Exposure to Toxoplasma gondii in Asian Elephants (Elephas maximus indicus) in Thailand

Toxoplasma gondii is the causative agent of toxoplasmosis in humans and various animal species worldwide. In Thailand, seroprevalence studies on T. gondii have focused on domestic animals, and information on infections in Asian elephants (Elephas maximus indicus) is scarce. This study was conducted to determine the seroprevalence of T. gondii infection in archival sera collected from 268 elephants living in Thailand. The serum samples were analyzed for anti-T. gondii immunoglobulin G antibodies using the latex agglutination test (LAT) and indirect enzyme-linked immunosorbent assay (iELISA) based on T. gondii lysate antigen (TLA-iELISA) and recombinant T. gondii dense granular antigen 8 protein (TgGRA8-iELISA). The prevalence of antibodies against T. gondii was 45.1% (121/268), 40.7% (109/268), and 44.4% (119/268) using LAT, TLA-iELISA, and TgGRA8-iELISA, respectively. Young elephants had a higher seropositivity rate than elephants aged >40 years (odds ratio = 6.6; p < 0.001; 95% confidence interval: 2.9–15.4). When LAT was used as the reference, TLA-iELISA and TgGRA8-iELISA showed a substantial (κ = 0.69) and moderate (κ = 0.42) agreement, respectively. Although our findings suggest the widespread exposure of Asian elephants to T. gondii in Thailand, the source of infection was not investigated. Therefore, investigation of the predisposing factors associated with toxoplasmosis is necessary to identify the potential risk factors for infection.


Introduction
Toxoplasma gondii is an obligate apicomplexan parasite that is widely prevalent in most warm-blooded animals, including humans [1,2]. The definitive hosts are domestic and wild felids, which excrete environmentally robust oocysts in their feces [3]. Intermediate hosts are infected via the fecal-oral route through the ingestion of food, water, or soil contaminated with sporulated oocysts, by consuming tissue cysts, or by congenital transmission [4].
Serological assays are essential for the detection of T. gondii infection in humans and animals [22]. Various techniques have been employed to screen for specific antibodies against T. gondii, such as the modified agglutination test (MAT), latex agglutination test (LAT), and indirect fluorescent antibody test (IFAT). Furthermore, an enzyme-linked immunosorbent assay (ELISA) based on soluble T. gondii lysate antigens (TLAs) has been used to detect T. gondii-specific antibodies in animals [23][24][25][26]. Recently, recombinant antigenic proteins of T. gondii have been widely used for serodiagnosis of toxoplasmosis in various animal species [27]. Several target antigens of T. gondii have been evaluated and applied in serological tests, such as the surface antigen, microneme antigen, rhoptry antigen, and dense granule antigen (GRA) tests. Antigens from the GRA group, such as GRA5, GRA6, GRA7, and GRA8, have demonstrated diagnostic potential for the serological detection of animal toxoplasmosis [27].
This study aimed to investigate the seroprevalence of T. gondii infection in Asian elephants living in Thailand using a commercial test kit and compared the results with those obtained using indirect ELISA (iELISA) based on TLA and a recombinant protein of T. gondii dense granular antigen 8 (TgGRA8).

Discussion
The seroprevalence of T. gondii in domestic and wild animals in Thailand has been investigated in the past [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. However, there are few reports on T. gondii infection in elephants. In this study, the seropositivity rate for T. gondii in elephants in captivity was >40%, which was higher than previously reported seropositivity rates in Asian elephants in Thailand, which were reported as 13% [28] and 25.6% [29] by LAT. Because the lifespan of elephants is similar to that of humans, the likelihood of T. gondii infection is higher than that in other animals. Moreover, some elephant owners release them to explore and forage for food in nearby forests [30], where they come into contact with plants contaminated with oocysts excreted by wild felids or domestic cats. Thus, a long-term study and risk factor association of T. gondii infection in elephants in Thailand is warranted.
We did not find a difference in T. gondii seropositivity between female and male elephants, which is consistent with the findings of a previous report that both male and female elephants were equally exposed to T. gondii infection [28]. The highest seroprevalence of T. gondii was detected in elephants aged 1-20 years and 21-40 years, which is similar to a previous report of high T. gondii seroprevalence in adult elephants [28]. However, our results showed a low prevalence of infection in elephants aged >40 years. This could be due to a possible bias and the high number of elephants of unknown age (60/268, 22.4%) in our study cohort. Because no data on congenital toxoplasmosis in elephants are available, further studies on the clinical manifestations of T. gondii infection in elephants should be performed.
We used LAT, TLA-iELISA, and TgGRA8-iELISA to detect T. gondii infection in elephant sera. Although LAT exhibited low sensitivity compared with MAT, it is widely used as a reference test for the seroprevalence of toxoplasmosis in various animal species, including elephants [28,29,[31][32][33]. ELISA is a common serological test that can be easily performed on a large scale, and many commercial kits are available to detect specific immunoglobulins produced in response to T. gondii infection. The conventional TLA-iELISA test shows a high degree of agreement with MAT, IFAT, or LAT for detecting antibodies against T. gondii in both humans and animals [25,34]. In the present study, TLA-iELISA demonstrated high sensitivity (78.5%) and specificity (90.4%), and the kappa value indicated a substantial agreement with LAT (κ = 0.69) for the detection of T. gondii infection in elephant sera. This indicates that TLA-iELISA could be an alternative serological test for T. gondii infection in elephants and other wild animals. Recombinant antigens are considered alternative diagnostic antigens to native antigens and have been used to improve the serodiagnosis of T. gondii [27]. Recombinant TgGRA8 antigen has been used in ELISA to detect toxoplasmosis in both humans and animals. High sensitivity and specificity for the detection of T. gondii have been reported using TgGRA8-iELISA in humans [35], goats [36], and domestic turkeys [37]. This is the first report of using recombinant protein TgGRA8 as a coating antigen to detect antibodies against T. gondii in elephants. The kappa value between LAT and TgGRA8-iELISA revealed a moderate agreement (κ = 0.42), with low sensitivity and specificity. Recently, multi-epitope or chimeric antigens have been used as an alternative approach to address the need for standardizing and increasing the sensitivity and specificity of serodiagnostic tests for animal toxoplasmosis [25,26,38]. Therefore, the application of a single recombinant protein or a mixture of recombinant proteins in an ELISA test to diagnose T. gondii infection in elephants needs to be developed and evaluated.
The long-term storage of serum affects the stability of several biochemical components and immunoglobulins in human and animal sera [39][40][41]. This could be impacted on the serological tests in our study because most of serum samples were stored for a long time. However, one study revealed that IgG antibodies against T. gondii in human sera can be reliably measured for 10 years of storage at −20 • C with no modification of interpretation of toxoplasmosis serologies [41]. To clarify whether long-term storage of such animal sera has an influence on the serological testing of T. gondii infection, further study will be necessary.
In conclusion, T. gondii infection is fairly common in Asian elephants in Thailand. The prevalence of the infection found in wild animals in Thailand could be of public health and conservation concern. Furthermore, the Asian elephants can be used as sentinels to monitor the potential contamination in the environment with T. gondii. However, updated information on the seroprevalence of this infection in elephants and a risk factor analysis should be performed to determine the actual situation and to identify the infection source and possible transmission routes.

Animal Samples
Archival elephant sera (n = 268) in this study were obtained from the project on animal DNA fingerprints in a cooperation between the Asian Elephant Foundation of Thailand and the Monitoring and Surveillance Center for Zoonotic Diseases in Wildlife and Exotic Animals, Faulty of Veterinary Science, Mahidol University. The serum samples were collected in various provinces of Thailand from 2009 to 2013 and in 2020 (Figure 1). For this study, the sample was divided into three aliquot parts for the serological tests and frozen at −30 • C. Data were recorded for each animal and included the sampling area, sampling date, sex, and age.

Animal Samples
Archival elephant sera (n = 268) in this study were obtained from the project on animal DNA fingerprints in a cooperation between the Asian Elephant Foundation of Thailand and the Monitoring and Surveillance Center for Zoonotic Diseases in Wildlife and Exotic Animals, Faulty of Veterinary Science, Mahidol University. The serum samples were collected in various provinces of Thailand from 2009 to 2013 and in 2020 (Figure 1). For this study, the sample was divided into three aliquot parts for the serological tests and frozen at −30 °C. Data were recorded for each animal and included the sampling area, sampling date, sex, and age.

Preparation of T. gondii GRA8 Antigen
Purified recombinant T. gondii dense granular antigen 8 (TgGRA8) was prepared as described previously [40]. Briefly, TgGRA8 (582 bp) was amplified using polymerase chain reaction and then ligated into a pET-21a vector using NdeI and XhoI as the cloning sites (General Biosystems, Durham, NC, USA) that were transfected into Escherichia coli

Preparation of T. gondii GRA8 Antigen
Purified recombinant T. gondii dense granular antigen 8 (TgGRA8) was prepared as described previously [40]. Briefly, TgGRA8 (582 bp) was amplified using polymerase chain reaction and then ligated into a pET-21a vector using NdeI and XhoI as the cloning sites (General Biosystems, Durham, NC, USA) that were transfected into Escherichia coli strain Rosetta (DE3) cells. TgGRA8 protein production was induced using isopropyl-β-Dthiogalactopyranoside to a final concentration of 1 mM with mild shaking at 20 • C overnight. Next, the cells were centrifuged at 4400× g for 20 min at 4 • C, and the resultant bacterial pellet was resuspended in 20 mL of prechilled lysis buffer. Then, the cells were disrupted by sonication on ice for 10 min. The total proteins in the soluble fraction were subjected to affinity purification using an anti-DYKDDDDK G1 affinity resin (GenScript, Piscataway, NJ, USA) according to the manufacturer's protocols. The purity and quantity of the proteins were checked using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The protein concentration was measured using the BSA assay (Pierce Biotechnology, Inc., Rockford, IL, USA).

Preparation of Toxoplasma Lysate Antigen
Tachyzoites of the T. gondii RH strain were cultivated in a monolayer of Vero cells (African green monkey kidney cells) with Dulbecco's Modified Eagle's Medium (GIBCO, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum. The pellets were harvested and washed twice in phosphate-buffered saline (PBS), followed by three freeze-thaw cycles, based on the standard procedure. The protein concentration in the TLA preparation was measured using Bradford reagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's recommendations, and the proteins were stored at −20 • C until use.

Latex Agglutination Test
We tested the elephant sera for T. gondii infection using a MAST Toxoreagent LAT (Mast Group, Liverpool, UK), according to the manufacturer's instructions. Samples were considered positive when agglutination was observed at a dilution of ≥1:64 [29].

IgG Indirect ELISA (iELISA)
iELISA was performed to detect T. gondii infection according to a previously described procedure [39], with slight modifications. Optimal dilutions were established using checkerboard titrations with dilutions of coating antigen and sera. The ELISA plates (Nunc, Roskilde, Denmark) were coated with 0.1 mL of TLA or TgGRA8 (1 µg/mL) diluted in a coating buffer (50 mM carbonate, pH 9.6) and were incubated at 4 • C overnight. Then, the plates were rinsed five times with PBS containing 0.05% Tween 20 (PBS-T), and nonspecific immune sites were blocked with 5% PBS-skimmed milk (PBS-SM) for 1 h at 37 • C. After washing with PBS-T, the control and sample sera were diluted to 1:100 in PBS-SM, and 100 µL of this mixture was added to each well in duplicate. After incubation at 37 • C for 1 h, the plates were washed and incubated with 100 µL of horseradish peroxidase-conjugated recombinant protein A/G (1:10,000 dilution; Thermo Fisher Scientific, Rockford, IL, USA), previously reported to bind elephant IgG [42,43], at 37 • C for 1 h. Next, the peroxidase activity was determined by adding 100 µL of 3,3 ,5,5 -tetramethylbenzidine (Invitrogen, Carlsbad, CA, USA), and the reaction was stopped by adding 100 µL of 1 N of HCl. The optical density (OD) was measured at a wavelength of 450 nm using an ELx808 ELISA microplate reader (Bio Tec Instruments, VT, USA). Positive and negative serum controls were confirmed using MAST Toxoreagent, a commercial LAT (Liverpool, UK), and were included in all plates. Serum samples were considered positive when the average OD > (OD mean [from the negative control sera] + 3 standard deviations from the negative control sera)].

Statistical Analyses
Data were analyzed using SPSS version 26.0 for Windows (IBM Corp., Armonk, NY, USA). The seroprevalence was calculated based on the ratio of positive results to the total number of tested animals. The association between T. gondii seropositive individuals and risk factors (age and sex) was analyzed using the chi-square test. A p value of <0.05 was considered statistically significant. The kappa values, specificity, sensitivity, and 95% confidence intervals were calculated using VassarStats (www.vassarstats.net (accessed on 25, October 2021)). The strength of agreement was graded according to the kappa values as fair (0.21-0.40), moderate (0.41-0.60), or substantial (0.61-0.80).