How studies on FPV diagnosis and management are helping answer some practical questions important to shelter medicine and beyond.

Feline panleukopenia virus (FPV), or feline parvovirus, is an important cause of disease in cats, particularly kittens.¹ It is frequently seen in animal shelters, which house a mixed and ever-changing population of animals, including kittens that are too young to be reliably protected by routine vaccination.^2,3 In puppies with the closely-related canine parvovirus (CPV) infection, clinical signs are often classic, with vomiting and bloody diarrhea being quite consistent. In cats, however, vomiting and diarrhea are not always seen, particularly early in the disease course. Bloody diarrhea is relatively uncommon, and the disease may manifest non-specifically as sudden death or acute signs of septic shock. In our experience, severity can also range from mild to severe, even within the same litter.

CPV RAPID ANTIGEN TESTS HAVE MIXED RESULTS

Practical point-of-care (POC) diagnostic testing has been largely limited to CPV rapid antigen tests, which are not optimized or labeled for FPV, and have yielded mixed results in studies.^4,5 Highly sensitive quantitative PCR tests can detect small amounts of virus DNA, but this may lead to false positive results, particularly after modified live virus (MLV) vaccination.⁶ It may also extend isolation times by detecting DNA from non-viable virus. Our studies7–9focused on several practical questions regarding FPV diagnosis and management:

• How does POC testing compare with quantitative PCR?
• Can rectal swabs or vomit samples be tested if diarrhea is not present?
• What does a weak positive POC test result mean?
• When do infected kittens stop shedding virus and when can they be released from isolation?
• How are test results affected by modified live vaccination?

We utilized and compared a rapid antigen POC test for CPV (IDEXX SNAP Canine Parvo ELISA; SNAP) and a real-time, quantitative PCR test for FPV.

WHAT DO RAPID ANTIGEN AND PCR TEST RESULTS MEAN?

In cats suspected of having clinical panleukopenia infection, sensitivity and specificity for the SNAP test, compared with PCR, were as follows:

• Fecal samples: 55% sensitivity and 99% specificity (n=102)
• Anal swabs: 30% sensitivity and 96% specificity (n=55)
• Vomit: 100% sensitivity and 100% specificity (n=17)

When fecal PCR was compared with anal swab PCR, the sensitivity was relatively low, at 77%. The lower sensitivity for anal swabs was most likely due to small sample volumes. The results for vomit should be considered preliminary, because only 17 samples were tested, and only two were positive.

This author had previously understood that our observed low sensitivity for the SNAP test arose from the fact that FPV must differ quite markedly from CPV. This is not, in fact, the case. The feline virus is genetically very stable,¹⁰ and differs from CPV by only six amino acid residues.^4,11 The low SNAP sensitivity compared with PCR is, in fact, a “tip of the iceberg” phenomenon. Many feline stool samples contain relatively low concentrations of virus, which are below the detection limit of the antigen test. Virus load can be expected to differ between groups and individuals, depending on factors like vaccination, husbandry, previous maternal exposure, and individual immunity.

In the short period since this study was conducted, this “tip of the iceberg” phenomenon has become familiar around the world thanks to the COVID-19 pandemic. By now, we are all aware that rapid antigen tests for the SARS-CoV-2 virus are positive when virus loads are high, but are negative for lower virus loads that still produce positive PCR results.¹²

The study confirmed previous findings that, in asymptomatic cat, any positive SNAP test, no matter how faint the positive color change, should be viewed as a true positive.^4,5 This is consistent with the manufacturer’s recommendations for CPV results.¹³

We concluded that, like the SARS-CoV-2 rapid tests, the FPVPOC antigen test is a useful screening test, but a negative result in a clinically suspicious patient is not reliable. In these cases, the SNAP can be repeated on a subsequent stool sample, or a sample can be submitted for PCR. Vomit samples can be attempted if no stool is available.

Through this study, we also gained a clearer understanding of what routine FPV PCR results mean. Results are currently reported by the diagnostic laboratory as “positive” or “negative”, with no quantitation reported. DNA quantitation is, however, part of the test methodology, and was provided for the study. Many “negative” samples contained some panleukopenia DNA. Small quantities of parvoviruses (which could be field virus and/or vaccine virus) can be detected in stool from many healthy dogs and cats.^14,15 Consequently, diagnostic laboratories set a technical cut-off value, in this case≥1.6 x 106 viral DNA copies per gram.¹⁶ Virus concentrations above the cut-off are reported as “positive”, and those below are “negative”. In cases where test interpretation is difficult, quantitative results can be obtained from the laboratory.

SUMMARY OF STUDY FINDINGS

Diagnostic testing⁷

• Stool samples remain the sample of choice for diagnosing feline panleukopenia, but vomit has potential for early diagnosis.
• Any positive SNAP result in a sick kitten should be considered a true positive, but a negative test is unreliable.
• PCR can be used to confirm the diagnosis where needed.

Viral shedding after clinical infection⁸

• The SNAP test has little utility for post-diagnosis confirmation that viral shedding has ceased.
• Our findings supported release from isolation >14 days after diagnosis; future virus isolation research might support even earlier release. Additional precautions must be in place after release from isolation.

Post-vaccinal positive tests⁹

• Post-vaccinal SNAP tests were rare, but positive PCRs were quite common, and typically occurred on Day 7 following the initial MLV vaccination.
• Judicious test submission and careful assessment are needed to avoid false positive diagnoses.

Panleukopenia is treatable in shelters if adequate resources are available, with 80% survival in our study.

HOW LONG SHOULD INFECTED CATS BE KEPT IN ISOLATION?

A follow-up study tested stool samples from naturally infected shelter kittens on Days 0 (day of diagnosis), 3, 7, 14 and 21. The goal was to optimize isolation times. Both FPV and CPV viral loads typically peak within the first few days, then rapidly decline.^15,18 However, older literature stating that FPV can be shed for up to six weeks¹⁷ continues to be quoted in reviews and book chapters.

Samples were available from 16/40 cats with clinical FPV, confirmed by PCR. SNAP tests were positive for 12/16 cats on Day 0, and 3/16 on Day 3. The SNAP was negative for all kittens on Day 7, and positive for one on Day 14 (a presumed false positive). In contrast, the PCR test remained positive for 13/16 on Day 3, 6/16 on Day 7 and 1/16 on Day 14. It was negative for 12/12 on Day 21, as was the SNAP. We concluded that the SNAP test is not reliable for detecting ongoing virus shedding. As a result of this study, we have removed our shelter’s previous requirement for a negative SNAP test after two weeks.

The study did not support using clinical measures as a proxy for virus shedding, with diarrhea and systemic signs (pyrexia, lethargy, inappetance, weight loss) all being unreliable indicators of likely PCR status. A negative PCR test was not accompanied by a resolution of diarrhea in many cases. This is unsurprising, given the severe intestinal damage caused by FPV,1 and the potential for secondary infections and dysbiosis.

Although we used PCR to determine shedding, it is important to note that this modality does not distinguish between nucleic acid from live and dead viruses. Virus isolation studies of SARS-CoV-2 showed that positive PCR results persisted well beyond the point that virus could be cultured.^19,20 In dogs with CPV, viable virus was shed for a maximum of eight to 14 days post-infection.^18,21 Virus isolation is the true test of virus viability and potential infectivity, and a follow-up study comparing PCR and virus isolation during recovery from FPV would be very useful.

Previous shelter recommendations were to hold recovered cats for three weeks after recovery, while more recentre commendations were to isolate them for five weeks after diagnosis.^22,23 Based on our findings, and previous research, we recommended that infected cats can be released from isolation >14 days after diagnosis.

Follow-up PCR tests are not routinely required to determine the timing of release from isolation. After considering multiple factors, and our own context and risk management, our current shelter protocol was adjusted to allow release from isolation from seven days after diagnosis, so long as the animal is bright, eating well and gaining weight.

Biological variation being a given, additional measures must be in place. These include vaccination upon intake, robust cleaning and sanitation, stress reduction, and housing recovered cats in areas containing only vaccinated adults. Of interest in this study was that 80% of the initial group of 40 panleukopenia-positive cats and kittens survived, compared with previously reported mortality of 50% to 90%.^24–26 No referral treatment was required. Of paramount importance was the ability to provide intravenous fluid support, when needed.

DO VACCINES RESULT IN FALSE POSITIVE RESULTS?

After MLV vaccination, vaccine parvovirus replicates in the intestine, and post-vaccinal shedding has been demonstrated in both dogs and cats.^27,28 Cats entering a shelter might experience poor appetite, vomiting and diarrhea for many reasons, such as stress, diet change, parasites or other infections. A false positive diagnosis of FPV in such animals could lead to unnecessary isolation, outbreak measures, or even euthanasia. Shelter guidelines recommend frequent vaccination of kittens to try to hit the “sweet spot” between vaccine interference by maternal antibodies and the time of maximum susceptibility to infection,²⁹ meaning that there could be more than one risk period for vaccine-related false positive PCR results.

The study above tested stool samples from healthy shelter cats and kittens, starting before or within 24 hours of FVRCP vaccination (Felocell 3, Zoetis). Cats with any previous vaccination history were excluded. Thirty-seven cats were followed to Day 14, and 17 to Day 21. There was one positive SNAP test on Day 7, confirming previous findings that vaccine-positive SNAP results are uncommon.³⁰ (In the previous study, the frequency of post-vaccinal positive results differed for different vaccines and point-of-care tests.³⁰)

Eight cats had nine positive PCR results, one on Day 3 and eight on Day 7. This is consistent with peak viral loads for both vaccine virus and natural infections.^6,15,18,28 The amount of viral shedding was significantly lower in the vaccine-positive samples, compared to cats with confirmed clinical infections. All the cats with positive results post-PCR were ≤6 months old, and most were from higher-risk environments, namely hoarding settings or community cat colonies. This could suggest subclinical shedding of field virus and/or lack of neutralizing antibodies to reduce vaccine virus concentrations, as might have occurred in older and previously exposed or vaccinated cats. No positives occurred seven days after the second vaccine in the 11 cats that had a second vaccine on Day 14.

To avoid false positive results in the initial period following vaccination, FPV PCR testing should be limited to cats with a high index of suspicion for FPV infection, and/or if there is high outbreak potential. History, vaccination dates, and clinical status should be taken into account when interpreting the results. Other supporting factors, such as neutropenia and quantitative DNA results (available from the reference laboratory), should also be assessed. If the diagnosis remains in doubt, shelters should manage the animal as a positive case.

References:

1. Barrs VR. Feline panleukopenia: a re-emergent disease. Vet Clin North Am Small Anim Pract. 2019;49(4):651670. doi:10.1016/j.cvsm.2019.02.006.
2. Jakel V, Cussler K, Hanschmann KM, et al. Vaccination against feline panleukopenia: implications from a field study in kittens. BMC Vet Res. 2012;8(May). doi:10.1186/1746-6148-8-62.
3. DiGangi BA, Levy JK, Reese MJ, Dingman PA, Tucker SJ, Dubovi EJ. Effects of maternally-derived antibodies on serologic responses to vaccination in kittens. J Feline Med Surg. 2012;14(2):118-123.doi:10.1177/1098612X11432239.
4. Abd-Eldaim M, Beall MJ, Kennedy MA. Detection of feline panleukopenia virus using a commercial ELISA for canine parvovirus. Vet Ther. 2009;10(4):E1-6. www.ncbi.nlm.nih.gov/pubmed/20425728.
5. Neuerer FF, Horlacher K, Truyen U, Hartmann K. Comparison of different in-house test systems to detect parvovirus in faeces of cats. J Feline Med Surg. 2008;10(3):247-251. doi:10.1016/j.jfms.2007.12.001.
6. Bergmann M, Schwertler S, Reese S, Speck S, Truyen U, Hartmann K. Antibody response to felinepanleukopenia virus vaccination in healthy adult cats. J Feline Med Surg. 2018;20(12):1087-1093.https://doi.org/10.1177/1098612X17747740.
7. Jacobson LS, Janke KJ, Giacinti J, Weese JS. Diagnostic testing for feline panleukopenia in a shelter setting: a prospective, observational study. J Feline Med Surg. 2021;23(12):1192-1199.doi:10.1177/1098612×211005301.
8. Janke KJ, Jacobson LS, Giacinti JA, Weese JS. Fecal viral DNA shedding following clinical panleukopenia infection in shelter kittens: a prospective, observational study. J Feline Med Surg. 2021; Epub ahead(June). doi:10.1177/1098612X211023056.
9. Jacobson L, Janke K, Ha K, Giacinti J, Weese J. Feline panleukopenia virus DNA shedding following modified live virus vaccination in a shelter setting. 2022. doi:https://doi.org/10.1016/j.tvjl.2021.105783.
10. Wang X, Carrai M, Van Brussel K, et al. Low intrahost and interhost genetic diversity of Carnivore protoparvovirus1 in domestic cats during a feline panleukopenia outbreak. 2022. doi:10.3390/v14071412.
11. Hueffer K, Parker JSL, Weichert WS, Geisel RE, Sgro J-Y, Parrish CR. The natural host range shift and subsequent evolution of canine parvovirus resulted from virus-specific binding to the canine transferrin receptor. J Virol.2003;77(3):1718-1726. doi:10.1128/jvi.77.3.1718-1726.2003.
12. Brümmer LE, Katzenschlager S, Gaeddert M, et al. Accuracy of novel antigen rapid diagnostics for SARS-CoV-2: a living systematic review and meta-analysis. Vol 18.; 2021. doi:10.1371/journal.pmed.1003735.
13. IDEXX Reference Laboratories. Frequently asked questions about SNAP parvo test. 2017.https://idexxcom-live-b02da1e51e754c9cb292133b-9c56c33.aldryn-media.com/filer_public/94/bb/94bb23b2-7d3a-4d40-a119-1b82cc0f145e/snap-parvo-test-faqs.pdf.
14. Meggiolaro MN, Ly A, Rysnik-Steck B, et al. MT-PCR panel detection of canine parvovirus (CPV-2): vaccine and wild-type CPV-2 can be difficult to differentiate in canine diagnostic fecal samples. Mol Cell Probes.2017;33:20-23. doi:10.1016/j.mcp.2017.02.007.
15. Jas D, Aeberlé C, Lacombe V, Guiot AL, Poulet H. Onset of immunity in kittens after vaccination with a non-adjuvanted vaccine against feline panleukopenia, feline calicivirus and feline herpesvirus. Vet J.2009;182(1):86-93. doi:10.1016/j.tvjl.2008.05.025.
16. Gizzi ABDR, Oliveira ST, Leutenegger CM, et al. Presence of infectious agents and co-infections in diarrheic dogs determined with a real-time polymerase chain reaction-based panel. BMC Vet Res. 2014;10:1-8.doi:10.1186/1746-6148-10-23.
17. Csiza CK, Scott FW, de Lahunta A, Gillespie VMD. Immune carrier state of feline panleukopenia virus-infected cats. Am J Vet Res. 1971;32(3):419-426.
18. Decaro N, Desario C, Campolo M, et al. Clinical and virological findings in pups naturally infected by canine parvovirus type 2 Glu-426 mutant. J Vet Diagnostic Investig. 2005;17(2):133-138.doi:10.1177/104063870501700206
19. Ben-Shmuel A, Brosh-Nissimov T, Glinert I, et al. Detection and infectivity potential of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) environmental contamination in isolation units and quarantine facilities. Clin Microbiol Infect. 2020;26(12):1658-1662. doi:10.1016/j.cmi.2020.09.004.
20. Mondelli MU, Colaneri M, Seminari EM, Baldanti F, Bruno R. Low risk of SARS-CoV-2 transmission by fomites in real-life conditions. Lancet Infect Dis. 2020. doi:10.1016/S1473-3099(20)30678-2.
21. Proksch AL, Unterer S, Speck S, Truyen U, Hartmann K. Influence of clinical and laboratory variables on faecal antigen ELISA results in dogs with canine parvovirus infection. Vet J. 2015;204(3):304-308. doi:10.1016/j.tvjl.2015.03.009.
22. Tuzio H. Feline panleukopenia. In: Hurley KF, Miller L, eds. Infectious Disease Management in Animal Shelters.Ames, Iowa, USA: Wiley-Blackwell; 2009:183-196.
23. Guidebook: Feline panleukopenia. Koret Shelter Medicine Program 2017.https://www.sheltermedicine.com/library/guidebooks/?r=feline-panleukopenia.
24. Kruse BD, Unterer S, Horlacher K, Sauter-Louis C, Hartmann K. Prognostic factors in cats with felinepanleukopenia. J Vet Intern Med. 2010;24(6):1271-1276. doi:10.1111/j.1939-1676.2010.0604.x.
25. Litster A, Benjanirut C. Case series of feline panleukopenia virus in an animal shelter. J Feline Med Surg.2014;16(4):346-353. doi:10.1177/1098612X13497738.
26. Porporato F, Horzinek MC, Hofmann-Lehmann R, et al. Survival estimates and outcome predictors for shelter cats with feline panleukopenia virus infection. J Am Vet Med Assoc. 2018;253(2):188-195. doi:10.2460/javma.253.2.188.
27. Bergmann M, Schwertler S, Speck S, Truyen U, Reese S, Hartmann K. Faecal shedding of parvovirus deoxyribonucleic acid following modified live feline panleucopenia virus vaccination in healthy cats. Vet Rec.2019:doi 10.1136/vr.104661. doi:10.1136/vr.104661.
28. Freisl M, Speck S, Truyen U, Reese S, Proksch AL, Hartmann K. Faecal shedding of canine parvovirus after modified-live vaccination in healthy adult dogs. Vet J. 2017;219:15-21.
29. Stone AES, Brummet GO, Carozza EM, et al. 2020 AAHA/AAFP feline vaccination guidelines. J Am Anim Hosp Assoc. 2020;56(5):249-265. doi:10.5326/jaaha-ms-7123.
30. Patterson E V., Reese MJ, Tucker SJ, Dubovi EJ, Crawford PC, Levy JK. Effect of vaccination on parvovirus antigen testing in kittens. J Am Vet Med Assoc. 2007;230(3):359-363. doi:10.2460/javma.230.3.359.

AUTHOR PROFILE

LINDA S. JACOBSON, BVSC, MMEDVET(MED) PHD, TORONTO HUMANE SOCIETY

Linda obtained her veterinary degree in Pretoria, South Africa, in 1986 and subsequently completed a residency in small animal internal medicine and a PhD on the pathophysiology of virulent canine babesiosis. She completed the University of Florida Online Graduate Certificate in Shelter Medicine in 2015. She is part of the College of Veterinarians of Ontario’s Standard of Care Advisory Group and served on Humane Canada’s shelter accreditation standard committee. She joined the Toronto Humane Society in 2010, where she is currently senior manager: shelter medicine advancement.