Veterinary Parasitology: Regional Studies and Reports 12 (2018) 61–68

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Veterinary Parasitology: Regional Studies and Reports

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High seroprevalence of Toxoplasma gondii in goats in Jharkhand state of T

India
Madhurendra Bachana, Asit Ranjan Deba, Biswa Ranjan Maharanab,c, Sudhakar N.R.b,
⁎
Vikrant Sudanb,d, B.C. Saravananb, Anup Kumar Tewarib,
a
Department of Veterinary Parasitology, Ranchi Veterinary College, Birsa Agricultural University, Kanke, Ranchi, Jharkhand 834006, India
b
Division of Parasitology, Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India
c
RVDEC, LUVAS, Uchani, Karnal, Haryana, 132001, India
d
Department of Parasitology, College of Veterinary Sciences and Animal Husbandry, Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go
Anusandhan Sansthan (DUVASU), Mathura, Uttar Pradesh 281001, INDIA

A R T I C L E I N F O A B S T R A C T

Keywords: Toxoplasmosis, caused by Toxoplasma gondii, is an important food borne zoonosis worldwide. Although goat
ELISA meat constitutes an important dietary protein source, improperly cooked meat is a potential source of infection
Goat to humans. Data on prevalence of toxoplasma in goat is scanty from India. Serological detection is the practical
IFAT option for prevalence studies on T. gondii, as no biological stage of the parasite is present in the clinical materials
Recombinant SAG1
from the intermediate hosts. The present study was undertaken in the Jharkhand state of India which is largely
Toxoplasma gondii
inhabited by economically weaker aborigine population, who depend largely on animal husbandry for liveli-
hood. A total of 445 serum samples were collected for testing, which represented goats under intensive and free
range system of rearing. T. gondii specific IgG antibodies were detected in 42.47% (n = 189) samples by rSAG1
based indirect ELISA. The seroprevalence data were analyzed in respect of age, sex, breed of the goats and
altitude of the study area as well as rearing conditions of the animals to establish correlation, if any. Though age
and sex of the animals had a direct correlation with infection, the same could not be established with the other
factors. The sensitivity and specificity of the diagnostic ELISA were compared with IFAT, as well as with a
commercially available ELISA kit. The rSAG1-ELISA had 92.66% sensitivity and 90.67% specificity with a po-
sitive predictive value of 86.77% and negative predictive value 94.92% when compared with IFAT, whereas
when compared with the commercial ELISA kit, 87.50% sensitivity and 90.91% specificity with a positive
predictive value of 91.30% and negative predictive value 86.96% were observed. Inter rater agreement (kappa)
was calculated. rSAG1-ELISA showed good agreement with IFAT (kappa = 0.824) and commercially available
ELISA Kit (kappa = 0.783). Receiver Operating Characteristics (ROC) curve analysis, revealed a larger area
under curve (AUC) of 0.99 (95%CI, 0.97–1.0) when compared with IFAT as gold standard and a highest relative
sensitivity 91.30 (95% CI 72–98.3) and specificity 1.0 (95% CI 85.2–100) for the cut off value of 0.6005. The
present study revealed high seroprevalence of T. gondii in goats from Jharkhand, which has public health sig-
nificance.

1. Introduction
Toxoplasmosis is a worldwide zoonosis caused by Toxoplasma
gondii, an obligate coccidian parasite. Virtually any warm-blooded an-
imals, including humans are intermediate hosts for the parasite while
felines serve as the definitive host (Dubey and Beattie, 1988; Tenter
et al., 2000; Dabritz and Conrad, 2010; Dubey and Jones, 2008). T.
gondii forms tissue cysts in the omnivores and carnivores, including
scavengers rendering the infected animals as potential sources of
infection to all (Tenter et al., 2000). Herbivores generally acquire in-
fections while grazing on pastures contaminated with environmentally
resistant oocysts shed only by cats (Yilmaz and Hopkins, 1972; Frenkel
et al., 1975; Dubey and Beattie, 1988; Lindsay et al., 2002, 2003).
Humans become infected typically through ingestion of raw or in-
adequately cooked meat containing live T. gondii tissue cysts (Weinman
and Chandler, 1954; Desmonts et al., 1965; Sacks et al., 1983; Choi
et al., 1997; Dubey et al., 2002, 2005; Belfort-Neto et al., 2007;
Robertson, 2007). Transmission through drinking raw goat milk has

⁎
Corresponding author.
E-mail address: anup_tewari@ivri.res.in (A.K. Tewari).

https://doi.org/10.1016/j.vprsr.2018.02.004
Received 13 July 2017; Received in revised form 29 December 2017; Accepted 13 February 2018
Available online 14 February 2018
2405-9390/ © 2018 Elsevier B.V. All rights reserved.
M. Bachan et al. Veterinary Parasitology: Regional Studies and Reports 12 (2018) 61–68

been documented (Patton et al., 1990; Riemann et al., 1975; Sacks districts of Jharkhand (n = 185) as well as from some selected villages
et al., 1982; Skinner et al., 1990). Though T. gondii infection may be of Ranchi (n = 260) where the goats are reared free-range. The sam-
innocuous in immunocompetent intermediate hosts, abortion or pre- pling was done during July to November 2010 from animals differing in
natal complication in the fetus is common during congenitally acquired age, sex and body weight.
infections (Buxton, 1998; Esteban-Redondo and Innes, 1997; Pereira-
Bueno et al., 2004). 2.2. Blood collection
Backyard rearing of goats is practiced throughout India and con-
tributes significantly to the rural economy. Data on seroprevalence of The blood samples (2–3 ml) were collected aseptically from the ju-
caprine toxoplasmosis from India are scanty; a high level of ser- gular vein using sterile syringes without anticoagulant for separation of
oprevalence reported in ruminants from India and neighbouring serum in wide mouth test tubes (Borosil). Kids < 6 months of age were
countries using native protein-based DAT, IFAT, ELISA and MDAT (Sah excluded from sampling to avoid measuring antibodies passively
et al., 2018; Khan et al., 2017; Satbige et al., 2016; Rahman et al., 2014; transferred. The blood was allowed to clot at room temperature; serum
Shahiduzzaman et al., 2011; Sharma et al., 2008; Malik et al., 2005; separated and then transported on ice to the Protozoology laboratory of
Vijaya Bharathi et al., 2003; Mirdha et al., 1999; Dubey et al., 1993) Parasitology Division, Indian Veterinary Research Institute, Izatnagar,
and using recombinant protein-based ELISA (Sudan et al., 2015; Singh where they were stored at −20 °C until use.
et al., 2015; Velmurugan et al., 2008). Diagnosis of toxoplasmosis in
animals by visualizing bradyzoites in situ using CT scans, though pos-
sible, is both cost-prohibitive and impractical. Currently, serological 2.3. Reference sera
detection of chronic or latent infections using native tachyzoite derived
proteins is practiced widely, but development and standardization of The reference positive and negative sera were kindly donated by Dr.
high throughput methodologies using the improved techniques remains J. P. Dubey, USDA, Beltsville, USA.
essential. The commonly used agglutination tests, viz. modified agglu-
tination test and dye test, require handling live tachyzoites with the 2.4. Expression of rSAG1
associated risk of infection to human handlers. Native protein removes
the risk of infections in the finished product but this antigen has its own The recombinant T. gondii surface antigen protein 1 (rSAG1) was
set of limitations. The difficulties associated with the mass production expressed as reported elsewhere (Velmurugan et al., 2008). In brief, the
and isolation of T. gondii tachyzoites from peritoneal fluid following complete 1183 bp ORF of SAG1 coding region was PCR amplified using
experimental infection in mice or from tissue culture and the sub- the primer pair (5′-GGTTGTATGTCGGTTTCGCTGAC-3′ and 5′-GATCA
sequent harvesting of native antigenic proteins from these tachyzoites CTCACGCGACACAAGCTGC-3′) for cloning into pGEM-T vector. A
affect the degree of sensitivity or specificity achieved in diagnostic as- truncated 958 bp SAG1 fragment was PCR amplified using the primer
says based on these antigen preparations. This constitutes the major pair containing the same forward primer and a new 27 nucleotide long
drawbacks with the use of commercially produced native protein used reverse primer 5′-CGTCAAGCTTCAGCCGATTTTGCTGGAC-3′ con-
in many diagnostic tests. taining a HindIII restriction site. The 958 bp PCR product was restric-
The seroprevalence of T. gondii in goats from the Jharkhand state of tion digested with BamHI and HindIII to yield an 814 bp product and
India, which is largely inhabited by economically weak tribal people, ligated to pET-32b(+) (Novagen) for expression in BL-21(DE3) pLysS
was examined in the present study. Jharkhand extends from 21° 55′ to (Novagen) E. coli. The overexpression of recombinant SAG1 protein was
25° 35′ North Latitude to 83° 20′ to 88° 02′ East Longitude. The state has achieved after 7 h induction with 1 mM isopropylthio-βgalactoside
a geographical area of 7.970 million hectares, of which about 0.1 mil- (IPTG) at 37 °C. The purity of fusion protein was checked by SDS-PAGE
lion hectares are pastures and other grazing lands, 0.3 million hectares (Fig. 1).
are cultivable wastelands and 1.795 million hectares are the net sown
area. The state comprises of a hilly undulating Chotanagpur Plateau
with predominantly tropical forests and tribal settlements. The Tropic
of Cancer passes through the middle of Ranchi City and the average
temperature of the state is 25 °C. The average annual rainfall is
1400 mm and > 80% precipitation occurs during June to September.
Jharkhand hosts a goat population of about 6.5 million, of which 6.3
million is reared under rural environments and about 0.18 million goats
are reared in the urban areas (19th Livestock Census of India, 2012). A
locally developed recombinant T. gondii surface antigen 1 (rSAG1) -
ELISA (Velmurugan et al., 2008) was used to establish the ser-
oprevalence of T. gondii in goats. The association between T. gondii
seropositivity to the host related variables viz. age, gender and breed, as
well as, to the type of production system of that region was also studied.
The diagnostic specificity and sensitivity of the rSAG1-based ELISA was
established with these caprine serum samples and results were com-
pared with IFAT as well as commercially available CHEKIT-Toxotest
enzyme immuno-assay (EIA) kit (IDEXX Lab.).

2. Materials and methods

2.1. Sampling

Stratified random sampling method was applied for the present
seroprevalence study. A total of 445 sera samples were collected from
goats of two different breeds viz. Black Bengal and Beetal from two
different organized government farms located in the Chatra and Ranchi
Fig. 1. Confirmation of rSAG1 protein in BL21 (DE3) pLysS E. coli cells by SDS-PAGE. The
12% polyacrylamide gel showing high level expression of rSAG1 at 47 kDa region; Lane
M: Pre-stained molecular weight marker, Lane 1, 2, 3, 4: Expression of r SAG1 protein 0,
4, 6 and 8 h post induction, respectively.

62
M. Bachan et al.
2.5. Enzyme linked immunosorbent assay (ELISA)
The test was performed following the method of Lin et al. (1992)
with minor modifications. The dilution of the reagents for ELISA was
optimized by checkerboard titration. Polystyrene plates (Maxisorp,
Nunc) were coated with 2 μg of rSAG1 protein diluted in 100μl carbo-
nate-bicarbonate buffer (pH 9.6) per well at 37 °C for 1 h and then at
4 °C for overnight. Excess unbound antigen was removed by washing
the plates thrice with PBS-T (pH 7.2, 0.05% Tween-20). The free
binding sites in the wells were blocked with 5% fat-free milk powder
(Amresco) prepared in PBS at 37 °C for 2 h. Following stringent
washing, test serum samples diluted 1:100 in 1% non-fat milk powder
was added to each well (100 μl per well) and incubated at 37 °C for 1 h.
Excess unbound antibodies were removed by washing with PBS-T be-
fore 100 μl of HRPO-labeled, rabbit anti-goat IgG (GCC Biotech, India)
diluted 1:40000 in 1% non-fat milk powder was added to each well and
allowed to react at 37 °C for 1 h. Following three final washes with PBS-
T, the wells were developed at room temperature in the dark with
100 μl of OPD substrate dissolved in citrate-phosphate buffer (pH 5.0)
containing H2O2. The reaction was stopped after 10 min by adding 50 μl
of 3 N HCl per well. Absorbance was read at 492 nm in an ELISA reader
(Microscan).
The commercial kit (IDEXX Laboratories) based ELISA was per-
formed following the protocol of the manufacturer. Briefly, 100 μl of
pre-diluted serum samples and controls (1:400) in CHEKIT washing and
dilution solution were dispensed into wells of the microtitre plate pre-
coated with antigen and incubated at 37 °C for 60 min after gentle
mixing. Following incubation, three washings with 300 μl of CHEKIT
Washing and Dilution solution were performed while avoiding the
formation of air bubbles. Following washing, 100 μl of Ruminant-IgG-
PO-Conjugate were dispensed in each well and incubated at 37 °C for
60 min in a humid chamber. Following incubation, the wells were
washed and 100 μl of CHEKIT-TMB-Substrate were added to each well
and incubated at 25 °C for 15 min. The reaction was stopped by adding
100 μl of CHEKIT Stopping-TMB-Solution, pre-warmed to room tem-
perature, and wells were read at 450 nm in the same ELISA reader used
above. Interpretation of results was made by taking the average OD of
sample, positive control serum and negative control serum using the
formula:
ODsample − ODnegative
Value = × 100%
ODpositive − ODnegative
The resulting values were interpreted as recommended by kit
manufacturer: < 20% - negative; 20%–30% - ambiguous; 30%–100% -
weak positive; and, > 100% - positive.
For analysis with commercial ELISA Kit, 46 serum samples, in-
cluding equal numbers of positive and negative samples determined
previously with rSAG1-based ELISA, were selected to represent a range
of OD values (high, medium or low); all samples were treated in du-
plicate. For determination of sensitivity and specificity, all the samples
that fell within the positive range (ambiguous to positive) were con-
sidered positive.
Further, by using these samples, cut off for optimal sensitivity and
specificity was determined and the area under curve (AUC) was cal-
culated to assess the performance of the rSAG1-ELISA in comparison to
cELISA and IFAT keeping IFAT as the gold standard for discriminating
positive and negative samples. The relative diagnostic sensitivity and
specificity of the ELISA were evaluated for different cut-offs.
2.6. Indirect fluorescent antibody test (IFAT)
Antigen slides were prepared, following the procedure outlined in
the USDHEW (U.S. Department of health, Education and Welfare)
Manual, 1976 with minor modification. Briefly, four small circles were
etched out on microscope slides before applying the antigen.
63
Veterinary Parasitology: Regional Studies and Reports 12 (2018) 61–68
Tachyzoites of T. gondii, isolated fresh from mouse peritoneal fluid
following experimental infection, were washed with PBS (pH 7.2), ap-
plied to the scribed regions and then fixed on the slides with chilled
acetone. The coated slides were incubated in presence of diluted test
sera for 30 min at 37 °C in a humidified chamber and then washed
3 × 5 min in PBS (pH 7.6) and air dried. Ten microlitres of rabbit anti-
goat IgG-FITC conjugate (Bangalore Genie, India), diluted 1:100 in 1%
Evans blue (PBS pH 7.6), was loaded onto each scribed region. The
slides were incubated, washed and air dried as before and mounted
immediately using a drop of buffered glycerol prior to examination
using an epifluorescent microscope at 400× magnification for detec-
tion of parasite-specific fluorescence. A titre of 1:64 or above was
considered as positive.
2.7. Ethical consideration
The study was approved by the animal ethics committee of the
Veterinary faculty, Ranchi Veterinary College, Birsa Agricultural
University, Kanke, Ranchi, Jharkhand. Prior permission was obtained
from animal owners before collection of blood and serum samples. The
samples were collected by a professional veterinarian, according to
guidelines for animal husbandry and to good animal practice stipulated
by OIE's terrestrial animal health code for the use of animals in research
and education.
2.8. Statistical analyses
Sensitivity and specificity, positive predictive value, negative pre-
dictive value and kappa were calculated using the online tool, http://
www.medcalc.org. Further, area under curve (AUC) for all three tests
and relative sensitivity and specificity of rSAG1 ELISA were calculated
in Sigma Plot 12.
Significant association between T. Gondii sero positivity and age,
gender, breed, type of production (farm or village) or geographic lo-
cation was tested by Chi-Square test. Results were considered statisti-
cally significant when the observed p-value was < 0.05.
3. Results
SAG1 was expressed using pET32-b (+) and BL21 (DE3) host cell.
Recombinant protein was purified to homogeneity by Ni-NTA chro-
matography and subsequently the over expressed protein was refolded
to assess its biological activity. Immunoreactivity of the expressed
proteins was confirmed by SDS-PAGE and western blot analysis which
resolve at 47 kDa (Fig. 1).
A total of 445 goat serum samples were collected from two different
farms and nearby villages of Jharkhand, India and were tested by
rSAG1 based ELISA. An operational cut-off value was established by
adding three standard deviations to the mean absorbance value for
negative control sera on all the plates during the analytical study. A
scatter plot of sample number versus OD values was drawn with the
calculated operational cut-off value of 0.577 (Fig. 2). Out of 445 sam-
ples tested, 189 samples (42.47%) were above this cut-off value and
considered positive.
Data on age, sex and breed of goats sampled as well as the farm/
region, rearing system and altitudes were examined in the context of
the observed seroprevalence of toxoplasmosis (Fig. 3; Table 1). Goats
were grouped into three different age groups (9–12 months,
13–24 months and > 24 months). In goats 9–12 months of age, 33 of
121 samples were seropositive (27.27%). In goats 13–24 months of age,
92 of 213 samples (43.19%) were seropositive. In adult goats over
24 months old, 64 of 111 samples (57.66%) were positive. A statisti-
cally significant (p < 0.05) association was observed between age and
seropositivity. Goats of 13–24 months of age or > 24 months of age
were 1.98 ± 0.497 or 3.54 ± 1.106 times, respectively, more likely to
be seropositive than goats of 9–12 months of age.
M. Bachan et al. Veterinary Parasitology: Regional Studies and Reports 12 (2018) 61–68

commercial ELISA Kit (kappa = 0.783) (Table 3).

Receiver operating characteristics (ROC) curve analysis, revealed a
larger area under curve (AUC) of 0.99 (95%CI, 0.97–1.0) when com-
pared with IFAT as standard (Fig. 4) and a highest relative sensitivity
91.30 (95% CI 72–98.3) and specificity 1.0 (95% CI 85.2–100) using a
cut-off value of 0.6005 (Fig. 5).

4. Discussion

Goat husbandry contributes significantly to the rural economy of

India. Jharkhand state is situated in the eastern part of India and hosts a
significantly high aborigine population (26% of the total population of
the state); about 91% of this population reside in villages as per
2011census of India. About 40% of population of the state is involved in
goat farming. The current investigation assumes its importance given
Fig. 2. The scatter plot showing the OD492nm of individual serum samples. A total of 445 the zoonotic nature of toxoplasma and its potential to cause abortion
goat serum samples were tested for Toxoplasma gondii specific antibody by rSAG1-ELISA. and foetal aberrations both in humans and small ruminants.
The absence of pathognomonic clinical signs, coupled with the
evasive nature of Toxoplasma gondii, especially in chronic infections,
Fewer serum samples from male goats (52/150, 34.66%) were
makes detection of infections in food production animals challenging.
tested seropositive using the rSAG1-ELISA than samples from female
Diagnosis of toxoplasmosis in the intermediate hosts through conven-
goats (137/295, 46.44%); however, there was no significant difference
tional parasitological techniques is difficult because typically available
in seropositivity between male and female goats when the observations
clinical specimens do not harbour the parasite. Tachyzoites are present
were controlled with respect to age. Similarly, the association of T.
in the circulation of an infected host only transiently and bradyzoites
gondii seropositivity with gender and breed of goats and the type of
develop extravascularly in a wide variety of organs and muscles.
production system followed was statistically insignificant (Table 1).
Serological techniques remain the mainstay of diagnosis in production
The sensitivity and specificity of the diagnostic ELISA were de-
animals because computer tomography (CT) scans, as practiced on
termined against IFAT as well as with commercial ELISA kit [CHEKIT-
humans, is not financially viable in most veterinary situations. Several
Toxotest enzyme immuno-assay (EIA) kit, IDEXX Lab.]. The sensitivity
studies have shown that serological recognition of T. gondii varies with
and specificity of the diagnostic rSAG1-ELISA were determined as
the stage of infection (Lappin et al., 1994; Tomavo et al., 1994), animal
92.66% (95% CI 87.77–96.03) and 90.67% (95% CI 86.54–93.87), re-
species investigated and the parasite haplotype(s) (Denkers et al., 1994)
spectively with a positive predictive value of 86.77% (95% CI
involved. Variation in the immune response has also been demonstrated
81.10–91.25) and negative predictive value 94.92% (95% CI
against different strains of T. gondii (Bohne et al., 1993). Variation in
91.47–97.27) against IFAT as standard (Table 2). Further, a subset of 46
the response may also occur depending on the stage of infection, viz.
samples, representing strong positive, weak positive and negative
chronic stage of infection caused by bradyzoite stage of the parasite
samples (based on mean OD values from the rSAG1-ELISA), was used to
(Torpier et al., 1993) or tachyzoite induced acute stage of infection
assess the specificity and sensitivity of the commercial ELISA kit. Sen-
(Dubey, 1995). Agglutination based serological tests such as the direct
sitivity to the extent of 87.50% (95% CI 67.64–97.34) and specificity of
agglutination test (DAT) or modified agglutination test (MAT) are fre-
90.91% (95% CI 70.84–98.88) with a positive predictive value of
quently employed for diagnosis of human and animal toxoplasmosis;
91.30% (95% CI 71.96–98.93) and negative predictive value 86.96%
both depend on the availability of whole tachyzoites grown in vivo. The
(95% CI 66.41–97.22) were established (Table 3). The rSAG1- ELISA
use of a recombinant protein in an ELISA for serological detection of T.
showed good agreement with IFAT (kappa = 0.824) (Table 2) and the
gondii infections (e.g. Sudan et al., 2015; Singh et al., 2015; Velmurugan

Fig. 3. The bar diagram represents the overall gender, age, breed, farm, farm/village, altitude wise variation in seroprevalence of T. gondii as determined by rSAG1-ELISA in goats.

64
M. Bachan et al. Veterinary Parasitology: Regional Studies and Reports 12 (2018) 61–68

Table 1
Correlation between the host and environmental factors with seroprevalence of toxoplasmosis in goat.

Factors Category No. of samples examined No. of samples Positive (%) Chi-square Degrees of freedom p value

Gender Male 150 52(34.66) 5.642 1 0.018

Female 295 137(46.44)
Age (months) 9–12 121 33(27.27) 21.962 2 < 0.001
13–24 213 92(43.19)
> 24 111 64(57.66)
Breed Black Bengal 325 136(41.84) 0.0631 1 0.8016
Beetal 120 53(44.17)
Farms Chatra 120 53(44.17) 0.0136 1 0.9071
Ranchi 65 28(43.08)
Farm/Village Farms 185 81(43.78) 0.0593 1 0.8075
Villages 260 108(41.53)
Altitude (feet above mean sea level) Chatra (1400) 120 53(44.17) 0.0631 1 0.8016
Ranchi (2140) 325 136(41.84)

et al., 2008) has several advantages including less costly, large scale
production of a specific protein using heterologous expression systems;
the reproducibility and uniformity of the resulting recombinant test
antigen can improve diagnostic specificity. Importantly, recombinant
antigen production removes the need for in vivo propagation of ta-
chyzoites with its associated animal welfare and human infection risks.
Reports of toxoplasmosis in goats from India are scanty. The present
rSAG1-ELISA-based serological survey revealed a high (42.47%) ser-
oprevalence of T. gondii in goats from Jharkhand. Previously, detection
of T. gondii-specific antibodies was reported in 22.7% of sheep and
12.5% of goats from Palampur and 81.2% of Pashmina goats and 89.7%
of local outbred goats from the Kumaon region (Dubey et al., 1993). An
IHAT based study showed 41.21% samples as positive, whereas 32.21%
samples were positive by MDAT (Vijaya Bharathi et al., 2003). High
seroprevalence of T. gondii was reported in 50% sheep, 41.26% goats
and 64.44 to 71.8% cattle from West Bengal, Uttar Pradesh and Kerala
states of India using heterologously expressed recombinant T. gondii
surface antigen based ELISAs (Sudan et al., 2015; Singh et al., 2015). In
a prevalence study spanning different agro-climatic zones, several fac-
tors like diagnostic techniques used for detection, number and fre-
quency of visits by felines on the farm, different climatic conditions in
various zones taken under study may contribute to the variability
(Sawadogo et al., 2005). The high seroprevalence in goats found in the
present study indicates high contamination level of the environment by
oocysts of Toxoplasma gondii as primary infection in goats occurs
through oral route with feed contaminated with oocysts of T. gondii
voided through cat faeces.
The present investigation established a positive correlation of T.
gondii infection with the age of the animal. The prevalence of infection
was significantly different (p ≤ 0.001) between young and old animals.
The goats were assigned in three different groups, viz. 9–12 months,
13–24 months and > 24 months for the convenience of analysis. One
hundred and twenty one samples were assayed in the age group of
9–12 months, of which 33 samples (27.27%) were positive. Two hun-
dred and thirteen samples were assayed from the 13–24 months of age
group, of which 92 samples (43.19%) were found positive. Out of 111
samples assayed from goats > 24 months of age, 64 samples (57.66%)
were found positive. The findings confirm previous reports demon-
strating a higher exposure risk associated with increasing age (Rahman
et al., 2014; Dubey et al., 1992).
As far as the correlation of infection with gender of the animals is
concerned, significantly higher prevalence in female goats was ob-
served in comparison to their male counterparts (p-value: 0.018). The
observation is in agreement with the previous reports of higher female
susceptibility to Toxoplasma gondii infections than males (Van der Puije
et al., 2000; Figliuolo et al., 2004 and Jittapalapong et al., 2005).
Earlier, Alexander and Stinson (1988) reported that the female an-
imals are more susceptible to protozoan parasites as compared to male.
It is now widely accepted that many hormones including the sex asso-
ciated hormones directly influence the immune system (Roberts et al.,
2001). Estrogen enhances antibody production and androgen suppress
both T-cell and B-cell immune responses (Da Silva, 1999). Relaxation of
immunity in females may also be correlated to factors, viz. pregnancy,
nutrition, age, and environmental factors. Non-significant gender as-
sociation was also reported (Jittapalapong et al., 2005; Teshale et al.,
2007), though number of positive samples has been shown to be greater
in females in comparison to males. However, Bisson et al. (2000) and
Cavalcante et al. (2008) observed that sex was not a significant de-
terminant for exposure to Toxoplasma gondii infection in goats. It may
be a reasonable conclusion that the exposure time determines occur-
rence of toxoplasmosis in animals. It is also an important factor that
since females are reared longer in comparison to males for the sake of
getting more kids over the years as well as milk, it might have influ-
enced on the generated data.
The correlation of other factors like breed, farms, rearing conditions
and altitude of the sampling area to T. gondii infection remain insig-
nificant (Table 1). In breed wise comparison, though Beetal showed
higher percentage seropositivity in comparison to Black Bengal goats,
the difference was non-significant (p-value: 0.8016). Similar findings
were reported by Jittapalapong et al. (2005). However, a significantly
higher seroprevalence of T. gondii in Beetal over Teddy goats was re-
ported from Pakistan (Tasawar et al., 2011). So far as the analysis of
samples from the organized farms of Chatra and Ranchi is concerned,
the number of seropositive samples was almost similar and the differ-
ence was statistically non-significant (p value: 0.9071). Further, no
significant variation was found between farm and villages (p value:
0.8075) as well as in altitude by comparison (p-value: 0.8016).
Toxoplasmosis is diagnosed in animals indirectly by serological

Table 2
The diagnostic sensitivity and specificity of rSAG1-ELISA determined against IFAT (standard) for detection of Toxoplasma gondii specific antibodies in goat serum.

rSAG1 ELISA IFAT Sensitivity Specificity Positive predictive value Negative predictive value Kappa value
(95% CI) (95% CI) (95% CI) (95% CI) (95% CI)
Positive Negative Total

Positive 164 25 189 92.66% 90.67% 86.77% 94.92% 0.824

Negative 13 243 256 (87.8–96.0%) (86.5–93.9%) (81.1–91.2%) (91.5–97.3%) (77.0–87.7%)
Total 177 268 445

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M. Bachan et al. Veterinary Parasitology: Regional Studies and Reports 12 (2018) 61–68

Table 3
The diagnostic sensitivity and specificity of rSAG1-ELISA determined against commercial ELISA kit (standard) for detection of Toxoplasma gondii specific antibodies in goat serum.

rSAG1 ELISA Commercial ELISA Sensitivity Specificity Positive predictive value Negative predictive value Kappa value
(95% CI) (95% CI) (95% CI) (95% CI) (95% CI)
Positive Negative Total

Positive 21 2 23 87.5% 90.9% 91.3% 87.0% 0.783

Negative 3 20 23 (67.4–97.3%) (70.8–98.9%) (72.0–98.9%) (66.4–97.2%) (0.603–0.962)
Total 24 22 46

the commercially available native protein based diagnostic tests.

Fig. 4. Receiver operating characteristics (ROC) analysis of T. gondii rSAG1-ELISA shows
an area under curve (AUC) of 0.99 and of cELISA 0.98 considering IFAT as standard.
tools. Although several serological tests have been described in litera-
ture, in the absence of standardized reagent or methodologies suitable
for a reference laboratory and for field use, sero-monitoring of tox-
oplasmosis using the improved techniques has not been widely em-
ployed or practiced. The native protein based serological methods are
often subject to limitations of sensitivity and specificity. The commonly
used agglutination tests, viz. modified agglutination test and dye test
require handling of live tachyzoites and there is a potential risk of in-
fection to human handlers. Difficulties associated with harvesting na-
tive antigenic proteins by mass production and isolation of the parasites
from peritoneal fluid through experimental infection in mice or from
tissue culture, as well as their variable degree of sensitivity or specifi-
city for use in diagnostic assays constitute the major drawbacks with
Therefore, development of recombinant protein based diagnostic
methods and their application for generating reliable epidemiological
data on the disease has been emphasized.
The sensitivity and specificity of the diagnostic rSAG1-ELISA was
determined against IFAT as well as with a commercial ELISA kit
[CHEKIT-Toxotest enzyme immuno-assay (EIA) kit, (IDEXX Lab.)].
All of the 445samples were taken into consideration to evaluate
sensitivity and specificity keeping IFAT as gold standard. The rSAG1
based ELISA showed 92.66% (95% CI 87.77–96.03) sensitivity and
90.67% (95% CI 86.54–93.87) specificity with a positive predictive
value of 86.77% (95% CI 81.10–91.25) and negative predictive value
94.92% (95% CI 91.47–97.27) against IFAT. The kappa value of 0.824
indicates its good agreement with IFAT.
While comparing with the commercial ELISA kit, 46 samples were
selected for evaluation of sensitivity and specificity. The rSAG1-ELISA
showed 87.50% sensitivity (95% CI 67.64–97.34) and 90.91% specifi-
city (95% CI 70.84–98.88) with a positive predictive value of 91.30%
(95% CI 71.96–98.93) and negative predictive value 86.96% (95% CI
66.41–97.22). The kappa value of 0.783 denotes its good agreement
with the commercial ELISA kit.
ROC analysis enabled comparison of various cut offs regarding re-
lative sensitivity and specificities of the ELISA and the area under cover
(AUC) of the diagnostic test represents a statistical summary of the
overall diagnostic performance of the test (Greiner et al., 2000). Ac-
cording to an arbitrary guideline, it should be possible to distinguish
between non-informative and perfect tests based on AUC values, viz.
AUC = 0.5 is non-informative and a value between 0.5 and 0.7 denotes
less accurate. However, values between 0.7 and 0.9 is moderately ac-
curate; between 0.9 and 1 is highly accurate (0.9 < AUC < 1) and
AUC value of 1 is indicative of perfect tests (Swets, 1988). Based on this
analysis, the AUC value of 0.99 in this study suggests that the rSAG1-
ELISA represents highly accurate test tool with good discrimination of
positive from negative samples. A cut off of 0.6005 revealed the most
appropriate sensitivity and specificity (91.30% and 100%).

Fig. 5. Relative sensitivity and specificities of the Toxoplasma gondii rSAG1-ELISA at different cut-offs. Vertical line shows the selected ELISA cut-off of 0.6.

66
M. Bachan et al.
Earlier we had described the immunodiagnostic potential of rSAG1-
ELISA, however the study was based on a small sample size
(Velmurugan et al., 2008). Wua et al. (2009) expressed a truncated
SAG1 gene in Escherichia coli. An ELISA kit based on the purified re-
combinant truncated SAG1 (rtSAG1) was developed which was used to
detect antibodies against T. gondii in human sera. The results showed
that the infection of T. gondii could be detected sensitively and speci-
fically by this serologic method. The positive concordance between
rtSAG1-ELISA and Western blot, the gold standard, was 93.9% (31/33).
However, the positive concordance between the commercial available
ELISA Kit 1 (Haitai, Zhuhai, China) and ELISA Kit 2 (DiaSorin ETI-
TOXOK-M reverse Plus, Italy) with Western blot was 79.5% (31/39)
and 91.2% (31/34), respectively. Comparatively, the positive con-
cordance of ELISA Kit 1 and 2 with Western blot was lower than
rtSAG1-ELISA, in particular, the ELISA Kit 1 (p < 0.01), which in-
dicated that the rtSAG1 protein could be used as the diagnostic antigen
for human toxoplasmosis.
This is the first report on seroprevalence of Toxoplasma gondii in-
fection in goats, which is based on diagnostically important re-
combinant SAG1 antigen based ELISA, from Jharkhand state of India.
The ELISA has potential for large through put application to generate
data on prevalence of caprine toxoplasmosis from other parts of India.
Conflict of interest
The authors declare that there is no competing interest.
Acknowledgement
The authors acknowledge Dr. J. P. Dubey, USDA, Beltsville, USA for
kindly providing the reference sera. The authors also acknowledge Prof.
J. R. Barta, Department of Pathobiology, Ontario Veterinary College,
University of Guelph, Ontario, Canada for his critical comments during
preparation of the manuscript. The authors acknowledge the Dean,
Ranchi Veterinary College and the Director, IVRI for the facilities pro-
vided. The authors acknowledge the Indian Council of Agricultural
Research, New Delhi for financial grant provided.
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