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Comparison of serological tests for detection of Mycoplasma gallisepticum antibodies in eggs and chicks hatched from experimentally infected hens
Veterinary Microbiology 60 Ž1998. 207–213
Comparison of serological tests for detection of Mycoplasma gallisepticum antibodies in eggs and chicks hatched from experimentally infected hens I. Kempf ) , F. Gesbert Centre National d’Etudes Veterinaires et Alimentaires, Unite´ Mycoplasmologie Bacteriologie, BP 53, Zoopole ´´ ´ Les Croix, 22440 Ploufragan, France Received 7 July 1997; accepted 7 January 1998
Abstract Specific pathogen free hens and males were experimentally infected with Mycoplasma gallisepticum. Eggs were then collected, and a part was incubated and set for hatching. Mycoplasma cultures were performed on infected adults and antibodies to MG were analysed by use of slide agglutination ŽSA. test and commercial ELISA tests on adults and chicks sera and on yolks from non incubated eggs. Both ELISA tests could detect antibodies in yolks from non incubated eggs laid three weeks after infection. SA and the three ELISA tests revealed positive sera in chicks hatched from eggs laid as soon as one week after infection. q 1998 Elsevier Science B.V. Keywords: Mycoplasma gallisepticum; Serology; Yolk; Chick
1. Introduction Mycoplasma gallisepticum ŽMG. is the aetiologic agent of chronic respiratory disease in chickens, resulting in reduced feed conversion, reduced egg production and significant downgrading of carcasses at slaughter ŽYoder, 1991.. Control programmes directed at eradication of the pathogen from breeder flocks are traditionally based on serological testing andror isolation of the organism. Because the main route of spread of MG )
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0378-1135r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 7 8 - 1 1 3 5 Ž 9 8 . 0 0 1 5 3 - 9
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208
I. Kempf, F. Gesbertr Veterinary Microbiology 60 (1998) 207–213
infection is egg transmission, it is highly desirable to eliminate infected breeder flocks. However because such a solution is not always economically feasible, it is important to be able to detect vertical infection in chicks. The present study was conducted in order to compare the efficacy of serological methods including slide agglutination ŽSA. test and different commercial enzyme-linked immunosorbent assay ŽELISA. kits for detection of antibodies in eggs and chicks hatched from MG experimentally infected hens.
2. Materials and methods 2.1. Bacterial strains The M. gallisepticum R-P10 strain ŽKempf et al., 1988a. was used. 2.2. Mycoplasma media FM4 broth and FM4 agar plates were prepared ŽFrey et al., 1968.. Transport medium was 2% buffered peptone water containing glycerin Ž1.2% volrvol. and bacterial growth inhibitor ŽPenicillin G, 1000 units per ml of medium.. 2.3. Infection of birds Forty-eight 57-week-old Specific Pathogen Free hens and six males were obtained from the Experimental Poultry Unit of CNEVA- Ploufragan, France and were reared in strict isolation. They were inoculated intratracheally with the R-P10 strain of M. gallisepticum Ž2 = 10 6 color-changing units per bird.. 2.4. Sampling On days 0, 10, 21, 30, 41, 49, 61 and 146 post-inoculation ŽPI., tracheal swabs were collected from 20 randomly selected hens, placed in transport medium and used for mycoplasma culture. On days 0, 10, 21, 30, 41, 49, 61, 100 and 146 PI, 20 to 46 blood samples were collected from hens for SA and ELISA tests for antibodies to MG. Eggs were collected from five days before challenge until day 133 PI. Five lots of eggs were set for hatching. The first lot comprised 60 eggs laid during the week before inoculation ŽW0., the second lot comprised 217 eggs laid from 0 to 20 days PI ŽW1–W3., the third lot comprised 169 eggs laid from 21 to 41 days PI ŽW4–W6., the fourth comprised 240 eggs laid from 42 to 64 days PI ŽW7–W9. and the fifth lot comprised 240 eggs laid from 65 to 73 days PI ŽW10–W11.. Egg yolk from a part of non incubated and sera from all hatched chicks were collected for MG antibodies analysis. 2.5. Mycoplasma cultures One hundred microliters from each tracheal swab were inoculated in 0.9 ml FM4 broth. Ten m l from each suspension were sown on to FM4 agar. Plates were incubated
I. Kempf, F. Gesbertr Veterinary Microbiology 60 (1998) 207–213
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for 28 days. Broths were incubated at 378C and subcultured on to mycoplasma agar when there was a color change or, in its absence, after one week of incubation. Mycoplasma isolates were identified as M. gallisepticum according to the immuno-binding method on membrane filtration ŽPoumarat et al., 1991.. 2.6. Serological testing SA tests were carried out on sera according to standard procedures ŽKempf et al., 1994.. M. gallisepticum stained antigens were obtained from Intervet International. Sera were first tested undiluted. Positive sera were retested after being heated and diluted 1:5 with phosphate-buffered saline pH 7. Sera that still reacted when diluted were considered positive. Sera that reacted only when undiluted were recorded as suspicious. ELISA tests were conducted on sera using chicken MG ELISA kits ŽProFLOK w . supplied by Kirkegaard and Perry Laboratories ŽKPL, Gaithersburg, MD, USA., MG FlockChek w antibody test kits ŽIdexx France. and, for a few chick sera, MG Svanovire kit ŽSvanova Biotech, Uppsala, Sweden.. ELISA tests were performed and interpreted as directed by their respective manufacturer. Optical densities were read with a Dynatech MR5000 ELISA reader. For KPL tests, a sample to positive ratio value ŽSp. was calculated for each sample by subtracting the average negative control absorbance from each sample absorbance. The difference was divided by the corrected positive control absorbance. Results were interpreted as directed by the manufacturer: negative ŽSp 0.200., suspicious Ž0.200 - Sp - 0.599. or positive Ž0.600 - Sp.. For Idexx tests, the relative level of antibody in each serum was determined by calculating a sample to positive ŽSrP. ratio. If this ratio was less than or equal to 0.5, the sample was considered negative. Sera with SrP ratios greater than 0.5 were considered positive. For the Svanovir blocking enzyme immuno-assay, sera with a percent inhibition ŽPI. greater than 40% were considered positive; sera with a PI between 30% and 40% were considered suspicious and sera with a PI lower or equal to 30% were considered negative. SA tests were performed on chloroformic extracted yolks, according to a previously published method ŽKempf et al., 1988b.. Briefly, all yolk material was removed and thoroughly mixed prior to removal of aliquots. A 0.5 ml sample was diluted with 0.5 ml phosphate-buffered saline pH 7. Two ml chloroform were added and thoroughly mixed and the mixture was stored overnight at room temperature before centrifugation at 250 = g for 20 min. The clear supernatants were collected and were tested by SA test. Extracted yolks which were positive were retested after dilution 1:5 with phosphatebuffered saline. The interpretation was as for sera. For KPL and Idexx ELISA tests, yolks were diluted 1:5 in phosphate buffered saline pH 7 and stored frozen. After thawing, diluted yolks were first diluted 1:10 with the different kit supplied buffers before dilution 1:2, or 1:10 in the ELISA wells for KPL or Idexx respectively. Thus the final dilutions of yolks were, as those of sera, 1:100 and 1:500 for KPL and Idexx tests respectively. 2.7. Data analysis The numbers of positive results according to the different serological tests were analysed using the x 2 test. A significance level of 5% was used.
210
I. Kempf, F. Gesbertr Veterinary Microbiology 60 (1998) 207–213
3. Results 3.1. Experimental infection Respiratory symptoms were observed on infected hens from 3 days after challenge and persisted for approximately ten days. Two hens died from accidental reason. A total of 2088 eggs was produced during the 133 days of the PI observation period. Because there was no non infected control group, the effect of MG challenge on egg production could not be evaluated. Fertility was low and egg mortality was high Ždata not shown., probably because of poor rearing and incubation conditions. 3.2. Mycoplasma cultures The results of mycoplasma cultures on tracheal swabs collected on adult birds are given in Table 1. 3.3. Serology The results of serological tests on hens and males sera are given in Table 1. SA and both ELISA tests yielded positive results as soon as 10 days PI. The numbers of positive sera for SA, KPL or Idexx tests were respectively 272r292 Ž93%., 245r293 Ž84%. and 233r271 Ž86%.. Thus SA test significantly detected more positive birds than ELISA tests. The numbers of positive sera for KPL or Idexx tests did not differ significantly. When non incubated eggs were tested, SA detected significantly less positive yolks Ž236r347, 68%. compared to KPL Ž264r334, 79%. or Idexx Ž268r334, 80%. tests respectively ŽTable 2.. The numbers of positive results for the two ELISA tests were not significantly different. First positive results were obtained on week 7 or 3 PI according
Table 1 Cultures and serological tests on hen sera Day PI
Culture
Serological tests Number tested
SA
KPL
IDEXX
0 10 21 30 41 49 61 100 146
0%a 100% 100% 85% 40% 70% 75% nd 100%d
20,19,19 b 46 46,47,47 46,45,45 45 45,45,33 45,45,33 25 28
0%c 100% 100% 98% 100% 100% 100% 100% 100%
0% 46% 94% 100% 100% 100% 100% 100% 100%
0% 67% 94% 98% 100% 100% 100% 100% 100%
nd: not done. a Percentage of positive cultures Ž20 samples tested.. b Number tested by SA, KPL or Idexx tests respectively Žwhen different.. c Percentage of positive results. d Ten swabs tested on day 146 PI.
I. Kempf, F. Gesbertr Veterinary Microbiology 60 (1998) 207–213
211
Table 2 Serological results on non incubated eggs Week PI
Number tested
SA
KPL
IDEXX
0 1 2 3 4 5 6 7 8 9 10 11 14 15 16 17 18 19 20 21
39 12 10 9 6 3 2 3 18 16 13 6 30 30 30 30 30 30 30,8,8 a 0,9,9
0%b 0% 0% 0% 0% 0% 0% 67% 83% 100% 100% 100% 97% 97% 77% 73% 83% 90% 97% nd
0% 0% 0% 33% 83% 100% 100% 100% 100% 100% 100% 100% 100% 97% 100% 100% 100% 97% 100% 100%
0% 0% 0% 67% 83% 100% 100% 100% 100% 100% 100% 100% 100% 97% 100% 100% 100% 100% 100% 100%
a
Number tested for SA, KPL or Idexx tests respectively Žwhen different.. Percentage of positive results. nd: not done.
b
to SA or both ELISA tests respectively. First suspicious results were obtained on the 5th week according to SA whereas KPL test detected one suspicious yolk as soon as the second week PI Ždata not shown.. When sera from one-day-old chicks were tested according to SA, KPL and Idexx tests, the numbers of positive results were respectively 88r114 Ž77%., 84r114Ž74%., 86r114 Ž75%. and did not differ significantly ŽTable 3.. Sixty-seven of the chick sera were tested with KPL, Idexx and Svanovir test and yielded 39, 40 and 30 positive results respectively Žnot significantly different.. For all tests, first positive sera were observed on chicks hatched from eggs laid one week after infection.
Table 3 Serological tests on chicks Hatch Žweek of lay.
Number tested
SA
KPL
Idexx
1st ŽW0. 2nd ŽW1–W3. 3rd ŽW4–W6. 4th ŽW7–W9. 5th ŽW10–W11.
7 17 21 22 47
0%a 94% 90% 100% 66%
0% 12% 76% 95% 96%
0% 18% 71% 100% 98%
a
Percentage of positive results.
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I. Kempf, F. Gesbertr Veterinary Microbiology 60 (1998) 207–213
4. Discussion Like the young of many other species, the newly hatched chick is relatively immuno-competent, but some protection against pathogens is afforded by the transmission of antibodies from the dam via the egg ŽRose and Orlans, 1981.. Levisohn et al. Ž1985. showed that embryo mortality due to virulent MG was completely blocked in eggs containing maternal antibody to MG but maternal antibody in the chicks confer very little protection against challenge according to Lin and Kleven Ž1984.. Yolk contains IgG and IgM and IgA are absent from the yolk but present in the white. The amounts of IgG in yolk have been reported to be 20–25 mgrml in the hen’s egg ŽRose and Orlans, 1981.. Patterson et al. Ž1962. demonstrated the derivation of IgG from serum and its selective transmission across the follicular epithelium of the ovary to the yolk of the hen’s egg. According to these authors, the pattern of rise and fall of antibodies in yolk is the same as in serum, but is delayed by 5–6 days, the time required for the maturation of the egg. IgG is thought to be transmitted from the yolk to the embryo serum via the vitelline and hepatic portal circulations. Most of the serum IgG present at hatching passes from the yolk sac during the last 5–6 days of embryonation. With increasing age of chick, the detectable antibodies decrease, resulting from catabolism of maternal Ig. Ig M and Ig A derived from oviduct secretions are present in unembryonated white, in 12 and 17 day amniotic fluid, and in the 19 day embryo intestine ŽRose and Orlans, 1981.. According to our serological results, the immunological response of experimentally infected hens was observed as soon as ten days PI Žfirst sampling day. and remained detectable until the end of the experiment. Use of egg yolk serological tests ŽSA, HI tests or ELISA. to detect antibody to MG has already been described by different teams ŽMohammed et al., 1986; Piela et al., 1984; Kempf et al., 1988b. and we decided to evaluate commercially available serological tests for detection of antibodies in eggs from infected hens. Antibodies could be detected much earlier with ELISA tests compared to SA test. This difference may be attributed to the SA test’s ability to detect mainly IgM which are absent from yolk, but we did not attempt to identify the classes of antibody involved in this study. All methods could detect antibodies on sera of one-day-old chicks hatched from the eggs laid as soon as the first week PI, but the number of positive samples according to SA was higher. On the following weeks Žweeks 4–9 PI., large numbers of chicks were found positive according to all methods. Thereafter the number of SA positive chicks seemed to decrease but this tendency could not be confirmed by subsequent sampling since the experiment had to be stopped.
5. Conclusion The results of this experiment on experimentally MG infected hens showed that, when it is not possible to sample breeders, in order to detect a vertical MG infection in hatching eggs, as soon as possible, the method of choice, because of its easiness and precocity, seems to be ELISA detection on yolks from non incubated eggs. Both ELISA and SA can detect MG antibody in one-day-old chicks.
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Acknowledgements The authors thank Y. Morin for skilled technical assistance.
References Frey, M.L., Hanson, R.P., Anderson, D.P., 1968. A medium for isolation of avian mycoplasma. Am. J. Vet. Res. 29, 2163–2171. Kempf, I., Cacou, P.M., Guittet, M., Ollivier, C., Morin, M., L’Hospitalier, Bennejean, G., 1988a. Mycoplasmose a` Mycoplasma gallisepticum: realisation d’un modele role ´ ` experimental; ´ ˆ de l’ammoniac comme facteur d’exacerbation. Avian Pathol. 17, 601–616. Kempf, I., Ollivier, C., Drouin, P., Guittet, M., Bennejean, G., 1988b. Detection des anticorps mycoplas´ miques dans le vitellus d’oeufs non incubes. ´ Rev. Med. ´ Vet. ´ 139, 837–841. Kempf, I., Gesbert, F., Guittet, M., Bennejean, G., Stipkovits, L., 1994. Evaluation of two commercial enzyme-linked immunosorbent assay kits for the detection of Mycoplasma gallisepticum antibodies. Avian Pathol. 23, 329–338. Levisohn, S., Glisson, J.R., Kleven, S.H., 1985. In ovo pathogenicity of Mycoplasma gallisepticum strains in the presence and absence of maternal antibody. Avian Dis. 29, 188–197. Lin, M.Y., Kleven, S.H., 1984. Transferred humoral immunity in chickens to Mycoplasma gallisepticum. Avian Dis. 28, 79–87. Mohammed, H.O., Yamamoto, T.E., Carpenter, T.E., Ortmayer, H.B., 1986. Comparison of egg yolk and serum for the detection of Mycoplasma gallisepticum and M. synoÕiae antibodies by enzyme-linked immunosorbent assay. Avian Dis. 30, 398–408. Patterson, R., Youngner, J.S., Weigle, W.O., Dixon, F.J., 1962. Antibody production and transfer to egg yolk in chickens. J. Immunol. 9, 272–278. Piela, T.H., Gulka, C.M., Yates, V.J., Chang, P.W., 1984. Use of egg yolk serological tests ŽELISA and HI. to detect antibody to Newcastle diseases, infectious bronchitis and Mycoplasma gallisepticum. Avian Dis. 28, 877–883. Poumarat, F., Perrin, B., Longchambon, D., 1991. Identification of ruminant mycoplasmas by dot immunobinding on membrane filtration ŽMF Dot.. Vet. Microbiol. 29, 329–338. Rose, E.M., Orlans, E., 1981. Immunoglobulins in the egg, embryo and young chick. Dev. Comp. Immunol. 5, 15–20. Yoder, H.W., 1991. Mycoplasma gallisepticum infection. In: Calnek, B.W., Beard, C.W., Barnes, H.J., Reid, W.M., Yoder, H.W., Jr. ŽEds.., Diseases of Poultry 9th 3dn. Iowa State Univ. Press, Ames, IA, pp. 198–212.
Comparison of serological tests for detection of Mycoplasma gallisepticum antibodies in eggs and chicks hatched from experimentally infected hens I. Kempf ) , F. Gesbert Centre National d’Etudes Veterinaires et Alimentaires, Unite´ Mycoplasmologie Bacteriologie, BP 53, Zoopole ´´ ´ Les Croix, 22440 Ploufragan, France Received 7 July 1997; accepted 7 January 1998
Abstract Specific pathogen free hens and males were experimentally infected with Mycoplasma gallisepticum. Eggs were then collected, and a part was incubated and set for hatching. Mycoplasma cultures were performed on infected adults and antibodies to MG were analysed by use of slide agglutination ŽSA. test and commercial ELISA tests on adults and chicks sera and on yolks from non incubated eggs. Both ELISA tests could detect antibodies in yolks from non incubated eggs laid three weeks after infection. SA and the three ELISA tests revealed positive sera in chicks hatched from eggs laid as soon as one week after infection. q 1998 Elsevier Science B.V. Keywords: Mycoplasma gallisepticum; Serology; Yolk; Chick
1. Introduction Mycoplasma gallisepticum ŽMG. is the aetiologic agent of chronic respiratory disease in chickens, resulting in reduced feed conversion, reduced egg production and significant downgrading of carcasses at slaughter ŽYoder, 1991.. Control programmes directed at eradication of the pathogen from breeder flocks are traditionally based on serological testing andror isolation of the organism. Because the main route of spread of MG )
Corresponding author. Tel.: [email protected]
q 33-2-96-76-01-05;
fax:
0378-1135r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 7 8 - 1 1 3 5 Ž 9 8 . 0 0 1 5 3 - 9
q 33-2-96-78-68-61;
e-m ail:
208
I. Kempf, F. Gesbertr Veterinary Microbiology 60 (1998) 207–213
infection is egg transmission, it is highly desirable to eliminate infected breeder flocks. However because such a solution is not always economically feasible, it is important to be able to detect vertical infection in chicks. The present study was conducted in order to compare the efficacy of serological methods including slide agglutination ŽSA. test and different commercial enzyme-linked immunosorbent assay ŽELISA. kits for detection of antibodies in eggs and chicks hatched from MG experimentally infected hens.
2. Materials and methods 2.1. Bacterial strains The M. gallisepticum R-P10 strain ŽKempf et al., 1988a. was used. 2.2. Mycoplasma media FM4 broth and FM4 agar plates were prepared ŽFrey et al., 1968.. Transport medium was 2% buffered peptone water containing glycerin Ž1.2% volrvol. and bacterial growth inhibitor ŽPenicillin G, 1000 units per ml of medium.. 2.3. Infection of birds Forty-eight 57-week-old Specific Pathogen Free hens and six males were obtained from the Experimental Poultry Unit of CNEVA- Ploufragan, France and were reared in strict isolation. They were inoculated intratracheally with the R-P10 strain of M. gallisepticum Ž2 = 10 6 color-changing units per bird.. 2.4. Sampling On days 0, 10, 21, 30, 41, 49, 61 and 146 post-inoculation ŽPI., tracheal swabs were collected from 20 randomly selected hens, placed in transport medium and used for mycoplasma culture. On days 0, 10, 21, 30, 41, 49, 61, 100 and 146 PI, 20 to 46 blood samples were collected from hens for SA and ELISA tests for antibodies to MG. Eggs were collected from five days before challenge until day 133 PI. Five lots of eggs were set for hatching. The first lot comprised 60 eggs laid during the week before inoculation ŽW0., the second lot comprised 217 eggs laid from 0 to 20 days PI ŽW1–W3., the third lot comprised 169 eggs laid from 21 to 41 days PI ŽW4–W6., the fourth comprised 240 eggs laid from 42 to 64 days PI ŽW7–W9. and the fifth lot comprised 240 eggs laid from 65 to 73 days PI ŽW10–W11.. Egg yolk from a part of non incubated and sera from all hatched chicks were collected for MG antibodies analysis. 2.5. Mycoplasma cultures One hundred microliters from each tracheal swab were inoculated in 0.9 ml FM4 broth. Ten m l from each suspension were sown on to FM4 agar. Plates were incubated
I. Kempf, F. Gesbertr Veterinary Microbiology 60 (1998) 207–213
209
for 28 days. Broths were incubated at 378C and subcultured on to mycoplasma agar when there was a color change or, in its absence, after one week of incubation. Mycoplasma isolates were identified as M. gallisepticum according to the immuno-binding method on membrane filtration ŽPoumarat et al., 1991.. 2.6. Serological testing SA tests were carried out on sera according to standard procedures ŽKempf et al., 1994.. M. gallisepticum stained antigens were obtained from Intervet International. Sera were first tested undiluted. Positive sera were retested after being heated and diluted 1:5 with phosphate-buffered saline pH 7. Sera that still reacted when diluted were considered positive. Sera that reacted only when undiluted were recorded as suspicious. ELISA tests were conducted on sera using chicken MG ELISA kits ŽProFLOK w . supplied by Kirkegaard and Perry Laboratories ŽKPL, Gaithersburg, MD, USA., MG FlockChek w antibody test kits ŽIdexx France. and, for a few chick sera, MG Svanovire kit ŽSvanova Biotech, Uppsala, Sweden.. ELISA tests were performed and interpreted as directed by their respective manufacturer. Optical densities were read with a Dynatech MR5000 ELISA reader. For KPL tests, a sample to positive ratio value ŽSp. was calculated for each sample by subtracting the average negative control absorbance from each sample absorbance. The difference was divided by the corrected positive control absorbance. Results were interpreted as directed by the manufacturer: negative ŽSp 0.200., suspicious Ž0.200 - Sp - 0.599. or positive Ž0.600 - Sp.. For Idexx tests, the relative level of antibody in each serum was determined by calculating a sample to positive ŽSrP. ratio. If this ratio was less than or equal to 0.5, the sample was considered negative. Sera with SrP ratios greater than 0.5 were considered positive. For the Svanovir blocking enzyme immuno-assay, sera with a percent inhibition ŽPI. greater than 40% were considered positive; sera with a PI between 30% and 40% were considered suspicious and sera with a PI lower or equal to 30% were considered negative. SA tests were performed on chloroformic extracted yolks, according to a previously published method ŽKempf et al., 1988b.. Briefly, all yolk material was removed and thoroughly mixed prior to removal of aliquots. A 0.5 ml sample was diluted with 0.5 ml phosphate-buffered saline pH 7. Two ml chloroform were added and thoroughly mixed and the mixture was stored overnight at room temperature before centrifugation at 250 = g for 20 min. The clear supernatants were collected and were tested by SA test. Extracted yolks which were positive were retested after dilution 1:5 with phosphatebuffered saline. The interpretation was as for sera. For KPL and Idexx ELISA tests, yolks were diluted 1:5 in phosphate buffered saline pH 7 and stored frozen. After thawing, diluted yolks were first diluted 1:10 with the different kit supplied buffers before dilution 1:2, or 1:10 in the ELISA wells for KPL or Idexx respectively. Thus the final dilutions of yolks were, as those of sera, 1:100 and 1:500 for KPL and Idexx tests respectively. 2.7. Data analysis The numbers of positive results according to the different serological tests were analysed using the x 2 test. A significance level of 5% was used.
210
I. Kempf, F. Gesbertr Veterinary Microbiology 60 (1998) 207–213
3. Results 3.1. Experimental infection Respiratory symptoms were observed on infected hens from 3 days after challenge and persisted for approximately ten days. Two hens died from accidental reason. A total of 2088 eggs was produced during the 133 days of the PI observation period. Because there was no non infected control group, the effect of MG challenge on egg production could not be evaluated. Fertility was low and egg mortality was high Ždata not shown., probably because of poor rearing and incubation conditions. 3.2. Mycoplasma cultures The results of mycoplasma cultures on tracheal swabs collected on adult birds are given in Table 1. 3.3. Serology The results of serological tests on hens and males sera are given in Table 1. SA and both ELISA tests yielded positive results as soon as 10 days PI. The numbers of positive sera for SA, KPL or Idexx tests were respectively 272r292 Ž93%., 245r293 Ž84%. and 233r271 Ž86%.. Thus SA test significantly detected more positive birds than ELISA tests. The numbers of positive sera for KPL or Idexx tests did not differ significantly. When non incubated eggs were tested, SA detected significantly less positive yolks Ž236r347, 68%. compared to KPL Ž264r334, 79%. or Idexx Ž268r334, 80%. tests respectively ŽTable 2.. The numbers of positive results for the two ELISA tests were not significantly different. First positive results were obtained on week 7 or 3 PI according
Table 1 Cultures and serological tests on hen sera Day PI
Culture
Serological tests Number tested
SA
KPL
IDEXX
0 10 21 30 41 49 61 100 146
0%a 100% 100% 85% 40% 70% 75% nd 100%d
20,19,19 b 46 46,47,47 46,45,45 45 45,45,33 45,45,33 25 28
0%c 100% 100% 98% 100% 100% 100% 100% 100%
0% 46% 94% 100% 100% 100% 100% 100% 100%
0% 67% 94% 98% 100% 100% 100% 100% 100%
nd: not done. a Percentage of positive cultures Ž20 samples tested.. b Number tested by SA, KPL or Idexx tests respectively Žwhen different.. c Percentage of positive results. d Ten swabs tested on day 146 PI.
I. Kempf, F. Gesbertr Veterinary Microbiology 60 (1998) 207–213
211
Table 2 Serological results on non incubated eggs Week PI
Number tested
SA
KPL
IDEXX
0 1 2 3 4 5 6 7 8 9 10 11 14 15 16 17 18 19 20 21
39 12 10 9 6 3 2 3 18 16 13 6 30 30 30 30 30 30 30,8,8 a 0,9,9
0%b 0% 0% 0% 0% 0% 0% 67% 83% 100% 100% 100% 97% 97% 77% 73% 83% 90% 97% nd
0% 0% 0% 33% 83% 100% 100% 100% 100% 100% 100% 100% 100% 97% 100% 100% 100% 97% 100% 100%
0% 0% 0% 67% 83% 100% 100% 100% 100% 100% 100% 100% 100% 97% 100% 100% 100% 100% 100% 100%
a
Number tested for SA, KPL or Idexx tests respectively Žwhen different.. Percentage of positive results. nd: not done.
b
to SA or both ELISA tests respectively. First suspicious results were obtained on the 5th week according to SA whereas KPL test detected one suspicious yolk as soon as the second week PI Ždata not shown.. When sera from one-day-old chicks were tested according to SA, KPL and Idexx tests, the numbers of positive results were respectively 88r114 Ž77%., 84r114Ž74%., 86r114 Ž75%. and did not differ significantly ŽTable 3.. Sixty-seven of the chick sera were tested with KPL, Idexx and Svanovir test and yielded 39, 40 and 30 positive results respectively Žnot significantly different.. For all tests, first positive sera were observed on chicks hatched from eggs laid one week after infection.
Table 3 Serological tests on chicks Hatch Žweek of lay.
Number tested
SA
KPL
Idexx
1st ŽW0. 2nd ŽW1–W3. 3rd ŽW4–W6. 4th ŽW7–W9. 5th ŽW10–W11.
7 17 21 22 47
0%a 94% 90% 100% 66%
0% 12% 76% 95% 96%
0% 18% 71% 100% 98%
a
Percentage of positive results.
212
I. Kempf, F. Gesbertr Veterinary Microbiology 60 (1998) 207–213
4. Discussion Like the young of many other species, the newly hatched chick is relatively immuno-competent, but some protection against pathogens is afforded by the transmission of antibodies from the dam via the egg ŽRose and Orlans, 1981.. Levisohn et al. Ž1985. showed that embryo mortality due to virulent MG was completely blocked in eggs containing maternal antibody to MG but maternal antibody in the chicks confer very little protection against challenge according to Lin and Kleven Ž1984.. Yolk contains IgG and IgM and IgA are absent from the yolk but present in the white. The amounts of IgG in yolk have been reported to be 20–25 mgrml in the hen’s egg ŽRose and Orlans, 1981.. Patterson et al. Ž1962. demonstrated the derivation of IgG from serum and its selective transmission across the follicular epithelium of the ovary to the yolk of the hen’s egg. According to these authors, the pattern of rise and fall of antibodies in yolk is the same as in serum, but is delayed by 5–6 days, the time required for the maturation of the egg. IgG is thought to be transmitted from the yolk to the embryo serum via the vitelline and hepatic portal circulations. Most of the serum IgG present at hatching passes from the yolk sac during the last 5–6 days of embryonation. With increasing age of chick, the detectable antibodies decrease, resulting from catabolism of maternal Ig. Ig M and Ig A derived from oviduct secretions are present in unembryonated white, in 12 and 17 day amniotic fluid, and in the 19 day embryo intestine ŽRose and Orlans, 1981.. According to our serological results, the immunological response of experimentally infected hens was observed as soon as ten days PI Žfirst sampling day. and remained detectable until the end of the experiment. Use of egg yolk serological tests ŽSA, HI tests or ELISA. to detect antibody to MG has already been described by different teams ŽMohammed et al., 1986; Piela et al., 1984; Kempf et al., 1988b. and we decided to evaluate commercially available serological tests for detection of antibodies in eggs from infected hens. Antibodies could be detected much earlier with ELISA tests compared to SA test. This difference may be attributed to the SA test’s ability to detect mainly IgM which are absent from yolk, but we did not attempt to identify the classes of antibody involved in this study. All methods could detect antibodies on sera of one-day-old chicks hatched from the eggs laid as soon as the first week PI, but the number of positive samples according to SA was higher. On the following weeks Žweeks 4–9 PI., large numbers of chicks were found positive according to all methods. Thereafter the number of SA positive chicks seemed to decrease but this tendency could not be confirmed by subsequent sampling since the experiment had to be stopped.
5. Conclusion The results of this experiment on experimentally MG infected hens showed that, when it is not possible to sample breeders, in order to detect a vertical MG infection in hatching eggs, as soon as possible, the method of choice, because of its easiness and precocity, seems to be ELISA detection on yolks from non incubated eggs. Both ELISA and SA can detect MG antibody in one-day-old chicks.
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Acknowledgements The authors thank Y. Morin for skilled technical assistance.
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