Zero Prevalence of Trichinella spp.

in Pigs Delivered in a Local

Slaughterhouse in Los Baos, Laguna
Alag, V.K.B.1, dela Cruz, C.P.P. 1, Manjares, M.L.D. 1, and Tupaen, S.P.1
1

Graduate Student, Graduate School, University of the Philippines Los Baos,

College 4031, Laguna, Philippines

Abstract
Trichinella spp., a known nematode infection, causes trichinellosis, a zoonotic
disease that is a public health hazard and an economic problem in swine production and
food safety. With the limited evidence of existence of Trichinella infection in the
Philippines, this study was conducted to check for its presence in pigs reared and
slaughtered in Los Baos, Laguna. A total of 30 diaphragm pillars were obtained from
pigs for slaughter in the local slaughterhouse of Los Baos, divided to 3 pools of 10
samples of 5g each sample. The pool samples were then artificially digested, and then
examined under a compound microscope. No Trichinella spp. larvae were observed in
all the samples. The result of our study suggests that Trichinella spp. has low prevalence
and is not endemic in Los Baos, Laguna.

Keywords: Trichinella, artificial digestion, pork, food safety

INTRODUCTION
Trichinella spp. are known to be
one of the world's most widely-distributed
groups of nematode infection because of
its ability to infect a broad spectrum of
mammalian hosts (Despommier et al.,
2005). It is the causative agent of
Trichinellosis, a zoonotic disease, which
is a public health hazard and an
economic problem in swine production
and food safety (Angi et al., 2014).
Trichinellosis is common in areas of
traditional agriculture where swine are
mostly grown in small private farms with

poor basic environmental sanitation (Beck

et al., 2005).
Trichinella can be transmitted to
humans via ingestion of meat that
contains viable Trichinella larvae. In the
case of Trichinella in pork, the larvae
grow and reproduce in the intestines and
the new larvae invade the hosts muscle
tissue (FAO, 2002).Trichinella species
normally accumulate in the diaphragm
and tongue of pigs (Kapel, 2005).
The most common vehicles of
human infections are mostly domestic
and wild pigs (Conlan, 2014). The
domestic pig become infected by
Trichinella by eating scraps from other
infected pigs, ingestion of infected rats,

tail-biting from infected pigs, and

ingestion of feces from pigs that had been
contaminated (1-2 days previously). The
domestic cycle is prevalent where pigs
are grown in small farms without
veterinary control or microbiological
barriers (Pozio, 2000).
In Southeast Asia, the tradional
practice of eating uncooked or partially
cooked pork places the people at risk of
acquiring food-borne parasitic diseases
like Trichinellosis. However, Conlan et al
(2011) stated that data on Trichinellosis of
wild and domestic animals in Southeast
Asia are scarce. The surveys in pigs
addressing
the
prevalence
of
Trichinellosis and burden infection are
limited, and only contemporary data are
documented in two small research
studies in Vietnam and Laos. Also,
according to Vu Thi et al., (2010), even in
backyard and free-range pigs, the
prevalence of Trichinellosis is relatively
low. In the study of Pozio (2007), it was
stated that Trichinella spp. infection has
never
been
documented
in
the
Philippines, supporting the study of Auer
(1995) that concludes that there is no
evidence of the occurrence of Trichinella
spiralis in the Philippines, specifically in
Mindoro.
With the limited data on the
existence of Trichenella spp. infection in
pigs in the Philippines, studies are
needed to examine the prevalence of this
parasite in the country. Therefore, the
objective of this study is to check for
presence of Trichenella infection in pigs
reared and slaughtered in the Municipality
of Los Banos, Laguna.

MATERIALS AND METHODS

Sample Collection
The meat (diaphragm pillar)
samples were obtained from pigs
slaughtered in the slaughterhouse in
Brgy. Bayug, Los Baos, Laguna. A total
of 30 samples were collected from pigs
selected using simple random sampling in
two collection dates (15 samples per
collection) within April of 2015, and were
stored in an ice chest with ice
immediately after collection. The samples
were then stored in a chiller in the Animal
Nutrition Laboratory for 4 days and 1 day
for samples collected on 1st day and
samples
collected
on
2nd
day,
respectively.
Sample Digestion
The study used the method of
artificial digestion using a magnetic stirrer
proposed by Gamble et al. (2000). 5g of
pure lean muscle were taken from each
sample weighed using a digital analytical
balance (KERN 770, Germany). The
examination of meat samples for
Trichinella spp. larvae was carried out
with the use of pooled sample digestion
method, with a total number of 3 pools of
10 samples each (50g per pool).
Each pooled sample was digested
using a digestive fluid comprising of 0.5%
pepsin and 0.2% of HCl. The digest fluid
together with the pooled meat samples
were put in a 2L glass beaker and stirred
for about 40 minutes, while being
maintained at temperature of 44C to
46C, using a hot plate magnetic stirrer.
The digestion fluid was then poured

through gauze to filter any undigested

meat while allowing the passage of
Trichinella larvae into a separatory funnel.
The primary sedimentation process was
allowed to occur for 30 minutes, then 40
ml was released into a glass tube, and
was allowed to sit again (secondary
sedimentation) for another 10 minutes.
Subsequently, 30 ml of the supernatant
was withdrawn, and the remaining 10 ml
was poured onto a Petri dish for
observation in a microscope.
Microscope Examination
The samples were brought to the
Parasitology Research Laboratory of the
Institute of Biological Sciences, UPLB for
examination. The samples contained in
the Petri dish was directly examined
under low power objective (x10
magnification)
using
a
compound
microscope (Nikon 1705, Japan) for
presence or absence of Trichinella spp.
larvae.

RESULTS AND DISCUSSION

Trichinella
spp.
are
considered zoonotic nematode worms
with global distribution (Markell et al.,
1999).Trichinella spp. are found in
virtually all warm-blooded carnivores
(Gamble, 1997; Markell et al., 1999), with
pigs having the most records among
domestic animals (Pozio, 2007). The
nematode has been associated with pork
products, and the understanding which
many people have about the need to
cook pork thoroughly is based on the risk

of becoming infected with this parasite

(Gamble, 1997). Outbreaks of Trichinella
spp. can be attributed to three factors: (1)
the area or country is highly endemic for
the parasite; (2) well-organized public
health system that enables precise
reporting of disease outbreak; and (3)
culinary habits that may provide
opportunities to eat undercooked meat
(Pozio, 2007). These factors are
important in estimating the actual
prevalence of the parasite in areas of
interest for epidemiological investigations.
The life cycle of Trichinella starts
when a susceptible mammalian host
consumes meat containing the encysted
larvae, commonly known as the cyst of
the parasite. The larvae are liberated
from the cysts when exposed to gastric
acid and pepsin in the stomach and
invade the small bowel mucosa where
they develop into adult worms. The
female adults begin to shed their larvae
after a week and these migrate to striated
muscles where they encyst. The life cycle
recommence
when
the
muscles
containing the encysted larvae are
ingested by another susceptible host
(Markell et al., 1999; Figure 1). Rodents
are primarily responsible for maintaining
the endemicity of Trichinella infection.
Carnivorous and omnivorous animals,
such as pigs or bears, feed on infected
rodents or meat from other animals.
Humans are accidentally infected when
eating improperly cooked meat with
encysted larvae (CDC).
This study aimed to detect the
presence of encysted larvae in pigs
slaughtered in a local slaughterhouse in
Los Baos, Laguna, Philippines. We

found zero prevalence among the

samples
examined
using
artificial
digestion coupled with optical microscopy
as the method of choice. According to
Pozio (2007), the absence of Trichinella
spp. in several countries, including the
Philippines, may be conclusive or may
only be present passively, that is, the
infection cannot be established in
mammalian hosts. Moreover, Gamble
(1997) has pointed out that sporadic
information is available on the prevalence
of trichinellosis in pigs in South America,
Africa, and Asia which includes the
Philippines. It was pointed out that the
prevalence of Trichinella spp. among
domesticated farm animals is generally
low. A recent study from Kupang City,
Indonesia showed prevalence lower than
1% following inspection of 1,650 grams of
masseter muscle tissues (Angi et al.,
2014). Similar cases have been observed
from northern Thailand and Chiang Mai,
China, showing very low prevalence rates
of the parasite in direct examination of
muscle tissues (Takashi et al., 2000).
Our study suggests that Trichinella
spp. has low prevalence among pigs in
Los Baos. We attribute this result to the
following factors: the method of choice,
the nature of occurrence, production
system from which the pigs were raised,
and the sample size used in this study.
Detection tests for Trichinella spp.
are categorized into two: (1) direct
detection of first-stage encysted larvae or
free juvenile in striated muscle tissue, and
(2) indirect detection of infection by tests
for specific antibodies (OIE Terrestrial
Manual, 2012). In this study, we used the
artificial digestion method which falls into

the direct detection category. Digestion

tests are able to detect more than 1 larva
per gram (lpg) of tissue, but at low levels
of infection, uneven distribution of larvae
within tissues is a limiting factor. This is
compensated by testing larger samples
per carcass, such as a minimum of 35 g
for pigs and 510 g for horses, game and
indicator wildlife species such as foxes
(OIE Terrestrial Manual, 2012). The
negative result in our study may have
been an effect of the low sensitivity of the
artificial digestion method used in
detecting Trichinella in the muscle
samples.
Moreover,
the
uneven
distribution of larvae within tissues could
have been of high influence, even though
we tried to compensate it by testing 5 g of
muscle from the diaphragm pillars per
carcass. Furthermore, the body location
where the samples are obtained
(diaphragm pillars) could have also
contributed to the negativity of the result.
The muscles of choice to be digested are
the pillars of the diaphragm, which show
a good balance between the number of
larvae per gram and the digestibility;
however, the muscle of the tongue
harbours a higher number of larvae
mainly in T. spiralis and T. britovi infected
pigs (Istituto Superiore di Sanit, 2006).
On the other hand, a serological
test would have been another promising
detection test with much higher sensitivity
than the artificial digestion method
(serological test [ELISA assay]: 1 larva
per 100 g of tissue; artificial digestion
method: 1 larva per g of tissue) (OIE
Terrestrial Manual, 2012). Serological
assays are the most common tests used
for indirect detection method. A low rate

of false-positive results has also been

reported for serological tests. The
specificity
of
enzyme-linked
immunosorbent assays (ELISA) for
Trichinella infection is directly linked to
the type and quality of the antigen
employed in the test. Secretory antigens
collected by short-term (1820 hours)
maintenance of Trichinella spp. muscle
larvae in vitro and synthetic carbohydrate
antigens currently provide the most
specific and economical source. It is
critical that appropriate positive and
negative control sera be used to ensure
that ELISAs are performing at a minimum
acceptable level of sensitivity and
specificity (OIE Terrestrial Manual, 2012).
The result of our study suggests
that Trichinella is not endemic in Los
Baos. Furthermore, no reports can be
found regarding human outbreaks of the
parasite in this area which supports the
idea that this parasite is not prevalent
among inhabitants in Los Baos. It can
be stated that a system for reporting of
Trichinella infection in Los Baos is not
active since the infection rate is low and
may not be considered as public health
emergency. Moreover, Filipinos prefer
eating well-cooked pork products and this
may be considered a good factor on why
Trichinella cannot establish in Filipino
population, such as in Los Baos.
Meanwhile, some countries in Southeast
Asia showed outbreaks of Trichinella. For
instance, a major trichinellosis outbreak in
Northern Laos was reported in 2003,
suggesting the high endemicity of the
parasite in the area. According to the
authors, consumption of uncooked or
fermented pork at funeral and wedding

ceremonies was the main source of

infection (Barennes et al., 2008).
The use of good production and
management
practices
for
swine
husbandry will preclude most risks for
exposure to trichinae in the environment
(Gamble, 2007). In Los Baos, the
predominant source of pigs delivered to
the local slaughterhouse usually comes
from intensive farming system. It is
notable that intensive system has good
herd health management and they
practice
acceptable
environmental
hygiene practices. These schemes can
minimize the persistence of infective
agents such as parasites belonging to the
genus Trichinella. Moreover, Trichinella
infections in domestic pigs are sporadic
when there is a direct transmission of
these pathogens from wild animals to
pigs. Free-roaming and backyard pigs are
those at higher risk for Trichinella
infection in the domestic habitat. Indeed,
they are in contact with wildlife such as
rodents (Istituto Superiore di Sanit,
2006).
Finally, the small sample size used
in this study may be a contributing factor
on why we found zero prevalence of
Trichinella in examined samples of pig
muscle tissue. Epidemiological studies
usually utilize a large sample size to
detect the presence of infective agents or
disease, especially in areas with low
endemicity (Thrusfield, 2003). It can be
deduced that our sample size did not
meet the required number of tissues due
for examination to detect the larval forms
in pooled muscle tissues.

RECOMMENDATIONS
We recommend the use of higher
sample size to increase the odds of
finding Trichinella larvae in the samples,
which, together with increasing the
amount per sample, may compensate the
uneven and sporadic distribution of larvae
within the tissues (OIE Terrestrial Manual,
2012), increasing the chance of detection
if larvae are present. Moreover, the use of
tongue as the source of sample could be
considered. According to Pozio (2007),
tongue, as a predilection site, tops as the
site where Trichinella species prefer to
accumulate. It is followed by diaphragm
pillars (which is the sample source in the
current study), and then by masseter.
However, the nature of the tongue muscle
makes artificial digestion method less
efficient. Furthermore, samples taken
from pigs reared in backyard or free
range system could be considered

instead of taking samples from pigs

reared in intensive systems, where
sylvatic animals, such as rodents, are
less likely to come in contact. We also
recommend the use of serological assays
for increased sensitivity and increased
specificity of detection for Trichinella spp.
in the muscle tissues of pigs.

ACKNOWLEDGEMENT
We would like to acknowledge the
assistance provided by the management
of the local slaughterhouse of Los Baos,
Laguna from where we obtained the
samples used in this study. We would
also like to express our gratitude to the
faculty and staff of Animal Dairy Sciences
Cluster, especially to Dr. EM Agbisit Jr.,
Dr. JMUP Hurtada, and Dr. RC Sulabo.