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Development and validation of an ELISA for detecting antibodies to Eimeria tenella in chickens
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Veterinary Parasitology 150 (2007) 306–313 www.elsevier.com/locate/vetpar
Development and validation of an ELISA for detecting antibodies to Eimeria tenella in chickens C.C. Constantinoiu a,*, J.B. Molloy b, W.K. Jorgensen b, G.T. Coleman a b
a School of Veterinary Science, University of Queensland, Brisbane, Qld. 4072, Australia Department of Primary Industries and Fisheries, 665 Fairfield Road, Yeerongpilly, Qld. 4105, Australia
Received 3 July 2007; received in revised form 6 September 2007; accepted 13 September 2007
Abstract The aim of this study was to develop and validate an ELISA for detecting chicken antibodies to Eimeria tenella. An initial comparison of merozoite and sporozoite antigen preparations revealed few differences in their ability to monitor the onset, kinetics and magnitude of the antibody response suggesting that both antigens would be equally useful for development of an ELISA. Furthermore the cross-reactivity of these antigens with sera from birds infected with chicken Eimeria species was similar. The merozoite antigen was selected for further evaluation because it was easier to prepare. Discrimination between sera from birds experimentally infected with E. tenella and birds maintained in an Eimeria-free isolation facility was excellent. In sera collected from free-range layers and commercial broilers there also appeared to be clear discrimination between infected and uninfected birds. The ELISA should prove useful for monitoring infectivity in vaccination programmes in layer and breeder flocks and for assessing the effectiveness of biosecurity measures in broiler flocks. # 2007 Elsevier B.V. All rights reserved. Keywords: ELISA; Eimeria; Chickens; Sporozoites; Merozoites; Antibodies
1. Introduction Eimeriosis is a major parasitic disease that costs the world poultry industry at least US$ 2400 million dollars annually (Shirley et al., 2005). Seven species of Eimeria infect chickens (Fernando, 1990). The Eimeria lifecycle is complex involving several phases of asexual development in the chicken followed by sexual stages that culminate in the release of oocysts in faeces. The immunity elicited by infections with Eimeria is species specific, but strong and relatively long lasting, making vaccination an attractive alternative to chemo-
* Corresponding author. Tel.: +61 7 336 29 514; fax: +61 7 336 29 429. E-mail address: [email protected] (C.C. Constantinoiu). 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.09.019
prophylaxis (Rose and Wakelin, 1990; Allen et al., 2005). The development of resistance to available coccidiostats and public concern over residues in meat and eggs are likely to result in increased use of live vaccines for the control of the disease (Chapman et al., 2002; Shirley et al., 2005). Live vaccines are based on either virulent or attenuated strains of Eimeria species. The most widely used method of attenuation is selection for precocious development in chickens (Williams, 2002). Precocious strains are characterized by shortened lifecycles in which the terminal generations of schizogony are either deleted or abbreviated (Shirley et al., 2005). Immunity is stimulated by the initial infection with the parasites contained in the vaccinal dose and is boosted and maintained by subsequent infections with oocysts from the litter. Thus, the recirculation of oocysts has been shown to be essential
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to the development of protective immunity (Chapman and Cherry, 1997; Williams, 2002). To successfully vaccinate a flock it is important that most, if not all, birds ingest a small dose of oocysts at the same time to ensure early uniform cycling of the parasites and development of flock immunity (Danforth et al., 1997; Chapman et al., 2005). However, most vaccine delivery methods do not result in all birds becoming infected (Chapman et al., 2005). A minority of birds do not receive the vaccinal dose and may suffer clinical coccidiosis if subsequently exposed to a large number of oocysts of vaccine or wild strains (Chapman et al., 2005). The lack of distinct clinical signs after infection with attenuated strains also means that there is no easy way for vaccine users to monitor the efficacy of their vaccine delivery method and the development of immunity. A simple method for monitoring vaccination outcomes is therefore required (Onaga et al., 2005). One way to assess the uniformity of vaccination is to transfer a sample of birds to cages and check their faeces for oocysts (Chapman and Cherry, 1997; Chapman et al., 2005) but this method is labour intensive and very few birds can be screened. Serological techniques, such as ELISA, that detect specific antibodies offer an alternative method for monitoring vaccine infectivity. ELISA is relatively inexpensive and has the capacity for high sample throughput (Onaga et al., 1989, 2005; Smith et al., 1993; Guzman et al., 2003). Antibodies are not thought to have a role in the development of protective immunity but it is likely that their development parallels the development of the protective cell-mediated response (Onaga et al., 2005). The detection of antibodies after vaccination therefore provides evidence that the birds have become infected and are likely to be immune. The asexual stages of Eimeria are thought to be more immunogenic than the sexual stages and most studies on the immune response of chickens have focused on them (Rose, 1987; Lillehoj, 2005). No assays for detecting chicken antibodies to E. tenella are commercially available but the possibility of using sporozoite and oocyst antigens in ELISA for detecting the antibody response in Eimeria infections has been investigated (Mockett and Rose, 1986; Onaga et al., 1986; Gilbert et al., 1988; Smith et al., 1993). However, none of these reports provided a full evaluation of an ELISA for detecting antibodies in E. tenella infections. The published tests are clearly capable of detecting antibodies to E. tenella but in no case was discrimination between infected and uninfected birds good enough for routine use. In this paper we report on a comparison of the antibody response against sporozoites and merozoites
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of an attenuated strain of E. tenella and the development and evaluation of an ELISA using a merozoite antigen preparation. The ELISA detects antibodies to E. tenella and most other economically important species of chicken Eimeria. It is quick and inexpensive and suitable for monitoring vaccination programmes and natural exposure of poultry flocks. To the best of our knowledge this is the first report of an ELISA based on E. tenella merozoite antigen. 2. Materials and methods 2.1. Birds Rhode Island Red/Rhode Island White cross-non-SPF birds were purchased from a commercial supplier as dayold chickens and housed in positive pressure isolators with HEPA filtered air until 3–4 weeks of age. While in the isolators the birds were maintained on feed-containing a commercial coccidiostat (Cycostat 66, Roche, Frenchs Forest, Australia) at 0.5 g/kg and faeces were tested weekly by the sugar flotation test to confirm freedom from Eimeria spp. For experimental work, the birds were transferred to suspended wire cages, in limited access, climate-controlled rooms. They were on a coccidiostat-free food for at least 2 days prior to inoculation of the parasites. All experiments involving live chickens were performed with the approval of the Animal Research Institute Animal Ethics Review Committee (approval number SA 2006/08/140). 2.2. Parasites The strains of chicken Eimeria used in these experiments are presented in Table 1. Oocysts were sporulated, separated from faeces and stored as described previously (Jorgensen et al., 1997). Five out of the seven strains used were attenuated by Table 1 The strains of chicken Eimeria used in this research Species
Line
Attenuated by selection for precocious development
E. E. E. E. E. E. E. E. E.
Redlands a Darten Ingten Medneca Medmaxa RAa Bowden Jorgensen Jorgensen
Yes No, wild strain No wild strain Yes Yes Yes No, already a mild strain Yes No, already a mild strain
a
tenella tenella tenella necatrix maxima acervulina brunetti mitis praecox
Components of Eimeriavax 4M1.
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selection for precocious development and have been previously characterized (Jorgensen and Anderson, 1999; Anderson and Jorgensen, 2004; Jorgensen et al., 2006). The E. tenella, E. necatrix, E. maxima, and E. acervulina strains are components of a commercially available live vaccine (Eimeriavax 4M1, Eimeria Pty, Werribee, Victoria, Australia). 2.3. Preparation of antigens from sporozoites and merozoites Sporozoites were obtained from sporulated oocysts of the Redlands strain of E. tenella as described by Shirley (1995). To obtain merozoites, 3-week-old birds inoculated by gavage with 2 106 sporulated oocysts of the Redlands strain of E. tenella were sacrificed 108 h post-infection (PI) and their intestines processed for merozoite isolation as described by Shirley (1995). The sporozoites and merozoites were purified on diethylaminoethyl (DEAE) cellulose (DE52, Whatman International Ltd., Maidstone, UK) columns using the method described by Shirley (1995). Purified parasites were disrupted by freezing/thawing five times and sonicating on ice (Sonifier 250, Branson, Danbury, CT, USA). The final suspension was centrifuged at 2000 g for 18 min at 4 8C. The supernatant was collected and the protein concentration determined by measuring the absorbance at 280 nm (BioPhotometer, Eppendorf, Hamburg, Germany). Antigens were stored in 200 ml aliquots at 80 8C until use. 2.4. ELISA Test wells of microtitre plates (MaxiSorp, Nunc, Roskilde, Denmark) were coated overnight, at 4 8C, with 1 mg of sporozoite or merozoite antigen in 100 ml of 0.05 M carbonate buffer (pH 9.6). Control wells were incubated overnight at 4 8C, with 100 ml of 0.05 M carbonate buffer (pH 9.6) only. After washing three times with phosphate-buffered saline (136.8 mM NaCl, 1.47 mM KH2PO4, 8 mM Na2HPO4 and 2.68 mM KCl, pH 7.4, PBS) containing 0.05% Tween 20 (PBST) non-specific binding sites were blocked by incubation with 200 ml of 2% skim milk powder (SMP) in PBST for 1 h at room temperature (RT). After three washes with PBST sera diluted 1:100 in 2% SMP in PBST were added to a test and control well and the plates incubated for 1 h at RT on a shaker. Positive and negative control sera were included on each plate. After incubation with sera the plates were washed five times with PBST and the wells filled with 100 ml of goat anti-chicken IgG peroxidase conjugate (Bethyl Laboratories Inc., Montgomery, TX,
USA) diluted in 1:50,000 in 2% SMP in PBST. After 1 h incubation at RT on a shaker the plates were washed five times and the enzyme reaction developed with 3,30 ,5,50 tetramethylbenzidine (TMB) (KPL, Gaithersburg, MD, USA) for 10 min in the dark. The reaction was stopped by the addition of 100 ml of 2M H3PO4 per well and the absorbance read at 450 nm with an ELISA Microplate Reader (Model 680, Bio-Rad Laboratories, Hercules, CA, USA). All sera were analysed in duplicate. For each serum, the average absorbance of the uncoated wells was then subtracted from the average absorbance of the antigen-coated wells. Serum from a bird infected with E. tenella Redlands strain produced one of the highest absorbances among the infected birds, and was chosen as the positive control. Absorbances of test sera were calculated as a percentage of the absorbance of the positive control and expressed as percent positivity (PP). 2.5. Time course of serum antibodies Two groups of six birds were used in this trial. The birds in the first group were inoculated by gavage, in the crop, with 400 oocysts of E. tenella Redlands strain (the approximate number of oocysts of the same strain found in one dose of Eimeriavax 4M1). The birds in the second group were inoculated with one dose of Eimeriavax 4M1 by placing a drop of vaccine into each chicken’s eye according to the manufacturer’s instructions. The birds were reared for the first 26 days after infection in cages placed in trays to facilitate oocyst recirculation. This treatment was meant to simulate conditions that occur in commercial farms where birds are reared on the floor. Oocyst recirculation was assessed by periodic examination of faeces by the sugar flotation test. Six birds reared in a separate room in cages on trays served as negative controls and were inoculated with distilled water only. All birds were wing bled weekly from 1 week PI for 10 weeks. The blood was allowed to clot for 1 h at RT and then overnight at 4 8C, centrifuged at 800 g for 10 min, aliquoted, and stored at 20 8C until use. 2.6. Cross-reactivity of antibodies to other species of chicken Eimeria Groups of six to eight birds, reared in cages with mesh flooring, were inoculated by gavage in the crop twice at 1month intervals with 5000 sporulated oocysts of each of the Eimeria species shown in Table 1 except the Ingten and Darten strains of E. tenella. Negative control groups of two birds housed under the same conditions, in a separate room were inoculated at the same time with distilled
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water only. Two weeks after the last inoculation all birds were anaesthetised with isoflurane (IsoFlo, Abbott Australasia, Botany, Australia), and blood was collected via cardiac puncture. The birds were then euthanised by cervical dislocation before recovery from anaesthesia. Serum was obtained and stored as described above. 2.7. Sera for ELISA validation Known positive sera were collected from 59 Rhode Island Red/Rhode Island White cross-birds 2 weeks after infection by gavage with attenuated (Redlands) or wild (Ingten and Darten) strains of E. tenella. Birds were inoculated twice at for and 6 weeks of age with 3000–5000 sporulated oocysts. Infection was confirmed by detection of parasites in the faeces. These birds were vaccinated against Marek’s disease (MD) only. Known negative sera were collected from 77 Arbor Acres broilers that were reared in cages with mesh floor in an isolation shed and fed a ration-containing coccidiostat as per industry standard. These birds were vaccinated against infectious bronchitis (IB) only.
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3. Results 3.1. Purity and yield of ELISA antigens The antigen preparations were derived from virtually 100% pure sporozoites and merozoites (Fig. 1a and b). Approximately 65 106 pure merozoites per bird were obtained and about 53–58 106 merozoites (or sporozoites) were processed to obtain one milligram of antigen. Merozoites recovered from one bird supplied enough antigens for testing at least 1000 sera. 3.2. Dynamics of the antibody response The dynamics of the antibody response to merozoite and sporozoite antigens following infection with E. tenella by gavage or vaccination with Eimeriavax 4M1 were compared. The antibody response to merozoite and sporozoite antigens was of similar intensity and followed the same pattern. For both antigens, high antibody levels were detected 2 weeks after the primary
2.8. Sera from commercial farms Sera were collected from 198 free-range pullets that were vaccinated with Eimeriavax 4M1 plus E. brunetti (Table 1). The birds were from two groups in separate sheds on the same farm. At the time of serum collection the pullets were 13 weeks old and they had been vaccinated against coccidiosis twice, at one and 42 days of age. They had also been vaccinated against IB, MD, newcastle disease (ND), fowl pox (FP), egg drop syndrome (EDS), fowl cholera (FC), Mycoplasma synoviae (Ms), Mycoplasma gallisepticum (Mg), avian encephalomyelitis (AE) and infectious laryngotracheitis (ILT). A total of 82 sera were also collected from five farms of free-range layers that were not vaccinated against coccidiosis. These birds were older than 37 weeks when the sera were collected. All free-range birds were maintained on a coccidiostat-free diet and were vaccinated against IB, MD, ND, FP, EDS, FC, Ms and Mg. In addition, 105 sera were collected at slaughter from 6-week-old commercial broilers that were reared on a diet containing a commercial coccidiostat and vaccinated against MD, IB and ND. 2.9. Statistical analyses Estimates of sensitivity and specificity and approximate 95% confidence limits were calculated according to Fleiss (1981).
Fig. 1. (a) Purified sporozoites of E. tenella Redlands strain. (b) Purified merozoites of E. tenella Redlands strain.
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3.4. Sensitivity and specificity of the merozoite antigen ELISA
Fig. 2. Comparison of the time course of the antibody response to sporozoite (~) and merozoite (*) antigens in birds infected with E. tenella by gavage (dotted line) or by eye drop vaccination with Eimeriavax 4M1 (continuous line). The reaction of sera from uninfected control birds with sporozoite (~) and merozoite (*) antigens is also shown. At each time point the arithmetic means and the standard error bars of absorbances of sera collected from six birds are shown.
infection and peaked 1 week later (Fig. 2). The antibody levels then declined slightly before reaching a plateau at 6–7 weeks PI. Control birds maintained under Eimeriafree conditions showed no increase in antibody levels throughout the experiment. 3.3. Cross-reactions of chicken antibodies to other Eimeria species with E. tenella antigens Sera from birds infected by gavage with the seven species of chicken Eimeria were tested in the ELISA using both sporozoite and merozoite antigen preparations (Fig. 3). Reactivity of antibodies with the two antigen preparations was similar. Antibodies in all birds infected with E. tenella or E. necatrix reacted strongly but cross-reactivity of antibodies to other Eimeria species was variable. Antibodies to E. acervulina, E. maxima and E. brunetti did cross-react with E. tenella antigens but the reactivity varied between birds. Sera from birds infected with E. mitis and E. praecox reacted weakly if at all.
Fig. 3. The reactivity of individual sera from birds infected with the seven species of chicken Eimeria with E. tenella sporozoite (S) and merozoite (M) antigen preparations.
Sensitivity and specificity were calculated using only the merozoite antigen because the antibody response to merozoites and sporozoites appeared similar and the merozoite antigen was easier to prepare. The distributions of ELISA PP values for the 59 sera from the birds experimentally infected with different strains of E. tenella and 77 birds reared under Eimeria-free conditions are shown in Fig. 4. There was clear discrimination between uninfected and infected birds. On the basis of these data the positive threshold was arbitrarily set at 10% of the absorbance of the positive control. In these sets of sera the sensitivity estimates of the ELISA were 100% (95% confidence limits: 92.4 and 100) while the specificity estimates were 100% (95% confidence limits: 94.1 and 100). 3.5. Performance of the test in the field The distribution of ELISA PP values for the 385 sera collected from commercial farms is presented in Fig. 5. The distribution for 196 of 198 vaccinated layers that were clearly positive in the ELISA (PP > 10%) resembled that of the birds experimentally infected with E. tenella (Fig. 4). The remaining two birds were clearly negative. The vast majority (97.5%) of 82 freerange layers also tested positive with the ELISA and the distribution of PP values also resembled that of experimentally infected birds. In contrast, only 1 of 105 sera from broilers reared under conditions that minimised exposure to Eimeria spp. and fed rations containing a commercial coccidiostat was positive. The distribution of PP values for the broilers was almost identical with that of cage-reared birds maintained under Eimeria-free conditions (Fig. 4).
Fig. 4. Frequency distribution of ELISA percent positivity (PP) values for sera of known antibody status (59 sera from E. tenella infected birds and 77 sera from uninfected birds).
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Fig. 5. Frequency distribution of ELISA percent positivity (PP) values for sera from 198 pullets vaccinated with Eimeriavax 4M1 plus E. brunetti and from 82 free-range layers and 105 broilers that had not been vaccinated against coccidiosis.
4. Discussion Antibodies raised in birds infected with E. tenella reacted similarly with sporozoite and merozoite antigens, suggesting that most antigens are common to the two-lifecycle stages. The dynamics of the antibody response to merozoite and sporozoite antigens in E. tenella-infected birds was also similar. There was no detectable antibody response at day 7 PI but a strong antibody response to both antigens was present at 14 days PI. Last generation merozoites are present about 4 days after sporozoites so antibodies against sporozoite antigens could be expected to precede antibodies against merozoites. This may have been the case but it was not apparent, probably because of the apparent antigenic similarity between the two stages. Antibody levels peaked at about 21 days after the primary infection then declined slightly before reaching a plateau and persisting at a high level for the duration of the experiment, presumably because of continuous exposure as a result of reinfection through recirculation of oocysts. This is consistent with previous reports that antibodies persist for extended periods in birds reared on the floor (Gilbert et al., 1988; Smith et al., 1993; Guzman et al., 2003). In our field trials high antibody levels were found in pullets 7 weeks after vaccination and in unvaccinated layers older than 37 weeks. The time course of the antibody response in vaccinated birds was similar to that in birds inoculated only with E. tenella, suggesting that the presence of extra species in the vaccine does not interfere with the development of the humoral response to E. tenella. High levels of antibodies were detected even after infections with as few as 400 oocysts, irrespective of the mode of inoculation (gavage or vaccination by eye drop). Most researchers (Mockett and Rose, 1986; Smith et al.,
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1993) used higher doses (10,000 oocysts) in the belief that higher doses result in better exposure and higher antibody titres (Gilbert et al., 1988; Onaga et al., 1989). Antibodies against most species of chicken Eimeria reacted with E. tenella sporozoite and merozoite antigens. These results are consistent with previous research that showed that the invasive stages of chicken Eimeria species share many antigens and that antibodies raised against one species cross-react with the antigens of other species (Sutton et al., 1989; Xie et al., 1992; Uchida et al., 1994; Talebi and Mulcahy, 1995; Shirley et al., 2005). The ELISA is therefore not capable of discriminating E. tenella infection from infections with most other Eimeria species. However this should not be a major drawback to using the test to monitor exposure in chickens following vaccination or natural infections. The strongest cross-reactivity in the ELISA was observed with E. necatrix, E. acervulina and E. maxima (Fig. 3), and these three species are commonly included in commercial vaccines. In commercial flocks, natural exposure to Eimeria is usually multi-species, the most common species being E. tenella, E. acervulina and E. maxima (Shirley et al., 2005), and our ELISA is capable of detecting antibodies in the majority of birds infected with these species. Furthermore, from the standpoint of broiler production, the primary goal is to determine whether or not a particular flock is infected with Eimeria species (Onaga et al., 1986). An ELISA based on an E. tenella antigen may therefore be suitable for monitoring exposure of chickens to Eimeria species either as a result of vaccination of natural infection. The ELISA may therefore prove to be a useful tool for monitoring the immune status of flocks because the development of the humoral response is thought to parallel the development of cell-mediated response. The merozoite antigen ELISA was selected for evaluation in the field because the antigen was easier to prepare. The level of discrimination between birds experimentally infected with E. tenella and uninfected birds was excellent and much higher than in previous reports (Onaga et al., 1989; Guzman et al., 2003). This is likely to reflect the high purity of the parasite preparation used for antigen production (Fig. 1b). Discrimination between sera collected in the field from vaccinated or naturally infected birds and uninfected birds was also excellent. On a commercial farm 99% of pullets were clearly positive when tested in the ELISA 7 weeks after vaccination. The levels of antibodies were high, suggesting that oocysts were recirculating and the birds were likely to be immune. Two birds were serologically negative. As they had been vaccinated twice and there was evidence of oocyst recirculation in
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the shed it is unlikely that they were not infected and it appears likely these individuals failed to mount a detectable antibody response. The intended application of this ELISA is to monitor flocks rather than individual birds so a negative response in such a low proportion of birds should not be a drawback. Most unvaccinated freerange layers were also positive and had uniformly high antibody levels, suggesting constant exposure to Eimeria. In contrast, in a broiler flock fed a dietcontaining coccidiostat only a few birds had low levels of antibody, suggesting little or no exposure. These results are consistent with previous data demonstrating minimal exposure in broiler flocks and uniform exposure of layer flocks (Onaga et al., 1986; Smith et al., 1993). The experimental broiler flock used as negative controls had been vaccinated against IB while the commercial broiler flock we tested had been vaccinated against MD, IB and ND. Both flocks were negative in the ELISA suggesting that there is no cross-reactivity between antibodies produced against these vaccines and E. tenella merozoite antigens. Cross-reactivity with other vaccines used by the poultry industry is possible but unlikely. The ELISA described here should be useful for monitoring exposure in layer flocks as a result of vaccination or natural infection and for monitoring the effectiveness of biosecurity measures in broiler flocks. Acknowledgements Special thanks to Jess Morgan for helpful suggestions while writing the manuscript, David Mayer for statistical analysis of the data, Grant Richards for supplying the vaccine, Tanya Nagle, Rod Jenner, Peter Trappett and Danny Singh for allowing access to flocks, Anthea Bruyeres, Sandy Jarrett, Jan-Maree Hewitson, Ryan O’Neil, Andrew Kelly and Ashley Ostrofsky for able technical assistance. References Allen, P.C., Jenkins, M.C., Miska, K.B., 2005. Cross protection studies with Eimeria maxima strains. Parasitol. Res. 97, 179–185. Anderson, G.R., Jorgensen, W.K., 2004. Live vaccines for three species of Eimeria. A report for the Rural Industries Research and Development Corporation. RIRDC Project DAQ-259J. RIRDC Publication no. 03/143, ACT, Australia. Chapman, H.D., Cherry, T.E., 1997. Eyespray vaccination: infectivity and development of immunity to Eimeria acervulina and Eimeria tenella. J. Appl. Poul. Res. 6, 274–278. Chapman, H.D., Cherry, T.E., Danforth, H.D., Richards, G., Shirley, M.W., Williams, R.B., 2002. Sustainable coccidiosis control in
poultry production: the role of live vaccines. Int. J. Parasitol. 32, 617–629. Chapman, H.D., Roberts, B., Shirley, M.W., Williams, R.B., 2005. Guidelines for evaluating the efficacy and safety of live anticoccidial vaccines, and obtaining approval for their use in chickens and turkeys. Avian Pathol. 34, 279–290. Danforth, H.D., Lee, E.H., Martin, A., Dekich, M., 1997. Evaluation of a gel-immunization technique used with two different Immucox vaccine formulations in battery and floor-pen trials with broiler chickens. Parasitol. Res. 83, 445–451. Fernando, M.A., 1990. Eimeria: infections of the intestine. In: Long, P.L. (Ed.), Coccidiosis of Man and Domestic Animals. CRC Press, Boca Raton, FL, pp. 63–77. Fleiss, J.L., 1981. Statistical Methods for Rates and Proportions, 2nd ed. Wiley, New York. Gilbert, J.M., Bhanushali, J.K., McDougald, L.R., 1988. An enzymelinked immunosorbent assay for coccidiosis in chickens: correlation of antibody levels with prior exposure to coccidia in the laboratory and in the field. Avian Dis. 32, 688–694. Guzman, V.B., Silva, D.A., Kawazoe, U., Mineo, J.R., 2003. A comparison between IgG antibodies against Eimeria acervulina, E. maxima, and E. tenella and oocyst shedding in broiler-breeders vaccinated with live anticoccidial vaccines. Vaccine 21, 4225– 4233. Jorgensen, W.K., Anderson, G.R., 1999. Attenuation and characterization of Eimeria spp. A report for the Rural Industries Research and Development Corporation. RIRDC Project DAQ-215J. RIRDC Publication no. 98, ACT, Australia. Jorgensen, W.K., Stewart, N.P., Jeston, P.J., Molloy, J.B., Blight, G.W., Dalgliesh, R.J., 1997. Isolation and pathogenicity of Australian strains of Eimeria praecox and Eimeria mitis. Aust. Vet. J. 75, 592–595. Jorgensen, W.K., Anderson, G.R., Jeston, P.J., Blight, G.W., Molloy, J.B., 2006. Selection and characterization of two attenuated vaccine lines of Eimeria tenella in Australia. Aust. Vet. J. 84, 89–94. Lillehoj, H.S., 2005. Immune Response to Cocccidia. In: Proceedings of the IXth International Coccidiosis Conference, Brazil. Mockett, A.P., Rose, M.E., 1986. Immune responses to Eimeria: quantification of antibody isotypes to Eimeria tenella in chicken serum and bile by means of the ELISA. Parasite Immunol. 8, 481– 489. Onaga, H., Saeki, H., Hoshi, S., Ueda, S., 1986. An enzyme-linked immunosorbent assay for serodiagnosis of coccidiosis in chickens: use of a single serum dilution. Avian Dis. 30, 658–661. Onaga, H., Togo, M., Kudo, Y., Motohashi, T., Ishii, T., 1989. The use of an enzyme-linked immunosorbent assay for estimation of the immune status of chickens artificially immunized against coccidiosis. Vet. Parasitol. 33, 199–205. Onaga, H., Kawahara, F., Umeda, K., Nagai, S., 2005. Field basis evaluation of Eimeria necatrix-specific enzyme-linked immunosorbent assay (ELISA) for its utility in detecting antibodies elicited by vaccination in chickens. J. Vet. Med. Sci. 67, 947–949. Rose, M.E., 1987. Eimeria, Isospora, and Cryptosporidium. In: Soulsby, E.J.L. (Ed.), Immune Responses in Parasitic Infections: Immunology, Immunopathology, and Immunoprophylaxis. CRC Press, pp. 275–312. Rose, M.E., Wakelin, D., 1990. Immunity to coccidiosis. In: Long, P.L. (Ed.), Coccidiosis of Man and Domestic Animals. CRC Press, Boca Raton, FL, pp. 281–306. Shirley, M.W., 1995. Eimeria species and strains of chickens. In: Eckert, J., Braun, R., Shirley, M.W., Coudert, P. (Eds.), Guide-
C.C. Constantinoiu et al. / Veterinary Parasitology 150 (2007) 306–313 lines on Techniques in Coccidiosis Research. European Commission, pp. 1–25. Shirley, M.W., Smith, A.L., Tomley, F.M., 2005. The biology of avian Eimeria with an emphasis on their control by vaccination. Adv. Parasitol. 60, 285–330. Smith, N.C., Bucklar, H., Muggli, E., Hoop, R.K., Gottstein, B., Eckert, J., 1993. Use of IgG- and IgM-specific ELISAs for the assessment of exposure status of chickens to Eimeria species. Vet. Parasitol. 51, 13–25. Sutton, C.A., Shirley, M.W., Wisher, M.H., 1989. Characterization of coccidial proteins by two-dimensional sodium dodecyl sulphatepolyacrylamide gel electrophoresis. Parasitology 99 (Pt 2), 175–187.
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Talebi, A., Mulcahy, G., 1995. Cross-reactivity among antisera raised against five avian Eimeria species in the natural host and in rabbits. Avian Pathol. 24, 533–544. Uchida, T., Hasbullah, Nakamura, T., Nakai, Y., Ogimoto, K., 1994. Cross reactivity of serum antibodies from chickens immunized with three Eimerian species. J. Vet. Med. Sci. 56, 1021–1023. Williams, R.B., 2002. Anticoccidial vaccines for broiler chickens: pathways to success. Avian Pathol. 31, 317–353. Xie, M., Gilbert, J.M., McDougald, L.R., 1992. Electrophoretic and immunologic characterization of proteins of merozoites of Eimeria acervulina, E. maxima, E. necatrix, and E. tenella. J. Parasitol. 78, 82–86.
Veterinary Parasitology 150 (2007) 306–313 www.elsevier.com/locate/vetpar
Development and validation of an ELISA for detecting antibodies to Eimeria tenella in chickens C.C. Constantinoiu a,*, J.B. Molloy b, W.K. Jorgensen b, G.T. Coleman a b
a School of Veterinary Science, University of Queensland, Brisbane, Qld. 4072, Australia Department of Primary Industries and Fisheries, 665 Fairfield Road, Yeerongpilly, Qld. 4105, Australia
Received 3 July 2007; received in revised form 6 September 2007; accepted 13 September 2007
Abstract The aim of this study was to develop and validate an ELISA for detecting chicken antibodies to Eimeria tenella. An initial comparison of merozoite and sporozoite antigen preparations revealed few differences in their ability to monitor the onset, kinetics and magnitude of the antibody response suggesting that both antigens would be equally useful for development of an ELISA. Furthermore the cross-reactivity of these antigens with sera from birds infected with chicken Eimeria species was similar. The merozoite antigen was selected for further evaluation because it was easier to prepare. Discrimination between sera from birds experimentally infected with E. tenella and birds maintained in an Eimeria-free isolation facility was excellent. In sera collected from free-range layers and commercial broilers there also appeared to be clear discrimination between infected and uninfected birds. The ELISA should prove useful for monitoring infectivity in vaccination programmes in layer and breeder flocks and for assessing the effectiveness of biosecurity measures in broiler flocks. # 2007 Elsevier B.V. All rights reserved. Keywords: ELISA; Eimeria; Chickens; Sporozoites; Merozoites; Antibodies
1. Introduction Eimeriosis is a major parasitic disease that costs the world poultry industry at least US$ 2400 million dollars annually (Shirley et al., 2005). Seven species of Eimeria infect chickens (Fernando, 1990). The Eimeria lifecycle is complex involving several phases of asexual development in the chicken followed by sexual stages that culminate in the release of oocysts in faeces. The immunity elicited by infections with Eimeria is species specific, but strong and relatively long lasting, making vaccination an attractive alternative to chemo-
* Corresponding author. Tel.: +61 7 336 29 514; fax: +61 7 336 29 429. E-mail address: [email protected] (C.C. Constantinoiu). 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.09.019
prophylaxis (Rose and Wakelin, 1990; Allen et al., 2005). The development of resistance to available coccidiostats and public concern over residues in meat and eggs are likely to result in increased use of live vaccines for the control of the disease (Chapman et al., 2002; Shirley et al., 2005). Live vaccines are based on either virulent or attenuated strains of Eimeria species. The most widely used method of attenuation is selection for precocious development in chickens (Williams, 2002). Precocious strains are characterized by shortened lifecycles in which the terminal generations of schizogony are either deleted or abbreviated (Shirley et al., 2005). Immunity is stimulated by the initial infection with the parasites contained in the vaccinal dose and is boosted and maintained by subsequent infections with oocysts from the litter. Thus, the recirculation of oocysts has been shown to be essential
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to the development of protective immunity (Chapman and Cherry, 1997; Williams, 2002). To successfully vaccinate a flock it is important that most, if not all, birds ingest a small dose of oocysts at the same time to ensure early uniform cycling of the parasites and development of flock immunity (Danforth et al., 1997; Chapman et al., 2005). However, most vaccine delivery methods do not result in all birds becoming infected (Chapman et al., 2005). A minority of birds do not receive the vaccinal dose and may suffer clinical coccidiosis if subsequently exposed to a large number of oocysts of vaccine or wild strains (Chapman et al., 2005). The lack of distinct clinical signs after infection with attenuated strains also means that there is no easy way for vaccine users to monitor the efficacy of their vaccine delivery method and the development of immunity. A simple method for monitoring vaccination outcomes is therefore required (Onaga et al., 2005). One way to assess the uniformity of vaccination is to transfer a sample of birds to cages and check their faeces for oocysts (Chapman and Cherry, 1997; Chapman et al., 2005) but this method is labour intensive and very few birds can be screened. Serological techniques, such as ELISA, that detect specific antibodies offer an alternative method for monitoring vaccine infectivity. ELISA is relatively inexpensive and has the capacity for high sample throughput (Onaga et al., 1989, 2005; Smith et al., 1993; Guzman et al., 2003). Antibodies are not thought to have a role in the development of protective immunity but it is likely that their development parallels the development of the protective cell-mediated response (Onaga et al., 2005). The detection of antibodies after vaccination therefore provides evidence that the birds have become infected and are likely to be immune. The asexual stages of Eimeria are thought to be more immunogenic than the sexual stages and most studies on the immune response of chickens have focused on them (Rose, 1987; Lillehoj, 2005). No assays for detecting chicken antibodies to E. tenella are commercially available but the possibility of using sporozoite and oocyst antigens in ELISA for detecting the antibody response in Eimeria infections has been investigated (Mockett and Rose, 1986; Onaga et al., 1986; Gilbert et al., 1988; Smith et al., 1993). However, none of these reports provided a full evaluation of an ELISA for detecting antibodies in E. tenella infections. The published tests are clearly capable of detecting antibodies to E. tenella but in no case was discrimination between infected and uninfected birds good enough for routine use. In this paper we report on a comparison of the antibody response against sporozoites and merozoites
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of an attenuated strain of E. tenella and the development and evaluation of an ELISA using a merozoite antigen preparation. The ELISA detects antibodies to E. tenella and most other economically important species of chicken Eimeria. It is quick and inexpensive and suitable for monitoring vaccination programmes and natural exposure of poultry flocks. To the best of our knowledge this is the first report of an ELISA based on E. tenella merozoite antigen. 2. Materials and methods 2.1. Birds Rhode Island Red/Rhode Island White cross-non-SPF birds were purchased from a commercial supplier as dayold chickens and housed in positive pressure isolators with HEPA filtered air until 3–4 weeks of age. While in the isolators the birds were maintained on feed-containing a commercial coccidiostat (Cycostat 66, Roche, Frenchs Forest, Australia) at 0.5 g/kg and faeces were tested weekly by the sugar flotation test to confirm freedom from Eimeria spp. For experimental work, the birds were transferred to suspended wire cages, in limited access, climate-controlled rooms. They were on a coccidiostat-free food for at least 2 days prior to inoculation of the parasites. All experiments involving live chickens were performed with the approval of the Animal Research Institute Animal Ethics Review Committee (approval number SA 2006/08/140). 2.2. Parasites The strains of chicken Eimeria used in these experiments are presented in Table 1. Oocysts were sporulated, separated from faeces and stored as described previously (Jorgensen et al., 1997). Five out of the seven strains used were attenuated by Table 1 The strains of chicken Eimeria used in this research Species
Line
Attenuated by selection for precocious development
E. E. E. E. E. E. E. E. E.
Redlands a Darten Ingten Medneca Medmaxa RAa Bowden Jorgensen Jorgensen
Yes No, wild strain No wild strain Yes Yes Yes No, already a mild strain Yes No, already a mild strain
a
tenella tenella tenella necatrix maxima acervulina brunetti mitis praecox
Components of Eimeriavax 4M1.
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selection for precocious development and have been previously characterized (Jorgensen and Anderson, 1999; Anderson and Jorgensen, 2004; Jorgensen et al., 2006). The E. tenella, E. necatrix, E. maxima, and E. acervulina strains are components of a commercially available live vaccine (Eimeriavax 4M1, Eimeria Pty, Werribee, Victoria, Australia). 2.3. Preparation of antigens from sporozoites and merozoites Sporozoites were obtained from sporulated oocysts of the Redlands strain of E. tenella as described by Shirley (1995). To obtain merozoites, 3-week-old birds inoculated by gavage with 2 106 sporulated oocysts of the Redlands strain of E. tenella were sacrificed 108 h post-infection (PI) and their intestines processed for merozoite isolation as described by Shirley (1995). The sporozoites and merozoites were purified on diethylaminoethyl (DEAE) cellulose (DE52, Whatman International Ltd., Maidstone, UK) columns using the method described by Shirley (1995). Purified parasites were disrupted by freezing/thawing five times and sonicating on ice (Sonifier 250, Branson, Danbury, CT, USA). The final suspension was centrifuged at 2000 g for 18 min at 4 8C. The supernatant was collected and the protein concentration determined by measuring the absorbance at 280 nm (BioPhotometer, Eppendorf, Hamburg, Germany). Antigens were stored in 200 ml aliquots at 80 8C until use. 2.4. ELISA Test wells of microtitre plates (MaxiSorp, Nunc, Roskilde, Denmark) were coated overnight, at 4 8C, with 1 mg of sporozoite or merozoite antigen in 100 ml of 0.05 M carbonate buffer (pH 9.6). Control wells were incubated overnight at 4 8C, with 100 ml of 0.05 M carbonate buffer (pH 9.6) only. After washing three times with phosphate-buffered saline (136.8 mM NaCl, 1.47 mM KH2PO4, 8 mM Na2HPO4 and 2.68 mM KCl, pH 7.4, PBS) containing 0.05% Tween 20 (PBST) non-specific binding sites were blocked by incubation with 200 ml of 2% skim milk powder (SMP) in PBST for 1 h at room temperature (RT). After three washes with PBST sera diluted 1:100 in 2% SMP in PBST were added to a test and control well and the plates incubated for 1 h at RT on a shaker. Positive and negative control sera were included on each plate. After incubation with sera the plates were washed five times with PBST and the wells filled with 100 ml of goat anti-chicken IgG peroxidase conjugate (Bethyl Laboratories Inc., Montgomery, TX,
USA) diluted in 1:50,000 in 2% SMP in PBST. After 1 h incubation at RT on a shaker the plates were washed five times and the enzyme reaction developed with 3,30 ,5,50 tetramethylbenzidine (TMB) (KPL, Gaithersburg, MD, USA) for 10 min in the dark. The reaction was stopped by the addition of 100 ml of 2M H3PO4 per well and the absorbance read at 450 nm with an ELISA Microplate Reader (Model 680, Bio-Rad Laboratories, Hercules, CA, USA). All sera were analysed in duplicate. For each serum, the average absorbance of the uncoated wells was then subtracted from the average absorbance of the antigen-coated wells. Serum from a bird infected with E. tenella Redlands strain produced one of the highest absorbances among the infected birds, and was chosen as the positive control. Absorbances of test sera were calculated as a percentage of the absorbance of the positive control and expressed as percent positivity (PP). 2.5. Time course of serum antibodies Two groups of six birds were used in this trial. The birds in the first group were inoculated by gavage, in the crop, with 400 oocysts of E. tenella Redlands strain (the approximate number of oocysts of the same strain found in one dose of Eimeriavax 4M1). The birds in the second group were inoculated with one dose of Eimeriavax 4M1 by placing a drop of vaccine into each chicken’s eye according to the manufacturer’s instructions. The birds were reared for the first 26 days after infection in cages placed in trays to facilitate oocyst recirculation. This treatment was meant to simulate conditions that occur in commercial farms where birds are reared on the floor. Oocyst recirculation was assessed by periodic examination of faeces by the sugar flotation test. Six birds reared in a separate room in cages on trays served as negative controls and were inoculated with distilled water only. All birds were wing bled weekly from 1 week PI for 10 weeks. The blood was allowed to clot for 1 h at RT and then overnight at 4 8C, centrifuged at 800 g for 10 min, aliquoted, and stored at 20 8C until use. 2.6. Cross-reactivity of antibodies to other species of chicken Eimeria Groups of six to eight birds, reared in cages with mesh flooring, were inoculated by gavage in the crop twice at 1month intervals with 5000 sporulated oocysts of each of the Eimeria species shown in Table 1 except the Ingten and Darten strains of E. tenella. Negative control groups of two birds housed under the same conditions, in a separate room were inoculated at the same time with distilled
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water only. Two weeks after the last inoculation all birds were anaesthetised with isoflurane (IsoFlo, Abbott Australasia, Botany, Australia), and blood was collected via cardiac puncture. The birds were then euthanised by cervical dislocation before recovery from anaesthesia. Serum was obtained and stored as described above. 2.7. Sera for ELISA validation Known positive sera were collected from 59 Rhode Island Red/Rhode Island White cross-birds 2 weeks after infection by gavage with attenuated (Redlands) or wild (Ingten and Darten) strains of E. tenella. Birds were inoculated twice at for and 6 weeks of age with 3000–5000 sporulated oocysts. Infection was confirmed by detection of parasites in the faeces. These birds were vaccinated against Marek’s disease (MD) only. Known negative sera were collected from 77 Arbor Acres broilers that were reared in cages with mesh floor in an isolation shed and fed a ration-containing coccidiostat as per industry standard. These birds were vaccinated against infectious bronchitis (IB) only.
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3. Results 3.1. Purity and yield of ELISA antigens The antigen preparations were derived from virtually 100% pure sporozoites and merozoites (Fig. 1a and b). Approximately 65 106 pure merozoites per bird were obtained and about 53–58 106 merozoites (or sporozoites) were processed to obtain one milligram of antigen. Merozoites recovered from one bird supplied enough antigens for testing at least 1000 sera. 3.2. Dynamics of the antibody response The dynamics of the antibody response to merozoite and sporozoite antigens following infection with E. tenella by gavage or vaccination with Eimeriavax 4M1 were compared. The antibody response to merozoite and sporozoite antigens was of similar intensity and followed the same pattern. For both antigens, high antibody levels were detected 2 weeks after the primary
2.8. Sera from commercial farms Sera were collected from 198 free-range pullets that were vaccinated with Eimeriavax 4M1 plus E. brunetti (Table 1). The birds were from two groups in separate sheds on the same farm. At the time of serum collection the pullets were 13 weeks old and they had been vaccinated against coccidiosis twice, at one and 42 days of age. They had also been vaccinated against IB, MD, newcastle disease (ND), fowl pox (FP), egg drop syndrome (EDS), fowl cholera (FC), Mycoplasma synoviae (Ms), Mycoplasma gallisepticum (Mg), avian encephalomyelitis (AE) and infectious laryngotracheitis (ILT). A total of 82 sera were also collected from five farms of free-range layers that were not vaccinated against coccidiosis. These birds were older than 37 weeks when the sera were collected. All free-range birds were maintained on a coccidiostat-free diet and were vaccinated against IB, MD, ND, FP, EDS, FC, Ms and Mg. In addition, 105 sera were collected at slaughter from 6-week-old commercial broilers that were reared on a diet containing a commercial coccidiostat and vaccinated against MD, IB and ND. 2.9. Statistical analyses Estimates of sensitivity and specificity and approximate 95% confidence limits were calculated according to Fleiss (1981).
Fig. 1. (a) Purified sporozoites of E. tenella Redlands strain. (b) Purified merozoites of E. tenella Redlands strain.
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3.4. Sensitivity and specificity of the merozoite antigen ELISA
Fig. 2. Comparison of the time course of the antibody response to sporozoite (~) and merozoite (*) antigens in birds infected with E. tenella by gavage (dotted line) or by eye drop vaccination with Eimeriavax 4M1 (continuous line). The reaction of sera from uninfected control birds with sporozoite (~) and merozoite (*) antigens is also shown. At each time point the arithmetic means and the standard error bars of absorbances of sera collected from six birds are shown.
infection and peaked 1 week later (Fig. 2). The antibody levels then declined slightly before reaching a plateau at 6–7 weeks PI. Control birds maintained under Eimeriafree conditions showed no increase in antibody levels throughout the experiment. 3.3. Cross-reactions of chicken antibodies to other Eimeria species with E. tenella antigens Sera from birds infected by gavage with the seven species of chicken Eimeria were tested in the ELISA using both sporozoite and merozoite antigen preparations (Fig. 3). Reactivity of antibodies with the two antigen preparations was similar. Antibodies in all birds infected with E. tenella or E. necatrix reacted strongly but cross-reactivity of antibodies to other Eimeria species was variable. Antibodies to E. acervulina, E. maxima and E. brunetti did cross-react with E. tenella antigens but the reactivity varied between birds. Sera from birds infected with E. mitis and E. praecox reacted weakly if at all.
Fig. 3. The reactivity of individual sera from birds infected with the seven species of chicken Eimeria with E. tenella sporozoite (S) and merozoite (M) antigen preparations.
Sensitivity and specificity were calculated using only the merozoite antigen because the antibody response to merozoites and sporozoites appeared similar and the merozoite antigen was easier to prepare. The distributions of ELISA PP values for the 59 sera from the birds experimentally infected with different strains of E. tenella and 77 birds reared under Eimeria-free conditions are shown in Fig. 4. There was clear discrimination between uninfected and infected birds. On the basis of these data the positive threshold was arbitrarily set at 10% of the absorbance of the positive control. In these sets of sera the sensitivity estimates of the ELISA were 100% (95% confidence limits: 92.4 and 100) while the specificity estimates were 100% (95% confidence limits: 94.1 and 100). 3.5. Performance of the test in the field The distribution of ELISA PP values for the 385 sera collected from commercial farms is presented in Fig. 5. The distribution for 196 of 198 vaccinated layers that were clearly positive in the ELISA (PP > 10%) resembled that of the birds experimentally infected with E. tenella (Fig. 4). The remaining two birds were clearly negative. The vast majority (97.5%) of 82 freerange layers also tested positive with the ELISA and the distribution of PP values also resembled that of experimentally infected birds. In contrast, only 1 of 105 sera from broilers reared under conditions that minimised exposure to Eimeria spp. and fed rations containing a commercial coccidiostat was positive. The distribution of PP values for the broilers was almost identical with that of cage-reared birds maintained under Eimeria-free conditions (Fig. 4).
Fig. 4. Frequency distribution of ELISA percent positivity (PP) values for sera of known antibody status (59 sera from E. tenella infected birds and 77 sera from uninfected birds).
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Fig. 5. Frequency distribution of ELISA percent positivity (PP) values for sera from 198 pullets vaccinated with Eimeriavax 4M1 plus E. brunetti and from 82 free-range layers and 105 broilers that had not been vaccinated against coccidiosis.
4. Discussion Antibodies raised in birds infected with E. tenella reacted similarly with sporozoite and merozoite antigens, suggesting that most antigens are common to the two-lifecycle stages. The dynamics of the antibody response to merozoite and sporozoite antigens in E. tenella-infected birds was also similar. There was no detectable antibody response at day 7 PI but a strong antibody response to both antigens was present at 14 days PI. Last generation merozoites are present about 4 days after sporozoites so antibodies against sporozoite antigens could be expected to precede antibodies against merozoites. This may have been the case but it was not apparent, probably because of the apparent antigenic similarity between the two stages. Antibody levels peaked at about 21 days after the primary infection then declined slightly before reaching a plateau and persisting at a high level for the duration of the experiment, presumably because of continuous exposure as a result of reinfection through recirculation of oocysts. This is consistent with previous reports that antibodies persist for extended periods in birds reared on the floor (Gilbert et al., 1988; Smith et al., 1993; Guzman et al., 2003). In our field trials high antibody levels were found in pullets 7 weeks after vaccination and in unvaccinated layers older than 37 weeks. The time course of the antibody response in vaccinated birds was similar to that in birds inoculated only with E. tenella, suggesting that the presence of extra species in the vaccine does not interfere with the development of the humoral response to E. tenella. High levels of antibodies were detected even after infections with as few as 400 oocysts, irrespective of the mode of inoculation (gavage or vaccination by eye drop). Most researchers (Mockett and Rose, 1986; Smith et al.,
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1993) used higher doses (10,000 oocysts) in the belief that higher doses result in better exposure and higher antibody titres (Gilbert et al., 1988; Onaga et al., 1989). Antibodies against most species of chicken Eimeria reacted with E. tenella sporozoite and merozoite antigens. These results are consistent with previous research that showed that the invasive stages of chicken Eimeria species share many antigens and that antibodies raised against one species cross-react with the antigens of other species (Sutton et al., 1989; Xie et al., 1992; Uchida et al., 1994; Talebi and Mulcahy, 1995; Shirley et al., 2005). The ELISA is therefore not capable of discriminating E. tenella infection from infections with most other Eimeria species. However this should not be a major drawback to using the test to monitor exposure in chickens following vaccination or natural infections. The strongest cross-reactivity in the ELISA was observed with E. necatrix, E. acervulina and E. maxima (Fig. 3), and these three species are commonly included in commercial vaccines. In commercial flocks, natural exposure to Eimeria is usually multi-species, the most common species being E. tenella, E. acervulina and E. maxima (Shirley et al., 2005), and our ELISA is capable of detecting antibodies in the majority of birds infected with these species. Furthermore, from the standpoint of broiler production, the primary goal is to determine whether or not a particular flock is infected with Eimeria species (Onaga et al., 1986). An ELISA based on an E. tenella antigen may therefore be suitable for monitoring exposure of chickens to Eimeria species either as a result of vaccination of natural infection. The ELISA may therefore prove to be a useful tool for monitoring the immune status of flocks because the development of the humoral response is thought to parallel the development of cell-mediated response. The merozoite antigen ELISA was selected for evaluation in the field because the antigen was easier to prepare. The level of discrimination between birds experimentally infected with E. tenella and uninfected birds was excellent and much higher than in previous reports (Onaga et al., 1989; Guzman et al., 2003). This is likely to reflect the high purity of the parasite preparation used for antigen production (Fig. 1b). Discrimination between sera collected in the field from vaccinated or naturally infected birds and uninfected birds was also excellent. On a commercial farm 99% of pullets were clearly positive when tested in the ELISA 7 weeks after vaccination. The levels of antibodies were high, suggesting that oocysts were recirculating and the birds were likely to be immune. Two birds were serologically negative. As they had been vaccinated twice and there was evidence of oocyst recirculation in
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the shed it is unlikely that they were not infected and it appears likely these individuals failed to mount a detectable antibody response. The intended application of this ELISA is to monitor flocks rather than individual birds so a negative response in such a low proportion of birds should not be a drawback. Most unvaccinated freerange layers were also positive and had uniformly high antibody levels, suggesting constant exposure to Eimeria. In contrast, in a broiler flock fed a dietcontaining coccidiostat only a few birds had low levels of antibody, suggesting little or no exposure. These results are consistent with previous data demonstrating minimal exposure in broiler flocks and uniform exposure of layer flocks (Onaga et al., 1986; Smith et al., 1993). The experimental broiler flock used as negative controls had been vaccinated against IB while the commercial broiler flock we tested had been vaccinated against MD, IB and ND. Both flocks were negative in the ELISA suggesting that there is no cross-reactivity between antibodies produced against these vaccines and E. tenella merozoite antigens. Cross-reactivity with other vaccines used by the poultry industry is possible but unlikely. The ELISA described here should be useful for monitoring exposure in layer flocks as a result of vaccination or natural infection and for monitoring the effectiveness of biosecurity measures in broiler flocks. Acknowledgements Special thanks to Jess Morgan for helpful suggestions while writing the manuscript, David Mayer for statistical analysis of the data, Grant Richards for supplying the vaccine, Tanya Nagle, Rod Jenner, Peter Trappett and Danny Singh for allowing access to flocks, Anthea Bruyeres, Sandy Jarrett, Jan-Maree Hewitson, Ryan O’Neil, Andrew Kelly and Ashley Ostrofsky for able technical assistance. References Allen, P.C., Jenkins, M.C., Miska, K.B., 2005. Cross protection studies with Eimeria maxima strains. Parasitol. Res. 97, 179–185. Anderson, G.R., Jorgensen, W.K., 2004. Live vaccines for three species of Eimeria. A report for the Rural Industries Research and Development Corporation. RIRDC Project DAQ-259J. RIRDC Publication no. 03/143, ACT, Australia. Chapman, H.D., Cherry, T.E., 1997. Eyespray vaccination: infectivity and development of immunity to Eimeria acervulina and Eimeria tenella. J. Appl. Poul. Res. 6, 274–278. Chapman, H.D., Cherry, T.E., Danforth, H.D., Richards, G., Shirley, M.W., Williams, R.B., 2002. Sustainable coccidiosis control in
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Talebi, A., Mulcahy, G., 1995. Cross-reactivity among antisera raised against five avian Eimeria species in the natural host and in rabbits. Avian Pathol. 24, 533–544. Uchida, T., Hasbullah, Nakamura, T., Nakai, Y., Ogimoto, K., 1994. Cross reactivity of serum antibodies from chickens immunized with three Eimerian species. J. Vet. Med. Sci. 56, 1021–1023. Williams, R.B., 2002. Anticoccidial vaccines for broiler chickens: pathways to success. Avian Pathol. 31, 317–353. Xie, M., Gilbert, J.M., McDougald, L.R., 1992. Electrophoretic and immunologic characterization of proteins of merozoites of Eimeria acervulina, E. maxima, E. necatrix, and E. tenella. J. Parasitol. 78, 82–86.