Research in Veterinary Science 129 (2020) 187–192

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Research in Veterinary Science

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Risk factors associated with the within-farm transmission of bovine viral T

diarrhea virus and the incidence of persistently infected cattle on dairy
farms from Ibaraki prefecture of Japan
Masataka Akagamia,b, Satoko Sekia, Yuki Kashimaa, Kaoru Yamashitaa, Shoko Oyaa, Yuki Fujiia,
Mariko Takayasua, Yuji Yaguchia, Atsushi Suzukia, Yoshiko Onoa, Yoshinao Ouchia,
Yoko Hayamac,
⁎

a
Ibaraki Prefecture Kenpoku Livestock Hygiene Service Center, Ibaraki, Japan
b
Ibaraki Prefecture Kennan Livestock Hygiene Service Center, Ibaraki, Japan
c
Viral Disease and Epidemiology Research Division, National Institute of Animal Health, National Agriculture and Food Research Organization, Ibaraki, Japan

ARTICLE INFO ABSTRACT

Keywords: For understanding the factors affecting bovine viral diarrhea virus (BVDV) transmission, this study investigated
Bovine viral diarrhea virus the distribution of BVDV and the epidemiological features of persistently infected (PI) cattle in Ibaraki Prefecture
Japan of Japan, and identified farm-level risk factors associated with BVDV infection, with a focus on within-farm
Persistently infected cattle transmission and PI animal detection.
Risk factors for within-farm transmission
Among all 377 dairy farms, forty-four PI cattle were identified on 22 farms. Thirty-eight and six PI cattle were
born on their current farms or purchased, respectively. Twenty-six PI cattle were born from pregnancies on their
current farms, seven from pregnancies in summer pastures, and eight from pregnancies on other farms. The
within-farm seroprevalence on farms with PI animals was significantly higher than that on farms without PI
cattle.
Of 333 farms holding homebred cattle without movement records, antibody-positivity in homebred cattle was
observed on 194 farms; these cattle were likely infected by within-farm transmission. Herd size, summer pas-
turing, and BVDV infection status of the nearest dairy farm were risk factors associated with within-farm
transmission. Likewise, herd size, summer pasturing, and the proportion of purchased cattle were related to PI
animal occurrence.
This study shows the risk of within-farm transmission and occurrence of PI animals after the introduction of
BVDV via purchasing and summer pasturing, and illustrates the significant role of PI cattle in circulating BVDV.
More effective measures for screening BVDV infection and PI animals, including intensive tests targeting moved
cattle and newborn calves, and bulk milk surveillance, are required to control the spread of BVDV in Japan.

1. Introduction
The genus Pestivirus of the family Flaviviridae comprises four re-
cognized species: bovine viral diarrhea virus (BVDV)-1, BVDV-2, clas-
sical swine fever virus (CSFV), and border virus (Simmonds et al.,
2012). Additionally, putative and unclassified species, including Giraffe
virus, Pronghorn virus, Bungowannah virus, and HoBi-like virus (also
referred to as BVDV-3 or atypical bovine pestivirus), have been iden-
tified recently (Bauermann and Ridpath, 2015; Blome et al., 2017).
Pestivirus infection in cattle, BVDV-1 and BVDV-2, are widespread
and represent major concerns throughout the world (Evans et al., 2019;
Gillespie et al., 1960; Houe, 1999). Bovine viral diarrhea (BVD) caused
by BVDV may take an inapparent course or lead to different clinical
signs such as fever, diarrhea, transient immunosuppression, decrease in
milk yield, respiratory signs, and reproductive disease (Houe, 1994;
Houe, 2003). The reduction of milk production following reproductive
disorders involving abortion and poor breeding performance, and in-
creased mortality of calves due to respiratory disease and weakness,
causes a significant economic impact on infected farms (Lindberg,
2003; Ridpath et al., 2010). Furthermore, if a pregnant dam is infected

⁎
Corresponding author at: Viral Disease and Epidemiology Research Division, National Institute of Animal Health, National Agriculture and Food Research
Organization, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan.
E-mail address: hayama@affrc.go.jp (Y. Hayama).

https://doi.org/10.1016/j.rvsc.2020.02.001
Received 26 July 2019; Received in revised form 30 December 2019; Accepted 10 February 2020
0034-5288/ © 2020 Elsevier Ltd. All rights reserved.
M. Akagami, et al.
with BVDV, persistently infected (PI) calves may result (Ridpath et al.,
2010). Many PI cattle suffer from ill-thrift and/or develop fatal mucosal
disease (Evans et al., 2019). Because PI cattle continue to excrete large
amounts of BVDV throughout their lives, PI cattle are an important
source of infection within and between farms (Lindberg, 2003).
In Europe, the spread of BVDV infection in the cattle population has
mostly been caused by BVDV-1 (Lindberg et al., 2006; Yesilbag et al.,
2017), although outbreaks of BVDV-2 have also been recently reported
in Germany and Poland (Gethmann et al., 2015; Polak et al., 2014;
Strong et al., 2018). In some European countries, national or regional
coordinated control and eradication programs for BVDV have already
been established. These programs, involving mandatory testing of
calves using tissue samples as well as bulk milk surveillance, have
contributed to decrease numbers of PI cattle and of BVDV infection
prevalence, and have accelerated the eradication of BVDV (Houe et al.,
2006). In Japan, BVDV infection of cattle is a notifiable disease, and
about 100–300 cattle are diagnosed every year (MAFF, 2019).
In Japan various genotypes of BVDV, such as 1a, 1b, 1c, and 2a, are
distributed throughout the country. BVDV-1 is the dominant strain (Abe
et al., 2016; Matsuno et al., 2007; Seki et al., 2008), while no evidence
of HoBi-like viruses have been observed according to an investigation of
cattle between 2012 and 2017 (Kozasa et al., 2018). Because of the
increased number of BVDV infection cases, the Japanese animal health
authority, the Ministry of Agriculture, Forestry, and Fisheries (MAFF)
strengthened the prevention and control measures against BVDV by
establishing BVDV control guidelines in 2016 (MAFF, 2016). This
program relies on accurate identification and prompt removal of PI
cattle as well as vaccination for prevention of the disease on a voluntary
basis. Based on the program, prefectural government officers guide
farmers to implement appropriate biosecurity for BVDV and encourage
them to participate in the inspection of cattle for screening BVDV in-
fection.
Risk factors associated with BVDV infection have been actively
studied in European countries. Several epidemiological studies have
reported that large-scale dairy farms, cattle purchases, summer pas-
turing, high density dairy farming, and production of dairy calves are
risk factors related to generating PI cattle (Amelung et al., 2018;
Graham et al., 2013; Presi et al., 2011). Additionally, detection of PI
cattle, presence of the pregnant cattle, and the total number of dairy
cattle on a farm have been identified as risk factors associated with
farm-level infection of BVDV (Humphry et al., 2012; Sarrazin et al.,
2013). Graham et al. (2016) also revealed that the presence of BVDV
infected farms in the neighborhood also posed a risk of local disease
spread.
In Japan, epidemiological studies of BVDV have mainly been con-
ducted in Hokkaido Prefecture, which is the largest dairy farming area
located in the northern part of Japan. Hokkaido Prefecture contains
almost 40% of dairy farms and 60% dairy cattle in the country (MAFF,
2018). Because of the flourishing dairy industry, selling pregnant hei-
fers to other prefectures and accepting young cattle from other pre-
fectures for rearing on summer grazing pastures are very common in
Hokkaido. To evaluate the control measures for BVDV in this region,
the effectiveness of mass vaccination, inspection prior to communal
pasture grazing in summer, and bulk milk herd screening tests have
been examined using a scenario tree model (Isoda et al., 2017; Isoda
et al., 2019). However, in prefectures other than Hokkaido Prefecture,
epidemiological studies for understanding the prevalence of BVDV and
occurrence of PI animals, as well as the risk factors associated with
BVDV infection, have been inadequate. According to European studies,
variation in the background factors affecting BVDV status among the
regions, such as BVDV prevalence, quality of veterinary service, and
density of dairy farms, may result in differences in regional risk factors
(Amelung et al., 2018; Graham et al., 2013). Considering the differ-
ences in dairy farming between Hokkaido Prefecture and other pre-
fectures in Japan, it is important to understand the factors that influ-
ence the risk of BVDV infection at the regional level.
188
Research in Veterinary Science 129 (2020) 187–192
Therefore, in this study, with a view to establishing effective BVDV
control measures that is suitable for dairy farm management, we firstly
conducted a survey of BVDV, targeting dairy cattle in Ibaraki
Prefecture, Japan, between 2014 and 2017. Next, based on the survey
data, the distribution of BVDV and epidemiological features of PI cattle
were descriptively analyzed. Furthermore, epidemiological analysis
was conducted to assess the farm-level risk factors associated with
BVDV infection and the occurrence of PI animals. We focused on the
risks associated with the infection of homebred cattle (i.e., within-farm
transmission). Epidemiological analysis was conducted with con-
sideration of herd management, biosecurity measures, and geo-
graphical features of the dairy farms.
2. Materials and methods
2.1. Study population and study design
In Japan, 1,380,000 dairy cattle are raised on 15,700 farms (MAFF,
2018). In this study, we targeted the dairy farms in the Ibaraki Pre-
fecture, located in eastern Japan. In Ibaraki Prefecture, almost 23,800
dairy cattle are raised on 361 farms, as of February 2018, and the
numbers of dairy cattle and farms in Ibaraki Prefecture is the eighth-
highest in the country (MAFF, 2018).
First, to determine the exposure status on all dairy farms in Ibaraki
Prefecture, serum samples of dairy cattle (≥12-month-old) from 377
dairy farms, which were collected by the local veterinary officers be-
tween April 2014 to March 2017, were studied. The serum samples
were randomly collected on each farm from a sample size that can
detect more than 10% sero-positive prevalence (26 samples per farm
with fewer than 100 animals, and maximum 35 samples per farm with
100 or more animals) (Cannon and Roe, 1982); thus, 9016 sera were
collected. Because these serum samples were collected randomly on
each farm, samples from cattle that did not have any movement records
(i.e., cattle were born and raised on the same farm) as well as samples
from cattle with movement records, including purchase from another
farm or sending to and returning from summer pastures, were included.
The collected serum samples were tested for anti-BVDV antibody using
blocking ELISA (VDPro BVDV ab ELISA, Median Diagnostics,
Chuncheon, Korea). The serum samples of all dairy cattle on 302 farms
that were found to be antibody-positive were subsequently tested for
BVDV antigen using sandwich ELISA (IDEXX BVDV ag ELISA, Japan,
IDEXX laboratories) (Masuda et al., 2017). BVDV antigen-positive cattle
were re-tested after 3 weeks, and cattle that were antigen-positive on
both tests were diagnosed as PI cattle. On the farms where PI cattle
were identified, after removal of the PI cattle from the herds, all calves
born in the ensuing 10 months underwent follow-up tests for BVDV
antigen as described above.
The BVDV genotype was distinguished by RT-PCR and restriction
fragment length polymorphism (RFLP) analysis on the PCR products
from PI cattle, based on the protocol of Yamaguchi et al. (1997) and
Harpin et al. (1995). This method rapidly discriminates the PCR pro-
ducts of pestivirus from CSFV, BVDV-1, and BVDV-2 using restriction
endonucleases (Bgl I and Pst I). Viral RNA was extracted from buffy coat
samples collected from PI animals using fully automated instrumenta-
tion with an in-tip nucleic acid extractor (magLEAD 12gC; Precision
System Science Co., Chiba, Japan). RT-PCR amplification of 5’-UTR was
carried out with the published primers 324 and 326 (Vilcek et al., 1994)
using the One Step RT-PCR Kit (QIAGEN, Hilden, Germany) according
to the manufacturer's instructions. RFLP analysis of amplified PCR
products was performed via two-step digestion (Bgl I followed by Pst I).
Briefly, viral RNA in which the Bgl I cleavage site was present was
designated as CSFV, and viral RNA that was not cleaved was designated
as BVDV. Among the BVDV samples, the viral RNA with the Pst I
cleavage site was determined to be BVDV-1, and the sample that was
not cleaved was BVDV-2.
M. Akagami, et al.
2.2. Categorization of farm-level status
This study focused on the risk of within-farm transmission of BVDV.
Thus, in this study, “within-farm transmission” was defined as BVDV
transmission to homebred cattle without movement records. Because
we could not determine the infection status of homebred cattle without
movement records on farms where all sampled cattle had movement
records, these farms were excluded from the epidemiological analysis.
Additionally, farms where BVD vaccination had been conducted were
excluded. Specifically, based on the data of 9016 sampled cattle, 44
farms, including 5 farms where vaccination was conducted; 22 farms
where all sampled cattle were purchased from other farms; and 17
farms where all sampled cattle had records of summer pasturing in
another region, were excluded, and the remaining 7969 cattle on 333
farms were used for the epidemiological analysis.
Farm-level status was categorized according to the results of anti-
body tests and antigen tests. When at least one homebred animal
without movements record was antibody-positive on a farm, the farm
was defined as a “BVDV-circulated-farm”. On a BVDV-circulated-farm,
the infected homebred cattle were recognized as having been infected
from an infection source on the farm. Among the BVDV-circulated-
farms, farms with PI cattle were defined as “PI-farms”. We defined
“BVDV-non-circulated-farms” as farms on which no seropositive ani-
mals were detected among the sampled cattle, except for those that
were vaccinated and those that had movement records of being pur-
chased or moved for summer pasturing. If all sampled cattle tested
antibody-negative, the farm was categorized as a “BVDV antibody-free
farm”.
2.3. Collection of epidemiological information
To investigate the risk factors associated with the within-farm
transmission of BVDV and the occurrence of PI animals, epidemiolo-
gical information on the dairy farms were collected, focusing on the
herd management, feeding and biosecurity measures, and geographical
features (Table 1). For herd management and biosecurity measures, the
farmers were interviewed. Herd management factors included herd size
(number of female cattle > 2 years old), proportion of purchased cattle,
summer pasturing, and presence of other animal species. The herd size
was categorized into large-sized or small-sized cattle farms, based on
the median number of adult cattle (the cut-off value for a small-sized
farm was fewer than 30 animals).
The use of communal summer pasturing is common in Japan, that
is, dairy cattle are temporarily sent to and reared on grazing farms in
areas with a cool climate suitable for dairy cattle during summer, such
as Hokkaido Prefecture. Some of the cattle are bred during summer
Table 1
Epidemiological information collected in the bovine diarrhea virus (BVDV)
survey on dairy cattle farms.
Herd management
Proportion of purchased cattle
Summer pasturing (Yes/No)
Grazing beef cattle or other species (sheep, goats, pigs, horses) (Yes/No)
Feeding and biosecurity measures
Disinfection of vehicle (Yes/No)
Use of footbath (Yes/No)
Somatic cell count in milk
Conception rate in cattle
Occurrence of abortion and stillbirth
Control of contact with wild animals
Geographical features
Distance from the nearest dairy farm (km)
Distance from the nearest beef farm (km)
Distance from the nearest sheep or goat farm (km)
Infection status of BVDV on the nearest dairy farm
189
Research in Veterinary Science 129 (2020) 187–192
pasturing, mostly via artificial insemination. Because many cattle are
gathered on the grazing farm, summer pasturing is considered to be a
potential risk factor for BVDV infection. Movement of cattle, such as
purchasing and summer pasturing, were obtained from the National
Database on Individual Cattle Identification System.
Feeding and biosecurity measure factors included disinfection of
vehicles, use of footbaths, the somatic cell count in milk, the conception
rate of the cattle, occurrence of abortion and stillbirth, and control of
contact with wild animals.
In terms of geographical features, geographical information about
the farms was obtained using the GIS system developed by the pre-
fectural government, and the distances from the nearest dairy farm,
beef farm, and sheep or goat farm, as well as the infection status of
BVDV on the nearest dairy farm, were examined.
Additionally, detailed information about PI cattle, including birth
date and place, age at diagnosis of PI, and the place where their dam
was impregnated (mostly by artificial insemination), were obtained.
2.4. Statistical analysis
To assess the risk factors associated with the within-farm trans-
mission of BVDV, BVDV-circulated-farms and BVDV-non-circulated-
farms were compared. Likewise, PI-farms and BVDV-antibody-free
farms were compared to assess the risk factors associated with the oc-
currence of PI animals. Initially, a univariable analysis was performed
using the chi-square or Fisher's exact test for categorical data and the
Mann–Whitney U test for numerical data. Only variables with a p-
value < .1 and with a pairwise correlation coefficient < 0.5 were
entered into the multivariable logistic regression models. The final
models were constructed using a manual and backward and forward
variable selection approach. The goodness-of-fit of the estimated
models was compared using Akaike's information criterion (AIC). The
models with the smallest AIC were deemed to be the best-fitting model.
All statistical analyses were conducted using R version 3.5.2 (R Core
Team, 2018).
3. Results
3.1. Identification and epidemiological features of PI cattle
Forty-four PI cattle were identified on 22 of 377 farms (5.8%).
According to the results of the RFLP analysis, 43 cattle were infected
with BVDV-1, and there was a single case of infection with BVDV-2.
These PI cattle were categorized by month of age: 20 animals (45.5%)
were calves < 6-months old, 7 (15.9%) were calves aged 6–12 months,
8 (18.2%) were heifers (12–24-months old) and 9 (20.4%) were milking
cows (> 24-months old). In the follow-up test on the PI detected farms,
10 newborn calves on 7 farms were diagnosed as being PI animals.
Fig. 1 shows the number of PI cattle alive, including 34 cattle identified
by the survey and 10 cattle identified in the follow-up test, during the
period between January 2011 and March 2018. At least 13 PI cattle
were present in the prefecture at the start of the survey (April 2014),
and a maximum of 19 PI cattle were alive during the study period. One
or more PI cattle were raised for seven years until the end of the period.
Table 2 shows the place of birth of PI cattle and their dam. Six PI cattle
(13.6%) were introduced from other farms, and 38 PI cattle (86.4%)
were born on their current farms. In terms of the dams of PI cattle, 4
(9.1%) and 7 (16.9%) were introduced from farms within or outside the
prefecture, respectively; and 33 (75.0%) were born on their current
farms. In terms of the location of impregnation of the dams of PI cattle,
2 (4.6%) and 13 (29.5%) were impregnated on farms within or outside
the prefecture, respectively. Among these, 7 were impregnated during
summer pasturing. Twenty-nine dams of PI cattle (65.9%) were im-
pregnated on their current farms.
M. Akagami, et al.
Fig. 1. Number of persistently infected (PI) cattle alive between January 2011
and March 2018.
3.2. Sero-prevalence of BVDV
Of 333 farms holding homebred cattle without movement records,
antibody-positive cattle were observed on 194 farms (58.3%), and these
farms were categorized as BVDV-circulated-farms. The remaining 139
farms (41.7%) were antibody-negative, except for some sampled cattle
that had movement records or vaccinated record, and were categorized
as BVDV-non-circulated farms. Among these, on 75 farms (22.5%),
BVDV antibody was not detected in any of the sampled cattle, and these
were categorized as BVDV antibody-free farms.
Individual level seroprevalence was 29.8% (95% CI: 28.8–30.8%)
(Table 3). The within-farm seroprevalence was significantly higher on
PI-farms (78.6%, 95% CI: 75.7–81.5%) than on farms in other cate-
gories (p < .001). In BVDV-non-circulated farms, the within-farm sero-
prevalence was significantly lower (9.0%, 95% CI: 8.0–10.0%)
(p < .001).
3.3. Factors associated with within-farm transmission of BVDV and PI
occurrence
In the analysis of within-farm transmission of BVDV, three varia-
bles—herd size, summer pasturing, and BVDV-infection status of the
nearest dairy farm—showed a positive relationship with the within-
farm transmission in univariable analysis (p-value < .1) and remained
in the final multivariable analysis model (Table 4). The results showed
that cattle on large-sized farms had a 2.09 times higher risk of being
serologically positive to BVDV via within-farm transmission (odds ratio
(OR) 2.09, p = .002). Farms that used summer pasturing also had an
increased risk of within-farm transmission of BVDV, which was 2.22
times greater compared to those that did not (p = .004). When the
nearest dairy farm was infected with BVDV, the risk of within-farm
transmission increased by 2.07 times (p = .003). According to the
depth-interviews conducted with the farmers, there was some co-
operation between farmers in the same neighborhood, with regards to
Table 2
Number of persistently infected (PI) cattle by their birthplace, their dam's birthplace, and the place of pregnancy.
Current farm
Other farms or grazing places outside of Ibaraki Prefecture
Other farms within Ibaraki Prefecture
Research in Veterinary Science 129 (2020) 187–192
looking after their cattle and sharing equipment. For example, they
reported helping each other with calving difficulties and the dehorning
of calves.
Moreover, the following five variables showed a relationship with
the occurrence of PI cattle, according to univariable analysis: herd size,
proportion of purchased cattle, summer pasturing, the BVDV-infection
status of the nearest dairy farm, and the distance to the nearest dairy
farm with BVDV infection. Multivariable analysis showed that, similar
to the results of within-farm transmission analysis, large-sized herds
(OR 8.42, p = .006) and using summer pasturing (OR 5.80, p = .019)
significantly increased the risk of the occurrence of PI cattle (Table 4).
Increasing the proportion of purchased cattle was also associated with
the occurrence of PI cattle (OR 1.06, p = .001).
4. Discussion
This study demonstrated the distribution of BVDV and epidemio-
logical features of PI cattle by surveying dairy cattle in the Ibaraki
Prefecture of Japan. Furthermore, risk factors associated with the
within-farm transmission of BVDV and the occurrence of PI cattle were
revealed by analysis of features of herd management, biosecurity
measures, and geography.
The survey showed that BVDV was circulating on 194 farms
(58.3%). On these farms, homebred cattle without movement records
appeared to contract infection from the infected cattle residing on the
farm. Among these BVDV-circulated farms, PI cattle were identified on
22 farms (5.8%). The incidence rate of PI cattle in this region was al-
most similar to that on country-level (7.6%, 95% CI:3.1%–16.4%)
(Kameyama et al., 2016). During the period of 2010–2019, one or more
PI cattle (maximum, 19 cattle) were raised on the farms in this region.
This result indicates that PI cattle are constantly present in this region
and that cattle on dairy farms are consistently at risk of exposure to PI
cattle and BVDV infection.
Because of the high level of virus shedding by PI cattle, once PI
cattle have been introduced to a farm, a cycle is initiated in which new
PI cattle are likely to be born within the farm, resulting in the continued
spread of BVDV on the farm (Lindberg, 2003; Yasutomi et al., 2004).
The results of our survey demonstrated that the within-farm ser-
oprevalence on PI farms (78.6%, 95% CI:75.7–81.5%) was significantly
higher than that on farms in other categories, suggesting that PI cattle
was a significant source of BVDV transmission. In the epidemiological
investigation of 44 PI cattle, 38 PI animals (86.4%) and 33 of their dams
(75.0%) were born at their current farm. This result suggests that PI
newborn calves were constantly being produced on these farms. Thus,
identifying PI cattle as early as possible and removing them from the
herd is important for the prevention and control of BVDV transmission
within a farm.
Multivariable analysis identified that a large herd size and a high
proportion of purchased cattle are significantly associated with the
occurrence of PI animals. Several previous studies have demonstrated a
significant association between herd size and the risk of BVDV infection
(Amelung et al., 2018; Bishop et al., 2010; Graham et al., 2016; Presi
et al., 2011). Additionally, the risk of introduction of disease posed by
purchasing animals is well-recognized (Amelung et al., 2018; Graham
et al., 2016; Kaiser et al., 2017). In Japan, dairy farms are growing in
farm size, and numerous pregnant cattle are introduced from Hokkaido,
the largest dairy farming area in the Japan. Our results suggest that
Birthplace of PI cattle Dam's birthplace Place of pregnancy
38 (86.4%) 33 (75%) 29 (65.9%)
4 (9.1%) 7 (15.9%) 13 (29.6%)
2 (4.5%) 4 (9.1%) 2 (4.5%)
190
M. Akagami, et al. Research in Veterinary Science 129 (2020) 187–192

Table 3
Individual level seroprevalence of bovine diarrhea virus (BVDV).
Farm type No. of farms No. of inspected animals No. of positive animals Prevalence (95% CI)⁎⁎

All farms 333 7969 2378 29.8% (28.8–30.8)a

BVDV-circulated-farm⁎ 172 4092 1479 36.1% (34.7–37.6)a
PI farm 22 790 621 78.6% (75.7–81.5)b
BVDV-non-circulated farm 139 3087 278 9.0% (8.0–10.0)c

95% CI, 95% confidence interval.

⁎
Persistent infection (PI) farms were excluded.
⁎⁎
Values sharing a letter are not statistically different.

there is a growing risk for the occurrence of PI animals due to the
movement of cattle accompanying the upsizing of dairy farms in the
Ibaraki Prefecture (Kadohira et al., 2006; Yasutomi et al., 2004).
Summer pasturing was found to be associated with both ser-
oconversion to BVDV in cattle without movement records via within-
farm transmission and the occurrence of PI animals in this study.
Summer pasturing was also thought to play an important role in
maintaining and transmitting BVDV in Switzerland (Presi et al., 2011).
During summer pasturing, many cattle from different farms are kept
together, with multiple opportunities for contact. Additionally, most
heifers return to their original farms after breeding on the grazing
farms. Thus, if infected or PI cattle were kept together on the pasturing
farm, transmission of BVDV is highly likely to occur, and cattle preg-
nant with PI calves return to their home farms. This result strongly
suggests that summer pasturing poses a high risk of infection with
BVDV and pregnancy with PI calves, causing within-farm transmission
and generation of PI cattle after returning to the original farms.
In terms of risk factors associated with within-farm BVDV trans-
mission, BVDV-infection of the nearest dairy farm was significantly
associated with within-farm infection, as was herd size and summer
pasturing. In a previous study, the risks posed by the presence of BVDV-
infected cattle on neighboring farms were identified (Graham et al.,
2016). Thus, it is possible that BVDV-infection on the neighboring
farms is a potential risk for introduction of the disease, although the
specific transmission pathways were not clarified. According to inter-
views conducted with the farmers in this study, some farmers in the
neighborhood cooperated in looking after their cattle and in sharing
equipment. Thus, these types of indirect transmission via movement of
persons or equipment might cause transmission of the disease among
neighboring farms.
However, BVDV-infection on the nearest dairy farm was not sig-
nificantly associated with the occurrence of PI animals. Because the
temporal window for PI of calves is limited (between 20 and 120 days
of gestation (Baker, 1987; Done et al., 1980), even if BVDV transmission
occurs between neighboring farms, it does not seem to relate clearly to
the occurrence of PI animals.
Clinical signs of BVDV infection, such as abortion and still birth,
were not significantly associated with the transmission of BVDV in-
fection within farms in this study. This result suggests that detection of
circulating BVDV within farms based on clinical signs alone would be
difficult (Sarrazin et al., 2013). Furthermore, although a risk of infec-
tion by other animals, such as beef cattle, sheep, and goats, were not
identified in our study, these factors should not be disregarded as a
potential source of the virus (Braun et al., 2014).
In this study, the risk of within-farm transmission following the
introduction of BVDV via the movement of cattle during purchase and
summer pasturing was revealed, and the significant role of PI cattle in
circulating BVDV within dairy farms were recognized. In order to
prevent the introduction and spread of BVDV by PI cattle, it is im-
portant to conduct inspections of cattle that are to be introduced to a
farm or are to be sent for summer pasturing. In the case of summer
pasturing in Hokkaido Prefecture, the cattle are required to undergo
voluntary inspection before they are sent to the grazing farms. Thus, the
risk of generating PI cattle during summer pasturing is likely to be re-
duced. On the other hand, if BVDV is maintained within a farm due to
the presence of an infection source, such as PI cattle on the farm itself,
inspection targeting only the cattle to be introduced will not be suffi-
cient to suppress the occurrence of PI animals. In European countries, to

Table 4
Results of multivariable logistic regression models for the within-farm transmission of bovine diarrhea virus (BVDV) and the occurrence of persistently infected (PI)
cattle.
Variable Levels No. of farms No. of positive (% positive)a Odds ratio 95% CIb p-value

Within-farm transmission of BVDV

Farm size Small (< 30 adult cattle) 167 81 (48.2%) 1 – –
Large(≥30 adult cattle) 165 113 (68.5%) 2.09 1.32, 3.33 0.002
Summer pasturing No 241 127 (52.7%) 1 – –
Yes 92 67 (72.8%) 2.22 1.30, 3.87 0.004
Infection status of BVDV on the nearest dairy farm Not infected 105 49 (46.7%) 1 – –
Infected 228 145 (63.6%) 2.07 1.28, 3.39 0.003

Occurrence of PI cattle
Farm size Small (< 30 adult cattle) 53 4 (7.5) 1 – –
Large (≥30 adult cattle) 44 18 (40.9) 8.42 1.03, 1.10 0.006
Percentage of purchased cattle (numerical variable)c – – 1.06 1.02, 1.10 0.001
Summer pasturing No 80 13 (16.3%) 1 – –
Yes 17 9 (52.9%) 5.8 1.37, 27.11 0.019
Distance from the nearest dairy farm (km) (numerical variable)d – – 0.73 0.45, 1.02 0.12
Infection status of BVDV on the nearest dairy farm Not infected 34 5 (14.7%) 1 – –
Infected 63 17 (27.0%) 3.24 0.78, 16.75 0.127

a
Positive means “seropositive farm” in the model of within-farm transmission BVDV, and “PI farm” in the model of occurrence of PI cattle.
b
95% CI, 95% confidence interval.
c
Percentages of purchased cattle in PI farm and non-PI farm (median, 2.5th–97.5th percentile) were 21.7% (0–98.0%) and 0% (0–47.3%), respectively.
d
Distances from the nearest dairy farm in PI farm and non-PI farm (median, 2.5th–97.5th percentile) were 1.5 km (0.1–11.0 km) and 0.9 km (0.09–9.1 km),
respectively.

191
M. Akagami, et al.
detect PI cattle, a surveillance program for calves, using tissue samples
(ear notch), has been implemented (Amelung et al., 2018; Houe et al.,
2006). However, this surveillance method is not applied in Japan, al-
though the practical use of bulk milk screening for BVDV on dairy farms
has been investigated (Kozasa et al., 2005; Saino et al., 2013). More
effective measures for screening BVDV infection and PI cattle, such as
intensive tests targeting movement cattle and newborn calves, or bulk
milk surveillance, are required to prevent and control the spread of
BVDV in Japan.
Declaration of Competing Interest
The authors declare that there are no potential conflicts of interest
with respect to the research, authorship, and/or publication of this
article.
Acknowledgments
We thank all of the livestock owners for their cooperation in this
study. This work was supported by grant projects related to the pro-
motion of regional research and development by the Ministry of
Education, Culture, Sports, Science and Technology in Japan.
References
Abe, Y., Tamura, T., Torii, S., Wakamori, S., Nagai, M., Mitsuhashi, K., Mine, J., Fujimoto,
Y., Nagashima, N., Yoshino, F., Sugita, Y., Nomura, T., Okamatsu, M., Kida, H.,
Sakoda, Y., 2016. Genetic and antigenic characterization of bovine viral diarrhea
viruses isolated from cattle in Hokkaido, Japan. J. Vet. Med. Sci. 78, 61–70.
Amelung, S., Hartmann, M., Haas, L., Kreienbrock, L., 2018. Factors associated with the
bovine viral diarrhoea (BVD) status in cattle herds in Northwest Germany. Vet.
Microbiol. 216, 212–217.
Baker, J.C., 1987. Bovine viral diarrhea virus: a review. J. Am. Vet. Med. Assoc. 190,
1449–1458.
Bauermann, F.V., Ridpath, J.F., 2015. HoBi-like viruses–the typical 'atypical bovine
pestivirus'. Anim. Health Res. Rev. 16, 64–69.
Bishop, H., Erkelens, J., Van, Winden S., 2010. Indications of a relationship between
buying-in policy and infectious diseases on dairy farms in Wales. Vet. Rec. 167,
644–647.
Blome, S., Beer, M., Wernike, K., 2017. New leaves in the growing tree of pestiviruses.
Adv. Virus Res. 99, 139–160.
Braun, U., Reichle, S.F., Reichert, C., Hassig, M., Stalder, H.P., Bachofen, C., Peterhans, E.,
2014. Sheep persistently infected with border disease readily transmit virus to calves
seronegative to BVD virus. Vet. Microbiol. 168, 98–104.
Cannon, R.M., Roe, R.T., 1982. Livestock Disease Surveys: A Field Manual for
Veterinarians. Australian Bureau of Animal Health, Canberra.
Done, J.T., Terlecki, S., Richardson, C., Harkness, J.W., Sands, J.J., Patterson, D.S.,
Sweasey, D., Shaw, I.G., Winkler, C.E., Duffell, S.J., 1980. Bovine virus diarrhoea-
mucosal disease virus: pathogenicity for the fetal calf following maternal infection.
Vet. Rec. 106, 473–479.
Evans, C.A., Pinior, B., Larska, M., Graham, D., Schweizer, M., Guidarini, C., Decaro, N.,
Ridpath, J., Gates, M.C., 2019. Global knowledge gaps in the prevention and control
of bovine viral diarrhoea (BVD) virus. Transbound. Emerg. Dis. 66, 640–652.
Gethmann, J., Homeier, T., Holsteg, M., Schirrmeier, H., Sasserath, M., Hoffmann, B.,
Beer, M., Conraths, F.J., 2015. BVD-2 outbreak leads to high losses in cattle farms in
Western Germany. Heliyon 1, e00019.
Gillespie, J.H., Baker, J.A., McEntee, K., 1960. A cytopathogenic strain of virus diarrhea
virus. Cornell Vet. 50, 73–79.
Graham, D.A., Clegg, T.A., Lynch, M., More, S.J., 2013. Herd-level factors associated with
the presence of bovine viral diarrhoea virus in herds participating in the voluntary
phase of the Irish national eradication programme. Prev. Vet. Med. 112, 99–108.
Graham, D.A., Clegg, T.A., Thulke, H.H., O'Sullivan, P., McGrath, G., More, S.J., 2016.
Quantifying the risk of spread of bovine viral diarrhoea virus (BVDV) between con-
tiguous herds in Ireland. Prev. Vet. Med. 126, 30–38.
Harpin, S., Elahi, S.M., Cornaglia, E., Yolken, R.H., Elazhary, Y., 1995. The 5′-un-
translated region sequence of a potential new genotype of bovine viral diarrhea virus.
Arch. Virol. 140, 1285–1290.
Houe, H., 1994. Bovine virus diarrhoea virus: detection of Danish dairy herds with per-
sistently infected animals by means of a screening test of ten young stock. Prev. Vet.
Med. 19, 241–248.
Houe, H., 1999. Epidemiological features and economical importance of bovine virus
diarrhoea virus (BVDV) infections. Vet. Microbiol. 64, 89–107.
Houe, H., 2003. Economic impact of BVDV infection in dairies. Biologicals 31, 137–143.
Houe, H., Lindberg, A., Moennig, V., 2006. Test strategies in bovine viral diarrhea virus
control and eradication campaigns in Europe. J. Vet. Diagn. Investig. 18, 427–436.
Humphry, R.W., Brulisauer, F., McKendrick, I.J., Nettleton, P.F., Gunn, G.J., 2012.
Prevalence of antibodies to bovine viral diarrhoea virus in bulk tank milk and
192
Research in Veterinary Science 129 (2020) 187–192
associated risk factors in Scottish dairy herds. Vet. Rec. 171, 445.
Isoda, N., Asano, A., Ichijo, M., Wakamori, S., Ohno, H., Sato, K., Okamoto, H., Nakao, S.,
Kato, H., Saito, K., Ito, N., Usui, A., Takayama, H., Sakoda, Y., 2017. Evaluation of
control measures for bovine viral diarrhea implemented in Nemuro District,
Hokkaido, Japan, using a scenario tree model. J. Vet. Med. Sci. 79, 1172–1181.
Isoda, N., Asano, A., Ichijo, M., Ohno, H., Sato, K., Okamoto, H., Nakao, S., Kato, H.,
Saito, K., Ito, N., Usui, A., Takayama, H., Sakoda, Y., 2019. Assessment of the cost
effectiveness of compulsory testing of introduced animals and bulk tank milk testing
for bovine viral diarrhea in Japan. J. Vet. Med. Sci. 81, 577–585.
Kadohira, M., Yasutomi, I., Markov, J., Tajima, M., 2006. Quantitative risk assessment of
bovine viral diarrhea virus infection in Yubetsu, Hokkaido, Japan. J. Vet. Epidemiol.
11, 32–39 (in Japanese).
Kaiser, V., Nebel, L., Schupbach-Regula, G., Zanoni, R.G., Schweizer, M., 2017. Influence
of border disease virus (BDV) on serological surveillance within the bovine virus
diarrhea (BVD) eradication program in Switzerland. BMC Vet. Res. 13, 21.
Kameyama, K., Konishi, M., Tsutsui, T., Yamamoto, T., 2016. Survey for detecting per-
sistently infected cattle with bovine viral diarrhea in Japan. J. Vet. Med. Sci. 78,
1329–1331.
Kozasa, T., Tajima, M., Yasutomi, I., Sano, K., Ohashi, K., Onuma, M., 2005. Relationship
of bovine viral diarrhea virus persistent infection to incidence of diseases on dairy
farms based on bulk tank milk test by RT-PCR. Vet. Microbiol. 106, 41–47.
Kozasa, T., Torii, S., Kameyama, K., Nagai, M., Isoda, N., Shiokawa, M., Aoki, H.,
Okamatsu, M., Sekiguchi, H., Saito, A., Sakoda, Y., 2018. Prevalence of HoBi-like
viruses in Japan between 2012 and 2017 based on virological methods and serology.
Jpn. J. Vet. Res. 66, 317–324.
Lindberg, A.L., 2003. Bovine viral diarrhoea virus infections and its control. A review.
Vet. Q. 25, 1–16.
Lindberg, A., Brownlie, J., Gunn, G.J., Houe, H., Moennig, V., Saatkamp, H.W., Sandvik,
T., Valle, P.S., 2006. The control of bovine viral diarrhoea virus in Europe: today and
in the future. Rev. Sci. Tech. 25, 961–979.
Masuda, T., Kuroda, M., Iwao, K., Ikemoto, C., Kodani, M., Masuda, Y., Kmeyama, K.,
Sakoda, Y., 2017. Epidemiological survey of bovine viral diarrhea virus isolated from
the offspring of cows moved to another prefecture and the eradication efforts in
Tottori prefecture. J. Jpn. Vet. Med. Assoc. 70, 575–579 (in Japanese).
Matsuno, K., Sakoda, Y., Kameyama, K., Tamai, K., Ito, A., Kida, H., 2007. Genetic and
pathobiological characterization of bovine viral diarrhea viruses recently isolated
from cattle in Japan. J. Vet. Med. Sci. 69, 515–520.
Ministry of Agriculture Forestry and Fisheries (MAFF), 2016. Guidelines for Prevention
and Control of Bovine Viral Diarrhea and Mucosal Disease. http://www.maff.go.jp/j/
syouan/douei/pdf/bvd_md_gl.pdf (in Japanese).
Ministry of Agriculture Forestry and Fisheries (MAFF), 2018. Statistical Survey on
Livestock. http://www.maff.go.jp/j/tokei/kouhyou/tikusan/index.html (in
Japanese).
Ministry of Agriculture Forestry and Fisheries (MAFF), 2019. Annual Statistics of
Domestic Animal Infectious Diseases. http://www.maff.go.jp/j/syouan/douei/kansi_
densen/kansi_densen.html (in Japanese).
Polak, M.P., Kuta, A., Rybaltowski, W., Rola, J., Larska, M., Zmudzinski, J.F., 2014. First
report of bovine viral diarrhoea virus-2 infection in cattle in Poland. Vet. J. 202,
643–645.
Presi, P., Struchen, R., Knight-Jones, T., Scholl, S., Heim, D., 2011. Bovine viral diarrhea
(BVD) eradication in Switzerland—experiences of the first two years. Prev. Vet. Med.
99, 112–121.
Ridpath, J.F., Fulton, R.W., Kirkland, P.D., Neill, J.D., 2010. Prevalence and antigenic
differences observed between bovine viral diarrhea virus subgenotypes isolated from
cattle in Australia and feedlots in the southwestern United States. J. Vet. Diagn.
Investig. 22, 184–191.
Saino, H., Kawauchi, K., Usui, A., Ohno, H., Sakoda, Y., Tajima, M., 2013. Implementation
and verification of the effectiveness of a regional control program for bovine viral
diarrhea virus infection in Hokkaido, Japan. J. Jpn. Vet. Med. Assoc. 66, 791–796 (in
Japanese).
Sarrazin, S., Veldhuis, A., Meroc, E., Vangeel, I., Laureyns, J., Dewulf, J., Caij, A.B.,
Piepers, S., Hooyberghs, J., Ribbens, S., Van Der Stede, Y., 2013. Serological and
virological BVDV prevalence and risk factor analysis for herds to be BVDV ser-
opositive in Belgian cattle herds. Prev. Vet. Med. 108, 28–37.
Seki, Y., Seimiya, Y.M., Motokawa, M., Yaegashi, G., Nagai, M., Hayashi, M., 2008.
Application of restriction fragment length polymorphism analysis to simple and rapid
genotyping of bovine viral diarrhea virus strains isolated in Japan. J. Vet. Med. Sci.
70, 393–395.
Simmonds, P., Becher, P., Collett, M.S., Gould, E.A., Heinz, F.X., Meyers, G., Monath, T.,
Pletnev, A., Rice, C.M., Stiasny, K., Thiel, H.-J., Weiner, A., Bukh, J., 2012. Family
flaviviridae. In: King, A.M.Q., Adams, M.J., Carstens, E.B., Lefkowitz, E.J. (Eds.),
Virus Taxonomy Ninth Report of the International Commitee on Taxonomy of
Viruses. Elsevier Academic Press, San Diego, pp. 1003–1020.
Strong, R., Graham, S.P., La Rocca, S.A., Raue, R., Vangeel, I., Steinbach, F., 2018.
Establishment of a bovine viral diarrhea virus type 2 intranasal challenge model for
assessing vaccine efficacy. Front. Vet. Sci. 5, 24.
Yamaguchi, O., Sakoda, Y., Sato, M., Nakamura, S., Fukusho, A., 1997. Gene detection
and discrimination of pestivirus strains by RT-PCR and restriction endonuclease
analysis. J. Jpn. Vet. Med. Assoc. 50, 639–644 (in Japanese).
Yasutomi, I., Okazawa, M., Hara, Y., 2004. Epidemiological investigation of bovine viral
diarrhea virus infection with evaluating risk factors. J. Vet. Epidemiol. 8, 77–83 (in
Japanese).
Yesilbag, K., Alpay, G., Becher, P., 2017. Variability and global distribution of sub-
genotypes of bovine viral diarrhea virus. Viruses 9, 128.