Refining the evolutionary tree of the horse Y chromosome.

The Y chromosome carries information about the demography of paternal lineages, and thus, can prove invaluable for retracing both the evolutionary trajectory of wild animals and the breeding history of domesticates. In horses, the Y chromosome shows a limited, but highly informative, sequence diversity, supporting the increasing breeding influence of Oriental lineages during the last 1500 years. Here, we augment the primary horse Y-phylogeny, which is currently mainly based on modern horse breeds of economic interest, with haplotypes (HT) segregating in remote horse populations around the world. We analyze target enriched sequencing data of 5 Mb of the Y chromosome from 76 domestic males, together with 89 whole genome sequenced domestic males and five Przewalski's horses from previous studies. The resulting phylogeny comprises 153 HTs defined by 2966 variants and offers unprecedented resolution into the history of horse paternal lineages. It reveals the presence of a remarkable number of previously unknown haplogroups in Mongolian horses and insular populations. Phylogenetic placement of HTs retrieved from 163 archaeological specimens further indicates that most of the present-day Y-chromosomal variation evolved after the domestication process that started around 4200 years ago in the Western Eurasian steppes. Our comprehensive phylogeny significantly reduces ascertainment bias and constitutes a robust evolutionary framework for analyzing horse population dynamics and diversity.

BoLA-DRB3 genetic diversity in Highland Creole cattle from Bolivia.

The genetic diversity of the BoLA-DRB3 gene has been reported in different cattle breeds owing to its central role in the immune response. However, it is still unknown in hundreds of cattle breeds, especially native populations. Here, we studied BoLA-DRB3 genetic diversity in Highland Creole cattle (CrAl) from Western Bolivia, raised at altitudes between 3800 and 4200 m. DNAs from 48 CrAl cattle were genotyped for BoLA-DRB3 exon 2 alleles using polymerase chain reaction-sequence-based typing (PCR-SBT). The results were compared with 1341 previously reported data from Tropical Creole cattle and other breeds raised in the region. Twenty-three BoLA-DRB3 alleles were identified in CrAl, including the BoLA-DRB3*029:02 variant previously detected in other Creole cattle. Observed and expected heterozygosity were 0.87 and 0.93, respectively. Nucleotide diversity and the number of pairwise difference values were 0.078 and 19.46, respectively. The average number of nonsynonymous and synonymous substitutions were 0.037 and 0.097 for the entire BoLA-DRB3 exon 2, and 0.129 and 0.388 for the antigen-binding site, respectively. Venn analysis and the review of the IPD-MHC database and the literature showed that 2 of 64 alleles were only detected in CrAl, including BoLA-DRB3*029:01 previously reported in African cattle and *048:01 detected in Philippine cattle. Two additional alleles, BoLA-DRB3*007:02 and *029:02, were only present in CrAl and Lowland Creole cattle. Principal Component Analysis (PCA) showed that Bolivian Creole cattle breeds were closely located but they were distant from the Colombian Hartón del Valle Creole. FST analysis showed a low degree of genetic differentiation between Highland and Lowland Bolivian Creole cattle (FST = 0.015). The present results contribute to increasing our knowledge of BoLA-DRB3 genetic diversity in cattle breeds.

Characterization of bovine MHC DRB3 diversity in global cattle breeds, with a focus on cattle in Myanmar.

BACKGROUND: Myanmar cattle populations predominantly consist of native cattle breeds (Pyer Sein and Shwe), characterized by their geographical location and coat color, and the Holstein-Friesian crossbreed, which is highly adapted to the harsh tropical climates of this region. Here, we analyzed the diversity and genetic structure of the BoLA-DRB3 gene, a genetic locus that has been linked to the immune response, in Myanmar cattle populations. METHODS: Blood samples (n = 294) were taken from two native breeds (Pyer Sein, n = 163 and Shwe Ni, n = 69) and a cattle crossbreed (Holstein-Friesian, n = 62) distributed across six regions of Myanmar (Bago, n = 38; Sagaing, n = 77; Mandalay, n = 46; Magway, n = 46; Kayin, n = 43; Yangon, n = 44). In addition, a database that included 2428 BoLA-DRB3 genotypes from European (Angus, Hereford, Holstein, Shorthorn, Overo Negro, Overo Colorado, and Jersey), Zebuine (Nellore, Brahman and Gir), Asian Native from Japan and Philippine and Latin-American Creole breeds was also included. Furthermore, the information from the IPD-MHC database was also used in the present analysis. DNA was genotyped using the sequence-based typing method. DNA electropherograms were analyzed using the Assign 400ATF software. RESULTS: We detected 71 distinct alleles, including three new variants for the BoLA-DRB3 gene. Venn analysis showed that 11 of these alleles were only detected in Myanmar native breeds and 26 were only shared with Asian native and/or Zebu groups. The number of alleles ranged from 33 in Holstein-Friesians to 58 in Pyer Seins, and the observed versus unbiased expected heterozygosity were higher than 0.84 in all the three the populations analyzed. The FST analysis showed a low level of genetic differentiation between the two Myanmar native breeds (FST = 0.003), and between these native breeds and the Holstein-Friesians (FST <  0.021). The average FST value for all the Myanmar Holstein-Friesian crossbred and Myanmar native populations was 0.0136 and 0.0121, respectively. Principal component analysis (PCA) and tree analysis showed that Myanmar native populations grouped in a narrow cluster that diverged clearly from the Holstein-Friesian populations. Furthermore, the BoLA-DRB3 allele frequencies suggested that while some Myanmar native populations from Bago, Mandalay and Yangon regions were more closely related to Zebu breeds (Gir and Brahman), populations from Kayin, Magway and Sagaing regions were more related to the Philippines native breeds. On the contrary, PCA showed that the Holstein-Friesian populations demonstrated a high degree of dispersion, which is likely the result of the different degrees of native admixture in these populations. CONCLUSION: This study is the first to report the genetic diversity of the BoLA-DRB3 gene in two native breeds and one exotic cattle crossbreed from Myanmar. The results obtained contribute to our understanding of the genetic diversity and distribution of BoLA-DRB3 gene alleles in Myanmar, and increases our knowledge of the worldwide variability of cattle BoLA-DRB3 genes, an important locus for immune response and protection against pathogens.

New evidence of bovine leukemia virus circulating in Myanmar cattle through epidemiological and molecular characterization.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle. BLV infects cattle worldwide and causes serious problems for the cattle industry. In this study, we examined the prevalence of BLV infection and the distribution of BLV genotypes in cattle in the northern, central, and southern parts of Myanmar. The prevalence of BLV infection among Myanmar cattle (37.04%) in this study was markedly higher than the prevalence (9.1%) observed in our earlier study in which BLV was detected from the limited number of cattle only from a small area of Myanmar. Phylogenetic analysis of partial env-gp51 sequence of the isolated BLV strains revealed that there are at least three BLV genotypes (genotype-1, genotype-6, and genotype-10) in Myanmar, which have also been detected in the neighboring countries. We performed this study to estimate the BLV proviral load, which is a major diagnosis index for determining the virus transmission risk. The cattle of the three test regions with warm, wet, and humid climatic conditions (upper Sagaing, Yangon, and Kayin) exhibited a high mean proviral load, while cattle of three other regions with low annual rainfall and very high temperature (Mandalay, Magway, and upper Bago) exhibited a low mean proviral load. Further, the level of proviral load and the prevalence of BLV infection in Myanmar native cattle (N = 235) were lower than that in the hybrid cattle (Holstein Friesian × Myanmar native) (N = 62). We also observed that the cattle with high risk for BLV transmission, which have high proviral load, may enhance the BLV infection rate. Hence, to control BLV transmission, it is necessary to eliminate these cattle with high-risk for BLV transmission and to diagnose BLV provirus in cattle in the remaining regions/states of Myanmar sharing a boundary with neighboring countries.

Genotype distribution and allele frequencies of the genes associated with body composition and locomotion traits in Myanmar native horses.

Myanmar native horses are small horses used mainly for drafting carts or carriages in rural areas and packing loads in mountainy areas. In the present study, we investigated genotype distributions and allele frequencies of the LCORL/NCAPG, MSTN and DMRT3 genes, which are associated with body composition and locomotion traits of horses, in seven local populations of Myanmar native horses. The genotyping result of LCORL/NCAPG showed that allele frequencies of C allele associated with higher withers height ranged from 0.08 to 0.27, and 0.13 in average. For MSTN, allele frequencies of C allele associated with higher proportion of Type 2B muscular fiber ranged from 0.05 to 0.23, and 0.09 in average. For DMRT3, allele frequencies of A allele associated with ambling gait ranged from 0 to 0.04, and 0.01 in average. The presences of the minor alleles of these genes at low frequencies suggest a possibility that these horse populations have not been under strong selection pressure for particular locomotion traits and body composition. Our findings of the presence of these minor alleles in Southeast Asian native horses are also informative for considering the origins of these minor alleles associated with body composition and locomotion traits in horse populations.

The molecular epidemiological study of bovine leukemia virus infection in Myanmar cattle.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle. BLV infects cattle worldwide and affects both health status and productivity. However, no studies have examined the distribution of BLV in Myanmar, and the genetic characteristics of Myanmar BLV strains are unknown. Therefore, the aim of this study was to detect BLV infection in Myanmar and examine genetic variability. Blood samples were obtained from 66 cattle from different farms in four townships of the Nay Pyi Taw Union Territory of central Myanmar. BLV provirus was detected by nested PCR and real-time PCR targeting BLV long terminal repeats. Results were confirmed by nested PCR targeting the BLV env-gp51 gene and real-time PCR targeting the BLV tax gene. Out of 66 samples, six (9.1 %) were positive for BLV provirus. A phylogenetic tree, constructed using five distinct partial and complete env-gp51 sequences from BLV strains isolated from three different townships, indicated that Myanmar strains were genotype-10. A phylogenetic tree constructed from whole genome sequences obtained by sequencing cloned, overlapping PCR products from two Myanmar strains confirmed the existence of genotype-10 in Myanmar. Comparative analysis of complete genome sequences identified genotype-10-specific amino acid substitutions in both structural and non-structural genes, thereby distinguishing genotype-10 strains from other known genotypes. This study provides information regarding BLV infection levels in Myanmar and confirms that genotype-10 is circulating in Myanmar.

Detection of antibodies against Fusobacterium necrophorum and Porphyromonas levii-like species in dairy cattle with papillomatous digital dermatitis.

Bovine digital epidermitis involves different pathologies, including PDD, interdigital dermatitis, and foot rot. Bacteriological and molecular biological studies suggest that these are multimicrobial infections. During our study on the isolation of treponemes from biopsies of PDD, colonies producing black pigment were isolated frequently from the primary cultures, suggesting that Porphyromonas species were present. Moreover, 16S rRNA genes of Fusobacterium necrophorum and Porphyromonas levii-like species were detected in the lesions. We therefore determined whether an immunological response could be elicited by a P. levii-like organism isolated from a PDD lesion, as well as two subspecies of F. necrophorum in the sera from cows with and without PDD. A total of 151 serum samples were collected from 85 cows with PDD lesions and 33 cows without lesions on 12 PDD-positive farms and from 33 cows on two PDD-free farms. ELISA data showed that IgG antibody levels against antigens of P. levii-like species and F. necrophorum subsp. necrophorum were significantly higher in cows on PDD-positive farms than in cows on PDD-free farms, regardless of the presence of PDD lesions in the cows on the PDD-positive farms. However, F. necrophorum subsp. funduliforme was present at low levels in both groups. The ELISA results were confirmed by western blot analysis. Furthermore, antigens of these bacteria were detected in PDD-biopsy sections examined by immunohistochemical staining. F. necrophorum subsp. necrophorum and P. levii-like species may be involved in the pathogenesis of PDD.

Analysis of the IgG immune response to Treponema phagedenis-like spirochetes in individual dairy cattle with papillomatous digital dermatitis.

Papillomatous digital dermatitis (PDD) is a major infectious disease of the foot skin in dairy cattle. Treponema phagedenis-like spirochetes have been consistently detected in PDD lesions, and antibodies against these organisms have been demonstrated in affected cattle. However, little is known about the dominant antigens recognized by the immune system of affected cattle. Here, we investigated the IgG immune response to T. phagedenis-like isolates by Western blotting with different sera using whole-cell lysates and extracted glycolipid from 18 and 8 isolates, respectively, including those from different cattle on the same or different farms, isolates from different lesions affecting a single cow, and different isolates from the same lesion affecting a single cow. The reactivity of sera in Western blot assays revealed different banding patterns or showed no bands, suggesting that considerable antigenic variations, including glycolipid, may exist among the isolates, even in those from single individuals. With use of a total of 151 serum samples collected from three groups of cattle, i.e., PDD-positive cows on PDD-positive farms (group A), PDD-negative cows on PDD-positive farms (group B), and cows on PDD-free farms (group C), the levels of IgG antibodies against four T. phagedenis-like isolates were measured by enzyme-linked immunosorbent assay (ELISA). The optical density in groups A and B was significantly higher than that in group C, even though the value varied among the antigens used. Therefore, combinations of multiple Treponema species should be used for serological analysis and the development of a suitable vaccine because of antigenic variations.

Detection of treponemes in canker lesions of horses by 16S rRNA clonal sequencing analysis.

Equine canker is a chronic pododermatitis of the hoof in horses. Although spirochetes are detectable histopathologically in the lesions, the precise etiology remains unclear. This study reports the 16S rRNA gene sequencing of randomly selected clones based on PCR with Treponema-specific primers, using the canker lesions from two horses and healthy frog and sole from a horse. A total of 114 clones were obtained from the lesions, but no clones were detected in the healthy hoof tissues. The clones from the canker lesions examined were grouped into 19 operational taxonomic units, such as treponemal phylotypes originating from papillomatous digital dermatitis lesions of dairy cattle and as-yet uncultured human oral treponemes, indicating the presence of multiple treponemes in the lesions.

Identification of candidate pathogens of papillomatous digital dermatitis in dairy cattle from quantitative 16S rRNA clonal analysis.

Although it is suspected that papillomatous digital dermatitis (PDD), an infectious foot disease of cattle, is caused by multiple bacteria, it remains unclear precisely which ones are involved in the etiology. To study the bacterial community, we used 16S rRNA gene sequencing of randomly selected clones based on PCR with minimum amplification cycles to search for organisms present in PDD lesions but not in healthy foot skin. The nucleotide sequences of 1525 clones from 5 PDD lesions (836 clones) and 4 samples of healthy foot skin (689 clones) were determined and grouped into 316 operational taxonomic units (OTUs) with a cut-off value of >99% sequence identity. Two OTUs, P-01 (143 clones; 100% nucleotide sequence identity with Treponema phagedenis) and P-02 (112 clones; 86% identity with Bacteroidetes), were detected most frequently in all PDD samples examined. In contrast, OTU N-01 (87 clones), showing 99% nucleotide sequence identity with Moraxella phenylpyruvica, was the most prevalent in the normal samples examined. Spirochaetes were detected in only 1 sample. Phylogenetic analysis showed that T. denticola-like and T. phagedenis-like spirochetes were the predominant groups in the PDD lesions. Detection of multiple treponemes and an unknown bacterium close to Bacteroides sp. at high rates by a culture-independent approach could be evidence of the association of these organisms with PDD.

The mode of biofilm formation on smooth surfaces by Campylobacter jejuni.

Many microorganisms produce extracellular polymers referred to collectively as "slime" or glycocalyx, and form biofilms on solid surfaces in natural ecosystems. Campylobacter jejuni, one of the most important foodborne pathogens, also has the ability to form biofilm on stainless steel, glass, or polyvinyl chloride in vitro. However, the issue of biofilm formation by Campylobacter species has not been extensively examined. The present study was performed to examine the mode of adhesion of C. jejuni to a smooth surface. When bacterial suspensions in Brucella broth were incubated in microplate wells with a glass coverslip, microcolonies 0.5~2 mm in diameter were formed on the coverslip within 2 hr from the start of incubation. These microcolonies gradually grew and formed a biofilm of net-like connections within 6 hr. Transmission electron microscopy indicated that massive amounts of extracellular material masked the cell surface, and this material bound ruthenium red, suggesting the presence of a polysaccharide moiety. Scanning electron microscopy indicated that the flagella acted as bridges, forming net-like connections between the organisms. To determine the genes associated with biofilm formation, aflagellate (flaA(-)) and flagellate but non-motile (motA(-)) mutants were constructed from strain 81-176 by natural transformation-mediated allelic exchange. The flaA(-) and motA(-) mutants did not form the biofilm exhibited by the wild-type strain. These findings suggest that flagella-mediated motility as well as flagella is required for biofilm formation in vitro.

Antimicrobial susceptibility of Treponema phagedenis-like spirochetes isolated from dairy cattle with papillomatous digital dermatitis lesions in Japan.

The minimal inhibitory concentrations (MICs) of 23 Treponema phagedenis-like spirochetes isolated from dairy cattle with papillomatous digital dermatitis (PDD) lesions in Japan were investigated by a broth microdilution method using 15 antimicrobial agents. Although all MIC values showed a monomodal distribution, the MICs of the antimicrobial agents for 90% (MIC(90)) of the isolates tested varied among the agents examined. The MIC(90) values for penicillin G, ampicillin, and erythromycin were <0.06 microg/ml. In contrast, the MIC(90) values for kanamycin, streptomycin, rifampicin, sulfamethoxazole, trimethoprim, and colistin were >128 microg/ml. Oxytetracycline, lincomycin, enrofloxacin, chloramphenicol, ceftiofur, and gentamicin showed intermediate values, i.e., 0.5~32 microg/ml. The present study suggested that no isolate had acquired resistance to the antimicrobial agents examined, although they may have natural resistance to some agents. Furthermore, the in vitro antimicrobial susceptibility data would provide helpful information for PDD treatment and the development of a selective medium for isolating the organism effectively.

Genetic heterogeneity among strains of Treponema phagedenis-like spirochetes isolated from dairy cattle with papillomatous digital dermatitis in Japan.

Papillomatous digital dermatitis (PDD) is an infectious foot disease of cattle that is prevalent throughout the world. Although it has been prevalent in Japan since the first case was reported in 1992, full epidemiological and bacteriological examinations have not been conducted. We collected 91 lesions of PDD from 80 dairy cattle on 12 farms in eight regions of Japan to isolate the spirochetes that are frequently detected in lesions. We isolated 40 strains of spirochetes from 24 cattle (30.0%) by a simple two-step culture technique, in which the biopsy samples were incubated at 4 degrees C for 48 to 72 h in an enrichment broth supplemented with antibiotics, which improved the rate of isolation, and then inoculated on selective agar plates. All spirochetes examined were catalase positive and oxidase negative and showed weak beta-hemolytic activity. Enzyme activities were identical to those of Treponema phagedenis ATCC 27087. Sequencing of the 16S rRNA gene showed that all strains isolated had >99% identity to those of the T. phagedenis type strain and of T. phagedenis-like strains isolated from PDD lesions in the United States and Europe. Pulsed-field gel electrophoresis and PCR-based random amplified polymorphism DNA methods revealed considerable diversity among strains isolated not only from different cattle but also from the same individuals. These findings may provide further evidence for the role of these treponemes in the pathogenesis of persistent PDD.