November 1, 2013 BIOL 303H Term Paper Jacquline Plyler
Investigating the Genetic Susceptibility of Feline Infectious Peritonitis within Purebred
Felines
As our scientific advancement progresses, the relationship between animals and humans in the microbial world has been thoroughly investigated and thus has become increasingly imperative with regards to understanding pathogenesis and disease transmission. Many pathogens that infect animals can infect humans, or have recently evolved to do so.
Coronaviruses are infamous for their capability to undergo tissue tropism, or changing their host, which results in new diseases and unpredictable changes in pathogenicity (Chang et al. 2012).
This trait has made all varieties of coronaviruses major public health concerns, as these tropic and genomic changes can be accompanied by cross-species transmissions, using a variety of intermediate hosts with very few limitations.
Feline coronaviruses are a prime example of the tropic qualities of the coronaviruses that virologists, veterinarians, and geneticists are trying to understand. Two pathotypes of feline coronaviruses exist within a controversial relationship: feline enteric coronavirus (FECV) and the feline infectious peritonitis (FIP) virus. FECV is nonvirulent, infecting as many as 90% of cats within a given population (variation under environmental conditions) at any given time. FIP, on the other hand, is a rare development of FECV, infecting between 1-5% of a cat population, but, it is virtually 100% lethal (Feline Infectious Peritonitis 2008). Chang et al. (2012) discovered that FIP occurs from a mutation of the 3c and S genes of the FECV viral genome.
The overwhelming result is that rather than infecting gastrointestinal epithelial cells, as seen in
FECV, FIP will inhabit and replicate within macrophages. This has made FIP a “death sentence” among felines due to the virus infecting and reproducing in macrophages, causing a large number of macrophages to burst, releasing the viruses. The increased macrophages at the site of
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November 1, 2013 BIOL 303H Term Paper Jacquline Plyler infection and their subsequent apoptosis induces severe inflammatory damage to serosal membranes of abdominal organs, kidneys, and brain.
A likely intrinsic factor of genetic susceptibility is one that requires further study, as documented cases of FIP were not confirmed until the 1950’s (Golovko et al. 2013), suggesting that domestic cats have not had a significant period of time to evolve immunity against the virus.
This also leads to the indication that purebred felines are genetically more susceptible to FIP than random bred felines.
The study performed by Pesteanu-Somogyi et al. (2006) was the first time that a predisposition to specific breeds had been examined. Over a course of sixteen years, Dr.
Pesteanu-Somogyi and her team evaluated a total of 11,535 cats of known breed (purebred or mixed). Breed and FIP diagnosis of all cats at the time of evaluation was recorded and compared using Fisher’s exact test, in which “ p ” values of less than or equal to 0.05 were considered statistically significant. Confidence intervals of 95% and odds ratios were also calculated for each variable to provide more supportive statistical analysis. It was found that 33 of the 9511 mixed breed and 27 (13 different purebreds) of the 2024 purebred cats contracted FIP (Pesteanu-
Somogyi et al. 2006). Figure 1 below is a graphical representation of the statistical difference in the amount of cats that contracted FIP in the mixed breed and purebred populations. Purebred cats had an obvious higher prevalence of FIP than mixed breed cats.
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November 1, 2013 BIOL 303H Term Paper Jacquline Plyler
Figure 1 (Pesteanu-Somogyi et al. 2006): Statistical prevalence of FIP contraction by population. In all of the cats in the sixteen-year study, the prevalence of FIP was approximately 0.5%, with the population of mixed breed cats being just below 0.4%. The asterisk (*) denotes the statistically significant FIP prevalence within the purebred cat population, approximately 1.4% - about triple the FIP prevalence of mixed breed cat population and the entire population evaluated.
The study also determined that some breeds were more susceptible to FIP than others were. The Abyssinian, Bengal, Birman, Himalayan, Ragdoll, and Rex breeds were found to be the most susceptible breeds compared to the other purebred data collected to contract FIP (Table
1) (Pesteanu-Somogyi et al. 2006).
Table 1 (Pesteanu-Somogyi et al. 2006) : Prevalence, Odds ratio, Confidence Interval, and Fisher’s exact test results for specific breeds with FIP diagnosis. Breeds with corresponding P-values are shown in bold showed statistically significant (P-value < 0.05) values. This statistical data displayed that the Abyssinian, Bengal, Birman,
Himalayan, Ragdoll, and Cornish and Devon Rex breeds were more susceptible to FIP than their other purebred counterparts.
Breed Cats diagnosed with FIP/total number cats seen (% affected with FIP)
Odds ratio
Confidence interval
P-value (Fisher's exact test)
Abyssinian 3/99 (3.0%)
1/8 (12.5%)
8.98
41.03
2.71–29.77
4.91–342.85
0.006
Bengal
Birman
Burmese
Exotic Shorthair
Havana Brown
4/18 (22.2%)
1/37 (2.7%)
1/62 (1.6%)
2/2 (100%)
82.06
7.98
4.71
–
26.66–262.44
1.06–59.91
0.63–34.98
–
0.028
<0.001
0.124
0.199
–
Himalayan
Manx
Persian
4/364 (1.1%)
1/67 (1.5%)
4/481 (0.5%)
2/13 (15.3%)
3.19
4.35
2.41
52.22
1.12–9.06
0.59–32.29
0.85–6.83
11.14–244.79
0.046
0.213
0.101
Ragdoll
Rex (Cornish and
Devon)
Russian Blue
Siamese
2/17 (11.7%)
1/39 (2.6%)
1/536 (0.2%)
38.29
7.56
0.54
8.42–174.15
1.01–56.68
0.07–3.93
0.001
0.002
0.130
1.00
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November 1, 2013 BIOL 303H Term Paper Jacquline Plyler
While Pesteanu-Somogyi et al.’s (2006) study provided statistically significant insight into breed being an essential host factor, diagnosis was performed by different veterinarians through various tests. Since the study, two new highly sensitive tests for FIP diagnosis have arisen and are now considered to be the “gold standard”: Direct Immunofluorescence (DIF) and
Immunohistochemistry (IHC). Worthing et al. (2012) conducted DIF and IHC tests on 868 samples submitted for FIP diagnosis at the Veterinary Pathology Diagnostics Services at The
University of Sydney, Australia. DIF was used to detect the FECV antigen within macrophages by infusing an effusion sample with fluorescent-marked anti-FECV antibodies. Should fluorescence be present in the slide of macrophages, the sample tested positive for FIP prevalence due to the fact that FECV mutates to FIP to infect macrophages. IHC was performed after initial histological tests by staining tissue slides by H-and-E staining methods and incubating the samples with both primary and secondary anti-FECV antibodies. Histological analysis was then performed, with a positive result displaying an immune response on the slide
(Worthing et al. 2012). Table 2 below shows the results of the new treatment methods:
Table 2 (Worthing et al. 2012) : Comparison of Immnotechniques for FIP Detection from submitted samples.
DIH was performed on effusion samples (80%), while IHC was performed on tissue samples (20%).
Direct
Immunofluorescence
Immunohistochemistry 90
Totals
Test Positive Result (+)
292
382 (44%)
Negative Result (-)
397
89
486 (56%)
Totals
689 (80%)
179 (20%)
868 (100%)
χ 2
analyses were calculated for positive and negative results in both the DIF and IHC treatment groups (Worthing et al. 2012). Figure 2 below displays the observed and expected percentages of FIP contraction based on χ 2
statistical calculations. As can be observed, there is a
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November 1, 2013 BIOL 303H Term Paper Jacquline Plyler statistically significant difference between the observed frequencies of FIP contraction in mixed and pure breeds, with purebreds having a higher observed frequency of FIP contraction.
Figure 2 (Worthing et al. 2012) : Observed and expected frequencies of FIP positive testing for domestic crossbred and purebred cats. The asterisk (*) denotes statistical significance in the observed frequencies of both the submitted domestic cross and purebred cat samples. The observed frequency of FIP contraction in the domestic crossbred population was significantly lower than expected, while the observed frequency of FIP contraction in the purebred population was significantly higher than expected. (P-value < 0.05)
Further breed comparisons were made for expected and observed frequencies based on the outcomes of the χ 2
tests. Figure 3 below displays the data of pedigree and the expected and observed frequency of FIP contraction results. It was found that the British Shorthair, Devon
Rex, Abyssinian, Persian, and Himalayan breeds were more genetically susceptible to FIP contraction than their other purebred counterparts.
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November 1, 2013 BIOL 303H Term Paper Jacquline Plyler
Figure 3 (Worthing et al. 2012) : Observed and expected frequencies of FIP contraction for each breed in the study. The asterisk (*) denotes statistical significance in the observed frequency of FIP contraction for that breed (Pvalue < 0.05). Breeds found to have a high prevalence of FIP within their pedigree in the study were the British
Shorthair, Devon Rex, Abyssinian, Persian, and Himalayan.
Due to more specific testing for FIP becoming available, the basic conclusions of the study performed by Pesteanu-Somogyi et al. (2006) was validated by Worthing et al.’s (2012) data: pedigree cats are significantly over-represented in FIP cases, indicating a genetic susceptibility among pedigreed cats that had yet to be understood.
Golovko et al. (2013) undertook a genome wide association study (GWAS) of the Birman cat breed using SNPs (single nucleotide polymorphisms) to provide genetic evidence for purebreds being more susceptible to FIP. The Birman breed was chosen due to its small population, high level of inbreeding, and high FIP incidence. The researches obtained 199 buccal swabs from 34 cases of confirmed FIP infection by attending veterinarians and 161 healthy cases from registered breeders and owners. Approximately 70% of the confirmed FIP infected samples were from cats ages 1.5 years or younger, with no infected cat greater than 5.5 years used in the study. The DNA samples were then genotyped using a Illumina Infinium HD feline 63K SNP
BeadChip
®
Array and the SNPs assigned to their specific chromosomes and positions based upon the International Cat Genome Sequencing Consortium (ICGSC) Felis_catus 6.2/felCat5 assembly on the University of California – Santa Cruz Genome Browser (Golovko et al. 2013).
To guarantee high-quality data, all SNPs were subjected to strict quality-control criteria under association analysis. When examining the DNA samples, fourteen DNA samples were excluded due to low genotyping, with 10% or more missing genotype. When analyzing the
SNPs, approximately 811 SNPs failed the missingness test (10% missing genotyping rate) and
25,484 failed the frequency test (less than 5% minor allele frequency), so only 36,862 SNPs were available for the GWAS analyses (Golovko et al. 2013).
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November 1, 2013 BIOL 303H Term Paper Jacquline Plyler
Two analyses were performed, the first without age stratification and the second with age stratification. The second analysis limited cases to cats most susceptible (1.5 years and younger) and limited the controls to cats most resistant (2 years and older), reducing the sample size to 108 cats, with 20 cases and 88 controls.
After the GWAS was completed, Golovko et al. (2013) selected the highest ranking SNPs were examined in both analyses, and potential candidate genes involved with the immune response were identified within 500 kb – 1Mb of each SNP. Using the mathematical formulation of –log
10
P
(raw)
, or the logarithm of the P-value of the odds ratio, SNPs were potentially significant if they possessed a calculated value higher than 5.0. A Bonferoni correction for genome wide significance was also performed using the mathematical formulation of –log
10
P
(raw)
= 5.8 to find statistically significant SNPs.
In analysis 1, the strongest associations were found to be on Chromosomes A2, C2, and
D4. However, after Bonferroni correction, only the highest ranked SNP on Chromosome A2 retained significance (Table 3). For analysis 2, the strongest associations were found to be on
Chromosomes A1, A2, A3, C2, and E2. However, after Bonferroni correction, the SNPs on
Chromosomes A1 and E2 retained significance (Golovko et al. 2013). The five proposed candidate genes based upon the data from the GWAS were ELMO 1, RRAGA, TNFSF10,
ERAP1 and ERAP2. Each gene is also referred to as an “immunogene” due to its known involvement in immune response (Golovko, et al. 2013).
Table 3 (Golovko et al. 2013) : Highest Ranking SNPs from Analysis 1 and 2 and their Candidate Genes. From Analysis 1, one SNP from Chromosomes A2, C2, and D2 were found to be significant based on the unadjusted P
(raw)
data. The corrected
P
(Bonf)
data suggested that the SNP on A2 is most statistically significant, and it correlates with the ELMO1 immunogene. From
Analysis 2, four SNPs from A1 and one SNP from Chromosomes A2, A3, C2, and E2 were found to be significant based on the unadjusted P
(raw)
data. The corrected P
(Bonf)
data suggested that three of the SNPs on A1 and the SNP on E2 are the most statistically significant, and the SNPs on Chromosome A1 correlate with immunogene ERAP1 and 2.
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November 1, 2013 BIOL 303H Term Paper Jacquline Plyler
Chr
Analysis-1
A2
C2
D4
A3
A1
A2
C2
Analysis-2
A1
A1
A1
E2
SNP
A2.191286425
C2.107344890
D4.37641174
A1.196617776
A1.206840008
Un.59861682
E2.65509996
A3.140889392
Un3.4893672
A2.191286425
C2. 107021201
SNP position
126618108
93184134
45868722
154265118
164728174
155715831
54165589
129597254
160942986
126618108
92907113
P
(raw)
1.3 × 10
−6
7.8 × 10
−6
6.6 × 10
−6
2.5 × 10
−7
4.1 × 10
−7
4.3 × 10
−7
1.2 × 10
−6
3.7 × 10
−6
8.2 × 10
−6
1.4 × 10
−5
1.2 × 10
−5
P
(Bonf)
0.048
0.288
0.243
0.009
0.015
0.016
0.043
0.137
0.259
0.399
0.347
Candidate gene
ELMO 1
TNFSF 10
RRAGA
ERAP 1,2
ERAP 1,2
ERAP 1,2
ERAP 1,2
ELMO 1
TNFSF 10
In Figure 4 below, a Manhattan plot of the visual significance of the highest ranking
SNPs can be seen, along with their proposed candidate genes.
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November 1, 2013 BIOL 303H Term Paper
(A)
Jacquline Plyler
(B)
(Ci)
(Cii)
(D)
Figure 4 (Golovko, et al. 2013) : Manhattan Plots of Chromosomes A2, D4, C2, and A1 with Candidate Genes Identified.
(A) Candidate gene ELMO 1 is recognized for having the highest ranking SNP on Chromosome A2 with an association value of about 6. (B) Candidate gene RRAGA is recognized for having the highest ranking SNP on Chromosome D4 with an association value slightly above 5. (C) Candidate gene TNFS10 is recognized for having the highest SNP in Analysis 1 and 2 on
Chromosome C2. (Ci, Cii) In both Analysis 1 and 2 TNFSF10 had an association value slightly above 5. (D) Candidate genes
ERAP1&2 is recognized for having the highest ranking SNP on Chromosome A1 with an association value of above 6.
The findings of candidate genes were reliant on the GWAS results, but candidate genes were selected based on differing prevalence as well. ELMO1 and ERAP1,2 were selected because both obtained genome-wide significance within their analyses. ERAP1,2 also was the center of a 10.5 Mb region of 31 genes formed by seventeen of the nineteen highest ranking
SNPs on Chromosome A1 (Golovko et al. 2013). Though ELMO1 only obtained genome-wide
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November 1, 2013 BIOL 303H Term Paper Jacquline Plyler significance in Analysis 1, it possessed strong associations in both Analysis 1 and 2. TNFSF10 possessed strong associations in both Analysis 1 and 2 as well. RRAGA obtained a strong association only in Analysis 1.
Though each candidate gene was selected using a variety of criteria, what is most important is that all of the genes possessed a strong association and are known to be immunogenes. ELMO1 is known as the “engulfment and cell motility 1” gene, and is involved in the cytoskeletal rearrangements required for cell motility and phagocytosis of apoptotic cells.
ELMO1 has even been associated with preventing the transcription of the HIV-1 RNA inside Tcells (Gegonke et al. 2006). TNFSF10, or TRAIL (TNF-related apoptosis-inducing ligand) is commonly referred to as “tumor necrosis factor superfamily 10.” TNFSF10 plays a vital role in apoptosis and is essential for a normal functioning immune system. These genes are expressed by Cytotoxic T-cells, dendritic cells, macrophages, and other immune system cells in which
Type I interferon and interferon regulatory factors regulate TRAIL in HIV-1 infected macrophages, inducing apoptosis of infected cells, for example (Falschlehner et al. 2009). “Rasrelated GTP-binding A”, or RRAGA, may also induce apoptosis through acting as a cellular target for adenovirus E3-14.7K, which is a known inhibitor of TNF-alpha (tumor necrosis factor
– alpha) functions. By acting as a target for the adenovirus, RRAGA in the cell in which it is made prevents the inhibition of TNF-alpha, allowing the immune system to continue producing these tumor necrosis factors that signal natural killer T-cells in the immune response (Li et al.
1997). ERAP1,2 is known for its involvement with the cleavage of viral and bacterial proteins into smaller fragments within the endoplasmic reticulum. “Endoplasmic Reticulum
Aminopeptidase 1 and 2” cleaves these pathogenic proteins in the endoplasmic reticulum within a macrophage, and then binds the “left-over” proteins to an MHC complex to transport it to the
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November 1, 2013 BIOL 303H Term Paper Jacquline Plyler cell surface (Cagliani et al. 2010). Once at the surface, the complex can be recognized by a T-cell as being foreign to the body, and subsequent immune response pathways are activated. All five candidate genes provide hints to how FIP functions in its cellular targeting and mechanisms.
The study performed by Golovko et al. (2013) was a series of genetic firsts. It was the first attempt to elucidate genes associated with complex disease susceptibility in cats using
GWAS. Also, this study was the first attempt to identify genes associated with FIP susceptibility, confirming the traits’ presumed complexity by the large number of candidate genes identified.
However, this genetic first calls for more GWAS and gene sequencing of these polymorphisms to gather further evidence for these gene associations with the FIP susceptibility phenotype.
Though the study may have made several milestones in virology, genetics, and animal science, there were some noticeable problems associated with the study. Firstly, multidimensional scaling (MDS) was used to more closely examine population stratifications between Denmark and US sample, and it was determined that although the two populations were closely related, they were geographically distinguishable. This factor was ignored in the results based upon the assumption of neutral selection of susceptibility traits since the breed’s formal recognition in 1925, indicating that there was not significant time for trait selection to change from the breed’s founding to present-day (Golovko et al. 2013). Therefore, no geographical stratification was applied to the population and the potential effects of geographical differences on FIP susceptibility were not taken into account when using two verified geographically distinguishable populations. Also, the two aforementioned studies both provided evidence for purebred genetic susceptibility, but the degree of significance for types of breeds differed in each. This was possibly due to the fact that Pesteanu-Somogyi et. al’s (2006) study occurred in the US and Worthing e. al’s (2012) occurred in Australia. Secondly, GWAS gene candidacy was
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November 1, 2013 BIOL 303H Term Paper Jacquline Plyler not one-hundred percent concordant with FIP susceptibility phenotype, as in there were major differences in association strength, Bonferroni correction data, SNP loci, gene length, etc
(Golovko, et al. 2013). These factors were somewhat taken into account when selecting candidate genes based upon different criteria, but they were not thoroughly investigated.
One question that remains to be answered is why does FIPV target a type of macrophage that exhibits a peculiar tropism for venules in the omentum, serosal surfaces, meninges, and uveal tract (Golovko et al. 2013)? Understanding not just cellular tropism but the mechanisms behind subset cellular tropism could lead to priceless microbiological breakthroughs in fighting against diseases caused by coronaviruses and other tropic pathogens. Additionally, the degree to which each candidate gene potentially plays its role in preventing infected macrophages from undergoing pathogen-induced apoptosis via intracellular or extracellular signaling needs to be better understood (Golovko et al. 2013). One polymoprhic gene may affect another’s performance, act independently of the others, or have a major or minor impact on the immune system.
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November 1, 2013 BIOL 303H Term Paper Jacquline Plyler
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