫩艾美耳球虫28SrRNA基因部分序列的扩增与分析

本研究应用PCR技术扩增来自广东的3株柔嫩艾美耳球虫的28S rRNA基因部分序列,并与GenBank^TM登录的柔嫩艾美耳球虫、堆型艾美耳球虫、鼠肉孢子虫和刚地弓形虫虫株的相应序列进行比对分析。试验结果显示,柔嫩艾美耳球虫3个样品均获得1172 bp的28S rRNA基因部分有效序列,不同虫株序列没有差异,与GenBank^TM登录的柔嫩艾美耳球虫相应序列只有一个碱基差异,显示种内序列高度保守,而与堆型艾美耳球虫、鼠肉孢子虫、刚地弓形虫相应的序列存在不同程度的差异。结果表明,28S rRNA基因部分序列可作为研究艾美耳球虫种间及其他顶复门原虫遗传变异的标记。     

牛瑟氏泰勒虫吉林株18S rRNA基因的扩增及系统发育研究

本研究根据GenBank上发表的牛瑟氏泰勒虫18S rRNA基因的核苷酸序列设计并合成1对特异性引物,对寄生于牛体内的瑟氏泰勒虫基因组DNA进行扩增,得到1 356 bp的18S rRNA基因片段,测序后blast分析表明该虫种属牛瑟氏泰勒虫。将该基因片段序列与GenBank中8种已知泰勒虫的相应序列进行比较分析,建立系统发育树。结果表明,牛瑟氏泰勒虫吉林分离株与水牛泰勒虫亲缘关系最近,与小泰勒虫亲缘关系较远。这一结果说明宿主因素对泰勒虫的基因型影响较大。