Tracking tick-borne diseases in Mongolian livestock using next generation sequencing (NGS).

The livestock industry in Mongolia accounts for 24% of national revenue, with one third of the population maintaining a pastoral lifestyle. This close connection between Mongolian population and livestock is a major concern for pathogen transfer, especially given the increase in vector-borne diseases globally. This study examines blood samples from livestock to assess the prevalence of tick-borne bacterial infections across three provinces in Mongolia (Dornogovi, Selenge, Töv). Whole blood samples from 243 livestock (cattle=38, camel=11, goat=85, horse=22, sheep=87) were analyzed with 16S metagenomics next-generation sequencing (NGS) to screen for bacterial pathogens. Positive-NGS samples for Anaplasma, Bartonella, Ehrlichia, Francisella, Leptospira, and Rickettsia were confirmed by conventional PCR and DNA sequencing. Prevalence rates of Anaplasma, Bartonella, and Ehrlichia were 57.6%, 12.8%, and 0.4%, respectively. A significant difference in the prevalence of Anaplasma spp. in livestock by province was observed, with a higher prevalence in Selenge (74.2%, p<0.001) and Töv (64.2% p = 0.006) compared to the semi-arid region of Dornogovi (39.8%). In contrast, no association was observed in Bartonella prevalence by provinces. All Anaplasma sequences (N = 139) were characterized as A. ovis. For Bartonella species characterization, phylogenetic analyses of gltA and rpoB genes identified three Bartonella species including B. bovis, B. melophagi and Candidatus B. ovis. Bartonella bovis was detected in all 22-positive cattle, while B. melophagi and Candidatus B. ovis were found in four and three sheep, respectively. This study identifies a high prevalence of tick-borne pathogens within the livestock population and to our knowledge, is the first time NGS methods have been used to explore tick-borne diseases in Mongolia. Further research is needed in Mongolia to better understand the clinical and economic burdens associated with tick-borne diseases in both livestock and pastoral herder populations.

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.

Detection of bacterial pathogens in Mongolia meningitis surveillance with a new real-time PCR assay to detect Haemophilus influenzae.

Since the implementation of Haemophilus influenzae (Hi) serotype b vaccine, other serotypes and non-typeable strains have taken on greater importance as a cause of Hi diseases. A rapid and accurate method is needed to detect all Hi regardless of the encapsulation status. We developed 2 real-time PCR (rt-PCR) assays to detect specific regions of the protein D gene (hpd). Both hpd assays are very specific and sensitive for detection of Hi. Of the 63 non-Hi isolates representing 21 bacterial species, none was detected by the hpd #1 assay, and only one of 2 H. aphrophilus isolates was detected by the hpd #3 assay. The hpd #1 and #3 assays detected 97% (229/237) and 99% (234/237) of Hi isolates, respectively, and were superior for detection of both typeable and non-typeable Hi isolates, as compared to previously developed rt-PCR targeting ompP2 or bexA. The diagnostic sensitivity and specificity of these rt-PCR assays were assessed on cerebrospinal fluid specimens collected as part of meningitis surveillance in Ulaanbaatar, Mongolia. The etiology (Neisseria meningitidis, Hi, and Streptococcus pneumoniae) of 111 suspected meningitis cases was determined by conventional methods (culture and latex agglutination), previously developed rt-PCR assays, and the new hpd assays. The rt-PCR assays were more sensitive for detection of meningitis pathogens than other classical methods and improved detection from 50% (56/111) to 75% (83/111). The hpd #3 assay identified a non-b Hi that was missed by the bexA assay and other methods. A sensitive rt-PCR assay to detect both typeable and non-typeable Hi is a useful tool for improving Hi disease surveillance especially after Hib vaccine introduction.

Genetic diversity of Anaplasma and Ehrlichia bacteria found in Dermacentor and Ixodes ticks in Mongolia.

Anaplasma and Ehrlichia are tick-borne bacterial pathogens that cause human granulocytic anaplasmosis, human monocytic ehrlichiosis, and are severe threats to livestock economies like Mongolia. In this study, ticks were collected, identified, and pooled (n = 299) from three distinct environments across central Mongolia. Each pool was initially tested for Anaplasma/Ehrlichia using a 16S rRNA PCR assay that detects both genera, and specific PCR testing was done to identify those positive samples. Maximum likelihood estimation (MLE) of infection rates of ticks collected from the environment in Selenge aimag (province) found infection rates of Ixodes persulcatus ticks to be 2.0% (95% CI: 0.7, 4.3%) for A. phagocytophilum and 0.8% (95% CI: 0.1, 2.5%) for both nonspecific Ehrlichia and Anaplasma. Ehrlichia muris was only detected in I. persulcatus ticks collected from the Selenge aimag, where the MLE was 1.2% (95% CI: 0.1, 2.5%). The calculated MLE infection rate of Anaplasma spp. in questing Dermacentor nuttalli ticks ranged from 1.9% (95% CI: 1.1, 9.1%) in the Tov aimag to 2.3% (95% CI: 1.3, 10.8%) in the Selenge aimag. However, when examining MLE in ticks removed from livestock, estimates increase substantially, ranging from 7.8% (95% CI: 4.2, 13.3%) in Dornogovi to 22.5% (95% CI: 14.3, 34.3%) in Selenge, suggesting that livestock play a key role in disease maintenance. Considering the collective economic losses that can result from these pathogens and the potential for illness in nomadic herdsmen, these results highlight the need for enhanced TBD surveillance and prevention measures within Mongolia.

Estimated seroprevalence of Anaplasma spp. and spotted fever group Rickettsia exposure among herders and livestock in Mongolia.

BACKGROUND: To better understand the epidemiology of tick-borne disease in Mongolia, a comprehensive seroprevalence study was conducted investigating exposure to Anaplasma spp. and spotted fever group (SFG) Rickettsia spp. in nomadic herders and their livestock across three provinces from 2014 to 2015. METHODS: Blood was collected from 397 herders and 2370 livestock, including sheep, goats, cattle, horses and camels. Antibodies against Anaplasma spp. and SFG Rickettsia were determined by indirect immunofluorescence using commercially available slides coated with Anaplasma phagocytophilum and Rickettsia rickettsii antigens. Logistic regression was used to determine if the odds of previous exposure differed by gender, location, and species, with or without adjustment for age. To examine the association between seroprevalence and environmental variables we used ArcGIS to circumscribe the five major clusters where human and animal data were collected. RESULTS: Anaplasma spp. exposure was detected in 37.3% (136/365) of humans and 47.3% (1120/2370) of livestock; SFG Rickettsia exposure was detected in 19.5% (73/374) humans and 20.4% (478/2342) livestock. Compared to the southern province (aimag) of Dornogovi, located in the Gobi Desert, humans were significantly more likely to be exposed to Anaplasma spp. and SFG Rickettsia in the northern provinces of Tov (OR=7.3, 95% CI: 3.5, 15.1; OR=3.3, 95% CI: 1.7, 7.5), and Selenge (OR=6.9, 95% CI: 3.4, 14.0; OR=2.2, 95% CI: 1.1, 4.8). CONCLUSION: The high seroprevalence of Anaplasma spp. and SFG Rickettsia in humans and livestock suggests that exposure to tick-borne pathogens may be common in herders and livestock in Mongolia, particularly in the more northern regions of the country. Until more is known about these pathogens in Mongolia, physicians and veterinarians in the countryside should consider testing for Anaplasma and SFG Rickettsia infections and treating clinically compatible cases, while public health authorities should expand surveillance efforts for these emerging infections.

Distribution and molecular characteristics of rickettsiae found in ticks across Central Mongolia.

BACKGROUND: Little is known regarding tick-borne diseases in Mongolia, despite having 26% of the population still living nomadic pastoral lifestyles. A total of 1497 adult unfed ticks: 261 Ixodes persulcatus, 795 Dermacentor nuttalli, and 441 Hyalomma asiaticum, were collected from three ecologically distinct regions in Central Mongolia. Tick pools (n = 299) containing ~5 ticks each, were tested for Rickettsia and Tick-borne encephalitis virus (TBEV) using nested polymerase chain reaction, reverse transcription-PCR, and quantitative real-time RT-PCR. RESULTS: Assays yielded pooled prevalence of 92.5% (49/53) and 1.9% (1/53) for pooled I. persulcatus testing positive for "Candidatus Rickettsia tarasevichiae" and TBEV, respectively, while Rickettsia raoultii was found in 72.8% (115/158) of pooled D. nuttalli samples. When calculating a maximum likelihood estimation, an estimated 46.6% (95% CI: 35.2-63.6%) of I. persulcatus ticks in the pooled sample were infected with "Candidatus R. tarasevichiae". CONCLUSIONS: Both "Candidatus R. tarasevichiae" and R. raoultii are recognized as emerging tick-borne pathogens, with this being one of the first reports of "Candidatus R. tarasevichiae" in Mongolia. Given that "Candidatus R. tarasevichiae" shares the same vector (I. persulcatus) as TBEV, and infections may present with similar symptoms, Mongolian physicians treating suspected cases of TBEV should include "Candidatus R. tarasevichiae" infection in their differential diagnosis and consider prescribing antimicrobial therapy.