Seroprevalence of Seven Reproductive Diseases in Beef and Dairy Cows from Three Provinces in Indonesia

Bovine reproductive diseases are endemic in Indonesia, but comprehensive information about their infectious causes is not available. Therefore, our aim for this study was to detect several infectious agents that cause reproductive diseases in Indonesian beef and dairy cows. A total of 152 cow serum samples collected by Faculty of Veterinary Medicine of Brawijaya University and Veterinary Disease Investigation Centre as a part of the mandatory and regularly surveillance system from three provinces during 2019–2020 were used. The samples were then sent to Indonesian Research Centre for Veterinary Science (IRCVS) for further detection of seven reproductive diseases by enzyme-linked immunosorbent assay (ELISA). Seven reproductive diseases to be tested in parallel are neosporosis, chlamydiosis, brucellosis, Q fever, bovine viral diarrhea (BVD), infectious bovine rhinotracheitis (IBR), and BHV-4 infection. The dominant reproductive diseases in Indonesian cows were BVD (45.69%), chlamydiosis (31.58%), IBR (20.53%), neosporosis (11.84%), and BHV-4 infection (10.53%). The seroprevalence of IBR, BHV-4 infection, neosporosis, and brucellosis varied significantly (P < 0.05) between dairy and beef cattle. The most dominant reproductive diseases in aborted cows were chlamydiosis (45%), BVD (41%), and neosporosis (10%). The conclusion drawn from this study is that the dominant reproductive diseases in Indonesian cows are BVD, chlamydiosis, IBR, neosporosis, and BHV-4 infection. Chlamydiosis, BVD, and neosporosis are common among aborted cow. Chlamydiosis, neosporosis, and BHV-4 infection should be included in the national priority list in Indonesia. Control and preventive measures should be focused on high-risk areas and animals like stray cat and dog.


Introduction
Loss of pregnancy in either beef or dairy cows causes a lot of economic loss. In general, the causes of loss of pregnancy in cows can be caused by infectious or noninfectious factors. percentage of abortion cases in dairy cows in Ethiopia which ranges from 2.20 to 14.60% [6]. Abortion cases in dairy cows in Australia and New Zealand are only 7% and 6%, respectively [7]. However, the cases of abortion in Balinese and Madurese cattle in Indonesia were reported to be 0.77%, respectively [8].
Comprehensive information of reproductive diseases like abortion are endemic in Indonesia, but their infectious causes are not known. Generally, the prevalence data for bovine reproductive diseases in Indonesia are dominated by brucellosis prevalence studies. However, brucellosis alone is not always responsible for reproductive diseases. Some other infectious agents that can cause loss of pregnancy in cows are Neospora caninum [9][10][11], Chlamydophila abortus, Chlamydophila pecorum [11,12], Coxiella burnetii [11,12], bovine viral diarrhea (BVD) [11][12][13], bovine herpesvirus type 1 (BHV-1) [7,13], and bovine herpesvirus type 4 (BHV-4) [11,14]. erefore, studies to determine the infectious causes of reproductive diseases are very important in Indonesia. Hence, the objective of this study was to detect several infectious agents that cause reproductive diseases in Indonesian beef and dairy cows.

Study Design and Samples History.
A total of 152 cow serum samples collected by Faculty of Veterinary Medicine of Brawijaya University (FVMBU) and Veterinary Disease Investigation Centre (VDIC) as a part of the mandatory and regularly surveillance system from three provinces during 2019-2020 were used. All samples were previously tested by FVMUB and VDIC with negative results for brucellosis using RBT (rose bengal test) and CFT (complement fixation test) methods. e samples were then sent to Indonesian Research Centre for Veterinary Science (IRCVS) for further detection of seven reproductive diseases by enzyme-linked immunosorbent assay (ELISA). Seven reproductive diseases to be tested in parallel are neosporosis, chlamydiosis, brucellosis, Q fever, bovine viral diarrhea (BVD), infectious bovine rhinotracheitis (IBR), and BHV-4 infection.
One hundred and fifty-two samples of bovine serum consisted of 88 samples from dairy cows and 64 samples from beef cattle. All samples from dairy cow are female Friesian Holstein (FH) originating from East Java (72 samples), West Java (48 samples), and Lampung (32 samples

Regional Distribution of Reproductive
Diseases. e dominant reproductive diseases in East Java province were IBR, BVD, and chlamydiosis with a prevalence of more than 10% (Table 1 and Figure 1). e seroprevalence of IBR (78.38%) and BVD (56.76%) was the highest in Pasuruan, East Java. In Batu, East Java, the dominant reproductive diseases were BVD (48%) and chlamydiosis (28%).
e seroprevalence of chlamydiosis in Lampung Province (77.78%) was significantly (P < 0.01) higher compared to other locations (5.26%-28%). e BVD seroprevalence in Lampung Province (11.11%) was significantly (P < 0.05) lower from the BVD seroprevalence in Pasuruan (56.76%) and West Bandung (64%) only. e reproductive diseases most commonly detected in five cities were BVD and chlamydiosis (Figure 1), followed Veterinary Medicine International by neosporosis (detected in four cities) and brucellosis and Q fever (detected in three cities), respectively. As for IBR and BHV-4 infection, although their seroprevalence was quite high, they were limited to one location only, so they were considered as a specific problem for that region. IBR was only dominant in Pasuruan, and BHV-4 infection was only dominant in Bogor.

e Proportion of Reproductive Diseases in Seropositive
Cows. Among the 64 beef cattle, 57 cattle were declared as seropositive for one or more reproductive diseases. In total, there were 78 cases of four reproductive diseases that were detected in 57 seropositive cows. e proportion of four reproductive diseases from the 78 seropositive cases in beef cattle was dominated by IBR (42%), BVD (33%), and chlamydiosis (23%), as shown in Figure 3(a).
In contrast, 63 of 88 dairy cows were stated seropositive for one or more reproductive diseases. In total, there were  114 cases of seven reproductive diseases that were detected in 63 seropositive cows. e proportion of seven reproductive diseases from 114 seropositive cases in dairy cows was dominated by BVD (36%), chlamydiosis (20%), neosporosis (16%), and BHV-4 infection (16%), as shown in Figure 3 e results of this study found that 192 cases of seropositive reproductive diseases were distributed among 96 healthy cows with no history of abortion, but had been declared seropositive to one or more reproductive diseases. Proportionally, the five most dominant reproductive diseases in healthy cows were BVD (35%), chlamydiosis (22%), IBR (19%), BHV-4 infection (9%), and neosporosis (9%), as shown in Figure 4(a). e three most dominant reproductive diseases in cows with the history of abortion were chlamydiosis (44%), BVD (40%), and neosporosis (10%), as shown in Figure 4(b). Table 2 provides the seroprevalence of reproductive diseases in cows with and without the history of abortion. e seroprevalence for BVD, chlamydiosis, IBR, BHV-4 infection, and neosporosis was 44.88%, 27.34%, 24.41%, 11.72%, and 11.72%, respectively, in cow without any history of abortion. However, the seroprevalences for chlamydiosis, BVD, neosporosis, and BHV-4 infection were 54.17%, 50%, 12.5%, and 4.17%, respectively, in cows with the history of abortion (Table 2). e seroprevalences for BVD and chlamydiosis were significantly different (P < 0.05) compared to those for neosporosis and BHV-4 infection in cows that had aborted. None of the cows that had aborted were

Reproductive Diseases in Aborted Cows.
e most prevalent infectious diseases associated with abortion in Indonesia are chlamydiosis (54.17%), BVD (50%), and neosporosis (12.50%). One study reported IBR, BVD, and neosporosis as the main causes of loss of pregnancy in cattle in Brazil [17]. Other studies reported that neosporosis was proportionally the main cause of loss of pregnancy in cattle with a prevalence of above 15%, while BVD had a prevalence of below 5% [9,11], and even chlamydiosis was only 0.83% [11]. ese results are expected to change the old perspective and belief that the main cause of abortion in Indonesia is B. abortus. e evidence we observed was contradictory, that brucellosis was not found in abortive cows as also reported in California, USA [9]. erefore, chlamydiosis, BVD, and neosporosis require more intensive attention in reproductive disease surveillance in Indonesia.

BVD.
BVD is considered a reproductive disease with moderate to high prevalence across the world [19]. Subclinical infections by BVDV in pregnant cows can cause abortion 10-90 days postinfection [13]. BVD is also suspected to be transmitted through the vector Stomoxys calcitrans as its genetic material was discovered on flies that fed on persistently infected cattle [20]. is could lead to a wider distribution area. Another disadvantage of BVDV infection is the various congenital defects on the fetus, especially on the central nervous system [19]. erefore, BVD does not only cause abortions but also congenital defects in various organ systems. e BVD seroprevalence in Indonesia (44.88%) was the highest. In Indonesia, there are no BVD vaccination programs, so seropositive results in this study and other studies likely originated from virus exposure in the field. Higher BVD prevalence has also been reported in imported cows (63%) that enter Indonesia with no vaccination documents [21]. Its similar prevalence is as found in Belgium and Serbia. e average BVD prevalence in Belgium was reported to be 36.4%, while in Serbia, it was 10.64-74% [22,23]. Australia, which is an endemic area for BVD, also reports its prevalence of BVD as 13% in South Australia, 65% in Victoria, 35% in Tasmania, and even reaching 92% in the Northern Territory [24].
Based on the results of this study, it was also revealed that BVD seroprevalence in dairy cow and beef cattle in Indonesia was 47.22% and 41.82%, respectively. is result was slightly lower than the results of other studies in Indonesia in dairy cows which ranged from 56.25 to 77% [25,26]. In contrast, the seroprevalence in beef cattle in this study was higher than that of other studies in Indonesia, ranging from 9.26 to 28% [26,27]. Overall, there is evidence that BVD prevalence in dairy cow in Indonesia is higher than in beef cattle. e results of this study are contrary to reports in Belgium which stated that BVD prevalence in dairy cow is not different from that of beef cattle, namely, 29.5% and 30.6% [22].
e role of C. pecorum in abortus is still a matter of debate among researchers. Some researchers claim that C. pecorum does not cause abortus but has been known to cause other diseases such as encephalomyelitis, polyarthritis, and keratoconjunctivitis [7,28,29]. Others claim that C. pecorum can cause abortus, although it is not dominant [12,[30][31][32].
C. abortus has an affinity for placental tissue and thus plays a key role in causing abortion and reproductive disorders, whereas C. pecorum is more commonly found in the gastrointestinal tract [29]. is is in line with the study on dairy cows in western Germany which revealed that the sorting order of Chlamydophila species causes abortus, i.e., C. psittaci, C. abortus, and C. pecorum with prevalences of 55.80%, 35.80%, and 8.40%, respectively [33].
Based on the results of this study, it was revealed that the cumulative prevalence of chlamydiosis in cows in Indonesia was 27.34%. is result is slightly higher than a previous study (25%) [34]. Similar finding was also reported from Zimbabwe 32.3% [35]. Chlamydiosis seroprevalence in dairy cows in Indonesia is similar to those of Sweden and Italy, which are 28% and 24%, respectively [36,37]. In contrast, Jordan and Ireland have lower chlamydiosis prevalences in dairy cows at 19.90% and 6.04%, respectively [38,39]. However, Indonesia's seroprevalence is still lower than that of Taiwan, which reported a chlamydiosis seroprevalence in abortive cows of 71.4% [40].

4.5.
Neosporosis. Neosporosis seroprevalence in Indonesia has been reported to be 5.80% in Balinese cattle [41]. Another study in 2015 reported that the neosporosis seroprevalences in West Bandung and Bogor were 26.52% and 30%, respectively [42]. Our observation revealed 32% and 6 Veterinary Medicine International 27.27% seroprevalences in West Bandung and Bogor, respectively. is evidence indicates that during the period 2015-2020, neosporosis in those two cities tends to be constant.
is study was the first to report a significant difference in the neosporosis seroprevalence between beef and dairy cows in Indonesia, which was 0% and 20.45%, respectively. In Argentina, a significant difference in neosporosis seroprevalence (P < 0.01) between beef and dairy cows was reported at 7% and 20.30%, respectively [43]. In Spain, the neosporosis seroprevalence in dairy cows (35.90%) was significantly higher (P < 0.01) than in beef cattle (17.90%) [44]. e neosporosis seroprevalence in abortive cows in Belgium was also reported to be significantly different (P < 0.05) between beef (14%) and dairy cows (28.60%) [45].

BHV-4 Infection and Brucellosis.
is study is the first to report the BHV-4 infection prevalence in cattle in Indonesia. In this study, the BHV-4 infection prevalence in cattle was 11.72%, which was three times higher than that of brucellosis and Q fever. e proportion of BHV-4 infection in aborted cows is 3% (Figure 4) with seroprevalence 4.17% (Table 2). e data are still lower than the USA and Turkey's, which were reported to be 16% and 28.78%, respectively [46,47]. Turkey reported in detail the BHV-4 infection seroprevalence in repeat-breeder dairy cows and cows with and without reproductive disorders, namely, 53.70% [48], 57.20%, and 44.90%, respectively [49]. e BHV-4 infection data in Indonesia are thought to be lower than the actual value due to the limited sample numbers and sampling area. erefore, it is advisable to increase the sample numbers and expand the sampling location to obtain more conclusive information.

Conclusion
e dominant reproductive diseases in Indonesian cows are BVD, chlamydiosis, IBR, neosporosis, and BHV-4 infection. Significant variations of the prevalence of the diseases exist among beef and dairy cows and regions. Chlamydiosis, BVD, and neosporosis are common among aborted cows in Indonesia with higher prevalence. Chlamydiosis, neosporosis, and BHV-4 infection are reproductive diseases that are not yet categorized as national priorities in Indonesia. We recommend including these three diseases into the list of strategic infectious diseases as a national priority. Prevention and control decisions like vaccination should be focused in regions with high risk.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.