Molecular characterization and phylogenetic analysis of lumpy skin disease in Egypt

Lumpy skin disease (LSD) is an infectious viral disease affecting cattle, cause severe economic losses. In the last few years, the disease is widely distributed in many countries in the Middle East, including Egypt. This study aimed to determine the genetic character of LSDV circulating in some governorates in Egypt and its relation with other LSDVs and vaccine strains in GenBank. A total of 50 skin nodules and 50 blood samples were collected from clinically affected cattle to detect LSDV using PCR targeting the P32 gene. The positive samples were characterized using PCR targeting the GPCR gene. The amplified products of four samples detected in the skin nodule of cattle from Alexandria and Kafr ElSheikh governorates were sequenced, and the phylogenetic tree was constructed. Out of 100 analyzed samples, 70 samples were positive for LSDV by PCR assay. In addition, the sequence alignment of the obtained LSDV strains detected in the Alexandria governorate showed high similarity to the LSDV genome (MN995838). In contrast, LSDV strains from Kafr ElSheikh governorate revealed high similarity and the previous Egyptian strain (MG970343), which was isolated from cattle in Sharkia governorate in 2016. Also, the phylogenetic analysis confirmed that one of the LSDV strain (LC601598) from Alexandria is closely related to the LSDV of Menofia/Egypt/2019 (MN271722), while another one (LC601597) is closely related to vaccine strains of LSDV. Moreover, the LSDV strains from Kafr Elsheikh showed closely related to previous LSDV strains isolated from Menofia (MG970343) and Dakahlia (KP071936) governorates and clustered with other LSDV strains in a distinct cluster. This information is for understanding the epidemiology of LSDV and supporting the establishment of an efficient control program for the disease.     

Phylogenetic analysis of the lumpy skin disease viruses in northwest of Iran

Lumpy skin disease (LSD) is a devastating viral disease of cattle which has recently spread from Africa into the countries of the Middle East. The aim of the present study was to investigate the relationships among lumpy skin disease viruses (LSDV) isolated from different regions of Iran and the origin and spread of these viruses. In this study, a total of 234 blood samples from clinically affected animals from four provinces in the northwest of Iran were screened for LSDV using polymerase chain reaction (PCR). From 80 positive samples for LSDV detected by PCR, the partial P32 gene (759 bp) of 12 isolates were sequenced and phylogenetically analyzed. LSD viruses were grouped in three subclusters with an overall 97.1–100% nucleotide identity. LSDVs isolated from Gilan showed lowest nucleotide identity with the other LSDVs. Four isolates of LSDV including KO-1, EA-1, EA-3, and WA-3 showed 100% similarity with each other and also with the Neethling strain. Phylogenetic analysis indicated that the identified LSDVs were closely related to each other and had high-sequence homology with other LSDV isolates from Africa. It was concluded that LSD outbreak probably occurred in the northwest of Iran by LSDVs entering the country from Iraq and P32 nucleotide sequence information obtained in the present study is a valuable resource in understanding the genetic nature and molecular epidemiology of local LSDV isolates which can be used for future vaccine development based on the circulating strains in the region.     

The isolation of lumpy skin disease virus and bovine herpesvirus-4 from cattle in Egypt

Lumpy skin disease (LSD) virus (LSDV) was isolated for the first time from cattle in Egypt in 2 disease outbreaks. Bovine herpesvirus-4 (BHV-4) and LSDV were detected in a pooled sample from the first outbreak (Suez). Only LSDV was isolated from the second outbreak (Ismalia). The capripoxviruses were identified as LSDV by neutralization with specific antiserum and by their ability to produce generalized LSD in experimentally inoculated cattle.     

Brazilian isolates of Trypanosoma (Megatrypanum) theileri: diagnosis and differentiation of isolates from cattle and water buffalo based on biological characteristics and randomly amplified DNA sequences

We detected and cultivated isolates of Trypanosoma (Megatrypanum) theileri from cattle and water buffaloes in S?o Paulo state, southeastern Brazil, which were characterized by comparing morphological, growth and molecular features. Although isolates from cattle and water buffalo were morphologically indistinguishable, they differed in their growth characteristics. A dendrogram based on randomly amplified polymorphic DNA (RAPD) patterns indicated close-genetic relationships among all isolates from both species, which were all tightly clustered together and distant from Megatrypanum species from wild mammals. In addition, isolates within the T. theileri-cluster were clearly segregated into two host-associated groups. The sequence of a synapomorphic RAPD-derived DNA fragment (Tth625), which was shared by all T. theileri trypanosomes from cattle and buffalo but not detected in any of 13 other trypanosome species, was used as target for a conventional T. theileri-specific PCR assay. We also defined RAPD fragments (Tthc606 and Tthb606) that distinguished cattle from buffalo isolates. Thus, distinct growth features and genetic variability distinguished between isolates from cattle and water buffaloes of the same geographic origin, suggesting an association of these isolates with their host species. The trypanosomes from water buffalo reported here are the first T. theileri-like isolates from the Asiatic buffalo (Bubalus bubalis) to be continuously cultured and compared with cattle isolates using biological and molecular methods.     

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.     

Incidence and molecular characterisation of lumpy skin disease virus in Zimbabwe using the P32 gene

Between January, 2013 and December, 2014, there was a lumpy skin disease (LSD) outbreak that affected cattle in different localities of Zimbabwe. The outbreak resulted in severe economic losses to the livestock industry. A retrospective study was conducted by examining stored veterinary records of the LSD outbreak at the Central Veterinary Laboratory (CVL) in Harare, Zimbabwe. Over the 2-year period, a total of 10,038 cases and 880 deaths (8.77 %) were recorded. LSD was reported from all regions of the country, with the highest incidence occurring in Mashonaland West (30.95 %) and Midlands province (14.59 %). The frequency of reported outbreaks was highest in March and April, with the lowest reported cases occurring in November. A total of 25 representative specimens (skin biopsies) were collected from nodular skin lesions of infected cattle, and after viral DNA isolation, the P32 gene was successfully amplified, by using PCR, in 88 % (22/25) of all assayed specimens. Out of the 22 samples that showed amplification, 16 (73 %) were selected for DNA sequencing, and from these, 13 sequences were submitted to GenBank and assigned accession numbers: KX033494, KX033495, KX033496, KX033497, KXO33498, KX033499, KX033500, KX033501, KX033502, KX033503, KX033504, KX033505 and KX033506. Phylogenetic analyses of the 13 sequences was done by using MEGA 7 and showed that the viruses formed two major clusters implying that at least two strains of LSDV are in circulation in Zimbabwe. This study provides the first report on the incidence and molecular characterisation of LSDV in Zimbabwe.     

Isolation of Mycobacterium spp. from milking buffaloes and cattle in Nepal

In Nepal, mycobacterial isolates obtained from the milk and feces of buffaloes and cattle that were positive for the single intradermal cervical tuberculin (SICT) tests were genetically identified. A total of 36 mycobacterial strains were isolated from 39% of the buffaloes (14 of 36) and 34% of the cattle (11 of 32). Of the 36 strains, 13 were identified as M. bovis, and these strains were isolated from 17% of the buffaloes (6 of 36) and 16% of the cattle (5 of 32). M. bovis was isolated from both the milk and feces of one buffalo and one cattle, the milk alone of three buffaloes and three cattle, and the feces alone of two buffaloes and one cattle. These results suggest that milking buffaloes and cattle infected with M. bovis exist in Nepal. The remaining 23 strains were atypical mycobacteria. A program for the elimination of bovine tuberculosis should be implemented as soon as possible, and the public health education and proper hygienic practices may be required.     

Orf virus circulation in cattle in Turkey

Orf virus (ORFV) causes contagious skin disease that mainly affects sheep and goats with zoonotic potential. However, there is not enough information about the association between ORFV and occurrence of skin disease in cattle. The present study describes outbreaks of ORFV infection in cattle in different provinces that are located in the Aegean, Central Anatolian and Mediterranean regions of Turkey. During the months of June and August 2017, vesicular fluid and scab samples were collected from cattle which had proliferative skin lesions. First, presence of lumpy skin disease virus (LSDV) and bovine herpesvirus 2 (BoHV-2, known as the causative agent of pseudo-lumpy skin disease) were investigated by real time PCR and PCR, respectively. Then, samples tested for the presence of parapoxviruses by PCR using primers specific to major envelope protein gene (B2L). Parapoxvirus DNA was detected in investigated samples whereas LSDV and BoHV-2 DNA were not detected. The analysis of the B2L gene sequences revealed that cattle were infected with ORFV. The isolates in the present study shared 100% sequence identity at the nucleotide and amino acid level when compared with previously characterised Turkish field ORFV isolates from goats in 2016. Results of the study show unusual infection of cattle with ORFV, and suggest that ORFV jumps the host species barrier from goats to cattle.     

Detection of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> in tissue samples of cattle and buffaloes

Tissue samples were collected at random from cattle (Bos taurus) and buffalo (Bubalus bubalis) from an abattoir of the district of Lahore and were analyzed for the presence of Mycobacterium avium subsp. paratuberculosis and Mycobacterium bovis through acid-fast staining and polymerase chain reaction (PCR). Body condition of animals and diarrhea were recorded. Most of the animals were emaciated. Diarrhea was noticed in 15.6% of buffaloes and 19.2% of cattle. Intestinal pathology was observed in 29% of buffaloes and 32.8% of cattle. Number of mesenteric lymph node (MLN) showing gross lesions was a bit higher (35.6%) in cattle than buffalo (31.2%). Acid-fast staining of tissue scraping smears revealed the presence of acid-fast bacilli (AFB) in 17.4% intestinal and 16.4% MLN tissue samples in buffalo, while in cattle 19.2% intestinal and 17.8% MLN were found positive for AFB. In buffaloes, PCR confirmed 12.8% intestinal and 12.4% MLN positive samples for M. avium subsp. paratuberculosis. However, in cattle, PCR analysis demonstrated 14.2% positive results for M. avium subsp. paratuberculosis in both MLN and intestinal tissue samples. PCR also confirmed M. bovis in 5.8% of cattle and 5% of buffalo MLN and intestinal tissues. PCR positive tissue samples for M. avium subsp. paratuberculosis were from those animals which were emaciated, having diarrhea, and severe gross lesions. AFB were also detected in tissue scraping smears of these animals. It is concluded that infection by various mycobacterium species can be differentiated by PCR, which is not possible by acid-fast staining technique.     

Molecular epidemiology of Echinococcosis from food producing animals in north India

Echinococcosis is an important medical, veterinary and economic concern in India. Ten cysts were randomly selected from each intermediate host species (cattle, buffalo, sheep, goat and pigs). Either the germinal layer (sterile cysts) or protoscoleces (fertile cysts) were collected for molecular characterization. A 434 base pair fragment of the mitochondrial cytochrome oxidase-1 gene was amplified using PCR from each isolate. Ten representative samples (2 from each intermediate host species) were sequenced in both the directions from which readable sequences were obtained from nine for phylogenetic analysis (NCBI, Blast). Phylogenetic analysis of cytochrome oxidase I gene revealed that seven (77.7%) isolates, from cattle (2), pigs (2), buffaloes (1) and goat (2) were clustered with the Indian Buffalo (G3) strain of Echinococcus granulosus, while two (22.2%) isolates from sheep were clustered with the sheep strain (G1) of E. granulosus. Phylogenetic analysis of the cytochrome oxidase-1 gene revealed that the buffalo strain (G3) and common sheep strain (G1) are cycling among livestock in north India and that these strains are highly adapted to cattle, buffalo, sheep, goats and pigs.     

Genetic diversity in Trypanosoma theileri from Sri Lankan cattle and water buffaloes

Trypanosoma theileri is a hemoprotozoan parasite that infects various ruminant species. We investigated the epidemiology of this parasite among cattle and water buffalo populations bred in Sri Lanka, using a diagnostic PCR assay based on the cathepsin L-like protein (CATL) gene. Blood DNA samples sourced from cattle (n = 316) and water buffaloes (n = 320) bred in different geographical areas of Sri Lanka were PCR screened for T. theileri. Parasite DNA was detected in cattle and water buffaloes alike in all the sampling locations. The overall T. theileri-positive rate was higher in water buffaloes (15.9%) than in cattle (7.6%). Subsequently, PCR amplicons were sequenced and the partial CATL sequences were phylogenetically analyzed. The identity values for the CATL gene were 89.6–99.7% among the cattle-derived sequences, compared with values of 90.7–100% for the buffalo-derived sequences. However, the cattle-derived sequences shared 88.2–100% identity values with those from buffaloes. In the phylogenetic tree, the Sri Lankan CATL gene sequences fell into two major clades (TthI and TthII), both of which contain CATL sequences from several other countries. Although most of the CATL sequences from Sri Lankan cattle and buffaloes clustered independently, two buffalo-derived sequences were observed to be closely related to those of the Sri Lankan cattle. Furthermore, a Sri Lankan buffalo sequence clustered with CATL gene sequences from Brazilian buffalo and Thai cattle. In addition to reporting the first PCR-based survey of T. theileri among Sri Lankan-bred cattle and water buffaloes, the present study found that some of the CATL gene fragments sourced from water buffaloes shared similarity with those determined from cattle in this country.     

The Epidemiology of Mycobacterium bovis in Buffalo in Iran

Mycobacterium bovis is the cause of bovine tuberculosis (bovine Tb) in animals and is considered to be zoonotic and accordingly it infects humans, although cattle are the main host. Buffalo can also be infected and develop bovine Tb. In Iran, almost half a million buffaloes are farmed, mainly in three provinces. In West Azerbaijan, which has the largest numbers of buffaloes, cattle and buffalo are often farmed together. According to the reports of the Iranian Veterinary Organization over the last 25 years, there have been no reports of bovine Tb in buffalo, although the disease is often reported in cattle in this province. Eighteen and 140 pathology specimens from cattle and buffalo, respectively, collected from West Azerbaijani abattoirs were cultured. From one buffalo specimen out of 140, M. bovis was recovered, whereas the pathogen was isolated from 13 cattle specimens. Spoligotyping showed a relatively higher polymorphism within these isolates compared with M. bovis isolated from other Iranian provinces.     

牛结节性皮肤病病毒实验室检测方法研究进展

牛结节性皮肤病(LSD)是由牛结节性皮肤病病毒(LSDV)引起的以发热、消瘦、皮肤水肿、局部形成坚硬的结节或溃疡、淋巴结肿大等为主要特征的急性、亚急性传染病.本病目前尚无特效治疗药物,疫苗接种是当前主要的防控措施,但我国目前并没有针对该病的疫苗,因此建立快速准确的检测方法显得尤为重要.本文从该病的病毒分离和鉴定、血清学...     

Analysis of the Differences in Temperature Adaptability Between Buffaloes and Yellow Cattle Based on Transcriptome Data of Skin Tissues

In order to find out signaling pathways that related to the differences in temperature adaptability between buffaloes and Yellow cattle, RNA-sequencing and bioinformatics analysis were performed on the skin tissues of 3 adult buffaloes and 3 adult Yellow cattle. The results showed that 59 369 167 and 69 837 009 reads were obtained from buffaloes and Yellow cattle skin, respectively. The percentages of the uniquely mapped reads in buffaloes and Yellow cattle were 70.76% and 75.55%, respectively. There were 1 611 differentially expressed genes in buffaloes and Yellow cattle skin, 801 of which were up-regulated in buffaloes skin and 810 were down-regulated in buffaloes skin. GO analysis showed that there were 316, 263 and 278 differentially expressed genes classified to the cellular components, molecular functions and biological processes, respectively. KEGG analysis showed that the differentially expressed genes were enriched in 33 signaling pathways, some of which such as neuroactive ligand-receptor interaction, oxidative phosphorylation, cholinergic synapse, serotonergic synapse, GABAergic synapse and calcium signaling pathway might related to the differences in temperature adaptability between buffaloes and Yellow cattle. In summary, the synapse and oxidative phosphorylation might be related to the differences in temperature adaptability between buffalo and Yellow cattle. This study would lay a foundation for further understanding the molecular mechanism of temperature adaptation differences between buffaloes and Yellow cattle and molecular breeding.     

牛结节性皮肤病病毒研究进展

牛结节性皮肤病(LSD)在全世界范围内均有流行和记载,对我国养牛业构成了严重的威胁。接种疫苗是目前防控LSDV最有效的方法,其中减毒活疫苗在许多国家均有使用。近年研究表明,LSDV活疫苗的使用会造成疫苗重组病毒的流行,接种后会引起动物不同程度的不良反应。对LSDV的病原学特征、分离鉴定方法、流行病学的情况、检测方法以及LSDV疫苗的研究进展进行了阐述,以期为牛结节性皮肤病的免疫预防以及鉴别诊断提供研究思路。     

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.     

基于皮肤组织转录组数据研究水牛与黄牛温度适应性的差异

为了找到与水牛和黄牛温度适应性差异相关的信号通路,本研究对3头成年水牛和3头成年黄牛的皮肤组织进行了转录组测序和生物信息学分析。结果显示,测序后水牛和黄牛皮肤组织分别得到59 369 167和69 837 009条reads,与参考基因组的唯一比对率分别为70.76%和75.55%。水牛和黄牛皮肤组织共有1 611个差异表达基因,其中801个在水牛皮肤中上调,810个在水牛皮肤中下调。GO分析结果显示,注释到细胞组分、分子功能、生物学过程的差异表达基因分别为316、263和278个。KEGG分析结果显示,差异表达基因共富集在33条信号通路上,其中神经活性配体-受体相互作用、氧化磷酸化、胆碱能突触、5-羟色胺能神经突触、GABA能突触、钙信号通路等信号通路可能与水牛和黄牛在温度适应性上的差异相关。综上所述,突触与氧化磷酸化可能与水牛和黄牛之间的温度适应性差异有关,本研究为进一步了解水牛和黄牛对温度适应性差异的分子机制和分子育种奠定基础。     

Prevalence and genetic profiles of Shiga toxin-producing Escherichia coli strains isolated from buffaloes, cattle, and goats in central Vietnam

We investigated the prevalence of Shiga toxin-producing Escherichia coli (STEC) in 568 healthy domestic animals (buffaloes, cattle, and goats) from 98 farms in the central region of Vietnam. The aims of this study were to determine if the prevalence of STEC in South East Asia is similar to that in other parts of the world, to characterize the virulence gene profiles from the recovered STEC and to determine if the recovered STEC belong to serotypes commonly associated with human disease. STEC and intimin-positive strains were recovered from 27% of buffaloes, 23% of cattle, and 38.5% of goats. Seventy percent of buffalo farms, 60% of cattle farms and 100% goat farms were positive for STEC. Of 170 STEC strains, 99 carried both stx1 and stx2 genes, 36 carried the stx2 gene, and 35 carried the stx1 gene. The eae gene was found in six caprine isolates, but not in buffalo or bovine isolates. Among 173 E. coli strains (170 STEC and 3 intimin-positive), 110 carried the ehxA gene, 106 possessed the saa gene. Further characterization of stx subtypes demonstrated that among 134 stx1-containing isolates, 107 belonged to the stx1c subtype and 27 were the stx1 subtype. Of the 132 stx2-containing isolates, 36 were stx2, 34 were stx2c, 43 were stx2d subtype, 3 belonged to stx2g, and 16 strains were stx2d(act). The stx2c variant was dominant in strains isolated from buffalo while the stx2d variant occurred more frequently in caprine isolates. Only 9 (5%) STEC strains contained genes encoding for serotypes O26, O91, O121, O145, and O157 LPS, which are more frequently associated with human infections. The results of this study provide data for understanding of epidemiology of STEC among domestic animals in Vietnam and indicate that buffaloes are also an important reservoir of STEC.     

一起境外输入性牛结节性皮肤病疫情

2020年11月，山东滨州市、东营市引进牛皮肤出现自限性皮肤结节、损伤以及结痂等症状，疑似发生牛结节性皮肤病（lumpy skin disease，LSD）。为确诊两地疫情及了解病原的遗传演化关系，利用荧光定量PCR方法进行诊断，以PCR方法扩增GCPR基因并进行核苷酸比对分析和遗传演化分析。检测结果显示，采集的样品中检测到牛结节性皮肤病病毒（lumpy skin disease virus，LSDV），两地疫情确诊为LSD疫情。GCPR基因核苷酸比对结果显示，China/SDBinzhou/2020、China/SDDongying/2020 GCPR基因存在12个核苷酸的插入，与疫苗毒株Neethling vaccine LW 1959株、Neethling-LSD vaccine-OBP株以及俄罗斯发现的疫苗样毒株Saratov株在GCPR基因的核苷酸插入序列相同，具备疫苗样毒株的特征。系统发育分析结果表明，China/SDBinzhou/2020、China/SDDongying/2020 GCPR基因与我国新疆发现的LSDV毒株GCPR基因处同一小分支中，亲缘关系较近。同时，我国LSDV毒株与Neethling vaccine LW 1959株、Neethling-LSD vaccine-OBP株以及Saratov株等共处于一大分支中。综上，确诊滨州市、东营市两地疫情为LSD疫情，这是山东省首次确诊输入性LSD疫情。     

Isolation of bovine herpesvirus-3 from African buffaloes (Syncerus caffer)

Eleven virus isolations were made from the blood of 45 free living healthy African buffaloes by long term cocultivation of their leucocytes with bovine thymus or spleen cells. The isolates were indistinguishable from each other or from herpesviruses isolated from a severely ill buffalo calf and from a dead buffalo. These viruses possessed the characteristics of the bovine herpesvirus-3 (BHV-3) group and were indistinguishable by serology and restriction endonuclease analysis from the BHV-3 type strains Movar 33/63 and DN599. There was a 93.6 per cent prevalence of indirect immunofluorescent antibody to BHV-3 in the sera of 94 buffaloes in the sample population. No clinical signs or viraemia were detected in five cattle inoculated with 10(8.7) log10 TCID50 of the isolate from the sick buffalo calf. Two of three cattle hyperimmunised with this virus resisted challenge with malignant catarrhal fever herpesvirus, which proved fatal for the other immunised animal and for three control cattle.