Development and validation of species-specific nested PCRs for diagnosis of acute sarcocystiosis in sheep

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Abstract

Sheep may be infected by four species of Sarcocystis. Two of these species, Sarcocystis tenella and Sarcocystis arieticanis, are pathogenic. They may cause abortion or acute disease during the early phase of infection, and chronic disease during the late phase of infection. Thus far, diagnosis of sarcocystiosis in sheep has been limited, because traditional diagnostic tests based on the detection of Sarcocystis-specific antibodies are only genus-specific and, thus, cannot differentiate between pathogenic and non-pathogenic species. In addition, most of these tests show a reasonable sensitivity only for the late phase of infection. Therefore, diagnosis of acute sarcocystiosis has been based mainly on post-mortem examination, i.e. after the animal had succumbed to the disease. Here we established species-specific nested PCR assays based on unique small subunit ribosomal RNA gene sequences of S. tenella and S. arieticanis. These PCR assays specifically detect DNA of the homologous species in blood samples of sheep. No cross-reactions were observed with the heterologous pathogenic species, the non-pathogenic species Sarcocystis gigantea, or the closely related coccidia Toxoplasma gondii and Neospora caninum. In sheep experimentally infected with S. tenella or S. arieticanis, positive PCR results were correlated with the early phases of multiplication (endopolygeny) of the parasites. By contrast, Sarcocystis-specific antibodies were detected by an enzyme-linked immunosorbent assay only during the terminal phase of endopolygeny or thereafter. Thus, the nested PCR assays developed here enable, for the first time, the diagnosis and differentiation of infections with S. tenella and S. arieticanis in living sheep during the acute phase of the disease and facilitate comprehensive studies on the epidemiology and importance of infections with pathogenic Sarcocystis species in sheep.

Introduction

Sarcocystis species are obligatorily heteroxenous tissue cyst-forming coccidia. Merogony and cyst formation take place in the intermediate host, and gamogony and sporogony in the definitive host 1, 2. Sheep are intermediate hosts of four Sarcocystis species: Sarcocystis tenella (synonym Sarcocystis ovicanis), Sarcocystis gigantea (synonym Sarcocystis ovifelis), and probably Sarcocystis arieticanis are distributed worldwide, while infections with Sarcocystis medusiformis have been reported only from Australia, New Zealand, and Iran 3, 4, 5. Sarcocystis gigantea and S. medusiformis are transmitted by felids and are non-pathogenic. Sarcocystis tenella and S. arieticanis are transmitted by canids and are pathogenic 1, 2, 6.

Sheep become infected with S. tenella or S. arieticanis by ingesting sporocysts with contaminated food or water. The asexual development of both species comprises two phases of multiplication by endopolygeny in vascular endothelial cells 7, 8, 9, 10, 11, 12, 13. First-generation meronts of both species occur in arterioles of mesenteric lymph nodes, and those of S. tenella also in vascular endothelial cells of the heart, skeletal muscles, liver, and kidney. Second-generation meronts of both species develop in capillaries of almost all internal organs. Merozoites of the second generation of endopolygeny initiate the formation of tissue cysts, which can be formed in probably all striated muscles. Cysts of S. tenella may also be formed in cells of the CNS or in Purkinje fibres of the heart. Further asexual reproduction of the parasites takes place by repeated endodyogeny within the tissue cyst. Mature tissue cysts containing several hundred thousands of cystozoites are the terminal life-cycle stage of the parasites in sheep 7, 8, 14, 15, 16. If these cysts are ingested by a canine definitive host, the cystozoites initiate the sexual phase of the life-cycle (gamogony), which leads to the formation of oocysts in the small intestine and the shedding of mature sporocysts into the environment with the faeces of the definitive host 7, 9, 12, 13, 17, 18, 19, 20.

The severity of disease in sheep caused by S. tenella and S. arieticanis depends on the dose of ingested sporocysts and the immune status of the host. Disease is usually most severe during the early multiplication of the parasites by endopolygeny, which takes place from about day 5 to day 35 p.i. During this phase, a primary infection with one of the pathogenic Sarcocystis species may lead to acute sarcocystiosis with encephalitis, encephalomyelitis, and haemorrhagic diathesis which can cause the death of the animal 8, 9, 12, 13, 16, 21, 22, 23. In pregnant sheep, acute sarcocystiosis frequently results in foetal death, abortion, or premature birth of the offspring 15, 24, 25. Animals surviving a primary Sarcocystis infection may acquire immunity that protects them against acute disease after challenge with the homologous species, but not after infection with the heterologous pathogenic species [6]. However, chronic sarcocystiosis during the late phase of infection, i.e. after the formation of tissue cysts which takes place from about 30 days p.i. onwards, may cause additional economic losses due to reduced quality and quantity of meat, milk, or wool 9, 26, 27, 28, 29.

Under natural conditions, sheep may be infected with different pathogenic and non-pathogenic Sarcocystis species at any time. Thus far, diagnosis of acute disease or abortion caused by S. tenella or S. arieticanis has been limited by the low specificity and sensitivity of traditional diagnostic methods. Serological tests currently used for the diagnosis of Sarcocystis infections are only genus-specific and cannot differentiate infections with pathogenic species from those with non-pathogenic species. In addition, most of these tests are based on cystozoite-derived antigen preparations that detect antibodies only during the late phase of infection 2, 30, 31, 32, 33, 34, 35. There is no clinical symptom that is specific for acute sarcocystiosis, and, thus, diagnosis of the disease to date has been based mainly on histological detection of meronts at post mortems.

Molecular biological tests based on the detection of S. tenella and S. arieticanis molecules that may circulate in the blood during the proliferative phase of the parasites in the vascular system, would be a great advance for the diagnosis of acute sarcocystiosis in living sheep. Recently, a comparison of ssrRNA gene sequences of a range of tissue cyst-forming coccidia led to the identification of six variable regions in this gene that may be targeted by species-specific oligonucleotides designed to amplify ssrRNA gene fragments by PCR 2, 36. Although there are only few nucleotide differences among the ssrRNA genes of tissue cyst-forming coccidia [37], single-step PCR assays based on these differences can differentiate genomic DNA templates of pathogenic Sarcocystis species from those of non-pathogenic Sarcocystis species infecting sheep or goats [36]. Here we report the development of diagnostic nested PCRs that are based on oligonucleotides corresponding to the variable regions of the ssrRNA genes of S. tenella and S. arieticanis and enable a sensitive and species-specific detection of these species in blood samples of sheep infected experimentally with either S. tenella or S. arieticanis.

Section snippets

Parasites and genomic DNA preparations

Cystozoites of S. tenella and S. arieticanis were isolated by tryptic digestion of muscle samples derived from sheep infected experimentally with either S. tenella (Berlin strain) or S. arieticanis (Berlin strain) 38, 39. Cystozoites of S. gigantea were isolated mechanically from the oesophagi of naturally infected sheep slaughtered at an abattoir in North Rhine-Westphalia, Germany [40]. Endozoites of Toxoplasma gondii were harvested from the peritoneal cavities of mice infected experimentally

Optimisation of single-step amplifications

Maximum sensitivity for the amplification of a large ssrRNA gene fragment from the genomic DNA template of S. tenella in a single-step PCR assay using the primer pair STA–ST3 was obtained with four of the 15 polymerases tested (i.e. the Taq DNA polymerases of Appligene, Biomol, and Promega; and the GoldStar DNA polymerase of Eurogentec). All of the following experiments were carried out using the Promega Taq DNA polymerase. This polymerase was provided with a buffer that allowed the use of

Discussion

Several immunological tests have been developed for serological diagnosis of Sarcocystis infections in sheep 1, 6, 49. The most commonly used tests are IFATs or ELISAs that employ cystozoite-derived antigen preparations. These tests have a high sensitivity for the late phase of the infection, i.e. after tissue cyst formation has been initiated. In Europe and Australia, seroprevalences of Sarcocystis infections in sheep as determined by IFAT or ELISA have been found to range between 77 and 97% 4

Acknowledgements

We thank Professor Dr A.O. Heydorn, Institut für Parasitologie und Tropenveterinärmedizin, Freie Universität Berlin, Germany, for providing the Berlin strains of S. tenella and S. arieticanis and Dr N. Müller, Institute of Parasitology, University of Berne, Switzerland, for the preparation of genomic DNA of N. caninum. We also wish to thank Ms Angelika Duttman and Mr K. Fafoutis for excellent technical assistance.

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