Genetic analysis of river,swamp and hybrid buffaloes of north‐east India throw new light on phylogeography of water buffalo (Bubalus bubalis)

This study analysed buffaloes from north‐east India and compared their nuclear and mitochondrial DNA variations with buffaloes of mainland India, China, Mediterranean and South‐East Asia. Microsatellite genotypes of 338 buffaloes including 210 from six north‐east Indian buffalo populations and three mainland Indian breeds were analysed to evaluate their genetic structure and evolutionary relationships. Phylogenetic analysis and multidimensional scaling plot of pairwise F_ST revealed the clustering of all swamp‐type buffaloes of north‐east India with Lower Assamese (significantly hybrid type) buffaloes in one plane and all the mainland river buffaloes in another plane while the upper Assamese buffaloes being distinct from both these clusters. Analysis of mtDNA D‐loop region of 530‐bp length was performed on 345 sequences belonging to 23 buffalo populations from various geographical regions to establish the phylogeography of Indian water buffalo. The swamp buffaloes of north‐east India clustered with both the lineages of Chinese swamp buffalo. Multidimensional scaling display of pairwise F_ST derived from mitochondrial DNA data showed clustering of upper Assamese, Chilika and Mediterranean buffaloes distinctly from all the other Indian buffalo populations. Median‐joining network analysis further confirmed the distinctness and ancestral nature of these buffaloes. The study revealed north‐east region of India forming part of the wider hybrid zone of water buffalo that may probably extend from north‐east India to South‐East Asia.     

A preliminary investigation of tuberculosis and other diseases in African buffalo (Syncerus caffer) in Queen Elizabeth National Park, Uganda

A survey to determine the prevalence of bovine tuberculosis caused by Mycobacterium bovis and certain other infectious diseases was conducted on 42 free-ranging African buffaloes, (Syncerus caffer) from May to June 1997 in the Queen Elizabeth National Park, Uganda. Using the gamma interferon test, exposure to M. bovis was detected in 21.6% of the buffaloes. One dead buffalo and an emaciated warthog (Phacochoerus aethiopicus) that was euthanased, were necropsied; both had miliary granulomas from which M. bovis was isolated. None of the buffaloes sampled in Sector A of the park, which has no cattle interface, tested positive for bovine tuberculosis (BTB) exposure. The prevalence and distribution of BTB does not appear to have changed significantly since the 1960s, but this may be due to fluxes in the buffalo population. Serological testing for foot-and-mouth disease (FMD) demonstrated positive exposure of 57.1% of the buffaloes sampled, with types A, O and SAT 1-3, which is the first known report of FMD antibodies to A and O types in free ranging African buffaloes. Foot-and-mouth disease virus types SAT 1 and SAT 3 were isolated from buffalo probang samples. Two percent of the buffaloes had been exposed to brucellosis. None of the buffaloes tested had antibodies to rinderpest, leptospirosis or Q fever.     

Full-term delivery of river buffalo calves (2n = 50) from in vitro-derived vitrified embryos by swamp buffalo recipients (2n = 48)

Field trials were carried out on the use of swamp buffaloes (2n = 48) as recipients of in vitro-derived vitrified river buffalo (2n = 50) embryos. River buffalo embryos were produced through a total in vitro production system using frozen semen from a top progeny-tested bull and slaughter-house-derived ovaries and cryopreserved by a vitrification technique. Recipient swamp buffaloes were Philippine carabaos selected on body condition, parity, and reproductive status and owned by village farmers. Eighty post-warmed riverine embryos were transferred non-surgically to 40 swamp buffalo recipients, of which three healthy riverine calves were born by normal delivery and one stillbirth, representing a 5.0% (4/80) full-term development and 10% (4/40) calving rate with 75% (3/4) normal and 25% (1/4) stillbirth. The results demonstrate that, under field conditions and despite the difference in chromosome number between the embryo and recipient, swamp buffaloes are potential surrogate mothers of imported, genetically superior river buffalo frozen embryos. Thus, embryo IVP-vitrification and transfer technology is a potential tool in the propagation of genetically superior buffaloes. Precautionary measures, however, are recommended to avoid dystocia and stillbirth.     

Cystic echinococcosis in water buffaloes: epidemiological survey and molecular evidence of ovine (G1) and buffalo (G3) strains

A survey of cystic echinococcosis (CE) in the water buffalo (Bubalus bubalis) of the Italian Mediterranean breed was carried out in Campania, a region of southern Italy. In addition, a molecular study was performed on 48 hydatid cysts coming from 48 water buffaloes in order to determine the Echinococcus granulosus strain(s) present in this host. Out of a total of 722 water buffaloes examined for CE, 76 (10.5%) were found infected. The average number of cysts per buffalo was 4.3 (minimum 1, maximum 45). Seventeen buffaloes had hydatid cysts only in the liver (with an average of 5 cysts/liver), 34 only in the lungs (with an average of 1.8 cysts/lungs), and 25 buffaloes had cysts both in the liver and in the lungs. Fertile cysts were found in 10 (13.2%) out of the 76 positive buffaloes. The sequencing of the mitochondrial cytochrome C oxidase subunit 1 (CO1) gene of the 48 hydatid cysts produced sequences of 419 bp for each sample analysed. For 33 samples, alignment of the obtained sequences with those present in GenBank showed a total homology with the common domestic sheep strain G1; for 15 samples, sequences obtained showed 100% homology with buffalo strain G3. The findings of the present survey represent the first epidemiological and molecular comprehensive studies on CE in water buffalo from an endemic area for E. granulosus.     

A review on breeding and genetic strategies in Iranian buffaloes (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>)

The aim of current study was to review breeding progress and update information on genetic strategies in Iranian buffaloes. Iranian buffalo is one of the vital domestic animals throughout north, north-west, south and south-west of Iran with measurable characteristics both in milk and meat production. The species plays an important role in rural economy of the country due to its unique characteristics such as resistance to diseases and parasites, having long productive lifespan and showing higher capability of consuming low-quality forage. In Iran, total production of milk and meat devoted to buffaloes are 293,000 and 24,700 tons, respectively. Selection activities and milk yield recording are carrying out by the central government through the Animal Breeding Centre of Iran. The main breeding activities of Iranian buffaloes included the estimation of genetic parameters and genetic trends for performance traits using different models and methods, estimation of economic values and selection criteria and analysis of population structure. Incorporating different aspects of dairy buffalo management together with improved housing, nutrition, breeding and milking, is known to produce significant improvements in buffalo production. Therefore, identifying genetic potential of Iranian buffaloes, selection of superior breeds, improving nutritional management and reproduction and developing the education and increasing the skills of practical breeders can be useful in order to enhance the performance and profitability of Iranian buffaloes.     

An experimental intratonsilar infection model for bovine tuberculosis in African buffaloes, Syncerus caffer

An infection model for Mycobacterium bovis in African buffaloes, Syncerus caffer, was developed, using the intratonsilar route of inoculation. Two groups of 11 buffaloes each, aged approximately 18 months, were infected with either 3.2 x 10(2) cfu (low dose) or 3 x 10(4) cfu (high dose) of M. bovis strain isolated from a buffalo. A control group of six buffaloes received saline via the same route. The infection status was monitored in vivo using the comparative intradermal tuberculin test, and in vitro by the modified interferon-gamma assay. All buffaloes were euthanazed 22 weeks post infection and lesion development was assessed by macroscopic examination, culture and histopathology. It was found that the high dose caused macroscopic lesions in nine out of 11 buffaloes. Mycobacterium bovis was isolated from all buffaloes in the high-dose group and from six out of 11 in the low-dose group.     

Ultrasonographic and macroscopic anatomy of the enucleated eyes of the buffalo (Bos bubalis) and the one-humped camel (Camelus dromedarius) of different ages

The ultrasonographic appearance and measurements of the normal buffalo and camel eye globes were described in 60 buffaloes (Bos bubalis) aged 1?year (28 eyes) and 10?years (32 eyes), and in 51 humped camels (Camelus dromedarius) aged 1?year (26 eyes) and 10?years (24 eyes). Ocular measurements were recorded by A- and B-scan ultrasonographic examination of 40 buffalo eyes (18 young and 22 adult eyes) and 34 camel eyes (14 young and 20 adult eyes) using a KANGH ultrasound scanner equipped with 10?MHz probe. For gross measurements, 20 buffalo and 16 camel eye globes were frozen and dissected and the same measurements were made using fine callipers macroscopically. The aqueous and vitreous humour of the buffalo and camel eyes appeared anechoic. The cornea, anterior and posterior lens capsule and iris appeared hyperechoic. The ocular measurements for the axial length, vitreous chamber depth (VCD), corneal thickness, lens thickness and scleroretinal rim thickness increase with the advance of age in both buffaloes and camels. Except for the anterior chamber depth, VCD and lens thickness, which were larger in adult camels than in adult buffaloes, no other differences between ocular dimensions were observed in both species. The results of this study are valuable for comparative ocular anatomy and will be useful for ultrasonographic evaluation of ocular diseases in buffaloes and camels.     

Infection of African buffalo (Syncerus caffer) and cattle with Theileria parva lawrencei after serial passage in cattle

The infectivity of a Theileria parva lawrencei stabilate, from a stock derived from an African buffalo (Syncerus caffer) in the Serengeti National Park, Tanzania, was investigated. In the first experiment a buffalo and three cattle were inoculated with a stabilate from a stock passaged three times in cattle. All cattle developed fatal theilerial infections. Isolations from the buffalo by tick feeding and cell culture isolation showed that it was infected with T p lawrencei at the time of inoculation, but the second isolation made 19 days after inoculation behaved like T p parva in cattle, developing a high parasitosis, while the third isolation made three months later behaved like T p lawrencei with low parasitosis. It was concluded that two biological types of T parva could exist in a buffalo at one time, but it was not shown that the buffalo had become a carrier of T p lawrencei adapted to cattle. In the second experiment two buffaloes and three cattle were inoculated with T p lawrencei (Serengeti) stabilate which had been passaged six times through cattle and ticks. The two buffaloes had mild theilerial infections and developed serological titres in the indirect fluorescent antibody test, but the cattle had fatal infections. Tick and cell culture isolations of T parva were possible during the clinical reactions of the buffaloes, but no carrier state was demonstrated. Theileria-infected cell lines were established from the buffaloes and the cattle and were examined using monoclonal antibodies against T parva schizonts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Infection of bovine immunodeficiency virus and bovine leukemia virus in water buffalo and cattle populations in Pakistan

A survey of antibodies to bovine immunodeficiency virus (BIV) known as bovine lentivirus and bovine leukemia virus (BLV) was conducted with samples from water buffalo and cattle populations in Pakistan. A total of 370 water buffaloes and 76 cattle were tested, and 10.3% and 15.8%, respectively, were found positive for anti-BIV p26 antibodies determined by Western blotting, while 0.8% of water buffaloes and no cattle were positive for anti-BLV antibodies determined by immunodiffusion test. BIV-seropositive water buffaloes and cattle were found to have BIV proviral DNA in the peripheral blood mononuclear cells determined by nested polymerase chain reaction. This is the first report of BIV infections in water buffaloes.     

Single Nuclease Polymorphism Analysis of Leptin Gene in Buffalo

This study was aimed to detect the polymorphisms of Leptin gene in buffalo that provided a fundamental for further study on marker assisted breeding in buffaloes.The single nuclease polymorphisms (SNP) of Leptin gene were identified and genotyped by using DNA pooled sequencing and high-resolution melting (HRM) method in three buffalo breeds with 182 buffalo individuals,respectively.The results showed that seven SNPs of Leptin gene were identified in studied population that were located in the intron 1,intron 2 and exon 3 regions,respectively.All the SNPs loci were moderate polymorphism except for the SNP5 and SNP6.The χ²-test indicated that all the SNPs loci were in agreement with Hardy-Weinberg equilibrium in Nili-Ravi population (P>0.05).Our findings revealed that seven SNPs of Leptin gene in buffalo were identified and genotyped in population,which provide the data support for further analyzing the associations between these polymorphism and production traits in buffaloes.     

水牛瘦素基因单核苷酸多态性分析

试验旨在检测水牛瘦素（Leptin）基因序列的多态性,为进一步开展水牛标记辅助育种研究奠定基础。运用DNA混合池结合直接测序及高分辨熔解曲线（HRM）法在182头奶水牛个体中进行Leptin基因SNP位点的筛选和分型。结果表明,在试验群体中Leptin基因共发现7个SNPs,位于内含子1、内含子3和外显子3区域,分别是A3123G、A3776G、A4154G、A5228G、A5524C、G5573T和A5751C。除了SNP5和SNP6位点在尼里－拉菲水牛群体中的多态信息含量（PIC）属于低度多态之外,所有SNPs位点在3个水牛群体中均属于中度多态。经χ²检验,所有SNPs位点的突变在尼里－拉菲水牛群体均达到Hardy-Weinberg平衡状态（P>0.05）。本研究成功筛查7个水牛Leptin基因SNPs位点并进行了基因分型,为下一步开展奶水牛Leptin基因的标记－性状关联分析奠定基础。     

Seroepidemiology of Neospora caninum and Toxoplasma gondii in cattle and water buffaloes (Bubalus bubalis) in the People's Republic of China

A seroepidemiological survey of Neospora caninum and Toxoplasma gondii in cattle and water buffaloes was carried out in the People's Republic of China. Serum samples were obtained from dairy (n=262, 9 herds in 9 provinces) and beef cattle (n=10, 1 herd) and water buffaloes (n=40) in China. All sera were tested for antibodies to N. caninum and T. gondii by an enzyme-linked immunosorbent assay (ELISA) and an indirect agglutination test (IAT), respectively. The overall seroprevalence of N. caninum in dairy cattle was 17.2% (45/262), and the herds seroprevalence of N. caninum was 88.9% (8/9), and antibodies to T. gondii were present in 6 cows (2.3%). None of the cows had antibodies against both T. gondii and N. caninum. Antibodies to T. gondii or N. caninum were not found in beef cattle or water buffaloes. The seroprevalence of N. caninum in aborting cows (20.2%) was higher than that in non-aborting cows (16.6%) with an odds ratio of 1.26 (95% CI, 0.54-2.95), but the difference was not statistically significant (P>0.05). There was no apparent association of N. caninum seropositivity with age or number of pregnancies. This is the first report on the seroprevalence of N. caninum in cattle and water buffaloes in China.     

Evaluation of genetic variability and mutation drift equilibrium of Banni buffalo using multi locus microsatellite markers

The present study was conducted to evaluate genetic diversity of Banni buffalo and its relationship/differentiation with Murrah using genotypic data on 24 heterologus bovine specific microsatellite marker loci. A total of 138 alleles were observed with a mean of 5.75 alleles/locus across two populations. The mean observed and expected heterozygosities were found to be 0.441 and 0.572 respectively in Banni buffaloes while it was 0.464 and 0.610 respectively in Murrah buffaloes. The average heterozygosity deficit was significantly positive with substantially higher values observed in Banni (22.3%) and Murrah (24%) buffalo populations. Banni buffalo population, when evaluated for mutation drift equilibrium revealed significant heterozygosity excess under IAM while no such excess was observed under SMM and TPM. The qualitative graphical test revealed a normal L-shaped distribution of allele frequencies indicating the absence of genetic bottleneck in Banni buffaloes. The mean estimates of F-statistics over all the loci were 0.376 for F_IT, 0.187 for F_ST and 0.232 for F_IS respectively. Analysis of molecular variance (AMOVA) revealed 18.95% of the total variation being explained by between breed differences while 14.36% of the variation explained differences between individuals within each breed. Genotype assignment test revealed distinct clustering of Banni and Murrah buffaloes. Genetic distance was estimated using three different methods, the results of which revealed considerable genetic differentiation between these two buffalo populations. The divergence time between Banni and Murrah buffaloes was estimated to be around 7286 years. The results of the present study may be helpful in decision making for conservation programs as Banni buffalo population is on decline.     

水牛同期发情技术的研究

采用不同药物处理和给药方式,对不同品种水牛在不同季节自然发情和同期发情配种后的受胎效果进行系统研究,以期建立一套稳定的适合水牛同期发情的处理方法。试验选取本地水牛(102头)、摩拉水牛(129头)、尼里/拉菲水牛(98头)和杂交水牛(326头)共655头,分为5个组进行比较试验。结果表明:河流型摩拉水牛和尼里/拉菲水牛的同期发情率和配种受胎率均高于杂交水牛和本地水牛(P<0.05),但同为河流型的摩拉和尼里/拉菲水牛之间没有差异(P>0.05),以本地水牛效果最差,其同期发情率和配种受胎率分别为74.51%和30.39%;应用不同的药物处理时,以GnRH+PGF2α+GnRH效果最好,同期发情率和受胎率分别为88.46%和46.38%,其同期发情率显著高于其他各药物组(P<0.05),而用PGc的效果最差(分别为79.10%和33.21%);用PMSG肌注+PGc灌注法,其同期发情率和受胎率效果最好,显著高于其他各组(P<0.05),其次是PMSG肌注+PGc肌注法,PGc肌注法效果最差;在冬季进行同期发情处理时,同期发情率和受胎率最高(分别为83.75%和43.28%),明显高于其他各季节(P<0.05);水牛自然发情的人工授精受胎率和胚胎移植的受胎率均比同期发情的高,差异显著(P<0.05)。     

Isolation and identification of bubaline herpesvirus 1 (BuHV-1) from latently infected water buffalo (Bubalus bubalis) from Iran

In order to isolate buffaloes herpesvirus 1 (BuHV-1) from latently infected water buffalo (Bubalus bubalis), 16 buffalo heifers were selected from a herd. At first, animals were bled and their sera were tested by virus neutralization (VN) test, using bovine herpesvirus 1 (BoHV-1). According to the results of VN test and dexamethasone injection (0.1 mg/kg BW) for 5 consecutive days, the examined buffaloes were divided into 4 groups. Vaginal and nasal swabs were daily collected from all buffaloes from day 0 to 10 days later. Based on the cytopathic effects in cell culture, a herpesvirus was isolated only from nasal swabs of three seropositive buffaloes which they had received dexamethasone. The nasal swabs of these three buffaloes were also positive in PCR, using primers specific for ruminant herpesviruses gD gene. The identity of the isolated viruses was determined according to partial amino acid sequences of gD, deduced from the nucleotide sequences of the PCR products. On the basis of sequence alignment, phylogenetic analysis, and genetic distances, the three buffalo virus isolates were more closely related to BuHV-1 and BoHV-5 than to BoHV-1.

     

Methanogen diversity in the rumen of Indian Surti buffalo (Bubalus bubalis), assessed by 16S rDNA analysis

The methanogenic communities in buffalo rumen were characterized using a culture-independent approach of a pooled sample of rumen fluid from three adult Surti buffaloes. Buffalo rumen is likely to include species of various methanogens, so 16S rDNA sequences were amplified and cloned from the sample. A total of 171 clones were sequenced to examine 16S rDNA sequence similarity. About 52.63% sequences (90 clones) had ≥ 90% similarity, whereas, 46.78% of the sequences (81 clones) were 75-89% similar to 16S rDNA database sequences, respectively. Phylogenetic analyses were also used to infer the makeup of methanogenic communities in the rumen of Surti buffalo. As a result, we distinguished 23 operational taxonomic units (OTUs) based on unique 16S rDNA sequences: 12 OTUs (52.17%) affiliated to Methanomicrobiales order, 10 OTUs (43.47%) of the order Methanobacteriales and one OTU (4.34%) of Methanosarcina barkeri like clone, respectively. In addition, the population of Methanomicrobiales and Methabacteriales orders were also observed, accounting 4% and 2.17% of total archea. This study has revealed the largest assortment of hydrogenotrophic methanogens phylotypes ever identified from rumen of Surti buffaloes.     

云南水牛mtDNA D-loop遗传多样性研究

[目的] 探究云南3个水牛品种(德宏水牛、滇东南水牛、盐津水牛)的mtDNA D-loop区的遗传多样性与母系起源。[方法]采用PCR扩增、测序及生物信息学方法。[结果] 本研究共分析了215条云南水牛mtDNA D-loop全序列,检测到107个多态位点,定义了86种单倍型。结果表明,云南3个水牛品种的mtDNA遗传多样性非常丰富,其中,滇东南水牛的遗传多样性最高(Hd: 0.946±0.017,Pi: 0.0162±0.0018),盐津水牛的遗传多样性最低(Hd: 0.805±0.063,Pi: 0.0141±0.0031)。系统发育分析结果表明,在3个云南水牛品种中检测到2个主要支系(A和B支系及其亚支系)及一个稀有支系C,其中A支系为主要支系(79.07%),且经历了强烈的群体扩张。[结论] 云南3个水牛品种具有丰富的mtDNA遗传多样性,主要有A与B两个母系起源。     

Evaluation of the specificity of the gamma-interferon test in Italian bovine tuberculosis-free herds

We investigated the specificity of the gamma-interferon test for bovine tuberculosis (TB) in 1,557 cattle in 30 paratuberculosis-free and officially certified TB-free dairy herds, located in three provinces of the Lombardy Region in Northern Italy. The TB-free status of the herds under examination was further confirmed by the tuberculin skin test, by an antibody assay and by post mortem examination of animals culled from the herds during the study period. The specificity of the gamma-interferon tests after a single test and a double sampling scheme were 88.8% and 95.4%, respectively. After a single test, 11.7% of dubious reactors were also detected, while most cattle (47.4%) were shown to be avian reactors, probably due to contamination from infected birds and/or forage. There was strong evidence that the specificity of the test could be related to the animals' interaction with environmental mycobacteria and/or ageing. To reduce the percentage of nonspecific bovine reactors under alleged TB-free conditions, test procedures might involve the use of more specific antigens and/or different reaction thresholds.     

Lymphocyte subset distribution in apparently normal and single intradermal test-positive water buffaloes analyzed by flow cytometry

Monoclonal antibodies (mAbs) against bovine lymphocyte cell surface antigens namely, MHC Class I, MHC class II (DP, DQ and DR), CD3, CD4, CD8, gamma delta TCR, WC1N1 and WC1N2, were tested for their reactivity on apparently normal buffalo mononuclear cells prepared from spleen, lymph nodes and peripheral blood. All the mAbs cross-reacted with the buffalo mononuclear cells. The mean (+/-SD) CD4:CD8 cell ratio in the peripheral blood of apparently normal buffaloes was 1.08+/-0.049 while in the spleen and lymph nodes it was 0.90+/-0.080 and 1.81+/-0.430, respectively. The lymphocyte subsets in the buffaloes positive for tuberculosis by the single intra dermal (SID) test was found to be altered; the CD4 cells were reduced while the CD8 and gamma delta cells were increased. The mean CD4:CD8 ratio in the SID positive buffaloes was 0.36+/-0.010.     

盱眙水牛遗传多样性及系统地位的研究

对江苏省盱眙县183头水牛外形特征及体尺指标进行了调查和测量,同时随机抽取48头母牛,用垂直平板聚丙烯酰胺凝胶电泳(PAGE)和水平淀粉凝胶电泳(SGE)对编码血液蛋白18个基因座的基因频率进行检测,同时引用相同方法所得东南亚21个水牛群体5个座位的研究资料,进行聚类分析,结果表明:成年公牛体高、体长、胸围和管围分别为129.16,147.69,198.66和23.12 cm,成年母牛分别为128.43,143.81,193.11和21.62 cm,估计体重公母牛分别为516.39和479.83 Kg。在所检测18个基因座中,有5个存在多型,多态位点百分比为27.78%。群体内遗传变异水平较低,平均杂合度和平均有效等位基因数分别为0.0793和1.1355。聚类分析表明:盱眙水牛与台湾和泰国水牛亲缘关系较近,孟加拉国为沼泽型水牛和河流型水牛的分界线,不同水牛群体的聚类结果基本与其地理分布吻合。