Nghiên cứu bệnh viêm ruột do parvovirus trên chó tại một số tỉnh thành đồng bằng sông cửu long tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL DISSERTATION Major: PATHOLOGY AND TREATMENT OF ANIMALS Major code: 62 64 01 02 NGUYEN THI YEN MAI RESEARCH ON CANINE PARVOVIRUS INFECTION IN DOGS IN SOME PROVINCES OF THE MEKONG DELTA Can Tho, 2020 i THE DISSERTATION WAS COMPLETED AT CAN THO UNIVERSITY Supervisor: Assoc Prof Dr Tran Ngoc Bich The dissertation is defendded in front of the University Examination Place: , Can Tho University Time:: Reviewer 1: Reviewer 2: Reviewer 3: Futher information of the dissertation could be found at: Learning Resource Center of Can Tho University National Library of Vietnam ii THE LIST OF PUBLISHED WORKS RELATED TO THIS DISSERTATION Nguyen Thi Yen Mai, Tran Ngoc Bich, Pham The Lam, Nguyen Phuc Khanh, 2016 Parvovirus disease in dogs at Can Tho Animal Health Clinic Office Journal of Agriculture and Rural Development No 11/2016, pages 151-155 Nguyen Thi Yen Mai, Tran Ngoc Bich, Tran Van Thanh, 2018 Situation of Parvovirus enteritis in dogs at Veterinary Offices of Tien Giang, Dong Thap and Can Tho City Science Journal of Can Tho University 54 (Agricultural Union No.), pages 136-142 Nguyen Thi Yen Mai, Tran Ngoc Bich, Tran Van Thanh, 2018 Situation of Parvovirus enteritis in dogs at Can Tho University Veterinary Clinic Journal of Veterinary Science and Technology, volume XXV Number 4, pages 36-41 Tran Van Thanh, Tran Ngoc Bich, Thai Quoc Hieu, Nguyen Thi Yen Mai, 2018 Situation of Parvovirus enteritis in dogs at Tien Giang Department of Livestock and Veterinary Medicine Journal of Agriculture and Rural Development No 24/2018, pages 102-107 Keovongphet Phuthavong, Tran Ngoc Bich, Nguyen Thi Yen Mai, Tran Van Thanh, 2018 Survey of Parvovirus enteritis in dogs at Can Tho University Veterinary Clinic Science Journal of Can Tho University 54 (Agricultural Union No.), pages 51-55 iii Chapter I: INTRODUCTION 1.1 Necessity of thesis Inflammatory bowel disease caused by Canine Parvovirus type (CPV-2) in dogs is an acute infectious disease (Kelly 1978; Appel et al., 1979; Hoelzer et al., 2008) CPV-2 is considered the most threatening to puppies from weaning to six months of age However, outbreaks of inflammatory bowel disease and dog adult mortality from CPV-2 have been reported (Decaro et al., 2008) The disease occurs in all breeds of dogs, all of ages and regardless of gender (Castro et al., 2007; Gombac et al., 2008) The disease progresses rapidly, making it difficult for dog owners to detect early, when it was detected that the dog was seriously ill and difficult to treat The main symptoms of the diseases include fever, vomiting, diarrhea, loose stools mixed with fresh blood with an unpleasant fishy odor that leads to dogs losing a lot of blood, water and electrolytes, metabolic disorders, cardiovascular collapse, kissing delirious and dead (Sherding, 2000; Joshi et al., 2001; Ramprabhu et al., 2002; Miranda et al., 2016) According to the Sub-Department of Livestock and Veterinary, Can Tho city, Dong Thap, Tien Giang and Ben Tre, at December 2018, in the Mekong Delta, the demand for raising pets of people is increasing; in which dog is one of the most favourite animal as pet For the elderly, lonely singles, children, as dog is friendly and consider pet as a companion every day, even dog is consider a part of the family Although the incidence of inflammatory bowel disease in dogs due to CPV-2 were 43.30% (Dung Nguyen Van et al., 2017) and the cure rate for symptoms were quite high at 86.30% (Le Minh Thanh, 2009) But when the pet was sicked, the days of treatment have not been cured or not cured, will be the unavoidable days that owners were worry, depression, Therefore, the study of this disease in the Mekong Delta is necessary to make the disease prevention and intervention more active in order to reduce economic losses and especially limit the emotional damage of dog owners Because of all the reasons above, thesis: “Research on inflammatory bowel disease caused by CPV-2 in the Mekong Delta region” was carried out 1.2 Thesis Objectives Genotype sequencing, establishment of phylogenetic trees, identification of infection rates, assessment of homologeneity of molecular genetic characteristics of disease-causing CPV-2 genotypes with genotypes of CPV-2 in vaccines on gene banks; evaluating the effect of treatment and protection ability of CPV-2 preventive vaccine, in order to help veterinary medicine that was oriented intervention to overcome disease symptoms, producing and using vaccines to prevent and fight against enteritis due to CPV-2 in dogs in the Mekong Delta that were effective treatment 1.3 The new distribution of thesis This is the first research project in Vietnam that basically and systematically studied a wide range of CPV-2 inflammatory bowel disease in dogs that is being widely adopted in some provinces and cities in the Mekong Delta Therefore, the result of the thesis was to establish a phylogenetic tree, determine the infection rate and the circulation of pathogenic genotypes of CPV-2 Assessing the homogeneity of molecular genetic characteristics of field CPV-2 with genotypes of CPV2 in vaccines on the gene banks and assessing protection ability of CPV2 vaccine in dogs currently circulating in some provinces in the Mekong Delta 1.4 Practical significance and applied capacity of thesis This thesis is the first basic information source of scientific foundation in Vietnam for researching about inflammatory bowel disease caused by CPV-2 in dogs in some provinces and cities in the Mekong Delta From there, this result actively supported local veterinarians to produced vaccines to prevent and against CPV-2, diagnosis, prognosis, effective treatment and scientific data for the following studies Chapter III: MATERIALS AND METHODS The thesis was carried out with contents: 3.1 Content 1: Determining the incidence of CPV-2 inflammatory bowel disease in dogs treated in some clinics of some provinces of the Mekong Delta 3.1.1 Experimental subjects: 380 dogs with symptoms of vomiting, diarrhoea, bloody diarrhea Making medical record sheets to monitor age, gender, breed and vaccination 3.1.2 Number of survey samples: 380 samples from the Veterinary Offices of Can Tho city, Tien Giang, Dong Thap and Ben Tre (each province selected two clinics), collecting samples according to the formula of Thrusfield (1995) P (1-P) 0.433 (1- 0.433) N = 1.962 = 377.262 n = (z)2 x 0.05 d2 In which: n= number of sample properties Z= value of thenormal distribution for confidence level of 95% P= expected prevalence d= absolute error of 5% The samples was presented in Table 3.1 Table 3.1: Number of samples Numerical order Place Numerical of samples Can Tho city 101 Dong Thap 90 Tien Giang 97 Ben Tre 92 Total 380 3.1.3 Research method Descriptive cross-sectional research, firstly study and analysis Collecting fecal samples of sick dogs with symptoms of vomiting, diarrhoea at Veterinary Clinics of some provinces above to identify dogs positive for CPV-2 inflammatory bowel disease by Antigen Rapid CPV test kit of American Bionote company monitors Monitoring and recording a chronic disease to prevention and treatment Every day, before the medication was gave, surveyed and recorded body temperature, diarrhea, dehydration, blood loss, fecal, recording the disease progression and make a medical record sheet for each infected dog After that, providing supportive drugs, the course of treatment for to days 3.1.4 Monitoring targets The rate of dogs with inflammatory bowel disease by CPV-2 on age, sex, breed and vaccination; frequency of the typical clinical manifestations and evaluation of treatment effect of this disease in dogs at the Veterinary Clinical Offices of the four provinces mentioned above were recorded 3.2 Content 2: Identification of genotypes of CPV-2 inflammatory bowel disease in dogs treated in some clinics of some provinces of the Mekong Delta 3.2.1 Test subjects: Selecting 80 samples with positive results for CPV-2 in content 3.2.2 Research methodology: Collecting 80 fecal samples from 80 individual dogs with clear positive results for CPV-2 in content Then, selecting each province into 20 samples (with time to appeared red line at position C that was earlier than minutes after the sample solution was put into the test and there were clear pink lines), conducted DNA extraction, performed PCR reaction with primers VP2 GMCOM (Gurpreet Kaur et al., 2015) were presented in Table 3.2 Table 3.2: Primer set VP2 GMCOM (Gurpreet Kaur et al., 2015) Primer Primer sequencing Accession Nucleotide name (5’ – 3’) No position VP2 2816GGTCAACCTGCTGT GMCOMF CAGAAA 2835 M19296.1 VP2 AGGTGCTAGTTGA 4525GMCOMR GATTTTTCAT 4503 F: forward; R: reverse Product length 1710bp Reaction conditions: 950C for minutes, followed by 950C for 30 seconds, 580C for 30 seconds, 720C for minute, this cycle was repeated 35 times, finally 720C in minutes After the PCR reaction was finished, the PCR product was tested on 1.5% agarose gel by electrophoresis method The purified DNA product was sent to Phu Sa Biochem Co., Ltd to decoded the VP2 gene sequence (by Sanger method on ABI system 3130 (USA) and check the order received by BioEdit software) When results of sequence analysis of CPV-2 genome were available, collating the genome sequence of CPV-2 of the sequenced samples in the above primers and compared with the nucleotide positions of the control samples which was determined CVP-2, CPV-2a and CPV-2b 3.2.3 Monitoring criteria: Determination of CPV-2 type and genotype of CPV-2: CPV-2a, CPV-2b, CPV-2c, new CPV-2a and new CPV-2b in dogs with inflammatory bowel disease treated at Veterinary clinics of Can Tho city, Dong Thap, Tien Giang and Ben Tre 3.3 Content 3: Determining and evaluating the homogeneity of molecular genetic characteristics of the genotypes of field CPV-2 (DBSCL) infections in dogs with CPV-2 genotypes on gene banks (Genbank); with CPV-2 genotypes in vaccines on gene banks of NCBI 3.3.1 Test subjects: Selecting 32 representative samples with clear and beautiful results in content for provinces and cities of the Mekong Delta (8 samples for each province) 3.3.2 Research methodology: After the research samples were sequenced, the complete sequences will be read and edit in Bioedit 7.0.5.3, this file contained at least sequence of each type and included all genotypes of CPV-2 (CPV-2a, CPV-2b, CPV-2c), original CPV-2 genotypes and recent CPV-2 genotypes were collected from NCBI's gene bank The sequences were fully aligned (Global alignment) by Clustal-W 2.0.11 software The CPV-2 segment of all sequences, after being sorted, it will be exported as a fasta file and used to draw a phylogenetic tree with MEGA software The phylogenetic tree was drew by the Neighbor Joining method with the nucleotide model (substitution model), the Maximum Composite Likelihood and 1000 samples of Boostrap relication The percentage of the number of occurrences of a group on the total number of sampled returns was an indicator of a group's Bootstrap support value The percentage difference between the nucleotide sequences of the cloned CPV-2 from the sample and the reference sequence was calculated by the MEGA software, thereby, determining the generation distance of the phylogenetic tree 3.3.3 Monitoring criteria: Determining and evaluating the homogeneity of molecular genetic characteristics of type CPV-2 causing inflammatory bowel disease in dogs in some provinces of Mekong Delta with CPV-2 genotypes on gene banks (Genbank), also with genotypes of CPV-2 in vaccines gene banks of NCBI 3.4 Content 4: Assessing the protective capacity of Parvovirus vaccine in circulating dogs in some provinces in the Mekong Delta 3.4.1 Experimental subjects: Foreign and domestic dogs breed at weeks of age, healthy, not vaccinated with Parvovirus These dogs were the offspring of mother dogs that have been vaccinated against CPV-2 enteritis vaccine 3.4.2 Experimental design: 15 foreign dogs breeds and 15 domestic dogs breeds were injected with selected vaccines (symbolized as sequencing areV1, V2 V3) 3.4.3 Research methodology: All of domestic and foreign dogs breeds above were cared out, nourished, prevented and treated during the same study period (only different in types of preventive vaccines) The first vaccination was at weeks of age, and repeated twice after the first weeks (according to manufacturer's recommendations) Collecting blood samples (each dog from 1-2 ml blood) at the time before vaccination; after repeated second vaccination: month, months, months, months and 12 months, centrifuging (3000 rpm) for minutes and extracting serum (stored at minus 20oC) Then, the antibody content was determined by ELISA kit (Asan Easy Test ® CPV Ab) of Korea 3.4.4 Recorded parameters: Comparing with the protective ability of types of CPV-2 preventive vaccines in circulating dogs in some provinces of the Mekong Delta and compared with domestic and foreign dogs breeds in this experiment 3.5 Statistical analysis: The first data was analyzed by Microsoft Excel 2010 software (to calculate data: total, percentage, ) the summary data of the report was analyzed by Chi square (χ2) and ANOVA twoway anlysis of variance of Minitab Statistical Software version 16.0 Chapter IV: RESULT AND DISCUSSION 4.1 The prevalence of CPV-2 inflammatory bowel disease in dogs treated in some clinics of some provinces of the Mekong Delta 4.1.1 The prevalence of CPV-2 infection in dogs The prevalence of CPV-2 infection in dogs was presented in Table 4.1 Table 4.1: The rate of dogs with inflammatory bowel disease caused by CPV-2 via CPV Ag test kit Number of dogs vomiting and diarrhea with bloody stool (n=380) Number of samples infected with CPV2 (n=123) Can Tho city 101 33 32.67 Dong Thap 90 30 33.33 Clinic Rate (%) Table 4.5: The prevalence of CPV-2 infection in dogs by vaccination Number of samples Number of samples Vaccination positive for CPV-2 Rate (%) observed (n=380) (n=123) Unvaccinated 281 109 38.79 Vaccinated 99 14 14.14 (P0.05) The average of CPV-2 symptomatically treatments in dogs with inflammatory bowel disease in these provinces were 85.37% 4.2 Results of genotype determination of CPV-2 infection in dogs The rate of genotypes of CPV-2 was presented in Table 4.8 11 Table 4.8: Results of genotype determination of CPV-2 infection in dogs (CPV 2a, CPV-2b, CPV-2c, New CPV-2a and New CPV-2b) Genotype CPV Total number of Total number of Rate (%) samples tested positive samples CPV -2a 80 01 1.25 CPV -2b 80 0 CPV -2c 80 79 98.75 New CPV-2a 80 0 New CPV-2b 80 0 (P