ABSTRACT
Feline leukemia virus (FeLV) infection causes immunosuppression, degeneration of the hematopoietic system, and fatal neoplasms. FeLV transmission occurs mainly by close social contact of infected and susceptible cats. Developing procedures for the diagnosis of feline retroviruses is crucial to reduce negative impacts on cat health and increase the number of animals tested. Blood collection requires physical or chemical restraint and is usually a stressful procedure for cats. Our objective was to evaluate the use of samples obtained from oral, conjunctival, and rectal mucosae for the molecular diagnosis of FeLV. Whole blood and oral, conjunctival, and rectal swabs were collected from a total of 145 cats. All samples were subjected to the amplification of a fragment of the gag gene of proviral DNA. Compared to blood samples used in this study as a reference, the accuracies for each PCR were 91.72, 91.23, and 85.50% for samples obtained by oral, conjunctival, and rectal swabs, respectively. The diagnostic sensitivity and specificity were 86.11 and 97.26% for the oral swabs, 90 and 92.59% for the conjunctival swabs, and 74.24 and 95.77% for the rectal swabs, respectively. The kappa values for oral, conjunctival, and rectal swabs were 0.834, 0.824, and 0.705, respectively. The diagnosis of these samples showed the presence of proviral DNA of FeLV in oral and conjunctival mucosae. In conclusion, mucosal samples for the molecular diagnosis of FeLV are an excellent alternative to venipuncture and can be safely used. It is faster, less laborious, less expensive, and well received by the animal.
INTRODUCTION
Feline leukemia virus infection was first described by Jarrett et al. (1) as an infectious agent related to the occurrence of lymphosarcoma in cats. The discovery of this agent, a Gammaretrovirus member of the Retroviridae family, has provided an excellent animal model to study virus-related cancers and, years later, for the study of other retroviruses, such as the human immunodeficiency virus and the feline immunodeficiency virus (FIV) (2, 3). The outcome of FeLV infection can vary according to the interplay between host and virus. Most frequently, the exposed cat suppresses viral replication as a result of a robust immune response and remains healthy. If, after exposure to the virus, proviral DNA is detectable in peripheral blood monocyte cells (PBMCs), with either the absence of antigenemia or transient antigenemia, this infection is categorized as regressive infection. However, if neither provirus DNA nor plasma virus RNA loads are detectable circulating after challenged by FeLV, this infection is described as abortive infection. In this stage, antibodies anti-FeLV are found. In a lower proportion of infected cats, the virus establishes progressive infection, which is marked by persistent viremia, with both proviral and plasma viral RNA loads with consequent development of severe FeLV-related diseases (4–6). The regressive status of infection develop into a “progressive infection” if the cat has detectable viral RNA and undergoes severe immunosuppression (4).
A number of methods are available to detect FeLV infection (7). Although it is useful for detecting transient or persistent viremia, enzyme-linked immunosorbent assay (ELISA) and immunochromatography tests are by far the most common diagnostic platforms used by clinicians. The immunofluorescent antibody (IFA) test is also used to detect the cell-associated viral core protein p27 in blood smears. The results of the IFA test and virus isolation (VI) have high concordance rates, but IFA testing is not recommended as a screening test because cats in early infection stages are capable of shedding the virus and thus may not be detected. However, the IFA test can be used for prognostic reasons or to confirm a positive or suspicious result (8). Although VI is considered the ultimate criterion for FeLV infection diagnosis (7), it is not practicable for laboratorial routine because it is difficult to perform and time-consuming, in addition to requiring special facilities. In cats that overcame viremia, the results from ELISA-based methods and IFA tests should be evaluated very carefully. Cats with the regressive status of the infection can still harbor FeLV proviral DNA, which contains the required information for viral reactivation, without present antigenemia (9, 10). Molecular methods, such as PCR, have been widely used to identify and quantify virus particles (5, 6, 9, 11). Although saliva (12), tissues (3), urine (13), and feces (14) have already been used to determine the FeLV status of cats, blood is the most common type of sample used.
Blood collection by venipuncture requires a secure and stable restraint of the patient to ensure the safety of both the animal and the veterinary staff. However, the responses of many individuals when in a fight-or-flight situation make blood collection difficult to perform, requiring practical training and patience on the part of the medical staff. The consequences of an arousal of this instinct can offer risks to the animal, the owner, and the veterinarian. Cat restraint during veterinary care prevents bite and scratch injuries, avoiding the transmission of some zoonotic diseases, such as sporotrichosis and rabies, and limiting patient movement during veterinary procedures (15). However, although important for human safety, restraint also increases fear, and cats can become more aggressive. These animals may experience fear and anxiety due to a real threat or an overreaction even when little or no threat is present (16).
For many cats, a veterinary visit is a stressful experience, and its negative impact can last for days. When is intense or long enough to exceed the individual’s adaptability, aversive stimuli can trigger a negative effect on their health and well-being (17). In addition, it can lead to unwanted behavioral changes. One of the consequences of the stress response is the suppression of immune system function and then the development of a new infection or the reactivation of an earlier one (18). According to Tanaka et al. (19), cats with high levels of stress are almost five times more likely to develop upper respiratory tract infection than cats with lower levels of stress. Stress has been associated with several gastrointestinal problems, such as diarrhea or vomiting (20, 21). These effects also play an important role in the development of feline interstitial cystitis, the most common diagnosis in cats with lower urinary tract disease (22, 23). One of the most serious behavioral changes related to stress in cats is anorexia, which can lead to potentially fatal hepatic lipidosis. Thus, less invasive and less stressful methods to collect samples from cats are critical to ensure the patient’s welfare and low-stress management.
The aim of this study was to evaluate whether molecular diagnosis of FeLV by conventional PCR is reliable using samples collected from oral, conjunctival, and rectal mucosae by noninvasive methods.
MATERIALS AND METHODS
Animals.We collected samples from 145 sheltered or domiciled cats of the municipality of Belo Horizonte. Sex, breed and origin of the animals were not considered as criteria of inclusion in this study. The youngest animal tested was 45 days old, and the oldest one was 12 years old. The clinical condition of the animal was not an excluding factor. This study was approved by the Ethics Committee on the Use of Animals of the Universidade Federal de Minas Gerais (CEUA/UFMG) under protocol number 384/2017.
Sample collection and processing.(i) Blood samples. Blood collection was performed by venipuncture of the cephalic vein in vacuum tubes containing EDTA and in tubes without anticoagulant for serum separation. No cat underwent sedation for collection but only physical restraint. At each collection, in addition to the blood, conjunctival, oral, and rectal mucosa were also swabbed. Blood samples were then centrifuged (23,000 × g, 10 min; centrifuge 5415R; Eppendorf, Hamburg, Germany). Serum, plasma, and PBMCs were segregated in different tubes. All samples were stored at –20°C until use. Prior to freezing, part of the plasma was used to perform the immunoassay test (SNAP Combo FeLV Ag/FIV antibody test; IDEXX, Inc.) to identify FeLV antigenemia and the presence of anti-FIV antibodies.
(ii) Conjunctival, oral, and rectal swabs. Samples were collected by rubbing the nylon-tipped rod against the conjunctival, oral, and rectal mucosae. Two swabs were used to collect conjunctival material (one for each eye), two were used for the collection of oral samples, one was used for each side of mouth (right and left), and one was used for the rectal sample.
The lower conjunctival mucosa was swabbed with a cotton rod from the lateral to the medial direction of each eye, with simultaneous rotational movement of the rod. The same procedure was performed for the upper eyelid mucosa. Oral mucosa was swabbed for 10 s per side. The upper and lower gums and the internal mucosa of buccinator muscle (cheeks) were traversed. To sample the rectal membrane, a swab was inserted into the anus of the animal, posterior to the sphincter, and rubbed gently over its mucosa in a circular motion of 360°. During this movement, the rod was rotated on its own axis to increase the amount of sample collected.
After collection, swabs were immediately placed in sterile 1.5-ml microcentrifuge tubes containing 400 μl of phosphate-buffered saline (PBS), the external tip was removed, and the tubes were closed. Swabs were then processed as previously described by Gomes-Keller et al. (12). Briefly, all samples were incubated at 42°C for 10 min. The tubes were subsequently centrifuged (8,000 × g, 1 min; centrifuge 5415R) to remove drops from the lid. Swabs were inverted and recentrifuged under the same conditions. Swabs were then discarded, and the final product was stored at –20°C.
Nucleic acid extraction.Total nucleic acids were extracted from PBMCs (blood samples) and from the eluate of swabs using the ReliaPrep blood and gDNA miniprep system (Promega), according to the manufacturer’s recommendation. An initial volume of 190 μl of PBMCs and of eluate were used for extraction of total nucleic acids. Each sample was eluted in a final volume of 100 μl of elution buffer. Extracted samples were stored at –20°C until analysis.
Detection of Feline and FeLV DNA by PCR.(i) GAPDH as a control of DNA extraction. To ensure the extraction quality, all of the extracted samples were subjected to amplification of the feline glyceraldehyde 3-phosphate dehydrogenase (GAPDH) constitutive gene fragment (24).
(ii) Molecular detection of FeLV proviral DNA. There is no consensus in the literature about which test should be used as a gold standard for FeLV diagnosis (25). Thus, in this study, the identification of FeLV proviral DNA in PBMCs by conventional PCR was considered the standard test. Primers were designed based on the gag gene sequence described previously, corresponding to the amplification of a 450-bp fragment common to all subtypes of exogenous FeLV (26). The same primers and amplification conditions were used for identification of a 450-bp fragment in the extracted DNA from the mucosae. Samples from the same individual that presented divergence of results between the PCR of PBMC and any PCR of mucosal samples were subjected to a nested PCR, as previously established (27).
Conventional PCR for the 450-bp fragment was performed in a final volume of 25 μl of the mixture, per sample, with the following reagents: 5 μl of 5× Green GoTaq Flexi Buffer (Promega), 1.0 μl of MgCl2+ (25 mM; Promega), 1.0 μl of each primer, 0.5 μl of dNTPs (10 mM, PROMEGA), 0.2 μl of GoTaq polymerase (500 U; Promega), and 14.3 μl of RNase/DNase-free ultrapure water. A total of 2.0 μl of extracted DNA was added as a template. The primer subsets are described in Table 1. Initial denaturation occurred at 94°C for 5 min, followed by 35 cycles of 1-min duration at 94°C (denaturation), 1 min at 54°C (annealing), and 1 min at 72°C (extension) per cycle. The final amplification process lasted 5 min at a temperature of 72°C. The products obtained were visualized in 1.5% agarose gel stained with ethidium bromide.
Primers used for normalizer gene and for feline leukemia proviral DNA identification
Nested PCR (nPCR) was used as the confirmation test of samples with divergent results. The reagent volumes and conditions for amplification of external and internal fragments of the gag gene were the same, with only the added primers varying (outer and inner primers). The following reagents were used: 5.1 μl of 5× Green GoTaq Flexi Buffer (Promega), 1.5 μl of MgCl2+ (25 mM; Promega), 1.5 μl of each primer (10 pmol/μl; Invitrogen), 0.5 μl of deoxynucleoside triphosphates (10 mM; Promega), 0.25 μl of GoTaq polymerase (500 U; Promega) and 9.65 μl of RNase/DNase-free ultrapure water. Then, 2.0 μl of DNA from the sample was added to the final product of the first reaction. For the second reaction, 2 μl of the amplified product of the first reaction was added. The two sets of primers were used to amplify the 770-bp (outer) and 601-bp (inner) segments equivalent to U3LTR and part of the gag region of the FeLV provirus. The primer subsets are described in Table 1. Initial denaturation occurred at 94°C for 2 min, followed by 35 cycles of 45 s at 94°C (denaturation), 30 s at 60°C (annealing), and for 1 min at 72°C (extension) per cycle. The final process, consisting of amplification, lasted 10 min at a temperature of 72°C. The electrophoretic run for all evaluations occurred in a 1.5% agarose gel stained with ethidium bromide.
(iii) Analytical sensitivity. The DNA templates used to determine the analytical sensitivity of each reaction were produced by cloning the fragments of interest from FeLV (from gag and U3LTR genes). These fragments were amplified from a positive sample under the same reaction conditions described above for the detection of the proviral DNA in samples. After amplification of the gag-450 and U3LTR-601, the amplicons were subjected to an electrophoretic run and the respective fragments were collected. The samples were purified by using a GenElute gel extraction kit (Sigma-Aldrich), and the final product was linked in the pGEM-T easy vector (Promega) by using T4 ligase (Promega) and propagated in electrocompetent Escherichia coli strain JM109. Subsequently, a miniprep DNA, RNA, and protein purification kit (Macherey-Nagel) was used according to the manufacturer’s protocol. The DNA concentrations were measured by a NanoDrop (Thermo Fisher Scientific), and the integrity of the cloned DNAs was analyzed in 1.5% agarose gel stained with 0.01% ethidium bromide.
The analytical sensitivity was estimated by the amplification of a 10-fold serial dilution of each template (i.e., cloned fragment), diluted 11 times (10−11). The corresponding PCRs were performed with samples in triplicate. The most diluted sample amplified by each PCR, identified by electrophoresis, was then 2-fold diluted and also amplified in three replicates.
Detection of antigenemia.The presence of circulating FeLV antigen (core protein p27) and anti-FIV antibodies was evaluated by a point-of-care (PoC) test (SNAP Combo FeLV Ag/FIV antibody test; IDEXX, Inc.) according to the manufacturer’s instructions. PBMC PCR, which has been previously described, was used as a confirmatory test of infection.
Statistical analysis.(i) Occurrence of FeLV in the evaluated population. The positivity and negativity rate for FeLV was determined for each methodology used.
(ii) Accuracy. To determine the accuracy of each methodology evaluated, the results obtained in each molecular methodology were compared to those obtained using PBMC-PCR.
The results coinciding with the positive result by PBMC-PCR were considered to be true positives. “False positives” were considered when a positive result, obtained by the technique evaluated, occurred in negative animals for the PBMC-PCR. The same occurred for the determination of true-negative results, i.e., results coinciding with a negative PBMC-PCR result. However, when any of the PCR results for swabs yielded negative results for samples considered positive by PBMC-PCR, they were considered “false negatives.”
(iii) Diagnostic sensitivity and specificity. The sensitivity and specificity of each PCR for the swabs were obtained in relation to the PBMC-PCR results. Since the immunoassay test identifies only viremic cats, only animals with concomitant positive PBMC-PCR and rapid-test results were considered true positive for the calculation of sensitivity and specificity. Cats with regressive infection were not considered true positives for this calculation. Since the targets and platforms are different, it is not possible to establish whether the absence of viremia means a misdiagnosis by the PoC test.
(iv) Kappa value. The level of agreement among the results obtained by each methodology was verified. For this purpose, the kappa coefficient (28) was calculated in comparisons between the PBMC-PCR and each of the PCRs using RStudio software. Interpretation of the level of agreement was based on the approach described by McHugh (29).
Sequencing.Samples from each category (PBMC, oral swabs [OS], conjunctival swabs [CS], and rectal swabs [RS]), considered positive by PCR, had their bands extracted from the agarose gel using a GenElute gel extraction kit (Sigma-Aldrich) according to the protocol established by the manufacturer. Nucleotide sequencing of the amplified fragment was performed using the Sanger method (30). The similarity of sequences obtained was compared to those available in the NCBI genome bank (BLAST [https://blast.ncbi.nlm.nih.gov]).
RESULTS
Animals and sampling.Before this study, the FIV and FeLV infection status was unknown for all animals examined here. Whole-blood, oral, and rectal mucosal swabs were collected from 145 animals. Only 114 cats were available for the collection of conjunctival-swab samples. None of the cats had to be sedated for the collection of samples.
Nucleic acid extraction quality.The quality of DNA extraction was tested by amplification of the feline’s GAPDH constitutive gene fragment. All (100%) of oral-swab (145/145) and conjunctival-swab (114/114) samples amplified a fragment of 709 bp of GAPDH, and 137 (137/145) of rectal swabs presented the same 709-bp fragment, representing 94.48% of all samples analyzed from this site.
Analytical sensitivity.From an initial concentration of 212 ng/μl of FeLV-gag-clone DNA (4.36 × 1011 copies), the conventional PCR was able to amplify until reaching a 10−4.5 dilution, corresponding to 4.36 × 106.5 copies of DNA/μl. For the nested PCR, the initial concentration of FeLV-U3LTR-clone DNA was 178.3 ng/μl (2.14 × 1011 copies), and this method was able to amplify until 10−9, corresponding to 2.14 × 102 copies of DNA/μl.
FeLV occurrence and phases of infection.Based on the standard test used here, PBMC-PCR, 72/145 (49.66%) animals were considered positive for FeLV in the population studied. For the PoC test, 33/145 animals were positive for the FeLV antigen, and only one cat tested positive for FIV (01/145). Of these 33 samples, only one was not considered a true positive based on the standard test.
Of the 145 animals, 32 (22.07%) positive results in both tests (i.e., both PoC test positive and PBMC-PCR positive) were classified as showing a “progressive infection.” Forty cats (27.59%) with a positive infection identified by PBMC-PCR but that were negative in the PoC test were classified as showing a “regressive infection” (Table 2). The status for each of the remaining 73 animals (50.34%), i.e., negative by both methodologies, was considered either an abortive infection or as no prior exposure to FeLV.
Infection presentation in the evaluated population (n = 145) according to PBMC-PCR and PoC test results
Occurrence of FeLV in the evaluated population according to the mucosa accessed.In addition to the occurrence values obtained by PBCM-PCR, the rates of positive results obtained by PCR of oral, conjunctival, and rectal mucosae and by immunoassay methodologies were also calculated (Table 3). The results obtained by the PoC test must be evaluated with caution because its target is the p27 viral capsid protein. Animals in the regressive stage of infection may not produce it and thus are not identified by this assay.
Occurrence of FeLV by different samples based on PCR and the PoC test
Diagnostic sensitivity and specificity of OS-PCR, CS-PCR, and RS-PCR.Sensitivity and specificity findings derived by using OS-PCR, CS-PCR, and RS-PCR relative to PBMC-PCR are shown in Table 4.
Diagnostic sensitivity and specificity of OS-PCR, CS-PCR, and RS-PCR relative to PBMC-PCR (standard test)
In OS-PCR, two samples considered positive by OS-PCR were determined to be negative in PBMC-PCR and then classified as false positives (Table 4). The number of false-negative samples (negative by OS-PCR but positive by PBMC-PCR) was ten. Thus, the PCR sensitivity of oral swabs was 86.11%, and the specificity was 97.26%. Following the same criterion adopted for OS-PCR, in CS-PCR, four animals were considered false positive, while another six were considered false negative. Thus, the PCR sensitivity of CS-PCR was 90.00%, and its specificity was 92.59% (Table 4). Regarding the RS-PCR, three animals had false-positive results, and 17 erroneously presented negative results (Table 4). Thus, the sensitivity of rectal-swab PCR was 74.24%, and the specificity was 95.77% (Table 4).
Agreement between tests according to stage of infection.Based on the stage of infection previously characterized, we evaluated how well each test was able to correctly perform FeLV diagnosis (Table 5). Among the categories, the evaluation of animals with regressive infection had the worst performance. As described above (see Materials and Methods), amplification of the GAPDH gene using DNA extraction for eight of the rectal samples failed, and for this reason the samples were discarded. Of these samples, two correspond to negative cats, two correspond to regressively infected cats, and four correspond to progressively infected cats (based on the PBMC-PCR results). For the OS-PCR, the number of samples in each category is equivalent to the PBMC-PCR. Regarding to the samples of CS-PCR, only 114 animals were available to be assessed. A total of 54 (54/114) were from FeLV negative animals, 37 (37/114) were from regressively infected cats, and 26 (26/114) were from progressively infected cats. A total of 63.15% of regressive infected animals were properly identified by rectal-swab PCR. When considering viremic cats (progressive infection), conjunctival-swab PCR was able to identify 100% of animals. In the overall evaluation, regardless of the form of presentation of infection, the accuracy of each test was verified in reference to results obtained by diagnosis using PBMCs (Table 6).
Agreement of each test with PBMC-PCR results, stratified by phase of infection
General accuracy of each PCR in comparison to PBMC-PCR results
The kappa correlation (Table 7) between the standard test and the results of the mucosa PCR revealed a strong level of agreement with PBMC-PCR and OS-PCR (κ = 0.834, P < 0.001) and with PBMC-PCR and CS-PCR (κ = 0.824, P < 0.001). A moderate level was found between PBMC-PCR and RS-PCR (κ = 0.705, P < 0.001). Considering the PBMC-PCR and the point-of-care test correlation, the kappa value shows a weak agreement (κ = 0.46). The percentages of agreement observed were 91.7% for OS-PCR, 91.2% for CS-PCR, 85.4% for RS-PCR, and 73.1% for the PoC test.
Kappa statistics values for test comparisons and interpretation for all methodologies applied
The general accuracy was calculated for each PCR evaluated based on the results of the standard test. Rectal-swab PCR was found to have the lowest value, 85.40%. OS-PCR and CS-PCR presented fairly similar values, 91.72 and 91.23%, respectively (Table 6).
Discordant results between PBMC-PCR and mucosal PCR.There was disagreement in the PCR results in 29 of the evaluated animals. The PBMC-PCR result, used in this study as a reference, is considered to determine the true status of infection. Thus, different results from the same animal were considered false positives or false negatives. In five animals (animals 5, 6, 10, 59, and 62), only PBMC-PCR was able to identify the presence of proviral DNA (Table 8). All samples with discordant results were analyzed by nPCR, as described above.
Discordant results of OS-PCR, CS-PCR, and RS-PCR compared to the standard test (PBMC-PCR), as confirmed by nPCR
DNA sequencing.Products obtained after FeLV-gag amplification by PCR were subjected to nucleotide sequencing. A range of 92.3 to 99% similarity was found between the sequence of the FeLV strain 1172MG gag polyprotein gene (GenBank accession no. EU048350.1) and the sequence of the complete genome (GenBank accession no. KP728112.1) of FeLV strain Glasgow-1; both are registered in GenBank (https://www.ncbi.nlm.nih.gov/).
DISCUSSION
A diagnosis of FeLV infection requires special attention by clinicians and cat owners. When infected, some cats are able to completely clear the infection (abortive infection) or reduce, or suppress, viral replication (regressive infection). Others, however, cannot inhibit viral replication, which characterizes a progressive infection (5, 6). In these cats, the production of new infectious viral particles is constant and high, and such virus particles are easily shed by fluids and body secretions. In addition to being virus transmitters, these viremic animals are more likely to develop diseases associated with FeLV infection, such as nonregenerative anemia and lymphomas (5, 7, 31, 32). The reduction or complete suppression of viremia tends to decrease or eliminate the presence of the capsid protein p27, the major biological marker of infection detected by many immunological tests, such as immunoassays (11). As a consequence, confirmatory tests aiming to identify the presence of the viral genome are crucial. Due to the high sensitivity and the possibility of quantification of the viral load, PCR has been widely used as a reference test for the diagnosis of feline viral leukemia (11, 33, 34). Quantitative PCR is also a useful tool to establish the prognosis of disease in infected animals and for the follow-up of treatment by periodically checking the viral load (3, 35, 36).
Felines are very sensitive to environmental stimuli, responding in an intense way to most of them. Handling a cat patient can be stressful and even dangerous to the animal and to the veterinary team as well. Physiological responses to stress associated with the laborious restraint process make blood collection difficult to perform, requiring practical training and patience on the part of medical staff. Sometimes, venipuncture is impossible in cats without sedation (37), which incurs extra costs to the owner and potential health risks for the cat. Nevertheless, routine diagnostic tests are almost exclusively performed on the basis of whole blood, serum, and/or plasma. The collection of swabs for execution of this study was shown to be a faster, less-laborious, and well-accepted procedure for animals compared to blood collection. This is corroborated by observations in previous studies (14). Although it also requires special care, the use of swabs enables owners themselves or veterinary technicians to collect material for FeLV laboratory diagnosis.
The choice to swab these mucosae was more than the ease of access to veterinarians or owners. In 2015, while evaluating the viral and proviral loads in different tissues of FeLV-positive cats, Helfer-Hungerbuehler et al. (3) observed that the gastrointestinal tract, salivary glands, and lymphoid tissues presented the highest proviral load for FeLV, followed by the mucosa and other glands, regardless of the course of infection. These findings are in line with results obtained by Gomes-Keller et al. (14) for the presence of viral DNA and RNA in FeLV-positive animals. Rojko et al. (38) demonstrated that infection of mucosa and glandular epithelium occurs within 4 weeks after infection. Thus, cellular material from oral, conjunctival, and rectal membranes are valuable samples for the diagnosis of FeLV. The performance of rectal swabbing, although more invasive than oral swabbing, is still less stressful and laborious than traditional venipuncture (39, 40). We also observed it for the collection of ocular mucosa material in this work. To our knowledge, this is the first study to evaluate the collection of conjunctival mucosa samples for the diagnosis of FeLV infection.
Our results reveal that the diagnosis of FeLV infection by swabbing these three mucosae had satisfactory sensitivity rates, i.e., 86.11% for OS-PCR, 90% for CS-PCR, and 74.24% for RS-PCR. Even in animals with regressive infection, which tend to have moderate or very low proviral loads (11, 13), there was concordance of results in 80% (32/40) of OS-PCR and 83.78% (31/37) of CS-PCR compared to the standard PBMC-PCR. However, when we evaluated the concordance of results between RS-PCR and PBMC-PCR in animals with regressive infection, we found an agreement of 63.15% (24/38) (Table 5). It is important to consider that, since the proviral load was not quantified in this study, false-negative results obtained may have occurred in animals whose proviral load was lower than the detection limit for conventional PCRs. This may explain the results for five animals (samples 5, 6, 10, 59, and 62) that were positive only for PBMC-PCR (Table 8).
The lower values of sensitivity found for the rectal swab (Table 4) may be associated with inherent conditions of the rectal cavity. Similar to our results, Gomes-Keller et al. (14) reported the absence of viral and proviral acid nucleic in rectal swabs of some regressively infected animals. The presence of feces at this site may be a limiting factor for the technique when collecting cellular material, since when adhering to the cotton region, feces can compete for the cotton surface and prevent cells from being collected, thus reducing the volume of cellular material collected by this route. It is also important to consider that PCR is an enzymatic reaction, and therefore false-negative results may have been obtained due to the presence of inhibitors commonly found in the gastrointestinal tract, such as calcium ions, lipids, bile salts, and urea (41–43). Inhibitors may also be introduced during the processing or extraction of nucleic acid. Ethanol residues and ionic detergents were used in the DNA extraction protocol of this study. However, we strictly followed the manufacturer’s recommendations, and the same treatment was used for all samples, including PBMC samples. This allows us to infer that the possible presence of inhibitors may be more related to the sample origin site than to the process applied to them. Inefficient amplification can occur if an insufficient amount of DNA is used or may be due to the presence of inhibitors in the extraction product. The presence of such inhibitors may reduce the efficiency and/or reproducibility of PCR, contributing to inaccurate results (42). Inhibitors generally exert their effects through direct interaction with DNA extracted even or by interfering with thermostable DNA polymerases. Direct binding to the DNA strand can prevent amplification and even cause the inhibitor to escape the inhibitor purification step that exists in the extraction process. Adding PBS to the samples from the oral cavity allowed us to reduce the action of saliva on the DNA present, as previously stated (44).
The presence of FeLV nucleic acids in the population of this study was similar to the occurrence found in 2011 by Coelho et al. (34), i.e., 47.5%, for the municipality of Belo Horizonte, Minas Gerais. However, the samples analyzed in 2011 were mostly from local veterinary clinics, which could lead to an overestimated rate of positivity since most of the animals were ill. Our concomitant evaluation of viremia and proviral DNA in the present study determined the rate of each stage of infection. Of the 145 PBMC samples evaluated, 73 (50.34%) were negative and 72 were positive (49.66%) (Tables 2 and 3). Since we did not evaluate the presence of anti-FeLV antibodies, negative animals were classified as never exposed to virus or as an abortive infection. Among the positive results, 32 animals had viremia detectable by the PoC test at the time of evaluation, corresponding to 22.07% of the total number of animals (32/145). We classified these 32 cats as showing progressive infection. The remaining 27.59% (40/145) of the positive animals only presented proviral DNA, with no antigenemia detected by the PoC test, and therefore the infection status was classified as regressive. These results stress the importance of molecular diagnosis for the confirmation of FeLV infection, especially in animals determined to be negative by rapid tests. It is not possible to compare these two diagnostic platforms since their targets are different. However, similar to the quantification of viral load by qPCR, the association of these two techniques provides clinicians and owners valuable information about the prognosis of infection. We identified one cat with positive results in the PoC test, which did not present proviral DNA in any of the PCRs used in this work. This allows us to conclude that the rapid test presented a false-positive result. Although it was an isolated case, p27 positivity in clinically healthy animals should also be tested for confirmation of infection.
Cats with regressive infection do not shed virus and thus are not considered a threat to other cats (8). For many years, the epidemiological role of animals that suppressed the infection was neglected, mainly because of a lack of techniques capable of detecting them. Thus, the role of proviral DNA in the transmission of FeLV is still poorly understood. The amplification of FeLV proviral DNA from mucosal samples in this study demonstrates that viral genomic material is readily shed to the environment and may represent a potential source of transmission of virus between a carrier and a susceptible individual. It is known, for example, that blood transfusion between a regressively infected donor, with only detectable proviral load, and a susceptible cat is capable of infecting this individual (7). The proviral DNA contains all the genetic information necessary for the synthesis of new viruses. Infection of a healthy cell by phagocytosis of infected cells has already been described for human T-cell lymphotropic virus (HTLV) (45). In an in vitro study, the authors showed that dendritic cells, which are antigen-presenting cells present in mucosae and peripheral blood, were satisfactorily infected when exposed to HTLV type 1. In addition, these authors found that infected cells produced new detectable viral particles in the supernatant. To confirm this finding, a cell culture was treated with reverse transcriptase inhibitors, leading to a reduction of 95% of HTLV proviral DNA. A similar evaluation should be performed for samples containing FeLV proviral DNA.
Considering only cats with progressive infection, 100% (26/26) of the conjunctival-swab samples evaluated were concordant with the reference test using PBMC. It shows that viremic animals are perfectly identified in this sample by PCR. Agreement for the oral swab was lower, with a value of 93.75%, but it was still extremely satisfactory. We observed that the concordance of RS-PCR with PBMC-PCR was the lowest, at 89.29% (Table 5). Although these are high values, we expected more significant results since the gastrointestinal tract is one of the main viral replication sites (14, 40). However, as discussed before, the presence of inhibitors of PCR commonly found in the gastrointestinal tract can exert negative effects on DNA amplification (41–43).
It is important to consider that in this study, we did not evaluate whether the viral RNA is also easily detected by the same mucosal PCRs. We classified the status of infection by detecting p27 and proviral DNA simultaneously in blood, but a follow-up is highly recommended for progressively infected animals. These cats are more likely to develop FeLV-related diseases, and most of them will die within a few years (8). Viremic cats also play an important role in the transmission of FeLV to other cats since they shed high loads of virus into the environment. Westman et al. (46) evaluated the use of saliva in three different PoC tests available in Australia and, although a high specificity found (100%), the sensitivity when using saliva was only 54%. Studies assessing the value of material from mucosa samples for viral RNA detection are required to complement the diagnosis of FeLV using samples from the mucosae assessed here. However, Gomes-Keller et al. (40) demonstrated that antigenemic cats consistently shed FeLV RNA in saliva, in a strong correlation to the presence of viral RNA in plasma. Thus, progressively infected cats could be identified by reverse transcription-PCR with a high level of agreement with the detection of the capsid protein p27 in plasma by ELISA (12). The authors also showed that FeLV RNA present in oral swabs was stable for more than 2 months at room temperature, adding more value to this type of sample. It is important to highlight that these alternatives proposed here and by other authors (12) should be considered, so far, as screening tests. Quantification of plasmatic proviral DNA and viral RNA loads is valuable from a clinical perspective since it can be used as a prognosis marker.
For negative animals, the agreement between PBMC-PCR and OS-PCR was 97.26%, the agreement between PBMC-PCR and RS-PCR was 95.77%, and the agreement between PBMC-PCR and CS-PCR was 92.59% (Table 5). The high values of specificity reveal that these samples are excellent alternatives for use in PCR screening tests. False-positive results among negative animals can be related to the occurrence of contamination during extraction or DNA amplification processes. However, we conducted all PCR mixture preparations under appropriate aseptic procedures (equipment and operator), and the addition of the DNA template of each sample into the tubes was carried out in an isolated installation to that of mixture preparation. Every amplification step had the internal controls checked (negative control, positive control, and blank control). False-positive results can also occur due to transient viremia or a proviral load under the assay’s limit. The positive result for PCR of mucosa samples in discordance with PBMC-PCR represents the complexity of FeLV infection pathogenesis. As shown by Helfer-Hungerbuehler et al. (3), the proviral load can be higher in tissues than in blood in some individuals, mainly in those presenting with regressive infection. This difference in detection can also be due to an early stage of infection or even a focal infection. Since we did not clinically follow-up these animals or retest them during the course of this study, this assumption could not be proved nor excluded. Interestingly, corroborating this complex dynamic, only PBMC-PCR was able to identify the presence of proviral DNA in five animals (5, 6, 10, 59, and 62) (Table 8).
Taken together, our results show that the use of oral, rectal, and conjunctival mucosal swabs is a promising tool for the diagnosis of feline viral leukemia, especially in animals with progressive infection and for places where the presence of a veterinarian is limited. Nonetheless, improvements in technique are needed. The kit used for extraction of total DNA does not cover the extraction of nucleic material from feces; the use of a kit specific for this type of sample would thus improve the quality of the extracted DNA in rectal mucosa. This may yield an improvement in the sensitivity and specificity values for these samples. Another limiting factor we observed using swabs is that, by the methodology applied, little or no collected material is available for a new acid nucleic extraction. The volume of sample remaining inside the microtubes after removal of the rods is variable. This is due to the retention of liquid by the material of which the rod is made. We believe that duplicated collection then becomes a valuable alternative to guarantee enough sample for a second evaluation.
To our knowledge, this is the first study evaluating the efficiency of FeLV diagnosis infection by PCR in three different mucosal samples. In addition to being easily applicable, the high sensitivities and specificities found show that the proposed sampling methods are excellent alternatives to the traditional venipuncture and can be performed by veterinarians, technicians, and owners. The rate of positive animals detected by rapid testing in relation to the total number of animals carrying the viral genomic material reinforces the importance of a confirmatory test for the determination of infection by FeLV. Our study also showed that animals with regressive infection are able to shed cells containing FeLV proviral DNA, revealing a potential source of transmission in these individuals.
ACKNOWLEDGMENTS
The authors declare that they have no conflicts of interest.
We acknowledge the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior–CAPES, the Conselho Nacional de Desenvolvimento Científico e Tecnológico–CNPq and the Fundação de Amparo à Pesquisa do Estado de Minas Gerais–FAPEMIG. J.K.P.D.R. is CNPq fellow recipient.
FOOTNOTES
- Received 31 July 2019.
- Returned for modification 30 August 2019.
- Accepted 8 November 2019.
- Accepted manuscript posted online 20 November 2019.
- Copyright © 2020 American Society for Microbiology.
