An innovative tool for non-invasive contact-free pathogen monitoring in animal saliva

Reviewer #1 (Public review):

Summary:

This manuscript describes the development and validation of a low-cost device to identify viruses from saliva samples of animals non-invasively. This device was tested under laboratory conditions to assess whether viruses could be recovered in different environmental conditions and after different durations of time. The devices were then used to sample mice and cats in shelters to assess utility.

Strengths:

Sampling animals is cost-effective and highly labour-intensive, and this device has the potential to substantially improve surveillance. The device is relatively low-cost, and the authors demonstrate that the virus can be obtained from these filter papers after different durations of time and in different environmental conditions.

Weaknesses:

The authors do not discuss if different volumes were obtained from different animals (for example, due to different behaviours or attractiveness of the odour baits). Additionally, it appears the virus results were cross-validated using the serological status of the animals. While I am not an expert on FIV, there seems that there could be potential for different levels of viral shedding, and it would be more prudent to cross-validate against blood or another gold standard sample. Finally, the statistical analysis could be improved as there appear to be relatively few replicates and limited analysis conducted.

Reviewer #2 (Public review):

Summary:

The study introduces an innovative device designed to collect non-invasive saliva samples from animals using disposable cassettes with odor attractants and filter paper. The authors aimed to validate this tool for pathogen monitoring, specifically by detecting pathogen RNA in animal models. While the concept is compelling and the problem statement well-framed, the validation of the device for pathogen detection was not achieved. For example, the rabies virus was not detected in the chosen model, and results were limited primarily to FeLV. The work highlights the potential of saliva-based sampling for microbiota analysis, but the rationale for virus selection and the experimental design require further clarification. Overall, the study presents a novel approach with promise, though its current scope is better suited to microbiota monitoring rather than pathogen surveillance.

Strengths:

The innovative design of the device, which enables non-invasive saliva collection through disposable cassettes with odor attractants, represents a creative and practical advance in sampling methodology. The authors undertook an extensive experimental effort, generating a substantial amount of data that highlights the feasibility of saliva-based monitoring. The rationale for exploring saliva as a medium is valid, and the work successfully shows that the device can be applied to microbiota profiling, where the strongest results were obtained. This methodological innovation could be valuable for expanding non-invasive approaches to animal health monitoring.

The authors acknowledge that metabarcoding sequencing has limitations; however, the study could be refocused on the microbiota in general rather than on pathogen detection. They could give greater prominence to the taxonomic composition of microorganisms in saliva using high-throughput sequencing. That is where they obtained the most results.

Weaknesses:

Despite the enormous experimental effort undertaken, the results fall short of the expected success of the proposed test. The rationale and criteria for virus selection are not clearly explained, leaving the experimental design insufficiently justified.

The central aim of validating the device for pathogen detection was not achieved, particularly in the case of the rabies virus. The mouse infection model used for the rabies virus does not seem to adequately replicate the natural course of the disease. This could explain, at least in part, the negative results obtained.

Of the three viruses evaluated, satisfactory results were obtained only for FeLV, and the sample size remains limited. According to the literature reviewed, this virus is not common in wild cats, so the applicability of the results would appear to be limited primarily to domestic cats.

The collected samples were stored at −80 {degree sign}C for later analysis, which likely contributed to the high Ct values observed with the device. The need to store samples at low temperatures may be a limitation to applying this technique in wildlife sampling scenarios where access to dry ice or liquid nitrogen tanks may be difficult.

Stating that the device can be used for pathogen monitoring in wild animals is not desirable, since the viruses for which results were obtained are not relevant in wild animals. On the other hand, claiming that this is a tool for monitoring diseases in endangered species is also misleading. Endangered species are typically scarce and therefore would not be the reservoirs that these surveillance efforts should target. In fact, groups such as wild rodents would be a better target for monitoring zoonotic pathogens.