Love for puppies is universal. When a young unvaccinated puppy arrives at the clinic and, instead of squirming and showering the attending vet with smelly puppy breath kisses, it lies listlessly on the examination table, all powers of quick and accurate diagnostics must be summoned. Dietary indiscretions, poisoning, and parasitic, bacterial, and viral agents can all cause signs of gastritis in previously healthy puppies. However, when an unvaccinated puppy is involved, thoughts immediately turn to the very contagious virus that can transform previously happy-go-lucky healthy puppies into critically or fatally ill animals in the space of days.
Parvovirus is an extremely hardy virus found almost everywhere, spread through direct or indirect ingestion of infected fecal matter. Preliminary diagnosis is made based on vaccination history, signalment, clinical signs and leukopenia on the complete blood count (CBC) test. In-clinic, standard rapid point-of-care (POC) tests, such as antibody or antigen detection, are used to confirm infection. While not perfect, POC antigen fecal lateral flow tests are quite specific but sensitivity varies between manufactures. This essentially means, “If it’s positive, you can be confident you’re dealing with a Parvoviral infection, but if it’s negative, the results may be incorrect”. Patients with parvovirus infections should be hospitalized in an isolation ward, and all staff who come in contact with these canines must wear personal protective equipment to prevent the virus from spreading to others. With such high stakes, additional diagnostic testing should be performed, especially if the results do not fit the clinical picture. The question is, “which tests can provide the most accurate results in the shortest amount of time?”
A recent article published in the Journal of Veterinary Internal Medicine by Segev et al., suggests the use of a novel sampling method in polymerase chain reaction (PCR) testing of suspected Parvoviral infections. In addition, the presence of Parvovirus-specific IgM in blood samples from sick puppies was discovered to be a good predictor of early infection in this study.
Because recent vaccination can interfere with accurate PCR analysis, Segev et al. set out to determine if it is possible to increase the sensitivity of the test by distinguishing between passive vaccinal shedding and the true state of disease. By examining the progress of the virus following initial infection, they decided to test pharyngeal and blood, as well as fecal samples for the presence of Parvovirus.
Four different groups of canines were included in the study; (i) confirmed cases of Parvo, (ii) healthy puppies prior to and during the stages of core vaccination (iii) adult dogs receiving core vaccine boosters and (iv) puppies presented with signs of gastritis not associated with Parvovirus but diagnosed with canine distemper. DNA was extracted from the clinical samples at various time points and analyzed with laboratory designed RealTime PCR (RT PCR). With the understanding that the average veterinarian clinic would have to outsource the samples for this procedure, they also tested the samples using the in-clinic POC PCR test kit (PCRun Parvovirus Molecular Detection Kit, Biogal Galed Labs Acs, Israel). Through repeated testing and careful calibration, they were able to develop a short protocol of less than 25 minutes that eliminated almost all of the false positives caused by recent puppy vaccination.
When the results from all of the sample types collected from Parvovirus diagnosed puppies were compiled, a 98% agreement was calculated between the RT PCR and the POC PCRun. Surprisingly more blood and pharyngeal samples were found to be positive for parvovirus than fecal samples (95%, 98% and 88% respectively). These findings suggest that PCR and antigen testing of fecal samples should be reassessed and reconsidered when used to diagnose Parvovirus infections.
All of the adult dogs in the booster group, as well as the distemper patients, tested PCR and PCRun negative for the presence of Parvovirus in all of the samples types. In contrast, PCR and PCRun analysis of blood, pharyngeal and fecal samples examined in the puppy vaccination group demonstrated decreasing levels of Parvovirus shedding up to day 20 post first vaccination. Twelve and 28 days following the second vaccination all the samples tested negative except for one dog that remained positive on the fecal sample. The quantitative findings revealed that, in the case of vaccine shedding, fecal samples were the least accurate for diagnosing CPV in dogs undergoing vaccination. If this is true then how can one diagnose a suspected case of parvovirus in puppies which are in the stages of core vaccination? To quote the article, “viremia and fecal shedding are common manifestations in pups only after the first vaccination, and thereafter positive results in dogs presenting compatible clinical signs should be considered as caused by field strain infection”.
Serological testing the CPV infected puppies was undertaken using in-clinic POC IgM and IgG dot ELISA kits (ImmunoComb Canine Parvovirus & Distemper IgM and VacciCheck IgG Antibody Test Kits, Biogal Galed Labs) to gain a more complete picture of the stage of the disease. Ninety-two percent of the dogs diagnosed with CPV had titers of IgM while the remaining 8% did not have a measurable titer but were PCR and PCRun positive. Of the 60 sick dogs included in this study group, 5 had negative IgM and IgG titers despite positive qRT-PCR, suggestive of an early infection before development of a humoral response sufficient to be measured by the serological test.
As IgM was undetectable in all puppies before vaccination and positive in 92% of the dogs diagnosed with CPV, IgM serology should be considered as highly sensitive and specific before vaccination and can aid in the diagnosis of CPV. Twenty days after vaccination, IgM serology was positive in all puppies thus should not be used to establish a diagnosis of CPV at that time point. According to the article, IgM testing in non-vaccinated puppies should be considered a highly sensitive and specific an aid in the diagnosis of CPV, but should not be used as a stand-alone test.
The take-home message from this article is that quantitative serology and PCR are both useful techniques for assisting in accurate diagnosis of canine Parvovirus infections and both tests can be performed in-clinic using POC kits.