Characterization of Egyptian Isolates of Canine Distemper and Canine Parvoviruses

This study was aimed to investigate the incidence of CD and CP viruses in Cairo, Egypt. In a private Vet. Clinic in Cairo, five local breed puppies of about 3-5 months of age were supposed to be infected with the virus of canine distemper (CDV) showing fever, runny nose, salivation and coughing. Another 6 German Shepard and 4 Gryphon puppies of about 6-9 months of age were suffered from fever and bloody diarrhea suspected to be infected with canine parvovirus (CPV). All of these puppies had no history of vaccination. Trials of virus segregation in Vero and MDCK cell lines and usage of virus neutralization test (VNT) using specific anti-CD and anti-CP sera revealed that 3 out of 5 nasal swabs were positive to CD virus and 3 out of 10 fecal swabs were positive to CPV. These results were confirmed by Rt-PCR showing positive amplification with CD and CP, respectively. So, we could say that VNT and Rt-PCR are essential assays to identify CD and CP viruses and puppy vaccination should not be neglected to protect them against such diseases. ـــــــــــــــــــــــــــــــــــــــــ


INTRODUCTION
Infectious diseases threaten dogs like other animal species. Viral infections represent the most dramatic diseases affecting dog populations as Canine Distemper (CD) and Canine Parvo (CP). They usually end with death if the infected dogs did not crescive the correct treatment and management.
Canine distemper virus infects the wild and domestic species of dogs, pandas, coyotes, wolves, foxes, skunks, raccoons, and large cats (Ikeda et al., 2001). CD affects several body systems, as well as the respiratory and gastrointestinal tract, the brain and spinal cord, with common signs that include feverish, eye inflammation, discharges from nose and eye, difficult or labored breathing, coughing, nausea, diarrhea, loss of zest, laziness and hardening of nose and footpads. The CD infection can be accompanied by secondary bacteriological infection and can present eventual earnest neurological signs (Ikeda et al., 2001).
Canine distemper virus (CDV) has RNA (single-stranded) and belongs to the family paramyxoviridae. The disease is highly contagious via inhalation with morbidity and deaths differ greatly between animal species, with up to 100% doom in nonvaccinated populations. The seriousness and duration of the disease are contingent on the animal's age and the immune state and the degree of virulence of the virus. The distemper signs fluctuate widely from no signs to moderate respiratory signs to severe acute pneumonia with nausea, diarrhea mixed by blood, and death. Other signs may be observed as a runny snoot, diarrhea, vomiting, lack of body fluids, excessive salivation, coughing and/or difficult breathing, loss of zest, and weight loss. Also, there are signs related to the central nervous system including confined involuntary twitching of muscles seizures with excessive salivation and palate movements commonly termed as "distemper myoclonus" or "chewing-gum fits" (Andreas et al., 2015).
The animal may exhibit sensibility to the lighting, incoordination, augmentation sensibility to voluptuous stimuli such as pain or touch, and retrograde motor capabilities. Less commonly, paralysis and blindness may have occurred (Green, 2012).

Reverse
transcription-polymerase chain reaction (RT-PCR) can be used to reveal viral RNA in respiratory tract secretions, cerebrospinal liquid, stool and urine. This test provides a quantitative measure of the CDV viral load, which is usually much higher during active infection compared to the level detected due to recent vaccination. A stand-alone test is too obtainable for quantitative distemper virus data from collected swabs of respiratory mucosa, preferably deep pharyngeal (University of Wisconsin Madison Shelter Medicine, 2010). The current work aimed to isolate and characterize CD and CP viruses recently affecting dogs in Cairo Province, Egypt.

Diseased dogs:
Five puppies of an age range 3-5 months (Local breed) showed fever (39-40 O C); a runny nose, excessive salivation, coughing and/or difficult breathing and loss of appetite suspected to be infected with canine distemper virus. Ten other puppies of an age range 6-9 months (6 German Shepard and 4 Gryphon) were suffered from high fever (41 O C), enteritis with vomiting and bloody diarrhea. Two of these puppies were dead within 3 days. Such signs are suspected to be CPV infections. These puppies were found in a private Veterinary Clinic in Cairo without vaccination history.

Type of Samples:
Five nasal swabs were obtained from suspected CD-infected puppies while ten fecal swabs were obtained from suspected CP-infected puppies and subjected to trials of virus isolations.

Cell cultures:
African green monkey kidney and Madin-Darby Canine Kidney Epithelial (MDCK) cell lines propagated with Minimum Essential Medium supplemented with 10% newborn calf serum were used for trials of isolation of CDV and CPV, respectively. 100 microgram of streptomycin and 100 IU of penicillin-G sodium/ml were added to all cell culture media.

Propagation, detection and isolation of CDV and CPV using cell cultures:
Isolation

Serologic Detection and Identification of CDV and CPV using of Virus neutralization test (VNT):
The obtained CD and CP virus isolates were identified through the application of VNT on the third virus passage of each virus using specific anti-CD and anti-CPV-2 sera according to Yoneda et al., (2008)

6.Primers used:
The used primers for detection of CD and CP viruses in the present study are tabulated in table (1)   Viral RNA (from Canine distemper isolates) was extracted from the purified virus (Sambrook et al., 1989), by QIA amp Viral RNA Mini Kit (Qiagen Germany, cat #52904), according to the manufacture instructions.

7.2.Extraction of CPV viral DNA
Viral DNA (from parvovirus isolates) was extracted from the purified virus (Sambrook et al., 1989) by Gene JET Genomic DNA Purification Kit (Thermo Scientific Cat. # K0721) as stated by the manufacturer's instructions and stored at -20°C till used.

QRT-PCR:
The amplification response was completed in MX3005P real-time PCR (Agilent, USA). The reaction mix per 25µl was 10 ng of the extracted RNA, 12.5µl of 2XBrillient II one step qRT-PCR master mix (Agilent cat # 600809), 50pmol of each primer and 100 pmol of each probe. The cycling condition was as adjusted at 50°C/30min for reverse transcription and initial denaturation phase at 95 °C for 10 minutes, 40 cycles of 95°C for 15 seconds, 50 °C for 20 seconds, and 70 °C for 20 seconds, the fluorescence emission for FAM was adjusted to be collected at the terminus of each extension stage.

CONCLUSION
Depending on the data of the present work, it could be concluded that neglection of puppy vaccination against infectious diseases; especially those of dramatic forms as CD and CP; resulted in puppy infection, which could spread the viruses in the environment and disease outbreaks. So, it could be stated that virus isolation and identification using specific and accurate assays as VN and RT-PCR are essential steps to reach correct disease diagnosis aiming to control infectious diseases. In addition, identifying the potential risk factors of CD and CPV infections may help apply preventive measures.

Declaration of Conflicting Interests
The authors revealed that there was no potential conflicts of interest.