DEVELOPMENT AND ASSESSMENT OF A COAGGLUTINATION TEST FOR DETECTING CANINE PARVOVIRUS IN CLINICAL SPECIMENS

Ahlam Kadiri 1 , Nadia Amrani 1 , Khalil Zro 2 and Jaouad Berrada 1 . 1. Institut Agronomique et Vétérinaire Hassan II. 2. Société de Productions Biologiques et Parmaceutiques Vétérinaires (Biopharma). ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Rapid diagnosis of canine parvovirus (CPV-2) infection is of key importance in management and control of the disease. The present study aims at the development and evaluation of an easy to use rapid test for the detection of canine parvovirus particles in clinical samples collected and tested during daily veterinary practice. For this purpose, the coagglutination test (COA) using Methylene blue stained anti-CPV2 antibodies sensitised staphylococcus aureus ATCC 12598 was performed to detect CPV-2 in 91 clinical samples (86 rectal swabs and 5 organ homogenates) collected between 2011 and 2015 from suspected canine clinical cases received in Moroccan veterinary clinics. The same samples were previously tested by Hemagglutination test and real-time PCR (Amrani et al., 2016), the results were compared and employed in the evaluation of COA. The results of the COA showed 79.12% (72/91) of tested samples to be positive and 20.87% (19/91) to be negative. The intensity of reaction in positive samples was scored high (3+) in 6 samples (6.6%), medium (2+) in 28 samples (30.7%) and low (1+) in 57 samples (62.6%) which occurred within a median time of 3min, 4min and 3min42s, respectively. In comparison with PCR, the COA test demonstrated a good agreement of 80.2 % and a sensitivity of 80%. The coagglutination test is a promising test allowing daily diagnostic of canine parvovirus in field, veterinary clinics and non-equipped laboratories with molecular techniques.

…………………………………………………………………………………………………….... Introduction:-
Digestive disorders including diarrhoea and vomiting are considered as major canine health problems and the most frequent cause for veterinary consultation (Rakha et al., 2015;Ylmaz et al., 2002). Among numerous aetiologies involved in these disorders, canine parvovirus (CPV-2) infection is the most frequent and serious pathology, characterized by high morbidity and mortality rates, especially in susceptible puppies of 6 weeks to 6 months of age (Prittie, 2004). The causative CPV-2 is a non-enveloped virus with icosahedral capsid enclosing a single stranded DNA of ~5000bp characterized by a high mutation rate similar to that observed for RNA viruses (Decaro et al., 2009). Since its emergence, CPV-2 has rapidly evolved giving rise to highly pathogenic variants CPV-2a, 2b and 2c (Decaro and Buonavoglia, 2012). Vaccination remains the main tool to control and prevent canine parvovirus 12 infection. However, vaccine neutralisation by long-lasting maternal antibodies (MDA) associated with poor or absence of cross-protection between CPV-2 antigenic variants, compromise seriously the success of vaccination (Decaro et al., 2005a(Decaro et al., , 2008. Early, rapid, accurate diagnosis and management of CPV-2 is of key importance in effective reduction of infected dog's morbidity and mortality (Kim et al., 2015). Molecular diagnostic tests, notably the real-time PCR, were demonstrated to be more sensitive than traditional methods (Decaro et al., 2005b;Desario et al., 2005). However, PCR-based tests are expensive, carried out in specialized laboratories only and are difficult to establish for standard practice in veterinary clinics. Therefore, development of rapid, simple, sensitive and affordable tests detecting CPV-2 particles in clinical samples would be convenient and can be conducted in veterinary clinical practice (Kantere et al., 2015). The coagglutination (COA) test was previously developed (Genovn and Ivanov, 2006;Singh et al., 1998) by exploiting the property of staphylococcus aureus membrane protein A to bind the Fc portion of anti-parvovirus IgG which recognizes CPV-2 antigen in positive tested samples. Compared with Haemagglutination test (HA), this test was reported specific and sensitive (Genovn and Ivanov, 2006).
The aim of the present study is to develop an easy to use and interpret coagglutination assay and to evaluate its performances (sensitivity, positive predictive value, overall agreement) in comparison with real-time PCR as gold standard test for canine parvovirus detection.

Bacteria:-
Lyophilised Staphylococcus aureus ATCC 12598 was reconstituted and revived following the manufacturer's recommendations. An isolated S. aureus colony was cultured into Brain and Heart broth (BHB) for 18h at 37°C in a shaking water bath. The resulting bacterial culture was washed 3 times in PBS and inactivated by adding 0.5% formaldehyde solution at 4°C for 18h and washed and heated at 80°C for 20min. Following inactivation, bacteria were washed three times and resuspended in PBS containing 0.5% Tween 20. Using spectrophotometry at 250nm, bacterial absorbance was fixed at 0.69, corresponding to 2.3 x 10 10 cells/ml. This suspension was stored at 4°C for maximum 3 months (Montassier et al., 1994).

Production of hyper immune serum:-
As shown in figure 1, Anti-parvovirus hyperimmune serum was produced on SPF, New Zealand, male rabbit weighting 3.5kg. Briefly, three CPV-2 vaccine doses (Primodog®, CPV strain C-780916 ≥ 105 .5 DICC 50 ) were inoculated by subcutaneous route on day 0, 15 and 30, respectively. At day 40, blood was collected from the hyperimmunized rabbit and serum was separated by centrifugation. After decomplementation at 56°C for 30 min, hyperimmune serum was mixed with the previously described S aureus suspension at 2:1 (V/V) and incubated for 3h at room temperature. To facilitate the test lecture, IgG−S.Aureus complexes were stained using 0.075% methylen blue stain. The produced reagent was stored at 4°C for use during one month.
Test procedure:-The coagglutination test was performed using a glass slide by homogenising equal volumes (50 µl) of clinical samples supernatants with stained IgG−S. Aureus reagent according to the method described previously (Genovn and Ivanov, 2006). The slide was examined macroscopically during 10 min for development of agglutinin particles. The time and the intensity of the reaction (1+, 2+, 3+) were recorded (figure 2). Positive and negative controls consisting of a positive and negative real-time PCR fecal samples collected from dogs were used. The overall procedure is summarised in figure 1.

Figure 1:-Reagent preparation and specimen testing protocol
The test performances were evaluated by calculating sensitivity and overall agreement in comparison with the gold standard test, real-time PCR, carried out previously for the same samples (Amrani et al., 2016).

Results:-
Obtained results and information about tested samples are presented in table 1.
14    Yoshimizu and Kimura, 1985). We investigated the potential use of COA to detect CPV-2 in clinical samples and evaluated its performances in comparison with the PCR test.
Previous COA assays required dark background or using microscope (x10) to be read macroscopically (Yoshimizu and Kimura, 1985) with the possibility to affect the test lecture and to determine its intensity. Thus, COA using methylen blue stained reagent provides an easy alternative and more objective lecture without the use of a microscope. A good agreement (80.2%) was found between COA and PCR as a gold standard test. In addition, no false positive samples was observed with COA test compared with haemagglutination test which was shown to be poorly sensitive, resulting in a high proportion of false-negative results. Hence, the COA test developed in the present study can be considered as a good alternative for use as a bench or penside test in clinical veterinary practice.

Summary:-
We report in the current study the development of a, simple, reliable and rapid test to detect canine parvovirus in daily veterinary practice. High performances, processing of many samples, easy procedure and results interpretation make the COA an effective tool especially in field conditions and laboratories that are not equipped with molecular technology platform.