Prophylactic efficacy of drugs against equine piroplasmidosis in Gorny Altai

The purpose of the research is to study the prophylactic efficacy of piroplasmicides and to elucidate the effect of prophylactic doses of Imidocarb Dipropionate on the pathogen viability.Materials and methods. Babezan 12% was intramuscularly injected to 150 horses based on the active substance of Imidocarb Dipropionate at 2.5 mg/kg of the body weight and Neosidin M was injected to 30 horses twice at a dose of 2.5 mL per 100 kg of the live weight with a 15-day interval. The control group of animals did not receive the drug. The clinical follow-up of the experimental and control animals’ condition was done for 72 days. Before the experiment and 14 days after the drugs, blood samples were examined by the nested PCR in the presence of genus-specific primers from the 18S rRNA gene sequence for the Babesia spp. / Theileria spp. DNA. Species identification and genotyping of the detected Piroplasma were performed by sequencing 18S rRNA gene fragments.Results and discussion. Among 12 examined horses, 8 animals (66.7%) had the Piroplasmida DNA found in the blood samples, of which 50.0% were identified as Theileria equi and 16.7% as Babesia caballi. A high T. equi DNA prevalence (over 50.0%) indicates an endemic course of equine theileriosis. Babesan 12% in early chemotherapy of the horses helped to prevent the incidence of theileriosis within 48 and 59 days. Double chemotherapy of the horses with Neosidin M with a 15-day interval prevented the morbidity for 52 days. Babesan 12% at the preventive dose had no effect on the viability of persistent T. equi stages

RESULTS OF RECONNAISSANCE STUDIES ON DETECTION OF BABESIOSIS FOCI IN THE IRKUTSK REGION

This article contains results of reconnaissance studies intended to find foci of babesia in the Irkutsk region. Babesia DNA were founded in two types of ticks (I. persulcatus, H. concinna) in the three districts of the Irkutsk region (Ekhirit-Bulagatsky, Irkutsky, Shelekhovsky). The analysis of 1220 nr length nucleotide sequences of 18S rRNA gene of five Babesia samples was conducted. It was shown that the identified Babesia sequenced differ from previously known, and refer to the two genetic groups. The first group included babesia, genetically most similar to the sheep pathogen Babesia crassa, second contains sample, nucleotide sequence of which is significantly different from all known (less than 95 % homology)

IDENTIFICATION OF HUMAN GRANULOCYTIC ANAPLASMOSIS AND MONOCYTIC EHRLICHIOSIS NATURAL FOCI IN THE KRASNOYARSK TERRITORY

Microbiological monitoring of the six districts of the Krasnoyarsk Territory was conducted in order to prove the existence of natural foci of human granulocytic anaplasmosis (HGA) and monocytic ehrlichiosis (HME) on its territory. Two-round PCR method was used for the detection of Anaplasma and Ehrlichia DNA. 374 samples from mites were analyzed with two-round PCR for the presence of Anaplasma and Ehrlichia DNA: 341 specimens of Ixodes persulcatus and 33 specimens of Haemaphysalis concinna. Ticks were collected from vegetation on the flag in the 6 districts of the Krasnoyarsk Territory: Yeniseisky, Achynsky, Sharypovsky, Ilansky, Minusinsky and Ermakovsky, belonging to different climate zones and have landscaped, geo-botanical and faunal features. The study showed that 5.6 % of taiga ticks infected with E. muris, 2,1 % with A. phagocytophilum. In 0.3 % of carriers we identified mixtinfected E. muris and A. phagocytophilum. These data indicate presence of HGA and HME foci in the central, western and southern parts of the Krasnoyarsk Territory

IDENTIFICATION OF THEILERIA EQUI IN HORSE BLOOD IN THE IRKUTSK REGION

This paper describes the detection of Piroplasmida DNA in the blood of horses. 166 blood samples of horses from five districts of the Irkutsk region were studied. The high piroplasms infection rate in horses (69 %) was found. For six positive samples nucleotide sequences were determined. The comparison of determined 18S rRNA gene sequences with previously published sequences showed that all revealed piroplasms DNA samples are Theileria equi. The data are presented on the lethal cases of equine piroplasmosis in the Irkutsk region in 2008

Species and Genetic Diversity of Representatives of the <i>Anaplasmataceae</i> Family Found in the Sympatry Zone of the <i>Ixodes, Dermacentor</i> and <i>Haemaphysalis</i> Genera Ticks

Introduction.On the territory of the Ekhirit-Bulagatsky district of the Irkutsk region zones of sympatry of four Ixodes ticks species are found, where the species and genetic diversity of infectious agents transmitted through tick bites may be more pronounced than in foci with a mono-dominant type of ticks’ population. In this connection, the study of the species and genetic diversity of representatives of the Anaplasmataceae family in the sympatry zone of the Ixodes ticks of closely related species was of scientific interest.Objective: To study the species and genetic diversity of members of the Anaplasmataceae family in the zones of sympatry of Ixodes ticks Ixodes persulcatus, Dermacentor silvarum, D. nuttalli and Haemaphysalis concinna, to identify the main carriers and potential reservoir hosts of ehrlichia and anaplasma.Methods. In the course of the study, 1106 specimens of adult ticks and 49 samples of small mammalian livers from the Ekhirit-Bulagatsky area were analyzed. Anaplasma and ehrlichia DNA were detected by two-round PCR in the presence of genus- and species-specific primers from the 16S rRNA gene region. The nucleotide sequences of the 16S rRNA gene and the fragment of the groESL operon were identified in some samples. Sequencing was carried out according to the Sanger method. Comparative analysis was performed using the BLASTN program and ClustalW method. Epidemiological data analysis was performed using parametric methods of statistical processing of the material.Results. The DNA of Ehrlichia muris and Anaplasma phagocytophilum were detected in all studied species of ticks in their sympatry area. However, the rate of infection of taiga ticks was significantly higher than that of H. concinna and Dermacentor spp. Potential reservoir hosts of the Anaplasmataceae family members can be classified as Microtus oeconomus, M. gregalis, Myodes rutilus and Sorex spp. When analyzing the nucleotide sequences of the 16S rRNA gene, three genetic variants of anaplasma were detected. The nucleotide sequences of the A. phagocytophilum groESL operon belonged to two genetic groups

Иксодовые инвазии лошадей, сезонная динамика и зараженность пастбищных клещей пироплазмидами в Горном Алтае

The purpose of the research is the characterization of species composition and abundance of ixodid ticks that parasitize on horses, seasonal dynamics of ticks inhabiting natural pastures and their piroplasmid infection. Materials and methods. The studies were performed in four Gorny Altai physiographic provinces. Ticks were manually collected from horses (1183 specimens). Ticks were counted on natural pastures every ten days with subsequent determination of the species composition (1109 specimens). Collected ticks of the genus Dermacentor (443 specimens) were examined by nested PCR in the presence of genus-specific primers for Babesia spp. and Theileria spp.; the species identification of discovered piroplasmas was determined by 18S rRNA gene sequencing. Results and discussion. Ixodid ticks of three genera (Dermacentor, Ixodes, Haemaphysalis) and 6 species: D. nuttalli, D. silvarum, D. reticulatus, D. marginatus, H. concinna and Ix. Persulcatus, were recorded in the collections from horses and pastures. The most numerous species was D. nuttalli (56.1%), and the most common, Ix. persulcatus, that was recorded in all districts except Kosh-Agachsky in South-Eastern Altai. D. silvarum accounted for 14.9%, and other species were represented to a lesser extent. The highest species diversity, 5 species, was observed in the Northern Altai pastures and only 1 species, D. nuttalli, was recorded in the South-Eastern Altai. The seasonal dynamics of ticks in the pasture was characterized by two peaks in numbers, namely, the spring peak in the 3rd decade of April and the autumn peak in the 2nd decade of October. The spring-summer period (March-June) had 87.1% of recorded ticks, and the autumn period (September-October) had 7.2%. Three species of the examined ticks (D. silvarum, D. marginatus, D. nuttalli) were found to have DNA of three piroplasmid species (Babesia caballi, Theileria equi and Babesia sp). Piroplasmids infection rate in ticks was 2.7-25.0%, and 2.7% on average in Gorny Altai.Цель исследований – характеристика видового состава и численности иксодовых клещей, паразитирующих на лошадях, сезонной динамики клещей, обитающих на природных пастбищах и зараженности их пироплазмидами. Материалы и методы. Исследования проводили в четырех физико-географических провинциях Горного Алтая. Осуществляли ручной сбор клещей с лошадей (собрано 1183 экз.). Подекадные учеты численности клещей проводили на природных пастбищах с последующим определением видового состава (1109 экз.). Собранных клещей рода Dermacentor (443 экз.) исследовали методом двухраундовой ПЦР в присутствии родоспецифичных праймеров на наличие ДНК Babesia spp. и Theileria spp., видовую принадлежность обнаруженных пироплазм устанавливали методом секвенирования фрагментов гена 18S рРНК. Результаты и обсуждение. В сборах с лошадей и на пастбищах зарегистрированы иксодовые клещи трех родов (Dermacentor, Ixodes, Haemaphysalis) и 6 видов: D. nuttalli, D. silvarum, D. reticulatus, D. marginatus, H. concinna и Ix. persulcatus. Наиболее многочисленным видом оказался D. nuttalli (56,1%), наиболее распространенным – Ix. persulcatus, который регистрировали во всех районах, кроме Кош-Агачского Юго-Восточного Алтая. На долю D. silvarum приходится 14,9%, остальные виды представлены в меньшей степени. Наибольшее видовое разнообразие отмечено на пастбищах Северного Алтая – 5 видов, в Юго-Восточном Алтае зарегистрирован только 1 вид – D. nuttalli. Сезонная динамика клещей на пастбище характеризуется двумя пиками численности, весенним – в 3-й декаде апреля и осенним – 2-й декаде октября. На весенне-летний период (март-июнь) приходится 87,1 % учтенных клещей, на осенний (сентябрь-октябрь) – 7,2%. У трех видов исследованных клещей (D. silvarum, D. marginatus, D. nuttalli) обнаружена ДНК трех видов пироплазмид (Babesia caballi, Theileria equi и Babesia sp). Зараженность клещей пироплазмидами составила 2,7–25,0%, а, в среднем, по Горному Алтаю – 2,7%

Профилактическая эффективность препаратов при пироплазмидозах лошадей в Горном Алтае

The purpose of the research is to study the prophylactic efficacy of piroplasmicides and to elucidate the effect of prophylactic doses of Imidocarb Dipropionate on the pathogen viability.Materials and methods. Babezan 12% was intramuscularly injected to 150 horses based on the active substance of Imidocarb Dipropionate at 2.5 mg/kg of the body weight and Neosidin M was injected to 30 horses twice at a dose of 2.5 mL per 100 kg of the live weight with a 15-day interval. The control group of animals did not receive the drug. The clinical follow-up of the experimental and control animals’ condition was done for 72 days. Before the experiment and 14 days after the drugs, blood samples were examined by the nested PCR in the presence of genus-specific primers from the 18S rRNA gene sequence for the Babesia spp. / Theileria spp. DNA. Species identification and genotyping of the detected Piroplasma were performed by sequencing 18S rRNA gene fragments.Results and discussion. Among 12 examined horses, 8 animals (66.7%) had the Piroplasmida DNA found in the blood samples, of which 50.0% were identified as Theileria equi and 16.7% as Babesia caballi. A high T. equi DNA prevalence (over 50.0%) indicates an endemic course of equine theileriosis. Babesan 12% in early chemotherapy of the horses helped to prevent the incidence of theileriosis within 48 and 59 days. Double chemotherapy of the horses with Neosidin M with a 15-day interval prevented the morbidity for 52 days. Babesan 12% at the preventive dose had no effect on the viability of persistent T. equi stages.Цель исследований – изучить профилактическую эффективность пироплазмоцидных препаратов и выяснить влияние профилактических доз имидокарба дипропионата на выживаемость возбудителей заболевания.Материалы и методы. 150 головам лошадей внутримышечно вводили бабезан 12% из расчета по действующему веществу имидокарба дипропионата 2,5 мг/кг массы тела и 30 головам двукратно вводили неозидин М в дозе по ДВ 2,5 мл на 100 кг живой массы с интервалом 15 сут. Контрольной группе животных препарат не вводили. Клиническое наблюдение за состоянием животных опытной и контрольной групп осуществляли в течение 72 сут. Перед началом опыта и спустя 14 сут после введения препаратов исследовали пробы крови методом двухраундовой ПЦР в присутствии родоспецифичных праймеров из области гена 18S рРНК на наличие ДНК Babesia spp. / Theileria spp. Установление видовой принадлежности и генотипирование обнаруженных пироплазм проводили посредством секвенирования фрагментов гена 18S рРНК.Результаты и обсуждение. Из 12 обследованных лошадей в пробах крови у 8 животных (66,7%) обнаружена ДНК пираплазмид, из них 50,0% идентифицированы как Theileria equi и 16,7% как Babesia caballi. Высокий уровень встречаемости ДНК T. equi (свыше 50,0%) свидетельствует об эндемичном течении тейлериоза лошадей. Применение бабезана 12% при ранней химиотерапии лошадей позволяет профилактировать заболеваемость тейлериозом в течение 48 и 59 сут. Двукратная химиотерапия лошадей неозидином М с интервалом 15 сут предотвращает заболеваемость в течение 52 сут. Бабезан 12% в профилактической дозе не оказывает воздействия на выживаемость персестирующих стадий T. equi

Detection of Babesia and Anaplasmataceae bacteria in small mammals from Irkutsk and Novosibirsk regions

Tissue samples from 228 small mammals captured from 2013 to 2015 in four districts of Irkutsk region and in one district of Novosibirsk region were examined for the presence of Anaplasmataceae bacteria and Babesia parasites by nested PCR method with subsequent sequencing of positive samples. In Ekhirit-Bulagatskiy District of Irkutsk region, Babesia microti DNA was found in 10.9 % of small mammals, Anaplasma phagocytophilum DNA - in 7.3 %, and Ehrlichia muris DNA - in 1.8 %. Infected mammals were not found in the other three examined districts of Irkutsk region. In Novosibirsk region, B. microti DNA was found in 8.9 % of small rodents, A. phagocytophilum DNA - in 8.9 % of small mammals, and Candidatus Neoehrlichia mikurensis DNA - in 8.9 % of small mammals. The identified infection agents corresponded to microorganism species and genetic variants previously found in Ixodes persulcatus ticks but not in ticks of other species

Identification of the etiological agent of equine piroplasmosis in Western and Eastern Siberia

Equine piroplasmosis is a natural tick-borne infection caused by hemoprotozoan parasites of the order Piroplasmida, Babesia caballi and Theileria equi. Animals that recover from piroplasmosis remain persistently infected carriers and can transmit pathogens to vector ticks. Cases of equine piroplasmosis are periodically observed in Siberia, however, no agent of equine piroplasmosis has yet been genetically characterized in Russia. The aim of this work was studying the prevalence of the infectious agents of piroplasmosis in horses from Siberia and genotyping the detected agents. Blood samples from 155 horses were examined for the presence of Babesia and Theileria DNA by nested PCR with the subsequent sequencing of positive samples. DNA of T. equi was found in blood samples from 57.9 %, 38.5 % and 65.0 % of horses from Novosibirsk province, Irkutsk province, and the Republic of Altai, respectively. T. equi DNA was found in the samples from almost all sampling sites included in this study, indicating that most of the studied sites are endemic for equine theileriosis. Surprisingly, DNA of B. caballi was not found in any of the samples examined, even though this agent had previously been detected in many regions in Russia, including Altai. The analysis of the determined 18S rRNA gene sequences demonstrated that T. equi samples belonged to two genetic groups, which differed significantly by the sequences of the variable (V4) region of the gene. All T. equi sequences from group B were identical and corresponded to T. equi sequences found in the blood of horses from China and Korea, while T. equi sequences from group A differed by 1–5 nucleotide substitutions and were identical to the sequences from the blood of horses from India and Brazil or differed from them by single mismatches. Notably, in this study the presence of etiological agent of piroplasmosis in blood samples from horses in Russia was genetically confirmed for the first time

Genetic heterogeneity of <i>Rickettsia helvetica</i> population

Background. To date, the genetic variability of Rickettsia helvetica has not been sufficiently studied.The aim. To study the prevalence and genetic variability of R. helvetica in Ixodes spp. collected in Western Siberia and the Russian Far East.Materials and methods. Ixodes  spp. collected from rodents in the Omsk province, Western Siberia (n = 280) and collected by flagging on Putyatin and Russky Islands in Primorsky Krai, Russian Far East (n = 482) were analyzed for the presence of Rickettsia spp. All positive samples were genotyped for the gltA gene fragment. For a number of R. helvetica samples, fragments of the 16S rRNA, ompA, ompB, sca4, htrA, and groEL genes and 23S–5S intergenic spacer were additionally sequenced.Results. Four Rickettsia species (R. helvetica, “Candidatus Rickettsia tarasevichiae”, “Candidatus Rickettsia uralica”, and “Candidatus Rickettsia mendelii”) were found. Of them, R. helvetica was identified in 72.2 % of Ixodes apronophorus and 18.8 % of  Ixodes trianguliceps from the Omsk province and in single Ixodes persulcatus from the Omsk province and Putyatin Island. This is the first finding of Rickettsia spp. in I. apronophorus. All known R. helvetica sequences from this study and the GenBank database belonged to four well supported monopheletic groups forming genetic lineages I–IV. Lineage I included European isolates from Ixodes ricinus, Western Siberian isolates from I. persulcatus, and some sequences from I. apronophorus. All R. helvetica sequences from I. trianguliceps from the Omsk province and I. persulcatus from  the  Komi Republic and one sequence from I.  apronophorus were assigned to  lineage  II. Most sequences from I.  apronophorus formed lineage  III; all known R. helvetica sequences from I. persulcatus from the Far East formed genetic lineage IV.Conclusion. The genetic heterogeneity of R. helvetica population was first demonstrated. Known isolates of R. helvetica are reliably assigned to four genetic lineages, but not in all cases the association of different lineages with a specific tick species or specific territory was observed