Posted on by L. M.

Evaluation of a commercial test for genetic resistance to feline infectious peritonitis

Original article: Evaluation of a commercial test for genetic resistance to feline infectious peritonitis, year 2015

Niels C. Pedersen, DVM PhD,
Professor Emeritus,
Center for Pet Health,
School of Veterinary Medicine,
University of California, Davis

The laboratory called AnimalLabs © in Zagreb, Croatia, offers a test that they say identifies cats that carry a genetic marker of resistance to FIP (http://www.animalabs.com/feline-infectious-peritonitis-dna-test-for-resistance/, accessed 7 October 2015). The same test is offered by Genimal Biotechnologies in France (https://www.genimal.com/dna-tests/cat/feline-infectious-peritonitis-resistance/, accessed 29 October 2015). AnimalLabs © has provided two links, one to a research article describing this marker1 , and the second review article on the FIP, which I published in 2009.2 The first article is freely available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041894/pdf/1297-9716-45-57.pdf. A summary of my 2009 review article is available at: https://sockfip.org/wp-content/uploads/2019/04/FIP_Synopsis_Jan13_09-1.pdf. I want to assure you that listing my review article does not support this test. In fact, I believe that the research behind this test was flawed in several respects and should be considered preliminary at best.

I would like to summarize the research that Hsieh and his colleagues did in the article.1 The researchers hypothesized that interferon-gamma is involved in protecting against FIP, a reasonable assumption based on previous research by other scientists. They sequenced the feline interferon-gamma gene (fIFNG) and identified differences between cats (i.e., genetic polymorphisms) at three different positions, which they designated 401, 408, and 428B. These polymorphisms included a single nucleotide polymorphism (SNP) at each of these positions. These SNPs were not located in the parts of the genes that code for the fIFNG protein (i.e., exons) but rather in the regions that surround the exons (i.e., introns). Intron regions are known to contain both nonsense sequences and genetic elements that regulate the level of protein expression by the gene. They then collected DNA from 66 healthy cats and 60 cats with FIP. They found no relationship between the differences in SNPs at positions +401 and +408, but there was a relationship with disease status for the SNP at position +428B. I quote their work – “of all SNPs tested; only fIFNG + 428C/T was shown to be significantly associated with infection outcome. At position +428, there was a higher frequency of the CT genotype in asymptomatic control cats (19.5 %) than in cats with FIP (6.3 %) and the data showed a significant correlation with disease resistance (p = 0.03) (Table 2).” It is noteworthy that among the 66 healthy and 60 cats with FIP, only cats that had CC or CT were present; no cats that had TT were present in either population. The table showing the relevant findings is given below:

Healthy catsFIP cats
+428 SNPs(% of cats)(% of cats)
CC66 (80.5)60 (93.8)
CT16 (19.5) 4 (6.3)
TT0 (0.0)0 (0.0)

The authors concluded that the difference between 16 healthy cats with CT (19.5 % healthy cats) was significantly different from the 4 FIP cats with CT (6.3 % cats with FIP). Using the odds ratio (OR), they calculated that the odds of CC in FIP cats were 3.6 times higher than in CT cats, and concluded that the T SNP is dominant over C. There were two problems with this conclusion, however. First, no cat in their study was TT, and given the fact that 20 cats in both populations carried the T SNP (20/126=15.8 %), one would expect 9 % (0.158 x 0.158=.092), or 11 cats out of 126 cats to be TT. This suggests that either TT is embryonic lethal or, more likely, the populations studied were not a random representation of all cats. Statistical comparisons are only valid when the populations are randomly selected, and special care should be taken when the significance level approaches the minimum P value of P ≤ 0.05 and the number of cases and controls is small. The null value for TT cats in the population also made it impossible to assess whether cats containing two copies of the “protective allele” also had significantly higher resistance than cats that were CC. One would expect that if T is dominant and CT is protective, TT would also be protective. Knowledge of the incidence of cats that are TT at +428 is crucial to the use of the test, because the only way to breed to the CT genotype, if it is the only genotype associated with resistance to FIP, is to maintain CC cats in the population. If TT cats are shown to have a similar degree of resistance to FIP as CT cats, then it is theoretically possible to breed for the T allele alone (CT and TT) assuming that the T allele is indeed associated with resistance. Therefore, the value of the test depends on the prevalence of the CC, CT and TT genotypes in the populations in which it will be used. It is likely that some breeds will be homozygous for the common C allele and therefore will not be covered by the test. Although unlikely, some breeds may have a high prevalence of T and again assuming that both CT and TT are protective, testing may not be cost-effective. If the prevalence of T is low, positive selection for T carries a risk of inbreeding (see discussion below). The bottom line is that breeders should not pay for this test until they have the information they need to use it properly in their breeding programs.

The authors had the biggest problem explaining the biological significance of the T SNP polymorphism. Most significant genetic mutations are found in exons (protein coding regions) and not in introns (non-protein coding regions). To provide biological relevance, the authors had to demonstrate that an intron mutation caused an increase in fIFNG production. The usual way to do this would be to isolate a normal gene with a CC SNP and an alternative gene with a CT SNP and show that there was a difference in the expression level of fIFNG between the two genes in a test system (ie in-vitro). The simplest test would be to identify a large group of cats that are CC, CT or TT, isolate their blood lymphocytes, stimulate them to produce IFNG, and then compare the levels of either specific RNA (by qRT-PCR) or the actual protein by ELISA or other assay. If their observations are correct, cat lymphocytes that are CT or TT should express more fIFNG than cats that are CC. Instead of this complex experiment, the authors decided to only measure fIFNG levels in the plasma of cats with FIP, arguing that cats with FIP and CT SNPs would have higher plasma levels of fIFNG than cats with FIP and CC SNPs. They found that 12/12 cats with FIP with CC SNP had very low levels of fIFNG, while all three cats with FIP with CT mutation had high levels. The conclusion was that the CT mutation allows for higher expression of fIFNG. There are several serious problems with this conclusion. First, it is paradoxical to conclude that a CT mutation confers resistance to FIP and that this resistance is associated with increased fIFNG expression, but then to prove this using plasma from CT cats that have suffered from FIP. The second problem is that there were only three cats in the CT group and 12 cats in the CC group, which makes any statistical comparison irrelevant. Finally, there is no good evidence that cats develop FIP because they are unable to produce sufficient levels of fIFNG. Several FIP studies have measured cytokine expression in cats with FIP.3 These studies were more concise when they showed an increase in FIFNG in the serum of cats with FIP and an increased production of blood lymphocytes in cats with FIP after stimulation. In fact, the general conclusion is that IFNG is stimulated rather than inhibited in cats with FIP.

Up to this point, I assume that this research is justified and that further research is needed. The problem is to make a huge leap from some weak research findings to practical applications. There is already ample evidence that FIP resistance cannot be attributed to a single polymorphism in a single gene. Our attempts to find the only gene responsible for FIP resistance in field studies with Burmese cats4 and randomly bred cats in our own experimental breeding program5 did not allow us to identify a single genetic marker of FIP resistance / susceptibility that we would consider significant. In fact, the only genetic risk factor we were able to identify for FIP resistance was kinship crossing itself.6 Breeding resistant cats with resistant cats did not increase the resistance of their offspring, but rather made them even more susceptible. This is exactly what you would expect from a genetic trait that includes many different genes and gene pathways. The more you try to select a single resistance factor, the more you inadvertently limit the role of multiple genes and gene pathways. Interestingly, the current recommendation is still the best, ie to avoid inbreeding and not keep cats that have fathered kittens with FIP. This recommendation is based on the theory that such cats carry a greater proportion of susceptibility factors than cats that do not produce kittens that have developed FIP and will produce kittens with an even higher proportion of risk factors when bred together. Based on these findings, selection for resistance to FIP using a single genetic marker is more likely to favor inbreeding and, in fact, increase the incidence of FIP. Finally, it should be recalled that heredity explains only 50 % susceptibilities to FIP, 7 the remainder being attributed to epigenetic (genetic changes that occur after birth) and environmental factors.

Although a simple genetic test that would significantly reduce the risk of FIP does not seem realistic, there is reason to continue to look for genetic explanations for why some cats may appear to be resistant to FIP and others succumb to it, and why some cats develop a wet form of FIP and in others a chronic dry form. These studies should focus on farms where FIP exists, where pedigrees are known and where genetic traits can be easily traced. However, the cost of such research should not be at the expense of breeders who order the test. The normal procedure for placing a genetic test for a disease trait on the market is to verify it before it is offered to the public. I don't think this test has been sufficiently researched.

Cited literature

  1. Hsieh LE, Chueh LL. Identification and genotyping of feline infectious peritonitis-associated single nucleotide polymorphisms in the feline interferon-γ gene. Vet Res. 2014, 45:57.
  2. Pedersen NC. A review of feline infectious peritonitis virus infection: 1963-2008. J Feline Med Surg. 2009, 11 (4): 225-58.
  3. Pedersen NC. An update on feline infectious peritonitis: virology and immunopathogenesis. Vet J. 2014, 201 (2): 123-32
  4. Golovko L, Lyons LA, Liu H, Sørensen A, Wehnert S, Pedersen NC. Genetic susceptibility to feline infectious peritonitis in Birman cats. Virus Res. 2013, 175 (1): 58-63.
  5. Pedersen NC, Liu H, Gandolfi B, Lyons LA. The influence of age and genetics onnatural resistance to experimentally induced feline infectious peritonitis. Vet Immunol Immunopathol. 2014, 162 (1-2): 33-40.
  6. Pedersen NC, et al. Immunity to feline infectious peritonitis virus infection is diminished rather than enhanced by positive selection for a resistant phenotype over three generations.Manuscript in preparation.
  7. Foley JE, Pedersen NC: Inheritance of susceptibility of feline infectious peritonitis in purebred catteries. Feline Practice, 1996, 24 (1): 14-22
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