Animal Infection, Morphological And Molecular Identification Of A New Piroplasma Pathogen

Piroplasma protozoa belongs to Apicomplexa, Sporozoa, Piroplasmida. Important pathogens in piroplasmida include Babesia spp. and Thelieria spp. Species in Genera Theileria and Babesia are known to infect both wild and domestic animals. And some species in Genus Babesia are important pathogens of human babesiasis. Previously, piroplasma were classified as hematocytozoonPiroplasma are transmitted by blood-sucking ticks. The sexual reproduction and sporogony are carried out in the invertebrate host, and asexual reproduction are in the vertebrate host. While taking vertebrate blood meal by ticks, Babeisia sporozoites are injected into the host and directly infect red blood cells. In contrast, Theileria sporozoites do not infect red blood cells but penetrate a lymphocyte (or macrophage) in which they develop into schizonts. The merozoites released from the schizonts enter red blood cells where they grow into piroplasms and multiply by budding into two or four daughter cells, forming tetrads, often in the shape of a Maltese cross.The tick becomes infected when ingesting blood cells containing piroplasms, which should probably be considered as gametocytes. They develop into male and female gametes in the tick gut. The microgametes fuse with macrogametes to form motile zygotes. The subsequent phase of development is different between Babesia and Theileria parasites. In Babesia the zygotes then multiply, which result invade numerous organs of the tick, including the ovaries. In this way, the infection passes through the ovary and the egg to the next tick generation. This is called transovarial transmission.In Theileria the zygotes do not multiply, but invade the hemolymph of the tick where they go towards the salivary glands. There is no invasion of other organs, and no passage through the ovaries and the eggs. When the next stage of the tick vector attaches to a new host, sporogony and maturation of the sporozoites in the salivary glands occur, and transmission takes place by the injection of infected saliva. This pattern is called transstadial transmission. Babesiosis represent in acute process, with a syndrome characterized by fever, anemia, hemoglobinuria, and icterus. It prevalent world wide, but endemic in rural, pasture, and field area with livestock and wild animals. The transmission is seasonal fluctuation. Several Babesis spp. are pathogens of human. The endemic of piroplasmosis results high mortality, which result in important problem of public health, and causes huge losses in animal husbandry and national economy.The taxonomy of piroplasma includes morphological and molecular biological classification. Morphologically, piroplasma parasites are round, pear, rod, oval, comma or amoeboid in shape on Giemsa-stained blood smears. The classical biological criteria, based on morphology, life cycle, and hosts are limited owing to changeable morphological feature, uncertainty of the life cycle, complexity in vector species.Present-day molecular tools make it possible to compare certain gene sequences of the genomes of various species, cryptic species, and gene types, and are of invaluable help in refining the relationships between various species.Theoretically, Theileria is not infective to human. But on searching the GenBank of NCBI, we found one DNA sequence that was amplified from genomic DNA of a febrile Chinese patient.In this study, we inoculated the blood taking from a febrile patient who was diagnosed as piroplasmosis, into several species of animals [KM strain and BALB/c mice, jirds (Meriones unguiculatus), and New Zealand white rabbits]to passage the pathogens. We made the blood smears regularly, conducted microscopic, electron microscopic observation and amplificating the partial fragment of18s rRNA of the infected blood.In blood smears,and ultrastructure sample made from the infected animal, we found that the piroplasma can form a lot of dense granules arround the neucleus of leucocyte. This, is similar to the character of Thelieria spp. that the schizogony in leukocyte, pointing out the pathogen is likely to be a related species of Thelieria spp. The organisms in erythrocytes can easily be overlooked in blood smear, they are small, usually single and variable in shape. We used four universal primers targeting the18S rRNA of Babesia spp. and Thelieria spp. according to reported reference papers, and desined the hemi-nested multiplex PCR to amplyfy the genomic DNA from the infected animal and patient. The seqences of amplified products from the three repeated PCR are highly homologic with Thelieria spp.. One364bp sequence from the cloned vectors have a maximum similarity (99%) with T. annulata.But, another seqence was obtained from two repeated PCR, on blasting the sequences from GenBank, we found it having a higher similarity with Colpodella than Babesia.The piroplasma of this study was derived from blood of a febril patient. Combining traditional morphological identification and molecular identification, the finding that the agamont appears in leukocytes, we conside the pathogen might be Thelieria spp.. From the results of several repeated PCR amplifications and sequencing, the sequence have a higher similarity with Colpodella sp. and Thelieria spp. than Babesia spp. Recently, there is one paper reported that Colpodella-like protozoa that might acting as a pathogen of human.So far in parasitological research, it is considerred that Babesia spp. are zoonosic pathogens of humans and animals babesiosis. Thelieria spp is obeserved only in animals. But we found that there is one Thelieria sp. sequence in GenBank which was amplified from human blood. Colpodella-like infection in human was also reported. While alignment3sequences of Colpodella, Thelieria and our sample7together, the sequence similarity were situated on both ends, with differences in middle sequence. The phylogenetic tree grouped our sequences more related to Thelieria and Colpodella. The results are difficult to explain, we can not judg whether the infection is caused by mixed causative agent or a new unknown pathogen. The taxonomic status of our pathogen might fall in Alveolata. In Alveolata, Colpodellids is a sister lineage with Apicomplexa, the later includes parasite of Plasmodium spp. Thelieria spp., and Babesia spp.. The exact taxonomic status needs further identification.This study characterised a emerging unknown human protozoan pathogen. It is not a Babesia sp. The results made our knowledges on this new pathogen one step forward, and these progresses might contribute great to the basic theory of prevention and control of this unknown piroplasma. This will also provide some referrences to identification this new pathogen in clinical diagnosis and epidemicological research. And it might be significant in prevention of this emerging pathogen.