Diagnosis of Canine Parvo Viral Infection in Dogs by Using Haemagglutination and Haemagglutination-Inhibition Tests
K.Basava Reddy1*, B. Shobhamani2, B.Sreedevi3, D.Rani Prameela4, B.Sudhakara Reddy5
1 Assistant Professor, Department of Veterinary Medicine, N.T.R.College of Veterinary Science, S.V.V.U., Gannavaram, Andhra Pradesh, India.
2 Professor, Department of Veterinary Medicine, C.V.Sc., S.V.V.U., Tirupati, Andhra Pradesh, India.
3 Professor, Department of Veterinary Microbiology, C.V.Sc., S.V.V.U., Tirupati, Andhra Pradesh, India.
4 Associate Professor, Department of Veterinary Microbiology, C.V.Sc., S.V.V.U., Proddatur, Andhra Pradesh, India.
5 Assistant Professor (Veterinary Medicine), Teaching Veterinary Clinical Complex, C.V.Sc., S.V.V.U., Proddatur, Andhra Pradesh, India.
Department of Veterinary Medicine,
N.T.R.College of Veterinary Science,
S.V.V.U., Gannavaram, Andhra Pradesh, India.
Article Type: Research Article
Received: February 01, 2015; Accepted: March 04, 2015;Published: March 05, 2015
Citation: K.Basava Reddy et al., (2015) Diagnosis of Canine Parvo Viral infection in Dogs by Using Haemagglutination and Haemagglutination- Inhibition Tests. Int J Vet Health Sci Res. 3(3), 46-48.
Copyright: K.Basava Reddy© 2015. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.
Canine parvo virus (CPV) is a significant worldwide canine pathogen. The present communication reports standardization of Haemagglutination Test (HA) and Haemagglutination-inhibition test (HI) for diagnosis of canine parvo viral infection in dogs in Tirupati, Andhra Pradesh, India.
3.Materials and Methods
4.Results and Discussion
CPV, Dogs, HA, HI, Tirupati.
Canine parvo viral (CPV) enteritis is an acute life threatening infection. It is highly contagious and represents one of the most common causes of acute hemorrhagic diarrhea in pet dogs. The virus is generally shed extensively for 7–12 days, but long-term excretion may occur as well. The disease is observed mainly in 6–12 week old pups and spreads from infected to susceptible dogs . Although the clinical cases of CPV are diagnosed by clinical signs, the confirmation must be done in the laboratory. HA and HI tests are simple, sensitive, relatively inexpensive and easy to perform . Hence, in the present study HA and HI tests were used for rapid diagnosis of CPV infection which is necessary for immediate treatment.
Materials and Methods
During the six months of study period faecal samples from 217 dogs with haemorrhagic gastroenteritis were collected from the clinics in the College of Veterinary Science, Tirupati (Figure.1 and Figure.2).
Haemagglutination Test (HA): The faecal samples or rectal swabs were emulsified in 1ml of 0.2M PBS of pH 7.0 and centrifuged at 6000 rpm for 20 min at 4°C. The supernatant was collected and stored at – 4°C until further use. The test was performed as per the Carmichael . Two fold serial dilutions of 50 μl of the sample were made in 0.2M Sorenson’s PBS of pH 7.0 in a 96 well ‘U’ bottom microtitre plates. To each well 50 μl of 1percent pig erythrocytes were added, mixed gently and allowed to settle at 4°C for 4hrs. One well, added with 50 μl of 0.2M Sorenson’s PBS of pH 7.0 and 50 μl of one percent pig erythrocytes, served as RBC control. The CPV vaccine virus (MegaVac- P) was used as a positive control and the faecal samples from healthy dogs were used as negative control. The highest dilution of sample showing complete haemagglutination was considered as the haemagglutination titre. In the present study the HA titre of 32 and above was considered as positive for CPV antigen.
Haemagglutination inhibition test (HI): Hyper immune serum against CPV was raised in the rabbits using the commercial vaccine (MegaVac-P) as per the method of Ramadass and Khader . One milliliter of emulsion prepared by mixing a single dose of MegaVac-P (Ag) vaccine and 1.0 ml of Freund’s adjuvant was injected subcutaneously to an apparently healthy rabbit. Another two injections of the freshly prepared emulsion of the antigen were given subcutaneously at an interval of 15 days. Freund’s complete adjuvant (FCA) was used for the first injection where as Freund’s incomplete adjuvant was used for subsequent injections. About 7 ml of blood was collected from the heart of the rabbit after 7 days of the last injection and the serum was separated and used for detection of parvo viral antigen. The hyper immune serum was inactivated at 56°C for 30 minutes in a water bath. Serum was centrifuged at 1500 rpm for 15mins. Then 50 μl of PBS was dispensed into each well numbered from 1 to 12 and 50μl of 1/10 diluted serum was taken in to first well with the help of micropipette and mixed well. Then 50 μl mixers were taken from first well and dispensed into the second well. The process was repeated till the last well and discarded 50μl from the last well and 50 μl of 4 HA dilution of antigen was added to each of the well. 50μl of the 1% pig RBC suspensions was added into all the wells. The plate was shaked and incubated at 4°C for 2hr. The HI titre was expressed as the reciprocal of highest dilution of serum inhibiting agglutination.
Results and Discussion
Out of 217 faecal samples screened for CPV antigen by HA test, 72 samples were agglutinated pig RBC with titres ranging from 32-1024 (Figure.3). All the 72 positive samples were further confirmed for CPV by HI test (Figure.4) (Table-1).
Although the clinical cases of CPV are diagnosed by clinical signs, the confirmation must be done in the laboratory. There are many diagnostic tests like virus isolation, electron microscopy, haemagglutination (HA), haemagglutination inhibition (HI), ELISA, polymerase chain reaction (PCR), immunochromatography, agarose gel precipitation test and immunoflourcent test (IFT) etc. however, a prompt diagnosis from the laboratory is beneficial to the clinical practitioner for immediate initiation of suitable therapy. The virus isolation is highly specific and gold standard, but time consuming and expensive method. The PCR has been applied to the detection of CPV-2 in faecal samples with high sensitivity and specificity but it is time consuming and expensive. Agarose gel precipitation test needs a higher concentration of virus for detection of antigen. HA and HI tests are simple, sensitive, relatively inexpensive and easy to perform, has its limitation in the requirement of continuous source of reactive RBC’s and the need to monitor specificity of low titred reaction with haemagglutination inhibition assay .
Faecal haemagglutination with pig RBC gave HA titres ranging from 32 to 1024. Previously Carmichael and Mathys stated that HA titre of 32 and above were diagnostic of CPV in dogs [3, 5]. In the present study all the faecal samples positive with the HA test were further confirmed by HI test. The appeal stated that inhibition of haemagglutination using specific antiserum was a convenient method for identification of CPV . HA and HI tests are simple and rapid as the test results can be obtained in 2-4 hours. Its limitation is the requirement of continuous source of reactive RBCs and the need to monitor specificity of low titred reaction with haemagglutination inhibition assay . In the present study also canine parvovirus took 2-4 hours to agglutinate the pig RBC at 4°C. These findings were correlated with the previous studies .
The present research was undertaken to diagnose the CPV by using HA, HI. The HA and HI tests were found to be simple and rapid screening tests for detection of CPV infection through faecal sample and also economical.
Authors are thanking full to the officers of the S.V.V.U for providing the facilities required for the present work. Corresponding author expressed special thanks to the Dr. B. Sudhakara Reddy for his cooperation while writing the articles.
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