Diagnosing Canine Parvovirus Infection

            Although many skilled clinicians deem the clinical signs associated with Canine Parvovirus infection as being nearly pathognomonic, precise diagnostic tests are required in order to differentiate Parvovirus infection from other diseases with a similar presentation.  One thing to keep in mind with each of the tests, however, is that each test possesses its own unique limitations, and are not always guarenteed to yield an accurate diagnosis.  In every scenario, a fecal sample is obtained from the animal suspected of being infected, and is assayed in order to determine if infection is present.  The methods of detection and diagnosing are:

 IDEXX Snap Parvovirus Test

            IDEXX Snap Canine Parvovirus Fecal Antigen Immunochromatography Assay

             The IDEXX Snap Test is a popular choice among many practicing veterinarians due to the fact that the test is rapid and can be performed in-clinic. 

             The main limitation of this method of testing is that large amounts of viral antigen are required from the patient in order to generate a true positive result on the test¹.  False negative results are common in animals that are not shedding large amounts of the virus, however, at the peak of clinical signs for parvovirus infection, sufficient amounts of viral antigen are being shed to yield a clear result on the test1.  Sensitivity is 100% and specificity is 99.9%2.

 
            Conventional and Real-Time Polymerase Chain Reaction (PCR)

Electrophoresis Gel     

            Conventional and real-time PCR are the most sensitive techniques for demonstrating the presence of parvoviral DNA.  These techniques utilize specific primers and enzymes to catalyze the replication of the Parvovirus genome from low to high concentrations.  The DNA product can then be analyzed by agarose gel electrophoresis to confirm that the patient is infected with Parvovirus1.

             Real-time PCR (RT-PCR) differs from conventional PCR in that RT-PCR is more sensitive and specific, more reproducible, and allows the detection and quantification of the Parvoviral genome within a few hours1.

             The central limitation of conventional and real-time PCR is that it is confined to specialized laboratories with the necessary equipment.  In addition, because the PCR can amplify very low levels of DNA, strict protocols are necessary in order to ensure that false positive results are avoided1.

             PCR is capable, however, of detecting the disease prior to the actual onset of clinical signs, and days before a positive result would be registered by the IDEXX immunochromatography assay3.  It is therefore a powerful tool in determining if a patient suffers from Parvovirus before the onset of clinical signs.  In addition to this, by employing specific techniques, PCR can yield the specific parvoviral serotype that an animal is afflicted with1.  Although this is not useful in the clinical treatment of the disease, it may affect the prognosis for the patient, as well as be important for research and epidemiological purposes.



ELISA Mechanism
          
          Double Antibody Sandwich (DAS) ELISA

             The DAS-ELISA employs monoclonal antibodies against a specific antigen of the Parvovirus4.  Primary antibodies trap the viral molecules while secondary antibodies conjugated to an enzyme system allowing quantification, "sandwich" the viral particles.

             This technique can allow quantification of the viral particles, while being sensitive and specific for Parvovirus.  The technique is relatively easy to perform, and can be adapted for the rapid processing of a large number of samples, which makes it advantageous when analyzing large groups of animals5.

             This technique, however, is also limited to a laboratory setting, and is not practical in a clinical setting, without the necessary laboratory personnel and equipment.

           

            Virus Isolation

Immunofluorescence

           
This technique is also limited to laboratory settings due to the need for specialized equipment.  It involves obtaining the supernatant from fecal  homogenates of suspected individuals and inoculating it into specific cell types.  Following an incubation period, the cells are transferred to glass slides and were treated with immunofluorescent (IF) antibodies against Canine Parvovirus.  Cells affected by Parvovirus will possess a characteristic fluorescent colour, indicating that the parvovirus suspect is positive1.

            Although this technique is highly accurate at diagnosing the disease, its main limitation is the time until a definitive diagnosis can be made.  Virus isolation and IF microscopy can take up to a week to complete, which can significantly reduce a patient's prognosis if treatment is delayed until the test result is obtained.




             Hemagglutination Assay
Hemagglutionation

             This technique utilizes a  property of parvoviruses when placed in solution with red blood cells.  The viral particles bind to molecules on the surface of red blood cells, preventing the cells from settling out of suspension 6.  A sample from an animal suspected of being affected with parvovirus is repeatedly diluted 1:2 with PBS.  This solution is mixed with erythrocytes, and after a 4 hour incubation at 4°C, the erythrocyte-viral mixtures are examined 1.  At dilutions in which agglutination of the erythrocytes is prevented by the presence of parvovirus, the red blood cells remain in solution (wells 1, 2, and 3 in the accompanying figure).  If the viral concentration is too low to prevent hemagglutination, a small disc of red blood cells will form (wells 4 through 10 in the accompanying figure).

             The method of testing is relatively inexpensive, is rapid to perform, and results are easy to interpret.  By running a standardized dilution of a known concentration of virus, and comparing the results of all of the patient's tested to the standard, an approximate viral titre can be determined.  The main limitation of the test is that the method of quantification is not accurate, and a positive result is not necessarily indicative of Parvovirus.  Several viral species can cause hemagglutination.  This assay, in combination with the appropriate clinical signs, is only indicative of Canine Parvovirus.


                References:
            

             1
Desario C, Decaro N, Campolo M, Cavalli A, Cirone F, Elia G, Martella V, Lorusso E, Camero M, Buonavoglia C.  Canine parvovirus infection: Which diagnostic test for
                virus?  Journal of Virological Methods.  2005.  126: 179-185.

             2 IDEXX Laboratories Inc.  Online Manual – http://www.idex.co.uk/animalhealth/testkits/parvo/.

             3 Ozkul A, Keles I, Karaogw T, Sabalar M, Burgu I.  Detection and RFLP Analysis of canine parvovirus (CPV) DNA by polymerase chain reaction (PCR) in a dog.  Turkey    
                Journal of Veterinary Animal Science.  2002.  26:1201-1203.

             4 Rimmelzwaan G, Juntti N, Klingeborn B, Groen J, UytdeHaag F, Osterhaus A.  Evaluation of  ELISAs based on monoclonal antibodies for the serology and antigen detection
                in canine parvovirus infection.  Veterinary Quarterly.  1990.  12(1): 14-20.

             5 De Iba ez R, Cortes E, Simarro I, Vela C, Casal I.  Development of new methods of canine parvovirus detection : ELISA DAS and immunocromatography one step.    
                
Medicina Veterinaria.  1996.  13(12):665-671.

             6 Sanna A, Fadda G, Turano A.  Viral hemagglutinins and viral hemagglutination.  L’igiene Moderna.  1967.  60(3):159-219.