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Usutu virus (USUV) is an emerging flavivirus that is maintained in an enzootic cycle with mosquitoes as vectors and birds as amplifying hosts. In Europe, the virus has caused mass mortality of wild birds, mainly among Common Blackbird (Turdus merula) populations. While mosquitoes are the primary vectors for USUV, Common Blackbirds and other avian species are exposed to other arthropod ectoparasites, such as ticks. It is unknown, however, if ticks can maintain and transmit USUV. We addressed this question using in vitro and in vivo experiments and field collected data. USUV replicated in IRE/CTVM19 Ixodes ricinus tick cells and in injected ticks. Moreover, I. ricinus nymphs acquired the virus via artificial membrane blood-feeding and maintained the virus for at least 70 days. Transstadial transmission of USUV from nymphs to adults was confirmed in 4.9% of the ticks. USUV disseminated from the midgut to the haemocoel, and was transmitted via the saliva of the tick during artificial membrane blood-feeding. We further explored the role of ticks by monitoring USUV in questing ticks and in ticks feeding on wild birds in the Netherlands between 2016 and 2019. In total, 622 wild birds and the Ixodes ticks they carried were tested for USUV RNA. Of these birds, 48 (7.7%) carried USUV-positive ticks. The presence of negative-sense USUV RNA in ticks, as confirmed via small RNA-sequencing, showed active virus replication. In contrast, we did not detect USUV in 15,381 questing ticks collected in 2017 and 2019. We conclude that I. ricinus can be infected with USUV and can transstadially and horizontally transmit USUV. However, in comparison to mosquito-borne transmission, the role of I. ricinus ticks in the epidemiology of USUV is expected to be minor.

Feed efficiency is an important trait of dairy production. However, assessing feed efficiency is constrained by the associated cost and difficulty in measuring individual feed intake, especially on pastures. The objective of this study was to investigate short-term feed efficiency traits of herbage-fed dairy cows and screening of potential biomarkers (n = 238). Derived feed efficiency traits were ratio-based (i.e., feed conversion ratio (FCR) and N use efficiency (NUE)) or residual-based (i.e., residual feed intake (RFI), residual energy intake (REI), and residual N intake (RNI)). Thirty-eight Holstein and 16 Swiss Fleckvieh dairy cows underwent a 7-d measurement period during mid- and/or late-lactation. The experimental data (n = 100 measurement points) covered different lactational and herbage-fed system situations: mid-lactation grazing (n = 56), late-lactation grazing (n = 28), and late-lactation barn feeding (n = 16). During each measuring period, the individual herbage intake of each cow was estimated using the n-alkane marker technique. For each cow, biomarkers representing milk constituents (n = 109), animal characteristics (n = 13), behaviour, and activity (n = 46), breath emissions (n = 3), blood constituents (n = 35), surface, and rectal temperature (n = 29), hair cortisol (n = 1), and near-infrared (NIR) spectra of faeces and milk (n = 2) were obtained. The relationships between biomarkers and efficiency traits were statistically analysed with univariate linear regression and for NIR spectra using partial least squares regression with feed efficiency traits. The feed efficiency traits were interrelated with each other (r: −0.57 to −0.86 and 0.49–0.81). The biomarkers showed varying R2 values in explaining the variability of feed efficiency traits (FCR: 0.00–0.66, NUE: 0.00–0.74, RFI: 0.00–0.56, REI: 0.00–0.69, RNI: 0.00–0.89). Overall, the feed efficiency traits were best explained by NIR spectral characteristics of milk and faeces (R2: 0.25–0.89). Biomarkers show potential for predicting feed efficiency in herbage-fed dairy cows. NIR spectra data analysis of milk and faeces presents a promising method for estimating individual feed efficiency upon further validation of prediction models. Future applications will depend on the ability to improve the robustness of biomarkers to predict feed efficiency in a greater variety of environments (locations), managing conditions, feeding systems, production intensities, and other aspects.

Exposure to direct solar radiation, high ambient temperature, lack of wind movement, coupled with own metabolic heat production, makes grazing dairy cows vulnerable to heat stress. In pastures, it would be beneficial to monitor heat stress by observable changes in behaviour. We hypothesised that grazing dairy cows exhibit behavioural changes due to increasing heat load in temperate climate. Over two consecutive summers, 38 full-time grazing Holstein dairy cows were investigated in 12 experimental periods of up to 3 consecutive days where the cows were repeatedly exposed to various levels of moderate heat load determined by the comprehensive climate index (CCI). The CCI defines the ambient climate conditions, combining air temperature, relative humidity, solar radiation and wind speed. Vaginal temperature (VT) was automatically measured as an indicator of heat stress. In addition, as a less invasive method, we investigated if reticular temperature (RET) can be indicative of heat stress on pastures. Walking activity, lying-, feeding, and ruminating durations were recorded continuously with sensors. Respiration rate (RR), proximity to and competition at the water trough, social licking, self-licking, inter-individual distance, and fly intensity were directly observed. Data were analysed in the morning (0900–1100 h) and during the hottest time of day when cows were on pasture (1230–1430 h). The VT and RET showed similar patterns in relation to the CCI, suggesting that RET can be suitable for continuous monitoring of heat stress on pastures. In the morning, the cow’s VT and RET did not relevantly react to the CCI. During the period 1230–1430 h, the cow’s mean VT (mean vaginal temperature (VTMEAN); range: 37.7–40.3 °C) and mean RET (mean reticular temperature; range: 37.0–41.1 °C) were positively related to the mean CCI (mean comprehensive climate index) in this period (mean ± SD: 25.9 ± 5.71 °C). For cows with greater VTMEAN, an increased mean RR and decreased durations of walking, lying, feeding, and ruminating were found. These cows were also more likely to be in proximity to the water trough and to have small inter-individual distances. Changes in these traits seem to reflect behavioural adaptations to heat stress in a temperate climate and could be used to detect the heat stress in individual dairy cows on pastures.

Usutu virus (USUV) is an emerging flavivirus that is maintained in an enzootic cycle with mosquitoes as vectors and birds as amplifying hosts. In Europe, the virus has caused mass mortality of wild birds, mainly among Common Blackbird (Turdus merula) populations. While mosquitoes are the primary vectors for USUV, Common Blackbirds and other avian species are exposed to other arthropod ectoparasites, such as ticks. It is unknown, however, if ticks can maintain and transmit USUV. We addressed this question using in vitro and in vivo experiments and field collected data. USUV replicated in IRE/CTVM19 Ixodes ricinus tick cells and in injected ticks. Moreover, I. ricinus nymphs acquired the virus via artificial membrane blood-feeding and maintained the virus for at least 70 days. Transstadial transmission of USUV from nymphs to adults was confirmed in 4.9% of the ticks. USUV disseminated from the midgut to the haemocoel, and was transmitted via the saliva of the tick during artificial membrane blood-feeding. We further explored the role of ticks by monitoring USUV in questing ticks and in ticks feeding on wild birds in the Netherlands between 2016 and 2019. In total, 622 wild birds and the Ixodes ticks they carried were tested for USUV RNA. Of these birds, 48 (7.7%) carried USUV-positive ticks. The presence of negative-sense USUV RNA in ticks, as confirmed via small RNA-sequencing, showed active virus replication. In contrast, we did not detect USUV in 15,381 questing ticks collected in 2017 and 2019. We conclude that I. ricinus can be infected with USUV and can transstadially and horizontally transmit USUV. However, in comparison to mosquito-borne transmission, the role of I. ricinus ticks in the epidemiology of USUV is expected to be minor. Author summary: Usutu virus (USUV) is a virus primarily circulating between birds and mosquitoes. Other blood-feeding arthropods, such as ticks, feed on birds. However, it is unknown whether ticks can be infected with USUV and transmit the virus to birds or other animals. Therefore, we infected a tick cell line and ticks collected from the field with USUV. We showed that the virus replicates in tick cells, as well as in field-collected ticks. Moreover, we showed that USUV disseminated through the tick body and was excreted with the saliva of ticks during artificial feeding. We further investigated if ticks are infected with USUV while feeding on wild birds and when searching for a host in the vegetation. We found that 7.7% of tick-infested birds carried USUV positive ticks, while USUV was not detected in questing ticks. Our findings provide evidence that ticks can be infected with USUV and might transmit the virus to another host, but the role of ticks in the transmission of USUV is expected to be minor. [ABSTRACT FROM AUTHOR]

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