An initial survey of 150 horses from Thailand for anti-Pythium insidiosum antibodies

Zin Mar Htun Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand Aree Laikul Department of Large Animal and Wildlife Clinical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Nakhon Pathom, Thailand Watcharapol Pathomsakulwong Equine Clinic, Kasetsart University Veterinary Teaching Hospital, Nakhon Pathom, Thailand Chompoonek Yurayart Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand Tassanee Lohnoo Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Wanta Yingyong Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Yothin Kumsang Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Penpan Payattikul Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Pattarana Sae-Chew Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Thidarat Rujirawat Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Chalisa Jaturapaktrarak Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Piriyaporn Chongtrakool Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand Theerapong Krajaejun (  mr_en@hotmail.com ) Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand https://orcid.org/0000-0003-0545-3765

Data on the epidemiology of pythiosis is limited. Since the rst case in 1985, human pythiosis has been increasingly reported from all over Thailand [2,10,[13][14][15][16][17][18][19], where P. insidiosum is ubiquitous in the environment [3,4]. A seroprevalence study estimated that ~32,000 Thai people have been exposed to the pathogen [20]. In the other endemic countries (i.e., Brazil, Costa Rica, the United States, and Australia), human pythiosis is relatively rare, whereas the disease in animals (i.e., horses and dogs) is much more prevalent [1,21]. It is unknown if it is host, pathogen, or environmental factors that contribute to the different prevalence of human and animal pythiosis.
Animal pythiosis was reported in Thailand in a horse in 2018 [22] and a dog in 2020 [23]. The relative paucity of reports of animal pythiosis in Thailand may result from under-recognition and under-diagnosis of the disease. To acquire evidence on the exposure of animals to the pathogen, we surveyed serum anti-P. insidiosum antibodies in 150 horses distributed across Thailand, using 3 established serological tests: a protein A/G-based enzyme-linked immunosorbent assay (ELISA); a protein A/G-based immunochromatographic test (ICT); and Western blot analysis [11,12,20]. This study is a rst step leading to a better understanding of the epidemiology of animal pythiosis in Thailand.

Materials And Methods
Serum samples from 150 healthy horses were made available from the Faculty of Veterinary Medicine, Kasetsart University, Thailand (Table 1). The horses resided in northern (n = 43), central (n = 86), eastern (n = 14), and southern (n = 7) regions of Thailand. Serum samples from a Thai horse with culture-proven pythiosis and a healthy blood donor served as the positive and negative controls, respectively. The serum sample were tested (in duplicate) to detect the anti-P. insidiosum antibodies using an established protein A/G-based ELISA [11] with some modi cations. Brie y, a 96-well polystyrene plate (Corning) was coated with 100 µl of 5 µg/ml culture ltrate antigen (prepared from the human-isolated P. insidiosum strain Pi-S [24]) in 0.1 M carbonate buffer (pH 9.6) and 1.5% NaCl (4 ºC overnight), washed 4 times with PBS and 0.05% tween-20 (PBS-T), blocked with 250 µl of 0.5% bovine serum albumin (Merck) in PBS (37 ºC for 80 min), and washed 4 times again with PBS-T. A diluted serum sample (1:1,600 in PBS; 100 µl) was incubated in each well (37˚C for 1 hr) and was washed 4 times with PBS-T. The peroxidase-conjugated protein A/G (Bio-Rad) (1:100,000 in PBS; 100 µl) was added to each well and incubated at 37˚C for 1 hr. After a washing step as above, the chromogenic substrate (Tetramethylbenzidine and H 2 O 2 ; 100 µl) (Thermo Scienti c) was added into each well and incubated in the dark at room temperature for 3 min. The enzymatic reaction was stopped by applying 100 µl of 0.5 N H 2 SO 4 . The optical density (OD) of every serum sample was measured (at 450-nm) by an In nite 200 Pro microplate reader (Tecan). The obtained OD minus the blank OD (PBS) was divided by that of the negative control to provide an ELISA value (EV).
The ELISA + cutoff point was determined to be the mean EV of all tested serum samples plus 3 SDs, as previously described by other investigators [12,20,25].
All ELISA-positive sera, together with an equivalent number of randomly-selected ELISA-negative samples, were tested for the presence of anti-P. insidiosum antibodies by ICT [12] and Western blot analysis [20]. The positive and negative control sera were tested in parallel. A protein A/G-based ICT strip was dipped in 100 ul of each serum sample, diluted 1:5,000 in 0.15 M PBS (pH 7.4), for 30 min. The appearance of both test and control lines indicated an ICT-positive result, while the presence of only the control line indicated an ICT-negative result. For Western blot analysis, CFA of P. insidiosum [24] was separated by SDS-PAGE (4% stacking and 12% resolving gels) using a Bio-Rad MiniProteon II apparatus (setting: 100v for 90 min) and blotted onto anitrocellulose membrane (Bio-Rad) using a Bio-Rad Mini Trans-Blot apparatus (setting: 100v for 60 min). The blotted membrane was blocked with 5% skimmed milk in PBS with 0.1% Tween-20 (PBS-T) at room temperature for 60 min, and incubated with a serum sample, diluted 1:2,000 in 1% skimmed milk in PBS-T, at room temperature for 3 hr. After a washing step, the A/G protein, conjugated with horseradish peroxidase, wasadded to the membrane and incubated at room temperature for 2 hr. Western blot signals were generated using 0.03% diaminobenzidinetetrahydrochloride, 0.05% cobalt chloride, and 0.06% hydrogen peroxide in PBS.
The provinces where the equine serum samples were obtained were plotted on an on-line Thailand map using the Microreact software [26]. The most recent numbers of registered horses and farms were derived from the Department of Livestock Development, Ministry of Agriculture and Cooperatives, Thailand (http://en.dld.go.th/). Means, standard deviations (SD), and distribution histogram of EVs from all equine serum samples were obtained by using the EXCEL program (version 16.35).

Results
Serum samples were obtained from 150 horses (on 27farms) distributed in 8 provinces across Thailand (Table 1).. The geographic locations of these provinces (i.e., Chiang Rai, Payao, Suphanburi, Kanchanaburi, Ratchaburi, Chachoengsao, Chonburi, and Trang) is plotted on a map (Figure 1),, which can be accessed online at https://microreact.org/project/f2QcmQktR. All serum samples (identi er [IDs]: #1-150) were screened for anti-P. insidiosum antibodies by protein A/G-based ELISA [11]. With the "mean+3SDs" ELISA cut-off point (EV = 40.  [20]. In the current study, serum samples were obtained from 150 out of 6,353 horses registered in Thailand (Table 1, Figure 1).. Based on the consensus results from 3 established serological tests (Figure 2), the seroprevalence of anti-P. insidiosum antibodies in 150 horses was 0.7% (or an estimated 42 in the entire Thai equine population). Such seroprevalence in Thai horses (0.7%) is 10 times higher than that estimated for Thai people (0.07%) [20] and 17 times lower than that of Brazilian horses (11.1%) [25]. A higher seroprevalence implies a higher exposure rate of a target population to P. insidiosum.
Lower seroprevalence (0.7 vs. 11.1%) and fewer horses than in the Rio Grande do Sul State of Brazil (6,353 vs. 550,000) [25,27] explain why the number of horses with pythiosis in Thailand is minimal with just one case reported to date [28] vs. hundreds of Brazilian horses reported with the disease [29][30][31][32][33][34][35][36]. The striking difference in seroprevalences of the Thai (0.7%) and Brazilian (11.1%) horses could imply that Thai people, by comparison, might have a lower chance of having environmental exposure to P. insidiosum. However, the reported cases of human pythiosis from Thailand are much higher than those from Brazil [10,13,13,16,16,[37][38][39]. A host or pathogen factors might contribute to such a different prevalence of human pythiosis: Thalassemia is a common underlying condition in Thai patients with pythiosis [2,40]; Genotypes of the Thai P. insidiosum strains are different from that of the Brazilian strains [41,42].

Conclusion
We report an initial survey of anti-P. insidiosum antibodies in 150 horses from Thailand, using 3 established serological methods (i.e., ELISA, ICT, and Western blot). The seroprevalence of the antibodies in the Thai equine population (0.7%) was markedly higher than that of the Thai human population (0.07%), but much lower than that of the Brazilian equine population (11.1%). The antibody surveillances reported by our group and other investigators were undertaken to promote a better understanding of the epidemiology and host susceptibility of pythiosis in different geographic locations. Larger studies will be necessary to obtain a complete picture of the epidemiology of pythiosis.

Limitations
Serum samples, used in this study, were derived from a limited number of horses (i.e., 150 out of 6,353 horses). Besides, the recruited horses were disproportionally distributed throughout Thailand. Therefore, the resulting seroprevalence is statistically limited.