Bluetongue virus detected in farm water and slaughterhouse wastewater before official outbreak reports

A new preprint shows that bluetongue virus RNA can be recovered from cattle drinking water, straw bedding and slaughterhouse wastewater during active BTV circulation. In Catalonia, passive wastewater samplers detected BTV more frequently than conventional grab samples, while retrospective testing identified viral RNA before some officially notified outbreaks. The results suggest that environmental RT-qPCR could be used as an aggregated screening tool to identify low-level circulation before targeted animal testing.

Bluetongue surveillance is generally based on clinical suspicion, entomological surveillance and laboratory testing of individual animals by RT-qPCR or serology. These methods remain necessary for confirmation and serotype identification, but they require direct animal sampling and may have limited sensitivity at herd level when only a small proportion of animals are viraemic.

Researchers therefore assessed whether shared environmental matrices could provide a pooled molecular signal of BTV circulation during the 2024 bluetongue outbreak in Catalonia. The study analysed 63 environmental samples collected from two cattle farms, one sheep farm and two slaughterhouses.

BTV RNA recovered despite low apparent within-herd prevalence

At farm level, the investigators tested drinking water, straw bedding, manure and environmental surface swabs after infection had been confirmed in animals.

BTV RNA was detected in 4 of 4 drinking-water samples, 2 of 2 environmental swabs, 6 of 9 straw-bedding samples and 3 of 14 manure samples. Drinking water yielded significantly more positive results than manure.

This occurred even though the proportion of RT-qPCR-positive animals on the three farms was only 0.90%, 0.49% and 0.16%. The results therefore indicate that viral RNA shed by a small number of infected animals can accumulate in matrices shared by the herd and become detectable without testing each animal individually.

Drinking water produced the most consistent farm-level signal

Drinking water was positive in all four samples analysed. The authors suggest that BTV RNA may enter trough water through saliva, nasal secretions or other oral material from infected animals. This interpretation is supported by the limited detection of bovine and ovine polyomaviruses used as indicators of faecal and urinary contamination. The BTV signal in drinking water therefore appeared unlikely to result simply from manure contamination.

From an operational perspective, trough water is also easier to collect repeatedly than individual blood samples and could potentially be incorporated into herd-level screening programmes.

Passive wastewater samplers improved BTV detection

The slaughterhouse component compared two sampling strategies. During intensive sampling in October 2024, researchers analysed 24 wastewater samples. Overall, BTV RNA was detected in 9 of 24 wastewater samples. However, the detection rate differed markedly: 66.7% of passive samplers were positive vs 20% of grab samples were positive. Passive sampling therefore yielded a substantially higher detection frequency.

Unlike a grab sample, which represents wastewater conditions at a single point in time, a passive sampler remains exposed for several hours or days and accumulates viral material continuously. This likely improves the probability of detecting intermittent shedding or low concentrations of viral RNA.

Passive sampling detected BTV when grab samples remained negative

On October 8, 12.82% of animals slaughtered originated from municipalities with reported BTV cases. All three grab samples were negative. In contrast, all three passive samplers deployed over the corresponding period tested positive. A similar discrepancy was observed on another sampling day, when only one of three grab samples was positive while passive sampling again detected BTV. These observations suggest that wastewater surveillance based on isolated grab samples may underestimate BTV circulation when viral RNA enters the effluent intermittently.

Wastewater RNA preceded some official outbreak notifications

Retrospective testing of archived slaughterhouse samples identified BTV RNA before some municipalities supplying animals to the facilities had reported outbreaks.

BTV was detected in September at one slaughterhouse, in July and August at the second slaughterhouse. At several of these time points, no BTV outbreak had yet been officially notified in the municipalities of origin of slaughtered animals. The result is compatible with earlier, clinically unrecognised transmission, particularly in cattle, where infection may remain inapparent. However, the study cannot determine the exact interval between infection and environmental detection, and it did not quantify how long BTV RNA remains detectable in wastewater.

The findings therefore support the use of wastewater as an early epidemiological signal, but not as a precise dating tool for virus introduction.

BTV-8 identified on one cattle farm

The molecular assays targeted BTV-4 and BTV-8, the main serotypes circulating in the region during the study period. Serotype identification was successful only on one cattle farm, where BTV-8 was confirmed. Positive wastewater and other environmental samples could not generally be serotyped. The authors attribute this mainly to low RNA concentrations, degradation of viral material and possible PCR inhibition.

Environmental RT-qPCR may indicate that BTV is circulating, but individual animal samples may still be required for serotype determination and full outbreak characterisation.

Manure showed lower analytical usefulness

Manure was the least productive farm matrix, with only 3 positive samples among 14 tested. This lower yield may reflect several factors, including heterogeneous distribution of viral RNA, degradation in faecal material and the presence of PCR inhibitors. The contrast with drinking water suggests that matrix selection will be critical if environmental BTV surveillance is to be incorporated into routine programmes.

Slaughterhouses could function as regional sentinel sites

Slaughterhouse wastewater has a different surveillance value from farm-level environmental samples. A slaughterhouse receives animals from multiple farms and municipalities, meaning that wastewater can integrate biological material from a geographically dispersed livestock population. A positive result therefore cannot identify the infected herd directly. Instead, it can indicate that BTV-positive animals are entering the slaughter stream from the surrounding catchment area. Combined with animal-movement records, this could allow authorities to narrow the geographic origin of the signal and prioritise follow-up testing. This is particularly relevant during periods when BTV is spreading through cattle populations with few clinical signs.

Environmental RT-qPCR would complement, not replace, animal testing

The study does not propose environmental sampling as an alternative to diagnostic testing of animals. A positive drinking-water or wastewater sample cannot identify which animal is infected, determine viraemia, establish disease status or provide all information required for official outbreak confirmation.

Its role would be to provide pooled evidence of BTV circulation across a herd or slaughterhouse catchment area. Such a signal could then trigger more targeted blood sampling, epidemiological investigation or entomological surveillance. This distinction is especially important in low-prevalence situations, where individual testing of large numbers of animals may be inefficient.

A proof of concept for pooled BTV surveillance

The Catalonian data provide a proof of concept for detecting BTV RNA outside the animal host. At farm level, drinking water and straw bedding produced the highest detection rates. At slaughterhouse level, passive wastewater sampling clearly outperformed grab sampling. The retrospective detection of BTV RNA before some official notifications also suggests that environmental matrices may reveal transmission before clinical surveillance identifies affected holdings.