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Published in final edited form as: Forensic Sci Int Genet. 2019 Oct 22;44:102160. doi: 10.1016/j.fsigen.2019.102160

A Novel Test for Determination of Wild Felid-Domestic Cat Hybridization

ES CHIU 1, K FOX 2, L WOLFE 2, S VANDEWOUDE 1
PMCID: PMC7062607  NIHMSID: NIHMS1544702  PMID: 31683165

Abstract

In October 2018, Colorado Parks and Wildlife seized an animal believed to be an illegally possessed bobcat. The owner claimed the animal was a bobcat/domestic cat hybrid, exempted from license requirements. Burden of proof lay with CPW to determine the lineage of the animal. Commercial microsatellite arrays and DNA barcoding have not been developed for identification of bobcat/domestic cat hybrids, and limited time and resources prevented development of such tests for this application. Instead, we targeted endogenous feline leukemia virus (enFeLV) to quickly and inexpensively demonstrate the absence of domestic cat DNA in the contested animal. Using this assay, we were able to confirm that the contested animal lacked enFeLV, and therefore was not a domestic cat hybrid.

Keywords: Bobcat, endogenous FeLV, qPCR, integration copy number, hybridization

Graphical Abstract

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Feline leukemia virus (FeLV) is a common retrovirus that can cause disease (feline leukemia) in domestic cats. This virus is believed to have been introduced to small cats shortly before the radiation of all Felis spp. through the predation of rodents infected with a related gammaretrovirus (Benveniste, Sherr et al. 1975, Willett and Hosie 2013). At some point prior to species evolution, Felis spp. also acquired endogenous FeLV (enFeLV). Endogenization of retroviruses occurs through germ line infections by retroviruses, leading to fixation of the gene, which is then transmitted under normal Mendelian inheritance (Tarlinton, Meers et al. 2006, Polani, Roca et al. 2010). Through this process, all members of the Felis genus (with the exception of F. bieti) now endemically harbor enFeLV, with multiple copies of the virus present within these species’ DNA (Roca, Pecon-Slattery et al. 2004, Polani, Roca et al. 2010). In domestic cats, full enFeLV genome copy number have been documented to vary from 8–12 copies per haploid genome, and individual integration sites may not be conserved (Chiu, Hoover et al. 2018). Solo long terminal repeats (LTRs), which are remnants of partial endogenous retroviral genomes resulting from partial genome excision during cellular mitosis, number between 22–57 copies per cell (Powers, Chiu et al. 2018). In contrast, non-Felis species, including bobcat (Lynx rufus) do not harbor enFeLV genomic sequences (Roca, Pecon-Slattery et al. 2004), thereby making enFeLV-LTR a convenient marker for wild felid-domestic felid hybridization. In summary, any animal that has been hybridized with a domestic cat should harbor multiple copies of the enFeLV-LTR, and any indication of enFeLV-LTR copies per cell greater than zero would indicate hybridization with domestic cats.

On 12 October 2018, Colorado Parks and Wildlife (CPW) confiscated what appeared to be a bobcat (Lynx rufus) from an owner keeping the animal as a pet. The owner claimed the animal was a bobcat/domestic cat hybrid. Under Colorado law (2 CCR 406–11), wild felids are illegal as pets without proper licenses (Resources 2017). However, under Article I #1103A, an exemption from license requirements includes “domestic cat (F. catus) including hybrids with wild felines.” As such, the burden of proof rested on CPW to prove the animal was not a hybrid.

Microsatellite panels do exist for determination of lynx/bobcat hybrids (Schwartz, Pilgrim et al. 2004), Oregon bobcats (Reding, Carter et al. 2013), and domestic cats (Lyons 2012) to which we could compare; however, time and resources did not allow for development and validation of a test specifically for this case. Furthermore, additional sampling of multiple individual animals from both species may have been necessary to overcome ascertainment bias (DeYoung, Demarais et al. 2003, Li and Kimmel 2013). In this report, we offer an affordable and time-efficient quantitative PCR assay to determine whether or not wild felids (excluding felids in the Felis genus) have been hybridized with domestic cats based on the presence or lack of one genetic element – enFeLV-LTR.

METHODS

Sample Collection

The contested animal was relinquished by the owner and housed at the Foothills Wildlife Research Facility in Fort Collins, CO. The animal was fractious and could not be handled for sample collection without sedation. Because sedation required consent from the owner, other options for sample collection were pursued. Ultimately, a cat toy was fashioned from an approximately 18-inch length of cotton rope tied on one end (Figure 1). The rope was purchased in a sealed package and was handled only with exam gloves. There was no known exposure to felid DNA other than the contested animal. The rope was held in front of the animal, which was observed biting the rope toy repeatedly. The rope was cut where the animal bit the rope. The single corded end was washed with 1 milliliter of phosphate buffered saline (PBS) serially to concentrate any cells washing off the rope (Smiley Evans, Barry et al. 2015), and then submerged in 15 mL of PBS and agitated to wash cells off the knot. The two preparations (rope wash and knot wash) were then centrifuged for 10 minutes at 1,000 rpm to pellet cells.

Figure 1. The rope used to collect sloughed buccal epithelia from the ‘hybrid’ bobcat.

Figure 1.

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The black arrows indicate where the animal bit the rope and where cells were washed during both preparations (i.e. rope and knot wash).

Control DNA (Table 1) consisted of dog DNA (courtesy of Dr. Anne Avery, CSU), Asian leopard cat (Prionailurus bengalensis)-domestic cat hybrid (Bengal cat) (Powers, Chiu et al. 2018), and 7 bobcats collected and processed as previously reported (Lee, Malmberg et al. 2017). Dog DNA was used as a negative control for feline C-C chemokine receptor type 5 (CCR5) and enFeLV. Cat DNA and Bengal cat hybrid DNA was used as positive controls for CCR5 and enFeLV. Bobcat DNA was used as a positive control for feline CCR5 and negative control for enFeLV.

Table 1. A contested hybrid bobcat DNA was positive for feline CCR5 (feCCR5), but negative for enFeLV-LTR, indicating the animal was not a domestic cat hybrid.

PCR results indicate Ct values in parentheses.

Samples/Controls feCCR5 (Ct) enFeLV-LTR (Ct) Comment
Dog - - Negative control for feCCR5 and enFeLV-LTR
Lynx rufus + (25.9 – 27.2) - Samples from verified L. rufus from biorepository (Lee, Malmberg et al. 2017). Positive control for feCCR5, negative control for enFeLV-LTR
Bengal cat + (25.5) + (20.7) Domestic cat-leopard cat hybrid acting as a positive for feCCR5 and enFeLV-LTR (Powers, Chiu et al. 2018).
Domestic cat + (26.4) + (22.0) Positive control for feCCR5 and enFeLV-LTR
‘hybrid’ bobcat -knot wash + (35.1) - Consistent with feline DNA but domestic cat enFeLV-LTR undetected
‘hybrid’ bobcat -rope wash + (32.9) - Consistent with feline DNA but domestic cat enFeLV-LTR undetected
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DNA Extraction and enFeLV-LTR Quantification

DNA was extracted using the Qiagen DNeasy Tissue and Blood DNA extraction kit per manufacturer directions. DNA concentration was measured by Qubit 2.0 (ThermoFisher Scientific, MA, USA). enFeLV LTRs were measured using previously described methods (Powers, Chiu et al. 2018). To determine copy number of enFeLV-LTR, we normalized enFeLV-LTR qPCR values against C-C chemokine receptor type 5 (CCR5), which occurs at two copies per cell (Howard, Reckling et al. 2010). The enFeLV-LTR assay contained 12.5 uL iTaq universal probes supermix (BioRad, CA, USA), 400 nM of both primers, 80 nM of the probe, up to 150 ng of DNA, and water making up a 25 uL reaction. Each CCR5 qPCR reaction contained 12.5 uL iTaq universal probes supermix, 200 nM of the forward primer and probe, 500 nM of the reverse primer, up to150 ng of DNA, and water making up a 25 uL reaction. Primer and probe sequences as well as cycling conditions are listed in Table 2. PCR assays were run simultaneously on the same plate to limit variation. Analysis of threshold values (Ct) were compared based on relative fluorescence.

Table 2.

Primer sequences and cycling conditions for assays described in Methods.

Assay (reference) Sequence Cycling conditions
CCR5 (Howard, Reckling et al. 2010) 95°C: 3 min;

40 cycles of 95°C: 5 sec;
60°C: 15 min;
Forward 5’-ACGTCTACCTGCTCAACCTGG-3’
Probe 5’-/56-FAM/TCCGACCTG/ZEN/CTCTTCCTCTTCACCCTCC/3IABkFQ/-3’
Reverse 5’-ACCGTCTTACACATCCCATCCC-3’
enFeLV-LTR (Powers, Chiu et al. 2018)
Forward 5’-GTCTTATCCTAAGTCCACCGTTTA-3’
Probe 5’-/56-FAM/CCTGGCCCT/ZEN/AAGATGGGAATGGAAA/3IABkFQ/-3’
Reverse 5’-CTAGGCTCATCTCTAGGGTCTATC-3’
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RESULTS

With the exception of dog DNA, all samples were positive for feline CCR5 gene with Ct values less than or equal to 35.1 in all positive samples and Ct values less than or equal to 27.2 in all cell-rich extractions (i.e., archived samples). Bengal hybrid cats and wild-type domestic cats were positive for enFeLV-LTR with Ct values lower than respective CCR5 Ct values (indicating >2 copies/cell). enFeLV was absent by qPCR in DNA extractions of cells from the contested animal, wild-type L. rufus, and dog DNA (Table 1).

DISCUSSION

All feline samples were positive for feline CCR5. This assay has been validated previously for domestic cats, pumas (Puma concolor), and bobcats (Powers, Chiu et al. 2018). A positive result therefore indicates that sufficient genomic DNA was present to detect target sequence at two copies per cell from a feline sample. Dog DNA was negative for feline CCR5 as anticipated, serving as a negative control. The qPCR threshold (Ct) values of CCR5 were lower for samples from control bobcat (Ct=25.9 – 27.2), Bengal cat (Ct=25.5), and domestic cat (Ct=26.4) than for samples from the contested animal (Ct=32.9–35.1). This is not surprising considering the quality of the samples, as the rope washes would be expected to contain less target material than blood or tissue samples extracted from control samples. Though Ct values were higher for the contested animals, they were still positive relative to the negative control dog sample, which remained negative after 40 PCR cycles. Because CCR5 is a diploid gene, it presents as 2 copies/cell, (Howard, Reckling et al. 2010) compared to >20 copies/cell recorded for enFeLV-LTR (Powers, Chiu et al. 2018), this predicts that any detection of CCR5 should denote adequate quality of the sample to detect enFeLV-LTR from a domestic cat sample.

enFeLV-LTR was detected in the domestic cat and domestic cat hybrid (Bengal cat) samples but not the known bobcat sample, as expected. The rope and knot washes obtained from the contested animal were negative for enFeLV-LTR, consistent with results from the known bobcat sample, and in contrast to results from the known domestic cat and known domestic cat hybrid (Bengal cat) samples. Clear inheritance of enFeLV-LTR in domestic cat-non-Felis hybrids was illustrated by inclusion of the domestic cat hybrid (Bengal cat) sample in this study in addition to previous work (Powers, Chiu et al. 2018). A 10-fold difference in copy number would be represented by a difference of 3.3 Ct values in qPCR assays with acceptable R-values (Svec, Tichopad et al. 2015), so had the contested animal been an F1 bobcat-domestic cat hybrid, we would have anticipated an enFeLV-LTR Ct value of approximately 32.2 from the sample with CCR Ct value of 35.5.

Further, if we consider a domestic cat harboring a conservative estimate of 20 enFeLV-LTR (normal range 22–57 copies/cell) unlinked loci that independently assort, there is approximately a one in a million chance of having no enFeLV-LTR inherited by a hybrid between the hypothetical domestic cat and a bobcat (Mendel 1865). Based on these findings, we concluded that the contested animal sample was derived from a felid that has not been hybridized with a domestic cat.

MANAGEMENT IMPLICATIONS

We propose this assay as an affordable and rapid method to determine domestic cat hybridization with wild felid species. This method will allow wildlife professionals to very sensitively determine whether domestic cat DNA has been hybridized to wild felids by determining absence of genetic markers, as opposed to categorizing specific genetic markers in microsatellite analysis which may be more subjective, and require testing of more loci. The determination of non-hybrid status does rely on interpretation of a negative result, which is always less desirable than reliance on a positive result. However, inclusion of appropriate positive and negative controls, as well as demonstration of adequate DNA quantity and quality, and the very low probability of false negative detection given the high copy number of enFeLV-LTR in domestic cats improves confidence in a negative result.

It is important to note that there are a few felids known to harbor enFeLV, including members of the Felis genus –jungle cat, sand cat, black-footed cat, and wildcat (F. chaus, F. margarita, F. nigripes, and F. silvestris, respectively) (Polani, Roca et al. 2010). The distribution of all other Felis spp., excluding the domestic cat, is limited to Africa, Asia, and Europe, therefore the possibility for hybridization with these species is likely to be limited in most applications for this test.

Highlights:

  • Felis catus genomes harbor multiple copies of endogenous feline leukemia virus (enFeLV)

  • Lynx rufus genomes do not harbor enFeLV

  • F. catus and L. rufus hybrids would have at least 1 copy enFeLV per cell

  • The contested animal lacks any enFeLV

  • The contested animal is not a domestic cat hybrid

Funding:

This work was supported by the Ecology of Infectious Diseases program – National Science Foundation (1413925); and the Office of the Director, National Institutes Of Health of the National Institutes of Health (F30OD023386). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Footnotes

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