Summary of doctoral thesis: Some epidemiological biology and of Fasciola sp. and the efficacy of anthelminthic treatments in cattle in the Mekong delta

Objectives: Identifying the species, distribution, biological characteristics and influential factors to the liver flukes infection rate in cattle the Mekong Delta. Suggesting the treatment methods for infected cattle in Mekong Delta.

MINISTRY OF EDUCATION AND TRAINING

CAN THO UNIVERSITY

SUMMARY OF DOCTORAL THESIS

Major: PATHOLOGY AND TREATMENT OF ANIMALS Major code: 62 64 01 02

Ha Huynh Hong Vu

Some epidemiological biology and of Fasciola sp and

the efficacy of anthelminthic treatments in cattle

in the Mekong delta

Can Tho- 2018

I

THIS THESIS WAS COMPLETED AT CAN THO

UNIVERSITY

Academic supervisor: Assoc Prof DR Nguyen Huu Hung

This thesis was defended against the Ph.D dissertation council at the university level

Thesis could be found at:

1 Learning Resource Center, Can Tho University

2 National Library of VietNam

II

PUBLISHED ARTICLES

Published Articles in journals

1 Ha Huynh Hong Vu, Nguyen Ho Bao Tran, Nguyen Huu Hung, 2014

Identification freshwater snail intermediate host of trematoda causing animal disease in Vinh Long and Dong Thap Province Journal of Science, Can Tho University, Special issue agriculture, pp 8-12

2 Ha Huynh Hong Vu, Nguyen Ho Bao Tran, Nguyen Huu Hung, 2015

Morphological and molecular characteristic of Fasciola sp infected in cattle

in Dong Thap province Journal of Science-Technique of Veterinary

Medicine, 6: 63-69

3 Ha Huynh Hong Vu, Nguyen Ho Bao Tran, Pham Duc Phuc, Nguyen

Huu Hung, 2016 Application of molecular marker-ITS-1 gene and

PCR-RFLP technique for determining large liver flucke (Fasciola sp.) in cattle in

Mekong river Delta, 2016 Journal of Science-Technique of Veterinary Medicine, 2: 85-92

4 Ha Huynh Hong Vu, Nguyen Ho Bao Tran, Nguyen Huu Hung, 2016

Large liver fluke (Fasciola sp.) infection of cattle in the Mekong Delta and

results of treatment trials Journal of Science, Can Tho University, Special issue agriculture, pp 17-22

5 Ha Huynh Hong Vu, Nguyen Ho Bao Tran, Nguyen Huu Hung, 2018

The surveillance on pathological characteristics of Fasciola gigantica

infected in Mekong delta Journal of Science, Can Tho University, Special issue agriculture, pp 12-17

Chapter I: INTRODUCTION 1.1 Rationale

According to the World Health Organization (WHO), Fascioliasis is one of the important diseases, which is found in humans and animals More than 2.4 million people in 70 countries were affected by the disease (WHO, 2015; Amer, 1016) In Vietnam, Fascioliasis in humans tends to increase gradually, from 2006 to 2010 In fact, 15,764 people and cases were

infected by Fasciola sp in 2006 and those cases increased to over 20,000

people in 2011 The disease in 52 provinces from North to South and

pathogenic species is determined mainly Fasciola gigantica (Nair et al

2012) Fasciolosis has been demonstrated and listed in zoonosis diseases The disease causes by the large liver flukes which require the intermediate host (freshwater snail species) to complete its life cycle The Mekong Delta possesses the geographic features such as innumerable canals, rivers, stream which is suitable to develop agriculture: paddy rice and vegetables

as well as provide the appropriate conditions for freshwater snail development Moreover, livestock husbandry also great develops because famers take advantages the source of by-product from agricultural processing However, most of husbandry farms are small-scale farms where people normally use by-products from agriculture and they do not have well knowledge about applying the techniques in animal husbandry and veterinary As the results, their livestocks expose high prevalence of helminthes infection Therefore, it is crucial to research about fasciolosis and how to manage the spreading of this disease in order to minimize the damage from it The study aimed to investigate “The epidemiological,

biological characteristics of Fasciola sp and the efficacy of anthelmintic

treatments in cattle in Mekong Delta”

1.2 Objectives

- Identifying the species, distribution, biological characteristics and influential factors to the liver flukes infection rate in cattle the Mekong Delta

- Suggesting the treatment methods for infected cattle in Mekong Delta

1.3 Scientific significance

- This is a systematic research about liver flukes Fasciola gigantica in

cattle: determining the prevalence of infection and influential factors to the pathogen Species were identified by morphological and molecular characteristics (PCR-RFLP, and sequencing)

- The life cycle of Fasciola gigantica in cattle in Mekong Delta were firstly

researched: identifying intermediate host (snails) Clinical symptoms and

anthelminthic testing would be useful for diagnosis and treatment

- This thesis provides documentations about Fasciola sp infected in cattle

(Mekong Delta), and supplies academic knowledge for veterinary parasitology books to education and training purposes

1.4 Practical significance

-The thesis results are the scientific background for recommending farmers

in effectively diagnosis, treatment and prevention liver flukes that minimizes the economic lose as well as contributes for the sustainable development of livestock husbandry

1.5 Innovative contributions of the thesis

This is the first research about Fasciola gigantica in infected cattle in

Mekong Delta which were identified by applying molecular biology techniques

This is also first research about the complete life cycle of Fasciola gigantica

Gross lesions and histopathological of Fasciolosis (causing by F.gigantica)

were completely described which were provided background for quickly diagnosis and treatments

Chapter III: CONTENT AND RESEARCH METHODOLOGY

3.1 The research contents

3.1.1 Determining the prevalence of liver flukes of cattle in the Mekong Delta provinces

- Determining the infection rate of liver flukes of cattle in the Mekong Delta provinces by the fecal examination and necropsy methods

3.1.2 Identifying the species of Fasciola sp in the Mekong Delta

provinces

- Determining the species of Fasciola sp by analyzing mophorlogical

molecular biology chacteristics and sequencing

3.1.3 Researching about life cycle of Fasciola gigantica

- Observing the development of the Fasciola gigantica egg outside the

definite host

- Observing the development of the larvaes of Fasciola gigantica in intermediate host (Lymnaea swinhoei and Lymnaea viridis) to stage cercaria infection

- Analyzing and recording the every development stage of Fasciola gigantica since embronated eggs to mature in definitive host

3.1.4 Fasciola gigantica Pathogen researching

-Determine the clinical symptoms, gross lesions and histopathology on

infected Fasciola gigantica cattle

3.1.5 Studying on treatments of Fasciola gigantica infected cattle

- Testing the efficacy of anthelminths: albendazole, mebendazole and

triclabendazole; and suggest the efficient treatment on Fasciola gigantica

infected cattle

3.2 Subjects, timeline, and researching areas

3.2.1 Subjects: cattle in six provinces: Dong Thap, An Giang, Vinh Long,

Tra Vinh, Ben Tre and Soc trang; liver flukes-Fasciola sp.; the snail Lymnaea spp

3.2.2 Timeline: from 11/2013 to 06/2017

3.2.3 Rearching areas

The cattle in 6 provinces (Mekong Delta), slaughter houses, histologic laboratory in the Department of Veterinary Medicine – College of Agricultural and Applied Biology, Can Tho University; Department of Clinical Veterinary Science and livestock - veterinary medicine – Nong Lam University, Anatomical pathology laboratory - University of medicine and pharmacy of Can tho

3.3 Materials and Chemistry

Necessary materials and chemistry for diagnosis and molecular biology

- Methods:

Fecal examination: modified sedimentation of Benedek (1943)

The autopsy methods: the operating SKRJABINE (1937)

-Observation targets

The overall infected rate of Fasciola sp in cattle, the infection rate of this

disease according to seasons, husbandry methods, ages, and ecogeographic areas; the intensity of the infection ( the number of species/ individual) -Statistical analysis: Chi-Square test /Minitab program version to compare

infection rates

3.4.2 Identification method

3.4.2.1 Identification method of trematode by morphology based on

documents written by David and Erasmus (1972), Soulsby (1980), Nguyen Thi Le (2000)

3.4.2.2 Identification method of live flukes by molecular techniques (PCR-RFLP) and sequence genes

Collecting and storing DNA extraction samples

Totally, 180 liver flukes were randomly collected from the liver and bile ducts in slaughter cattle from 6 surveyed provinces Specimens was stored

in physiological saline and brought to DNA laboratory

DNA extraction

DNA concentration measurement

PCR-RFPL techniques

PCR reaction:

Table 3.1 Primer sequence corresponding to the target gene

Gene Primer Primer sequence (5’ – 3’)

Annealing temperature -Tm ( o C)

Reference

ITS1

F/ITS1-R

ITS1-ITS1-F: TTG CGC

CCC TGITS1-R: TTG GCT GCG CTC TTC ATC

PCR products were incubated with restriction enzymes RsaI (5 IU)

overnight at 37oC Then the products were run on 1.5% agarose gel with Ethidium bromides in 80 votage in 30 minutes The gels were visualized under camera (Geldoc)

Table 3.2 Prediction the restriction patterns of the enzyme RsaI Enzyme restriction patterns in the region of ITS1 Fasciola sp

enzyme

Temperature, incubation time

Location cuting

Predicted length of cutting (bp)

5’ GT↓AC 3' 3' CA↑TG 5'

367, 104 (68, 59,

Sequencing positive samples

- Totally 12 liver fluke samples (Fasciola sp.) were collected from 6

provinces in Mekong Delta, specifically 2 samples/one province PCR products were purified and sent to Macrogen (Korea) to sequence (using Sanger sequence method)

Observation targets :

- Identification of Fasciola sp were done by morphological and

molecular biology chacteristic

- Comparasion the nucleotide sequence of target gene ITS-1 of

Fasciola sp among surveyed provinces and Fasciola sp collected

worldwide in Genbank

Analyzing data :

- BLAST the ITS-1 sequence (in NCBI) was used to identify the specific species, and comparing the level of similarity among multisequence by CLUSTAL OMEGA, analyzing Pairwise alignment/Calculate identity/Similarity for sequences (Bioedit)

3.4.3 Studies of life cycle of Fasciola gigantica

3.4.3.1 Identification of freshwater snails

Basing on the classification system of the freshwater snails was described by John (1982), Dang Ngoc Thanh and his colleagues (1980)

From this backgound knowledge, the snails Lymnaea were carefully

collected and feeded in the laboratory environment to produce the clean

snail generation Miracidium from embronated eggs (Fasciola gigantica)

were infected to clean snail generation

3.4.3.2 Observe the development of Fasciola gigantica egg in in vitro

a Reseach objects:

Fasciola gigantica eggs, Lymnaea swinhoei and Lymnaea viridis snails is

"clean snail"

b Experimental design

Table 3.3 Experiment designing for Fasciola gigantica eggs development

Experiment 1: Fasciola sp egg in petri disk with water levels of 0, 5 cm, no illumination, pH from 6-8, the temperature from 26-29 0 C Experiment 2: Fasciola

sp egg in petri disk above 0, 5 cm, lighting 4 hours/day, pH from 6-8, the

temperature from 26-29 0 C Experiment 3: Fasciola sp egg in petri without water

c Observation targets

- The length of time from eggs to develope to miracidium

- The length of time from the eggs hatching into miracidium to 50% eggs

were hachted and miracidia liberating from the egg shells

3.4.3.3 Observing the length of time of alive miracidia in water

a Research objects

Fresh miracidia have just liberated from the egg shells Those miracidia

were observed to identify the their longevity in water

b Experimental design

After miracidium infected to the snails, infected snails were collected and

necropsied at time points: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42 PI days in order to detect cercaria- free swimming stage in water and transforming to

Adolescaria For each time points, 10 of Lymnaea swinhoei and 10 Lymnaea viridis snails were surgery to the stage of development of the larva (redia and cercaria, sporocyst) in 2 species of snail Lymnaea

Table 3.4 Identifying the longevity of miracidia in water

3.4.3.4 Observing the development of larva stage of Fasciola in intermediate host Lymnaea spp snails

In this experiment, 960 miracidia were collected and infected to 240 Lymnaea swinhoei, and 240 Lymnaea viridis

Table 3.5 The invasion of Fasciola micracidia to Lymnaea snails

3.4.3.5 Cattle infected by Fasciola larvae

a Research objects

In this experiment, 8 cattle at the age of 7 months-12 months old were bought from the local farmers in the surveyed areas Before infecting, cattle were dewormed by albendazole and carefully tested the presence of liver fluke eggs as well as other helminths

b The cercaria infection causing lab layout for experimental cattle

Six cattle were divided into 3 different groups which were received 3 doses

100, 150, and 200 cercaria Those cattle were infected by ceraria through

oral adminstration Non-infected group was considered as negative control Observing the presence of liver fluke eggs in feces of infected cattle

The feces examination were conducted after 11weeks post infection and then feces samples were collect and check everyday until detecting the eggs

of Fasciola sp The sedimentation methods was applied to diagnose The

results were futher confirmed by necropsy method

3.4.4 Clinical symptoms and gross and histopathological changes on

cattle infected with Fasciola

3.4.4.1 Symptoms of cattle infected with Fasciola

- Physical and clinical examination were done in 60 infected cattle with

Fasciola and 6 infected cattle in infection experiment (3.4.3.5 )

3.4.4.2 Researching about the gross lesions and histopathology in liver

tissue causing by Fasciola gigantica infection

-Objects: livers from Fasciola gigantica infected cattle in this experiment and 45 livers from Fasciola gigantica infected cattle in slaughter houses in

Mekong Delta

3.4.5 Studying the prevention and treatment Fasciolosis in cattle

105 crossbred Sind cattle having in high infectious intensity from 2+ to 3+ were collected to test the efficacy of albendazole, mebendazole, triclabendazol The number of cattle were divided into 2 experiments and 5 cattles in control group

Experiment 1: treatment dosage- following the manufacturer's instructions Experiment 2: increasing the treatment dosage (higher dose the manufacturer’s recommendation)

Control group: not use any treatment

Table 3.7 The efficiency of albendazole, mebendazole and triclabendazole

on Fasciola sp infected cattle

(mg/kg body weight)

Number

of cattle treatment

Adminstration Route

Table 4.1 The prevalence of liver flukes infected cattle in Mekong Delta

Province Examined cattle Infected cattle Prevalence (%)

a,b in the same row showed the statistically significant difference at P< 0.05

Table 4.1 showed that the overall infected cattle by Fasciola sp 20.50% In

particular, cattle in An Giang province had the highest infectious rate of

Fasciola sp 25.87%, following by cattle in Dong Thap (25.23%) and Vinh Long with 24.57% The infectious rate of Fasciola sp in cattle in Ben Tre,

Tra Vinh and Soc Trang was 15.97%, 15.78% and 14.33%; respectively Most of infected cattle had the low intensity of infection (1+) which oocupied of 78.80%, following by the (2+) intensity with 17.62%, and (3+)

with 8.68% The infectious rate of Fasciola sp in cattle in An Giang, Dong

Thap and Vinh Long had statistically significant higher than those in Tra Vinh, Ben Tre and Soc Trang (p<0.05) The above results were explained that An Giang and Dong Thap province is located at the upstream of the Tien and the Hau River, Vinh Long is by the sides of the rivers Due to the geographic features such as innumerale canals and waterways, it is the appropriate conditions for the development of intermediate hosts (snail) of Fasciolosis Therefore, the infectious rate of Fasciolosis in cattle in An Giang, Dong Thap ang Vinh Long had the higher than those in Tra Vinh, Ben Tre and Soc Trang (the latter provinces locate near the sea and have the saltwater and blrackish water areas – not appropriate for intermediate hosts (snails) development This result confirmed that the geographic climate and the ecological conditions had great influence on the growth of intermediate hosts of Fasciolosis and by the way influence on the infectious rate of Fasciolosis in cattle This studying results is compatible with the researches of Pham Van Khue and Phan Luc1996); Phan Dich Lan et al (2002); Nguyen Thi Kim Lan et al (2008)

Table 4.2 The infection rate of liver fluke egg in cattle according to the breed

Provinces

Gender Dairycow

Prevalence (%)

Crossbred Sind Prevalence (%)

Local cattle Prevalence (%)

a,b in the same row showed the statistically significant difference at P< 0.05

The results in table 4.2 showed that the infectious rate of Fasciolosis in local cattle were 21.55%, that was higher than crossbred Sind 20.86% and the lowest infection rate in dairy cows (9.15%) Through statistical analysis

we found that the infection rates of dairy cows and crossbred Sind as well

as local cattle had statistical significant difference Meanwhile, local cattle and crossbred Sind had with no difference According to breed factor, the infectious rate of Fasciolosis in cattle among provinces also provided the same patterns

Table 4.3 showed that the prevalence of Fasciola infectious rate in cattle

increased according to ages; namely under 1 year old cattle (8.24%); closely by from 1 to 2 year-old-cattle (19.06%); and over 2 year-old cattle