Isolation and characterization of Dichelobacter nodosus from ovine and caprine footrot in Kashmir, India

Footrot is a highly contagious and economically important disease of sheep and goats, caused by Dichelobacter nodosus, a slow growing anaerobic Gram-negative rod. The current Australian antigenic classification system, based on variation in the fimbriae, classifies D. nodosus into at least 10 serogroups (A-I and M) and 18 serotypes. This investigation was intended to determine the serological diversity of D. nodosus in this region of Kashmir, India. Exudates of footrot lesions were collected from 24 naturally infected sheep and 42 goats located in the Kashmir valley. Of these 66 samples, 24 yielded evidence of D. nodosus by PCR using 16SrDNA specific primers. Multiplex PCR using serogroup specific primers revealed the presence of serogroup B in all the samples except two, which showed the presence of serogroup E D. nodosus. This study also documents the isolation of D. nodosus and detection of serogroup E for the first time in India.     

Occurrence of ovine herpesvirus type-2 infection in sheep and cattle in Samsun Province, Turkey

July 2004, a cow with clinical signs of ovine herpesvirus type-2 infection which is known as sheep associated malignant catarrhal fever (SA-MCF) was reported in Samsun Province in Turkey. Blood samples were collected from the suspected cow, 10 sheep housed with it, and from 150 healthy sheep and 29 healthy cattle randomly selected from different places in Samsun Province. Nested polymerase chain reaction (n-PCR) was used to detect ovine herpesvirus type-2 (OvHV-2) DNA in the suspected cow and competitive- ELISA (c-ELISA) kits were used to detect antibodies against OvHV-2. The suspected cow was found to be n-PCR positive and c-ELISA negative. The serological results were as follows: All 10 (100%) of sheep housed with the suspected cow and 18 of 29 (62%) of the randomly selected cattle were found seropositive. All 150 randomly selected healthy sheep were seronegative. The overall percentage of seropositivity was 14.7% (28/190). OvHV-2 DNA was detected in the peripheral blood leucocyte (PBL) samples of the cow and of the 10 sheep housed with the suspected cow.     

Transmission of ovine herpesvirus 2 among adult sheep

Previous studies from this laboratory have defined the pattern of acquisition of ovine herpesvirus 2 (OHV-2) in lambs under natural flock conditions. This study examined the question of whether OHV-2 could be transmitted between adult sheep. Two potential routes of transmission were examined: (1) direct inoculation of either viable leukocytes or whole blood from OHV-2 positive sheep, and (2) horizontal transmission through natural contact with OHV-2 positive sheep. Two groups of OHV-2 negative adult sheep were inoculated with material from infected sheep, one with 5x10(8) viable peripheral blood leukocytes (PBL), and the other with 100 ml of whole peripheral blood. No PCR signals were detected in any of the three sheep inoculated with the PBL during the 20 weeks following inoculation. In the group of five sheep inoculated with whole blood, two became PCR-positive at 7 and 8 weeks post-inoculation, respectively, and the remaining three sheep maintained their negative status until termination of the experiment at 20 weeks post-inoculation. In two experiments conducted in different flocks, a total of 20 adult sheep were used to examine horizontal transmission by contact; all animals became PCR-positive within 12 months of mixing the uninfected and infected animals. The results of these experiments support two conclusions. First, the susceptibility to OHV-2 is not limited to young lambs; adult sheep remain fully susceptible. Second, the fact that whole blood, but not PBL, from infected sheep was able to transmit the infection to only two of five inoculated sheep suggests that the infection in peripheral blood cells may be largely non-productive.     

Effect of passive transfer of maternal immune components on infection with ovine herpesvirus 2 in lambs

OBJECTIVE: To define the role of passively tranferred immunity in protection against early infection with ovine herpesvirus 2 (OvHV-2) in lambs. ANIMALS: 15 adult sheep and 34 lambs. PROCEDURES: 2 groups of animals were used, including 15 lambs born to OvHV-2-free ewes and 19 lambs born to OvHV-2-positive ewes. After nursing colostrum, all lambs and their dams were introduced into a flock positive for OvHV-2. Blood was obtained from the lambs every 2 weeks and examined by PCR assay and competitive inhibition ELISA. RESULTS: None of the animals had positive results by PCR analysis for samples obtained approximately 2 weeks after introduction into the flock. In the group of lambs from OvHV-2-infected ewes, 5 of 19 had positive results at 1 month of age and 17 of 19 by 5 months of age. In the group of offspring from OvHV-2-negative ewes, only 1 of 15 had positive results at 1 month of age, and the number reached 12 of 15 by 5 months of age. All lambs in both groups had positive results by 6 months. An active antibody response to the virus was detected in animals within 3 weeks after viral DNA became detectable in the blood. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis suggests that passively transferred immunity does not play an important role in the delay of infection with OvHV-2 in lambs. Age also does not seem to influence susceptibility. The rate of infection in young lambs may simply be a reflection of the intensity of viral exposure in their environment.     

Characterization of ovine herpesvirus 2-induced malignant catarrhal fever in rabbits

Malignant catarrhal fever (MCF) is a frequently fatal lymphoproliferative disease syndrome primarily of ruminant species, caused by gammaherpesviruses in the genus Macavirus. Ovine herpesvirus 2 (OvHV-2), carried by sheep, causes sheep-associated MCF worldwide, while Alcelaphine herpesvirus 1 (AlHV-1), carried by wildebeest, causes wildebeest-associated MCF, mainly in Africa. Diseases in rabbits can be induced by both viruses, which are clinically and pathologically similar; however, recent studies revealed different expression of viral genes associated with latency or lytic replication during clinical disease between the two viruses. In this study, we further characterized experimentally induced MCF in rabbits by nebulization with OvHV-2 from sheep nasal secretions to elucidate the course of viral replication, along with in vivo incorporation of 5-Bromo-2'-Deoxyuridine (BrdU), to evaluate lymphoproliferation. All six rabbits nebulized with OvHV-2 developed MCF between 24 and 29 days post infection. OvHV-2 DNA levels in peripheral blood leukocytes (PBL) remained undetectable during the incubation period and increased dramatically a few days before onset of clinical signs. During the clinical stage, we found that predominantly lytic gene expression was detected in PBL and tissues, and both T and B cells were proliferating. The data showed that the viral gene expression profile and lymphoproliferation in rabbits with OvHV-2 induced MCF were different from that in rabbits with AlHV-1 induced MCF, suggesting that OvHV-2 and AlHV-1 may play a different role in MCF pathogenesis.     

Respiratory disease in foals and the epizootiology of equine herpesvirus type 2 infection

The epizootiology of equine herpesvirus type 2 (EHV-2) infection was investigated in Thoroughbred foals on a stud farm which in previous years had suffered economic loss due to respiratory disease. Sixteen pairs of foals and their dams were selected for this study and all of the foals became infected with EHV-2 by two to four months of age. These animals responded serologically to the virus infection as detected by an enzyme-linked immunosorbent assay (ELISA). EHV-2 infection persisted in these foals for two to six months with constant or intermittent virus recovery. This persistent infection stimulated continuous production of antibodies against EHV-2. As soon as the antibody levels reached their peak at five to six months, the isolation rate of EHV-2 from the nasal cavity of these animals decreased, and eventually by nine months of age virus could no longer be recovered. Respiratory disease was observed in ten of the 16 foals; and two severely affected animals died at two months of age. EHV-2 was isolated from both foals at ante and/or post mortem examination. It is postulated that EHV-2, either as an initiating agent or by means of immnunosuppression, caused the respiratory disease observed in these foals.     

Molecular biology of bovine herpesvirus type 4.

Bovine herpesvirus type 4 (BHV-4) is a ubiquitous virus of cattle. Its genome is a 144 +/- 6 kb double-stranded DNA consisting of a unique central part (L-DNA) flanked at both ends by tandem repeats called polyrepetitive DNA (prDNA or H-DNA). The overall arrangement of genes has been obtained by the analysis of homologies between short BHV-4 DNA sequences and corresponding genes of Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS). The gene expression is temporally regulated. Glycoprotein precursor p (gp10/gp17) is expressed as gamma 1 polypeptide. Glycoproteins gp1, gp8, gp11 and their precursors are gamma 2 proteins. The analysis of strain variations allows the definition of two types of strains, based on the DNA patterns: the Movar 33/63-like and the DN 599-like strains. Only the M40 strain, isolated in India, fails to fit this classification. The genomic variations have been compiled to build a dendrogram showing three levels of divergence between BHV-4 strains or isolates. The available molecular data indicate that the BHV-4 genome shares much similarity with the DNA of EBV and HVS, two representative members of the gammaherpesvirinae. BHV-4 may therefore be classified in the subfamily gammaherpesvirinae.     

新型猪细小病毒感染的分子流行病学调查

近几年来,世界范围内的猪群出现了多种新型猪细小病毒(porcine parvovirus,PPV)的感染,其中我国南方地区的猪群也出现了不同程度的感染。本研究利用PCR方法对实验室保存的2008—2013年期间的送检病料,进行猪细小病毒2型(PPV2)、PPV3、PPV4、猪类博卡病毒(PBo-likeV)和猪博卡病毒(PBoV)感染的分子流行病学调查,结果显示阳性率依次为46.4%、21.1%、6.8%、12.3%和5.5%。结合PRRSV和PCV2检测结果,并对新型细小病毒与这2种病毒共感染情况进行初步分析,结果显示PPV3和PRRSV、PBo-likeV和PCV2混合感染的几率较高,提示它们在感染过程中可能存在着协同作用。本研究为细小病毒流行病学和致病机理的研究奠定了一定的基础。     

Epidemiology of ovine helminthiasis in Haryana,India

Tracer lambs were used to study the pasture contamination with infective stages of helminth parasites during one annual cycle in a subtropical climate. Post-mortem worm counts indicated that low infections with Haemonchus contortus occurred throughout the year except in June. However, twenty five or more H. contortus per lamb were recorded in January, April, May and August. Trichostrongylus colubriformis infection was detected throughout the year and 150 or more worms per lamb were recorded during January to May and in August. Anoplocephalids were recorded from the lambs throughout the year but had no seasonal pattern. Low infections with Oesophagostomum columbianum and Trichuris ovis were observed. The faecal egg counts from the permanent flock with whom the tracer lambs were grazed revealed heavy to mild worm burdens throughout the year. Coproculture indicated that H. contortus predominated from the second fortnight of May to December except in the second fortnight of July. Infection with T. colubriformis was more severe from January to the first fortnight of May and in the second fortnight of July. Negligible infections with O. columbianum, Bunostomum trigonocephalum, Gaigeria pachyscelis and Dictyocaulus filaria were also observed. Biohythergraphs prepared for H. contortus and T. colubriformis showed differences between observed and expected results. It is suggested that for realistic biohythergraphs related parameters in addition to rainfall and temperature should also be considered.     

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.     

Serological survey of bovine herpesvirus type 1 infection in China

To understand the nationwide seroprevalence of bovine herpesvirus type 1 (BoHV-1) infection of cows in China, 1344 sera of dairy cows from 29 provinces and 765 sera from 6 herds in Hubei province were collected with stratified random sampling. Another 483 sera from imported cows were included. The serum antibody was tested by BoHV-1 gG ELISA. The results demonstrated that the overall nationwide seroprevalence was 35.8% (481/1344), while the prevalence for individual province ranged from 12.1% to 77.8%. Although each province had positive samples, the prevalence was clustered in areas based on the cow population size. In Hubei Province, the overall seroprevalence was 22.2% (170/765) while the prevalence for individual farms varied greatly from 0.0% to 41.5%. The sera from imported cows had a moderate prevalence of 21.7% (105/483).     

重庆地区牛附红细胞体分子流行病学调查

根据重庆地区不同的地理条件以及不同的养殖模式,采用16S rRNA-PCR检测方法对重庆市进行了牛附红细胞体的分子流行病学调查。结果显示:重庆地区存在牛附红细胞体感染,平均感染率为11%。其中规模化养殖平均感染率(6%)低于散养(16.7%),丘陵地区平均感染率(2%)低于高山地区(25.6%)。     

Uterine infection with bovine herpesvirus type 4 in dairy cows

Diseases of the reproductive tract are a frequent problem in dairy herds. Herpesviruses are uterine pathogens also involved in other clinical diseases; for example, bovine herpesvirus type 4 BoHV‐4 induces abortion, enteritis, metritis, pneumonia and vaginitis, but it can also be detected in healthy cows. The role of BoHV‐4 in the development of clinical endometritis (CE) or subclinical endometritis (SE) has not clearly been described. Therefore, the objective of this study was to describe the prevalence of uterine BoHV‐4 infection and its relationship with clinical, bacteriological and cytological findings in dairy cows 20–30 days after calving. The experiment was performed as a completely randomized block design, with farm (n = 10) as blocking criterion and with cow (n = 397) as the experimental unit. Logistic regression models were used to assess the effect of BoHV‐4 infection on CE, SE and reproductive performance. Proportion of cows infected with BoHV‐4 was 5.8% (n = 23/397). BoHV‐4 was isolated in 11.0% (n = 12/109), 4.8% (n = 4/84) and 3.6% (n = 7/194) of cows diagnosed as CE, SE or healthy, respectively. A logistic model revealed that BoHV‐4 infection showed a tendency to increase the risk for CE (AOR = 2.17; p = .10) but significantly reduced both, the odds for artificial insemination within 80 days post‐partum (dpp) (AOR = 0.37; p = .035) and for pregnancy within 200 dpp (AOR = 0.13; p = .004). Furthermore, BoHV‐4 infection increased the chance for intrauterine infection with Trueperella pyogenes (AOR = 5.55; p < .001) and vice versa (AOR = 5.79, p < .001). In conclusion, BoHV‐4 infection is associated with reduced chances for insemination and pregnancy by 200 dpp and showed a trend to be associated with increased risk for CE. Furthermore, BoHV‐4 and Trueperella pyogenes infections are strongly related.     

Experimental aerosol infection of cattle (Bos taurus) with ovine herpesvirus 2 using nasal secretions from infected sheep

Infection of clinically susceptible ruminants, including domesticated cattle and American bison, with ovine herpesvirus 2 (OvHV-2) can result in the fatal lymphoproliferative and vasculitis syndrome known as malignant catarrhal fever (MCF). A reliable experimental infection model is needed to study the pathogenesis of MCF and to develop effective vaccination strategies to control the disease. An experimental aerosol infection model using sheep, the natural carriers of OvHV-2, has been developed (Taus et al., 2005). Using the protocol and OvHV-2 inoculum established in the previous study, eight calves were nebulized with four different doses of OvHV-2 in nasal secretions from infected sheep. Two control calves were nebulized with nasal secretions from uninfected sheep. Infection status of all calves was monitored using competitive inhibition ELISA, PCR and clinical parameters. Six of eight nebulized calves became infected with OvHV-2. One calf receiving the highest dose of virus developed typical clinical, gross and histological changes of MCF. This study showed that nasal secretions collected from sheep experiencing OvHV-2 shedding episodes were infectious for cattle and capable of inducing MCF. The data also indicate that cattle are relatively resistant to disease following infection. The use of more susceptible species as experimental animal models, such as bison and selected cervid species should be examined.