Two cases of hydrophobia in the Republic of Tatarstan: In vivo and postmortem laboratory diagnosis

The results of rabies in vivo and postmortem laboratory detection in two cases registered in the Republic of Tatarstan are reported: a victim bitten by a wolf in 2002 and another one bitten by a stray dog on Goa Island, India, in 2013. In the patient bitten by a wolf cornea imprints studies using the method of fluorescent antibodies (MFA) showed rabies-positive result 6 days before the patient's death. The results were confirmed by postmortem examination of different parts of the brain and salivary glands using the MFA, enzyme-linked immunosorbent assay (ELISA), optical microscopy, and bioassay methods. In the patient bitten by a stray dog the rabies virus specific antigen was detected by eye cornea studies using the MFA method and saliva studies using the ELISA. The rabies virus genome was also isolated from saliva and tear fluid using nested reverse-transcription polymerase chain reaction (RT-PCR) 9 days before the patient's death. The in vivo studies results were consistent with the postmortem study of different parts of the brain using the MFA, enzyme-linked immunosorbent assay (ELISA), optical microscopy, and bioassay methods. All the infection-positive results of both in vivo and postmortem studies were consistent with the clinical studies, i.e. rabies diagnosis was confirmed. The analysis of the rabies virus gene G fragment nucleotide sequence of 238 nd length showed a slight difference between the studied isolates (2 rabies) and the RABV AY956319 (1.68%), difference by 10.5% from the Vnukovo-32 vaccine strains and by 10.9 % from the SAD B19 rabies strain, respectively (rabies viruses of 1st genotype). It was also significantly different from the lissaviruses of 2,4,5, and 6 genotypes (21.0-32.7%). The obtained results indicate phylogenetic closeness of the studied isolates (2 rabies) with the RABV AY956319 rabies virus strain belonging to the 1st genotype

ВЕКТОРНАЯ КОМПЕТЕНТНОСТЬ И СПОСОБНОСТЬ НАСЕКОМЫХ – ПЕРЕНОСЧИКОВ ИНФЕКЦИЙ

The most important parameters of infection transmission: vector competence and vector ability of transmitters, elements of genesis of vector-borne diseases and intrabody pathology of insects are briefly described. Emergent occurrence and spread of vector-borne exotic diseases in nonendemic areas can be considered as a precursory warning of pandemic threat caused by infectious agents including pre-viously unknown agents of new areas and ecological zones. Epidemiological sig-nificance of arboviral mosquito-borne infection is being discussed.Кратко охарактеризованы важнейшие параметры трансмиссии - векторная компетентность и векторная способность переносчиков, элементы генеза трансмиссивной инфекции и внутриорганизменной патологии насекомых. Эмерджентное возникновение и распространение трансмиссивных экзотических инфекций на неэндемичных территориях служит пандемическим предвестником прогрессирующей экспансии возбудителями, в том числе ранее неизвестными, новых территорий и экозон. Обсуждается эпидемиологическое значение арбовирусных инфекций насекомых

ДНК-ДИАГНОСТИКА АНАПЛАЗМОЗА КРУПНОГО РОГАТОГО СКОТА

Objective of research: The purpose of our research was to develop a DNA diagnostic method for anaplasmosis in cattle. Materials and methods: Blood samples were obtained from the tail vein with the use of ethylenediaminetetraacetic acid as an anticoagulant. The extraction of DNA was performed with the kit Sorb-M. To analyze the gene msp4 the following sequences belonging to different isolates Anaplasma marginale were used. To select primers the conserved elements of sequences were detected with the server СlustalW2. The specificity of primers was checked by a BLASTN search. The results of polymerase chain reaction (PCR) were estimated using 2% agarose gel electrophoresis. Electrophoresis was performed within 40 minutes at the field intensity 5 V/cm. Fragments of gene msp4 obtained as a results of polymerase chain reaction (PCR) were purified, ligated and cloned in E. coli cells. Transformation was performed using the heat shock method. Search for E. coli colonies containing pGEM-msp4 plasmid was conducted by the PCR method using standard M13 primers with the following analysis of PCR results by electrophoresis. Target colonies of E. coli were cultured overnight at 37 °С in 2 ml LB medium containing ampicillin in a 100 mcg /ml concentration. Sequencing of received plasmids pGEM-msp4 was carried out by Sanger method and the genetic analyzer Applied Biosystems 3130.  Results and discussion: Development and approbation of primers on the basis of the MSP4 gene of Anaplasma marginale to perform DNA diagnostics of anaplasmosis in cattle by PCR method were described. Due to PCR sensitivity along with the use of primers, it is possible to identify 100 and more gene copies. TheЦель исследования – разработка метода ДНК-диагностики анаплазмоза крупного рогатого скота.  Материалы и методы. Пробы крови отбирали из хвостовой вены с использованием ЭДТА в качестве антикоагулянта. ДНК выделяли с помощью набора Sorb-M. Для анализа гена msp4 были использованы соответствующие последовательности, принадлежащие разным изолятам Anaplasma marginale. Выявление консервативных участков последовательностей для подбора праймеров проводили с помощью сервера СlustalW2. Видоспецифичность праймеров проверяли с использованием алгоритма BLASTN. Результаты полимеразной цепной реакции (ПЦР) оценивали методом электрофореза в 2%-ном агарозном геле. Электрофорез проводили в течение 40 минут при напряженности поля 5 В/см. Полученные в результате ПЦР фрагменты гена msp4 были очищены, лигированы и клонированы в клетках E. coli. Трансформацию проводили методом теплового шока. Поиск колоний E. coli DH5α, содержащих плазмиду pGEM-msp4, проводили методом ПЦР с использованием стандартных праймеров M13 с последующим анализом результатов ПЦР методом электрофореза. Целевые колонии наращивали в течение ночи при 37 °С в 2 мл среды LB, содержащей ампициллин в концентрации 100 мкг/мл. Секвенирование полученных плазмид pGEM-msp4 осуществляли по методу Сэнгера и генетического анализатора Applied Biosystems 3130. Результаты и обсуждение. Описаны разработка и апробация праймеров к гену msp4Anaplasma marginale для ДНК-диагностики анаплазмоза крупного рогатого скота методом ПЦР. Чувствительность ПЦР с использованием этих праймеров позволяет выявить 100 и больше копий гена. Проведенные испытания свидетельствуют о 100%-ной повторяемости и воспроизводимости данного метода

Vector competence and insect capacity of infection transmission

The most important parameters of infection transmission: vector competence and vector ability of transmitters, elements of genesis of vector-borne diseases and intrabody pathology of insects are briefly described. Emergent occurrence and spread of vector-borne exotic diseases in nonendemic areas can be considered as a precursory warning of pandemic threat caused by infectious agents including pre-viously unknown agents of new areas and ecological zones. Epidemiological sig-nificance of arboviral mosquito-borne infection is being discussed

Two cases of hydrophobia in the Republic of Tatarstan: In vivo and postmortem laboratory diagnosis

The results of rabies in vivo and postmortem laboratory detection in two cases registered in the Republic of Tatarstan are reported: a victim bitten by a wolf in 2002 and another one bitten by a stray dog on Goa Island, India, in 2013. In the patient bitten by a wolf cornea imprints studies using the method of fluorescent antibodies (MFA) showed rabies-positive result 6 days before the patient's death. The results were confirmed by postmortem examination of different parts of the brain and salivary glands using the MFA, enzyme-linked immunosorbent assay (ELISA), optical microscopy, and bioassay methods. In the patient bitten by a stray dog the rabies virus specific antigen was detected by eye cornea studies using the MFA method and saliva studies using the ELISA. The rabies virus genome was also isolated from saliva and tear fluid using nested reverse-transcription polymerase chain reaction (RT-PCR) 9 days before the patient's death. The in vivo studies results were consistent with the postmortem study of different parts of the brain using the MFA, enzyme-linked immunosorbent assay (ELISA), optical microscopy, and bioassay methods. All the infection-positive results of both in vivo and postmortem studies were consistent with the clinical studies, i.e. rabies diagnosis was confirmed. The analysis of the rabies virus gene G fragment nucleotide sequence of 238 nd length showed a slight difference between the studied isolates (2 rabies) and the RABV AY956319 (1.68%), difference by 10.5% from the Vnukovo-32 vaccine strains and by 10.9 % from the SAD B19 rabies strain, respectively (rabies viruses of 1st genotype). It was also significantly different from the lissaviruses of 2,4,5, and 6 genotypes (21.0-32.7%). The obtained results indicate phylogenetic closeness of the studied isolates (2 rabies) with the RABV AY956319 rabies virus strain belonging to the 1st genotype

Two cases of hydrophobia in the Republic of Tatarstan: In vivo and postmortem laboratory diagnosis

The results of rabies in vivo and postmortem laboratory detection in two cases registered in the Republic of Tatarstan are reported: a victim bitten by a wolf in 2002 and another one bitten by a stray dog on Goa Island, India, in 2013. In the patient bitten by a wolf cornea imprints studies using the method of fluorescent antibodies (MFA) showed rabies-positive result 6 days before the patient's death. The results were confirmed by postmortem examination of different parts of the brain and salivary glands using the MFA, enzyme-linked immunosorbent assay (ELISA), optical microscopy, and bioassay methods. In the patient bitten by a stray dog the rabies virus specific antigen was detected by eye cornea studies using the MFA method and saliva studies using the ELISA. The rabies virus genome was also isolated from saliva and tear fluid using nested reverse-transcription polymerase chain reaction (RT-PCR) 9 days before the patient's death. The in vivo studies results were consistent with the postmortem study of different parts of the brain using the MFA, enzyme-linked immunosorbent assay (ELISA), optical microscopy, and bioassay methods. All the infection-positive results of both in vivo and postmortem studies were consistent with the clinical studies, i.e. rabies diagnosis was confirmed. The analysis of the rabies virus gene G fragment nucleotide sequence of 238 nd length showed a slight difference between the studied isolates (2 rabies) and the RABV AY956319 (1.68%), difference by 10.5% from the Vnukovo-32 vaccine strains and by 10.9 % from the SAD B19 rabies strain, respectively (rabies viruses of 1st genotype). It was also significantly different from the lissaviruses of 2,4,5, and 6 genotypes (21.0-32.7%). The obtained results indicate phylogenetic closeness of the studied isolates (2 rabies) with the RABV AY956319 rabies virus strain belonging to the 1st genotype

Spread Dynamics of Leucosis in Cattle in Livestock Farms of the Russian Federation for 2000–2018

Leucosis occupies a leading position in the modern nosological structure of cattle infectious diseases. This is due to the high infection of livestock and large economic damage in affected farms [1–3]. Leucosis is a chronic tumor disease caused by an RNA-containing virus of the Retroviridae family. The disease is characterized by uncontrolled reproduction of immature hematopoietic cells [4, 5]. The objective is to assess in dynamics the indicators of cattle infection with the leucosis virus and the incidence of leucosis in farms of all categories of the Russian Federation in the period of 2000–2018. The data of the analysis of the epizootic situation for leucosis in cattle in farms of all categories of the Russian Federation for 2000-2018 are presented. Over this period of time, the number of diagnostic serological studies in the immunodiffusion reaction increased 1.88 times in all subjects. According to the results of epizootological monitoring in farms of all categories of the Russian Federation, the rates of infection of the bovine leucosis virus decreased 1.89 times, the incidence rates decreased 2.02 times. At present, the epizootic situation has improved markedly, but so far there are problems with incomplete release of farms from leucosis in cattle. It is necessary to continue work on the elimination of this dangerous chronic disease in farms of all categories in the territory of the Russian Federation

DNA DIAGNOSTICS OF ANAPLASMOSIS IN CATTLE

Objective of research: The purpose of our research was to develop a DNA diagnostic method for anaplasmosis in cattle. Materials and methods: Blood samples were obtained from the tail vein with the use of ethylenediaminetetraacetic acid as an anticoagulant. The extraction of DNA was performed with the kit Sorb-M. To analyze the gene msp4 the following sequences belonging to different isolates Anaplasma marginale were used. To select primers the conserved elements of sequences were detected with the server СlustalW2. The specificity of primers was checked by a BLASTN search. The results of polymerase chain reaction (PCR) were estimated using 2% agarose gel electrophoresis. Electrophoresis was performed within 40 minutes at the field intensity 5 V/cm. Fragments of gene msp4 obtained as a results of polymerase chain reaction (PCR) were purified, ligated and cloned in E. coli cells. Transformation was performed using the heat shock method. Search for E. coli colonies containing pGEM-msp4 plasmid was conducted by the PCR method using standard M13 primers with the following analysis of PCR results by electrophoresis. Target colonies of E. coli were cultured overnight at 37 °С in 2 ml LB medium containing ampicillin in a 100 mcg /ml concentration. Sequencing of received plasmids pGEM-msp4 was carried out by Sanger method and the genetic analyzer Applied Biosystems 3130.  Results and discussion: Development and approbation of primers on the basis of the MSP4 gene of Anaplasma marginale to perform DNA diagnostics of anaplasmosis in cattle by PCR method were described. Due to PCR sensitivity along with the use of primers, it is possible to identify 100 and more gene copies. Th