Hematological and biochemical aspects of cats naturally infected with feline immunodeficiency virus and feline leukemia

Copyright Lacerda et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License which permits unrestricted non-commercial use, distribution, and reproduction in any medium provided the original work is properly cited. Hematological and biochemical aspects of cats naturally infected with feline immunodeficiency virus and feline leukemia Aspectos hematológicos e bioquímicos de gatos naturalmente infectados pelos vírus da imunodeficiência felina e da leucemia felina


Introduction
The feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are two retroviruses that promote a progressive imbalance in the metabolism and immune system of the affected animals (Hosie et al., 2009;Hartmann, 2011;Teixeira et al. 2019). Diagnosis of FIV/FeLV infections is performed by associating the clinical signs presented by the cats to complementary laboratory tests (Teixeira et al., 2007;Little et al., 2020). Diagnosis is necessary for the practice of preventive measures such as early vaccination and the initiation of necessary care in the case of positive animals (Levy et al., 2008;Westman et al., 2016).
Considering the severity of these infections, information about laboratory changes in positive FIV and FeLV animals can be useful in determining the prognosis of infected felines. This study aimed to determine the laboratory alterations of domiciled cats naturally infected by FIV and/ or FeLV in the microregion of Ilhéus-Itabuna, Bahia, Brazil.

Location of the study and sampling design
Between February 2012 and April 2013, an epidemiological study of analytical cross-sectional design was conducted in the cities of Ilhéus (14°47"S; 39°02"W) and Itabuna (14°47"S; 39°16"W), which are within the microregion of Ilhéus-Itabuna, Bahia, Brazil. Through non-probability sampling, 200 cat owners were selected from the records of veterinary clinics in the region. The inclusion criteria were as follows: 1) Asymptomatic cats, i.e., clinical examination did not show any abnormalities suggestive of systemic disease such as vomiting, diarrhea, weight loss, nasal secretion, or neoplasia (Collado et al., 2012); and 2) cats aged 6 months or over. The study was conducted in accordance with the principles of bioethics and animal welfare, under protocol number 011/12 (CEUA/UESC).

Serological tests for FIV and FeLV
Three milliliters of blood was collected by venous, cephalic, or jugular puncture from each cat, where 1 mL was reserved for performing the complete blood count (CBC) and extracting DNA, and 2 mL for analysing the biochemical and serological profiles. The samples were tested for retroviruses using the commercial immunochromatography test: FIV ac/FeLV Ag Test Kit (Alere®), following the manufacturer's recommendations.

Extraction of genomic DNA and polymerase chain reaction (PCR) for FIV and FeLV
Genomic DNA was extracted from total blood using a commercial kit (QIAamp DNA Blood Minikit; QiagenTM), following the manufacturer's recommendations. All samples were subjected to nested PCR for detection of FIV and FeLV. The samples that presented negative results were subjected to GAPDH gene amplification in order to check the integrity of DNA and the absence of potential inhibitors. Table 1 presents the primers used for each PCR (FIV, FeLV, and GAPDH).
For amplification of the proviral DNA of FIV, nested PCR with two reactions were set up, each with a final volume of 25 µL composed of 10X buffer, 2.0 mM MgCl 2 , 0.2 mM of each dNTP, 1 µM of each primer, and 1.25 U Taq DNA polymerase. In the first reaction, 5 µL DNA of sample DNA was added and then 2 µL of the amplification product from this reaction was used in the second reaction. Sterile ultrapure water was used to complete the final reaction volume. The two reactions followed the same amplification protocol: initial denaturation at 95 °C for 5 min, followed by 30 cycles at 95 °C for 45 s, 58 °C for 45 s, and 72 °C for 45 s, and a final extension at 72 °C for 5 min. The composition of the reactions and amplification protocols was adapted from a previously described report (Hohdatsu et al.,1998).
To assess the proviral DNA of FeLV, nested PCR was performed. Both reactions presented final volumes of 25 µL, containing 10X buffer; 2.0 mM MgCl 2 ; 0.2 mM of each dNTP; 0.4 µM of each primer; and 1.25 U Taq DNA polymerase. In the first reaction, 5 µL of the sample DNA was added, and 2 µL of the amplification product from this reaction was used in the second reaction. Sterile ultrapure water was used to complete the final reaction volumes. The amplification protocol consisted of initial denaturation at 94 °C for 7 min, followed by 33 cycles at 94 °C for 55 s, 55.3 °C for 55 s in the first reaction and 59.5 °C for 55 s in the second reaction, 72 °C for 70 s, and a final extension at 72 °C for 7 min. The composition of the reactions and amplification protocols was adapted from a previosuly described report (Miyazawa & Jarrett, 1997).
For GAPDH gene amplification, final reaction volume of 25 µL was set up comprising of 10X buffer, 2.0 mM MgCl 2, 0.2 mM of each dNTP, 1.0 µM of each primer, 1.25 U Taq DNA polymerase, and 5 µL of sample DNA. The amplification protocol consisted of initial denaturation at 95 °C for 5 min, followed by 40 cycles at 94 °C for 30 s, 52 °C for 1 min, and 72 °C for 1 min, and a final extension at 72 °C for 5 min. The composition of the reactions and amplification protocols was adapted from a previously described report (Birkenheuer et al., 2003).

Hematological and biochemical analysis
The CBC was performed on an automatic counter (ABX VET, Horiba ™, Montpellier, France). Total plasma protein concentrations were determined using a manual clinical refractometer, and for specific leukometry blood smears stained with Giemsa stain (Merck S/A, Rio de Janeiro, Rio de Janeiro, Brazil) were performed and examined under an optical microscope at 100X magnification. The reference values used were according to the report by Jain (1993).

Statistical analysis
To perform the bivariate analysis, the results of hematologicaland biochemical analyses of cats positive and negative for FIV or FeLV (outcome variables) were categorized into the following variables: has anemia (yes or no); leukopenia (yes or no); neutropenia (yes or no); lymphopenia (yes or no); thrombocytopenia (yes or no); hyperproteinemia (yes or no); increased ALT (yes or no); increased AST (yes or no); increased GGT (yes or no); increased urea (yes or no); increased creatinine (yes or no); increase in total bilirubin (yes or no); increase in indirect bilirubin (yes or

Results
Of the 200 cats evaluated in the present study using nested-PCR and commercial immunochromatographic test, 6% (12/200) and 3% (6/200) tested positive for FIV and FeLV, respectively. No cat was positive for both viruses.
Hematological and biochemical results were analyzed for possible associations with FIV positive status (Table 2). A greater proportion of FIV positive cats showed an increase in the concentration of bilirubins (total, direct, and indirect) in relation to FIV negative cats (p<0.05) and showed a tendency toward hyperproteinemia. Due to the low number of FeLV positive cats, it was not possible to perform a statistical analysis, only a descriptive analysis. Of the six positive animals, none had anemia, leukopenia, neutropenia, lymphopenia, and three (50%) had thrombocytopenia. Among biochemical parameters, two cats showed an increase in serum ALT (17.6%) and AST (17.6%), respectively, whereas none of the cats presented hyperproteinemia and increased concentrations of urea and creatinine. Furthermore, two cats presented an increase in total and indirect bilirubin concentrations (33.3%).

Discussion
To the best of our knowledge, this is the first study in Northeastern Brazil to assess hematological and biochemical parameters in cats naturally infected with FIV/FeLV. These retroviruses are associated with a variety of laboratory findings related to decreased animal immunity (Gleich & Hartmann, 2009;Teixeira et al., 2019).
The presence of anemic conditions has already been well described in FIV positive cats due to the inflammatory process triggered by the virus, whichblocks the uptake of iron (Shelton et al. 1990) as well as in cats infected with FeLV-C subtype, leading to secondary infections and neoplastic cell formation (Gleich & Hartmann, 2009;Hartmann, 2011;Cristo et al., 2019). In our study, we observed a low risk for the development of hematological changes for both viruses. We believe that this is due to the stage of infection (subclinical) presented by these animals at the time of collection. Collado et al. (2012) observed that FeLV positive cats with clinical signs were more likely to have lower total red blood cell counts than those without signs.
There was no statistical association between the leukogram results and retroviral infection, reinforcing the hypothesis of the absence of bone marrow involvement. Changes in the total leukocyte count are related to the reduction in the number of circulating neutrophils, mainly in the acute phase of infections and later in the immunosuppression phase, due to insufficient myelopoiesis (Shelton et al., 1990;Costa et al., 2017;Turinelli & Gavazza, 2018). Lymphopenia is related to the onset of immunosuppression, with a decrease in the number of CD4 and CD8 cells (Hofmann-Lehmann et al., 1997;Novo et al., 2016;Little et al., 2020).

VARIABLES FIV
Odds ration  Regarding biochemical changes, we observed a tendency towards hyperproteinemia in FIV positive cats. Hartmann (2011) and Liem et al. (2013) reported an increase in plasma globulins in cats as a result of the polyclonal expansion of B lymphocytes in response to FIV. Hyperbilirubinemia is associated with the presence of FIV and has already been described in cases of immune-mediated hemolytic anemia in cats facing infectious diseases, including those caused by retroviruses (Kohn et al., 2006;Tasker et al., 2010). However, in the present study, the occurrence of hemolysis in an immune-mediated manner did not cause anemia in cats.
The results of the present study suggest that FIV/FeLV positive cats without clinical signs selected for cross-sectional studiesmay not initially show laboratory alterations compatible with immunosuppression. We believe that the hyperbilirubinemia presented by FIV positive cats should be evaluated with caution, and further studies are needed to assess the role of FIV in animal metabolism.