Background
Jaguarundis (
Puma yagouaroundi) are small diurnal felids that have several unpatterned color morphs – brownish-black, gray and reddish-yellow fur – and are protected across most of their range. The species occurs at low densities and has a decreasing population in the wild, despite being widely distributed throughout South and Central America and occupying a broad range of habitats [
1]. Mainly due to displacement from nature and, to a lesser extent, to captive breeding, jaguarundis are commonly found in zoos and similar captive settings in Brazil [
2].
The feline leukemia virus (FeLV) is an exogenous, oncogenic, immunosuppressive gammaretrovirus that can establish persistent infections in domestic cats
(Felis catus) [
3]. The prevalence of FeLV ranges from 1% to 8% in healthy cats almost everywhere in the world and is usually higher when sick cats are included [
4]. FeLV is naturally transmitted by oronasal exposure to virus-containing secretions, mainly saliva, but also in feces and urine [
5‐
7]. The effects of FeLV are cytoproliferative diseases including lymphomas and myeloproliferative disorders; degenerative illnesses, such as anemia and leukopenia; and immunosuppressive diseases associated with opportunistic infections [
3,
8].
FeLV infections tend to be rare or absent in many nondomestic felid species [
9‐
14], except for the European wildcat
(Felis silvestris silvestris), a species very closely related to the domestic cat, in which FeLV appears to be endemic [
15‐
17]. However, documentation of FeLV is becoming more common in wild felid species less closely related to the genus
Felis, which highlights the omnipresent threat that FeLV represents to the conservation of wild felids worldwide. FeLV has been shown to represent a major threat to the survival of critically endangered populations of Iberian lynxes
(Lynx pardinus) in Europe [
18,
19] and to Florida panthers
(Puma concolor coryi) in North America [
20,
21]. In Brazil, antibodies against FeLV have been detected in two free-ranging pumas
(Puma concolor) and two jaguarundis; FeLV DNA was detected in the following captive felids: an ocelot
(Leopardus pardalis), an oncilla
(Leopardus tigrinus) and two jaguarundis [
22‐
24].
For domestic cats, the detection of the structural viral protein FeLV p27 in serum or plasma is used as a marker of infection and, in most cases, as a parameter for viremia. The outcomes of FeLV infection vary according to infectious challenge dose, route of challenge, and possibly host susceptibility and virus strain. The classification of FeLV outcomes in the domestic cat has been refined using sensitive molecular assays that detect and quantify proviral FeLV DNA and viral FeLV RNA, in addition to traditional serological and virological methods [
25‐
30]. In this regard, the main host response categories of FeLV infection in domestic cats have been redefined as abortive, regressive, and progressive infection. In short, those cats that abort infection do not show any evidence of virus infection, except for seroconversion. Cats with regressive infection overcome viremia after an undetectable or transient initial phase by means of efficient cellular and humoral immune responses. These cats seroconvert, permanently harbor low to moderate FeLV proviral loads integrated in mononuclear cells, i.e., lymphocytes, and may or may not clear their plasma viral RNA loads [
25,
27,
31]. Cats with progressive infection are constantly viremic, have low or no antibodies to FeLV, show elevated FeLV proviral and viral loads in peripheral blood cells and plasma and may develop FeLV-associated disease [
25,
26,
28,
29].
In previous surveillance work, we detected FeLV infection in two captive-born jaguarundis (#1 and #4) as well as previous exposure to FeLV in two other captive-born jaguarundis (#2 and #22) among a population of 23 jaguarundis held at Fundação Parque Zoológico de São Paulo (FPZSP), Brazil [
23]. Jaguarundis #1 and #4 tested positive for FeLV p27 antigen by sandwich ELISA. FeLV proviral DNA was detected in blood from both animals by quantitative real-time polymerase chain reaction (qPCR). The sampling for this previous study occurred between 2003 and 2004 (Table
1).
Table 1
Investigation of feline leukemia virus (FeLV) for captive jaguarundis (Puma yagouaroundi) and putative infection outcomes
#1 | 20761 | Nege
| Neg | Neg | Posf
| Pos | Pos | Pos | Pos | Pos | Pos | Pos | Pos | Neg | Progressive |
#2 | 20762 | Posg
| Nth
| Pos | Neg | Neg | Nt | Neg | Nt | Nt | Neg | Nt | Nt | Neg | Abortive with seroconversion |
#3 | 21566 | Neg | Neg | Neg | Neg | Neg | Nt | Neg | Nt | Nt | Neg | Nt | Nt | Neg | Non-infected |
#4 | 21810 | Neg | Neg | Neg | Pos | Neg | Pos | Pos | Nt | Nt | Pos | Nt | Nt | Neg | Progressive |
#5 | 22096 | Neg | Neg | Neg | Neg | Neg | Neg | Neg | Nt | Neg | Neg | Nt | Neg | Neg | Non-infected |
#6 | 23120 | Neg | Nt | Neg | Neg | Neg | Nt | Neg | Nt | Nt | Neg | Nt | Nt | Neg | Non-infected |
#7 | 23851 | Neg | Nt | Neg | Neg | Neg | Nt | Neg | Nt | Nt | Neg | Nt | Nt | Neg | Non-infected |
#8 | 23911 | Neg | Nt | Neg | Neg | Neg | Nt | Neg | Nt | Nt | Neg | Nt | Nt | Neg | Non-infected |
#9 | 23956 | Neg | Nt | Neg | Neg | Neg | Nt | Neg | Nt | Nt | Neg | Nt | Nt | Neg | Non-infected |
#10 | 24142 | Neg | Nt | Neg | Neg | Neg | Nt | Neg | Nt | Nt | Neg | Nt | Nt | Neg | Non-infected |
#11 | 24189 | Neg | Nt | Neg | Neg | Neg | Nt | Neg | Nt | Nt | Neg | Nt | Nt | Neg | Non-infected |
#12 | 24226 | Neg | Nt | Neg | Neg | Neg | Nt | Neg | Nt | Nt | Neg | Nt | Nt | Neg | Non-infected |
#13 | 24227 | Neg | Nt | Neg | Neg | Neg | Nt | Neg | Nt | Nt | Neg | Nt | Nt | Neg | Non-infected |
#14 | 24958 | Neg | Neg | Neg | Neg | Neg | Neg | Neg | Nt | Nt | Neg | Nt | Nt | Neg | Non-infected |
#15 | 25244 | Neg | Nt | Neg | Neg | Neg | Nt | Neg | Nt | Nt | Neg | Nt | Nt | Neg | Non-infected |
#16 | 25845 | Neg | Nt | Neg | Neg | Neg | Nt | Neg | Nt | Nt | Neg | Nt | Nt | Neg | Non-infected |
#17 | 25846 | Neg | Neg | Neg | Neg | Neg | Nt | Neg | Nt | Nt | Neg | Nt | Nt | Neg | Non-infected |
#18 | 26005 | Neg | Nt | Neg | Neg | Neg | Nt | Neg | Nt | Nt | Neg | Nt | Nt | Neg | Non-infected |
#19 | 27088 | Neg | Nt | Neg | Neg | Neg | Nt | Neg | Nt | Nt | Neg | Nt | Nt | Neg | Non-infected |
#20 | 27155 | Neg | Nt | Neg | Neg | Neg | Nt | Neg | Nt | Nt | Neg | Nt | Nt | Neg | Non-infected |
#21 | 27156 | Neg | Nt | Neg | Neg | Neg | Nt | Neg | Nt | Nt | Neg | Nt | Nt | Neg | Non-infected |
#22 | 27689 | Neg | Nt | Pos | Neg | Neg | Nt | Neg | Nt | Nt | Neg | Nt | Nt | Neg | Abortive with seroconversion |
#23 | 28018 | Neg | Nt | Neg | Neg | Neg | Nt | Neg | Nt | Nt | Neg | Nt | Nt | Neg | Non-infected |
Positive results/total tested | 1/23 | 0/6 | 2/23 | 2/23 | 1/23 | 2/4 | 2/23 | 1/1 | 1/2 | 2/23 | 1/1 | 1/2 | 0/23 | |
By analogy, we hypothesized here that FeLV infection in P. yagouaroundi mimics the main outcomes observed for the domestic cat. Thus, the aim of this study was to perform additional serological and molecular tests and monitor the population of jaguarundis at FPZSP for FeLV infection and development of FeLV-related diseases for 5 years (2003–2007).
Discussion
We detected evidence of FeLV exposure in four (#1, #2, #4 and #22) out of 23 jaguarundis in the FPZSP (17%; 95% CI 5–39%). The remaining 19 jaguarundis were negative for FeLV in all serological and molecular tests performed. The population of jaguarundis presented clinical disorders that were common at FPZSP, as in any other captive setting in Brazil [
2,
12], and could be associated with a multitude of causes. Although the FeLV-positive jaguarundis also presented some conditions that could be related to FeLV [
8], our data were insufficient to prove a causal association.
Two captive-born male jaguarundis, the geriatric #1 and the mature adult #4, presented serological and molecular FeLV test results similar to the progressive FeLV infection outcome in domestic cats [
25]. Jaguarundis #1 and #4 were both antigenemic. Antigenemia is usually used as a measure for viremia in domestic cats and is consistently found in domestic cats with a progressive FeLV infection outcome [
26,
28]. Additionally, blood, buffy coat, serum and tissue samples from both jaguarundis were positive for FeLV proviral DNA and viral RNA with high proviral and viral loads, respectively (Table
4). While the presence of proviral DNA attests to the integration of provirus into blood cells, the detection of viral RNA usually indicates replicating virus. Once the infection outcome has been established, high proviral and FeLV RNA loads are characteristically found in cats with progressive infection, while low loads are detected in cats with regressive infection that are provirus positive but not viremic at the time [
25,
29,
50]. FeLV proviral and viral RNA loads were measured in the two FeLV-positive jaguarundis #1 and #4. Comparison of the FeLV tissue loads of jaguarundi #1 revealed loads similar to those reported earlier for domestic cats with progressive infection using identical methods. Viral RNA loads in serum from the two FeLV-positive jaguarundis were comparable to those described earlier in persistently antigenemic cats [
30]. The viral RNA load in the clinically healthy jaguarundi #1 was slightly lower than that in jaguarundi #4, which showed weight loss, vomiting, anorexia and diarrhea before death. This is also similar to previous findings from a study in cats, comparing viremic healthy and viremic ill cats [
51]. Moreover, consistent with findings in domestic cats with a progressive FeLV infection, no antibodies to FeLV antigens were detected in jaguarundis #1 and #4.
FeLV RNA and FeLV provirus were also detected in the saliva of jaguarundi #1. This finding is indicative of virus shedding; thus, jaguarundi #1 was a potential source of FeLV infection to other felids. Jaguarundi #1 was euthanized in good clinical condition as a biosafety measure. However, upon necropsy, histopathologic evaluation revealed splenic lymphoid depletion and muscular atrophy of the hind limbs; these findings may be associated with immunosuppression and neurological impairment in this animal. Jaguarundi #4 died presenting weight loss and gastrointestinal disorders including vomiting, anorexia and diarrhea. Histopathology revealed splenic alterations and evidence of enteritis (Table
3). Although these conditions are commonly associated with FeLV in domestic cats, it was not possible to ascertain whether they were caused by FeLV infection in this case.
Two captive-born jaguarundis, #2 and #22, presented test results similar to those reported for domestic cats with abortive FeLV infection and seroconversion as the only marker of FeLV exposure [
28]. The female jaguarundi #2 had a history of direct contact with the two jaguarundis with progressive FeLV infection, #1 and #4: she was a sibling of jaguarundi #1 and had mated with the male #4. This would explain how she was exposed to FeLV, either at the same time as her sibling #1 or during mating with #4. Subsequently, jaguarundi #2 gave birth to jaguarundi #23, who did not show any signs of FeLV exposure (Tables
1,
2,
4). This may well be the case, since the mother, #2, had developed an antibody response and probably had never shed FeLV (abortive infection). It is unknown for how long and at what virus challenge conditions jaguarundi #2 was exposed, as animals were naturally and not experimentally infected in the present study. Overall, we speculate that jaguarundi #2 developed an abortive FeLV infection for similar (and possibly several) reasons that may drive FeLV abortive infections in some domestic cats. The parents of male jaguarundi #22 were the FeLV-negative male jaguarundi #5 and the female #7. Thus, it is unknown at what time point jaguarundi #22 was exposed to FeLV.
Overall, we provide evidence of progressive and abortive FeLV infection in
P. yagouaroundi. Similar results have been found in Florida panthers
(Puma concolor coryi), which belong to the felid lineage 6 (genus
Puma), the same phylogenetic lineage as the jaguarundis [
52]. In Florida panthers, infection outcomes resembled those of domestic cats with progressive, regressive and abortive infection (previously persistent, regressive, and latent infection) [
20,
21]. We speculate that FeLV infection in jaguarundis is similarly unpredictable, and it is influenced for the same diverse aspects cited above for domestic cats.
Complimentary to the fact that FeLV causes various tumors in domestic cats [
53], the literature has reported a FeLV-associated multicentric T-cell lymphoma in a captive non-domestic cheetah
(Acinonyx jubatus) [
54] and non-FeLV-associated T- and B-cell lymphomas in geriatric African lions
(Panthera leo) [
55]. These data motivated the search for possible FeLV involvement in the appearance of the neoplastic intra-abdominal mass in jaguarundi #5. Although jaguarundi #5 had not shown any evidence of FeLV infection
intra vitam (Table
1), we further investigated whether FeLV antigens or proviruses were present in intestinal lymphoid cells and whether these cells produced FeLV viral RNA locally without it entering the bloodstream – as would be expected in a case of sequestered FeLV infection [
4]. However, no FeLV antigens were detected by immunohistochemistry and no FeLV provirus or viral RNA were detected by molecular assays. Infection with FIV, which is a lentivirus associated with lymphomagenesis in domestic cats [
56], was not detected by the commercial immunoassay, and only antibodies specific to FIV p24 capsid proteins were detected by Western blot, which may be indicative of an early or very late FIV infection or may have resulted from unspecific cross-reactivity. The FIV RT-PCR assays performed from whole blood were negative; however, this may be due to low viral loads or lack of specificity of the assays due to sequence diversity of different FIV isolates. In conclusion, jaguarundi #5 had developed a non FeLV-associated intestinal B-cell lymphoma; involvement of FIV in the development of the neoplasia cannot completely be ruled out.
Notably, a spleen sample from jaguarundi #5 had been previously analyzed for the presence of the FeLV receptor fTHTR1, which permits the virus to enter the cell. The fTHTR1 complementary DNA (cDNA) from that animal had shown 99% nucleotide and amino acid identity to the fTHTR1 cDNA sequences of the domestic cat and other wild felid species, such as the lynx
(Lynx pardinus), African lion
(P. leo bleyenberghi), Asiatic lion
(P. leo persica), and European wildcat
(F. silvestris silvestris) [
57]. In addition, fTHTR1 was quantified by real-time PCR in a few tissues from two jaguarundis where tissue was available (the viremic jaguarundi #1 and jaguarundi #5, which developed a tumor, but was not FeLV-infected).These limited results showed no significant difference between the fTHTR1 tissue loads in cat and jaguarundi tissue (results not shown). However, it also needs to be mentioned that the real-time PCR assay was designed for the domestic cat THTR1 and not the jaguarundi THTR1. Two-point mutations can be found in the region of the assay: one-point mutation in the middle of the forward primer and one in the probe. Thus, although similar fTHTR1 expression levels were found in jaguarundi tissue compared with cat tissue, we cannot exclude differences in the efficiency of the real-time PCR assay. These findings support the perspective that the first phase of the FeLV virus cycle – viral entry – might be similar among domestic cats and other wild felid species.
We did not detect enFeLV in the jaguarundis by qPCR. This is consistent with earlier data [
58] in which enFeLV was not detected in jaguarundis. Accordingly, FeLV-B, which has greater pathogenicity than FeLV-A in domestic cats and arises by recombination of exogenous FeLV-A with enFeLV sequences [
59], was not found in the two FeLV-positive jaguarundis #1 and #4. Moreover, the highly virulent FeLV-C was not detected. In FeLV-infected Iberian lynxes, enFeLV sequences were also not detected, but their FeLV-A variants were shown to be highly virulent, suggesting that the mechanisms inducing disease in these wild felids might be distinct from those in domestic cats [
19].
The
env sequence obtained from the FeLV infections of jaguarundis #1 and #4 [KR349469] clustered with the highest identity with the FeLV-FAIDS and FeLV-3281 strains, which represent members of a highly conserved group of horizontally transmitted, minimally pathogenic FeLV-A present in all naturally occurring infections in domestic cats. Both FeLV strains were originally isolated from cats in the United States of America (USA) [
60,
61]. Interestingly, the surface unit of the
env sequence of FeLV detected in the jaguarundis also showed 97% identity to those of the
env gene of FeLV isolated from the critically endangered Iberian lynxes [EU293175 to EU293194] in Spain, whose small wild population suffered from an outbreak of FeLV with several deaths [
18].
Both PCR-positive jaguarundis, #1 and #4, were infected with a FeLV with an identical
env sequence, indicating a common origin of the virus. It is unknown at what time point each of the jaguarundis became infected. The reproductive success achieved for jaguarundis in captivity is probably a result of the breeding program conceived for small felids at FPZSP [
62] and a number of measures for improving the well-being of the animals, including the location of the jaguarundi enclosures in an isolated and forested area of the zoo away from the public. Nonetheless, domestic cats commonly are seen invading the zoo, especially the more isolated areas, and they were probably the source of infection of FeLV for the jaguarundis. Domestic cats are the main reservoir for FeLV worldwide and greatly outnumber FeLV-infected wild felids. Given this, it is prudent to prevent the presence of domestic cats in nature reserves, zoos and other settings with captive wild felids and to reinforce the importance of control measures for FeLV in domestic cats, such as testing and vaccination.
A severe FeLV outbreak occurred in a previously naïve population of Florida panthers
(Puma concolor coryi) in North America from 2002 to 2005 in which five FeLV antigen-positive panthers died [
20,
21]. During a six-month period in 2007, six provirus-positive antigenemic Iberian lynxes
(Lynx pardinus) died in Spain [
18,
19]. In both situations, FeLV vaccination programs were initiated. We recommend safe recombinant subunits or inactivated FeLV vaccines for captive jaguarundis, especially considering that invading domestic cats, potential sources of infection, are a frequent problem faced by zoos. In addition, we recommend the inclusion of FeLV testing in breeding programs for jaguarundis.
Acknowledgments
We are deeply indebted to all veterinarians and staff of Fundação Parque Zoológico de São Paulo (FPZSP). We thank J. D. L. Fedullo, S. H. R. Corrêa, M. G. Bueno, A. P. Setzer, R.H.F. Teixeira, D.M. Soares, H. S. Barbosa, P. M. Bressan and J. B. Cruz for providing all the necessary support. The laboratory work was performed using the logistics of the Department of Pathology at School of Veterinary Medicine and Animal Sciences, University of São Paulo (USP); the Center for Clinical Studies at the Vetsuisse Faculty, University of Zurich; and the Institute of Veterinary Pathology, University of Giessen. We thank Dr. Maria Lúcia Zaidan Dagli, Patrícia Matsuzaki, Dr. M. Meli, Dr. V. Cattori, E. Gönczi, T. Meili, and B. Weibel for the excellent laboratory assistance and helpful support. We are grateful to Fábio Okutani Kozu, Míriam Halásk Vask, and Leandro Badiglian for ultrasonography performed at FPZSP. We also express our appreciation to the National Center of Research and Conservation of Carnivores (CENAP) of the Chico Mendes Institute for Conservation of Biodiversity (ICMBio), the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA).