This retrospective study provides an analysis of the prevalence and detectability of canine distemper virus (CDV), feline leukemia virus (FeLV), and feline immunodeficiency virus (FIV) in ocelots (Leopardus pardalis) sheltered in a wild animal recovery center in Guayaquil, Ecuador. Blood samples of 19 rescued ocelots from 2019–20 were analyzed using FeLV p27 antigen enzyme-linked immunosorbent assays (ELISA) and commercial insulated isothermal reverse transcriptase PCR (iiRT-PCR) kits. Using this PCR we detected positive results for CDV (4/ 17; 23.5%) and FeLV (14/16; 87.5%), but not for FIV (0/8). Three previously positive cases of CDV and two of FeLV showed negative results on retesting 6 mo later. Moreover, a third analysis was conducted and was negative for CDV. Our results suggest that ocelots can recover from the local CDV and FeLV strains. An ELISA for the FeLV p27 antigen showed no capability to detect FeLV in ocelots that were confirmed positive by iiRT-PCR. Regional lineages, viral virulence, and host immune response capabilities should be addressed in further research to inform management and decision making for wildlife conservation.

To maintain the health of wild felids in captivity and perform successful reintroductions in their original ecosystem, health issues caused by pathogens are gaining scientific attention due to the risk for individuals, the spread of diseases, and outbreaks in animal populations (Jessup et al. 1993; Meli et al. 2010). Canine distemper is a highly contagious illness caused by canine distemper virus (CDV), a pleomorphic RNA virus of the Morbillivirus genus, family Paramyxoviridae, that affects a broad range of carnivores including ocelots, Leopardus pardalis (Filoni et al. 2003; Sykes 2013; Nikolin et al. 2017). Infection may result in severe multisystemic illness with gastrointestinal, respiratory, and neurologic effects, but 50–70% of infections are asymptomatic (Sykes 2013). Pathogenicity and neuron targets vary depending on the CDV strain, and clinical effects may remain long after recovery from acute illness (Sykes 2013; Nikolin et al. 2017).

Feline leukemia virus (FeLV) is a gamma-retrovirus infecting many members of the Felidae family worldwide, including ocelots (Hoover and Mullins 1991; Jessup et al. 1993). Infection with different FeLV strains can cause malignant transformation or depletion of lymphocyte and hematopoietic cells, producing several symptoms associated with immunosuppression (Hoover and Mullins 1991). Feline immunodeficiency virus (FIV) is a lentivirus analogous to human immunodeficiency virus. It occurs in species-specific strains in felids worldwide, including domestic cats and ocelots (Troyer et al. 2011).

We analyzed the prevalence, detection, and monitoring of CDV, FeLV, and FIV in ocelots housed in the Mansión Mascota veterinary clinic in Guayaquil (–2°09′46.8″S, 79°54′10.4″W), Ecuador, a veterinary clinic adapted for wildlife treatment, rehabilitation, and conservation research with permission from the Ministry of Water and Environment of Ecuador. The wildlife guards managed by the Ministry of Environment of Ecuador rescue animals from the illegal market, traffic accidents, or agricultural activities, among others. Ocelots are rescued from nine localities around the Guayas province, mainly located in forests or agricultural lands. Before transfer to shelters or rescue centers, the ocelots are housed, clinically evaluated, and treated at Mansión Mascota. During 2019 and 2020, blood samples were collected from 19 ocelots during the day of arrival at Mansión Mascota clinic. Serum was extracted for monitoring and diagnosis of infectious diseases. When necessary, 7–10 mg/kg ketamine hydrochloride (Ketaset®, Fort Dodge Laboratories, Fort Dodge, Iowa, USA), 0.1 mg/kg of acepromazine (Acedan, Lab Holliday, Buenos Aires, Argentina), and 2 mg/kg propofol (Aculife Healthcare Pvt. Ltd., Sachana, India) were administered to ocelots via the cephalic vein to provide enough immobilization to complete the necessary procedures. Then, 5 mL of blood were collected from the medial femoral or jugular vein and stored at –2 to 8 C before being sent to a local laboratory, Diagnovet, in Guayaquil, Ecuador, within 3 h of collection, where serum was separated by centrifugation and analyses were carried out the same day. Additional tests were performed during the second sampling event or third sampling event on each individual (see Tables 13). No clinical signs of CDV, FeLV, or FIV (Sykes 2013) were reported in the ocelots.

Table 1

Summary of results from testing of 19 wild ocelots (Leopardus pardalis) at a rehabilitation center in Ecuador, by commercial laboratory-based enzyme-linked immunosorbent assay (ELISA) for the p27 antigen of feline leukemia virus (FeLV) and iiRT-PCR testing for canine distemper virus (CDV), FeLV, and feline immunodeficiency virus (FIV). np = not performed; 0 = negative; 1 = positive.

Summary of results from testing of 19 wild ocelots (Leopardus pardalis) at a rehabilitation center in Ecuador, by commercial laboratory-based enzyme-linked immunosorbent assay (ELISA) for the p27 antigen of feline leukemia virus (FeLV) and iiRT-PCR testing for canine distemper virus (CDV), FeLV, and feline immunodeficiency virus (FIV). np = not performed; 0 = negative; 1 = positive.
Summary of results from testing of 19 wild ocelots (Leopardus pardalis) at a rehabilitation center in Ecuador, by commercial laboratory-based enzyme-linked immunosorbent assay (ELISA) for the p27 antigen of feline leukemia virus (FeLV) and iiRT-PCR testing for canine distemper virus (CDV), FeLV, and feline immunodeficiency virus (FIV). np = not performed; 0 = negative; 1 = positive.
Table 2

The time sequence of canine distemper virus (CDV) findings by iiRT-PCR in repeatedly tested ocelots (Leopardus pardalis) at a rehabilitation center in Ecuador, showing three animals (173A, 539A, and 552A) initially positive and later negative. Cohoused relatives of the positive individuals, a brother of 539A (540A) and daughter of 551A (552A), remained PCR-negative throughout. np = not performed; 0 = negative; 1 = positive.

The time sequence of canine distemper virus (CDV) findings by iiRT-PCR in repeatedly tested ocelots (Leopardus pardalis) at a rehabilitation center in Ecuador, showing three animals (173A, 539A, and 552A) initially positive and later negative. Cohoused relatives of the positive individuals, a brother of 539A (540A) and daughter of 551A (552A), remained PCR-negative throughout. np = not performed; 0 = negative; 1 = positive.
The time sequence of canine distemper virus (CDV) findings by iiRT-PCR in repeatedly tested ocelots (Leopardus pardalis) at a rehabilitation center in Ecuador, showing three animals (173A, 539A, and 552A) initially positive and later negative. Cohoused relatives of the positive individuals, a brother of 539A (540A) and daughter of 551A (552A), remained PCR-negative throughout. np = not performed; 0 = negative; 1 = positive.
Table 3

The feline leukemia virus (FeLV) iiRT-PCR results for two ocelots (Leopardus pardalis; 539A and 552A) at a rehabilitation center in Ecuador that were initially positive for FeLV and later tested negative (6.6 mo and 2.6 mo between evaluation, respectively). Cohoused relatives, a brother (540A) and daughter (551A), respectively, always showed positive results. 0 = negative; 1 = positive.

The feline leukemia virus (FeLV) iiRT-PCR results for two ocelots (Leopardus pardalis; 539A and 552A) at a rehabilitation center in Ecuador that were initially positive for FeLV and later tested negative (6.6 mo and 2.6 mo between evaluation, respectively). Cohoused relatives, a brother (540A) and daughter (551A), respectively, always showed positive results. 0 = negative; 1 = positive.
The feline leukemia virus (FeLV) iiRT-PCR results for two ocelots (Leopardus pardalis; 539A and 552A) at a rehabilitation center in Ecuador that were initially positive for FeLV and later tested negative (6.6 mo and 2.6 mo between evaluation, respectively). Cohoused relatives, a brother (540A) and daughter (551A), respectively, always showed positive results. 0 = negative; 1 = positive.

Serum was tested by enzyme-linked immunosorbent assay (ELISA) for FeLV p27 antigen using the BioPronix FeLV Ag ELISA 96 Kit (Agrolabo, Turin, Italy) with a specific monoclonal antibody. Positive results typically indicate progressive FeLV infections and active viral replication (Beall et al. 2021).

The POCKIT™ Nucleic Acid Analyzer point-of-need PCR detection tool (Genereach Biotechnology, Taichung, Taiwan) was used for CDV, FeLV, and FIV detection using insulated isothermal reverse transcriptase PCR (iiRT-PCR) following the manufacturer's instructions. The iiRT-PCR has been confirmed as a useful technique to identify CDV infection (Elia et al. 2006; Sykes et al. 2013). Additionally, iiRT-PCR and ELISA have historically been used to detect FIV and FeLV in wild felids and domestic cats (Brown et al. 2008; Momtaz et al. 2012; Nicholin et al. 2017; Wilkes et al. 2018), but different levels of ELISA sensitivity for retroviruses such as FIV and FeLV have been reported in other studies, depending on the felid species analyzed (Osofsky et al. 1996; Franklin et al. 2007). The commercial POCKIT used an advanced iiRT-PCR and primers designed by the manufacturer, commonly successfully used to detect viral nucleic acids of CDV, FIV, and FeLV in samples from domestic cats and dogs. Primer sequences for the virus used by the POCKIT commercial kits were not provided by the manufacturer, but they stated that the N gene was the target gene used for CDV detection. Positive CDV results from iiRT-PCR indicate the presence of viral nucleic acids in the body and thereby indicate active canine distemper infections. However, positive FeLV results from iiRT-PCR can indicate active or previous FeLV infections, because FeLV is a retrovirus and remains integrated into the host chromosome even if the virus is not actively replicating (Beall et al. 2021). In the clinical performance evaluation of the FeLV POCKIT iiRT-PCR reagent set using domestic cat Felis catus blood samples in the study by Wilkes et al. (2018), comparison with reference quantitative PCR resulted in a 98.82% agreement, (κ=0.97), and the study concluded that iiRT-PCR assays can serve as reliable method for point-of-need FeLV detection. In the same study, samples were p27 antigen-positive on ELISA, indicating high agreement between POCKIT iiRT-PCR, real-time PCR, and ELISA (Wilkes et al. 2018). In addition, Hartmann (2001) reported a high sensitivity in the detection of FeLV through p27 antigen ELISAs in cases of domestic cats, and a high level of agreement was found in studies based on assays on previously isolated viruses. To avoid p27 immunoassay false positives and achieve 100% diagnostic sensitivity, Hartmann (2001) and Wilkes (2018) recommend combining it with a technique such as iiRT-PCR, which is designed to not react with other feline pathogens. However, in our study, FeLV p27 ELISA analyses produced no positive results in ocelot blood samples in contrast to iiRT-PCR results (Table 1). Similarly, other studies based on the FeLV p27 ELISA from Central and South America, Costa Rica (n=10) and Brazil (n=38), found no positive results in ocelots (Filoni et al. 2003; Blanco et al. 2011).

Thus, in the case of our western Ecuadorian ocelot samples, the BioPronix FeLV p27 ELISA was demonstrated to be unable to detect FeLV, with repeated test results being unable to match any positive result obtained by iiRT-PCR. We suspect that these ocelots may be infected with an undescribed local (Ecuadoran or South American) variant of FeLV. After an initial iiRT-PCR-positive result, results on subsequent testing could be due to recovery from infection or the presence of an amount of the pathogen below detection limits (Madewell and Jarrett 1983). Companions, a brother (540A) and a mother (551A), respectively, continued to test as positive (Table 3) under the same circumstances and time interval in the shelter.

In our study, FeLV-positive individuals (14/ 16 animals; 87.5%) were more abundant than CDV-positive animals (4/17 animals; 23.5%). For both viruses, negative results were registered after subsequent tests in the same animals months later (2/4; 50% and 3/3; 100%). The disappearance or undetectability of CDV after a positive result has been described in dogs (Sykes 2013) but has not been previously seen in wild felids. Canine distemper virus has been identified as the main factor in fatal epidemics in big cats, but with significant differences in regional virulence due to regional endemic evolution (Nikolin et al. 2017).

Negative results on FIV suggest either that FIV is not present in the area, or that variation of the RNA sequence in the FIV in ocelots in this area compared to FIV in domestic cats is sufficient to prevent detection of the virus with the PCR used (Troyer et al. 2005, 2011; Franklin et al. 2007).

The high observed prevalence of FeLV indicates that this may be a common circulating pathogen in the study area. The suspected recoveries from CDV found in all cases repeatedly analyzed suggest that these three ocelots were able to eliminate the virus. Similarly, two animals tested negative for FeLV after initial positive results, indicating possible recovery from the infection. The possible recovery of ocelots from FeLV and CDV is an important finding that should be included in decision making for wildlife conservation and wildlife shelters regarding treatment, management, and release into the wild. Further molecular research on regional variants or strains of the viruses, as well as differences in viral virulence and host immune response capabilities, are recommended.

We acknowledge the Ministry of Environment of Ecuador (MEE) for their collaboration on the agreement for diagnosis and research of biologic samples, Proyecto Fundación Sacha for their support, workers from Clínica Mansion Mascota for their daily work, Diagnovet Laboratory for their attention, to Yoyo for his patience and hard work for the recovery of the individuals, and Ana Patricia Mendoza, University of Missouri, for the suggestions and support.

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