Lack of evidence for arthropod or vertical transmission of Hepacivirus bovis in a persistently infected dairy herd

Abstract

Hepacivirus bovis (BovHepV) has been detected globally since its initial description in 2015, but its transmission routes remain largely unknown. Arthropod-borne or vertical transmission is a common route for several livestock viruses, including Rift Valley fever virus and bluetongue virus. Moreover, many members of the genus Orthoflavivirus (family Flaviviridae) are transmitted by hematophagous arthropods, raising the possibility that hepaciviruses such as BovHepV, which also belongs to the Flaviviridae family, could share similar transmission routes. In this study, we examined possible transmission routes of BovHepV, including the involvement of mosquitoes, hematophagous flies, biting midges, and mites. Arthropods were collected on a dairy farm with documented circulation of BovHepV genotypes 1 and 2 and were subsequently tested for the presence of BovHepV RNA by quantitative real-time PCR. Furthermore, multiple bovine sample types were examined for the presence of BovHepV RNA. To assess local spread, serum samples from cattle on a neighboring farm within flight range of stable flies and mosquitoes were analyzed. Potential vertical transmission was evaluated by testing serum samples from 20 calves and 20 young heifers. None of the analyzed samples tested positive for BovHepV RNA. However, BovHepV was detected in blood samples collected for bovine herpesvirus 1 (BoHV-1) surveillance one month after the arthropod collection periods in both 2022 and 2023, confirming ongoing virus circulation on the farm. Under the conditions investigated, no evidence was found to support transmission via arthropods or vertical transmission. However, rare transmission events or low-prevalence vector involvement cannot be excluded and warrant further investigation.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1007/s00705-026-06747-6.

Introduction

Since the first discovery of a nonprimate hepacivirus in dogs in 2011 [35], the virus has been reported in six continents [4, 8, 12, 15, 19, 40, 55, 68], with a host spectrum ranging from various mammals [7, 23, 36, 50, 66] to ducks [13], sharks [60], and snakes [17]. Hepacivirus is closely related to hepatitis C virus (HCV) and belongs to the Flaviviridae family, which also includes the Orthoflavivirus, Pestivirus and Pegivirus genera [49]. The genus currently comprises 14 species that infect a variety of host organisms [https://ictv.global/taxonomy, accessed on 12 January 2026]. This includes Hepacivirus bovis (BovHepV), previously referred to as Hepacivirus N, that infects cattle and was shown to have a widespread distribution following its initial discovery in 2015 in both Ghana [15] and Germany [4]. The virus has been classified into two genotypes, of which genotype 1 has been further divided into eleven subtypes (A-K). Whereas it is mainly found in cattle, it has also been detected in wild boar, raising questions about its host range and possible zoonotic risk [2, 5, 17, 41]. Infected animals can become persistently viremic, but infections have not been associated with clinical symptoms or elevated liver enzyme levels [5]. Although BovHepV is hepatotropic, with high viral loads found in the liver and associated lymph nodes, and the detection of viral RNA by fluorescent in-situ hybridization in hepatocytes, a clear link between infection and acute liver disease has not been established [4, 5, 26].

The transmission routes for BovHepV remain unknown, although possible routes, including tick-borne transmission, sexual or vertical transmission and iatrogenic transmission by the reuse of needles during vaccination have been proposed [26, 69]. Experimental studies with the related Hepacivirus equi (EqHV) demonstrated successful infection of naïve horses by infusion of EqHV-positive plasma [48, 51]. Other members of the Flaviviridae family, such as Pegivirus and Pestivirus, can be transmitted via placental transfer or direct contact with infectious material [39, 45, 56, 61], but BovHepV was not detected in secretions or excretions other than blood from BovHepV-positive cows, suggesting, despite the limited sample size, that alternative transmission routes may be involved [31]. Several members of the Orthoflavivirus genus, which also belong to the Flavivirdae family, are transmitted by arthropods, including mosquitoes, e.g. West Nile virus (Orthoflavivirus nilense) and yellow fever virus (Orthoflavivirus flavi), and ticks, e.g. tick-borne encephalitis (Orthoflavivirus encephalitidis) and Kyasanur forest disease virus (Orthoflavivirus kyasanurense) [6], but arthropod transmission studies for BovHepV have not been reported to date.

In 2020, a dairy farm in Lower Saxony, Germany reported cases of severe respiratory illness in cattle, including pneumonia, bronchitis and/or rhinitis, as well as fever of unknown origin, decreased milk production and peracute deaths. Next Generation Sequencing (NGS) analysis resulted in the detection of BovHepV genotype 1 (subtypes 1 A and F) and genotype 2 in bovine blood samples collected as part of the annual bovine herpesvirus 1 (BoHV-1) surveillance program, but no association with the observed clinical symptoms could eventually be established. No other viral sequences were detected in these samples. Subsequent annual screening for BovHepV revealed an ongoing circulation of BovHepV on the farm, with new infections occurring in animals that previously tested negative, raising questions about the possible transmission route(s) within the herd [31].

To investigate potential modes of BovHepV transmission, arthropods from the affected farm and various biological samples from infected cattle were analyzed. Diagnostic samples of calves developing clinical symptoms during the study time were additionally screened for BovHepV to assess the possibility of vertical transmission. All collected samples were subsequently tested for the presence of H. bovis by quantitative real-time PCR (qRT-PCR). To our knowledge, this is the first study investigating potential routes of transmission for BovHepV.

Materials and methods

Farm structure and Hepacivirus bovis circulation history

The farm maintained approximately 500 cattle distributed across two sites (A and B), located about 2.5 km apart. At Site A, located next to the owner’s residence, approximately 250 lactating and dry cows, predominantly of the Holstein Friesian and Simmental breeds, were kept. After birth, calves were housed individually before being transferred either to a straw-bedded pen with an automatic calf feeder or to group igloos. Female calves aged six to nine months were routinely transferred to Site B, where they were serviced by a bull from the farm’s own herd at 15 to 18 months of age. Animals at Site B were repeatedly examined as part of the BoHV-1 monitoring program. Gestating heifers were subsequently transported back to Site A before calving and integrated into the existing herd after parturition. Heifers kept at Site B consistently tested negative for BovHepV. However, 67 of 162 previously negative animals tested positive for BovHepV in subsequent years following their return to Site A, suggesting the existence of an effective transmission route within the housing system at Site A. As previously reported, genetically identical viruses could be found in different animals, while identical virus strains could persist within the same host for months to years. In addition, reinfections with novel BovHepV variants were observed, including viruses belonging to different subtypes or representing genetically distinct strains within the same subtype [31].

Collection of clinical samples

Following the identification of H. bovis in the herd in October 2020, additional samples were collected in December 2020 from seven cows with the highest virus loads to enable further veterinary diagnostic analyses and to investigate possible transmission routes. This was prompted by the observation that infection was initially confined to animals kept at Site A of the farm, while several previously negative heifers from Site B tested positive after being transferred to Site A. Collected materials included nasal, ocular and buccal swabs, rectally collected faecal samples, pooled quarter milk samples, serum and EDTA blood samples. As the young stock presented with clinical signs including nasal discharge and fever, additional sampling was conducted in February 2021 to exclude BovHepV infection as a potential cause. Serum samples, and occasionally nasal swabs, were collected from 20 calves aged up to five months and 20 heifers aged between five and 24 months.

Furthermore, in 2023, a neighboring bull fattening farm located approx. 550 m from the affected dairy farm was also screened for BovHepV infections. With the farmer’s consent, blood samples collected as part of the annual BoHV-1 surveillance were forwarded by the Lower Saxony State Office for Consumer Protection and Food Safety (LAVES) Oldenburg to the Friedrich-Loeffler-Institute (FLI) for BovHepV analysis.

Collection of ectoparasites using UV light traps

From May to September 2022, three BG-Pro UV light traps (Biogents, Regensburg, Germany) were positioned both in- and outside the cattle stable near the animals. All traps were installed 1 m above ground level for one week at the end of each month, from 7 pm to 7 am. Traps were emptied each morning. Captured arthropods were cooled in a refrigerator to induce immobility, facilitating the subsequent transfer to 80% ethanol with minimal damage to the specimens. The insects were subsequently identified using standard taxonomic keys [21, 58]. The feeding status of haematophagous arthropods was determined by visual inspection of abdominal blood content using light microscopy and classified as blood-fed, unfed, indeterminate, or damaged. Pools of 25 biting midges from each group were put into separate Eppendorf tubes. For Stomoxys flies, the heads and abdomens of half of the specimens were separated and pooled into groups of five per tube. Mosquitoes and black flies (Simulium spp.) were individually stored in Eppendorf tubes. All arthropod specimens and tissues were preserved in 80% ethanol. Half of the samples were sent to the FLI for RNA extraction and BovHepV analysis. The remaining half were retained as backup samples at the Freie Universität Berlin (FUB). To verify whether BovHepV was still circulating in the herd at the time of arthropod sampling, blood samples that were collected as part of the mandatory annual BoHV-1 screening were forwarded by the LAVES in Oldenburg to the FLI in October 2023, for H. bovis testing using qRT-PCR with the farmer’s permission.

Collection of ectoparasites using aerial sweep nets

Flies were collected with an aerial sweep net from Monday to Friday during the last week of each month between May and September 2022, between 12 pm and 1 pm. Captured specimens were immobilized by cooling, transferred to sample containers filled with 80% ethanol, and subsequently identified using standard taxonomic methods. Half of the collected S. calcitrans flies were dissected, and their heads and abdomens were placed into separate Eppendorf tubes in pools of five. The remaining Stomoxys specimens were left intact and stored in Eppendorf tubes, also in pools of five. Horseflies were individually stored in Eppendorf tubes. All tubes were filled with 80% ethanol for preservation. Half of the samples were sent to the FLI for RNA extraction and H. bovis analysis using qRT-PCR. The remaining samples were retained as backup at the FUB.

Examination for lice and mite infestation

In September 2023, 25 animals were randomly selected and examined for ectoparasites due to an increasing incidence of pruritus and crust formation at the tail base within the herd. The forehead, neck and tail base of each animal were combed using a lice comb and inspected for the presence of ectoparasites, such as sucking and biting lice.

In addition, diagnostic skin scrapings were taken from the base of the tail. Half of the collected material was stored in Eppendorf tubes with 80% ethanol, while the other half was stored dry. The dry samples were examined under a stereomicroscope, and motile mites were transferred to Eppendorf tubes containing 80% ethanol. Half of these samples were sent to the FUB for species identification, while the other half was sent to the FLI for RNA extraction and BovHepV analysis.

A retrospective analysis revealed that seven of the 25 animals tested positive for H. bovis in October 2022. In October 2023, eight additional animals tested positive, all of which had been negative the previous year.

RNA extraction and RT-PCR analysis

Dry swabs from the nasal mucosa, conjunctiva and oral mucosa, as well as rectal stool samples, were incubated and shaken for 30 min at room temperature in 1 ml of ZB5d cell culture medium with penicillin/streptomycin and amphotericin [54]. Mixed quarter-milk samples, serum, and EDTA blood were used directly for nucleic acid extraction. Arthropod samples were homogenised in 600 µl of cell culture medium and a 5 mm steel ball in the TissueLyser II (Qiagen, Hilden, Germany) for 3 min at 30 Hz. Stomoxys, horseflies, biting midges, Simulium and mosquitoes were pooled for RNA extraction. All other vectors were extracted and analyzed individually. The viral RNA was extracted from 100 µl of the liquid phase using the NucleoMagVET kit (Macherey-Nagel, Düren, Germany) on the semi-automated KingFisher platform (KingFisher Flex Magnetic Particle Processor, Thermo Fisher Scientific) and eluted into 100 µl of elution buffer. To verify the success of the RNA extraction, an internal control RNA (IC-2 RNA) was added during the extraction process.

The existing BovHepV-1- and BovHepV-2-specific PCR assays were used to analyze the various sample matrices and bovine samples in general [31]. As it was anticipated that only small amounts of viral RNA would be detectable in the vectors under investigation, a highly sensitive panBovHepV RT-qPCR assay was developed for the general screening of BovHepV RNA. The new panBovHepV RT-qPCR utilizes the highly conserved 5´-UTR (untranslated region) of BovHepV. When selecting the primers and probe, BovHepV sequences of genotypes 1 and 2 were used, including those publicly available at the time in 2023 and those generated in-house. The following oligonucleotides were combined for the panBovHepV-Mix11 assay: BoHepV-172-F (5´-GRC CTC YTC GGT AGA TTT GG-3´), BoHepV-284-R (5´-CAG CCG CAC CCA CRG ATC-3`) and BoHepV-237-FAM (5´-FAM-TCC ACA CAG CCT GAT AGG GTC CT-BHQ1-3´). The selected primers amplify a 113 bp fragment from the 5´UTR of all BovHepV, including both genotype 1 and genotype 2 strains. The sensitivity and specificity of the panBovHepV-Mix11 assay were verified by silico analyses and confirmed by comparative analyses of known BovHepV-positive and -negative bovine samples (Table S1).

The composition of the RT-qPCR reactions was similar to that of the previously published BovHepV-1 and BovHepV-2-specific RT-qPCR assays [31]. Briefly, the RT-qPCR reactions contained 1.25 µl RNase-free water, 6.25 µl 2x RT-PCR buffer, 0.5 µl RT-PCR enzyme mix, 1 µl panBovHepV primer probe mix 11 (10 pmol/µl primer-for, 10 pmol/µl primer-rev, 2.5 pmol/µl FAM probe, in 0.1x Tris-EDTA (pH = 8.0)), 1 µl EGFP mix1-HEX (2.5 pmol/µl primer-for, 2.5 pmol/µl primer-rev, 2.5 pmol/µl HEX probe, in 0.1x Tris-EDTA (pH = 8.0)) and 2.5 µl of template RNA. All RT-qPCRs were performed on the CFX 96 real-time PCR cycler (Bio-Rad, Hercules, CA, USA) using AgPath ID™ One-Step RT-PCR reagents from Applied Biosystems™ (Waltham, USA). The temperature profile used was 10 min at 45 °C, 10 min at 95 °C, followed by 45 cycles of 15 s at 95 °C, 30 s at 56 °C, and 30 s at 72 °C. Fluorescence values (FAM, HEX) were recorded during the annealing step. Samples were considered positive if the quantification cycle value (Cq) was < 45.

Results

Screening of different sample types for BovHepV RNA

In December 2020, two months after the initial identification of H. bovis various samples were collected from seven cows with the highest virus loads (BovHep-1 cq values: 28.75 to 30.65; BovHep-2 cq values: 29.37 to 33.54) and subsequently analyzed via real-time RT-PCR to assess possible transmission routes. All nasal, ocular and buccal swabs, as well as milk and faecal samples, tested negative for BovHepV, whereas serum and EDTA samples collected from the same animals tested positive. Since serum samples contained higher viral loads in comparison to EDTA samples, serum was selected for subsequent virus detection from other animals shown in Table 1.

Table 1.

Comparison of viral load in serum and EDTA blood samples

Animal	Matrice	BovHep Mix 5 FAM	BovHep2 Mix 4 FAM
1	serum EDTA	32.79 no Cq	no Cq no Cq
2	serum EDTA	29.37 32.64	no Cq no Cq
3	serum EDTA	no Cq no Cq	31.07 32.66
4	serum EDTA	28.65 30.47	no Cq no Cq
5	serum EDTA	no Cq no Cq	33.51 35.48
6	serum EDTA	28.46 32.77	no Cq no Cq
7	serum EDTA	35.18 35.24	no Cq no Cq

Screening of serum samples from calves and young heifers

Twenty serum samples each from calves and young heifers all tested negative for H. bovis using RT-qPCR. Cultural analysis of the nasal swabs confirmed the presence of Bovine Respiratory Syncytial Virus (BRSV) and Pasteurella multocida.

Screening of serum samples from a neighboring farm

Seventy-five blood samples collected as part of the annual BoHV-1 surveillance collected from a neighboring fattening farm, located approx. 550 m from the BovHepV-positive farm all tested negative for the presence of H. bovis by qPCR.

Collection of arthropods

During the study period, UV-light traps primarily captured Culicoides biting midges (n = 9,428), followed by mosquitoes (n = 99), black flies (Simuliidae, n = 86) and stable flies (S. calcitrans, n = 14). Aerial sweep netting was successful for the collection of stable flies (n = 1,101), as well as a small number of biting midges (n = 6) and horseflies (Tabanidae, n = 3) (Table 2). An overview of the feeding status of the collected arthropod species is shown in Table 3. The majority of the mosquitoes were identified as culicine species; only four of the 99 specimens were identified as Anopheles spp. No sucking or biting lice were detected, but all 25 examined cows were found to be infested with Chorioptes mites. A detailed overview of the specimens collected using UV-light traps is shown in Table S2 and a detailed overview of species and their feeding status is shown in Table S3.

Table 2.

Overview of arthropods collected using UV-light traps or aerial sweep nets

	collected with UV-light traps				collected with aerial sweep nets
	Stomoxys	Mosquitoes	Biting midges	Simulium	Stomoxys	Horseflies	Biting midges
May 2022	1	3	504	-	10	-	3
June 2022	2	10	2,238	-	217	1	-
July 2022	3	59	3,605	1	303	2	1
August 2022	7	25	2,999	85	404	-	2
September 2022	1	2	82	-	167	-	-
Total number	14	99	9,428	86	1,101	3	6

Table 3.

Overview of species and feeding status of the collected arthropods

Month	Arthropod Species	Blood-fed	Indeterminate	Unfed	Damaged
May 2022	Stomoxys	-	11	-	-
	Biting midges	40	433	34	-
	Culicine mosquitoes	-	3	-	-
June 2022	Stomoxys	45	174	-	1
	Biting midges	76	1794	368	-
	Culicine mosquitoes	-	10	-	-
	Horseflies	-	1	-	-
July 2022	Stomoxys	76	230	-	-
	Biting midges	98	2532	976	-
	Culicine mosquitoes	-	55	-	-
	Anopheline mosquitoes	-	4	-	-
	Horseflies	-	2	-	-
	Simulium	-	1	-	-
August 2022	Stomoxys	84	327	-	-
	Biting midges	61	2618	322	-
	Culicine mosquitoes	-	25	-	-
	Simulium	-	85	-	-
September 2022	Stomoxys	36	132	-	-
	Biting midges	4	62	16	-
	Culicine mosquitoes	-	2	-	-

Screening of arthropod samples by RT-qPCR and concurrent detection of BovHepV in cattle

None of the analyzed arthropod samples (n = 727) tested positive for H. bovis by high-sensitive panBovHepV-Mix11. In contrast, analysis of the 246 blood samples collected during the mandatory BoHV-1 surveillance demonstrated ongoing circulation of BovHepV on the farm in 2023, with 83 animals testing positive for BovHepV-1 and nine animals testing positive for BovHepV-2. Compared with the previous two years of testing, an increase in infection prevalence was observed. In October 2022, 72 of the 245 animals tested positive for BovHepV 1 while seven out of 245 tested positive for BovHepV-2. In September 2021, 53 out of 264 animals were positive for virus BovHepV-1 and three out of 264 for virus BovHepV-2 [31]. Out of 246 cattle blood samples analyzed, 100 cattle were tested positive using the panBovHepV-Mix11. Additionally, 83 samples were positive in the BovHepV-1 Mix5, while 8 samples tested positive in the BovHepV-2 Mix4. In comparison, 10 samples were only positive in the panBovHepV Mix11, with all of them having a quantification cycle value (Cq) greater than 36. Notably, no sample was specifically positive for either BovHepV-1 or BovHepV-2 and negative for panBovHep-Mix11 PCR. Data shown in Table S1.

Discussion

Nearly ten years after the first description of H. bovis, its transmission routes remain unknown. Because hematophagous arthropods, including ticks, mosquitoes and biting flies transmit various members of the Flaviviridae family [11, 29], vector-borne transmission of BovHepV is considered plausible. Arthropod-borne spread may occur either through biological transmission, in which the virus replicates within a competent vector, or through mechanical transmission, in which infectious material is passively transferred between hosts, for instance via contaminated mouthparts.

Mosquito-borne viruses within the Orthoflavivirus genus, such as West Nile virus (WNV) [14] and Japanese encephalitis virus [47], illustrate the importance of biological vector competence within the Flaviviridae family. In addition, mechanical transmission of WNV by S. calcitrans has been demonstrated under experimental conditions [22], and the interrupted feeding behavior of stable flies may facilitate blood-borne transmission [57]. However, evidence for arthropod involvement in hepacivirus transmission remains scarce. Jogalong virus, a hepacivirus related to duck hepacivirus, was detected in a single Culex annulirostris mosquito in Australia, although this likely reflected residual virus from a prior blood meal rather than replication within the mosquito [67]. Similarly, H. equi RNA was not detected in any of the 5,338 mosquitoes, grouped into 566 pools, collected across Austria [3].

In the present study, none of the analyzed arthropod samples tested positive for BovHepV RNA and so these findings do not support a major role for mosquitoes, biting midges or stable flies in H. bovis transmission within the investigated herd. However, several limitations must be considered. First, the number of collected mosquitoes was relatively low, limiting the ability to detect rare infection events. Second, pooled sample analysis may reduce sensitivity when viral loads are low, particularly if only a single infected specimen is present in a pool [64]. Although no Minimum Infection Rate (MIR) could be calculated due to the absence of positive pools, the sample size nevertheless allows exclusion of high-prevalence vector infection under the study conditions. In our initial study, the prevalence of BovHepV-1 and BovHepV-2 was 24.6% (95% confidence interval [CI]: 19.2–30.6%) and 11.3% (95% CI: 7.5–16.1%), respectively [31]. Compared with other arthropod-borne infections such as bluetongue virus (BTV), for which within-herd seroprevalence has been reported to range from 96.2% to 100% (95% CI) [9], or Schmallenberg virus (SBV), with a within-herd seroprevalence of 86.3% (95% CI: 84.75–87.71) [42], the seroprevalence observed in our study was considerably lower. The prevalence of BTV, SBV, but also for WNV, and Rift Valley fever virus (RVFV) in their respective vectors varies considerably depending on environmental conditions, climatic factors, vector species composition, and geographic distribution. Reported prevalences for BTV range from 0.5% to 37.8% [10, 30, 33, 63], while SBV prevalence ranges from 0.25% to 18.6% [10, 25, 37, 52, 53, 70]. WNV prevalence in vectors has been reported to be between 0.07% and 9.2% [1], while RVFV prevalence in mosquitoes has been observed at levels up to 25% [24]. In contrast, none of the arthropod samples investigated in the present study tested positive for H. bovis. Given the high sensitivity of the applied pan-Hepacivirus-PCR, which would likely have detected low-prevalence infections like those reported for WNV or SBV, these findings strongly suggest the absence of viral RNA in the examined insects, indicating that they are unlikely to play a role as vectors. However, low-prevalence infections or sporadic mechanical transmission events cannot be completely ruled out. Importantly, PCR-based detection primarily assesses the presence of viral RNA and does not directly evaluate vector competence or short-term contamination of mouthparts immediately after feeding. Arthropod specimens were preserved in 80% ethanol and previous studies, including two that specifically used 80% ethanol and successfully detected viral RNA in insects [18, 70], have demonstrated reliable RT-PCR detection of arboviral RNA from hematophagous insects stored in ethanol concentrations ranging from 70% to 99.5% [34, 62, 65], indicating that the use of 80% ethanol in the present study is unlikely to have compromised viral RNA detectability.

Horseflies were collected only in small numbers, preventing meaningful assessment of their role. As recognized mechanical vectors of several livestock pathogens [20, 27, 28, 32], their potential involvement cannot be excluded and would require targeted investigation. Tick-borne transmission was considered unlikely in this specific herd because all animals were housed indoors without pasture access and no tick infestation was observed. Although H. bovis has previously been detected in Rhipicephalus microplus ticks collected from cattle in China [59, 69], this finding may reflect residual blood rather than replication within ticks.

In September 2023, the herd exhibited increasing signs of itching and crust formation at the base of the tail. Twenty-five animals were preselected based on clinical signs of mange and examined for the presence of ectoparasites. These animals were retrospectively matched with records of individuals previously identified as infected with BovHepV. Seven of twenty-five animals tested positive for H. bovis in October 2022. Eight more animals tested positive in October 2023 but had been negative the previous year. The identified Chorioptes mites tested negative for the BovHepV genome. Chorioptes mites acquire nutrients by superficially scraping the host’s skin with their chelicerae and absorbing tissue fluids, a feeding process that leads to irritation and pruritus in affected animals [46]. As these mites live on the skin and are non-burrowing [38] it seems unlikely that they are involved in the transmission of BovHepV, even if the number of samples was limited.

Collectively, these data provide no evidence that the investigated arthropods play a major role in BovHepV transmission within this herd. The additional finding that animals from a neighboring farm within the flight range of stable flies, mosquitoes and biting midges tested negative supports the absence of frequent vector-mediated spread under local conditions, although this observation alone does not exclude occasional transmission events.

Evidence for vertical transmission remains limited. While BovHepV RNA was previously detected in pooled calf sera [55], none of the calves or young heifers examined in the present study tested positive. These findings argue against frequent vertical transmission in this herd, but do not exclude rare events or transient perinatal infection. Longitudinal studies of well-characterized dam-calf pairs, ideally including sampling at birth before colostrum intake, would be required to conclusively address this question. This would be analogous to bovine viral diarrhea virus (BVDV), where fetal immune tolerance during the first trimester of gestation can lead to lifelong virus persistence [43].

Given the continued circulation of H. bovis within the herd and the occurrence of newly infected animals over multiple years, alternative transmission routes must be considered. The exclusive detection of viral RNA in blood, but not in other matrices, supports the hypothesis of blood-associated transmission. Analogous to the related HCV in humans, transmission through direct blood contact is biologically plausible [44]. Iatrogenic spread via reuse of needles during vaccination or intravenous treatments has been previously proposed [4, 26] and is consistent with the known mechanical transmission of other bovine viruses, such as BTV, through shared needles [16]. Husbandry procedures associated with potential blood exposure, such as hoof trimming, should also be evaluated as possible risk factors. Experimental studies in horses have demonstrated successful transmission of H. equi via plasma or serum transfusion [48, 51], further supporting blood-borne transmission as a biologically plausible pathway for BovHepV. Sexual transmission, for instance via contaminated semen or embryo transfer equipment, also warrants consideration [26]. However, this route appears unlikely in this case, given the negative findings in all calves and heifers.

In conclusion, our data provides no evidence supporting a major role for the investigated arthropods or frequent vertical transmission in H. bovis spread within this dairy herd. The findings instead favor blood-associated transmission as the most plausible mechanism under the conditions studied. Therefore, management practices should be considered when evaluating disease prevention strategies. Effective farm management of cattle herds is essential to minimize infection risks from blood contact, such as that caused by contaminated needles or syringes used during routine procedures. Nevertheless, rare vector-mediated events, low-prevalence infections, or additional transmission pathways cannot be definitively excluded and should be addressed in future experimental and epidemiological studies.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conceptualization, NH, CvH, KD, BH and AMN; methodology, NH, CvH, KD, BH and AMN; formal analysis, NH, BH, AMN; investigation, NH; resources, NH, CvH, KD, BH and AMN; data curation, AMN and BH; writing - original draft preparation, NH; writing - review and editing, NH, CvH, KD, BH and AMN; supervision, AMN and KD; project administration, NH, AMN and BH; funding acquisition, NH, CvH, BH, and AMN. All authors have read and agreed to the published version of the manuscript.

Funding

Open Access funding provided by Freie Universität Berlin.

Data availability

All data generated or analysed during this study are included in this published article and its supplementary information files.

Declarations

Ethics approval and consent to participate

Only surplus sample material from screening programs or samples collected as part of veterinary diagnostics were used for the study.

Consent for publication

Both farmers whose farms were included in this study provided consent for the publication of this work.

Competing interests

The authors declare no competing interests.