MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL THESIS Major: PATHOLOGY AND TREATMENT OF ANIMALS Major code: 62 64 01 02 NGUYEN THI BE MUOI SURVEY OF LEPTOSPIRA AND LEPTOSPIROSIS IN DOGS IN SOME PROVINCES IN THE MEKONG DELTA Can Tho-2019 THIS THESIS WAS COMPLETED AT CAN THO UNIVERSITY Academic supervisor: Assoc Prof PhD HO THI VIET THU The thesis was defended at the university examination committee At.………………………………………., Cantho University At……… hour ….…, on date…… month… … year…… Reviewer 1: Reviewer 2: Reviewer 3: The dissertation is available in Libraries Central library of Can Tho University National library of Vietnam Chapter 1: INTRODUCTION 1.1 The imperativeness of thesis Leptospirosis is a common infectious disease to many species of animals and it is caused by several species of pathogenic spirochetes (pathogenic Leptospira species) The disease is prevalent throughout the world, especially in hot, humid tropical areas, which are conducive to the presence out of environments (Evangelista and Coburn, 2010) Pathogenic germs exist in the renal tubules and they are excreted in the urine to contaminate the environment (Adler and Moctezuma, 2010) and in naturals, rats are used as a source of spirulina and a source of transmission for the disease for dogs, humans, and other animals (Syke et al., 2011) In Vietnam, there have been many studies on the Leptospira infection in animals and the results show that the prevalence of Leptospira infection is variable, depending on the survey’s animals, time and places by some of the researchers (such as Vu Dinh Hung, 1995; Nguyen Dinh Ngan, 2000; Le Huynh Thanh Phuong, 2001; Hoang Manh Lam, 2002 and Hoang Kim Loan, 2002) However, research on this disease in dogs is very limited, especially in the Mekong Delta, where there is no study on Leptospira and Leptospirosis in dogs Thus, the thesis “Survey of Leptospira and Leptospirosis in dogs in some provinces in the Mekong Delta” was carried out 1.2 The aim of thesis - Survey of the spirochete Leptospira circulating in dogs and rats - To study the changes of pathogenic Leptospirosis in dogs - To study the effective treatments for Leptospirosis in dogs 1.3 The new distribution of thesis This is the first research discovered two species Leptospira interrogans and Leptospira fainei presence in unvaccinated dogs Leptospirosis in some provinces in the Mekong Delta 1.4 Scientific significance of the study This is the first research about the epidemiological of Leptospirosis in dogs, as well as a scientific basis for the construction process and treatment of the dog, limiting disease in humans and other animals Chapter III: CONTENTS AND METHODOLOGY 3.1 Content 1: Determination of Leptospira infection in dogs and rats 3.1.1 Research subjects Survey of Leptospia circulation is done on dogs health and dogs diseases, which are from months old and above, unvaccinated Leptospirosis dogs, from farms in provinces (Can Tho, Vinh Long, An Giang and Ca Mau) and three animal clinics (including healthy dogs and dogs diseases) in Can Tho city The survey of Leptospia circulation in rats from being trapped in the dog sampled households surveyed 3.1.2 Methodology a The method of blood sample Serum samples of dogs: cross-sectional serological analysis was carried out on 1.433 serum samples of healthy and dogs diseases, unvaccinated Leptospirosis dogs according to breed, age, sex and farming methods The intensity of infection was mainly concentrated at low antimicrobial antibody titration values of 1: 200 to 1: 800 in the Mekong Delta Sample size: estimated sample size should be based on the Fosgate (2009) epidemiological formula for each of the survey province Table 3.1 Quantity of dogs serum P(1-P).Z2 0.2(1-0,2) 1.962 n= = = 245 d2 0.052 Quantity (individual) Can Tho 650 Vinh Long 256 The sample size which needs to be An Giang 263 collected in each province is 245 samples Ca Mau 264 of dogs serum or more and is shown in Total 1.433 Table 3.1 Serum samples of rats: cross-sectional serological analysis was carried out on 647 serum samples rats in four provinces in the Mekong Delta Sample size: the estimated sample size should be based on the formula Location Table 3.2 Quantity of rats serum Locations Quanlity P(1-P) Z2 0.071(1-0,071) 1.962 (individual) n= = = 101 Can Tho 195 d2 0.052 Vinh Long 140 An Giang 185 The sample size required for each Ca Mau 127 province is 101 rats serum samples and is Total 647 shown in Table 3.2 Total 647 rats, in which rats (Rattus norvegicus) is 285, muskrats (Suncus murinus) is 152 and small rats (Mus musculus) is 210 b Implementation methods - The reaction MAT (microscopic Agglutination Test) is used to detect specific antibodies against Leptospira in dogs and rats with serogroup 18 by the Pasteur Institute in Ho Chi Minh City supply (according to the process of the WHO, 2003) - The reaction was considered positive when agglutination titer ≥ 1: 200 in dogs serum samples and ≥ 1: 20 in rats serum samples c Survey criteria - The prevalence of Leptospira in dogs and rats at survey sites - The prevalence of serogroup Leptospira in dogs and rats - The intensity of serogroup Leptospira infection in dogs and rats - The prevalence of Leptospira in dogs according to breed, age and farming methods - The correlation coefficient (R2) between the positive rate of Leptospira serogroup in rats and dogs, the linear regression equation y = a + bx 3.2 Content 2: Detection of Leptospira from urine The detection of Leptospira from urine is performed on the same 63 dogs examined the biochemistry physiology index of blood and urine from 111 dogs MAT ≥ 1: 400 3.3.1 Detection of Leptospira from urine by PCR Implementation methods: urine is collected midway through the urethra or directly collected at eliminating the process with urine volume of 6-20 ml, divided into parts for analysis - Part 1: ml of urine taken no centrifuge to analyze the physiological indicators of urine - Part 2: ml of urine taken after centrifugation for DNA extraction - Part 3: ml of urine taken after centrifugation to culture on the environment Ellinghausen-McCullough-Johnson (EMJH) The implementation of PCR Sixty-three dog urine sample used for DNA extraction and PCR reaction used on the basis of 16S rRNA gene amplification Table 3.3 Nucleotide sequence using primers 16S rRNA gene amplification region Gene Total Nucleotide sequence bp primers M-27F 5’-AGA GTT TGA TCM TGG CTC AG-3’ 1500bp 16S M-1492R 5’-TAC GGY TAC CTT GTT ACG ACT T-3’ The total primers are based on the work of Weisburg et al (1991) Table 3.4 PCR reaction components Components Volume/1 Concentration/1 reaction reaction 2X Master Mix 25X 1X Primer M-27F (10 µM) µL 0.4 µM Primer M-1492R (10 µM) µL 0.4 µM DNA tách chiết 1-2 µL 50-100 ng Bi.H20 Đủ 50 µL Table 3.5 Heating process in PCR Temperature Time Cycle o 95 C phút 95oC 30 giây 55oC 30 giây 30 72oC phút 45 giây 72oC phút 3.3.2 Detection of Leptospira from cultures Method for culture Leptospira - Leptospira cultured according to the WHO (2003) on environments EMJH, observed under dark-field microscopic examination and identified to species level by PCR and sequencing - PCR was performed as section 3.3.1 Sequence the 16S rRNA gene of Leptospira - The PCR’s products were then purified and decoded in nucleotide at company Macrogen (South Korea) - The sample was sequenced 16S rRNA automatically on the ABI 3130 Sequencer with two primers 27F and 1492R - If the homogeneity of the nucleotide sequence in the 16S rRNA gene is ≥ 95%, it can be determined to be a genus and ≥ 99%, which can be determined by the species (based on the level of homology description of La Scola et al., 2006) - Phylogeny is based on nucleotide sequences using MEGA 7.0 software (Kumar et al., 2016) Looking up the results on BLAST SEARCH and comparing a wide range of bacterias available in GeneBank and database on NCBI are to identify the isolated species and two species of the spirochete Leptospira reference used for comparison in the analysis of species arising: >Icterohaemorrhagiae NR_116542.1 Leptospira interrogans serovar icterohaemorrhagiae strain RGA 16S ribosomal RNA gene, partial sequence >Hurstbridge NR_043049.1 Leptospira fainei serovar hurstbridge strain BUT 16S ribosomal RNA gene, partial sequence 3.3 Content 3: Survey on the pathogenic changes of Leptospira in dogs The pathological changes in dogs diseases surveyed base on physiological biochemical index of blood and urine of 13 dogs positive for Leptospira by PCR and MAT ≥ 1: 400 a Blood analysis The testing of physiological blood was investigated by hematology (Cell Dyn 3200, USA), including leukocyte, neutrophile%, lymphocytes%, erythrocytes, hemoglobin, hematocrit, platelet and biological of blood was investigated monitoring by blood chemistry analyzer (AU604, Japan): ure, creatinine, AST, ALT and bilirubin and compared to standards of the Merck (2010) b Urine analysis The testing of the physiological index of urine surveyed by urine analyzer urine (U120, USA) include: red blood cells, white blood cells, proteins, and bilirubin and compared with Merck's standards (2010) and Khorami et al (2010) c Survey criteria - The change of biochemical and physiological index of blood and urine in dogs having Leptospirosis - Clinical signs of suspected Leptospirosis in dogs: fever, reflux, vomiting, hemorrhage, 3.4 Content 4: Study on the treatment of Leptospirosis in dogs Study subjects: healthy dogs and dogs diseases were brought to medical examination and treatment at the veterinary clinic in Can Tho city - Set up a collaborative regimen treatment with the owners - Sixty-three dogs in the content titer antibodies against Leptospira ≥ 1: 400, with the presence of Leptospira in urine by dark-field microscopic examination (is randomized with 32 dogs did not show clinical symptoms and 31 dogs have some manifestation clinical symptoms suspected Leptospirosis: vomiting, eating less, sad, sedentary, kidney failure, liver failure, ascites, skin hemorrhages and jaundice) Table 3.6 Experimental design Treatment 1: Shoptapen 20 0.2 mg/kgP, Intramuscular or 7-14 (Penicillin each three Subcutaneous days G+Streptomycin) days injection Treatment 2: 22 22 mg/kgP, Take medicine 7-14 Amoxicillin twice a day days Treatment 3: 21 mg/kgP, Take medicine 7-14 Doxycycline twice a day days Monitoring criteria is to assess effectiveness of treatment based on Goldstein, (2010); Sykes et al (2011); Francey and Schweighauser, (2012) and Schuller et al (2015) 3.5 Data analysis and processing - Data was processed for the statistics using Microsoft Excel 2013 software - The physiological, biochemical index of blood and urine are presented as ±SE - The investigation of factors related to Leptospira infection including breeds, age, sex, and farm method χ2 in Minitab 16.0 - The regression was used to analyze the correlation of Leptospira in rats and dogs with the equation of y=a + bx, where a and b are constant, y and x are positive rate in rats and dogs Chapter RESULTS AND DISCUSSION 4.1 Survey results of Leptospira infection in dogs and rats 4.1.1 The prevalence of Leptospira in dogs in some provinces in Mekong Delta Table 4.1 The prevalence of Leptospira in dogs Province Number of serum Number of Positive rate samples positive serum (%) samples Can Tho 650 159 24.46a Vinh Long 256 69 26.95a An Giang 263 53 20.15a Ca Mau 264 50 18.94b Total 1.433 331 23.10 Data in the same column with different caps are statistically different (P