Introduction
Myiasis disease caused by przhevalskiana spp. larvae are known as goat warble-fly infestation (GWFI). Goats reared in tropical, subtropical, and non-tropical areas are infected by warble flies of the przhevalskiana spp. It causes economic losses in animals, such as meat and milk losses ( Oryan et al., 2009 ). Regarding previous and recent studies, przhevalskiana spp. cause severe skin damage in Iran ( Rahbari and Ghasemi, 1997 ; Tavassoli et al., 2010 ). The infection in the early stages of the disease cannot be detected on the examination of hypodermosis in goats through touching the nodules under the skin of the backs and flanks of the livestock. Therefore, infections are commonly underestimated and neglected ( Sinclair and Wassall, 1983 ). Some immunological methods have solved this problem and can significantly detect the infection even before the appearance of warbles on the back of animals ( Sinclair and Wassall, 1983 ). There is a need to find methods in order to treat the infestation before economic losses incurred ( Yadav et al., 2012 ). Immunological tests have been developed as one alternative method in order to perform clinical, parasitological, and post mortem examination to detect the infection ( Navidpour et al., 2007 ). In the same line, some serological tests have been developed in order to diagnose the bovine hypodermosis created by Hypoderma bovis and H. lineatum. Moreover, an enzyme-linked immunosorbent assay (ELISA) test prepared with HC antigen extracted from H. lineatum L1 is commonly used in some countries for the serodiagnosis of hypodermosis ( Otranto et al., 2005 ). The ELISA is a beneficial method for the serodiagnosis of the GWFI, and it can be used as baseline data in order to control and develop a future eradication program for this economically important parasite ( Faliero et al., 2001 ; Jan et al., 2014 ). A competitive ELISA is intrinsically more specific than a sandwich ELISA since the positive findings are dependent upon the displacement of specific antibody/antigen binding ( Webster et al., 1997 ) Therefore, considering the importance of this disease in goats, the present study aimed to develop a competitive ELISA system for the diagnosis of GWFI.
Material and Methods
Preparation of the Larval Antigen. In order to prepare the larva antigen, 200 first instar larvae of przhevalskiana were obtained from infested goats in Khorramabad slaughterhouse, Khorramabad, Iran, from September 2017 to March 2018. The obtained larva was washed in 200 ml sterile phosphate-buffered saline in pH=7.2 and in room temperature for 4 times as described by Webster et al. (1997) . The samples were subsequently homogenized in Griffiths tubes containing carbonate buffer 0.1M in pH=9.6 and slowly stirred at 4 °C for 16 h. The homogenate samples were centrifuged in 28000 g at 4 °C for 15 min. Furthermore, Macro Lowry test was used to measure the protein concentration absorbance at 750 nm by Bovine Serum Albumin (BSA) standards. The samples were maintained at -20 °C ( Webster et al., 1997 ).
Preparation of the Hyperimmune Rabbit Sera. In order to prepare the hyperimmune rabbit sera, 5 six-month-old New Zealand white rabbits were subcutaneously inoculated with a total of 500 µg/ml of przhevalskiana antigen in 4 sites. The animals were administered with a 1:1 emulsion in Freund’s complete adjuvant in the first instance and then administrated with a 1:1 emulsion in Freund’s incomplete adjuvant 14 and 28 days later. The blood samples were collected 11 days after the last inoculation through a cardiac puncture. Finally, the sera were separated and stored in small aliquots at -20 °C ( Webster et al., 1997 ).
Collection of the Sera Samples from Goats. In order to prepare the positive and negative serum samples from September 2017 to March 2018, the blood samples were collected from infected slaughtered goats (by direct observation of different instars of przhevalskiana larvae in the subcutaneous tissues of a slaughtered goat) and non-infected goats. Finally, 37 and 46 positive and negative blood samples were taken, respectively, from the goats slaughtered in Khoramabad slaughterhouse. The sera samples were separated after blood clotting, centrifuged at 2000 rpm for 5 min, and stored in small aliquots at -20 °C.
Enzyme-Linked Immunosorbent Assay. Immuno module polysorb F12 strips (Nunc) were covered by przhevalskiana antigen in 1, 2.5, 5, 10, and 20 µg/ml in carbonate buffer 0.1M, pH=9.6 overnight at 4 °C (100 µl/well) as described by Webster et al. (1997) . The wells were washed 3 times by Phosphate Buffered Saline Tween-20 (PBST) (phosphate 0.01M, sodium chloride 0.15 M, pH=7.2, and 0.05% Tween 20) and then blotted dry. In total, 250 µl PBST 5% normal horse serum (NHS) was added into each well for blocking and then incubated in a shaker for 1.5 h at 37 °C. The wells were washed 4 times by PBST and blotted dry. Goat serum at dilutions of 1/200 in competition with hyperimmune rabbit serum at 1/3000 in PBST 1% NHS (50 µl per well) was applied in order to test per sample. The samples were incubated on a shaker at 37 °C for 45 min, and then the wells were washed four times with PBST. Extra wells were considered as ''rabbit serum only" and "goat serum only" control. Hyperimmune rabbit serum diluted in ratio of 1:3000 was added into all wells (50 µl per well). Goat serum was only considered as the control well, and then the samples were incubated at 37 °C for 30 min. Subsequently, the wells were washed before the addition of the conjugate. Conjugate serum, mouse anti-rabbit horseradish peroxidase, was diluted in ratio of 1/1000 in PBST 1% NHS and was added into all wells (100 µl per well) and then incubated on a shaker at 37 °C for 75 min. The wells were washed, and the reaction was observed through the inclusion of 100 µl of Chromogen BM blue (Roch) per well and incubated for 20 min in a dark place. The reaction was stopped after nearly 5 min by the inclusion of sulfuric acid 10% (50 µl per well), and optical densities were spectrophotometrically evaluated through assessing the absorbance at 450 nm.
Determination of the Cut-off. In order to determine the cut-off, initially, the percentage of inhibitions of the goat sample sera was calculated according to the following equation:
Inhibition % = 100[1-(x-Min)÷(Max-Min)]
Cut-off = Mean PI ± 3SD
Where "x" represents the absorbance values of each sample at 450 nm, "Min" refers to absorbance values with no inhibition, and "Max" denotes the absorbance values with 100% inhibition. Accordingly, the percentage of inhibition and mean percentage of inhibition of the goat sample sera were calculated and the cut-off between positive and negative samples was determined by calculating the mean percentage of the inhibition of the panel positive sera minus three standard deviations of the mean.
Determination of the Sensitivity and Specificity. The Tijssen (1993) formula was used to evaluate the sensitivity and specificity of the samples.
Sensitivity=True positive×100/true positive+false negative
Specificity= True negative×100/true negative+false positive
True positive was evaluated through the direct examination of different instars of przhevalskiana larvae in the subcutaneous tissue of slaughtered goats, whereas uninfected goats were considered as true negative.
Evaluation of the Enzyme-Linked Immunosorbent Assay Precision. In order to capture the precision or replicability of the immunoassay test results, two measures including inter- and intra-assay coefficients of variability (CV) were calculated in this study. Inter-assay variation was assessed by analyzing four control sera tested in quadruplicate in four separate assays conducted on different days. The intra-assay variation was assessed by testing the same control samples twice in quadruplicate on one day. The percentage of CV (%CV) was calculated according to the following equation:
% CV= standard deviation of the samples’ means/mean of the samples’ means×100
The mean %CV of the samples was calculated as inter- and intra-assay. For the intra-assay, %CV of less than 10 is generally acceptable, whereas, for inter-assay, the %CV should be less than 15 ( Webster et al., 1997 ).
Results
Concentration of Larval Antigen. The extracted antigen from larvae was tested spectrophotometrically by absorbance at 750 nm and the Macro Lowry method using the BSA standards. The larval antigen concentration 2.5mg/ml was obtained and used for the ELISA test.
Enzyme-Linked Immunosorbent Assay. Various samples of goat serum and hyperimmune rabbit serum were tested by five concentrations of the extracted antigen from the first instar larvae of przhevalskiana. The results indicated that the dilutions of 1/200 goat serum and 1/3000 hyperimmune rabbit serum, as well as the concentration of 5µg/ ml antigen, were proper for the implementation of the ELISA test. Only rabbit serum control was considered as 100%. Based on these dilutions, and out of 83 goat serum samples, 37 positive and 46 negative samples were tested by the ELISA in order to detect the anti-przhevalskiana antibodies in the goat serum samples. Increasing the antibodies in positive sera and connecting them to the antigen leads to less binding of the labeled antibodies. Therefore, the optical density (OD) values of the positive samples were obtained less than OD values of the negative samples which resulted in a reverse standard curve in the competitive ELISA. The OD values of each sample were obtained at 450 nm, and based on the results, the mean percentage of the inhibition and cut-off value of the goat serum samples were also obtained in this study. When the percentage of the inhibition of goat serum sample was more than the cut off value obtained at dilution 1/200, it was considered to be positive. On the other hand, if the percentage of the inhibition of goat serum sample was less than the cut off value obtained at dilution 1/200, the sample was considered to be negative (Table 1).
| 46.33% | 51.39% | 24.65% | 25.70% | 49.11% | 36.44% | 48.35% | 37.34% | 52.61% | 46.50% | 39.62% |
| 24.50% | 21.31% | 51 % | 39.46% | 36.28% | 50.25% | 21.39% | 20.50% | 48.37% | 50.22% | 47.24% |
| 47.20% | 36.76% | 33.12% | 50.25% | 49.33% | 37.50% | 31.40% | 53.37% | 36.24% | 25.56% | 52.65% |
| 51.85% | 48.14% | 21.15% | 34.59% | 47.15% | 38.53% | 49.55% | 32.68% | 50.75% | 28.30% | 46.13% |
| 37.65% | 28.10% | 51.72% | 21.43% | 20.31% | 48.19% | 20.31% | 21.53% | 46.78% | 38.50% | 47.12% |
| 28.45% | 48.22% | 20.23% | 46.12% | 47.58% | 48.06% | 37.55% | 46.05% | 21.80% | 50.65% | 39.52% |
| 50.75% | 30.35% | 49.50% | 20.10% | 48.65% | 46.20% | 32.47% | 29.78% | 33.52% | 51.70% | 34.02% |
| E 42.53% | D 6.88% | C 2.14% | B 29.87% | A 48.95% | 21.60% | 50.56% | 30.11% | 46.23% | 34.23% | 46.50% |
| A: Mean percentage of inhibition of positive goat serum samples at 1/200 dilution; B: Mean percentage of inhibition of negative goat serum samples at 1/200 dilution; C: Standard deviation of positive goat serum samples at 1/200 dilution; D: Standard deviation of negative e goat serum samples at 1/200 dilution; E: Cut-off 3SD serum sample | ||||||||||
Determination of the Cut-off. The mean inhibition percentage in positive goat serum samples in ratio of 1/200 dilution was obtained at 48.95%, and cut-off percentage for the pair of positive and negative samples was evaluated through measuring the mean percentage of inhibition of the panel positive sera minus three standard deviations of the mean. Based on that cut-off, the percentage for the pair of positive and negative samples in3SD was obtained at 42.53% (Figure1).
Figure 1. The results of mean percentages of inhibition and cut-off percentage for the pair of negative and positive serum samples
Sensitivity and Specificity of the Enzyme-Linked Immunosorbent Assay. Due to the collection of different samples of positive and negative goat sera in autumn and winter, some false negative and positive samples were observed with decreased sensitivity and specificity which can be related to differences in the sample collection season. Moreover, the small size of the first instar larvae of przhevalskiana (less than 1 mm during emerging from the egg) led to recording some infected cases as negative goat samples and were reported as false negative in a serological study. Therefore, after testing the negative and positive sera and comparing the results ( Kothgassner et al. ) with cut-off, the sensitivity and specificity were obtained at 89.18% and 84.78%, respectively.
Enzyme-Linked Immunosorbent Assay Precision. The inter-and intra-assay CVs to express the precision or repeatability of ELISA kit results were 5.33% and 2.81%, respectively. It suggests the great accuracy and acceptable replicability of this method in testing the serum samples (Tables 2 and 3).
| Samples | Group 1 | Group 2 | Mean | SD | CV% |
|---|---|---|---|---|---|
| Sample1 | 24.56% | 25.89% | 25.27 | 0.87 | 3.44% |
| Sample2 | 37.44% | 38.49% | 37.96 | 0.74 | 1.94% |
| Sample3 | 39.34% | 40.05% | 39.69 | 0.5 | 1.25% |
| Sample4 | 21.51% | 22.97% | 22.24 | 1.03 | 4.63% |
| Intra assay | 2.81% | ||||
| Time | Time1 | Time2 | Time3 | Time4 | Mean | SD | CV% |
|---|---|---|---|---|---|---|---|
| Sample1 | 24.65% | 23.85% | 24.12% | 22.05% | 23.66 | 1.12 | 4.73% |
| Sample2 | 37.44% | 36.64% | 39.11% | 35.01% | 37.05 | 1.7 | 4.58% |
| Sample3 | 39.34% | 43.87% | 41.31% | 39.17% | 40.92 | 2.19 | 5.35% |
| Sample4 | 21.51% | 23.97% | 22.14% | 20.45% | 22.01 | 1.47 | 6.67% |
| Inter assay | 5.33% | ||||||
Discussion
The tests in this study were conducted by the ELISA method and the L1 antigen of przhevalskiana. The development of the antibodies approved the correlation between the parasitological results and alterations in the antibody contents. In the current study, due to the small size of the first instar larvae of przhevalskiana and undetectable nature of them in the goats, parasitological examination of the body detected antibodies in the goat samples as negative, whereas the ELISA detected it as positive through the detection of przhevalskiana larvae antibodies in the goat serums. Previous studies have shown that antibody titer was increased by the migratory phase of the first instar larvae (L1) in case the samples were obtained at this time. The sensitivity and specificity of the test could be increased up to 100%. The antibodies were deleted from the circulation of the infected animals about 98 days after the disappearance of the infection ( Sinclair and Wassall, 1983a ; Boulard et al., 1996 ). In the current study, different samples of positive and negative goat sera were collected in autumn and winter; accordingly, there were some false negative and false positive samples with decreased sensitivity and specificity which can be related to differences in sample collection season. Moreover, the small size of the first instar larvae of przhevalskiana (less than 1 mm during emerging from the egg) led to recording some infected cases as negative goat samples and was reported as false negative in a serological study. In addition, the results of calculating the percentage of inhibition using the competitive ELISA showed a high level of dependence and specificity of the antibodies which can easily be used to detect the serodiagnosis of hypodermoosis infected samples. This can be attributed to the combination of hyperimmune rabbit serum with samples and the competition of its antibodies with the antibodies in the serum of goats for binding the larval antigen of the first stage of the przhevalskiana flies. According to the observations recorded by ( Navidpour et al., 2009 ), the sensitivity and specificity of the competitive ELISA for the detection of przhevalskiana larvae antibodies in the goat sera were 81.25% and 87.57% at the cut-off 2CD and 84.37% and 80.95% at the cut-off 3SD, respectively. Our findings approved the ability of the L1 antigen of przhevalskiana in ELISA kit in order to detect the anti-przhevalskiana antibodies. Furthermore, the sensitivity and specificity at cut-off 3SD were 89.18% and 84.78%, respectively. The two measures of inter- and intra-assay coefficients of variability to capture the precision or replicability of ELISA kit results were 5.33% and 2.81%, respectively. These figures suggest the great accuracy and acceptable replicability of this method for testing the goat serum samples and studying in an epidemiological survey, especially when the disease has started. Regarding the life period of larvae and with the use of blood samples taken in the early months of infestation when the first instar larvae are still in the migratory stage and have not yet caused damage to the skin and before the animal slaughter, it is possible to detect the infestation early using the competitive ELISA. Following that, the seroepidemiology of the disease in the area can be investigated after parasite removal by the administration of a parasiticide.
Ethics
We hereby declare all ethical standards have been respected in preparation of the submitted article.
Authors’ Contribution
Study concept and design: Bagheri, A.; Madani,
Acquisition of data: Bagheri, A.; Madani,
Analysis and interpretation of data: Madani, R.; Navidpour, S.; Hoghooghi-Rad, N.
Drafting of the manuscript: Bagheri, A.; Madani,
Critical revision of the manuscript for important intellectual content: Madani, R.; Navidpour, S.; Hoghooghi-Rad, N.
Statistical analysis: Bagheri, A.; Madani, R.; Navidpour, S.; Hoghooghi-Rad, N.
Administrative, technical, and material support: Bagheri, A.; Madani, R.; Navidpour, S.; Hoghooghi-Rad, N.
References
- Boulard C, Villejoubert C, Moiré N. Cross-reactive, stage-specific antigens in the Oestridae family. Vet Res. 1996; 27(4-5):535-44.
- Faliero SM, Otranto D, Traversa D, Giangaspero A, Santagada G, Lia R, Puccini V. Goat warble fly infestation by Przhevalskiana silenus (Diptera: Oestridae): immunoepidemiologic survey in the Basilicata region (southern Italy). Parassitologia. 2001; 43(3):131-4.
- Jan S, Lateef M, Abbas F, Maqbool A, Jabbar MA, Kakar H. Sero-epidemiological studies on goat hypodermosis in northern upland Balochistan, Pakistan. Pak J Zool. 2014; 46(1): 153-160.
- Kothgassner OD, Felnhofer A, Hlavacs H, Beutl L, Palme R, Kryspin-Exner I. Salivary cortisol and cardiovascular reactivity to a public speaking task in a virtual and real-life environment. Comput Hum Behav. 2016; 62:124-35.
- Navidpour S, Madani R, Golchinfar F, Bakhshandeh N. Serodiagnosis of goat warble fly (GWF)(Diptera: Oestridae) infestation using by a competitive ELISA. Biochem Cell Arch. 2009; 9(1):93-6.
- Navidpour S, Madani R, Goodarzi MA, Paykari H. Study of antibodies development in naturally infested goats by Przhevalskiana sp. using by ELISA kit. J Exp Zool. 2007; 10(2):301-3.
- Oryan A, Razavi SM, Bahrami S. Occurrence and biology of goat warble fly infestation by Przhevalskiana Silenus (Diptera, Oestridae) in Iran. Vet Parasitol. 2009; 166(1-2):178-81.
- Otranto D, Zalla P, Testini G, Zanaj S. Cattle grub infestation by Hypoderma sp. in Albania and risks for European countries. Vet Parasitol. 2005; 128(1-2):157-62.
- Rahbari S, Ghasemi J. Study on economic aspects of goat grubs in Iran. Trop Anim Health Pro. 1997; 29: 243-244.
- Sinclair I, Wassall D. Enzyme-linked immunosorbent assay for the detection of antibodies to Hypoderma bovis in cattle. Res Vet Sci. 1983; 34: 251-252.
- Tavassoli M, Tajik H, Yaghobzadeh-Khangahi R, Javadi S. Prevalence of Goat Warble Fly, Przhevalskiana spp. (Dipetra: Oestridae), in West Azarbaijan, Iran. Iran J Vet Sci Technol. 2010; 2: 33-38.
- Tijssen P. Practice and theory of enzyme immunoassays, Elsevier, Amsterdam; 1993.
- Webster KA, Giles M, Dawson C. A competitive ELISA for the serodiagnosis of hypodermosis. Vet Parasitol. 1997; 68(1-2):155-64.
- Yadav A, Katoch R, Khajuria JK, Katoch M, Rastogi A. Economic impact of Przhevalskiana silenus infestation in native goats of Northern India. Trop Anim Health Prod. 2012; 44(3):581-7.
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