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 epidemiological survey and genetic characterization of ovine gammaherpesvirus-2 in Mongolian livestock.

Sheep-associated malignant catarrhal fever (SA-MCF), caused by ovine gammaherpesvirus-2 (OvHV-2), is a fatal disease in all ruminants. The epidemiological survey and molecular characterization of OvHV-2 in Mongolian livestock were performed. Of 928 blood samples, 14 were positive for OvHV-2 in sheep and native cattle from Tsenkher County and in sheep from Lun County. Phylogenetic analyses revealed that the tegument gene of OvHV-2 sequences from Mongolian animals is identical to that in animals from Egypt, India, and Turkey, and is 98.0% similar to that in animals from Germany and Brazil. To our knowledge, this is the first confirmed report of OvHV-2 in Mongolian livestock, and could provide useful information for controlling SA-MCF.

Molecular Epidemiological Survey and Genetic Characterization of Anaplasma Species in Mongolian Livestock.

Anaplasma species are obligate intracellular rickettsial pathogens that cause great economic loss to the animal industry. Few studies on Anaplasma infections in Mongolian livestock have been conducted. This study examined the prevalence of Anaplasma marginale, Anaplasma ovis, Anaplasma phagocytophilum, and Anaplasma bovis by polymerase chain reaction assay in 928 blood samples collected from native cattle and dairy cattle (Bos taurus), yaks (Bos grunniens), sheep (Ovis aries), and goats (Capra aegagrus hircus) in four provinces of Ulaanbaatar city in Mongolia. We genetically characterized positive samples through sequencing analysis based on the heat-shock protein groEL, major surface protein 4 (msp4), and 16S rRNA genes. Only A. ovis was detected in Mongolian livestock (cattle, yaks, sheep, and goats), with 413 animals (44.5%) positive for groEL and 308 animals (33.2%) positive for msp4 genes. In the phylogenetic tree, we separated A. ovis sequences into two distinct clusters based on the groEL gene. One cluster comprised sequences derived mainly from sheep and goats, which was similar to that in A. ovis isolates from other countries. The other divergent cluster comprised sequences derived from cattle and yaks and appeared to be newly branched from that in previously published single isolates in Mongolian cattle. In addition, the msp4 gene of A. ovis using same and different samples with groEL gene of the pathogen demonstrated that all sequences derived from all animal species, except for three sequences derived from cattle and yak, were clustered together, and were identical or similar to those in isolates from other countries. We used 16S rRNA gene sequences to investigate the genetically divergent A. ovis and identified high homology of 99.3-100%. However, the sequences derived from cattle did not match those derived from sheep and goats. The results of this study on the prevalence and molecular characterization of A. ovis in Mongolian livestock can facilitate the control of infectious diseases in livestock.

Molecular detection and characterization of bovine viral diarrhea virus in Mongolian cattle and yaks.

Bovine viral diarrhea virus (BVDV) is classified into two species, namely, Bovine viral diarrhea virus 1 and Bovine viral diarrhea virus 2, and affects cattle worldwide, resulting in significant economic loss. The prevalence of BVDV-1 and BVDV-2 infections and its genotypes in Mongolian animals has not been studied. In this study, we surveyed BVDV infection in dairy cattle and yaks from Bornuur and Bulgan counties by RT-PCR, and the average infection rate in the sampling sites was 15.8 % and 20.0 %, respectively. In addition, molecular features of the 5'-UTR region of the BVDV genome in Mongolian cattle and yaks were identified as belonging to the subtypes BVDV-1a and BVDV-2a, respectively. Determining the prevalence, geographical distribution, and molecular diversity of BVDV-1 and BVDV-2 in various host species in Mongolia is important for further studies and process control programs.

Detection of bovine leukemia virus and identification of its genotype in Mongolian cattle.

Epidemiological studies have indicated that bovine leukemia virus (BLV) infection is globally distributed. However, no information regarding the disease and genetic diversity of the virus in the cattle of Mongolia is currently available. In this study, the prevalence of BLV was assessed using PCR, and the genetic diversity was analyzed through DNA sequencing. Of the 517 samples tested, 20 positives were identified. Phylogenetic analysis showed that six, one, and four isolates were classified into genotype 4, 7, and 1, respectively. Most isolates were clustered with isolates from Eastern Europe and Russia. This study is the first to investigate the BLV genotype in Mongolia.

A preliminary survey of the seroprevalence of Mycobacterium avium subspecies paratuberculosis in Mongolian cattle.

Johne's disease is a chronic infection with Mycobacterium avium susp. paratuberculosis (MAP), which causes huge economic losses to cattle industry. The seroprevalence of MAP in cattle of Mongolian was estimated by an ELISA assay using 356 serum samples which were collected from eleven provinces and Ulaanbaatar city. Out of these samples, 3 (0.84%) were found to be seropositive for MAP, originating from Tsenkher sum of Arkhangai province, Murun sum of Khuvsgul province, and Bornuur sum of Tuv province in Mongolia. This study represents first conformation of Johne's disease in Mongolian cattle. These findings provide vital information that can be used for the planning and execution of control measures for Johne's disease in the Mongolian cattle industry.

Detection of Trypanosoma brucei in field-captured tsetse flies and identification of host species fed on by the infected flies.

The prevalence of trypanosome infections in tsetse flies in the Chiawa area of Lower Zambezi in Zambia, with endemic trypanosomosis, was determined by a polymerase chain reaction (PCR) method that allowed the detection of trypanosome DNA and determination of the type of animal host fed on by the tsetse fly Glossina pallidipes, using tsetse-derived DNA extracts as templates. Ninety G. pallidipes (82 females and 8 males; 18.3%) of the 492 flies captured by baited biconical traps tested positive for the presence of Trypanosoma brucei species genomic DNA. Of the 90 T. brucei-positive flies, 47 (52.2%) also tested positive for vertebrate mitochondrial DNA. Sequence analysis of the vertebrate mitochondrial DNA amplicons established that they originated from 8 different vertebrate species, namely, human (Homo sapiens), African elephant (Loxodonta cyclotis), African buffalo (Syncerus caffer), waterbuck (Kobus ellipsiprymnus), roan antelope (Hippotragus equinus), greater kudu (Tragelaphus strepsiceros), warthog (Phacochoerus africanus), and goat (Capra hircus). Furthermore, to investigate the prevalence of trypanosome infections in domestic goats in the same area where trypanosomes had been detected in tsetse files, a total of 86 goats were randomly selected from 6 different herds. Among the selected goats, 36 (41.9%) were found to be positive for T. brucei species. This combined detection method would be an ideal approach not only for mass screening for infection prevalence in tsetse populations, but also for the prediction of natural reservoirs in areas endemic for trypanosomosis.

Cytokine responses in camels (Camelus bactrianus) vaccinated with Brucella abortus strain 19 vaccine.

In the present study, we determined the levels of cytokines produced by camel (Camelus bactrianus) peripheral blood mononuclear cells (PBMCs) in response to live attenuated Brucella abortus (B. abortus) S19 vaccine. Seven camels were vaccinated with commercial B. abortus S19 vaccine, and their cytokine responses were determined using a real-time PCR assay. Cytokine responses to B. abortus S19 were examined at 6 hr, 48 hr and 1, 2 and 3 weeks post-vaccination. Serological tests were performed to further confirm these immune responses. The results revealed that IFN-gamma and IL-6 were upregulated during the first week post-vaccination. Low level expressions of IL-1alpha, IL-1beta, TNFalpha and IL-10 and no expression of IL-2 and IL-4 were observed compared with the control camels. The findings showed that B. abortus stimulates cell-mediated immunity by directly activating camel Th1 cells to secrete IFN-gamma. This quantification of cytokine expression in camels is essential for understanding of Camelidae disease development and protective immune responses. This is the first report of in vivo camel cytokine quantification after vaccination.

Molecular cloning, sequencing and phylogenetic analysis of inflammatory cytokines of swamp type buffalo contrasting with other bubaline breeds.

The current research concerned in the cloning, sequencing and phylogenetic analysis of inflammatory cytokine (IL-1alpha, IL-1beta, IL-6 and TNF-alpha) genes from swamp buffalo and two bubaline breeds, CB (cross between swamp and riverine type buffalo) and the Bulgarian Murrah buffalo. Multiple sequence comparison showed a high homology between the bubaline breeds, which ranged from 99.3% to 100.0% similarity, whereas from 98.6% to 99.0% compared to cattle. The phylogenetic analysis had confirmed and justified the degree of relationship between these bubaline species and their distinctness to each other by the bootstrap value (%) generated. These findings were discussed with particular attention to the diversity of the inflammatory cytokine proteins within closely related species. The result of this study concluded that a small difference in the cytokine structures might be the reason behind or has a contributory factor on the previous reports about the existence of disease resistance. However, in-depth study is necessary to further qualify these findings.

Complete cDNA sequences and phylogenetic analyses of the Th1 and Th2 cytokines of the bactrian camel (Camelus bactrianus).

The complementary DNAs of the Th1 (IL-2, IL-12p35, and IFN-gamma) and Th2 (IL-4, IL-10 and IL-13) cytokine genes of the bactrian camel (Camelus bactrianus) were cloned, sequenced, and analyzed. IL-2, IL-4, IL-10, IL-12p35, IL-13, and IFN-gamma were found to have 465, 402, 537, 669, 411, and 501 bp length open reading frames with 154, 133, 178, 222, 136, and 166 amino acid encodings, respectively. The homology ranged from 58.8% to 100% between the nucleotide sequences of the camel cytokine genes and the published sequences of other mammalian genes, including the llama, pig, cow, horse, human, and mouse. The cDNA had highest homology with orders Artiodactyla (pigs and cattle) and Perissodactyla (horses), especially to the recently cloned llama sequences.

Comparative assessment of Th1 and Th2 cytokines of swamp type buffalo and other bubaline breeds by molecular cloning, sequencing and phylogenetics.

Comparative assessment of Th1 and Th2 cytokines of three bubaline breeds namely swamp buffalo, its crossbreed with riverine buffalo (CB), and the improved breed of Bulgarian Murrah buffalo (BMB), was done by molecular cloning, sequencing and phylogenetic analysis. The Th1 cytokines analyzed included IL-2, IL-12p35, IL-12p40, and IFN-gamma while Th2 cytokines included IL-4 and IL-10. Both groups showed strict conservation in the putative secondary structures and amino acid residues within the tribe Bovini, which indicated functional cross-reactivity. Nucleotide sequence homology ranged from 98.6 to 100.0% and was lowest for IL-12p35. With regard to amino acid sequence, the lowest homology was observed in IL-4 with 97.8%. This substitution was mainly due to differences in mRNA splicing. The phylogenetic relationship of the buffalo breeds was analyzed and showed them as a cluster comprised mainly of species belonging to the order Artiodactyla, including cattle and pigs. A deeper knowledge of these cytokine structures will favor understanding of water buffalo immunology and how much it differs from its closest subspecies and other animals.

Molecular cloning and phylogenetic analysis of inflammatory cytokines of Camelidae (llama and camel).

We cloned, sequenced and analyzed the cDNAs encoding Camelidae inflammatory cytokines, including llama (lama glama) interleukin (IL)-1alpha, IL-1beta, IL-6, tumor necrosis factor (TNF)-alpha and camel (Camelus bactrianus) IL-6 and TNF-alpha. The similarity levels of the deduced amino acid sequences of IL-1alpha, IL-1beta, IL-6 and TNF-alpha from llama (camel) to those from other mammalian species, ranged from 60.7% to 87.7%, 52.8% to 75.3%, 41.4% to 98.6%, and 72.9% to 99.6%, respectively. Phylogenetic analyses based on nucleic acid sequences showed that llama IL-1alpha, IL-1beta, IL-6 and TNF-alpha were more closely related to those of camel, pig, cattle, sheep and horse than to those of human, dog, cat, mouse and rat.

Cloning and sequence analysis of llama (lama glama) Th2 (IL-4, IL-10 and IL-13) cytokines.

This paper describes the cloning and sequence analysis of the cDNAs encoding the T helper (Th) 2 cytokines of llama including interleukin-4 (IL-4), IL-10 and IL-13. The cDNAs encoding for IL-4, IL-10 and IL-13 were amplified using specific primers designed from reported sequences of bovine cytokine genes. The cDNAs for llama IL-4, IL-10 and IL-13 were found to be 402, 537 and 411 bp in length, with open reading frames encoding 133, 178 or 136 amino acids, respectively. Homology analyses of nucleotide and deduced amino acid sequences of llama IL-4, IL-10 and IL-13 and phylogenetic analysis based on their nucleotide sequences indicated the close relationship in these cytokine genes between llama and eutherian mammalian order Artiodactyla (pig, cattle) and Perissodactyla (horse).

Quantification of llama inflammatory cytokine mRNAs by real-time RT-PCR.

We have developed a method by which llama cytokine mRNAs can be quantified using real-time reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted from lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) of llama, reverse transcribed to cDNA, and cytokine profiles for interleukin (IL)-1alpha, IL-1beta, IL-6 and tumor necrosis factor (TNF) alpha were quantified by real-time PCR. The expressions of mRNAs of inflammatory cytokines IL-1alpha, IL-1beta, IL-6 and TNFalpha were upregulated upon stimulation with LPS in a dose- and time-dependent manner. Incubation of PBMCs with 100 and 1,000 pg/ml of LPS for 3 to 6 hr resulted in the acceleration of the mRNA levels of inflammatory cytokines. Here, we describe a highly sensitive and reproducible method to quantify the transcription of llama cytokine mRNAs by real-time RT-PCR with the double-stranded DNA-binding dye SYBR Green I.

Cloning and sequence analysis of llama cytokines related to cell-mediated immunity.

In order to characterize the T helper 1 (Th1) cytokines of llama, we have cloned several llama cytokine genes and compared them to those of other mammalian species. The cDNAs encoding for interleukin (IL)-2, interferon (IFN)gamma, IL-12p35 and IL-12p40 were amplified using specific primers designed from reported sequences of bovine cytokine genes. The cDNAs for llama IL-2, IFN-gamma, IL-12 p35 and IL-12p40 were found to be 465, 501, 669 or 993 bp in length, with open reading frames encoding 154, 166, 222 or 330 amino acids, respectively. Homology analyses of nucleotide and deduced amino sequences of llama IL-2, IFN-gamma, IL-12p35 and IL-12p40 and phylogenetic analysis based on their nucleotide sequences indicated the close relationship in these cytokine genes between llama and eutherian mammalian order Artiodactyla, which includes pig and cattle.