Epidemiological survey of bovine leukemia virus in beef cattle in the Tohoku region of Japan

Abstract

The incidence of enzootic bovine leukosis, which is due to bovine leukemia virus (BLV), has been increasing in Japan. However, large-scale surveys have not been conducted in recent years, and the impact of cattle harboring high BLV proviral loads on within-farm BLV prevalence remains unclear. To clarify the situation, questionnaires and PCR and serological testing for BLV were conducted between 2022 and 2024 on 95 beef breeding farms (3,082 cattle) and 9 Japanese Black fattening farms (140 cattle) in the Tohoku region. We also examined the effects of various factors on within-farm proportions of BLV-positive cattle. The proportions of PCR-positive and seropositive cattle were 34.3% and 35.9% in beef breeding cattle and 15.7% and 15.0% in Japanese Black fattening cattle, respectively. Among the 74 farms with at least one PCR-positive animal, the proportion of PCR-positive cattle was significantly higher on farms with at least one animal with a high proviral load (≥400 copies/10 ng DNA) than on farms without any such cattle. Similarly, farms where high-proviral-load cattle accounted for≥10% of the herd had significantly higher proportions of PCR-positive cattle. In addition, farms that used public pastures, practiced on-farm grazing, or had experienced enzootic bovine leukosis within the past 5 years had significantly higher proportions of PCR-positive cattle. These findings suggest that BLV prevalence in beef cattle in the Tohoku region remains comparable to that reported approximately 10 years ago, indicating that BLV transmission in this region may be under control. Furthermore, cattle with high proviral loads may adversely affect within-farm BLV prevalence, indicating that targeted control measures focusing on such animals are essential for effective BLV control.

INTRODUCTION

Enzootic bovine leukosis (EBL) is caused by bovine leukemia virus (BLV). Most infected cattle display no clinical signs, and only a few percent of infected cattle develop B-cell lymphoma, which typically results in death [35]. Since EBL was designated as a notifiable infectious disease in 1998 in Japan, the number of reported cases has steadily increased nationwide, with more than 4,000 cases reported annually since 2019 [13]. Serological surveys of BLV in Japanese beef cattle reported seroprevalence of 7.4% in 1980 and 6.0% in 1982 [7]. However, a nationwide survey conducted between 2009 and 2011 revealed that the seroprevalence in beef breeding cattle had increased to 28.7% [21]. A few regional studies have since been reported. For example, a survey in Ibaraki Prefecture conducted between 2018 and 2022 reported a BLV seroprevalence of 43.0% among beef breeding cattle [5]. However, no large-scale survey has been conducted since the 2009–2011 study. Given the reported increase in EBL cases in recent livestock hygiene statistics in Japan, follow-up studies are needed to assess the current prevalence of BLV.

Like other members of Retroviridae, BLV is integrated into the host genome as a provirus [35]. Proviral load (PVL) influences transmissibility, and cattle harboring high PVLs are considered more likely to cause both horizontal [17] and vertical [16] transmission than animals with low PVLs. Although preferential culling of cattle with high PVLs is well known to reduce within-farm proportions of BLV-positive cattle [28, 30] or even achieve BLV-free status [2, 33, 36], the impact of high-PVL cattle on the within-farm proportions of BLV-positive cattle is unclear. Moe et al. [19] reported an association between the presence of high-PVL cattle and increased within-farm proportions of BLV-positive cattle among native and crossbred cattle. The conditions associated with high PVL and its impact have not yet been fully elucidated. Further research that considers breed, production type (dairy vs. beef), and management systems is warranted.

Comprising six prefectures—Aomori, Akita, Iwate, Yamagata, Miyagi, and Fukushima—the Tohoku region had approximately 335,400 beef cattle in 2024, representing 12.6% of the national total of about 2.672 million [12]. These data underscore Tohoku’s status as one of Japan’s leading livestock-producing areas. Consistent with national trends, the number of reported cases of EBL in the Tohoku region has continued to increase, with 507 cases reported in 2024. This corresponds to approximately 11.5% of the total reported EBL cases in Japan in 2024 (4,420 cases) and is roughly proportional to the number of cattle raised in the Tohoku region (12.6%) relative to the national herd [13]. More beef cattle are raised than dairy cattle in this region, making it one of the major beef production areas in Japan. In addition, although not publicly available, internal data from the Miyagi Prefecture Agricultural Mutual Aid Association suggest that EBL occurs more frequently in beef cattle than dairy cattle. Furthermore, compared with dairy farms, beef cattle farms are more numerous and their management practices are more diverse, thus facilitating the investigation of factors that influence within-farm BLV prevalence. Given these considerations, our study focused on beef breeding farms and Japanese Black fattening farms. The large-scale regional study we report here used PCR and antibody testing for BLV, and questionnaires, to determine the proportions of BLV-positive beef breeding cattle and Japanese Black fattening cattle raised on cooperating farms between 2022 and 2024. In addition, these large-scale datasets were used to assess the effects of high PVL on the within-farm proportions of BLV-positive cattle.

MATERIALS AND METHODS

Animals

Cattle from 95 beef breeding farms and 9 Japanese Black fattening farms that were located in the Tohoku region and consented to participate in this study were enrolled. The geographic distribution of the farms is shown in Fig. 1. On the beef breeding farms, a total of 3,082 animals—all of which were adult cows and female calves retained as replacement breeding stock—were included (Table 1). On the Japanese Black fattening farms, 140 fattening cattle at approximately 20 months old were investigated. Most of these cattle were born in the Tohoku region and were introduced into the study farms at approximately 10 months of age (Table 2).

Fig. 1.

Locations of inspected farms. The shaded area indicates the Tohoku region of Japan. Red circles indicate beef breeding farms and blue circles represent Japanese Black fattening farms.

Table 1. Numbers of farms and cattle and breeds inspected at beef cattle breeding farms in the Tohoku region.

Prefecture	No. of farms	No. of cattle	Breed*
			JB	F1	JS	Unknown
Aomori	3	556	345	197	10	4
Akita	5	168	162	6	–	–
Iwate	36	799	799	–	–	–
Yamagata	10	399	357	42	–	–
Miyagi	21	719	719	–	–	–
Fukushima	20	441	441	–	–	–
Total	95	3,082	2,823	245	10	4

* JB, Japanese Black; F1, crossbred; JS, Japanese Shorthorn.

Table 2. Numbers of cattle inspected at Japanese Black fattening farms in the Tohoku region.

Prefecture of fattening farm	Prefecture of birth							Total
	Aomori	Akita	Iwate	Yamagata	Miyagi	Fukushima	Others
Miyagi	7	–	24	5	79	–	17	132
Fukushima	–	–	–	–	–	8	–	8

Before sample collection, informed consent regarding sampling procedures and the handling of test data was obtained from participating farmers through Agricultural Mutual Aid Associations in the Tohoku region. PCR tests were performed at the Livestock Clinical Training Institute of the Miyagi Prefectural Agricultural Mutual Aid Association, whereas BLV antibody testing was conducted at Iwate University. The study protocol was approved by the Animal Experiment Committee of Iwate University (Approval No. A202028), and the study was conducted in accordance with the Iwate University Regulations for Animal Experiments.

Blood sampling and BLV testing

The study period was from June 2022 through March 2024. Blood samples were collected from the jugular veins of the cattle by using EDTA-containing tubes (Venoject II VP-DK052K; Terumo, Tokyo, Japan) and serum-separating tubes (Venoject II VP-AS109K; Terumo). Whole blood was used for the detection of PVL, and serum was used for the detection of anti-BLV antibodies. For PVL analysis, DNA was extracted from whole blood by using an automated DNA extraction system (Magtration System 12GC PLUS; Precision System Science, Chiba, Japan). The concentration of extracted DNA was measured by using a NanoDrop One (Thermo Fisher Scientific, Waltham, MA, USA) to assess the absorbance at a wavelength of 260/280 nm. Real-time PCR analysis was performed by using a Bovine Leukemia Virus qPCR Detection Kit (RC202A; Takara Bio, Kusatsu, Japan) on a StepOne Plus Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Foster City, CA, USA) [27]. PVL was calculated as the number of viral copies per 10 ng of DNA used in the qPCR reaction. According to previous reports [16], samples were categorized into three groups: PVL undetectable (negative); PVL 0 to <400 copies/10 ng DNA (low PVL); and PVL ≥400 copies/10 ng DNA (high PVL). For antibody detection, an ELISA kit (IDEXX Leukosis Serum X2 Ab Test; IDEXX Laboratories, Westbrook, ME, USA) was used according to the manufacturer’s instructions. Samples with false-positive results were included in the negative category.

To evaluate BLV infection status on beef breeding farms by age, proportions of PCR-positive cattle and PVL-based categories were calculated for each age group, and the correlation between age and PVL was analyzed. Furthermore, to assess the impact of high-PVL cattle on the within-farm proportions of positive cattle, BLV-positive breeding farms on which at least one PCR-positive animal was raised were divided into two groups—“with high-PVL cattle” and “without high-PVL cattle”—and the proportions of PCR-positive cattle were compared. Similarly, BLV-positive farms were categorized as “less than 10%” or “10% or more” according to the within-farm proportion of high-PVL cattle among tested cattle, and the proportions of PCR-positive cattle were compared.

Questionnaire survey for beef breeding farms

At the beef breeding farms included in the study, a questionnaire (Table 3) was used to collect information on management practices, including husbandry methods and measures against blood-sucking insects. The responses for each survey item were used to categorize the farms, and the proportions of PCR-positive cattle were compared across the resulting groups.

Table 3. Questionnaire for beef breeding farms.

Questions regarding		Option
General husbandry practices
	External cattle intake	Yes / No
	Stall type	Individual / Group
	Paddock	Yes / No
	On-farm grazing	Yes / No
	Public pasture	Yes / No
Control measures against blood-sucking insects
	Number of horseflies	Absent / Few / Many
	Number of stable flies	Absent / Few / Many
	Insect net	Yes / No
	Horsefly trap	Yes / No
	Insecticide or repellent	Yes / No
Others
	Maternal colostrum feeding to calves	Yes / No
	Change of plastic sleeves for rectal palpation	Yes / No
	Change of needles	Yes / No
	Disinfection of instruments during invasive procedures (castration, dehorning)	Yes / No
	Occurrence of EBL within the previous 5 years	Yes / No

EBL; enzootic bovine leukosis.

Statistical analysis

Data aggregation and figure preparation were performed by using Excel (Microsoft, Redmond, WA, USA). The proportions of ELISA-positive and PCR-positive cattle were calculated separately for breeding and fattening cattle. Correlations were evaluated by using Spearman’s rank correlation coefficient. To assess the association between age and the proportions of PCR-positive cattle, the Cochran–Armitage trend test was used, treating age as an ordered variable. Median values were compared by using the Mann–Whitney U test. Statistical analyses were performed with EZR (version 1.54; Saitama Medical Center, Jichi Medical University, Saitama, Japan), and P-values of less than 0.05 were considered statistically significant.

RESULTS

The proportion of BLV-positive beef breeding cattle was 34.3% (95% confidence interval [CI]: 32.6–36.0%) by PCR and 35.9% (95% CI: 34.3–37.6%) by ELISA. In contrast, the proportions of BLV-positive Japanese Black fattening cattle were 15.7% (95% CI: 9.7–21.7%) by PCR and 15.0% (95% CI: 9.1–20.9%) by ELISA. The proportions of BLV-negative, low-PVL, and high-PVL cattle were 65.8%, 23.2%, and 11.1%, respectively, among the beef breeding cattle and 83.6%, 10.0%, and 6.4%, respectively, among the Japanese Black fattening cattle (Fig. 2).

Fig. 2.

Proportions of categories based on proviral load (PVL) of bovine leukemia virus in beef breeding cattle and Japanese Black fattening cattle. Negative, low PVL, and high PVL indicate undetectable PVL, <400 copies/10 ng DNA, and ≥400 copies/10 ng DNA, respectively.

Among the 95 beef breeding farms, 21 had no cattle positive for BLV by PCR (the largest category), whereas a total of 23 farms had proportions of 70–100% BLV-positive animals (Fig. 3). Herd size showed no association with the proportion of PCR-positive cattle at the farm level (Spearman’s ρ=0.06, P=0.558; Fig. 4). In addition, the proportions of PCR- and ELISA-positive beef breeding cattle showed a significant increasing trend with age (P<0.001; Fig. 5). No correlation was found between PVL and age among PCR-positive cattle (Spearman’s ρ=0.03, P=0.301; Fig. 6).

Fig. 3.

Histogram of farm-level proportions of cattle PCR positive for bovine leukemia virus (BLV) on beef breeding farms.

Fig. 4.

Relationship between herd size and proportion of cattle PCR positive for bovine leukemia virus (BLV) on beef breeding farms. No association emerged between herd size and within-farm positivity (Spearman’s ρ=0.06, P=0.558).

Fig. 5.

Proportions of bovine leukemia virus (BLV)-positive cattle by PCR and ELISA, and numbers of cattle in each category based on proviral load (PVL) by age on beef breeding farms. Negative, low PVL, and high PVL indicate undetectable PVL, <400 copies/10 ng DNA, and ≥400 copies/10 ng DNA, respectively. Both proportions of positive cattle showed a significant increasing trend with age (Cochran–Armitage trend test: P<0.001).

Fig. 6.

Relationship between age and proviral load (PVL) in bovine leukemia virus (BLV)-positive beef breeding cattle. No correlation was found between age and PVL (Spearman’s ρ=0.03, P=0.301).

Comparison of farms with no high-PVL animals (n=13) and those with at least one high-PVL animal (n=61) revealed significantly higher proportions of PCR-positive cattle on farms with at least one high-PVL animal (P<0.001; Fig. 7a). Furthermore, classifying farms according to the within-farm proportion of high-PVL cattle (<10%, n=35; ≥10%, n=39) showed that farms with≥10% high-PVL cattle had significantly higher proportions of PCR-positive cattle than those with <10% (P<0.001; Fig. 7b). The questionnaire results indicated that the proportions of PCR-positive cattle were significantly higher on farms that used public pastures (P<0.05), practiced on-farm grazing (P<0.05), and had had cases of EBL within the previous 5 years (P<0.001) (Fig. 8). No significant differences emerged for the other questionnaire items.

Fig. 7.

Comparison of the proportions of bovine leukemia virus (BLV)-positive cattle among the 74 BLV-positive breeding farms, classified according to the (a) presence of cattle with high proviral loads (PVLs) and (b) proportion of high-PVL cattle. In each box plot, the central line represents the median, the box indicates the interquartile range (25th–75th percentiles), and the whiskers represent the minimum and maximum values. The proportions of BLV-positive cattle were significantly higher on farms with at least one high-PVL animal than on farms with no high-PVL animals. Furthermore, the proportions of BLV-positive cattle were significantly higher on farms with ≥10% high-PVL animals than on farms with <10% high-PVL cattle. ***P<0.001 (Mann–Whitney U test).

Fig. 8.

Comparison of the proportions of bovine leukemia virus (BLV)-positive cattle according to questionnaire items. In each box plot, the central line represents the median, the box indicates the interquartile range (25th–75th percentiles), and the whiskers represent the minimum and maximum values. The proportions of positive cattle were significantly higher on farms that used public pastures, practiced on-farm grazing, and had had at least one case of enzootic bovine leukosis (EBL) during the previous 5 years. *P<0.05; ***P<0.001 (Mann–Whitney U test).

DISCUSSION

Nationwide surveys of BLV infection in Japan have been conducted twice to date: the first in the 1980s [7] and the second during 2009–2011 [21]. These reports revealed that the proportion of BLV-infected cattle at the national level increased approximately fourfold over a 30-year period, causing considerable concern within the Japanese cattle industry. The second nationwide survey [21] also reported regional prevalence, with a 39.9% prevalence among beef breeding cattle in the Tohoku region. More than a decade has passed since that regional survey, and because the number of EBL cases has continued to increase nationwide [13], it was assumed that the current BLV prevalence in Tohoku would be even higher. Nevertheless, the present study showed that 34.3% of cattle were PCR positive— a rate comparable to that reported in the previous survey.

In addition, the current study represents the first investigation of BLV infection in Japanese Black fattening cattle in the Tohoku region. Previous data on BLV infection in Japanese Black fattening cattle in Japan include a survey conducted at a slaughterhouse in Tokyo, which reported an average prevalence of 14.4% during 2005–2007 [31], and another survey conducted at a slaughterhouse in Miyazaki Prefecture, which reported an average prevalence of 22.6% during 2015–2016 [24]. Because cattle slaughtered at Tokyo abattoirs are sourced from across the country, the BLV prevalence observed there is considered to reflect the national average [31]. The proportion of PCR-positive cattle among Japanese Black fattening cattle in the current study was 15.7%, indicating that the current BLV prevalence among beef fattening cattle in the Tohoku region is similar to the national level observed about 20 years ago. Although the precise reasons for this trend remain unclear, several potential contributing factors include an increasing number of farms implementing BLV control measures in response to the widespread dissemination of BLV and the rising incidence of EBL; the commercial availability of BLV genetic testing kits that enable early detection of high-PVL cattle with a high risk of transmission; and the establishment of a more accessible testing environment. Because of these efforts, the number of reported cases of EBL in the Tohoku region since 2020 has remained relatively stable at approximately 500–550 cases annually, compared with the increasing trend previously [13]. Furthermore, in the six prefectures of the Tohoku region, collaborative BLV control programs and educational outreach activities conducted over approximately the past decade by Agricultural Mutual Aid Associations, prefectural livestock health authorities, and universities likely contributed to this outcome.

Among BLV-positive beef breeding farms, the proportions of PCR-positive cattle were significantly increased on farms with at least one high-PVL animal and where high-PVL cattle accounted for 10% or more of the herd. The primary routes of horizontal BLV transmission are iatrogenic transmission [4] and transmission via blood-sucking insects [3, 14, 26]. In addition, infected cattle with high PVL are more likely to cause horizontal transmission to other cattle [17] and vertical transmission [16]. According to Moe et al. [19], the presence of high-PVL cattle among native and crossbred cattle was associated with increased BLV prevalence at the farm level. In the present study, high PVL was defined as at least 400 copies/10 ng DNA. Japanese Black cattle, a highly valuable beef breed, require particularly careful consideration in the establishment of PVL thresholds and culling guidelines. High-PVL cattle are at increased risk of developing EBL [9, 18]. Furthermore, in dairy cattle, high PVL has been associated with impaired mammary immunity, leading to more severe mastitis [34], an increased prevalence of subclinical mastitis and greater economic losses [22], and reduced carcass weight [23]. In beef cattle, high PVL is associated with a shortened productive lifespan [1]. Therefore, because keeping high-PVL cattle entails not only an increased risk of BLV transmission but also substantial economic risks, prioritizing the isolation or early culling of high-PVL cattle may effectively reduce BLV-associated losses at the farm level.

Herd size and proportions of BLV-positive cattle showed no association at the farm level in the current study, similar to previous reports [10, 11]. Although increasing the number of cattle per farm is a global trend driven by efforts to improve economic efficiency, it can increase the risk of infectious disease outbreaks. Because BLV is transmitted primarily via blood [4], the risk of within-herd transmission can be reduced through appropriate control measures, including preventing iatrogenic transmission and implementing effective control strategies against blood-sucking insects. Consequently, although infectious diseases generally show higher positivity in large-scale farms, our results suggest that herd size may not necessarily be a major risk factor for BLV infection. In contrast, some studies have reported an association between larger herd size and higher BLV positivity [20, 29], suggesting that further investigation is needed to clarify the roles of management practices, cattle breeds, and other contributing factors.

Although an increasing trend of BLV positivity with advancing age occurred, no association between age and PVL was found. These results are consistent with previous reports [6, 20]. Because BLV infection is lifelong, our findings reflect the cumulative increase in infection prevalence with age. At the same time, they indicate that increasing age does not necessarily correlate with increased PVL. Therefore, comprehensive analyses incorporating host genetic factors, such as bovine leukocyte antigen (BoLA) genes, are required to further elucidate the determinants of PVL. BoLA-DRB3 polymorphisms reportedly influence immune responses against BLV infection, with some alleles associated with resistance to disease progression and lower proviral load but others linked to susceptibility and higher PVL [6, 18, 32]. Several field studies—including a case report of successful BLV eradication on a highly infected farm—suggest that combining intensive control measures with genetic and management strategies may substantially reduce or eliminate within-herd BLV infection [2, 36].

Analysis of the questionnaire results indicated that farms using public pastures or practicing on-farm grazing tended to have higher BLV positivity. Mountainous areas used as public pastures or grazing lands harbor large populations of blood-sucking insects [8] that can transmit BLV. In addition, public pastures receive cattle from multiple farms, increasing the likelihood that infected cattle are present and thereby elevating the risk of horizontal transmission. Measures to control BLV and blood-sucking insects vary among public pastures, and factors such as the window period during which recently infected cattle test negative [15] and the co-mingling of infected and uninfected cattle during turnout or transport make complete prevention of BLV transmission difficult. Kobayashi et al. identified loose housing systems and the presence of horseflies as factors that increase within-farm transmission risk [10], and Ohno et al. reported that group housing systems such as free-stall and feedlot systems are associated with the presence of high-PVL cattle [25]. Farms implementing BLV control measures need to consider these factors when managing cattle, particularly in relation to the use of public pastures or on-farm grazing. Practical and feasible strategies under such conditions include confirming in advance whether appropriate BLV control measures are implemented at the public pasture or on-farm grazing and conducting quarantine and BLV testing after cattle return from these conditions, before reintegration into the home herd. Although we did not identify any significant association between BLV positivity and questionnaire items related to blood-sucking insects within farms, further investigations that include assessments of blood-sucking insects, stocking density, and housing systems on public pastures and on-farm grazing environments are warranted.

BLV positivity was increased on farms that had EBL cases within the previous 5 years. This finding suggests that, by the time EBL is diagnosed, BLV transmission within the farm is already advanced. Many farms are unaware of their BLV infection status and, in practice, the presence of infected cattle is often recognized only after EBL occurs. However, once EBL is detected, BLV may already be widespread within the herd, and implementing countermeasures at that stage requires considerable time, effort, and cost. Therefore, to reduce BLV prevalence and achieve herd-level BLV-free status, farms need to assess their BLV infection status before any EBL cases occur.

A limitation of this study is that the farms surveyed were not selected randomly; rather, they voluntarily participated from among the farms under the jurisdiction of local veterinarians. Therefore, it is important to consider the potential for selection bias, such as a higher likelihood of participation by farms with a particular interest in BLV or concerns about EBL. Furthermore, although we analyzed associations between questionnaire items and within-farm BLV positivity, we did not assess relationships among questionnaire variables and the potential impact of high-PVL cattle on BLV transmission. These aspects should be addressed in future studies.

Our findings suggest that the previous rapid increase in BLV prevalence among beef cattle in the Tohoku region has plateaued. Moreover, the results suggest that the presence of high-PVL cattle influences BLV prevalence at the farm level and that farms using public pastures, practicing on-farm grazing, or having experienced EBL cases tend to have higher BLV prevalence. Currently, approximately 4,000 cases of EBL occur annually nationwide in Japan, with roughly one-eighth (500 cases) occurring in the Tohoku region [13]. To reduce the economic impact of EBL, it is essential that the relevant organizations continue their collaborative efforts to decrease the numbers of BLV-infected cattle.

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

Funding Statement

This research was partly supported by a JRA livestock promotion project (Grant No. 2022-23) and JSPS-KAKENHI (Grant No. 25K09433).