Nghiên cứu nhiễm giun tròn đường tiêu hóa, bệnh giun lươn (strongyloidosis) trên lợn tại tỉnh bắc giang và biện pháp phòng trị tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING
THAI 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


The dissertation has been completed at:
AT 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


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1. INTRODUCTION
1.1. The urgency of the project
Bac Giang is a province with a quite development of pig husbandry. For
many years, pig production has made an important contribution to hunger
eradication, poverty reduction and enrichment for farmers in the province.
According to the Department of Animal Science and Veterinary Medicine of Bac
Giang 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 of
pigs, farmers have applied scientific and technical advances in pig farrming
practice, thus bringing significant economic benefits to them. However, pig
farming in Bac Giang province has faced with many difficulties, including
disease problems. In addition to common infectious diseases, gastrointestinal
nematode infections in pigs ofen occur chronically. Although nematode
infections do not cause high mortality of pigs as infectious diseases, nematode
infections 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 that
diseases caused by gastrointestinal nematodes were very common diseases and

was 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 Giang
province, there have been no studies on the situation of nematode infections
and Strongyloidosis in pigs, so there are no measures for effective
prevention 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 gastrointestinal
nematodes in pigs;
- Identification of some epidemiological, pathological and clinical
characteristics, the prevention and control measures of Strongyloidosis in
pigs in Bac Giang province.


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3. Scientific and practical significance of the topic
3.1. Scientific significance
The project provides scientific information about the situation of
gastrointestinal nematode infection in pigs in Bac Giang province;
Epidemiological,
pathological
and
clinical
characteristics
of

Strongyloidosis, 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 pig
producers applying preventive measures and treatment of nematode
infection in general, and Strongyloidosis in particular in order to limit the
prevalence of nematode infection in pigs and limit damage caused by
Strongyloidosis, making contribution to sustainable development of pig
farming in Bac Giang province.
4. New contributions of the project
- The project is the first systematic study on the
situation of gastrointestinal nematode infection in pigs in
Bac Giang province including ; Epidemiological, pathological
and clinical characteristics of pig threadworm infection
( strongyloidosis)
- Effective procedures for the prevention and treatment of
Strongyloidosis in pigs have been designed, recommended and applied
widely 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 the
results of the project. PhD student has referred to 151 references (including
68 published in recent years ( past five years).


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Chapter 1
DOCUMENT OVERVIEW
Nguyen Thi Le et al. (1996) indicated that nematodes in pigs are
distributed all over the world. In particular, in the tropic and subtropic regions
there is hot and humid climate, which is a very favourable condition for eggs
and nematode larvae to develop in the outside, making the gastrointestinal
nematode 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 infected
with strongyloides are characterized by diarrhea, loose stools with blood
due to hemorrhagic intestine, anemia, emaciation and sudden death. In
addition, when the larvae enter the alveoli, they can cause pneumonia with
fever at temperature of 40 - 41.5oC, coughing a lot, difficulty breathing.
According to Nguyen Thi Kim Lan (2011), In order to prevent
Strongyloidosis in pigs effectively, it is necessary to implement integrated
prevention measures, including: hygiene of pig pens, cleaning livestock
instruments and equipments; Pig manure and waste must be collected daily
and properly composted at the stipulated places; implement disinfection of
pig pens regularly, do not keep pigs at different age together, using a
reasonable 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 activities
for 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. )


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2.1.2. 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, and
Veterinary Medicine
- Bac Giang Agriculture and Forestry University; Bac Giang General
Hospital and Preventive Medicine Center of Province; Institute of Ecology
and 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 different
age; samples from sediment of pig house floor, wastewater samplesfrom pig
pens, samples from surface soil in garden for growing feed crops; samples from
nematodes 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, olympus
CX221 microscope, scanning electron microscope; stool test kits; blood
microsampling device; Hematology Analyzer.Erma PCE 210 and TC - Matrix;
Microtome Equipment, automatic DNA sequencing machine; ABI Prism 3130
Genetic Analyzer; centrifuge machine, micropipetes, electrophoresis

machine,UV Transilluminator, PCR machine, DNeasy Tissue Kit (QIAgen)
Chemicals used for preparing smear slide from specimens, saturated brine
solution; 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.
2.3. Contents of study
2.3.1. Study on prevalence and intensity of gastrointestinal nematode
infection on pigs in Bac Giang province
- Investigation of situation of gastrointestinal nematode prevention for
pigs in Bac Giang.
- Identification of gastrointestinal nematode species and their
distribution in Bac Giang
- Dissection of pigs to determine:


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+ The prevalence and intensity of gastrointestinal nematode infection
in locations
+The prevalence and intensity of infection of various gastrointestinal
nematode species in pigs
- Stool examination to determine:
+ The prevalence of gastrointestinal nematode species infection in
various locations
+ The prevalence and intensity of gastrointestinal nematode species
infection 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 Bac
Giang through dissection.
- Results from designation of Strongyloides by using morphological
technique, 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 and
treatment of Strongyloidosis in pigs
2.4. Methods of study
2.4.1. Study on prevalence and intensity of gastrointestinal nematodes
infection on pigs in Bac Giang
2.4.1.1. Investigation of actual situation of prevention and control of
gastrointestinal nematode infection in pigs in Bac Giang province:


6
Prevention and control of gastrointestinal nematode infection in pigs in Bac
Giang province was carried out by direct observation, using questionaire for pig
farminghouseholds 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 pigs
were collected then, the nematode smear slide were fixed using method as
discribed by De Grisse A. T. (1969). Species designation was made
according to species identification key of Phan The Viet et al. (1977), De
Ley 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 egg
counting technique for counting eggs per 1 gram of faeces in Mc master
counting 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 the
identification key of Phan The Viet et al. (1977), De Ley P. and Blaxter M. (2004). At
the same time, the superstructural morphology of Strongyloides spp. was observed
based 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 sequences
compared with the DNA sequences on the gene bank using MEGA software
version 6.0 (Tamura K. et al., 2013), the phylogenetic tree analysis and drawing

were performed by using the Maximum Likelihood (ML) method. with the most
appropriate model. The group confidence interval was assessed by bootstrap
values with 1000 replicate samples.


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2.4.2.3.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 each
experiment , the start of the experiment was labeled and dated. The samples were
placed at normal temperature and humidity.
On the first day, every 2 hours, samples were taken for testing by using
Fulleborn 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 to
identify the time duration for larvae to develop into infective larvae.
Infective larvae were determined by observing the morphology of
larvae under light microscope to recognize infective larvae based on
morphology 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 pathological

characteristics of the disease; experimental infection 2 was used for testing efficacy
of specific antheminthic drugs for treatment of Strongyloidosis: In phase 1: 15 pigs
were used and divided into 3 groups, group1, experimentally gastrointestinal
infection was caused ; Group 2: skin infection was caused ;and group 3 for control
also 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 pigs
were observed, compared to pigs in the control group.


8
Hematological indices were determined using Erma PCE - 210 and TC Matrix hematology analyzers.
Macropic lesions score were Identified by dissection of pigs
experimentally infected with Strongyloides, observing with the naked eye and
magnifying glass lesions in liver, lungs and gastrointestinal tract.
Study on microscopic lesions score using preparation of slides from
paraffin embedded tissues stained with Haematoxilin – Eosin, The slides were
observed 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 at
dosage of 5 mg/kg B.W, thiabendazole at dosage of 6.5 mg/kg B.W, ivermectin, at

dosage of 0.3 mg/kg B.W and experimental study on preventive measures for the
diseases 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 Office
software, 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 Giang
province had applied measures for prevention and control of nematodiasis


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in 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 for
prevention of diseases, but only 305 households implemented, accounting for
32.11%; 10.74% of households that applied the periodical disinfection of farm
equipment and tools. In term of manure collected and composted using
bioheating, there were only 7.16% of the households applying; periodical
deworming for pigs accounted for 14.84%, there were only 5.05% of
households applying combination of 2 preventive measures for prevention of
nematodiasis in pigs; In addition, up to 30.11% of households did not
implement 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)
Intensity of
Number Number
infection
Location
of
of
Percent
(Number of
(District) dissected infected age (%)
worms /pig min
pigs (pig) pigs (pig)
÷ max)
Viet Yen
264
155
58.71ab
5 - 1002
Hiep Hoa
269

147
54.65ab
2 - 905
Lang Giang
261
114
43.68b
1 - 467
Yen Dung
258
140
54.26ab
3 - 863
Son Đong
273
187
68.50a
8 - 1026
Total
1325
743
56.08
1 - 1026


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The results are presented in Table 3.3: 743/1325 dissected
pigs infected with gastrointestinal nematodes , accounting for
56.08%, 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 8-1026
worms/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 of
nematode infection in pigs between Son Dong and Lang Giang districts was
statistically significant (P <0.05).
Table 3.4. Prevalence and intensity of various gastrointestinal nematode
species in pigs (through dissection)
Index

Nematoda species

1
2
3
4

A. suum
S. ransomi
O. dentatum
T. suis
S.ransomi and other
roundworms ( Mixed
infections)
other roundworms
( Mixed infections)

5
6
Total

Intensity of
Number of Number of
worm infection
Percentage
dissected
infected
(Number of
(%)
pigs (pig) pigs (pig)
worms giun/pig
min - max

92
296
52
97

6.94c
22.34a
3.92cd
7.32c

1 - 22
4 - 1.026
2 - 87
3 - 650

194

14.64b

13 - 712

12

0.91d

7 - 125

743

56,08

1 - 1026

1325

1.325

*Note: within column percentage of infection bearing the different letter are
significantly different (P<0.05)).

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

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absence of Strongyloides accounting for 0.91% and infection intensity was
7. - 125 worms / pig.
3.1.3.3.Prevalence of gastrointestinal nematode infection in pigs (through
faeces examination) in the some locations of Bac Giang province
Table 3.5. Prevalence of gastrointestinal nematode infection in pigs
(through faeces examination) in some locations of Bac Giang province
Number of
Number of
Location
pigs(pig)
Percentage
infected pigs
(District)
examinated
(%)
(pig)
(pig)
Viet Yen
980
604
61.63ab
Hiep Hoa
985
580
58.88ab
Lang Giang
983

478
48.63b
Yen Dung
978
555
56.75ab
Son Đong
994
709
71.33a
Total
4.920
2.926
59.47
*Note: within column percentage of infection bearing the different letter are
significantly different (P<0.05)).

The results are presented in Table 3.5: pigs raised in 5 districts of BacGiang
province were all infected with nematodes, the prevalence of infection ranged from
48.63% -71.33%. Pigs raised in Son Dong district, the prevalence of gastrointestinal
nematode infection was the highest (71.33%); In Viet Yen, Hiep Hoa and Yen Dung
districts the prevalence of infection was 61.63%, 58.88% and 56.75% respectively;
Pigs raised in Lang Giang district the prevalence of infection was the lowest (48.63%)
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). Pigs
infected with four gastrointestinal nematode species with light and moderate infection
intensity were predominated (49.69% and 29.63% respectively).
3.1.3.5. Changes in gastrointestinal nematode infection depending on pig
age ( through faeces examination)


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The results are shown in table 3.7, pigs of all ages were infected with
gastrointestinal nematodes. Pigs under 2 months of age were infected with the highest
prevalence (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
of
age (27.23%). Thus, the infection prevalence of gastrointestinal nematodes in pigs
decreased gradually with the pig's age.
3.1.3.6.The prevalence of gastrointestinal nematode infection in pigs
depending on farming methods
The results are shown in Table 3.8: Pigs raised by traditional farming methods,
Infection prevalence of nematodes was the highest (85.59%); followed by
prevalence of infection in semi industrial pig farming (72.35%); In industrial pig
farming prevalence of infection was the lowest (28.64%) (P <0.05). The above
results showed that the farming methods had a significant effect on the prevalence of
gastrointestinal nematode infection in pigs.
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 with
gastrointestinal nematodes, the prevalence of infection varied from 40.70% - 73.80%.
In particular, pigs raised in summer, infection prevalences of nematodes were the
highest (73.80%), in autumn and spring infection prevalences were 65.66% and

56.82% respectively. In Winter Prevalence of gastrointestinal nematode infection was
the lowest (40.70%). The difference in nematode infection prevalence between
Summer 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
in pigs
Table 3.10. Results from dissection of pigs to collect and detect Strongyloides
Intensity of
Number of Number of pigs
Percentage
Location
infection
infected with
dissected
of infection
(District)
(min - max
pigs (pig)
(%)
S.ransomi (pig)
worm/pig)
264
Viet Yen
110
41.67a
9 - 1.002
269
Hiep Hoa

102
37.92ab
7 - 905
261
Lang Giang
54
20.69b
4 - 467


13
Yen Dung
Son Đong
Total

258
273

1.325

87
137
490

33.72ab
50.18a
36.98

6 - 863
11 - 1.026

4 - 1.026

*Note: within column percentage of infection bearing the different letter are
significantly different (P<0.05)).

The results are shown in table 3.10: There were 490 of total 1,325 pigs
dissected, infected with Strongyloides, accounting for 36.98%, the overall infection
intensity was 4 - 1,026 worms/pig. The prevalence and intensity of Strongyloides
infection varied among the investigated districts: The highest Strongyloides
infection prevalence and intensity were in pigs raised in Son Dong district
(50.18% and 11 - 1,026 worms/pig respectively); the lowest prevalence and
intensity of Strongyloides infection were in pigs raised in Lang Giang
district(20.69% and 4 - 467 worms/pig respectively).
3.2.1.2. Results from identification of Strongyloides by morphological technique
The results are shown in table 3.11. and figures 3.12a, 3.12b, 3.12c. 3.12d,
3.12e, 3.13, 3.14a, 3.14b, 3.14c: The parasitic female Strongyloides was in thread
shape, average size was 4.8 mm in length, average size of the largest body was of
0.055 mm with narrow anterior, conical obtuse tail. The esophagus was 0.99 ±
0.022 mm long. The anus was 0.065 ± 0.002 mm from the tail. The vulva was a
horizontal cleft, with a pair of labia located in the middle of posterior part of the
body, 1.913 ± 0.024 mm long from the tail. The ovary was meandering. Two
ovaries are thin shelled that start near the genital opening, one ovary pointed
upwards, towards the body and the other pointed down the tail. The uterus
contained 1 - 10 eggs. The eggs were ovoid in shape with thin shell containing
larvae, that average size was 0.051mm x 0.028 mm.
Observation of morphology of S. ransomi by using Hitachi S-4800 Scanning
electron Microscope (SEM) showed that this nematode species displayed
characteristic morphological features as follows. The oral cavities were symmetrical
bell shaped. Area surrounding the mouth, fell into 8 lobes of which 4 tongue-shaped
lobes protruding from the surface of the oral cavity at the end poit of each lobe. At the

end of the lobe there was oil glands protruding in the buccal cavity to form esophageal
spines, 4 lobes surrounding the oral cavity. The vulva was labia which was lip- like
structure The; body size was 0.032 mm wide, The tail was blunt and the tail tip was
in splited form. The buccal cavity superstructure of S. ransomi parasites in pigs in
Bac Giang was consistent with the description of Sato H. et al. (2008).
3.2.1.3. Results from identification of strongyloides species parasitic in pigs by
using molecular biology technique
863
bp


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Figure 3.15. The electrophoresis image of PCR products
of Molecular cloning and sequencing of 18S rDNA
gene fragments on agarose gel agar 1.0%
The results from electrophoresis image of PCR products of Molecular cloning
and sequencing of 18S rDNA gene fragments from 5 samples of Strongyloides
collected in 5 districts of Bac Giang province. Figure 3.16 showed that the
corresponding gel electrophoresis band was approximately 863bp (Figure 3.16).
* Relationship among Strongyloides ransomi species found in pigs in
Bac Giang province

Figure 3.16: The phylogenetic tree was constructed using 18S rDNA
gene sequence by method based on Maximum Likelihood
The phylogenetic tree was constructed using data from 18S rDNA gene
sequence by method based on Maximum Likelihood (ML) (Figure 3.16) .The
results showed that S. ransomi species gene sequence from Bac Giang,
Vietnam was 100% identical to the KU724126 of S. ransomi from Cambodia,
and all the sequences of S. ransomi formed a common branch close to S.

venezuelensis, with a genetic distance of 0.3%. On the other hand, 18S DNA
gene sequencing and analyzis of the hypervariable region- HVR-I showed that
the strongyloides found in pigs in Bac Giang province belongs to S. ransomi
species.
3.2.2. Study on strongyloides infection in pigs through faeces examination
3.2.2.1.The prevalence and intensity of Strongyloides infection in pigs in some
locations


15

Figure 3.17 Chart depicting prevalence of Strongyloides infection in pigs
in different locations
The results were presented in table 3.13 and figure 3.17: in all of 5 districts of Bac
Giang province, there were pigs infected with Strongyloides, the overall prevalence of
infection was 40.75%. the highest prevalence of Strongyloides infection was in Pigs in
Son Dong district and the lowest was in Pigs in Lang Giang district (52.01%, 26.04%
respectively). In term of the intensity of infection: pigs were infected with Strongyloides in
different levels, of which pigs were infected at light level, at moderate level, at heavy level
and very heavy level was 47.58%; 28.28%, 15.16% and 8.98% respectively.
3.2.2.2.The prevalence and intensity of Strongyloides infection depeding on
pig age

Figure 3.19. Graph depicting nematode infection with pig's age
The results are shown in table 3.14 and figure 3.19: The strongyloides infection
prevalene varied with age from 14.99% - 63.02%. Pigs under 2 months of age were
the highest infected (63.02%) and were infected at a very heavy level and very heavy
level with high percentage (20.60% and 13.98% respectively); followed by pigs 2 - 4
months of age (46.20%); The infection prevalence decreased significantly in pigs at 4 6 months of age (33.84%); The lowest prevalence was in pigs over 6 months of age
(14.99%) and pigs mostly were infected at light and moderate level. Thus, the

prevalence and intensity of Strongyloides infection decreased with age of pigs.
3.2.2.3. Prevalence and intensity of Strongyloides infection depending on
seasons


16

Figure 3.20. Graph depicting prevalence of Strongyloides in pigs varied
with seasons
The results are shown in table 3.15 and chart in Figure 3.20: pigs were
infected with Strongyloideis all year round, but in Summer and Autumn
seasons, Strongyloides infection was the highest accounting for 50.67% and
47.38% of the pigs tested respectively) and severely infected; In spring, the
prevalence of infection was 37.53%; In winter, was 26.80%, significantly
lower than that in Summer and Autumn (P <0.05), and pigs were infected
only at light level ( 63.75%). Thus, pigs infected with Strongyloides all year
round but pigs raised in Summer, Autumn, and Spring the infection of pigs
was higher than that in Winter.
3.2.2.4.The prevalence and intensity of Strongyloides infection in pigs
depending on farming methods

Figure 3.21. Graph depicting prevalence of strongyloides infection in
pigs in realation to pig farming methods
The results are shown in table 3.16 and figure 3.21: The highest
prevalence of Strongyloides infection in pigs raised under traditional methods,
using by products as pig feed (61.55%); and pigs in semi intensive production,
the prevalence of strongyloides infection was 47.63%; pigs in industrial
production the infection prevalence was 19.83%. In term of intensity of
infection: pigs raised under traditional
methods were infected with

Strongyloides at heavy level accounting for 18.98% and at very heavy level
12.88% higher than the other two methods.


17
3.2.3. Study on contamination of Strongyloides eggs and larvae in the
environment.
3.2.3.1. Study on contamination of Strongyloides eggs and larvae in pig
pens, wastewater pits, soil in garden for growing feed crops for pigs
The results are shown in table 3.17: 36.65% of the samples collected from
wastes in pig pen floors, 23.44% of the samples of wastewater from pig pens and
12.19% of the soil samples from land for growing feed crops, Strongyloides eggs
and larvae were detected. Prevalence of egg contamination differs significantly
between sampling locations (P <0.05). On the other hand, Strongyloides eggs
and larvae were found only in samples collected from pig farming households
infected with Strongyloides.
3.2.3.2. The duration from Strongyloides eggs hatching and developing into
pathogenic larvae in pig feces at laboratory temperature.
Table 3.18. The duration from Strongyloides eggs hatching and developing
into pathogenic larvae in pig feces at laboratory temperature
Developmental
duration needed to
Observed
become pathogenic
Season
number of
larvae
samples(sample)
( X ±m x )
(day)

Spring
15
47.13 ± 1,31
13.73b ± 0,43
3.40b ± 0.16
Summer
15
57.29 ± 093
5.53c ± 0,23
2,.56c ± 0.10
Autumn
15
5404 ± 1.05
6.27c ± 0.26
2.80c ± 0.07
Winter
15
41.24 ± 1.34
16.40a ± 0.27
4.40a ± 0.22
*Note: within column the numbers bearing the different letter are
significantly different (P<0.05)).
Number of
eggs/
microscopic
field

Duration from
eggs to hatch
into larvae

( X ±m x )
(hour)

The results are shown in table 3.18: In Summer and Autumn, it was warm,
so duration for eggs to hatch into larvae faster ( it took 5.53 hours and 6.27 hours
respectively, and the average length of time for larvae to develop into infective
larvae. (2.56 days and 2.8 days respectively) was also significantly faster than
that of Spring and Winter (P <0.05).
3.2.3.3. The survival time of infective larvae in pig feces in laboratories


18
The results are presented in table 3.19: The survival time of larvae
depended on the temperature and humidity of the specific environment: in
Summer and Autumn after 25 days larvae died completely. In Winter and Spring,
after 35 days, the larvae died completely in pig faeces. Therefore, In Summer
and Autumn in the North of our country temperature is higher, water evaporates
faster, and feces is dry which is a harmful condition for the survival of infective
larvae, so they die faster. Our study results were consistent with those of
Cavalcante M. M. A. S. et al. (2014).
Thus, infective Strongyloides larvae can survive for 25 - 35 days in pig
manure, they can spread from manure to the environment. Therefore, the
collection of manure for composting to kill eggs and Strongyloides larvae is
an important measure in preventing Strongyloidosis in pigs.
3.2.4. Study on pathological and clinical characteristics of
Strongyloidosis in experimentally infected pigs and naturally infected pigs
in the field
3.2.4.1 Study on experimental infection of pigs
* Result from experimental infection of pigs
The results are shown in Table 3.20: Pigs infected experitally from gastrointestinal

tract needed about 6-8 days to complete the life cycle; pigs infected experimentally through
the skin took about 8-11 days to complete the life cycle.
* Studying clinical progression in pigs infected experimentally with Strongyloides
The results are presented in tables 3.21 and 3.22: all of 10 pigs became sick
with the main clinical signs as follows: anorexia, poor weight gain, dry skin,
ruffled hair coats, pale mucous membranes, coughing, some pigs had fever,
digestive disorder sometimes loose stool, sometimes slime stool. Pigs infected
experimentally trough skin showed skin rashes.
* Study on some blood indices in experimentally
Strongyloides infected pigs.

Table 3.23. Changes in some red blood cell
indices in experimentally Strongyloides
infected pigs
RBC indices

Control pigs
( X ±m x )

Experimentally
infected pigs
( X ±m x )

Number of blood samples (sample)

10

20

19
RBC counts (milion/mm3)
Hemoglobin content (Hb) (g%)
Hematocrit (%)
Mean corpuscular volume (µm3)
Mean corpuscular hemoglobin
concentration (MCHC) (%)
Mean corpuscular hemoglobin (MCH
Hb

6.12a ± 0.13
10,93a ± 0.26
34,65a ± 0,28
54,15a ± 0,35

4,37b ± 0,09
9,44b ± 0,13
25,15b ± 0,19
41,25b ± 0,14

35,13a ± 0,43

34,98a ± 0,22

18,61a ± 0,35

17,95a ± 0,19

* *Note: within rows numbers bearing the different letter are significantly
different (P<0,01).

Table 3.24. Changes in WBC counts and leucogram in experimentally
Strongyloides infected pigs
Control pigs

Blood indices

( X ±m x )

Number of blood samples (sample)
WBC counts (thousand/mm3)

Leucogram

Experimentally
infected pigs
( X ±m x )

10
15,91b ± 0,32

20
22,14a ± 0,19

Neutrophils

43,11a ± 0,61

33,13b ± 0,50

Eosinophils (%)

3,69b ± 0,22

11,83a ± 0,21

Basophils(%)

1,33a ± 0,03

1,34a ± 0,03

Lymphocytes (%)

48,79b ± 0,21

49,95a ± 0,32

Large monocytes (%)

3,08b ± 0,16

3,75a ± 0,17

*Note: within rows numbers bearing the different letter are significantly
different (P<0,01)

Table 3.25. Changes in some blood
experimentally Strongyloides infected pigs
Blood indices

biochemical

Control pigs

indices

in

Experimentally
infected pigs


20
( X ±m x )

( X ±m x )

Number of blood samples
(sample)

10

Blood glucose levels (mmol/l)

5.46a ± 0,35

3,85b ± 0,14

Total serum protein (g/l)

66.57a ± 0,46

51,86b ± 0,37

Albumin (g/l)
Globulin (g/l)
A/G Ratio

35,64a ± 0,45
30,08b ± 0,48
1,19a ± 0,03

27,58b ± 0,37
39,03a ± 0,45
0,71b ± 0,01

20

* *Note: within rows numbers bearing the different letter are significantly
different (P<0.01)).

The results are shown in tables 3.23, 3.24, 3.25: WBCs were increased
(leukocytosis) in pigs infected with Strongyloidiasis especially increase of
eosinophils (eosinophilia), RBC, hemoglobin concentration, glucose levels,
protein content, albumin content in the blood of experimentally infected pigs
were lower, globulin content was higher than those of the control pigs.
* Macropic and microscopic lesions in pigs infected experimentally with
Strongyloides
(*) Macropic lesions in pigs infected experimentally with S. ransomi

Picture 2: pneumonia with ,
Picture 1: Small inestine with
congestion and petechiae
catarrhal enteritis, congestion
and hemorrhage
The results are shown in table 3.26: the macropic lesions caused by S.
ransomi in pigs in both experimental groups were clearly visible in the parasitic
sites of the worms and organs through which larvae migrated, including: small
intestine mucosa, especially catarrhal enteritis found in the Duodenum, with
hematomas and petechiae, and many parasitic worms were found in the small


21
intestine submucosa. Pneumonia with hematomas occurred. There were
hematomas in the skin. Lesions in other organs such as liver, kidneys, heart were
not clearly found.
(*) Microscopic lesions in some organs of pigs affected by
Strongyloidosis by experimental infection
The results are shown in table 3.27: Microscopic lesions were
characterized by degeneration of mucosal surface of the small intestine; the
surface sloughing off with Inflammatory cell infiltration, lymphoid hyperplasia.
Images of adult form, larvae and eggs of Strongyloides were seen in intestinal
slices; Hemorrhagic lungs with inflammatory cell infiltration, congestion of
pulmonary veins; slightly enlarged heart muscles inflammatory cell infiltration of
renal parenchyma and glomeruli were found.
3.2.4.2. Study on pigs naturally infected with Strongyloides in the field
* Clinical signs of pigs naturally infected with Strongyloides in the
locations
The results are shown in table 3.28: There were 346 of 1,265 infected pigs

manifesting clinical signs, accounting for 27.35%. Clinical signs of infected pigs in
the field were similar to those of experimentally infected pigs.
* Macropic lesions score in pigs infected with Strongyloides in the
locations.
There were 85 of 296 pigs examined for infection of Strongyloides
depicting lesions score, accounting for 28.72%. The lesions were
concentrated mainly in the small intestine, lungs, and skin (the site, where
the larvae migrated to and the parasitic site for adult worms). Lesions were
the same as those in experimentally infected pigs.
3.2.5. Study and recommendation of preventive measures and treatment
of swine Strongyloidosis
3.2.5.1. Testing of anthelmintic drugs for deworming strongyloides in pigs
* Determination of efficacy and safety of anthelmintic drugs used for
experimentally infected pigs
The results are shown in Table 3.30: All of 3 tested drugs including
fenbendazole at dosage of 5 m / kg B.W, thiabendazole, at dosage of 6.5
mg / kg B.W, ivermectin at dosage of 0.3 mg / kg B.W I.M administered
were highly effective over 90% and safe for pigs.
* Efficacy of anthelmintics used in large scale and narrow scale of
strongyloidiasis in pigs in the field
The results of efficacy of the anthelmintic drugs used for deworming
Strongyloides on a narrow scale and large scale in the field are shown in


22
tables 3.31 and 3.32 (in the full version of dissertation): The drug testing in
a narrow and wide scale in the field showed that All of 3 drugs had
activities against Strongyloidosis in pigs with high efficacy of over 90%
and safe for pigs, of which ivermectin had the highest efficacy of
deworming Strongyloides (98.46%).

3.2.5.2. Study on preventive measures and treatment of
Strongyloidosis in pigs
* Study on some disinfectant activites against Strongyloides larvae
Study results from some activities of disinfectants against Strongyloides
larvae were shown in Table 3.33 (in the full version of the dissertation)
Laboratory test results showed that all of disinfectants: Chloramin B,
Good Farm L, P.V.D iodin 10% killed S. ransomi s larvae.
* Study on rational and effective times of anthelmintic drugs used for
prevention of Strongyloidiasis in pigs
The results from Table 3.35 showed that 1 month after the
experimental prophylactic deworming of Strongyloides in pigs by using
ivermectin
in experimental 1 and 2, the infection prevalence of
Strongyloides was very low (4.72% and 6.36% respectively). In the control
group that anthelmintic drugs were not used for preventiion , the prevalence
of Strongyloidosis was significant higher (35.29%).
(*) The prevalence and intensity of Strongyloides infection in pigs 3
months after the experiment:
The results are shown in Table 3.36: 3 months after the experiment,
pigs in the control group infected with Strongyloides was quite high,
accounting for 41.58%. pigs in experimental group 2 the Strongyloides
infection prevalence was the lowest ( 2.73%).
3.2.5.3. Recommendation of preventive measures and treatment of pig
threadworm infection( Strongyloidosis)
From the study results of the project, we recommend the
preventive measures for pig threadworm infection as follows: Pigs
can be dewormed by using 1 of 3 anthelmintic drugs: fenbendazole at
dosage of 5 mg/kg B.W, thiabendazole atdosage of 6.5 mg/kg B.W.,
or ivermectin at dosage of 0.3 mg/kg B.W treatment of manure to
destroy pathogens; cleaning pigpends and

pig keeping areas;
enhancing care and nutrition provided for pigs
CONCLUSIONS AND RECOMMENDATIONS


23
1. Conclusions
From the study results from the project we draw some conclusions as
follows :
1.1. Characteristics of gastrointestinal nematode infection in pigs in Bac
Giang province:
The present situation of nematode prevention in Bac Giang province is
not good. 30.11% of households still do not apply preventive measures.
4 gastrointestinal nematode species in pig have been detected in Bac
Giang province icluding : A. sum, O. dentatum, T. suis and S. ransomi
Pigs in Bac Giang province have been infected with gastrointestinal
nematode at high prevalence (56.08% through dissection and 59.47%
through stool analysis).
The prevalence and intensity of nematode infection decreased with
age of pigs, pigs under 2 months old were the highest infected with
nematodes (78.84%); In traditional pig farming practices, the prevalence
and infection intensity of nematodes are higher than that of intensive pig
farming; In Summer and Autumn gastrointestinal nematode infections in
pigs are higher than those in Winter and Spring.
1.2. Pig thread worm infection ( swine Strongyloidosis)
* Results from identification of Strongyloides in pigs
By using morphological technique, the nematodes parasitic in small
intestine of pigs in Bac Giang province are S. ransomi consistent with
Schwartz and Alicata finding, 1930.Analysis of HVR - I in 18S rDNA
sequences from 5 samples of Strongyloides, shows completely identical to

the sequence (KU724126) of S. ransomi in Cambodia.
* Epidemiological survey of Strongyloidosis in pigs.
- The prevalence of Strongyloides in pigs in Bac Giang province is
40.75%, the heavy intensity infection and very heavy intensity infection is
15.16% and 8.98% respectively.
- The pig pen floors, wastewater pits from pig pens, the garden for
growing feed crops for pigs are contaminated with Strongyloides eggs and
Strongyloides larvae
- In Summer and Autumn Strongyloides eggs hatch into larvae faster
and develop into infective larvae significantly faster than in Spring and
Winter; infective nematode larvae persist for 25 to 35 days in pig feces in