Serological detection of Toxocara canis infection in dogs with recombinant

Cathepsin-L1, arginine kinase and TES-26 of T. canis

Outline of Research work (ORW)

for
Ph.D. Degree
By
Anju Varghese
Roll No. 1280
Division of Veterinary Parasitology

To

Deemed University
INDIAN VATERINARY RESEARCH INSTITUTE
IZATNAGAR BAREILLY-243122

Introduction
Toxocara canis is a common helminth parasite of dogs, a causative agent of canine
toxocarosis with worldwide distribution.The parasite is prevalent in all locations that harbour
domestic dogs particularly the puppies and other canids (Glickman and Schantz,1981).
Toxocarosis is a serious condition and a global zoonotic disease(Glickman and Magnawal,
1993). Toxocara canis is a common round worm of dogs and various mammals including
rabbits, mice, pigs, foxes, birds, etc. may act as paratenic hosts which may also play role in
its epidemiology (Xi and Jin, 1998).
Dogs are the reservoir for T. canis but puppies pose the greatest risk of spreading the
infection to humans (Cruz et al., 2008). Dogs are associated with more than 60 zoonotic
diseases of which helminthic parasites in particular pose serious public health concerns
(Khante et al., 2009). T. canis is considered as one of the main enteropathogens of dogs,
especially in newly whelped or neonates (Blagburn et al.,1996) and is responsible for
important zoonotic disease, visceral larva migrans (VLM). Infection in adult dogs is
characterized by encysted second stage larvae. However, these larvae can get activated in
pregnant females and cross the placental barrier to infect the pups, with vertical transmission
also occurring through milk (Akao and Ohta, 2007) Infectious mothers and puppies under
five weeks old, pass eggs in their faeces. A close relationship between humans and dogs may
represent a potential public health risk, since natural transmission of parasitic infections from
dogs to man may occur via environmental factors. Soil contamination of gardens and public
grounds by infectious parasitic forms constitutes a significant zoonotic risk (Habluetzel et
al., 2003).
Human toxocarosis caused by larvae of T. canis pose a greater risk of infection to
young children because of their habit of playing at public places and a tendency to place
contaminated objects and dirt in their mouth (Nagakura et al.,1990). Seroprevalence is higher
in developing countries, sometimes over 50% but infection has been reported in the first
world countries as well (Bachmeyer et al., 2003), with sero-prevalence in children in
developed countries ranging from 7 to 30% (Kayes, 1997). Generally, human toxocarosis is

caused by ingestion of eggs with contaminated soil, however, a cultural dietary preference for
eating raw or undercooked animal tissues may also enhance the risk of infection, as food
animals act as paratenic hosts (Done et al., 1960; Galvin, 1964; Nagakura et al., 1989). In
human or other non-canid hosts, these larvae wander aberrantly throughout the internal
organs viz. eyes, lungs, brain, heart, muscles, liver etc. These wandering larvae do not
develop further in these hosts but can cause severe local reactionsdue (Helwigh et al., 1999;
Xi and Jin, 1998). T.canis eggs require several weeks of incubation period outside the host
before becoming infective, so freshly laid unembryonated eggs cannot cause toxocarosis
(Amir et al., 1995). These eggs can remain infectious for years, as they are very resistant to
the effects of chemicals, as well as to changes in temperature.
Visceral larva migrans (VLM), ocular larva migrans (OLM) and covert toxocarosis
are three main clinical forms of toxocarosis that are traditionally described. Examination of
stools has no role in the evaluation of human toxocarosis. Finding Toxocara larvae within a
patient is the only definitive diagnosis for toxocarosis, however, biopsies to look for second
stage larvae in humans are generally not very effective and therefore, cannot be relied
(Bachmeyer et al., 2003). Other laboratory investigations are leukocytosis and marked
eosinophilia, hyper gammaglobulinemia (particularly IgM) and elevated isohemagglutinin
titres for antigens in blood groups A and B (Magnaval and Glickman, 2006). Serological test
using ELISA is recognized as an effective approach for diagnosis (Magnaval et al., 2001).
Molecular techniques, such as DNA hybridization, polymerase chain reaction (PCR) and
restriction fragment length polymorphism (RFLP) have been used to diagnose infection (Rai
et al., 1997) and PCR has proved to provide specific identification and analyze genetic
variation in this nematode (Zhu et al., 2001)
Immunodiagnosis is a valuable adjunct for VLM where the conventional
parasitological methods fail. Highly specific and sensitive diagnostic antigens are, therefore,
warranted for detection of infection in pets and humans. A number of antigens of T. canis
have been expressed and evaluated for their utility in diagnosis of human and canine
toxocarosis. These include T. canis cathepsin-L cysteine proteases (Tc-cpl-1), arginine kinase,
surface associated glycoproteins of molecular weights 32, 55,70,120 kDa, Toxocara
excretory-secretory antigen (TES-57), recombinant Toxocara excretory-secretory antigen

(rTES-120, rTES-26, rTES-30USM) (Suharni et al., 2009). Amongst these diagnostic

antigens, arginine kinase has been identified in several parasites like Trypanosoma cruzi, T.
brucei (Pereira et al., 1999, 2000 & 2002), Ctenocephalides felis (Werr et al., 2009)
Cathepsin-L enzymes have been identified as potential targets for drug or vaccine
development in many parasites, as their functions are essential in a variety of important
biological processes within the host, such as molting, cuticle remodelling, embryogenesis, feeding
and immune evasion. Cathepsin-L, a major cysteine proteinase secreted by the parasite plays a
pivotal role in various aspects of its pathogenecity. The enzyme takes part in nutrient acquisition by
catabolizing host proteins to absorbable peptides, facilitates the migration of the parasite through
the host intestine and liver by cleaving interstitial matrix proteins such as fibronectin, laminin and
native collagen and is implicated in the inactivation of host immune defences by cleaving
immunoglobulins. Accordingly, the protease has been recognized as an important target at which
parasite intervention strategies should be directed. Cathepsin-L cysteine proteinases are also
reported as sensitive and specific markers for the immunodiagnosis of various parasitic diseases
(Varghese et al., 2012). Cathepsin-L is secreted by all stages of the developing parasites and is highly
antigenic in infected animals. The purified or recombinant cysteine proteinase antigens have been used
for the diagnosis of human and /or animal paragonimiosis, clonorchiosis, schistosomosis and
fasciolosis (Raina et al., 2005).
One of the important phosphagen kinases of T. canis is arginine kinase, catalyzing the
reversible transfer of phosphate from MgATP to arginine yielding phosphoarginine and
MgADP, maintaining the normal cellular processes such as contractility, motility and
membrane transport (Meyer et al., 1984). Arginine kinase is emerging as a diagnostic target
in Toxocara infection and has recently been isolated in T.canis and characterized by
Wickramasinghe et al., 2007 & 2008 ). As this enzyme is not present in mammals, it might
be a useful diagnostic marker for VLM and can also be exploited in the immunoprophylaxis.
The sensitivity and specificity of the serological tests have been compromised due to
the issues of cross-reactivity of different antigens of helminths parasitizing a dog. Therefore,
evaluating new target molecules for their higher sensitivity and specificity is needed for
developing a reliable serological test for T. canis infection in human and canines. Present

work is therefore, envisaged to express some of the recombinant antigens of this parasite that
have been shown in earlier works with a high potential in the detection of occult infection
due to tissue embedded larvae in humans. However, no studies have been conducted on these
two antigens on their diagnostic potential in canines. T. canis arginine kinase has been
expressed and evaluated in earlier study (Bagde et al., 2013) for its potential in the detection
of T. canis infection in dogs that has shown this antigen detecting infection in canines with a
reliable sensitivity. Keeping this background in view, the present study is aimed to express
the above three antigens in a bacterial system and study them for their usefulness in the
development of a reliable diagnostic assay for T. canis infection in dogs.
The following objectives of the research programme are envisaged

Expression of Toxocara canis cathepsin-L-1, TES-26 and arginine kinase

recombinant proteins in a prokaryotic system.

Evaluation of sensitivity and specificity of these recombinant antigens for

detection of Toxocara canis infection in canines.

Review of Literature
Human toxocarosis
H.C. Wilder was the first researcher to describe toxocarosis in humans, when he
published a paper in 1950 describing ocular granulomas in patients thought to have
retinoblastomas. Beaver et al.(1952) published the presence of Toxocara larvae in granulomas
removed from patients with symptoms similar to those in Wilders patients (Despommier, 2003;
Holland and Smith, 2009). Since then a several research publications have been generated on
toxocarosis in human. The prevalence of human toxocarosis in tropical regions has been found to be
higher than in the temperate regions with serologic surveys in different countries revealing a
seropositivity of 19% in the Netherlands, 2.5% in Germany, 39% in Brazil, 5.8-36% in the Czech
Republic, 0-37% in Spain, 5.2% in Cuba, 10.9 % in Jordan, 47.5% in Colombia, 81% in Nepal,
and 13% in Slovak Republic (Tolan and Laufer, 2010).
Indian Scenario
A sero-epidemiological study was conducted in Kashmir where blood samples were
collected at random from children of six districts of the valley. ELISA was used for detection of IgG
antibodies against Toxocara excretory-secretory antigen. The study found more number of male
children infected (41.9%) than females (20.9%), with a total seroprevalence of 32.8%. The risk
factors of the infection were found as family background, living conditions, awareness etc (Dar et
al., 2008). In another serological study conducted in Chandigarh in north India, the antibody
response to the T. canis excretory-secretory antigen by ELISA in subjects residing in rural areas
near Chandigarh and in clinically suspected patients was studied. A positive antibody response to TES
antigen in 6.4% subjects and 23.3% of suspected patients indicated that human toxocarosis
may be endemic in certain regions of northern India (Malla et al., 2002). A related factor for the
prevalence of Toxocara infection in humans was the condition and status of vegetables taken by the
subjects. The children who were of the habit of eating raw vegetables were more prone to infection
(36.5%) than those who did not eat raw vegetables (20.3%). Children who were of the habit of
geophagia were also more prone to infection (36.5%). Water pretreatment was a significant risk

factor for the prevalence of toxocarosis (Dar et al., 2008). Contact with dogs or presence of pet in
house also constituted a high risk factor for Toxocara infection. In their study, the prevalence of
Toxocara infection was more in people using water from streams, rivers, ponds and wells than those
using tap water. In the Kashmir valley a higher rate of infection was reported in the population than in
the subjects residing in a rural area near Chandigarh. This may be due to low standards of hygiene,
frequent contact with the contaminated soil and lack of education (Ahmad et al., 2002).
Toxocarosis in dogs
Recent studies have indicated the following prevalence rates of toxocarosis in dogs in
different countries, 13.7% in Tanzania (Swai et al., 2010), 40% in Pakistan (Chattha et al., 2009),
60% in Iran (Daryani et al., 2009), 12.5% in Japan (Yamamoto et al., 2009), 12.8% in Northern
Greece (Papazahariadou et al., 2007), 17.7% in Spain (Martinez-Moreno et al., 2007), 6.5% in
Czech Republic (Dubna et al., 2007), 21% in Ethiopia (Yacob et al, 2007), 3.1% in Finland
(Pullola et al., 2006), 11.6% in Argentina (Fontanarrosa et al., 2006), 0.9% in Korea (Kim and
Huh, 2005), 33.6% in Italy (Habluetzel et al., 2003), 79% in Denmark (Engbaek et al., 1984) .
An epidemiological study in Spain, comparing a rural area with an urban one, revealed similar
T. canis infection rates of about 30% in dogs and a higher soil contamination in the rural (9%) than in
the urban (3.7%) zone (Conde Garcia et al., 1989). In a study conducted in the former East Germany
on infection rates in dogs, a higher T. canis prevalence was reported in rural (25.5%) than in urban
(15.2%) dogs (Knaus and Betke, 1986).
A survey on gastrointestinal parasites of non-descript dogs in Tanzania on coprological
examination revealed a higher prevalence of Toxocara eggs in young dogs compared to adults (Swai et
al., 2010). The overall prevalence of 59.3% (11% for Toxocara) of gastrointestinal tract parasite
eggs / oocysts in dogs in this study showed that there were frequent infections of indigenous dogs
with different species of helminths and protozoa. The relatively high level of parasitism recorded in
this study was probably related to the lack of improvement in animal health management
programmes or non-adoption of the modern animal health care programmes by dog owners.

An estimation of T. canis prevalence in dogs, in the Marche region of Italy revealed the
highest infection rate in rural hunting dogs (64.7%) and the lowest in urban companion dogs
(22.1%) (Habluetzel et al., 2003). The 26.2% T. canis positivity estimated for urban dogs in
this study was in agreement with a previous investigation in the same area, which recorded
28% egg prevalence in faecal samples collected in public green areas (Palliola et al., 1986).
Twice as many dogs from rural areas (48.4%) harboured egg shedding nematodes compared
to the urban (26.2%) ones. This difference may result from more frequent antihelminthic
treatments of urban dogs, due to a greater health consciousness of their owners (Poglayen et
al., 2000).
A study on the role of dogs in the transmission of gastrointestinal parasites in Assam,
India, revealed that nearly all (99%) dogs harbored one or more zoonotic species of
gastrointestinal parasites, with a total prevalence of 11% for T . canis infection (Traub et al.,
2002). Faecal samples of 642 stray dogs of Jodhpur city revealed a total of 88%
gastrointestinal parasite infection with a prevalence of 4.9% for Toxocara (Subhash and
Tanwar, 2007). In a study at Bareilly, prevalence rate was found high in less than 6 months
old stray (55.7%) and pet (34.5%) dogs with an overall prevalence of 23.08% (Samanta and
Ghorui, 1998). Another study conducted in certain pockets of Bihar revealed that T. canis
prevalence in dog population was 21.3% (Samanta et al., 1989). Examination of 816 soil
samples from different parts of Puducherry city viz. public places, residential areas, some
school play grounds revealed overall 2.2% contamination of soil with the ova of Toxocara.
Besides Toxocara, ova of non-hookworm strongyles (0.74%), Ancylostoma (1.23%),
Trichuris (0.74%), Taenia (0.12%) and Moniezia (0.86%) were also recorded. A few
ascarid eggs, mites and their eggs and larvae of free-living nematodes were commonly
encountered in the soil (Das et al., 2009).
To determine the prevalence of T. canis infection in dog population in and
around Bareilly, Uttar Pradesh, India, a total of 558 faecal samples both from stray
and owned dogs were screened, with 24.3% dogs positive for T. canis infection. Age
of the dogs had a considerable influence on prevalence, with a much higher
proportion of younger dogs being infected. The higher rate of prevalence of this
parasite in dogs could be the source of soil contamination for transmission of T. canis
to human (Sahu et al., 2013).
8

Soil contamination
Work done on the threshold recovery of T. canis eggs from soil suggested that the
centrifuge-flotation technique is efficient for the detection of Toxocara eggs, even in samples
containing low egg numbers (Xavier et al., 2010). Mohammad et al. (2010) studied the soil
contamination with Toxocara spp. eggs in the public parks from three areas of Khorram Abad,
Iran. A total of 285 soil samples from January to March 2009 in 18 public parks were
collected to test for soil contamination with Toxocara eggs using sucrose floatation method.
Distribution of Toxocara spp. eggs in samples collected from public parks was 63.3%.
The highest number of eggs recovered from 200 g of soil was 128. Their investigation
showed that public parks were contaminated with Toxocara eggs in Khorram Abad,
suggesting for exercising care when using public parks (Mohammad et al., 2010).
In a study conducted on the prevalence of parasites in soil and dog faeces, soil samples
were collected from 25 public squares in Seropdica, Brazil. The technique described by
Dunsmore et al. (1984) and an adaptation of the Ruiz et al. (2000) method were used to
recover parasites in soil. Seven squares were found to be contaminated and the most prevalent
parasites were Ancylostoma spp (13.6%) and Toxocara spp (4.0%). The prevalence of
zoonotic parasites in soil has implications for the spread of human disease in these areas
(Mandarino-Pereira et al., 2010). A study conducted by Teixeira et al. (2008) on prevalence
of T. canis infection in public squares of the Concrdia City, Santa Catarina, Brazil, reported
that the schools, squares and parks where sand is present can constitute an important
transmission source for several parasitic zoonoses, representing a potential risk, mainly for
children of school age playing at these places. Amongst several zoonoses, visceral larva
migrans (VLM) is an important pathology characterized by the migration of the larval
stages of Toxocara spp. in human tissues leading to immuno-allergic type of reactions. The
objective of their work was to determine the occurrence of parasite eggs in the samples of sand
from public squares of Concordia city; Centro, Nazare, Vista Alegre and Industriarios. A
significantly higher prevalence of T.canis eggs was due to the high number of canine
population in the municipal district and their easy access to these places (Teixeira et al.,
2008).
In a study on the contamination of soil with feline and canine ascarid eggs in public
parks, backyards and sand pits in Prague, Czech Republic, soil samples from shelters and
rural areas were analyzed. The highest rate of contamination (45%) was found in backyards
9

inhabited by feral cats. The eggs of Toxocara spp. were found in 20.4% of parks, 10% of
shelters and 5% of rural samples. Mean egg density per sample from Prague parks was 6.2
eggs / 100 g of soil. In 126 composite samples, the prevalence of Toxocara eggs was 11.9%.
The number of eggs in positive samples varied from 2 to 22 (per 100 g) (Dubna et al.,
2007).
In a Study conducted in Punjab on the prevalence of Toxocara spp. eggs in soil,
out of 208 soil samples examined 41 (19.7%) were positive for Toxocara (at least 1 egg in
a sample). They were present in a wide variety of examined places (urban, suburban and rural)
i.e. public parks, play grounds, backyards, streets, roadsides, canal sides, public and private
gardens. The urban public places were slightly less contaminated as compared to rural areas
and childrens play grounds, backyards and parks were comparatively more contaminated
than other selected sites. The soil in the public places was found to be comparatively more
contaminated than private places (Harbindar et al., 1997).
To determine the possible extent of soil contamination at different public
places with Toxocara spp. eggs in and around Bareilly,U.P., a total of 327 soil
samples were examined from different locations which are of public health
importance like public parks, playgrounds, door mat dusts, Sidewalks or road sides, in
Bareilly, Uttar Pradesh, to establish the prevalence of Toxocara eggs. 42 samples out
of 327 (12.84%) were found to be contaminated with the Toxocara spp. eggs and
public parks were more contaminated than the other sites. Prevalence of this zoonotic
parasite in soil has implications for the spread of human disease in these areas. The
authors believe that this may constitute a significant health risk, particularly to
children (Sudhakar et al., 2013a).
A pilot study was conducted to determine the presence of T. canis infection in
dog population in and around Bareilly, Uttar Pradesh, India. A total of 558 faecal
samples both from stray and owned dogs were screened and overall 24.3 % dogs were
found positive for T. canis suggesting a higher prevalence in the latter group. The age
of the dogs had a considerable influence on prevalence, with a much higher
proportion of younger dogs being infected. Among the stray dogs, the infection rate
was much higher (62.79 %) in pups, as compared to 7.8 % in adult. Similarly, of the
owned dogs screened 41.74 % pups were infected while the infection rate in adults
was only 3.38 %. The higher rate of prevalence of this parasite in dogs could be the
10

source of soil contamination for transmission of toxocarosis which is of public health

importance in this region (Sahu et al., 2013).
Characterization of Toxocara canis antigens
Characterization of the proteins that comprise adult T. canis worms will prove useful
in the development of vaccines and diagnosis of infection and in studies on the biology of
and immunity to nematodes and other parasites. The identification of specific antigens of
T. canis is important in order to develop better diagnostic techniques The excretorysecretory proteins and associated carbohydrates of the third stage larvae of T. canis
(Maizels et al., 2006) and the proteins of adult Ascaris suum (Frontera et al., 2003;
Takashima et al., 2003), a relative of T. canis were examined in some detail by these
authors.
Morales and co workers identified T. canis antigens by western blot in experimentally
infected rabbits. They infected ten rabbits orally with a dose of 5000 T. canis embryonated
eggs. Western blot assay was performed using E/S antigen of T. canis second stage larvae.
Results of WB indicated that in the first month after infection specific antibodies against 200,
116, 92 and 35 kDa antigens were detected; antibodies against 92, 80, 66, 45, 31 and
28 kDa antigens appeared later. All positive sera in the ELISA were also positive in WB.
Two antigen bands, 92 and 35 kDa were identified since the beginning and throughout the
course of infection. They reported that these antigens merit further evaluation as candidates
for use in diagnosis (Morales et al., 2002).
Sun and co-workers did a study on comparison of the protein constituents of the
major body compartments of T. canis. An analysis of the protein profiles of intact worms and
isolated tissues of adult male and female T. canis worms was conducted. Soluble proteins
recovered from homogenized whole specimens and dissected tissues (body wall, reproductive
tract, oesophagus and intestine) of T. canis adults from several different canine hosts were
separated by size using gradient sodium dodecyl sulphate electrophoresis (SDS-PAGE). SDSPAGE profiles of worms from different hosts were found to be virtually identical, irrespective
of sex or tissue type. Recovered proteins ranged in size from 3.4 to 325 kDa. The largest
number of recovered proteins was present in the female reproductive tract extracts (Sun et
al., 2007).

11

The excretory-secretory antigens of the infective stage larvae of T. canis (TES) have
been examined in detail by several workers. These TES proteins have been
characterized as mucins (glycosylated proteins of high molecular weight), 120, 4045
kDa, lectins (sugar-binding proteins), 70 and 32 kDa as well as several other proteins
at 26 and 55 kDa. There are molecular weight moieties observed in adult worms that
may be related to the larval TES products. A study by Badley et al. (1987) shows T. canis
excretorysecretory proteins from larvae, which may relate to those found in T. canis adults at
molecular weight bands 34 (34.7), 53 (50.8), 57 (59.4), 66 (65.3), 76 (74.4) and 94 kDa
(92.8 kDa) (Maizels et al., 2006).
A study conducted by Nawal and Mona, 2008 on serodiagnosis of human
toxocarosis using adult antigens of T. canis showed reactive excretory-secretory (TC-ES)
antigens of 8 polypeptide bands at 127, 94, 88.9, 50, 40.7, 33.7, 30 and 14.05 kDa,
respectively. Immunoblot profile of TC-ES antigen with rabbit TC-ES antisera showed 3
common reactive bands (Mona et al., 2004). Morales, et al., 2002 suggested that 92 and
35 kDa polypeptides of T. canis second stage larvae E/S antigen are specific to Toxocara
infection while 57 kDa fraction of the larval T. canis E/S antigen (TC-ES -57) has shown
specificity to T. canis infection and does not cross-react with sera of other related
infections (Iddawela et al., 2007). In their study on the immunoblotting profile of TC-ES
antigen, positive human and rabbit TC-ES anti-sera showed 5 common immuno-reactive
bands at 47.4, 40.6, 30.5, 21.9 & 19.3 kDa. So, they suggested that 58.739 and 29.19
kDa polypeptides of the adult T. canis E/S are specific for Toxocara infection in human and
these antigens merit further evaluation as candidates for use in the diagnosis of human
toxocarosis (Nawal and Mona., 2008).
Excretory-secretory antigens of adult T. canis were evaluated by Sudhakar et
al. 2013b. The specific reactivity of the T. canis excretory- secretory (TC-ES)
proteins was checked by Western blotting. The immuno-reactivity of the naturally
infected dog sera with the TC-ES antigens showed five bands at 43, 57,105, 139 and
175 kDa. The immuno-reactivity of the hyper immune serum raised in rabbits against
TC-ES antigens was observed with ten polypeptides of 21, 25, 30, 37, 45, 50, 57, 69,
77 and 105 kDa. Common antigen bands were observed at 57 and 105 KDa. These
antigens merit further evaluation as candidate for use in diagnosis of toxocarosis in
humans and adult dogs.
12

Serological characterization
Due to occult nature of human toxocarosis, serodiagnosis is recognized as an effective
approach to the laboratory diagnosis of Toxocara infection. It is also useful for epidemiological
studies on the prevalence of the disease. Woodruff and Bradstreet (1968) first used skin
testing to detect Toxocara specific IgE antibodies. Any pediatric patient with an
unexplained febrile illness and eosinophilia should be suspected of having VLM.
Hepatosplenomegaly and evidence of multisystem disease and history of pica make
the diagnosis of VLM more likely. Other indicators of infection include hypergammaglobulinemia and an elevated isohemagglutinin titre. Thus, a constellation of
clinical disease described above, a history of pica, eosinophilia and positive serology
strongly point to the diagnosis. Liver biopsy may reveal a granuloma surrounding a
larva but a successful diagnosis using this approach is fortuitous at best and not
recommended. Diagnostic tests for VLM are primarily immunological. The enzymelinked immunosorbent assay (ELISA), which employs antigens secreted by the
second-stage larvae, has sufficient specificity to be the best indirect test for
diagnosing this infection. Recombinant antigens have been produced from the
second-stage larvae of T. canis that promises to add even greater specificity to an
already-reliable test (approximately 92%) employing ELISA. The ELISA has a
reasonably high degree of sensitivity.
De Savigny (1977) reported diagnosis of genus specific Toxocara infection with a
passive haemagglutination test using Toxocara excretory-secretion antigens. The standard
serological test for confirming toxocarosis is an ELISA. Western blot analysis, which is as
sensitive as ELISA and relatively specific, should be used as a confirmatory test after
screening by ELISA (Magnaval et al., 1991). In a study on evaluation of dot-ELISA in
comparison with standard ELISA for the immunodiagnosis of human toxocarosis, the
sensitivity and specificity of the dot-ELISA were found to be 100 and 95%, respectively
(Roldn et al., 2006). An indirect antibody competition ELISA employing specific rabbit
IgG anti-TES antigen as the competition antibody was found to improve toxocarosis diagnosis
with a sensitivity of 60.2% and a specificity of 98% in humans (Carmago et al., 1992;
Nunes et al., 1999). Yokoi et al. (2002) produced a monoclonal antibody to the 120 kDaTES antigen of T. Canis larvae which may be useful for determining the parasite burden in
early infection. El-Massry (1999) showed that SDS-PAGE of T. canis adult somatic antigens
13

had bands at 125.3 and 117.7 kDa which may be used as specific markers for T. canis
adults. Magnaval et al. (1991) demonstrated four low Mr bands (24, 28, 30 and 35 kDa)
and three high Mr bands (132, 147 and 200 kDa) by Western blotting using TES.
Sarimehmetoglu et al. (2001) used TES antigens to diagnose VLM and found immunoreactive bands at 24, 28 and 48 kDa in infected mice and bands at 24, 28, 30, 35, 132, 147
and 200 kDa in humans.
An investigation of antibodies against T. canis was carried out in 15 naturally
infected puppies by ELISA, using four different antigens- eggs containing second
stage larvae, larvae, adult worms and excretory-secretory products of larvae. The
ELISA values against four different antigens increased with the age of the pups. The
correlation between age and ELISA values was significant in the positive direction
but a significant negative co-relationship between the number of worms and the
ELISA values was observed. The ELISA values in the puppies having unfertilized
eggs were greater than those having fertilized eggs. These findings suggest that in the
naturally infected puppies there are intensive relationships among the antibody levels,
age, number of worms and the condition of egg fertilization (Matsumura and Endo,
1982).
Matsumura et al. (1983a) studied correlation of antibody production and age
of the dogs with T. canis infection. In the sera of dog fetuses and newborn puppies, no
antibody to T. canis was found but an elevation of the circulating toxocaral antigens
(CTA) was detected. In the mother dogs, detectable IgG and IgM antibodies were
found. The IgG antibodies in the new born puppies gradually increased in the course
of this observation. The most elevated levels of IgA antibodies were observed one
month after birth. The same was true for the CTA levels. There was no placental
transfer of the antibodies nor could they be transferred from the mother dog to the
offspring via the colostrum.
In order to interpret the relation between the IgM antibody activities to T.
canis and the parasitological status, sera from puppies and adult dogs were measured
by ELISA. Autopsy was performed on these dogs for counting the number of T. canis
worms and stool examination was carried out for observing the fertilization of eggs.
The rates of infection by T. canis in the dogs decreased with age. The IgM antibody
activities gradually increased until 3 months of age. Thereafter, the significant levels
14

were maintained even at the adult age. However, the IgM antibody levels were not
associated with the age and the number of worms (P>0.05) when the correlation
coefficients were calculated between them. In contrast to the IgG antibodies, the
results of this study indicated that the IgM antibody activities do not relate to the age,
the number of worms and the fertilization of eggs (Matsumura et al., 1983b).
The surface of infective larvae of T. canis, reveals relatively few exposed surface
proteins which can be recovered in soluble form. The major components identified by surface
labelling have molecular weights of 32, 55, 70 and 120 kDa and are all significantly glycosylated.
All are recognized by the immune response in definitive (canine) and paratenic (murine or
human) hosts. Expression of these antigens on the parasite surface begins after the larvae hatch
from infective ova in vitro and presumably in vivo. Each of these molecules may also be
found in the set of secreted (ES) glycoconjugates released by larval parasites cultivated in
vitro. Analysis with monoclonal antibodies (MAbs) confirmed the identity of surface and
ES molecules and these MAbs showed differing patterns of binding to the epicuticle, the cuticular
matrix and to the oral orifice (Maizels and Page, 1990).
Loukas and co-workers, 1998 characterized cathepsin L-like cysteine protease
from T. canis infective larvae. Cysteine proteases play vital biological roles in both
intracellular and extracellular environments. A cysteine protease migrating at 30 kDa was
identified in somatic extracts of T. canis larvae (TEX) by its binding to the biotinylated
inhibitor Phe-Ala-CH2F. TEX proteases readily cleaved the cathepsin-L and B-specific
peptide substrate Z-Phe-Arg-AMC and to a lesser extent, the cathepsin B-specific peptide
Z-Arg-Arg-AMC. Excretory / secretory (TES) products of T. canis larvae did not cleave
either substrate. Partial sequence encoding the 5' end of a cysteine protease cDNA from
infective T. canis larvae was then obtained from an expressed sequence tag (EST) project.
The entire cDNA (termed Tc-cpl 1) was subsequently sequenced and found to encode a preproenzyme similar to cathepsin L-like proteases (identities between 36 and 69%), the closest
homologues being two predicted proteins from Caenorhabditis elegans cosmids, a cathepsin
L-like enzyme from Brugia pahangi and a range of parasite and plant papain-like proteases.
The recombinant protease (rTc-CPL-1) was expressed in bacteria for immunization of mice
and the subsequent antiserum shown to specifically react with the 30 kDa native protease in
TEX. Sera from mice infected with the parasite also contained antibodies to rTc-CPL-1, as
did sera from nine patients with proven toxocarosis (Loukas et al., 1998).
15

Cathepsin-L, a lysosomal endopeptidase expressed in most eukaryotic cells, is a

member of the papain-like family of cysteine proteinases (Barrett and Kirschke, 1981; Roth,
2000). Cathepsin-L plays a major role in antigen processing, tumor invasion and metastasis,
bone resorption and turnover of intracellular and secreted proteins involved in growth regulation
(Kane and Gottesman, 1990). Although commonly recognized as a lysosomal protease,
cathepsin-L is also secreted. This broad-spectrum protease is potent in degrading several
extracellular proteins (laminins, fibronectin, collagens I and IV, elastin, and other structural
proteins of basement membranes) as well as serum proteins and cytoplasmic and nuclear
proteins (Louise et al., 2009).
Cathepsin-L proteases are secreted by several parasites including T. solium and
constitute important antigens for immunodiagnosis. A protein fraction with cathepsin-L activity
was purified from the cysticercus fluid by size exclusion and ion-exchange chromatography.
The purified protein fraction included antigens of 53 and 25 kDa that were tested in a Western
immunoblot and in ELISA for detection of human cysticercosis. The sensitivity of the Western
blot was 96% for patients infected with multiple cysts and 78% for patients with a single
cyst. Specificity was 98%. The sensitivity of the ELISA was 98% in patients with multiple cysts
and 84% in patients with a single cyst. Specificity was 92.7% (Mirko et al., 2009).
A cDNA encoding a cathepsin-L cysteine protease (TsCL-1) from T. solium
metacestode was identified and characterized for the biochemical properties of the
recombinant enzyme. The cloned cDNA of 1216 bp encoded 339 amino acids with an
approximate molecular weight of 37.6 kDa. Sequence alignments of TsCL-1 showed low
sequence similarity of 27.3- 44.6% to cathepsin L-like cysteine proteases from other
helminth parasites (Lia et al., 2006).
Cathepsin-L, a major cysteine proteinase secreted by Fasciola species are also
reported as sensitive and specific markers for the immunodiagnosis of fasciolosis in ruminants.
A diagnostic ELISA with recombinant F. hepatica and F. gigantica cathepsin L-like
protease as antigen was developed to detect antibodies against F. hepatica in sheep and
cattle ( Raina et al., 2005; Varghese et al., 2012). Expression and purification of an active
cysteine protease of Haemonchus contortus using C. elegans as an expression system
showed optimal expression of Hc-cpl-1 under the control of the promoter of the
homologous C. elegans cpl-1 gene (Linda et al., 2007). Molecular cloning and functional
characterization of a cathepsin L-like proteinase from the fish kinetoplastid parasite
16

Trypanosoma carassii was carried out. A cathepsin L-like proteinase from T. carassii
was cloned and the nucleotide sequence of 1371 bp translated into a preproprotein of 456
amino acids. The findings suggest that the T. carassii cysteine proteinase is highly
conserved within the trypanosomatidae with respect to structure and activity but is not a major
protective antigen in carp (Aleksandra et al., 2008). Several studies have shown high
potential for cysteine proteinases of several helminths in the diagnosis of the infection and
as potent vaccine candidates.
Tetteh et al. (1999) studied identification of abundantly expressed novel and
conserved genes from the infective larval stage of T. canis by an expressed sequence tag
strategy. They undertook a random sequencing project from a larval cDNA library to
characterize the most highly expressed transcripts. In all, 266 clones were sequenced, the
high-abundance transcripts include a mucin and a C-type lectin, which are both major
excretory-secretory antigens released by parasites. Four highly expressed novel gene
transcripts, termed ant (abundant novel transcript) genes were found. Together, these four
genes comprised 18% of all cDNA clones isolated but no similar sequences occur in the
C. elegans genome. The discovery of these abundant, parasite-specific genes of newly
identified lectins and mucins, as well as a range of conserved and novel proteins provided
defined candidates for future analysis of the molecular basis of immune evasion by T. canis
(Tetteh et al., 1999).
Loukas and co-workers (2000) studied novel C-type lectin secreted by a tissuedwelling parasitic nematode. Many parasitic nematodes live for surprisingly long periods in
the tissues of their hosts, implying sophisticated mechanisms for evading the host immune
system. The nematode T. canis survives for years in mammalian tissues and when
cultivated in vitro, secretes antigens such as TES-32. From the peptide sequence, they
cloned TES-32 cDNA, which encodes a 219 amino-acid protein that has a domain
characteristic of host calcium-dependent (C-type) lectins, a family of proteins associated
with immune defence. Homology modeling predicted that TES-32 bears remarkable structural
similarity to mammalian immune-system lectins. Native TES-32 acted as a functional lectin in
affinity chromatography. Unusually,

it bound both mannose and galactose-type

monosaccharides, a pattern precluded in mammalian lectins by a constraining loop adjacent to

the carbohydrate-binding site. In TES-32, this loop appeared to be less obtrusive, permitting a
broader range of ligand binding. The similarity of TES-32 to host immune cell receptors
17

suggests a hitherto unsuspected strategy for parasite immune evasion (Loukas et al.,
2000).
Gems and Maizels (1996) worked on an abundantly expressed mucin-like
protein from T.canis infective larvae, the precursor of the larval surface coat
glycoproteins. They reported that evasion of host immunity by T. canis infective larvae is
mediated by the nematode surface coat, which is shed in response to binding by host antibody
molecules or effector cells. The major constituent of the coat is the TES-120 glycoprotein
series. They isolated a 730-bp cDNA from the gene encoding the apoprotein precursor of
TES-120. The mRNA is absent from T. canis adults but hyper abundant in larvae, making
up approximately 10% of total mRNA and is trans-spliced with the nematode 5' leader
sequence SL1. It encodes a 15.8 kDa protein (after signal peptide removal) containing a
typical mucin domain: 86 amino acid residues, 72.1% of which are Ser or Thr, organized
into an array of heptameric repeats, interspersed with proline residues. At the C-terminal
end of the putative protein are two 36-amino acid repeats containing six Cys residues, in a
motif that can also be identified in several genes in C. elegans. Although TES-120 displays
size and charge heterogeneity, there is a single copy gene and a homogeneous size of
mRNA. The association of over expression of some membrane-associated mucins with
immunosuppression and tumor metastasis suggests a possible model for the role of the
surface coat in immune evasion by parasitic nematodes (Gems and Maizels, 1996).
In a study conducted by Maizels and co workers (2000) to understand the ability of
T. canis larvae to survive in the immuno-competent host, they undertook molecular analyses
of the major genes expressed at this stage. By a combination of protein sequencing, gene
identification and expressed sequence tag (EST) analysis they characterized a range of
potentially important gene products from this parasite. Some of these are homologues of
prominent mammalian proteins such as C-type lectins (represented by the secreted
products TES-32 and TES-70) and mucins (TES-120) and additional products show
strong similarities to known cysteine proteases, phosphatidyl ethanolamine-binding
proteins and other ligands. A number of these proteins include a conspicuous 36-amino
acid motif containing six cysteines. This domain (termed NC6 or SXC) appears to be an
evolutionarily mobile module, which in T. canis is combined with a spectrum of diverse
functional domains in different genes. In addition, they identified a set of novel gene
sequences that show no resemblance to any genes encoded by the free-living nematode C.
18

elegans. Four of these are designated abundant novel transcripts and collectively these
account for nearly 20% of the cDNA isolated from the arrested infective stage. Such parasitespecific genes expressed at a high level by a stage that shows remarkable endurance may
represent critical products necessary for the success of the parasitic mode of life (Maizels
et al., 2000).
Yamasaki et al. (2000) developed a recombinant T.canis second-stage larva antigen
corresponding to the 30-kDa protein of the TES and tested its sensitivity and specificity.
The

specificity

of

the

recombinant

T.canis

antigen

developed

for

the

immunodiagnosis of human toxocarosis was compared with that of the excretorysecretory antigen from T. canis second-stage larvae by ELISA. A total of 153 human
serum samples from patients infected with 20 different helminths, including 11 cases
of toxocarosis, were examined. No false-negative reactions were observed for the
toxocarosis cases. When the TES was used at concentrations of 0.5 and 0.125 mg/ml,
cross-reactions were observed in 79 (55.6%) and 61 (43.0%) of 142 cases,
respectively. In contrast, when the recombinant antigen was tested at a concentration
of 0.5 mg/ml, cross-reactions were observed in 19 (13.4%) of 142 cases. At a
concentration of 0.125 mg/ml, however, the cross-reaction rate decreased sharply to
only 2.1%, corresponding to 3 of 142 cases. The cross-reactions occurred with one
case each of gnathostomosis, paragonimosis with Paragonimus miyazakii, and
spirometriosis, in which high antibody titres were detected. In addition, the
recombinant antigen showed negative reactions with serum samples from patients
infected with Ascaris and hookworms, the most common human parasites. These
findings were also supported by experiments with animals infected with Ascaris and
hookworm. The authors suggested the recombinant antigen is highly specific for
T.canis infection and may provide more reliable diagnostic results than other
methods(Yamasaki et al., 2000)
Wickramasinghe et al. (2007) reported molecular cloning, characterization,
expression and comparison of the kinetics of cDNA-derived arginine kinase of T. canis.
Arginine kinase (AK) is a member of a highly conserved family of phosphagen kinases.
Alignment of the cDNA-derived amino acid sequence of T. canis AK with other
phosphagen kinase sequences showed high amino acid identity with other nematode AKs and
phylogenetic analysis placed it as a distinct branch within a nematode AK cluster. Analysis of
19

the N-terminus sequence of T. canis AK revealed the presence of a signal targeting peptide
presumably targeting this protein to cytosol or endoplasmic reticulum. This was the first study
which reported description of arginine kinase in a zoonotic nematode. The determination of T.
canis AK and its phosphagen biosynthetic pathway, which is completely different from those
in mammalian host tissues, suggests this enzyme as a possible novel chemotherapy target for
VLM syndrome in humans (Wickramasinghe et al., 2007).
The same authors in the subsequent year (2008) reported expression of the
arginine kinase recombinant protein in a prokaryotic system. A recombinant arginine
kinase from the infective second-stage larvae was generated and a highly sensitive (100% at
1:100 and 1:500 dilutions and 97% at 1:1,000 dilution) IgG-ELISA for antigen capture in the
mouse model was developed (Wickramasinghe et al., 2008). This was the first
demonstration of diagnostic potential of T.canis arginine kinase in toxocarosis. The
authors suggested that the recombinant- AK based IgG-ELISA could be applied for
immunodiagnosis of human toxocarosis. However, it is necessary to evaluate the
specificity of this recombinant antigen with similar geohelminth infections.
O-Methylated glycans from Toxocara are specific targets for antibody binding in
human and animal infections. Antibodies generated to helminth infections other than T. canis
were unreactive with the glycans, except antibodies to other members of the Toxocara genus.
Hence, the carbohydrate structures represent immunogenic, genus-specific antigens
(Schabussova et al., 2007).
The use of recombinant antigens is promising for improving the specificity of the diagnosis
of toxocarosis. Toward this goal, an IgG4 ELISA involving three recombinant antigens: rTES30USM, rTES-26, and rTES-120 were developed. The diagnostic potential of each purified
recombinant antigen was tested with various immunoglobulin classes (IgG, IgM, and IgE) and
IgG subclasses. The IgG4 ELISA was described to have the highest specificity and was
further evaluated using a panel of serum samples. The rTES-26 IgG4 ELISA showed 80.0%
(24/30 samples positive) sensitivity and both the rTES-30USM IgG4 ELISA and rTES-120
IgG4 ELISA had 93.0% (28/30) sensitivity. Combined use of rTES-120 and rTES-30 IgG4
ELISA for the diagnosis of toxocarosis provided 100% sensitivity. The specificities of rTES26, rTES-30USM, and rTES-120 antigens were 96.2%, 93.9%, and 92.0%, respectively.
These results indicate that a diagnostic test based on these three recombinant antigens will
allow for more-accurate detection of toxocarosis (Suharni et al., 2009).
20

Ishiyama et al. (2009) attempted to establish a diagnostic method for detecting

circulating Toxocara canis antigen using a sandwich-ELISA. Monoclonal antibodies
were produced against the excretory-secretory (ES) antigen of second stage T. canis
larvae. The cross-reactivity of the sandwich-ELISA against thirteen different kinds of
parasite antigens was examined. The results revealed that the antibody reacted with T.
canis ES antigen, T. canis female antigen and T. canis second-stage larvae antigen but
did not react with any other antigens. The method was applied to suspected
toxocarosis patients and examined the circulating antigen in their sera. Out of nine
serum samples collected from patients with suspected toxocarosis based on both their
clinical symptoms and high antibody titers, five sera showed antigen-positive
reactions while the remaining four were negative. These results indicated that about
44.0% of the antibody-positive patients were antigen-negative with no active
infection (Ishiyama et al., 2009).
Jin et al. (2013) investigated the serodiagnostic efficacy of ELISA using
somatic larval antigen of T. canis larvae (TCLA). Serum specimens of 64 clinically
confirmed toxocarosis, 115 healthy controls and 119 other tissue-invading helminthic
infections were screened by ELISA using TCLA that showed 92.2% sensitivity and
86.6% specificity. Its positive diagnostic predictivity was 78.7% and negative
predictivity 97.8%. Serum samples from patients with Anisakis spp. (45.5%),
Gnathostoma spp. (19.2%), Clonorchis spp. (15.8%), Sparganum spp. (11.1%), and
Cysticercus (6.3%) infections cross-reacted. Immunoblot analysis on TCLA
recognized antigenic proteins of 28- and 30-kDa bands with a strong blotting
reactivity. The authors observed that the ELISA with TCLA antigen was relatively
acceptable for making differential diagnosis for patients with any sign of organ
infiltration and eosinophilia.
The sensitivity and specificity of the serological tests developed so far have
been compromised due to the issues of cross-reactivity of different antigens of
helminths parasitizing a dog. Therefore, evaluating new target molecules for their
higher sensitivity and specificity is required for developing a reliable serological test.
Present work is therefore, envisaged to express some new target antigens of this
parasite and exploit them for achieving higher sensitivity and specificity for detecting
T. canis infection in canines and subsequently in humans.
21

22

Technical Programme
Expression of Toxocara canis recombinant cathepsin-L1, arginine kinase and TES-26 proteins

Pups naturally infected with Toxocara canis adult worms will be treated with a suitable
anthelminthic for expulsion of the worms from the host.

Adult worms will be retrieved, washed and teased for release of the eggs from gravid uterus
in sterile tap water. A culture of the eggs will be set up for the development of 2nd stage (L2)
larvae in sterile water at 28oC in a BOD incubator.

Development of the larvae to L2 stage will be followed microscopically. Hatching of the

infective L2 larvae from the embryonated eggs will be carried out by treatment of these eggs
with 2% sodium hypochlorite, followed by mechanical disruption of these eggs. Live
infective larvae will be isolated from the unembryonated eggs, dead and unhatched larvae by
Baermann technique.

Total RNA from the L2 larvae will be isolated using Trizol reagent as per the standard
protocols and reverse transcribed to cDNA using oligo-dT primer.

cDNA synthesized from the total RNA will be PCR amplified for cathepsin-L1 cysteine
proteinase, arginine kinase and TES-26 genes using gene specific primers.

Authenticity of the above three genes will be confirmed by sequencing and cloned in suitable
prokaryotic expression vectors for expression of recombinant proteins in Escherichia coli.

The recombinant proteins will be purified using Nickel affinity chromatography.

Sero-diagnostic assays

Sera from Toxocara canis pups positive for faecal eggs will be retrieved and used as positive
control for evaluating the sensitivity of the three recombinant antigens. Pups free from T.
canis infection as determined by their faecal examination and ELISA for antibodies will be
used as healthy control animals for standardization of the diagnostic assay.

23

Mice will be infected with varying doses of T. canis infective (L2) eggs under laboratory
conditions and infection allowed to develop into visceral larva migrans. The three antigens
will be screened for their detection sensitivity in this laboratory model.

Likewise, five pups, 5-7 month old, free from T. canis infection will be experimentally
infected with varying doses of T. canis infective (L2) eggs under laboratory conditions and
allowed to develop into tissue dwelling larvae. The three antigens will be screened for their
diagnostic sensitivity in these experimentally infected pups.

Sera from adult dogs brought to the IVRI polyclinic for routine health check up and also
from stray dogs will be screened for T. canis antibodies using these recombinant proteins.

The study will be extended to evaluating the utility of these three antigens in the serodetection of T. canis infection in humans. Human sera will be obtained from the
opthalmology department, AIIMS, New Delhi from T. canis suspected patients with ocular
granuloma for detection of anti-T. canis antibodies.

Comparative diagnostic sensitivity of these antigens will be evaluated in ELISA, DOTELISA and latex agglutination test.

Cross-reactivity of these antigens with common canine nematodes viz. Ancylostoma

caninum and Dirofilaria immitis will be studied to determine specificity.

Data will be analyzed for comparative sensitivity and specificity of these three recombinant
antigens in the diagnosis of canine toxocarosis.

24

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