DOI: 10.2478/s11686-014-0245-z © W. Stefański Institute of Parasitology, PAS Acta Parasitologica, 2014, 59(2), 305–309; ISSN 1230-2821
Seroprevalence of Neospora caninum infection in dairy cows in Northern provinces, Thailand Tawin Inpankaew1,2*, Sathaporn Jittapalapong2, Thomas J. Mitchell3, Chainirun Sununta4, Ikuo Igarashi1 and Xuenan Xuan1 1
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan; 2 Department of Parasitology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, 10903. Thailand; 3Animal Health Laboratories, Department of Agriculture and Food Western Australia, 3 Baron-Hay Court, South Perth, Western Australia 6151, Australia; 4Chiang Rai Livestock Provincial Office, Department of Livestock Development, Chiangrai, Thailand
Abstract Neospora caninum, an obligate intracellular protozoan parasite, is the causative agent of neosporosis, recognized as a major cause of bovine abortion around the world. Thailand is a developing agricultural country located in Southeast Asia. Livestock developments particularly in dairy cows of this country have been hampered by low productivity including milk and slow growth rate due to the impact of many pathogens including N. caninum. Currently, there is no effective method for control of neosporosis since there is less information regarding current status of infections. The objective of this study was to investigate the seroprevalence of neosporosis in dairy cows of the northern part of Thailand. During 2006–2007, the sera of 642 cows from 42 small farm holders with the top three highest consensus of dairy farms in the northern provinces, such as Chiang Rai, Chiang Mai and Lumpang were collected and performed tests. Antibodies to N. caninum were assayed by enzyme-linked immunosorbent assay (ELISA) with recombinant N. caninum surface antigen 1 (NcSAG1) and indirect fluorescent antibody test (IFAT). The overall prevalence of N. caninum infection in this study was 46.9% (301/642) by ELISA and 34.3% (220/642) by IFAT.
Keywords Neospora caninum, dairy cows, NcSAG1, IFAT, Thailand
Introduction Neospora caninum, an obligate intracellular protozoan parasite, is the causative agent of neosporosis recognized as a major cause of bovine abortion around the world and known to have a detrimental effect on bovine pregnancy and on milk production. N. caninum is an apicomplexan protozoan parasite which was originally identified in tissues of clinically paralyzed dogs (Dubey et al. 1988). The molecular search for diagnostic antigens for N. caninum infection has been focused on the identification of immunodominant antigens that are recognized by sera from infected animals (Chahan et al. 2003). The surface protein of all obligatory intracellular parasites are believed to play critical roles in infection (Nishikawa et al. 2001) and considered as potential candidates for developing effective diagnostic reagent. For example, the surface
antigen 1 of N. caninum (NcSAG1) is an important candidate for developing a diagnostic tool of neosporosis (Hemphil et al. 1997; Howe et al. 1998). Thailand is a developing agricultural country located in Southeast Asia. Livestock development particularly in dairy cows of this country have been hampered by low production including milk and meat production due to the impact of many pathogens including N. caninum. There were a few reports of neosporosis in cattle (Suteeraparp et al. 1999) with inconclusive results since the cost of damages were not truly estimated or represent the real losses of Thai farmers. More information will be beneficial for reducing cost of animal owners. The objective of this study was to determine the seroprevalence of Neospora infection among dairy cows of Northern provinces, Thailand by using enzyme-linked immnunosorbent
*Corresponding author: [email protected]
306
assay (ELISA) using recombinant NcSAG1 and indirect fluorescent antibody test (IFAT).
Materials and Methods Animals and study areas A total of 642 Holstein-Friesian cows were collected from 42 small farm holders of the top three highest consensuses of dairy farms in the North of Thailand such as Chiang Mai (150 samples), Chiang Rai (392 samples) and Lumpang (100 samples). All dairy cows were female and bred for milk production between 1 to 12 year olds. Blood was collected from the caudal or jugular vein from dairy cows. Sera were separated after sedimentation of blood cells and were stored at –20°C until used. Parasite N. caninum tachyzoites of the Nc-1 strain (Dubey et al. 1988) were grown in monolayers of Vero cells cultured in a minimum essential medium supplemented with 8% fetal bovine serum and 50 µg/ml kanamycin at 37°C with 5% CO2 in tissue culture flasks. Parasites were harvested by scraping the monolayer at peak growth and purified by filtration using a 5 µm filter (Millipore, USA). Cloning and expression of the gene encoding truncated NcSAG1 The gene encoding truncated NcSAG1 (NcSAG1t) was cloned and expressed as previous reported (Chahan et al. 2003). Briefly, the purified N. caninum tachyzoites (1X108) were lyzed in 0.1 M Tris-HCl (pH 8.0) containing 1% sodium dodecyl sulfate (SDS), 0.1 M NaCl, and 10 mM EDTA and then treated with proteinase K (100 µg/ml) at 55°C for 2 h. The genomic DNA pellets were extracted by phenol/chloroform followed by ethanol precipitation. The DNA pellets were dissolved in a TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA) and used as a template DNA for PCR. The NcSAG1t gene without sequences encoding a hydrophobic signal peptide and a C-terminus was amplified by PCR using oligonucleotide primers, 5’-ACGAATTCAGAAAAATCACCT-3’ and 5’-ACGAAT TCGACCAACATTTTCAGC-3’, which both contains introduced EcoRI sites to facilitate cloning. The PCR product was digested with EcoRI and then cloned into the EcoRI site of the bacterial expression vector, pGEX-4T-3 (Promega, USA). The resulting plasmid was designated as pGEX/NcSAG1t. The pGEX/NcSAG1t was expressed as a glutathione S-transferase (GST) fusion protein (GST-NcSAG1t) in E. coli (DH5 α strain). E. coli transformed with pGEX/NcSAG1t was cultured at 37°C until the OD600 nm level reached 0.5. Synthesis of GST-NcSAG1t was induced with 0.5 mM isopropylthio-β-galactoside (IPTG) at 37°C for 4 h. The resulting E. coli was harvested by centrifugation and treated by sonication in a TNE buffer (50 mM
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Tris-HCl, pH 7.5, 0.1 M NaCl, 2 mM EDTA) containing lysozyme (100 µg/ml) and 1% Triton X-100. After centrifugation, the supernatant was harvested, and the GST-NcSAG1t was purified with Glutathione Sepharose 4B, according to the manufacturer’s instructions (Amersham Pharmacia Biotech, USA). The recovered supernatant was identified as purified GSTNcSAG1t and was used as antigen in ELISA. ELISA The ELISA was performed in flat-bottom 96-well microplates (Nunc, Denmark). The purified GST-NcSAG1t was diluted to an optimal concentration (5 µg/ml for NcSAG1) in a 50 mM carbonate-bicarbonate buffer (pH 9.6), of which 50 µl was added separately to duplicate wells for each sample. Coated plates were incubated at 4°C overnight. After the unabsorbed antigen was discarded, the wells were blocked with PBS containing 3% skim milk (blocking solution, 100 µl/well) at 37°C for 1 h. Then the plates were washed once with PBS containing 0.05% Tween 20 (PBS-T). Fifty microliters of serum diluted in the blocking solution (1:100) was added to each well and incubated at 37°C for 1 h. After incubated the wells were washed six times with PBS-T and subsequently incubated with 50 µl of goat anti-bovine IgG-horseradish peroxidase conjugate (ICN Biochemical, USA) (1:4,000) at 37°C for 1 h. After six washes, 100 µl of substrate solution [0.05% 2,2’-azino-bis (3-ethylbenzthiazoline-6-sulphonic), 0.2 M sodium phosphate, 0.1 M citric acid, 0.003% H2O2] was added to each well. After 1 h reaction at room temperature, the optimal density (OD) was read at 415 nm by using an MTP-120 ELISA reader (Corona Electric, Japan). The ELISA titer was expressed as the reciprocal of the maximum dilution that showed an ELISA value equal to or greater than 0.1, which is the difference in absorbance between that for the antigen (GST-NcSAG1t) well and that for the control antigen (GST) well. The cutoff point of 0.1 was the mean OD for negative sera plus four standard deviations. IFAT All samples were examined by IFAT to confirmed and compared with ELISA results. Briefly, Neospora antigen slides were incubated at 37°C with diluted cow sera (1:100) in 5% bovine serum albumin (BSA) with phosphate buffer saline (PBS) for 1 hr and then, add a fluorescein isothiocyanate-labeled goat anti-bovine IgG (1:200 in 5% BSA with PBS). Positive and negative control sera were prepared in each slide. Slides were examined under fluorescence microscopy and only a bright, linear peripheral fluorescence of the tachyzoites was considered positive. Statistical analysis To estimate sensitivity (Se), specificity (Sp) positive predictive value (PPV) and negative predictive value (NPV), results for the two techniques were categorized into positive and negative
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variables, presented in cross-tabulations, and compared for equal possibilities of being positive by using McNemar’s test with 95% confidence interval (CI). Moreover, IFAT technique was used as a gold standard.
Table III. Briefly, the sensitivity of ELISA was 95.5% and specificity was 78.4%.
Discussion Results Table 1 shows the seropositive for antibodies to N. caninum in dairy cows in northern Thailand. Nearly 47.0% (301/642) were positive by ELISA with recombinant NcSAG1. Lumpang province has the highest seroprevalence (64.0%), while Chiang Rai and Chiang Mai were found the seropositive at 41.6% and 49.3%, respectively (Table I). However, 220 from 642 samples (34.3%) were positive by IFAT. In the comparison between ELISA with recombinant NcSAG1 and IFAT, 210 positive sera were matched with both techniques. However, 91 positive sera were observed only by ELISA while, 10 samples were positive only by IFAT (Table II). In additional, the calculated sensitivities, specificities PPV and NPV with 95% conference interval (CI) are shown in
The seroprevalence of N. caninum in cattle or dairy cows had been studied in many countries (Gondim et al. 1999; Atkinson et al. 2000; Bae et al. 2000; Huong et al. 1998; Jenkins et al. 2000; Ooi et al. 2000). The prevalences were varied among countries, regions, herds and even at different times of the same herds (Yu et al. 2007), but there were a few reports on bovine neosporosis in Southeast Asia (Huong et al. 1998; Suteeraparp et al. 1999; Kashiwasaki et al. 2001; Jittapalapong et al. 2008; Arunvipas et al. 2012). The present study revealed that the seroprevalence of N. caninum infections in dairy cows in northern Thailand were 46.9% by ELISA and 34.3% by IFAT, which was higher than previously reported in Thailand (6% by Suteeraparp et al. 1999; 10% by Jittapalapong et al. 2008; 11.7% by Arunvipas et al. 2012a) and Vietnam (5.5% by Huong et al. 1998).
Table I. The seroprevalence of N. caninum infections in dairy cows by ELISA and IFAT NcSAG1 Provinces No. of sample
No. of positive sample
Chiang Rai
392
163
Chiang Mai
150
74
Lumpang
100
64
Total
642
301
IFAT Sero-prevalence (95%CI)
No. of sample
No. of positive sample
392
117
150
55
100
48
642
220
41.6 (36.7–46.5) 49.3 (41.3–57.3) 64 (54.6–73.4) 46.9 (43.1–50.8)
Sero-prevalence (95%CI) 29.8 (25.3–34.3) 36.7 (28.9–44.4) 48 (38.2–57.8) 34.3 (30.6–38.1)
Table II. A comparison of N. caninum positive results between ELISA-NcSAG1 and IFAT ELISA-NcSAG1 IFAT Positive Negative Total
Total Positive
Negative
210 91 301
10 331 341
220 422 642
Table III. Sensitivity, specificity, positive and negative predictive value of NcSAG1-ELISA using IFAT as a gold standard
Technique
Seroprevalence (95%CI)
Sensitivity (%) (95% CI)
Specificity (%) (95% CI)
IFAT ELISA
34.3 (30.6–38.1) 46.9 (43.1–50.8)
100 95.5 (91.6–97.7)
100 78.4 (74.1–82.2)
Positive predictive value (%) (95% CI) 100 69.8 (64.2–74.8)
Negative predictive value (%) (95% CI) 100 97.1 (94.5–98.5)
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During the host-parasite interaction, the surface of parasite cells are the main targets of host immune response; therefore, the surface antigens of parasites should be the potential targets for diagnosis and vaccine. NcSAG1 is identified as a surface antigen of N. caninum and are considered to be an important diagnostic candidate for the detection of antibodies to N. caninum in animals (Nishikawa et al. 2001; Chahan et al. 2003). The present results have shown that the seroprevalence among serum samples taken from cows in 3 provinces, Thailand by the ELISA using recombinant NcSAG1 ranging from 41.6 to 64%. About 70% of ELISA-positive samples were confirmed by IFAT. The results indicated that the ELISA using recombinant NcSAG1 was sensitive for detection of N. caninum in cattle and can be a useful diagnostic method for the serodiagnosis of N. caninum infection. In many dairy farms in Thailand, dogs can be found roaming around farms and have the close contact with the cattle. Besides the transplacental transmission in cattle, these dogs might be the source of infection, contributing to the high prevalence of N. caninum antibodies in cattle by contaminating the farm with oocysts. In this present study, most farms had at least one dog in nearby environment. Hence, the presence of dogs on a farm has been a potential risk to provide the increasing chance of horizontal transmission (Jittapalapong et al. 2008; Arunvipas et al. 2012b). In conclusion, this report describes the high seroprevalence of N. caninum infection in dairy cows in Thailand and other countries such as Brazil (14.1%, Gondim et al. 1999), Australia (24.0%, Atkinson et al. 2000), USA (16.0%, Rodriguez et al. 2002) and Vietnam (5.5%, Huong et al. 1998). Further serological surveys in dogs, lived in the surrounding area of infected farm need to be accomplished and also seroprevalence study in other regions, as well as, the association between seropositive status and bovine abortions in this country. Acknowledgements. We thank the provincial veterinary officers in the Department of Livestock Development in Chiang Rai, Chiang Mai and Lumpang provinces for their helps in collecting blood samples. We also thank the dairy cow owners to cooperate in this study. This project was financially funded by Faculty of Veterinary Medicine, Kasetsart University Research Development Institute (KURDI), the Japan International Cooperation Agency (JICA), and the Program of Founding Research Center for Emerging and Re-emerging Infectious Diseases, MEXT Japan.
References Arunvipas P., Inpankaew T., Jittapalapong S. 2012a. Seroprevalence and Risk Factors of Neospora caninum Infection among Dairy Cows in the Western Provinces of Nakhon Pathom, Ratchaburi and Kanchanaburi, Thailand. Kasetsart Journal (Natural Science.), 46, 64–70. Arunvipas P., Inpankaew T., Jittapalapong S. 2012b. Risk factors of Neospora caninum infection in dogs and cats in dairy farms in Western Thailand. Tropical Animal Health and Production, 44, 1117–1121. DOI: 10.1007/s11250-011-0048-2.
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Atkinson R.A., Cook R.W., Reddacliff L.A., Rothwell J., Broady K.W., Harper P., Ellis J.T. 2000. Seroprevalence of Neospora caninum infection following an abortion outbreak in a dairy cattle herd. Australian Veterinary Journal, 78, 262–266. Bae J.S., Kim D.Y., Hwang W.S., Kim J.H., Lee N.S., Nam H.W. 2000. Detection of IgG antibody against Neospora caninum in cattle in Korea. The Korean Journal of Parasitology, 38, 245–249. Chahan B., Gaturanga I., Xiaohong H., Liao M., Fukumoto S., Hirata H., Nishikawa Y., Suzuki H., Sugimoto C., Nagasawa H., Fujusaki K., Igarashi I., Mikami T., Xuan X. 2003. Serodiagnosis of Neospora caninum infection in cattle by enzyme-linked immunosorbent assay with recombinant truncated NcSAG1. Veterinary Parasitology, 118, 177–185. Dubey J.P., Carpenter J.L., Speer A., Topper M.J., Uggla A. 1988. Newly recognized fatal protozoan disease of dogs. Journal of the American Veterinary Medical Association, 198, 1269– 1285. Gondim L.F., Sartor I.F., Hasegawa M., Yamane I. 1999. Seroprevalence of Neospora caninum in dairy cattle in Bahia, Brazil. Veterinary Parasitology, 86, 71–75. Hemphil A., Felleisen R., Connolly B., Gottstein B., Hentrich B., Muller N. 1997. Characterization of a cDNA-clone encoding Nc-p43, a major Neospora caninum tachyzoite surface protein. Parasitology, 115, 581–590. Howe D.K., Crawford A.C., Lindsay D., Sibley L.D. 1998. The p29 and p35 immunodominant antigens of Neospora caninum tachyzoites are homologous to the family of surface antigens of Toxoplasma gondii. Infection and Immunity, 66, 5322– 5328. Huong L.T.T., Ljungstrom B.L., Uggla A., Bjorkman C. 1998. Prevalence of antibodies to Neospora caninum and Toxoplasma gondii in cattle and water buffaloes in southern Vietnam. Veterinary Parasitology, 75, 53–57. Jenkins M.C., Caver J.A., Björkman C., Anderson T.C., Romand S., Vinyard B., Uggla A., Thulliez P., Dubey J.P. 2000. Serological investigation of an outbreak of Neospora caninum-associated abortion in a dairy herd in southeastern United States. Veterinary Parasitology, 94, 17–26. Jittapalapong S., Sangvaranond A., Inpankaew T., Phasuk C., Pinyopanuwat N., Chimnoi W., Kengradomkij C., Saengow S., Pumhom P., Arunwipat P., Anakewit T., Robertson I.D. 2008. Seroprevalence of Neospora caninum infections of dairy cows in the North-east of Thailand. Kasetsart Journal (Natural Science), 42, 61–66. Kashiwasaki Y., Pholpark S., Charoenchai A., Polsar C., Teeverapanya S., Pholpark M. 2001. Postnatal neosporosis in dairy cattle in northeast Thailand. Veterinary Parasitology, 94, 217–220. Nishikawa Y., Kousaka Y., Tragoolpua K., Xuan X., Makala L., Fujisaki K., Mikami T., Nagasawa H. 2001. Characterization of Neospora caninum surface protein NcSRS2 based on Baculovirus expression system and its application for serodiagnosis of Neospora infection. Journal of Clinical Microbiology, 39, 3987–3991. Ooi H.K., Huang C.C., Yang C.H., Lee S.H. 2000. Serological survey and first finding of Neospora caninum in Taiwan, and the detection of its antibodies in various body fluids of cattle. Veterinary Parasitology, 90, 47–55. Rodriguez I., Choromanski L., Rodgers S.J., Weinstock D. 2002. Survey of Neospora caninum antibodies in dairy and beef cattle from five regions of the United States. Veterinary Therapeutics, 3, 396–401. Suteeraparp P., Pholpark S., Pholpark M., Charoenchai A., Chompochan T., Yamane I., Kashiwazaki Y. 1999. Seroprevalence of antibodies to Neospora caninum and associated abortion
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in dairy cattle from central Thailand. Veterinary Parasitology, 86, 49–57. Yu J., Xia Z., Liu Q., Liu J., Ding J., Zhang W. 2007. Seroepidemiology of Neospora caninum and Toxoplasma gondii in cattle and water buffaloes (Bubalus bubalis) in the People’s Republic of China. Veterinary Parasitology, 143, 79–85.
Received: October 16, 2013 Revised: January 15, 2014 Accepted for publication: March 13, 2014
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Seroprevalence of Neospora caninum infection in dairy cows in Northern provinces, Thailand Tawin Inpankaew1,2*, Sathaporn Jittapalapong2, Thomas J. Mitchell3, Chainirun Sununta4, Ikuo Igarashi1 and Xuenan Xuan1 1
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan; 2 Department of Parasitology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, 10903. Thailand; 3Animal Health Laboratories, Department of Agriculture and Food Western Australia, 3 Baron-Hay Court, South Perth, Western Australia 6151, Australia; 4Chiang Rai Livestock Provincial Office, Department of Livestock Development, Chiangrai, Thailand
Abstract Neospora caninum, an obligate intracellular protozoan parasite, is the causative agent of neosporosis, recognized as a major cause of bovine abortion around the world. Thailand is a developing agricultural country located in Southeast Asia. Livestock developments particularly in dairy cows of this country have been hampered by low productivity including milk and slow growth rate due to the impact of many pathogens including N. caninum. Currently, there is no effective method for control of neosporosis since there is less information regarding current status of infections. The objective of this study was to investigate the seroprevalence of neosporosis in dairy cows of the northern part of Thailand. During 2006–2007, the sera of 642 cows from 42 small farm holders with the top three highest consensus of dairy farms in the northern provinces, such as Chiang Rai, Chiang Mai and Lumpang were collected and performed tests. Antibodies to N. caninum were assayed by enzyme-linked immunosorbent assay (ELISA) with recombinant N. caninum surface antigen 1 (NcSAG1) and indirect fluorescent antibody test (IFAT). The overall prevalence of N. caninum infection in this study was 46.9% (301/642) by ELISA and 34.3% (220/642) by IFAT.
Keywords Neospora caninum, dairy cows, NcSAG1, IFAT, Thailand
Introduction Neospora caninum, an obligate intracellular protozoan parasite, is the causative agent of neosporosis recognized as a major cause of bovine abortion around the world and known to have a detrimental effect on bovine pregnancy and on milk production. N. caninum is an apicomplexan protozoan parasite which was originally identified in tissues of clinically paralyzed dogs (Dubey et al. 1988). The molecular search for diagnostic antigens for N. caninum infection has been focused on the identification of immunodominant antigens that are recognized by sera from infected animals (Chahan et al. 2003). The surface protein of all obligatory intracellular parasites are believed to play critical roles in infection (Nishikawa et al. 2001) and considered as potential candidates for developing effective diagnostic reagent. For example, the surface
antigen 1 of N. caninum (NcSAG1) is an important candidate for developing a diagnostic tool of neosporosis (Hemphil et al. 1997; Howe et al. 1998). Thailand is a developing agricultural country located in Southeast Asia. Livestock development particularly in dairy cows of this country have been hampered by low production including milk and meat production due to the impact of many pathogens including N. caninum. There were a few reports of neosporosis in cattle (Suteeraparp et al. 1999) with inconclusive results since the cost of damages were not truly estimated or represent the real losses of Thai farmers. More information will be beneficial for reducing cost of animal owners. The objective of this study was to determine the seroprevalence of Neospora infection among dairy cows of Northern provinces, Thailand by using enzyme-linked immnunosorbent
*Corresponding author: [email protected]
306
assay (ELISA) using recombinant NcSAG1 and indirect fluorescent antibody test (IFAT).
Materials and Methods Animals and study areas A total of 642 Holstein-Friesian cows were collected from 42 small farm holders of the top three highest consensuses of dairy farms in the North of Thailand such as Chiang Mai (150 samples), Chiang Rai (392 samples) and Lumpang (100 samples). All dairy cows were female and bred for milk production between 1 to 12 year olds. Blood was collected from the caudal or jugular vein from dairy cows. Sera were separated after sedimentation of blood cells and were stored at –20°C until used. Parasite N. caninum tachyzoites of the Nc-1 strain (Dubey et al. 1988) were grown in monolayers of Vero cells cultured in a minimum essential medium supplemented with 8% fetal bovine serum and 50 µg/ml kanamycin at 37°C with 5% CO2 in tissue culture flasks. Parasites were harvested by scraping the monolayer at peak growth and purified by filtration using a 5 µm filter (Millipore, USA). Cloning and expression of the gene encoding truncated NcSAG1 The gene encoding truncated NcSAG1 (NcSAG1t) was cloned and expressed as previous reported (Chahan et al. 2003). Briefly, the purified N. caninum tachyzoites (1X108) were lyzed in 0.1 M Tris-HCl (pH 8.0) containing 1% sodium dodecyl sulfate (SDS), 0.1 M NaCl, and 10 mM EDTA and then treated with proteinase K (100 µg/ml) at 55°C for 2 h. The genomic DNA pellets were extracted by phenol/chloroform followed by ethanol precipitation. The DNA pellets were dissolved in a TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA) and used as a template DNA for PCR. The NcSAG1t gene without sequences encoding a hydrophobic signal peptide and a C-terminus was amplified by PCR using oligonucleotide primers, 5’-ACGAATTCAGAAAAATCACCT-3’ and 5’-ACGAAT TCGACCAACATTTTCAGC-3’, which both contains introduced EcoRI sites to facilitate cloning. The PCR product was digested with EcoRI and then cloned into the EcoRI site of the bacterial expression vector, pGEX-4T-3 (Promega, USA). The resulting plasmid was designated as pGEX/NcSAG1t. The pGEX/NcSAG1t was expressed as a glutathione S-transferase (GST) fusion protein (GST-NcSAG1t) in E. coli (DH5 α strain). E. coli transformed with pGEX/NcSAG1t was cultured at 37°C until the OD600 nm level reached 0.5. Synthesis of GST-NcSAG1t was induced with 0.5 mM isopropylthio-β-galactoside (IPTG) at 37°C for 4 h. The resulting E. coli was harvested by centrifugation and treated by sonication in a TNE buffer (50 mM
Tawin Inpankaew et al.
Tris-HCl, pH 7.5, 0.1 M NaCl, 2 mM EDTA) containing lysozyme (100 µg/ml) and 1% Triton X-100. After centrifugation, the supernatant was harvested, and the GST-NcSAG1t was purified with Glutathione Sepharose 4B, according to the manufacturer’s instructions (Amersham Pharmacia Biotech, USA). The recovered supernatant was identified as purified GSTNcSAG1t and was used as antigen in ELISA. ELISA The ELISA was performed in flat-bottom 96-well microplates (Nunc, Denmark). The purified GST-NcSAG1t was diluted to an optimal concentration (5 µg/ml for NcSAG1) in a 50 mM carbonate-bicarbonate buffer (pH 9.6), of which 50 µl was added separately to duplicate wells for each sample. Coated plates were incubated at 4°C overnight. After the unabsorbed antigen was discarded, the wells were blocked with PBS containing 3% skim milk (blocking solution, 100 µl/well) at 37°C for 1 h. Then the plates were washed once with PBS containing 0.05% Tween 20 (PBS-T). Fifty microliters of serum diluted in the blocking solution (1:100) was added to each well and incubated at 37°C for 1 h. After incubated the wells were washed six times with PBS-T and subsequently incubated with 50 µl of goat anti-bovine IgG-horseradish peroxidase conjugate (ICN Biochemical, USA) (1:4,000) at 37°C for 1 h. After six washes, 100 µl of substrate solution [0.05% 2,2’-azino-bis (3-ethylbenzthiazoline-6-sulphonic), 0.2 M sodium phosphate, 0.1 M citric acid, 0.003% H2O2] was added to each well. After 1 h reaction at room temperature, the optimal density (OD) was read at 415 nm by using an MTP-120 ELISA reader (Corona Electric, Japan). The ELISA titer was expressed as the reciprocal of the maximum dilution that showed an ELISA value equal to or greater than 0.1, which is the difference in absorbance between that for the antigen (GST-NcSAG1t) well and that for the control antigen (GST) well. The cutoff point of 0.1 was the mean OD for negative sera plus four standard deviations. IFAT All samples were examined by IFAT to confirmed and compared with ELISA results. Briefly, Neospora antigen slides were incubated at 37°C with diluted cow sera (1:100) in 5% bovine serum albumin (BSA) with phosphate buffer saline (PBS) for 1 hr and then, add a fluorescein isothiocyanate-labeled goat anti-bovine IgG (1:200 in 5% BSA with PBS). Positive and negative control sera were prepared in each slide. Slides were examined under fluorescence microscopy and only a bright, linear peripheral fluorescence of the tachyzoites was considered positive. Statistical analysis To estimate sensitivity (Se), specificity (Sp) positive predictive value (PPV) and negative predictive value (NPV), results for the two techniques were categorized into positive and negative
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variables, presented in cross-tabulations, and compared for equal possibilities of being positive by using McNemar’s test with 95% confidence interval (CI). Moreover, IFAT technique was used as a gold standard.
Table III. Briefly, the sensitivity of ELISA was 95.5% and specificity was 78.4%.
Discussion Results Table 1 shows the seropositive for antibodies to N. caninum in dairy cows in northern Thailand. Nearly 47.0% (301/642) were positive by ELISA with recombinant NcSAG1. Lumpang province has the highest seroprevalence (64.0%), while Chiang Rai and Chiang Mai were found the seropositive at 41.6% and 49.3%, respectively (Table I). However, 220 from 642 samples (34.3%) were positive by IFAT. In the comparison between ELISA with recombinant NcSAG1 and IFAT, 210 positive sera were matched with both techniques. However, 91 positive sera were observed only by ELISA while, 10 samples were positive only by IFAT (Table II). In additional, the calculated sensitivities, specificities PPV and NPV with 95% conference interval (CI) are shown in
The seroprevalence of N. caninum in cattle or dairy cows had been studied in many countries (Gondim et al. 1999; Atkinson et al. 2000; Bae et al. 2000; Huong et al. 1998; Jenkins et al. 2000; Ooi et al. 2000). The prevalences were varied among countries, regions, herds and even at different times of the same herds (Yu et al. 2007), but there were a few reports on bovine neosporosis in Southeast Asia (Huong et al. 1998; Suteeraparp et al. 1999; Kashiwasaki et al. 2001; Jittapalapong et al. 2008; Arunvipas et al. 2012). The present study revealed that the seroprevalence of N. caninum infections in dairy cows in northern Thailand were 46.9% by ELISA and 34.3% by IFAT, which was higher than previously reported in Thailand (6% by Suteeraparp et al. 1999; 10% by Jittapalapong et al. 2008; 11.7% by Arunvipas et al. 2012a) and Vietnam (5.5% by Huong et al. 1998).
Table I. The seroprevalence of N. caninum infections in dairy cows by ELISA and IFAT NcSAG1 Provinces No. of sample
No. of positive sample
Chiang Rai
392
163
Chiang Mai
150
74
Lumpang
100
64
Total
642
301
IFAT Sero-prevalence (95%CI)
No. of sample
No. of positive sample
392
117
150
55
100
48
642
220
41.6 (36.7–46.5) 49.3 (41.3–57.3) 64 (54.6–73.4) 46.9 (43.1–50.8)
Sero-prevalence (95%CI) 29.8 (25.3–34.3) 36.7 (28.9–44.4) 48 (38.2–57.8) 34.3 (30.6–38.1)
Table II. A comparison of N. caninum positive results between ELISA-NcSAG1 and IFAT ELISA-NcSAG1 IFAT Positive Negative Total
Total Positive
Negative
210 91 301
10 331 341
220 422 642
Table III. Sensitivity, specificity, positive and negative predictive value of NcSAG1-ELISA using IFAT as a gold standard
Technique
Seroprevalence (95%CI)
Sensitivity (%) (95% CI)
Specificity (%) (95% CI)
IFAT ELISA
34.3 (30.6–38.1) 46.9 (43.1–50.8)
100 95.5 (91.6–97.7)
100 78.4 (74.1–82.2)
Positive predictive value (%) (95% CI) 100 69.8 (64.2–74.8)
Negative predictive value (%) (95% CI) 100 97.1 (94.5–98.5)
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During the host-parasite interaction, the surface of parasite cells are the main targets of host immune response; therefore, the surface antigens of parasites should be the potential targets for diagnosis and vaccine. NcSAG1 is identified as a surface antigen of N. caninum and are considered to be an important diagnostic candidate for the detection of antibodies to N. caninum in animals (Nishikawa et al. 2001; Chahan et al. 2003). The present results have shown that the seroprevalence among serum samples taken from cows in 3 provinces, Thailand by the ELISA using recombinant NcSAG1 ranging from 41.6 to 64%. About 70% of ELISA-positive samples were confirmed by IFAT. The results indicated that the ELISA using recombinant NcSAG1 was sensitive for detection of N. caninum in cattle and can be a useful diagnostic method for the serodiagnosis of N. caninum infection. In many dairy farms in Thailand, dogs can be found roaming around farms and have the close contact with the cattle. Besides the transplacental transmission in cattle, these dogs might be the source of infection, contributing to the high prevalence of N. caninum antibodies in cattle by contaminating the farm with oocysts. In this present study, most farms had at least one dog in nearby environment. Hence, the presence of dogs on a farm has been a potential risk to provide the increasing chance of horizontal transmission (Jittapalapong et al. 2008; Arunvipas et al. 2012b). In conclusion, this report describes the high seroprevalence of N. caninum infection in dairy cows in Thailand and other countries such as Brazil (14.1%, Gondim et al. 1999), Australia (24.0%, Atkinson et al. 2000), USA (16.0%, Rodriguez et al. 2002) and Vietnam (5.5%, Huong et al. 1998). Further serological surveys in dogs, lived in the surrounding area of infected farm need to be accomplished and also seroprevalence study in other regions, as well as, the association between seropositive status and bovine abortions in this country. Acknowledgements. We thank the provincial veterinary officers in the Department of Livestock Development in Chiang Rai, Chiang Mai and Lumpang provinces for their helps in collecting blood samples. We also thank the dairy cow owners to cooperate in this study. This project was financially funded by Faculty of Veterinary Medicine, Kasetsart University Research Development Institute (KURDI), the Japan International Cooperation Agency (JICA), and the Program of Founding Research Center for Emerging and Re-emerging Infectious Diseases, MEXT Japan.
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Received: October 16, 2013 Revised: January 15, 2014 Accepted for publication: March 13, 2014
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