الفهرس 
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Abstract
Canine parvovirus (CPV) is the causative agent of fatal, acute hemorrhagicenteritis and myocarditis in dogsbetween 6 weeks and 4 months old.CPV-2 was emerged as a new virus infecting dogs in 1978, and it was probably derived as a variant of Feline Panleukopenia virus. CPV-2 was subsequently replaced by three antigenic variants CPV-2a, 2b and 2c which now coexist in dog populations worldwide.
In Egypt,little information is presently available on the prevalence and evolution of CPV. This study aimed to provide detailed information about thediseaseincidence andto know the circulating subtypes of CPV-2 and to understand the diversity and evolution of the emergent subtypesin Egypt.
To achieve this goal a total of 124 fecal samples from diseased dogs belong to 7 breeds were collected from veterinary clinics and animal shelters in Ismailia, Port Said, Suez, Cairo and Giza and tested for CPV by PCR and rapid chromatographic immunoassay.PCR was shown to be more sensitive than rapid chromatographic immunoassay with a total positivity percentage of 79.03 and 71.77 respectively.
Ismailia recorded the highest infection rate of CPV with a rate of 39.52% more than Suez, Cairo, Giza and Port Said with percentages of 19.35%, 12.1%, 4.03 and 4.03 % respectively. Dogs of three month age are highly susceptible to CPV infection when examined by rapid strip test and PCR with positivity percentages of 43.54% and 48.4 % respectively.
At one month and 2 month age, the susceptibility of dogs to CPV appears to be low with a 1.61 % and 14.51 % respectively.Mortalities and infection rates were higher in female dogs than male (25.8 % in female and 16.9% in male). Cross breeds are more susceptible to CPV infection than pure and native dog breeds.
To validate the real time and conventional PCR protocol for diagnosis of CPV in fecal samples, different annealing temperature were optimized (49oC, 50oC, 51oC, 52oC, 53oC, 54oC, 55oC, 56oC, 57oC, 58oC, 59oC and 60oC) to select the proper annealing temperature in PCR protocol. The optimal annealing temperature is 60oC for 15 sec. at which optical signals produced by probe hydrolysis and converted to CT value during the reaction.
Out of 124 fecal samples, 98 positive samplesgive a predicted band to CPV-2, Twenty three DNA extracts were amplified in conventional PCR based method using specific set of foreward and reverse primers to VP-2 of CPV. Amplified 908bp segment of VP-2 gene were generated,purified and sequenced and the sequence were trimmed and cleaned to get 424 bp length of VP-2 gene. This segment includes the sequence of the strategic amino acid residue at position 426 that was the basis for CPV-2 classification as different subtypes (subtype CPV-2a, CPV-2b and CPV-2c).
Based on the nucleotide sequence and amino acid present at the strategic position 426.The 23 sequences were analysed to characterize CPV-2 subtypes and to discriminate between vaccine and field strains. The sequence analysis of VP-2 showed, 17 out of 23 CPV isolates were subtype 2b and 3 isolates subtype 2a and 3 isolates of subtype 2c with percentages of 74%, 13% and 13% respectively.
CPV-2b is more prevalent in Ismailia dogs (10 isolates) than Suez (3 isolates), Port Said (2 isolates) and Cairo province (2 isolates). All CP-2a (3 isolates) were in Ismailia dogs while, CP2c in Ismailia (2 isolates) and Cairo (1 isolates). Local dog breed are infected with the three subtypes of CPV-2 and showed to be more resistantto infection than pure and mixed breeds as all infected animals were recovered.
The obtained results revealed that the three subtypes were currently circulating in the five provinces of Egyptwith the predominance of CPV-2b over 2a and 2c. Baladi dog breed are infected with the three subtypes of CPV-2 (one 2a, three 2b and one 2c) and showed to be more resistant to CPV infection as 4 dog are recovered and only one dog died. Twelve dogs of German breeds either German pure or mixed breeds are susceptible to infection with the three subtypes of CPV-2 (one 2a, ten 2b and one 2c) with a variable clinical outcome (8 dogs died and 4 recovered). Each of the 23 examined dogs infected only with one CPV-2 subtype, no dog coinfected with 2 or 3 CPV-2 subtypes at the same time.
Concerning to amino acid substitutions at residues rather than 426 in 2 isolates of CPV-2b and 1 isolates CPV-2c, it is found that isolate code 9 and code 59 of CPV-2b have amino acid substitution at residue 304 and 393 from glutamine (G) to leucine (L) and from glutamic acid (E) to lysine (K) respectively while other strain of CPV-2c code 29 have amino acid substitutions at residue 373 from aspartic acid (D) to glycine (G). No amino acid substitutions rather than 426 residues in CPV-2a isolates.
High incidence of nucleotide point mutations in CPV-2a or CPV-2b or CPV-2c strongly suggested that these strains are field variant strains rather than vaccine strain. Sequence analysis of the VP-2 gene of CPV-2a, 2b and 2c revealed 100% amino acid identity with the strains previously published in gene bank. Continuous evolution of CPV in Egypt requires aperiodic checkup with monoclonal antibody based assays in addition to nucleic acid based assays.