Occurrence of Feline Immunodeficiency Virus and Feline Leukemia Virus Infection in Cats

Problem statement: The objective of the present study was to evaluate occurrence of the Feline Immunodeficiency Virus (FIV) and the Feline Leukemia Virus (FeLV) infection in asymptomatic domestic cats in the metropolitan regi on of Belo Horizonte, Minas Gerais, using the Polymerase Chain Reaction (PCR) for FIV detection a nd SNAP Combo Plus for FeLV and FIV detection. Approach: Blood samples were collected from the jugular vein of 78 healthy cats, mixed breeds and ages and both sexes. Specific primers we re designed for PCR to amplify a 244 bp fragment of FIV gag gene. Results: Five animals (6.41%) were positive by PCR and thre e animals (3.85%) were positive by SNAP Combo Plus for FIV an d 14 animals were positive for FeLV (17.95%). Conclusion: These results suggest that there is a significant o ccurrence of asymptomatic infected animals which may serve as potential trans mitters of FIV and FeLV.


INTRODUCTION
The Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) belong to the family Retroviridae, they are distributed worldwide and induce persistent infections in domestic cats and other small felids (Hosie et al., 1989;Pedersen et al., 1987;Yamamoto et al., 1989). Between 1.0-20.0% of domestic cats are infected with FIV in diverse geographic areas of the world, with an overall prevalence of infection between 1.0-15.0% in healthy cats and from 3.0-44.0% in sick cats (Bendinelli et al., 1995;Courchamp and Pontier, 1994;Ueland and Lutz, 1992). FeLV infection prevalence rates vary from 1.0-38.0% (Hosie et al., 1989). FIV infection is characterized by an impairment of immune functions and a progressive depletion of CD4 + cells. The most common clinical signs of FeLV infection are immune suppression, anemia and lymphoma (Hardy et al., 1976). FIV and FeLV infected cats may remain asymptomatic for years; however, eventually succumb due to direct viral effects or, more commonly, to secondary infections resulting from virus-induced immunosuppression (Pedersen et al., 1987;Bendinelli et al., 1995;Hardy et al., 1976;Johnson et al., 1994;Norsworthy et al., 1993;Okada et al., 1994). The saliva of infected cats is the main form of transmission of FIV and FeLV. The transmucosal infection via vaginal and rectal route can also occur, as well as through milk (Hardy et al., 1976;Bachmann et al., 1997;Carpenter et al., 1998). The rate of FIV and FeLV infection is influenced by gender (males greater than females due to roaming behavior); age (older cats are more likely to be infected) and health (sick cats are more likely to be infected, due to an impaired immune system) (Courchamp and Pontier, 1994).
The methods currently used for detection of FIV infection in domestic cats include virus isolation, immunological tests for detection of specific antibodies and molecular tests for detection of viral genomic sequences (Barlough et al., 1991;Deacon and Leah, 1989;Hohdatsu et al., 1992;Hosie and Jarrett, 1990;Rimstad and Ueland, 1992). Diagnosis of FeLV infection is based on clinical history and viral p27 protein detection in plasma, serum, or saliva of allegedly positive animals. Indirect immunofluorescense (IFA) and immunoenzimatic assays are the most used diagnostic methods (Miyazawa, 2002) and PCR is currently being used (Herring et al., 2001).
The objective of the present study was to evaluate the occurrence of the feline immunodeficiency virus and the feline leukemia virus infection in asymptomatic cats in the metropolitan region of Belo Horizonte, Minas Gerais using Polymerase Chain Reaction (PCR) for FIV detection along with SNAP Combo Plus (IDEXX ® , Westbrook, ME, USA) for FeLV and FIV detection.

MATERIAL AND METHODS
Animals: Blood samples were collected from the jugular vein of 78 healthy cats from shelters and private owners, mixed breeds and ages and both sexes. Samples were collected using two sterile disposable vacuum collection tubes, one for serologic analysis and the other one containing anticoagulant (EDTA) for DNA extraction. Animals were clinically examined and clinical signs were documented. All procedures and protocols used in this study were approved by the Universidade Federal de Minas Gerais Animal Experimentation Ethics Committee (101/09). A sample from a positive cat tested by SNAP Combo Plus and by PCR was used as a positive control. PCR reaction with no template was used as a negative control.
DNA extraction: DNA was isolated from whole blood using the NucleoSpin® Blood Columns kit (Machereynagel, Neumann-Neander, Düren, Germany), following the manufacturers protocol. DNA samples were quantified in the NanoDrop (Thermo Fisher Scientific, Wilmington, Delaware, USA) and stored at 20°C for further analysis Polymerase Chain Reaction (PCR) for FIV gag gene: Specific primers were designed for gel-based PCR using the sequences described by Hohdatsu et al. (1992) 5`-CTAGGAGGTGAGGAAGTTCA-3' (forward) and 5`-CTGCTTGTTGTTCTTGAGTT-3` (reverse) to amplify a FIV gag gene fragment of 244 bp. DNA was submitted to PCR in accordance with Hohdatsu et al. (1992) with some modifications as follows: an initial incubation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 2 min, extension at 72°C for 2 min and a 5 min final extension at 72°C. A 25 µL reaction consisted of 10X PCR-buffer (Invitrogen Life Technologies, Carlsbad, CA, USA), 3 mM of MgCl 2 (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 mM each dNTP, 1 pmol of each primer and 1 unit of Taq-DNA polymerase (Invitrogen Life Technologies, Carlsbad, CA, USA) and 2µL of DNA (approximately 150 ng). PCR products were analyzed in a 8 % polyacrylamide gel stained by silver nitrate.

FIV and FeLV antibody test:
Animal's serum samples were used to detect FIV and FeLV antibody, using SNAP Combo Plus kit (IDEXX ® , Westbrook, ME, USA), following the manufacturers protocol with no modification.     All infected animals were more than 2 years old and were asymptomatic at the time of the sample collections. PCR results revealed FIV gag gene detection in four cats (5.13%) (Fig. 1). In addition, SNAP Combo Plus results showed antibody detection against FIV in three (3.8%) cats, while for FeLV in eleven (14.10%). No animal was positive for both FIV and FeLV simultaneously considering results of FIV-PCR and FeLV SNAP, however, only one animal was positive for FIV and FeLV in the SNAP.

DISCUSSION
In the present study occurrences of 6.41% (PCR) and 3.85 % (SNAP) for FIV and 17.95% (SNAP) for FeLV infection were found in Belo Horizonte's metropolitan area. These results were different from the results found for FIV in Sao Paulo (14.7%) (Lara et al., 2008), Rio Grande do Sul (37.5%) (Caldas et al., 2000), Rio de Janeiro (20.2%) (Souza et al., 2002) and Minas Gerais (2.7%) (Caxito et al., 2006). Results found for FeLV were similar to those found in different Brazilian states. Reche et al. (1997) and Souza et al. (2002) found 8 and 17% of positive cats in Sao Paulo and Rio de Janeiro, respectively (Souza et al., 2002;Reche et al., 1997). The variability of serological and molecular diagnosis of feline immunodeficiency virus infection have been reported lately in others researches, in agreement with our study (Bienzle et al., 2004;Mortola et al., 2004). Seronegative samples with PCR positive results may be also found in cats living in close contact with FIV infected seropositive animals, becoming infected without developing antibodies or disease (Dandekar et al., 1992). However, such cats will usually seroconvert weeks to months later (Hosie et al., 2009).
FIV infection rate of 6.41% found in the present study in asymptomatic animals differ from the rate of 1.47% found by Caxito et al. (2006), in healthy animals in the same state, Minas Gerais. Authors also found an infection rate of 13.95% in sick cats, which confirms other data that demonstrate a higher susceptibility of sick animals to FIV infection (Hosie et al., 1989;Ishida et al., 1989). Minas Gerais and Sao Paulo states revealed that FIV infection was more prevalent in male (4.38% and 20.3%, respectively) than in female cats (0.90 and 9.7%, respectively) (Caxito et al., 2006). However, in present results FIV and FeLV infection rate in male and female could not be differentiated, in agreement with similar results described in Rio de Janeiro (Souza et al., 2002;Teixeira et al., 2007).

CONCLUSION
Additionally, FIV and FeLV infection in housed cats was more elevated than in sheltered cats and these results are in accordance with the results found by Caxito et al. (2006) and Lara et al. (2008) in which housed cats (3.05% and 23.1%, respectively) had a higher FIV infection percentage comparing to street cats (1.11%, 5.9%, respectively). However, high FIV and FeLV infection rates are associated to cats with free street access, which frequently show more aggressive behavior (Hosie et al., 1989;Hosie and Jarrett, 1990;Ishida et al., 1989;Bandecchi et al., 1992). According to animals age, 3.85 and 14.10% of FIV and FeLV infected cats were more than two years old, which are in the age group with the higher infection incidence. Hosie et al. (1989), showed that infection frequently occurs after the first year of age and its prevalence is higher in elderly cats. Serological studies showed the presence of anti-FIV antibodies in Sao Paulo (20.5%) (Reche et al., 1997) and Rio de Janeiro (16.7 and 21.0%) state (Souza et al., 2002;Reche et al., 1997;Mendes-de-Almeida et al., 2004). The higher number of anti-FIV cats in Sao Paulo and Rio de Janeiro comparing to Belo Horizonte, it's probably because serological methods are less specific and can generate false positive results (Hosie and Jarrett, 1990). However, obtaining a reliable diagnosis is important because FIV is a transmissible and infectious disease. Serological tests have limitations, so confirmation of positive ELISA results by western blot should be recommended (Hosie et al., 2009).
Epidemiological studies of FIV and FeLV infection are essential for prevention of virus dissemination in domestic cats. The diagnosis is an important tool to determine the environment of FIV and FeLV circulation and positive cats should be segregate from uninfected ones. The early diagnosis of FIV and FeLV infection is also important for its zoonotic aspect, since immunossupressed animals are likely to eliminate larger quantities of pathogens (O'Neil et al., 1991), such as Toxoplasma gondii (Witt et al., 1989), or Cryptococcus neoformans (Malik et al., 1992). In conclusion, FIV and FeLV accurate diagnosis at an early stage for the identification of subclinically infected cats, that contribute to the diffusion of disease, is required to quarantine any infected animals and to prevent the spread of the virus.

ACKNOWLEDGEMENT
This study was supported financially by CNPq, FAPEMIG and INCT de Informaçao Genético-Sanitária da Pecuária Brasileira. The authors would like to thank Dr.Elizabeth Maria Biondini Jardim from veterinary clinic "Recanto Animal"; Prof. Dr. Cleuza Maria de Faria Resende, Plínio Ferreira Mantovane, Prof. Dr. Adriane Pimenta da Costa Val and Shemina Romano Diniz from Veterinary School of Universidade Federal de Minas Gerais, for providing animal samples.