Mongolian Isolates Research Articles

First molecular characterization of Burkholderia mallei strains isolated from horses in Mongolia

Glanders, a highly contagious and often fatal disease affecting equids, is caused by Burkholderia mallei. Although sporadic cases of equine glanders have recently been documented in Mongolia, genome sequencing and molecular studies of the bacteria within this region are lacking. This study provided the first molecular characterization of B. mallei isolated from four native Mongolian horses from two different provinces in 2019 and 2022 by applying whole-genome sequencing with two SNP types (previously developed genotyping with 15 SNP markers that provide global coverage of the B. mallei population and the core genome coding SNP typing developed in this study). The Mongolian isolates were located within the L3B1 cluster, which was previously associated with the V-120 strain from Russia. Within the L3B1 cluster shared by neighboring countries, they were in a unique subbranch. In this study, specific SNP markers unique to the Mongolian strains were identified to track these strains using a high-resolution melting analysis (HRMA). This study revealed the unique phylogenetic background of Mongolian strains isolated from the eastern part of Mongolia. HRMA specific to the Mongolian subbranch may contribute to the molecular epidemiological monitoring of glanders in Mongolia and surrounding countries.

Experimental Infection of Domestic Pigs with African Swine Fever Virus Isolated in 2019 in Mongolia.

African swine fever (ASF) is an infectious viral disease caused by African swine fever virus (ASFV), that causes high mortality in domestic swine and wild boar (Sus scrofa). Currently, outbreaks are mitigated through strict quarantine measures and the culling of affected herds, resulting in massive economic losses to the global pork industry. In 2019, an ASFV outbreak was reported in Mongolia, describing a rapidly progressing clinical disease and gross lesions consistent with the acute form of ASF; the virus was identified as a genotype II virus. Due to the limited information on clinical disease and viral dynamics within hosts available from field observations of the Mongolian isolates, we conducted the present study to further evaluate the progression of clinical disease, virulence, and pathology of an ASFV Mongolia/2019 field isolate (ASFV-MNG19), by experimental infection of domestic pigs. Intramuscular inoculation of domestic pigs with ASFV-MNG19 resulted in clinical signs and viremia at 3 days post challenge (DPC). Clinical disease rapidly progressed, resulting in the humane euthanasia of all pigs by 7 DPC. ASFV-MNG19 infected pigs had viremic titers of 108 TCID50/mL by 5 DPC and shed virus in oral secretions late in disease, as determined from oropharyngeal swabs. Whole-genome sequencing confirmed that the ASFV-MNG19 strain used in this study was a genotype II strain highly similar to other regional strains. In conclusion, we demonstrate that ASFV-MNG19 is a virulent genotype II ASFV strain that causes acute ASF in domestic swine.

Molecular identification and risk factor analysis of the first Lumpy skin disease outbreak in cattle in Mongolia.

Lumpy skin disease (LSD) is a transboundary viral infectious disease in cattle caused by a Capripoxvirus. LSD has been recently introduced in some Asian countries. However, in Mongolia, no report of LSD is publicly available. We clinically examined LSD symptoms in 1,034 cattle from 4 soum (district) in Dornod province in Mongolia. Sixty-one cattle of them were confirmed with symptoms of LSD and then viral P32 gene was detected by a PCR. The overall prevalence of LSD in cattle was 5.9%. Females odds ratios (OR)=2.27 than males, adults (>2.5-years-old, OR=3.68) than young (1–2.5-years-old) and calves (<1-year-old) were at higher risks for LSD cases in Mongolia, while locations near the tube well and pond water are major risk areas for viral transmission due to density of insects often is high. For virus isolation, skin nodule tissue samples of 4 cattle located in four distinct soums were used for viral propagation using the MDBK cell line. Internal terminal repeat region and RPO30 gene of 4 Mongolian isolates were amplified and sequenced. In the phylogenetic trees, Mongolian LSDVs (2021) were clustered together with the Chinese (2020) and Vietnamese isolates (2020). This is the first report alarming the LSD outbreak in Mongolia that was confirmed by our study. The newly isolated viruses would be a useful base for developing diagnostic tools and inactivated vaccine technology. A large-scale study of LSD is next priority for establishing successful control strategy of further disease outbreak.

Molecular Survey of Babesia microti (Aconoidasida: Piroplasmida) in Wild Rodents in Turkey.

Babesia microti (Aconoidasida: Piroplasmida) (Franca, 1910) is an important tick-borne zoonotic parasite with rodents serving as reservoir hosts. In the present study, 536 rodents were captured from Burdur, Bartin, Giresun, and Yozgat provinces of Turkey between the years 2010 and 2012, and blood samples were examined for the presence of Babesia spp. using conventional PCR which targeted the 18S rRNA gene. The sequence analysis of PCR amplicons was tested for B. microti as well as for Hepatozoon spp., and Sarcocystis spp. Overall, 5.8% of the rodents were positive for B. microti: 41% in Myodes glareolus, 7.7% in Chionomys roberti, and 2% in Apodemus spp., whereas no Babesia DNA was detected in Mus macedonicus and Microtus spp. Six rodents were positive for Hepatozoon spp. and one rodent was positive for Sarcocystis spp. Overall, 14.9 and 4.5% of rodents captured from Bartin and Giresun provinces, respectively, were PCR positive for B. microti, whereas none of rodents captured in Burdur and Yozgat were positive for Babesia spp. The sequence data of B. microti from rodents revealed that all sequences belonged to the zoonotic genotype. Sequences of B. microti obtained from rodents of the Bartin province were genotypically closer to European isolates, whereas those obtained from rodents of the Giresun province were closer to Russian and Mongolian isolates.

Molecular epidemiology, associated risk factors, and phylogenetic analysis of anaplasmosis in camel

Camel Anaplasmosis is caused by members of family Anaplasmatacae, a tick transmitted, obligate intracellular bacteria. The etiological bacteria are transmitted by ixodid tick species. The species have multi host range distribution that is why it is crucial to diagnose it timely. The aim of present study was to investigate the molecular epidemiology i.e. prevalence and risk factors analysis of camel anaplasmosis. Furthermore, variations in hematological standards were also evaluated. The study found an overall 13.33% prevalence in camels. The confirmation of PCR positive samples for Anaplasma spp. was made through sequencing, the study isolatesshowed high homology with Iranian, Chinese, Philippines and South African isolates of Anaplasmatacae (Accession numbers'; KX765882, KP062964, KY242456, LC007100 and U54806) on BLAST queries. The phylogenetic analysis revealedthree study isolates of present study clustered with each other and the cluster was found closer to Chinese isolate of A. phagocytophilum (KY242456), A. marginale (KU586048), and Mongolian isolates of A. ovis (LC194134). Two of the isolates resembled Iranian isolate of Candidatus Anaplasmacamelii (KX765882), while one isolate resembled with Chinese isolates of A. Platys (KX987336) and Croatian isolates of A. Platys (KY114935). The key risk factors odds ratio (OR>1) identified for occurrence of camel anaplasmosis using regression model found sex and age of animal, previous tick history, tick infestation and tick control status, housing type, cracks in walls, rearing system and other species in surrounding as the key risk factors. The hematological parameters like lymphocytes, monocytes, granulocytes and platelets count were significantly decreased (p < 0.05) in diseased camels than healthy. This is the first ever molecular data on camel anaplasmosis in Pakistan. The disease should be monitored unceasingly as the etiologies have multi host distribution. Prompt attention should be offered to animals because neutropenia, lymphopenia and thrombocytopenia can exacerbate the disease by making the animal predisposed to otherdiseases.

Ancient and modern genome shuffling: Reticulate mito-nuclear phylogeny of four related allopatric species of Gyrodactylus von Nordmann, 1832 (Monogenea: Gyrodactylidae), ectoparasites on the Eurasian minnow Phoxinus phoxinus (L.) (Cyprinidae).

Phylogenetic analyses including four allopatric species of Gyrodactylus von Nordmann, 1832 on the Eurasian minnow Phoxinus phoxinus (L.) (Cyprinidae) revealed incongruence between the nuclear ITS1-5.8S-ITS2 and mitochondrial cox1 phylogenies due to ancient hybridisation. Gyrodactylus pannonicus Molnár, 1968 was sampled close to its type-locality, the upper reaches of River Tisza, tributary of Danube in the Black Sea Basin. Faunistic search detected three new related species with maximum composite likelihood distances in cox1 between 16.8-23.2% (tentatively 1.3 to 1.8 My of divergence). Gyrodactylus albolacustris n. sp. recorded in the White Sea Basin, eastern Baltic Basin and Mongolia was close to G. pannonicus in the nuclear ITS (divergence of 0.9%), but diverged in cox1 by 19.8%. The Mongolian isolate of G. albolacustris n. sp. diverged from the European isolates in cox1 by 8.9%, suggesting 0.7My of isolation. The two other new species differed from G. pannonicus by>4% in ITS and some large indels in ITS1, and by>20% in cox1. Gyrodactylus danastriae n. sp. was found in River Strwiąż, a tributary of the River Dniester (Black Sea Basin) and was characterised by smaller size of anchors and by 29-41bp dimorphic insertion in ITS1. Gyrodactylus botnicus n. sp. is considered endemic in the Baltic Basin, but was also found in the White Sea Basin as a postglacial immigrant, where it had hybridised with G. albolacustris n. sp. in spite of the high divergence in ITS (3.9%) and cox1 (22%). The discordant nuclear and mitochondrial phylogenies revealed an ancient mitochondrial introgression: G. albolacustris n. sp. was derived from a hybridisation combining proto-pannonicus ITS with proto-danastriae mitochondria, perhaps 1.3 My ago. The postglacial hybridisation of G. albolacustris n. sp. (as the donor of mtDNAalb and ITSalb) and G. botnicus n. sp. (donor of the ITSbot) offered a model of shuffling of the genomic components: the process of the homogenisation and stabilisation of nuclear ITS (concerted evolution) and the lineage sorting has hardly begun.

Comparable High Rates of Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli in Birds of Prey from Germany and Mongolia

Frequent contact with human waste and liquid manure from intensive livestock breeding, and the increased loads of antibiotic-resistant bacteria that result, are believed to be responsible for the high carriage rates of ESBL-producing E. coli found in birds of prey (raptors) in Central Europe. To test this hypothesis against the influence of avian migration, we initiated a comparative analysis of faecal samples from wild birds found in Saxony-Anhalt in Germany and the Gobi-Desert in Mongolia, regions of dissimilar human and livestock population characteristics and agricultural practices. We sampled a total of 281 wild birds, mostly raptors with primarily north-to-south migration routes. We determined antimicrobial resistance, focusing on ESBL production, and unravelled the phylogenetic and clonal relatedness of identified ESBL-producing E. coli isolates using multi-locus sequence typing (MLST) and macrorestriction analyses. Surprisingly, the overall carriage rates (approximately 5%) and the proportion of ESBL-producers among E. coli (Germany: 13.8%, Mongolia: 10.8%) were similar in both regions. Whereas bla CTX-M-1 predominated among German isolates (100%), bla CTX-M-9 was the most prevalent in Mongolian isolates (75%). We identified sequence types (STs) that are well known in human and veterinary clinical ESBL-producing E. coli (ST12, ST117, ST167, ST648) and observed clonal relatedness between a Mongolian avian ESBL-E. coli (ST167) and a clinical isolate of the same ST that originated in a hospitalised patient in Europe. Our data suggest the influence of avian migratory species in the transmission of ESBL-producing E. coli and challenge the prevailing assumption that reducing human influence alone invariably leads to lower rates of antimicrobial resistance.

Phylogenetic analysis of the five internal genes and evolutionary pathways of the Greek H3N8 equine influenza virus

To amplify the NS, NP, PB1, PB2 and PA internal genes of two equine H3N8 influenza A viruses isolated in Greece in 2003 and 2007 five primer pairs were designed. The derived sequences were analysed from a phylogenetic point of view and compared with the evolutionary patters of the HA and NA proteins. Comparison of nucleotide sequences of the five internal genes of the Greek strains showed high similarity (99.3% - 99.7%) to strains isolated from outbreaks in Europe and Asia during 2002-2008. A total of 11 amino acid substitutions of the surface protein NA and the RNP complex proteins were identified in the Greek strains compared to those of progenitor viruses circulating up to 2003. These substitutions were repeated in Chinese and Mongolian isolates from outbreaks in 2007-2008. Notably NS1 protein did not acquired amino acid substitutions and moreover, a stop codon introduced at position 220 was stably maintained in the Greek strains. Phylogenetic trees of the five internal genes did not show the same separation in clades. Greek strains classified them into the American sublineage (as for the PA) Florida clade II (as for the NP, NS1 and PB1) and among Chinese strains of 2007-2008 outbreaks (as for the PB2). Additionally, evolutionary profiles of these internal proteins, except PB2, indicated a parallel evolution fashion to the HA protein, suggesting the possible occurrence of genetic reassortment between H3N8 viruses of district evolutionary lineages. In conclusion, phylogenetic analysis of the internal genes reported in this study could establish a candidate framework for future scientific communications on the phylogenetic diversity and evolution of the equine influenza viruses.

Phylogenetic relationships of Mongolian Babesia bovis isolates based on the merozoite surface antigen (MSA)-1, MSA-2b, and MSA-2c genes

We conducted a molecular epidemiological study on Babesia bovis in Mongolia. Three hundred blood samples collected from cattle grazed in seven different districts were initially screened using a previously established diagnostic polymerase chain reaction (PCR) assay for the detection of B. bovis-specific DNA. Positive samples were then used to amplify and sequence the hyper-variable regions of three B. bovis genes encoding the merozoite surface antigen (MSA)-1, MSA-2b, and MSA-2c. The diagnostic PCR assay detected B. bovis among cattle populations of all districts surveyed (4.4–26.0%). Sequences of each of the three genes were highly homologous among the Mongolian isolates, and found in a single phylogenetic cluster. In particular, a separate branch was formed only by the Mongolian isolates in the MSA-2b gene-based phylogenetic tree. Our findings indicate that effective preventative and control strategies are essential to control B. bovis infection in Mongolian cattle populations, and suggest that a careful approach must be adopted when using immunization techniques.

An H5N1 highly pathogenic avian influenza virus that invaded Japan through waterfowl migration.

In 2010, an H5N1 highly pathogenic avian influenza virus (HPAIV) was isolated from feces of apparently healthy ducks migrating southward in Hokkaido, the northernmost prefecture of Japan. The H5N1 HPAIVs were subsequently detected in domestic and wild birds at multiple sites corresponding to the flyway of the waterfowl having stopovers in the Japanese archipelago. The Hokkaido isolate was genetically nearly identical to H5N1 HPAIVs isolated from swans in the spring of 2009 and 2010 in Mongolia, but less pathogenic in experimentally infected ducks than the 2009 Mongolian isolate. These findings suggest that H5N1 HPAIVs with relatively mild pathogenicity might be selected and harbored in the waterfowl population during the 2009-2010 migration seasons. Our data provide "early warning" signals for preparedness against the unprecedented situation in which the waterfowl reservoirs serve as perpetual sources and disseminators of HPAIVs.

몽골에서 최초로 분리된 뉴캣슬병 바이러스의 분자생물학적 특성

몽골 산란계 농장에서 발생한 뉴캣슬병 사례로부터 뉴캣슬병 바이러스를 분리하고 그 특성을 조사하였다. 그 결과, 발생 농장 산란계 폐사계의 뇌 및 폐 조직으로부터 뉴캣슬병 바이러스 MN1/10 주가 분리되었다. 이 바이러스는 F 단백질 분절 부위가 특징적인 병원성 motif(RRQKRF)를 가지고 있었으며 종란 평균 치사 지수(MDT)가 54.7시간으로 강독형 NDV이었다. 또한 발생 농장 내 생존하고 있는 산란계에서 고역가의 NDV 특이항체가 검출되었다. 유적학적 계통 분석을 실시한 결과, 몽골 분리주는 Class II에 속하는 genotype VIId 바이러스로 확인되었다. 유전학적 계통 분석 결과, 몽골 분리주는 몽골과 인접한 중국에서 유행하는 바이러스 그룹(CN2)에 속하는 것으로 분류되었다. 우리의 연구 결과는 몽골에서의 뉴캣슬병 최초 발생은 동북아시아 지역에서 유행하는 강독형 NDV의 유입에 의해서 이루어졌음을 말해 준다. The outbreak of Newcastle disease occurred for the first time at a commercial chicken farm near Ulaanbaatar, Mongolia in August 2010. Newcastle disease virus (NDV) obtained from infected chickens in Mongolia was characterized by biological and molecular biological approches. Mongolian NDV isolate killed all of chicken embryos within 60 h in the mean death time assay, indicating virulent for chicken. A genomic region of 695 nts between nts 1055 of the M gene and 508 of the F gene was amplified by RT-PCR and sequenced. The deduced amino acid sequence of the F protein cleavage site was <TEX>$^{112}RRQKRF^{117}$</TEX>, which is a typical sequence of velogenic strains of NDV and is agreement with the result of the MDT assay. The sequence of the partial F gene (nts 47 to 435) was used for genotyping by phylogenetic analysis. The phylogenetic analysis showed that the Mongolian isolate was of genotype VII within class II of NDV. Further phylogenetic analysis on the genotype VII strains revealed that the isolates placed in a genetic sublineage of VIId and most closely related with velogenic strains of NDV circulating in Far-east Asian region especially China, suggesting the introduction of velogenic NDV into Mongolia from neighboring countries.

Characterization of Mumps Viruses Circulating in Mongolia: Identification of a Novel Cluster of Genotype H

Although mumps virus is still causing annual epidemics in Mongolia, very few epidemiological and virological data have been reported. We describe here the first phylogenetic analysis data on the mumps viruses circulated in Mongolia in 2009. We detected 21 mumps virus cDNAs and obtained a virus isolate from 32 throat swabs of mumps patients in Ulaanbaatar, the capital of Mongolia. The phylogenetic analyses based on the 316 nucleotides of the small hydrophobic gene show that these sequences form a single cluster, with the closest relatedness to the viruses belonging to genotype H. According to the recommendation of the World Health Organization, Mongolian mumps viruses could be classified into a novel genotype because the divergence between new sequences and genotype H reference viruses is >5% (6.3 to 8.2%). However, additional analyses based on the fusion gene, the hemagglutinin-neuraminidase gene, and the whole-genome indicate that the divergences between the Mongolian isolate and other genotype H strains never exceed the within-genotype divergences of other genotypes. These results suggest that Mongolia strains should be included in genotype H and that the current criteria for mumps virus genotyping should be revised. We propose here that the Mongolian viruses should be classified as a new subgenotype termed H3. Since previous epidemiological studies suggested that genotypes H may be associated with central nervous system diseases, we evaluated the neurovirulence of the Mongolian isolate in the neonatal rat system. However, the virus does not exhibit prominent neurovirulence in rats.

Multilocus sequence typing of Mycoplasma agalactiae

Mycoplasma agalactiae is the main cause of contagious agalactia, a serious disease of sheep and goats, which has major clinical and economic impacts. We have developed a multilocus sequence typing (MLST) scheme using the sequenced genomes of the M. agalactiae strains PG2 and 5632. An MLST scheme based on the genes gltX, metS, gyrB, tufA and dnaA was designed and in total 3468 bp of sequence were analysed for each strain. MLST offers a highly discriminatory typing method for M. agalactiae and was capable of subdividing 53 strains into 17 distinct sequence types, largely according to geographical origin. MLST detected unexpected diversity in recent isolates from Spain, identifying two novel outliers, and enabled typing of novel Mongolian isolates for the first time. Genetic diversity in the sequenced regions was largely due to mutation, with recombination playing a much smaller role. A web-accessible database has been set up for this MLST scheme for M. agalactiae: http://pubmlst.org/magalactiae/. MLST offers a robust, objective molecular epidemiological tool for M. agalactiae that that enables interlaboratory comparison of data.

Genetic analyses of H5N1 avian influenza virus in Mongolia, 2009 and its relationship with those of eastern Asia

In May and August 2009, 14 highly pathogenic H5N1 isolates were obtained from migratory birds in central Mongolia. To trace the genetic lineage of the isolates, nucleotide sequences of all eight genes were determined and phylogenetically analyzed. Hemagglutinin and nucleoprotein genes were clustered in clade 2.3.2. The polymerase acidic gene was related to the isolates of South Korea and Japan obtained in 2003 and 2004 outbreaks, and a migratory duck isolate from Jiangxi, China. The neuraminidase and other internal genes were closely related to those of clade 2.3.4 viruses. The results indicate evolving genetic diversity of the hemagglutinin gene and acquisition of different polymerase acidic gene in the 2009 Mongolian isolates, likely via bird migration. Prevention of potentially wider outbreak in domestic poultry and accurate monitoring of H5N1 genetic mutation will require continuous monitoring for H5N1 in both domestic and wild birds, and will necessitate international cooperation with neighboring countries sharing migratory flyways.

Previous H1N1 influenza A viruses circulating in the Mongolian population.

Four influenza A viruses of the subtype H1N1, isolated from Mongolian patients in Ulaanbaatar between 1985 and 1991, were analysed by sequencing of various RNA segments. The isolate from 1985 was found to be highly related in all genes sequenced to strains isolated from camels in the same region and at about the same time. These camel isolates were presumably derived from a UV-light inactivated reassortant vaccine (PR8 x USSR/77) prepared in Leningrad in 1978 and used in the Mongolian population at that time [19]. The human isolate from 1988 was also found to be a derivative of a reassortant between PR8 and USSR/77; in contrast to the 1985 isolate, however, it contained an HA closely related to PR8. One of the Mongolian isolates from 1991 (111/91) was in all genes sequenced closely related to PR8, while the other isolate from 1991 (162/91) was closely related to H1N1 strains isolated around 1986 in other parts of the world. About 12% of 235 convalescent sera collected in various parts of Mongolia contained antibodies against PR8, while none of German control sera contained such antibodies. The mutational and evolutionary rates of the Mongolian strains seem to be significantly lower when compared to the rates of human influenza A strains isolated in other parts of the world. This might indicate that these rates depend to a certain extent on the population density. Thus, viruses from remote areas might keep the potential to reappear in the human population after several years to cause a pandemic as it had happened in 1977.

A new filamentous virus in shallot

Serological analysis (ELISA, immunoblotting, and immunoelectron microscopy) of the partially purified virus preparations obtained from shallot plants (selection sample no. 83) inoculated with the Mongolian isolate of onion yellow dwarf potyvirus (OYDV) revealed neither an OYDV coat protein antigen nor virions decorated by the corresponding antiserum. At the same time, these preparations contained many flexuous filamentous virus particles (FFVP) with a modal length of 780 nm. It is suggested that shallot no. 83 is resistant at least to the Mongolian isolate of OYDV, while the discovered FFVPs are the virions of some other viral agent. The nature and origin of a new virus are now under study in this laboratory