Molecular characterization of bovine foamy virus and its association with bovine leukemia virus infection in Vietnamese cattle

The detection of bovine foamy virus (BFV) in Vietnamese cattle was performed using conventional PCR targeting pol and gag genes. Out of 243 tested samples, ten (4.1%) and eight (3.3%) samples were positive for BFV gag and pol DNA, respectively. The prevalence of bovine leukemia virus (BLV) estimated by detection of proviral DNA using nested PCR targeting env gene was 26.7% (65/243). The results of nucleotide sequence alignment and the phylogenetic analysis suggested that Vietnamese BFV strains showed high homology to isolates belonging to either European or non-European clades. There was no significant correlation between BLV and BFV. This study provides information regarding BFV infection and confirms the existence of two BFV clades among Vietnamese cattle for the first time.     

Horizontal transmission and phylogenetic analysis of bovine leukemia virus in two districts of Miyazaki,Japan

Horizontal transmission is recognized as a major infection route for bovine leukemia virus (BLV), and cattle with high viral loads are considered to be a major infectious source in a herd. However, a correlation between viral loads and the risk of infection has been insufficient to use as a foundation for BLV control strategies. In this report, we examined the epidemiology of BLV infection and the infectious source in a local area. In 2013–2014, BLV infection was investigated in 1,823 cattle from 117 farms in two adjacent districts, Miyazaki, Japan. Seropositive samples for BLV were detected with 88 cattle and in 14 farms. Phylogenetic analysis revealed that 94% of the isolates clustered into genotype I and the remaining isolate into genotype III. Among genotype I, genetically distinct strains were spread at each farm, and cattle infected with less than 3 copies/100 cells did not transmit BLV to other cattle for more than thirty months. This is the first report of concrete data of viral load in relation to viral horizontal transmission under the field condition. The data facilitate farmers and veterinarians understanding the status of BLV infected cattle. This research contributes to BLV infection control and the development of effective BLV eradication programs.     

The recent prevalence of bovine leukemia virus (BLV) infection among Japanese cattle

A seroepidemiological survey of bovine leukemia virus (BLV) infection was conducted in Japan in 2007 using an enzyme-linked immunosorbent assay (ELISA) and an agar gel immunodiffusion (AGID) test. A total of 5420 cattle (dairy, 3966; breeding beef, 797; fattening beef, 657) from 209 farms in seven prefectures in Japan were tested. The overall prevalence of BLV infection was 28.6%. The prevalence of BLV infection in dairy cattle (34.7%) was higher than for both fattening beef cattle (7.9%) and breeding beef cattle (16.3%). Age-specific prevalence showed that BLV prevalence increased with age in all types of cattle and was notably different between dairy and beef cattle under 1 year of age. Among 207 farms, 141 herds (68.1%) had one or more positive animals. The proportion of these positive farms was significantly higher among dairy farms (79.1%) than among beef breeding farms (39.5%) and beef fattening farms (51.9%) (P < 0.001). Dairy farms (40.5%) also showed a significantly higher within-herd prevalence than beef breeding (27.4%) and fattening (14.9%) farms (P = 0.001). This study indicated that BLV is more widely spread in dairy cattle than in beef breeding cattle in Japan. Given the prevalence of BLV infection in dairy and beef cattle was 8- and 1.7-fold higher, respectively, than rates previously found in 1980–1982, BLV appears to be spreading particularly among the dairy cattle population during the last two decades. Further investigation is required to determine the risk factors necessary to control BLV infection that take into account the different farming practices that exist between dairy and beef sectors.     

鹅源鸭瘟与小鹅瘟二联弱毒疫苗的研究（初报）

鹅的鸭瘟和小鹅瘟是当前严重威胁养禽业的两种传染病,虽然已分别有了预防疫苗,但鸭瘟疫苗对鹅的鸭瘟效价较低,免疫期短,效果不一,特别是接种两种疫苗时,需多次注射,使用不便。根据鹅源鸭瘟病毒和小鹅瘟病毒可同时在同一鸭胚复制增殖,未发现干扰作用的存在,特进行二联疫苗的研制,以便提高疫苗质量和满足防疫需要。 研究结果显示,二联苗对成鹅具有良好的安全性和免疫原性,免疫鹅对鸭瘟强毒攻击的半数有效免疫剂量(10~(6.5)ED_(50)/1ml)、免疫鹅血清对小鹅瘟病毒的中和指数(NI=7586)分别略高于鸭瘟疫苗(10~(6.5)ED_(50)/1ml)和小鹅瘟疫苗(NI=3163),免疫鹅的鸭瘟中和抗体反应和小鹅瘟沉淀抗体反应与相应的鸭瘟疫苗和小鹅瘟疫苗基本相同,从而初步研制成功二联弱毒疫苗。二联苗免疫鹅对鸭瘟有免疫力,其后代可免遭小鹅瘟侵袭,具有双重作用,由于使用同一鸭胚生产二联苗,可简化制苗工艺,降低成本,便于现场应用,减少对鹅的应激作用。     

Evaluation of a bulk-milk ELISA test for the classification of herd-level bovine leukemia virus status

The results of a commercial bulk-milk enzyme-linked immunosorbent assay (ELISA) test for herd-level bovine leukemia virus (BLV) status were compared to results obtained from individual agar-gel immunodiffussion (AGID) testing on sampled cattle. A positive herd was defined as a herd having one or more AGID-positive animals. The estimated true herd status was based on the sensitivity and specificity of the AGID test and the number of cattle sampled per herd. Ninety-seven herds were used, with a mean of 13 cows sampled per herd. The AGID test indicated an apparent herd prevalence of 70.1%. After accounting for the number of cows sampled and the sensitivity and specificity of the AGID test, the estimated true herd prevalence of BLV was 52.3%. The ELISA test identified 79.4% of herds as positive for BLV, and had an apparent sensitivity and specificity of 0.97 and 0.62, respectively. However, after accounting for the sensitivity and specificity of the AGID test in individual animals, the specificity of the ELISA test was 0.44. The ELISA test was useful for identifying BLV-negative herds (i.e., ruling out the presence of BLV infection in test negative herds). With the moderately low specificity, herds identified as positive by the ELISA test would require further testing at the individual or herd level to definitively establish their BLV status.     

Production and related variables in bovine leukaemia virus-infected cows

A newly developed milk dot blot test was used to detect anti-bovine leukaemia virus (BLV) antibody in milk samples from 2079 lactating adult cows from among 61 herds. The milk dot blot test was highly repeatable; the concordance rate, compared with the agar gel immunodiffusion test performed on serum, was 83.5%. All herds contained BLV-positive cows; the prevalence rate was 36%. BLV-positive cows tended to come from larger herds and were older and more often later in lactation. Fourteen production and related variables (herd size, age, days open, days in milk, milk somatic cell count, milk, fat, and protein produced in the current lactation, projected production of milk, fat, and protein, and breed class average deviations for milk, fat, and protein) were compared between BLV-positive and BLV-negative cows. Although somatic cell count, milk produced, and projected production of milk and protein were related significantly to BLV status using simple tests of association, once the variables herd size, age and days in milk were controlled, these differences were removed. Further analyses using logistic (outcome: individual cow BLV status) and least-squares regression (outcome:herd proportion of BLV-positive cows) failed to show an association between any of the measured production or related variables and BLV-positivity. We concluded that the effect of BLV on production and related variables in dairy cows was below the sensitivity of our analytical techniques or was non-existent.Abbreviations ABCA herd average breed class average for milk, fat, and protein production - AVGAGE average age of the herd - ADIM herd average for days in milk - AGID agar gel immunodiffusion - AVGSCC herd average milk somatic cell count - BCA breed class average, a milk, fat and protein production index calculated by comparing a cow's actual 305-day lactation production to the corresponding BCA standard for the same breed, age, and month of calving - BLV bovine leukaemia virus - CALVINT calving interval - COWAGE cow age - DBCA breed class average deviation for milk, fat, and protein production, the difference between an individual cow's BCA and the herd average - DIM days in milk - HS herd size corresponding to the number of lactating cows in a herd - LACT actual amount of milk, fat, and protein produced in a cow's lactation - ODHIC Ontario Dairy Herd Improvement Corporation - PCTPOS percentage of herd that is BLV-positive - PROJ projected 305-day production for milk, fat, and protein by fitting to a standard lactation curve adjusted for days in milk and age at calving - RHBCA rolling herd average for breed class average for milk, fat, and protein production, the average for all cows that completed a lactation (cows must have completed a 305-day lactation) during the previous 12 months - SCC milk somatic cell count     

Seroprevalence of enzootic bovine leukosis in Trakya district (Marmara region) in Turkey

In this study, 481 cattle belonging to 77 farms from nine localities in Trakya district in the Marmara region of Turkey were blood sampled and serologically tested for enzootic bovine leukosis (EBL). Antibodies to bovine leukosis virus (BLV) were detected in 51 cattle sera (11%) belonging to nine farms in five localities. Cattle tested were mostly female Holstein or Brown Swiss of ages ranging between 18 months and 10 years. Analysis of the relationships between age, breed or sex and seropositivity to EBL in seropositive herds indicated no significant associations (p>0.05). The relationship between seropositivity and haematological changes was also studied, and seropositive cattle had higher lymphocyte percentage and lower neutrophil percentage than seronegative cattle (p<0.001).     

Associations between farm management practices, productivity, and bovine leukemia virus infection in Ontario dairy herds

The objective of this study was to investigate the associations between herd-level bovine leukemia virus (BLV) status and herd-level management and production variables. The study population consisted of 1330 cows sampled from 102 Ontario dairy herds. The individual-cow prevalence of BLV infection in the population (based on AGID testing) was 23%, with 69.6% of herds having one or more positive animals. The herd-level explanatory variables were divided into two datasets containing winter housing variables and all non-seasonal variables, and summer housing variables and all non-seasonal variables. In both datasets, multivariable analyses found a negative association between herd-level milk production and BLV status, and positive associations between weaning age and purchasing animals from outside sources, and BLV status. Housing pre-weaned calves in hutches or separate calf buildings in either season was associated with an increased risk of BLV. The model containing winter housing variables also included positive associations between contact with older animals and BLV status, and between BLV status and the facilities used to house dry cows in the winter.     

Risk factor analysis of bovine leukemia virus infection in dairy cattle in Egypt

Identification of the risk factors associated with Enzootic bovine leukosis (EBL) is essential for the adoption of potentially prevention strategies. Accordingly, our objectives were to determine the geographic distribution of Bovine Leukemia Virus (BLV) infection and identify the risk factors associated with cow-level BLV infection in the Egyptian dairy cattle. A cross-sectional study was conducted on 1299 mixed breed cows distributed over four provinces in the Nile Delta of Egypt in 2018. The randomly selected cows on each farm were serologically tested for BLV, and the cow’s information was obtained from the farm records. Four variables (geographic location, herd size, number of parities, and age) were used for risk analysis. A total of 230 serum samples (17.7 %) were serologically positive for BLV. The highest prevalence of BLV infection was associated with parity (OR = 3.4, 95 %CI 2.4–4.9) with 80 % probability of being BLV-positive at parity ≥5, followed by herd size (OR = 1.8, 95 %CI 1.4–2.2). However, geographic location seems to have no impact on the prevalence of BLV infection in Egypt. Our findings strongly indicate that the intensive surveillance and effective prevention strategies against BLV infection in Egypt should be provided to multiparous cows with ≥5 parities and live in large farm with more than 200 cows.     

流行性牛白血病的病原及传播途径研究进展

流行性牛白血病是由反转录病毒科丁型反转录病毒属的牛白血病病毒所致的一种肿瘤性疾病。在病毒的囊膜中含有数种糖蛋白(gp) ,在芯髓有几种非糖基化的蛋白 (p) ,其中gp51和 p2 4的抗原活性最高 ,可用作流行性牛白血病的特异性血清学诊断。此病的病毒可通过垂直和水平传播 ,其中水平传播的途径很多 ,主要包括血源性传播、分泌性传播、接触性传播、寄生性传播、精液性传播、乳源性传播和管理性传播等     

An international comparison of different laboratory tests for the diagnosis of bovine leukosis: suggestions for international standardization

A collaborative study was conducted to compare the detection limit of different laboratory tests for antibodies against bovine leukemia virus (BLV). Serum and milk samples were tested in agar gel immunodiffusion (AGID), different modifications of indirect ELISA, blocking ELISA and ELISA procedures using monoclonal antibodies to BLV gp51 or BLV p24. The detection limit of reference serum E4 diluted 2-fold in negative serum gave a median value of 1:16 in AGID, indirect ELISA, and monoclonal ELISA p24, 1:128 in monoclonal ELISA gp51, and 1:1024 in blocking ELISA. The detection limit of a 4% immunoglobulin preparation of E4 diluted in negative milk showed median values of 1:800 in indirect ELISA, 1:1000 in monoclonal ELISA, and 1:2400 in blocking ELISA. None of the ELISA procedures could detect all the positive individual milk samples diluted 1:50. The AGID test is the official reference test for detection of antibodies against BLV. Reference serum E4 diluted 1:10 in negative serum must be scored positive in the AGID test. It is suggested that an international reference serum standard be established rather than an official recommendation of a particular ELISA test.     

Evidence for transmission of bovine leukemia virus by rectal palpation in a commercial dairy herd

The risk of Bovine Leukemia Virus (BLV) transmission by rectal examination was determined over 22 months in a commercial dairy herd. All 167 BLV seronegative cattle, of breeding age or greater, were divided randomly into two groups and identified by neck-chain color. In the treatment group, routine rectal palpation occurred after a BLV infected animal and without a change of sleeve, while in the other group, palpation occurred in a similar manner with the exception that sleeves were changed between animals. When BLV seronegative cattle in either group were palpated after BLV infected cattle, the event and identification of the cattle involved were recorded. Serologic testing was performed eight times during the 22 month study to determine the number of animals that became infected following a palpation (an event). Thirty-one animals seroconverted during the study; 24 in the treatment (no sleeve change) group and seven in the sleeve change group. Sixteen of the animals in the treatment group that seroconverted had been palpated prior to their seroconversion. A hazard ratio (relative risk) for BLV seroconversion was determined between the two groups. Cows palpated with no sleeve change had a 2.8-fold increase in risk (confidence interval 1.1–6.8) of BLV infection. The increased risk of BLV infection associated with rectal palpation may have been affected by the presence of some highly infectious cows in the herd. This study confirms that rectal palpation without a change of sleeve may be a significant risk factor in some herds, and if efforts are made to decrease the spread of BLV in a herd, the potential for rectal sleeve transmission must be considered.     

A serological survey of bovine syncytial virus in Ontario: associations with bovine leukemia and immunodeficiency-like viruses, production records, and management practices.

Of the 920 cows tested, 56.7% showed antiretroviral serological reactivity. Prevalence rates (95% confidence interval) of antiretroviral antibodies among individual dairy cows in Ontario were: BIV 5.5% (4.0-7.0), BLV 25.7% (22.9-28.6), and BSV 39.6% (36.4-42.8). The following percentages of cows showed serological reactivity against the specified retroviruses: BIV 2.3%, BLV 14.0%, BSV 27.5%, BIV and BSV 1.3%, BIV and BLV 0.9%, BLV and BSV 9.9%, BIV and BLV and BSV 0.9%. These rates of sero-positivity are similar to those found in other countries. Serological test results were not adjusted for sensitivity and specificity. The prevalence rates of antibodies to the three retroviruses (BIV, BLV, and BSV) were significantly different, but no associations were observed between specific retroviral serological test results among individual cows. The prevalence rates of BIV and BSV seropositivity were constant across Ontario, whereas, there was a significant trend for the prevalence rate of BLV seropositivity to decrease going from southwestern to eastern Ontario; cows in eastern Ontario had approximately half the prevalence rate of those in southwestern Ontario. Cows that were seropositive for BSV were significantly older than BSV seronegative cows. There was no association between culling rate and BSV serology. Significant negative associations were found with winter or summer housing of calves separate from adults and summer outdoor exercise for dry cows. The use of calf hutches in the summer had a significant positive association with BSV seropositivity. Regression analyses were done to assess the association of retroviral (BIV, BLV, and BSV) seropositivity on calving interval, milk somatic cell count, and milk production. Serological test results for BIV, BLV, and BSV were entered into all models and all models were adjusted for intra-cluster (intraherd) correlation. Herd size and age were found to be important confounding variables. BIV seropositivity was not associated with any changes in production using this approach, however when considered in isolation BIV seropositivity remained associated with decreased milk production. BLV seropositivity was significantly associated with longer calving intervals and higher somatic cell counts in older cows. As well, in older cows, BSV seropositivity was significantly associated with higher milk production.     

Evidence of bovine immunodeficiency virus (BIV) infection: serological survey in Argentina

This is the first report of serological evidence for bovine immunodeficiency virus (BIV) infection in Argentina. The analysis was performed in 589 dairy bovine sera samples, applying indirect enzyme-linked immunosorbent assay (I-ELISA) using a synthetic antigen (transmembrane peptide, TM) and Immunofluorescent assay (IFA). In this study, 9 dairy herds from 4 Argentinian provinces were evaluated and 12% of the animals tested positive for BIV. Seven of the 9 herds tested were BIV seropositive and the percentage of BIV seropositive animals in the herds ranged from 2% to 42%. Direct detection of BIV provirus applying nested PCR was not conclusive. Antibody detection against bovine leukemia virus (BLV) in all sera was also performed applying immunodiffusion (ID) assay and 59% resulted seropositive. Statistical analysis of the results was carried out and possible evidence of association between BIV and BLV infection was considered. Future studies should be performed including local field isolates strains of BIV.     

Reliability in somatic cell count measurement of clinical mastitis milk using DeLaval cell counter

Somatic cell counts (SCC) measurements are typically performed using quantitative methods, such as the Breed method (Breed) and the Fossomatic method (FSCC). The DeLaval cell counter (DCC) developed recently is a quantitative somatic cell counter with a low initial cost and superior portability. However, since the DCC was specifically developed for measuring SCC of ≤ 4 × 10⁶ cells/mL milk from bulk tanks or individual cows, its reliability for estimating SCC that exceed this concentration has not yet been clarified. This study therefore examined whether it is possible to accurately measure SCC by diluting milk samples with initial SCC of 4 × 10⁶ cells/mL, as seen in clinical mastitis milk. We collected milk samples from 99 quarters of 99 Holstein cows with clinical mastitis. These milk samples were diluted 10‐fold with saline and thoroughly mixed before performing SCC measurement with the DCC. The correlation coefficients of SCC measured by the FSCC, Breed and DCC methods indicated strong correlations between each pair of methods. The findings showed that DCC can be used to identify bovine clinical mastitis milk and is useful as a quantitative SCC measurement device on farm sites.     

Development of a PCR to diagnose BLV genome in frozen semen samples

The sanitary and economic impact of BLV infection is associated with the interference in the international movement of cattle and their germ plasm. Although experimental data support the improbability that semen from BLV-positive bulls could infect recipient cows, restriction for commercialization of semen from infected animals is still present. The objective of this work was to standardize a PCR assay to diagnose the presence of BLV genome in frozen semen samples. The developed methodology involves the amplification of an internal fragment of gag gene. The limit of detection of this technique was six viral particles, using gag-PCR followed by hybridization analysis. Frozen semen samples from seropositive bulls were analyzed. It was possible to detect proviral DNA in 9 out of 173 samples. Additionally, a biological test in susceptible sheep was performed in order to evaluate the transmission of BLV genome by semen from seropositive animals. This data strongly suggest that semen from seropositive bulls that resulted negative by PCR can be used for artificial insemination (AI), accompanied by proper collection protocols. The development of this PCR assay constitutes a valuable diagnostic tool to determine the BLV-free status of frozen semen samples used for AI.     

Should the domestic buffalo (Bubalus bubalis) be considered in the epidemiology of Bovine Herpesvirus 1 infection?

Only limited information is available on the epidemiology and pathogenesis of Bovine Herpesvirus 1 (BoHV-1) in domestic buffalos. In this study, a virulent BoHV-1 field strain isolated from cattle was inoculated into buffaloes to evaluate their susceptibility to the virus and to investigate the establishment of viral latency through clinical, virological and serological investigations. Latency was also studied by attempting viral reactivation using pharmacological induction. Six of seven male, 5 months old buffaloes were intranasally inoculated with BoHV-1; the other animal was kept as negative control. The animals were clinically monitored during the post-infection (P.I.) and the post-pharmacological induction (P.P.) periods. During these periods, nasal and rectal swabs, and blood samples, with and without anticoagulant, were collected at 2–3 day intervals. On culling the animals, 206 days P.I., their trigeminal ganglia and tonsils were collected. No clinical signs referable to BoHV-1 were observed throughout the experimental period. However, seropositivity was detected in all infected animals within day 20 P.I., using BoHV-1 glycoprotein E and glycoprotein B competitive ELISAs (IDEXX) and virus neutralisation test. In real-time PCR (RT-PCR), five of these animals were positive, at least once, for nasal or rectal swabs, during the P.I. period. The sixth infected animal was found positive only in the trigeminal ganglia after culling. Ganglia were also positive for two other animals. Virus isolation in permissive cell-lines was successful for a part of the RT-PCR positive samples. The detected viruses were confirmed by genetic analysis as identical to the inoculated strain. No evidence of infection was observed in the negative control. This study represents the first experimental transmission of BoHV-1 in buffaloes, confirming their susceptibility to infection and their possible role as host/reservoirs of BoHV-1.     

Economic implications of bovine leukemia virus infection in mid-Atlantic dairy herds

OBJECTIVE: To determine the baseline costs of bovine leukemia virus (BLV) infection, including costs of clinical disease and subclinical infection, in a dairy herd representative of the mid-Atlantic region and compare these costs with the cost of a test-and-manage BLV control program. DESIGN: Stochastic spreadsheet model. SAMPLE POPULATION: A commercial Holstein dairy herd with 100 milking cows. PROCEDURES: A spreadsheet model was developed. The overall cost of infection included the cost of clinical disease (ie, lymphosarcoma [LS]) and the effects of subclinical infection on milk production and premature culling. Model input values and distributions were designed to reflect economic conditions in the mid-Atlantic region. Relative costs of infection and control were calculated for infection prevalences of 20, 50, and 80%. RESULTS: Estimated mean cost to the producer per case of LS was 412 dollars; for a herd with a 50% prevalence of BLV infection, annual incidence of LS was 0.66. Mean annual cost of subclinical infection at a 50% prevalence of infection was 6,406 dollars. Mean annual cost of a test-and-manage control program was 1,765 dollars. The cost of clinical disease and subclinical infection varied substantially with the prevalence of infection, whereas the cost of control varied with herd size. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that a basic BLV control program may be economically beneficial in herds in which the prevalence of BLV infection is > or = 12.5%. Farm-specific considerations may factor prominently when weighing the costs and benefits of an individual BLV control program.     

Genomic characterization and distribution of bovine foamy virus in Japan

Bovine foamy virus (BFV) is distributed through worldwide cattle herds. Although the biological features of BFV are not well understood, appearance of clinical manifestation by superinfection with other microorganisms is inferred. In Japan, reports of genomic characterizations and epidemiology of this virus are limited. In this study, we performed whole genomic sequencing of BFV strains Ibaraki and No.43, which were isolated in this country. Additionally, we investigated BFV in geographically distant four daily farms in Japan, to estimate the distribution of BFV and its correlation to bovine leukemia virus (BLV). BFV was distributed throughout Japan; the average positive rate was 12.7%. The nucleotide sequence identities of the isolates were 99.6% when compared with BFV strain isolated in the USA. The phylogenetic tree using env gene sequence showed strains Ibaraki, No.43 and Kagoshima were sorted in the same cluster including the USA and Chinese strains, while Hokkaido strain was in the other cluster including European strains. Although no clear correlation between BFV and BLV could be found, BFV and BLV infections were likely to increase with ages. Our data on epidemiology and characteristics of BFV will provide important information to reveal biological features of BFV.