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Comparative Evaluation of Immunochromatographic and Reverse Transcriptase Polymerase Chain Reaction based tests for Diagnosis of Canine Distemper.

Introduction

Canine distemper virus (CDV) is an enveloped, single-stranded, negative sense, RNA virus belonging to genus Morbillivirus in Paramyxoviridae family. It causes a highly systemic disease with prominent respiratory, gastrointestinal and nervous signs in dogs (Patel and Heldensb, 2009). The virus primarily replicates in lymphatic tissues of respiratory tract and subsequently reaches various organs, including cells of lower respiratory and gastrointestinal tracts, lymphoid organs, urinary bladder and central nervous system (Appel, 1987). This results in sub-clinical infection or combination of respiratory, ocular, gastrointestinal, neurologic and cutaneous signs or lesions (Greene and Appel, 1998). It's a highly immunosuppressive virus and thus increases host's susceptibility to several opportunistic infections, which are a main cause of distemper associated deaths (Pawar et al., 2011). Canine distemper is widely prevalent in tropical countries like India. The disease is characterized by rapid onset of severe leucopenia and loss of lymphocyte proliferation ability. Various laboratory techniques like dot ELISA, virus isolation and characterization have been attempted in the past for its diagnosis (Parthiban et al., 2000; Ramadass and Latha, 2001; Pawar et al., 2011). CDV related disease in canine population around the world seems to have increased in last decades either because of use of inappropriate vaccines (Kommonen et al., 1997) or due to increasing trend in illegal commercial sale of pups (Elia et al., 2006).

Laboratory confirmation of CDV infection is necessary as there are multiple clinical signs of disease which are overlapping with other respiratory and enteric diseases of dogs, thus hampering clinical diagnosis (Jones et al., 1997). A sensitive, specific and rapid method is desirable to detect even small amounts of virus early in infection owing to highly contagious nature of CDV, its pathogenesis and mortality rates. Reverse-transcriptase-polymerase chain reaction (RT-PCR) has been applied successfully for detection of canine distemper virus (Agnihotri et al., 2017; Frisk et al., 1999; Saito et al., 2006; Shin et al., 2004) in various clinical samples such as faeces, whole blood, serum, cerebrospinal fluid etc. However, molecular tests in spite of their sensitivity and specificity are time consuming, labour intensive and require expertise (Pereira et al., 2000). This has led to development of various rapid field level diagnostic test kits based on the principle of immunochromatography (IC) (Tayib, 2014). These tests are rapid and have a user-friendly format (Vakili et al., 2014). This study is an attempt to compare polymerase chain reaction based molecular tests with commercially available IC based test for diagnosis of Canine distemper by testing rectal swabs and serum samples respectively from dogs suffering from gastroenteritis and suspected for Canine distemper.

Materials and Methods

Fifty gastroenteric dogs suffering from vomition and diahorrea less than one year of age were included in the study. Faecal samples from affected dogs were collected with sterile swabs in sterile PBS. Viral RNA was extracted using Trizol method followed by which c-DNA was prepared. Published primers targeting N gene (Frisk et al., 1999) as well as designed primer pair targeting amplification of H gene of CDV was used for screening of faecal samples separately. Primers were designed using Bio-edit sequence alignment software and primer 3 plus software. In this, sequences of different isolates related to CDV were retrieved from Genbank and aligned for achieving the maximum conserved region. Primers of length 20-24 bp having G-C content within the range of 40-60 percent were selected. The specificity of sequences was obtained and nucleotide variations and amino acid variations with respect to H gene sequences of CDV were determined using BLAST (Basic Local Alignment Search tool). cDNA of extracted faecal RNA samples was prepared using 'Revert Aid first strand cDNA synthesis kit' by Thermo-scientific (K1622) and was stored at -20 (o)C till further use. To standardize the optimum annealing temperature for maximum amplification of H gene of CDV, gradient PCR was run using positive control with designed primers set of CDV (Table 1) i.e. p (CD/ H/ F) and p(CD/H/R) at temperatures 46[degrees]C, 48[degrees]C, 50[degrees]C, 52[degrees]C, 54[degrees]C and 56[degrees]C. Maximum amplification of desired H gene was obtained at 50[degrees]C with PCR product of size 665bp. Commercially available live attenuated multi component vaccines for CDV (Vencomax (a)) served as positive control.

PCR was performed in Thermal cycler (Veriti, Applied Biosystem) in 12.5 [micro]l reaction containing 3 [micro]l of template DNA, 6.25 [micro]l of Mastermix 2X concentration and 2.25 [micro]1of nuclease free water. Cyclic conditions used for pCD/ N primers were as described by Frisk et al., 1998. Cyclic conditions for pCD/ H primers included one cycle of initial denaturation at 94[degrees]C for 5 min, followed by 40 cycles of 94[degrees]C for 30 sec, annealing temperature of 50[degrees]C for 1 min and extension at 72[degrees]C for 1 min and a final extension at 72[degrees]C for 15 min. The PCR products were analyzed in 1.5 percent agarose gel, visualized under UV transilluminator in Gel documentation system (BIO-RAD) and documented by photography for further analysis. Immunochromatographic test was carried out using CD antigen detection kit (Scanvet Distemper (b)), following the manufacturer's instructions. The serum samples were gently mixed with equal quantity of assay diluents provided with the kits. Four drops of sample-diluent mix was added into the sample hole. The results were interpreted within 10 min. The appearance of a color band was suggestive of proper working of test. The left section of the result window indicated test results (T). The presence of only one band the right of result window (C) indicated a negative result. The presence of two color bands (T and C) within the result window indicated a positive result.

Results and Discussion

A total of 50 faecal samples from dogs suspected of viral gastroenteritis were screened for presence of CDV using published primers (Frisk et al., 1999), targeting N gene and designed primers targeting H gene. Only single sample (2%) was found positive out of 50 screened samples on targeting both N (Fig. 1) and H gene (Fig. 2) of CDV showing a band size of 287bp amplicon and 665bp amplicon respectively with RT-PCR. On the other hand, using IC kits, thirteen out of fifty serum samples (26 percent) of gastroenteric dogs were found positive (Fig. 3) for canine distemper infection including the same sample which was found positive with RT-PCR.

Diagnosis of canine distemper virus by immunofluorescence (IF) is applied to various specimens, including conjunctival, nasal and vaginal smears, using polyclonal or monoclonal antibodies. Immunofluorescence test is not sensitive and can detect CDV antigens only within three weeks after infection (Appel, 1987). Serological methods, such as seroneutralization (SN) assays have little diagnostic value as they give false positive results and diagnostic techniques like virus isolation are fastidious and time-consuming (Shin et al. (1995); Frisk et al., 1999; Kim et al., 2001). Frisk et al., 1999 used RT-PCR to detect viral nucleocapsid protein in various fluids like serum, whole blood and CSF using three different sets of primers, instead of using faecal samples. With the most sensitive primer pair set, they detected CDV nucleocapsid protein gene in 25 of 29 (86 percent) serum samples and 14 of 16 (88 percent) whole blood and CSF samples.

The nucleic acid detection system applied in present study proved to be only slightly sensitive for detection of CDV in faecal samples. However, Shin et al. (1995) and Frisk et al. (1999) reported that sensitivity of RT-PCR varied between selected primers, depending on their position in the gene. In present study, the reason for detection of only one positive faecal sample from diarrheic dogs for CDV using RT-PCR could be because of incapability of RNA virus to remain stable in faecal material due to activities of endogenous RNases and lack of accessibility of partially degraded RNA which may have influenced sensitivity of RT-PCR or it may be due to intermittent shedding of virus in faeces. However, use of RT-PCR with different body fluids (serum, CSF, ocular swabs, urine and whole blood) can increase sensitivity as CDV- RNA shows heterogeneous distribution in different body compartments (Frisk et al., 1999; Pawar et al., 2011).

In another study using Immunochromatography for detection of CDV, An et al., 2008 reported sensitivity and specificity of IC assay was maximal i.e. 100 percent and 100 percent, respectively relative to nested PCR, when conjunctival swabs were tested. They observed that conjunctival swab specimens were easy to obtain in early phase of CD infection and were the most suitable specimens for early antemortem diagnosis of CDV, because there is persistent shedding of CDV in eye, unlike in other bodily compartments. Conjunctival swabs from CD suspected dogs have to be collected in early phase of infection (Kim et al., 2006). However, An et al., 2008 also observed that when blood lymphocytes and nasal samples were tested, the IC assay was slightly less sensitive (89.7 percent and 85.7 percent, respectively) and specific (94.6 percent and 100 percent, respectively) than nested PCR.

A number of studies have been conducted on finding the sample types which are most appropriate for use with PCR-based methods to detect CDV. Using nested PCR analysis, one study revealed that of 22 blood, 20 urine, 25 saliva and 27 nasal swab samples from dogs suspected to have CD, 81.8 percent, 75 percent, 56 percent and 70.3 percent tested positive, respectively (Shin et al., 2004). A similar study using nested PCR that detected CDV NP RNA revealed that of 29 serum, 16 whole blood, and 16 cerebrospinal fluid (CSF) samples, 86 percent, 88 percent and 88 percent were positive, respectively (Frisk et al., 1999; Moritz et al., 2000). In contrast, hospitalized dogs with neurological disturbances but lacking typical findings of distemper were tested, RT-PCR analysis failed to detect CDV-RNA in any of the serum samples while only 1/5 (20 percent) whole blood and 2/5 (40 percent) CSF samples were positive. However, 4/5 (80 percent) urine and all 5 (100 percent) CSF samples were found positive (Amude et al., 2006).

C tests on the other hand have their limitations in terms of amount of antigen detection. It requires large amount of viral antigen to produce a clearly visible band. As a result, interpretation of results may be affected by subjectivity of test operator (An et al., 2008). These tests have an advantage over many other field diagnostic techniques because of their simplicity and rapidity. Due to these advantages, IC assays detecting specific antibodies are available for other Veterinary diseases, including CPV (An et al., 2008; Oh et al., 2006; Mohyedini et al., 2013).

In conclusion, PCR based tests are highly sensitive and specific than IC based tests; sensitivity of the molecular tests for detection of CDV depends upon several factors including sample for detection i.e. serum, blood, conjunctival or nasal discharges, urine etc., day of sampling, primers specificity and sensitivity etc. Our study is in confirmity with the observations of An et al., 2008 that IC based tests have an advantage of being rapid and easy to use without the need for special instruments. In the present study it was observed that IC test kits (Scanvet Distemper (b)) were more sensitive than RT-PCR based assays when serum samples are used for detection of CDV. The ready availability of IC assay would help to reduce the morbidity and mortality of the disease by allowing appropriate initiation of the treatment before clinically evident symptoms.

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Divya Agnihotri (1), Sushila Maan (2), Kanisht Batra (2) and V.K. Jain (3)

Veterinary Clinical Complex College of Veterinary and Animal Sciences Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS) Hisar - 125004 (Haryana)

(1.) Corresponding author. E-mail: dr_divya_agnihotri@ yahoo.co.in

(2.) Department of Animal Biotechnology

(3.) Veterinary Clinical Complex

(a) - Brand of Vencofarma Ltd., Brazil

(b) - Brand of Intas Animal Health, Ahmedabad
Table 1: Published and designed primers used for amplification of N and
H gene respectively of Canine distemper virus (CDV).

S.   Primers      Sequence                      Product  Reference
No.                                             Size

1.   pCD /N/ (F)  ACA GGA TTG CTG AGG ACC TAT   287bp    Published
     pCD /N/(R)   CAA GAT AAC CAT GTA CGG TGC            primers (Frisk
                                                         et al., 1999)
2.   pCD/H/F      AAT ATGGAATTTRGCAGATTG        665bp    Designed
     pCD/ H/R     CCCACTGCGATAGTACARAC                   primers
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Title Annotation:Research Article
Author:Agnihotri, Divya; Maan, Sushila; Batra, Kanisht; Jain, V.K.
Publication:Intas Polivet
Date:Jul 1, 2018
Words:2860
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