NGUYEN THI HUONG GIANG The thesis title STUDY ON THE PREVALENCE OF GASTROINTESTINAL NEMATODE INFECTION AND STRONGYLOIDIASIS ON PIGS IN BAC GIANG PROVINCE, THE PREVENTIVE MEASURES AND TR
Trang 1THAI NGUYEN UNIVERSITY
PhD CANDIDATE NGUYEN THI HUONG GIANG
The thesis title
STUDY ON THE PREVALENCE OF GASTROINTESTINAL NEMATODE INFECTION AND STRONGYLOIDIASIS ON PIGS IN BAC GIANG PROVINCE, THE PREVENTIVE MEASURES AND TREATMENT OF THE DISEASE
Speciality: Veterinary parasitology and
microbiology Code: 9.64.01.04
DISSERTATION IN VETERINARY MEDICINE
Thai Nguyen - 2020
Trang 2AT COLLEGE OF AGRICULTURE AND FORESTRY
THAI NGUYEN UNIVERSITY
Supervisers: 1 Professor Doctor Nguyen Thi Kim Lan
2 Doctor Nguyen Van Quang
Reviewer1:
Reviewer2 Reviewer3:
The dissertation will be defended at the Dissertation
committee in National level COLLEGE OF AGRICULTURE AND FORESTRY - TNU
Time date month year 2020
The dissertation can be found at
- Viet Nam national library:
- Learning Resource Centre in Thai Nguyen
university
- Library of Thai Nguyen university of Agriculture and Forestry
Trang 31 INTRODUCTION
1.1 The urgency of the project
Bac Giang is a province with a quite development of pig husbandry Formany years, pig production has made an important contribution to hungereradication, poverty reduction and enrichment for farmers in the province.According to the Department of Animal Science and Veterinary Medicine of BacGiang province, there were 1,043,749 pigs in 2017 in the whole province, in
2018 there were 1,105,291 pigs Along with the rapid increase in the number ofpigs, farmers have applied scientific and technical advances in pig farrmingpractice, thus bringing significant economic benefits to them However, pigfarming in Bac Giang province has faced with many difficulties, includingdisease problems In addition to common infectious diseases, gastrointestinalnematode infections in pigs ofen occur chronically Although nematodeinfections do not cause high mortality of pigs as infectious diseases, nematodeinfections usually occur in chronic form, making pigs stunted, growing slowly,reducing resistance of their bodies, and susceptible to other diseases
Phan Dich Lan et al (2005), Pham Sy Lang et al (2011) reported thatdiseases caused by gastrointestinal nematodes were very common diseases andwas one of reasons resulting in stunted pigs, reducing weight gain by 15-20%,and were susceptible to infectious diseases such as rotavirus infection,paratyphoid leading to more severe diarrhea syndrome
Although there is quite development of pig farming in Bac Giangprovince, there have been no studies on the situation of nematode infectionsand Strongyloidosis in pigs, so there are no measures for effectiveprevention and treatment of these diseases
From the urgent requirements of animal farming practice and disease
prevention for pigs, we conducted the entitled: "Study on the prevalence of gastrointestinal nematode infection and Strongyloidiasis on pigs in Bac Giang province, the preventive measures and treatment of the disease".
2 Objective of the project
- Determining prevalence and intensity of infection of gastrointestinalnematodes in pigs;
- Identification of some epidemiological, pathological and clinicalcharacteristics, the prevention and control measures of Strongyloidosis inpigs in Bac Giang province
Trang 43 Scientific and practical significance of the topic
3.1 Scientific significance
The project provides scientific information about the situation ofgastrointestinal nematode infection in pigs in Bac Giang province;Epidemiological, pathological and clinical characteristics ofStrongyloidosis, on the basis of which, set up a process of prevention and
treatment of strongyloidosis for pigs with high efficiency.
3.2 Practical significance
The results from the project are the basis for recommending the pigproducers applying preventive measures and treatment of nematodeinfection in general, and Strongyloidosis in particular in order to limit theprevalence of nematode infection in pigs and limit damage caused byStrongyloidosis, making contribution to sustainable development of pigfarming in Bac Giang province
4 New contributions of the project
- The project is the first systematic study on thesituation of gastrointestinal nematode infection in pigs inBac Giang province including ; Epidemiological, pathologicaland clinical characteristics of pig threadworm infection( strongyloidosis)
- Effective procedures for the prevention and treatment of
Strongyloidosis in pigs have been designed, recommended and appliedwidely to farms and pig farming households in Bac Giang province and the
neighboring provinces.
5 The dissertation structure
The dissertation consists of 129 pages (excluding references list):introduction 03 pages; overview of literatures 31 pages; objects, materials,contents and methods of the study 22 pages; study results and discussion
72 pages; Conclusions and recommendations 02 pages In the disertation,there are 36 tables, 13 charts and graphs, 120 colour pictures depicting theresults of the project PhD student has referred to 151 references (including
68 published in recent years ( past five years).
Trang 5Chapter 1 DOCUMENT OVERVIEW
Nguyen Thi Le et al (1996) indicated that nematodes in pigs aredistributed all over the world In particular, in the tropic and subtropic regionsthere is hot and humid climate, which is a very favourable condition for eggsand nematode larvae to develop in the outside, making the gastrointestinalnematode infection in pigs occur through out the years
According to Pham Sy Lang et al (2011), Gomathi M et al (2016),
pigs infected with threadworms though gastrointestinal tract and though
skin, piglets can be infected with S ransomi very early from the colostrum
so it is possible to see adult worms from 4 days old piglets Pigs infectedwith strongyloides are characterized by diarrhea, loose stools with blooddue to hemorrhagic intestine, anemia, emaciation and sudden death Inaddition, when the larvae enter the alveoli, they can cause pneumonia withfever at temperature of 40 - 41.5oC, coughing a lot, difficulty breathing.
According to Nguyen Thi Kim Lan (2011), In order to preventStrongyloidosis in pigs effectively, it is necessary to implement integratedprevention measures, including: hygiene of pig pens, cleaning livestockinstruments and equipments; Pig manure and waste must be collected dailyand properly composted at the stipulated places; implement disinfection ofpig pens regularly, do not keep pigs at different age together, using areasonable diet for pigs
According to Bui Thi Tho et al (2015), Nguyen Tai Nang et al.(2016), there are many new anthelmintic drugs that have a good activitiesfor dewornming nematodes, including drugs of ivermectin , benzimidazole,imidazothiazole groups
Chapter 2 OBJECTS, MATERIALS, CONTENTS AND METHODS OF STUDY 2.1 Object, time period and places of study
2.1.1 objects of study
- Pigs reared in various locations of Bac Giang province
- Gastrointestinal nematode species of pigs
- Pigs affected by Strongyloidosis (caused by Strongyloides spp )
Trang 62.1.2 Time period and place of study
* Study period: from 2016 - 2019
* Study places
- The project was implemented in 5 districts of Bac Giang province
including: Viet Yen, Hiep Hoa, Lang Giang, Yen Dung and Son Dong
- Sample testing places: Laboratory of Faculty of Animal Science, andVeterinary Medicine
- Bac Giang Agriculture and Forestry University; Bac Giang GeneralHospital and Preventive Medicine Center of Province; Institute of Ecologyand Biological Resources; National Institute of Hygiene and Epidemiology
2.2 Study materials:
*Experimental animals: pigs raised in 5 districts of Bac Giang province
* Types of samples for studying: fresh feces samples from pigs of differentage; samples from sediment of pig house floor, wastewater samplesfrom pigpens, samples from surface soil in garden for growing feed crops; samples fromnematodes collected from infected pigs; blood samples from pigs infected with
Strongyloides spp and healthy pigs; samples collected from small intestines,
heart, lungs, liver, kidneys of pigs affected by Strongyloidosis
* Instruments, equipments and chemicals: light microscope, olympusCX221 microscope, scanning electron microscope; stool test kits; bloodmicrosampling device; Hematology Analyzer.Erma PCE 210 and TC - Matrix;Microtome Equipment,automatic DNA sequencing machine; ABI Prism 3130Genetic Analyzer; centrifuge machine, micropipetes, electrophoresismachine,UVTransilluminator, PCR machine, DNeasy Tissue Kit (QIAgen)Chemicals used for preparing smear slide from specimens, saturated brinesolution; Barbagallo solution; solution for drawing water from nematode bodies;QIAquick PCR purification kit Disinfectants: P.V.D iodin 10%; Chloramin B;Good Farm L anthelmintic drugs for deworming Strongyloides in pigs:including fenbendazole, ivermectin and thiabendazole
Trang 7+ The prevalence and intensity of gastrointestinal nematode infection
in locations
+The prevalence and intensity of infection of various gastrointestinalnematode species in pigs
- Stool examination to determine:
+ The prevalence of gastrointestinal nematode species infection invarious locations
+ The prevalence and intensity of gastrointestinal nematode speciesinfection in pigs through faecal examination
+ The prevalence of gastrointestinal nematode infection depending pig
age, Farming methods and seasons
2.3.2 Study on Strongyloidiasis in pigs (Swine Strongyloidosis)
* Identify the species of Strongyloides in pigs
- Results from detection and collection of Strongyloides in pigs in BacGiang through dissection
- Results from designation of Strongyloides by using morphologicaltechnique, and by molecular biology engineering
* Studying Strongyloidiasis infection through stool analysis
- The prevalence and intensity of Strongyloides infection depending
on locations
- The prevalence and intensity of Strongyloides infection depending
on age of pig, season, and farrming method
* Study on contamination of strongyloides eggs and larvae in the environment
* Study on pathological and clinical characteristics of Strongyloidosis in experimental infected pigs and naturally infected pigs
* Study on preventive measures for strongyloidosis in pigs
-Testing and determination of anthelmintic drugs fo deworming
Strongyloides with high efficacy and safe for pigs
- Study and recommendation of procesdures for prevention andtreatment of Strongyloidosis in pigs
Trang 8Prevention and control of gastrointestinal nematode infection in pigs in BacGiang province was carried out by direct observation, using questionaire for pigfarminghouseholds and investigation results were filling out the paper form.
2.4.1.2.Method of study on prevalence and intensity of
gastrointestinal nematodes infection in pigs
- Method of descriptive epidemiology as discribed by (Nguyễn Nhu
Thanh et all, 2001) was applied;
- Minimum sample size was calculated using Win episcope 2.0 software;
- Samples were collected using method of.Stratified sampling;
- Pigs were dissected using methods of incomplete helminthological dissection for gastrointestinal organs as described by Skrjabin (1928);
Samples of parasitic nematodes in the gastrointestinal tract of pigswere collected then, the nematode smear slide were fixed using method asdiscribed by De Grisse A T (1969) Species designation was madeaccording to species identification key of Phan The Viet et al (1977), DeLey P and Blaxter M (2004);
- Nematode eggs in pigs were detected by using lleborn's flotation technique.
The intensity of nematode infection was determined by using Mc Master eggcounting technique for counting eggs per 1 gram of faeces in Mc mastercounting chamber (According to Hansen J and Perry P., 1994)
2.4.2 Method of study on Strongloidosis in pigs
2.4.2.1 Species identification made by using morphological method
Faecal smears of pigs infected with Strongyloides were fixed by using method
of De Grisse A T (1969), the morphology of Strongyloides was observed under the
light microscope, along with plotting to identify the species according to theidentification key of Phan The Viet et al (1977), De Ley P and Blaxter M (2004) Atthe same time, the superstructural morphology of Strongyloides spp was observedbased on the method of Sato H et al (2008)
2.4.2.2.Molecular technique for identification of Strongyloides
- PCR technique was used for testing 5 samples of Strongyloides collected
from 5 studied districts of Bac Giang province The obtained DNA sequencescompared with the DNA sequences on the gene bank using MEGA softwareversion 6.0 (Tamura K et al., 2013), the phylogenetic tree analysis and drawingwere performed by using the Maximum Likelihood (ML) method with the mostappropriate model The group confidence interval was assessed by bootstrap
values with 1000 replicate samples
Trang 92.4.2.3 Methods for assessing the environment contaminated with Strongyloides eggs and larvae
Soil samples for Strongyloides eggs were examined using techique discribed by Romanenko N A (1968) Strongyloides larvae in soil samples
were determined by using Baerman nmethod for larvae isolation
* Methods of identifying duration of Strongyloides eggs and larvae development and survival in faeces of pigs in laboratory
4 experimental groups were designed in 4 seasons: Spring, Summer,Autumn and Winter Faeces were collected from pigs severe infected with
Strongyloides A plastic pot in 15- 20 cm diameter was used for eachexperiment , the start of the experiment was labeled and dated The samples wereplaced at normal temperature and humidity
On the first day, every 2 hours, samples were taken for testing by usingFulleborn flotation technique and Bearmann method for larvae isolation to identify
the duration time when Strongyloides eggs hatch to larvae Then, about 3-5 grams of
faeces were taken daily for testing using Bearmann method for larvae isolation toidentify the time duration for larvae to develop into infective larvae
Infective larvae were determined by observing the morphology oflarvae under light microscope to recognize infective larvae based onmorphology as described by Viney M E and Lok J B (2015)
2.4.2.4 Establishing experimental infection of pigs for studying Strongyloidosis.
2 experimental groups were designed by using healthy crossbred pigs (♂Yorkshire x ♀ Mong Cai) at 1 - 2 months of age Pigs were divided into 3 phases:Phase1: experimental infection was caused in order to study the pathologicalcharacteristics of the disease; experimental infection 2 was used for testing efficacy
of specific antheminthic drugs for treatment of Strongyloidosis: In phase 1: 15 pigswere used and divided into 3 groups, group1, experimentally gastrointestinalinfection was caused ; Group 2: skin infection was caused ;and group 3 for controlalso set up The infecting dose was at 10,000 larvae phase 2 was designed for
experimental Strongyloides infection of 20 pigs which were divided into 4 groups 5
pigs for each Each pig was ingested 10,000 infective larvae
2.4.2.5 Study on pathological and clinical characteristics of Strongyloidosis in experimentally infected pigs and naturally infected pigs in the field
* Study on infected pigs
- Method of assessing clinical symptoms: the symptoms of infected pigswere observed, compared to pigs in the control group
Trang 10Hematological indices were determined using Erma PCE 210 and TC Matrix hematology analyzers.
Macropic lesions score were Identified by dissection of pigsexperimentally infected with Strongyloides, observing with the naked eye andmagnifying glass lesions in liver, lungs and gastrointestinal tract
Study on microscopic lesions score using preparation of slides fromparaffin embedded tissues stained with Haematoxilin – Eosin, The slides wereobserved under a microscope to identify microscopic lesions
* Methods of study on Strongyloidosis in pigs in the locations
Methods of identifying clinical symptoms, signs and macropic lesions score
in pigs infected with Strongyloides in the field were performed by using the same
methods as conducted in experimentally infected pigs
2.4.2.6 Experimental design for testing efficacy of some selected
anthelminthic drug against Strongyloides in pigs
Efficacy and safety of 03 anthelminthic drugs including fenbendazole atdosage of 5 mg/kg B.W, thiabendazole at dosage of 6.5 mg/kg B.W, ivermectin, atdosage of 0.3 mg/kg B.W and experimental study on preventive measures for thediseases were conducted by using - group experimental design, comparing them by
testing for differences between them.
2.4.5.Method for analysis of Experimental data
Experimental data were analyzed by using method of biological statistics(according to documents of Do Duc Luc et al 2017), on Microsorf Officesoftware, Excel 2010 program and minitab software version 16.0
Chapter 3 RESULTS AND DISCUSSION
3.1 Study on the prevalene and intensity of gastrointestinal nematode infection in pigs in Bac Giang
3.1.1 Situation of prevention of gastrointestinal nematodiasis for pigs in Bac Giang province
The findings are shown in table 3.1 :
Many of 950 investigated households in 5 districts of Bac Giangprovince had applied measures for prevention and control of nematodiasis
Trang 11in pigs, but the results from implementation were not high, namely:maintaining hygiene and sanitation practices such as providing housing,cleaning feed, animal farm tools and equipment are the easiest measures forprevention of diseases, but only 305 households implemented, accounting for32.11%; 10.74% of households that applied the periodical disinfection of farmequipment and tools In term of manure collected and composted usingbioheating, there were only 7.16% of the households applying; periodicaldeworming for pigs accounted for 14.84%, there were only 5.05% ofhouseholds applying combination of 2 preventive measures for prevention ofnematodiasis in pigs; In addition, up to 30.11% of households did notimplement preventive measures for nematodiasis in pigs
3.1.2.Composition of gastrointesstinal nematode parasites in pigs
Four nematode species were detected in the gastroitenematodiasisstinal tract
of pigs including A suum, S ransomi, O dentatum and T suis Ditected nematode
species were commonly distributed in 5 districts with the frequency of occurrence
of 100%
3.1.3.Prevalence and intensity of infection with gastrointesstinal nematodes in pigs
3.1.3.1 Prevalence and intensity of gastrointestinal nematode species in
pigs in the locations through dissection
Table 3.3 Overall prevalence and intensity of gastrointestinal nematode
species in pigs in the locations (through dissection)
Location
(District)
Number of dissected pigs (pig)
Number of infected pigs (pig)
Percent age (%)
Intensity of infection (Number of worms /pig min
Trang 12The results are presented in Table 3.3: 743/1325 dissectedpigs infected with gastrointestinal nematodes , accounting for56.08%, the overall intensity varied from
1 - 1026 worms / pig Among the investigated districts, the prevalence and intensity
of infection in pigs raised in Son Dong district was the highest (68.50% and 8-1026worms/pig respectively); the lowest was those in Lang Giang district (43.68% and 1– 467 worms / pig respectively) The difference in the prevalence and intensity ofnematode infection in pigs between Son Dong and Lang Giang districts wasstatistically significant (P <0.05)
Table 3.4 Prevalence and intensity of various gastrointestinal nematode
species in pigs (through dissection)
Index Nematoda species Number of dissected
pigs (pig)
Number of infected pigs (pig)
Percentage (%)
Intensity of worm infection (Number of worms giun/pig min - max
The results are shown in table 3.4: 4 nematode species were detected,
of which prevalence and intensity of S ransomi infection was the highest
(22.34% and 4 - 1,026 worms / pig respectively), prevalence and intensity
of T suis and A suum infection were significantly lower than those of S ransomi (P <0.05), the lowest was those of O dentatum (3.92% and 2 - 87
worms/pig respectively) Number of pigs infected with a mixture of
Strongyloides and other nematodes was 14.64% and 13-712 worms / pig
respectively There were only 12 cases of other nematode infection in the
Trang 13absence of Strongyloides accounting for 0.91% and infection intensity was
(District)
Number of pigs(pig) examinated (pig)
Number of infected pigs (pig)
Percentage (%)
3.1.3.4.Infection Prevalence and intensity of various gas trointestinal nematode species (through feces examination)
The results are shown in Table 3.6: The overall infection prevalence was
59.47% 4 nematode species were detected, of which prevalence of S ransomi infection was the highest (25.71%), the infection prevalence of A suum and T suis was lower (6.87% and 7.13% respectively), the lowest was prevalence of O dentatum infection (3.78%) Number of pigs infected with a mixture of Strongyloides and other
nematodes accounted for 15.04%, of which 0.93% were infected with a mixture of
nematodes in the absence of a Strongyloides The prevalence of S ransomi infection in
pigs compared with other nematode species was significantly different ( P < 0.05) Pigsinfected with four gastrointestinal nematode species with light and moderate infectionintensity were predominated (49.69% and 29.63% respectively)
3.1.3.5 Changes in gastrointestinal nematode infection depending on pig age ( through faeces examination)
Trang 14The results are shown in table 3.7, pigs of all ages were infected withgastrointestinal nematodes Pigs under 2 months of age were infected with the highestprevalence (78.84%), followed by pigs from 2 - 4 months old (69.71%), pigs from 4 -
6 months old (55.82%), the lowest prevalence of infection was in pigs over 6 months
3.1.3.7 The prevalence of gastrointestinal nematode infection in pigs depending on season
The results are shown in Table 3.9: in all four seasons, pigs were infected withgastrointestinal nematodes, the prevalence of infection varied from 40.70% - 73.80%
In particular, pigs raised in summer, infection prevalences of nematodes were thehighest (73.80%), in autumn and spring infection prevalences were 65.66% and56.82% respectively In Winter Prevalence of gastrointestinal nematode infection wasthe lowest (40.70%) The difference in nematode infection prevalence betweenSummer and Autumn was quite significant (P <0.05)
3.2 Study on Swine Strongyloidosis
3.2.1.Results from identification of Strongyloides species
in pigs in Bac Giang
3.2.1.1 Results from dissection of pigs to collect and detect Strongyloides
Number of pigs infected with
S.ransomi (pig)
Percentage
of infection (%)
Intensity of infection (min - max worm/pig)