Leukemia Virus, Feline

Diagnostic testing undertaken included p27 testing with three different point-of-care (PoC) FeLV antigen kits, p27 testing with a laboratory-based non-proprietary ELISA (in use since before PoC FeLV testing became available), semi-quantitative real-time proviral PCR (qPCR) testing to detect FeLV proviral DNA in leukocytes, semi-quantitative qRT-PCR testing to detect FeLV RNA in plasma, and testing for anti-FeLV neutralising antibodies (NAb). The same panel of FeLV tests was performed for all cats, on the same blood samples collected at a single time point, as outlined below. Occasionally, insufficient sample volume precluded all tests from being performed.
Whole blood samples were collected into two EDTA tubes from each cat and transported on ice to the UoS within 6 hours of collection. One tube was centrifuged for 3 min at 12,000× g and harvested plasma was aliquoted into two plain tubes using a sterile pipette and stored at −80 °C. At the end of the sampling period, one aliquot of frozen plasma from each cat was transported on dry ice to the University of Zurich for qRT-PCR testing and laboratory-based p27 ELISA testing, while the other aliquot of frozen plasma was thawed and transported on ice to the University of Glasgow for NAb testing. PoC testing with three FeLV kits (used concurrently) was performed with whole blood samples at the UoS, within 24 h of blood collection using the second EDTA tube, and DNA extraction from leukocytes was performed using sample collected in the same EDTA tube within 72 h of collection and stored at −80 °C for batch qPCR testing at the UoS.
Results from these analyses enabled classification of cats as FeLV-uninfected or FeLV-infected, using the following definitions (Table 1). FeLV-uninfected cats included FeLV-unexposed cats (p27-negative, qPCR-negative, NAb-negative) and FeLV-abortive infections (p27-negative, qPCR-negative, NAb-positive). FeLV-infected cats included cats with a ‘presumptively regressive infection’ (p27-negative, qPCR-positive, NAb negative or positive) and cats with a ‘presumptively progressive infection’ (p27-positive, qPCR-positive, NAb negative or positive). FeLV-vaccinated cats uncommonly produce NAb following vaccination without exposure [26 (link),27 (link),28 (link)], and instead, FeLV vaccination is thought to ‘prime’ the immune system and boost the NAb response following exposure [7 (link)]. Consequently, FeLV-vaccinated/FeLV-uninfected cats that tested NAb-positive were considered FeLV-vaccinated cats that had come into contact with the virus and were classified as ‘presumptively abortive infections’ (versus FeLV-unvaccinated/FeLV-uninfected cats that tested NAb-positive and were considered as ‘definitively abortive infections’). ‘Presumptively’ regressive and progressive infections were described because, in the absence of repeated testing, a transient viraemia may have been present in some cats, resulting in some regressive infections being incorrectly classified as progressive infections [1 (link),2 (link),29 (link)]. Detection of vRNA by qRT-PCR testing was not included in the definition for progressive infections, as has been suggested elsewhere, since residual plasma was not available from all FeLV-infected cats for testing [17 (link),29 (link)].

First, the associations between the presence or absence of WW and serological FeLV or FIV infection were determined by using the chi-square test [25 (link)]. Next, multivariate analysis (logistic analysis) [26 (link)] was performed to test the association between “serological FeLV detection” and the above clinical examination items. The sensitivity, specificity, positive predictive value and likelihood ratio were calculated between FeLV serology and WW positivity according to a previous report [11 (link)].

To identify diseases present in cats with wavy whiskers (WW) on their faces, we recorded information from examinations of cats visiting Niihama Animal Hospital in Ehime Prefecture, Japan, during 2006–2013. A Significant wavy change was defined as more than two regions with bends in one whisker, and a cat possessing more than two whiskers with wavy changes was determined to be a WW-positive cat (Fig. 1A) with the agreement of 3 clinicians (MM, YM, and IH). To accumulate enough WW-positive cats, WW-negative cats were also randomly recorded. A total of 358 cats, including 56 with WW, were enrolled in the present study. Some of the available case information about clinical diagnosis, especially FeLV-related disease or not, was recorded.