Abstract
The effect of an inactivated vaccine on the horizontal transmission of Mycoplasma gallisepticum was quantified in a transmission model. Twenty non-vaccinated and 20 vaccinated 23-week-old specific pathogen free hens were housed in pairs, while five individually housed hens acted as a negative control group. Each pair consisted of a challenged chicken (104 colony forming units intratracheally) and a non-challenged susceptible contact bird. Infection was monitored by serology, quantitative polymerase chain reaction and culture. All non-vaccinated and vaccinated in-contact chickens became infected with M. gallisepticum. The 95% confidence interval of the reproduction ratio, R (a measure of transmission defined as the average number of secondary cases caused by one infectious individual) was 4.48 to ∞ in both groups. However, the logarithm of the area under the curve in the vaccinated group was 0.51 lower (P=0.02) than in the non-vaccinated group, indicating that there was an effect of vaccination on the levels of potential shedding of M. gallisepticum. Nevertheless, the results of this study indicate that the use of an inactivated M. gallisepticum vaccine will not reduce the horizontal transmission of M. gallisepticum between laying hens.
Effet de la vaccination avec un vaccin inactivé sur la transmission horizontale de Mycoplasma gallisepticum
L'effet d'un vaccin inactivé sur la transmission horizontale de Mycoplasma gallisepticum (Mg) a été quantifié à l'aide d'un modèle expérimental. Des poules SPF, âgées de 23 semaines, 20 vaccinées et 20 non vaccinées, ont été hébergées par paire. Cinq autres poules ont été hébergées individuellement et ont servi de témoin négatif. Chaque paire était constituée d'un sujet éprouvé (104 colonies formant unité [CFU] par voie intra trachéale) et d'un sujet sensible non éprouvé contact. L'infection a été contrôlée par sérologie, amplification en chaîne quantitative (Q-PCR) et culture. Tous les sujets en contact, vaccinés et non vaccinés, ont été infectés par Mg. L'intervalle de confiance 95% du rapport de reproduction, R, qui mesure la transmission, défini comme le nombre moyen de cas secondaires causés par une infection individuelle a été de 4,48 à ∞ dans les deux groupes. Cependant, le logarithme de l'aire sous la courbe (AUC) dans le groupe vacciné a été de 0,51 inférieur à celui du groupe non vacciné (P=0.02). Ceci indique qu'il y a eu un effet de la vaccination sur les niveaux de l'excrétion potentielle de Mg. Néanmoins, les résultats de cette étude indiquent que l'utilisation d'un vaccin Mg inactivé ne réduit pas la transmission de Mg entre les poules pondeuses.
Effekt einer Vakzination mit einem Bakterin auf die horizontale Übertragung von Mycoplasma gallisepticum
Der Effekt einer inaktivierten Vakzine auf die horizontale Übertragung von M. gallisepticum (MG) wurde mit Hilfe eines Übertragungsmodell quantifiziert. 20 nicht vakzinierte und 20 vakzinierte 23-wöchige SPF-Hennen wurden paarweise eingestallt, während fünf einzeln gehaltene Hennen als negative Kontrollen dienten. Jedes hennenpaar bestand aus einem belastungsinfizierten Huhn (104 Kolonie bildende Einheiten (KFU) intratracheal) und einem nicht infizierten, empfänglichen Kontakttier. Die Infektion wurde mittels Serologie, quantitativer Polymerasekettenreaktion (Q-PCR) und Kultivierung überprüft. Alle nicht vakzinierten und vakzinierten Kontakttiere infizierten sich mit MG. Das 95%ige Vertrauensintervall des Reproduktionsverhältnis R als Maß für die Übertragung, definiert als Mittelwert der durch ein infektiöses Individuum verursachten Sekundärfälle, betrug in beiden Gruppen 4,48 bis ∞. Der Logarithmus des unter der Kurve liegenden Bereichs (AUC) war in der vakzinierten Gruppe jedoch 0,51 niedriger (p = 0,02) als in der nicht vakzinierten Gruppe, was auf einen Effekt der Vakzination auf die potentielle Höhe der MG-Ausscheidung hinweist. Nichtsdestoweniger zeigen die Ergebnisse dieser Studie, dass der Einsatz einer inaktivierten MG-Vakzine nicht die horizontale Übertragung von MG zwischen Legehennen reduziert.
Efecto de la vacunación con una bacterina en la transmisión horizontal de Mycoplasma gallisepticum
Se cuantificó el efecto de una vacuna inactivada en la transmisión horizontal de M. gallisepticum (Mg) en un modelo de transmisión. Un total de 20 aves SPF vacunadas y no vacunadas de 23 semanas de edad fueron alojadas en pares, mientras que cinco gallinas alojadas individualmente se utilizaron como grupo control negativo. Cada par consistía en un pollo inoculado experimentalmente (104 unidades formadoras de colonias (CFU) intratraquealmente) y un pollo susceptible, no inoculado, en contacto. La infección fue monitorizada mediante serología, técnica de reacción en cadena de la polimerasa cuantitativa (Q-PCR) y cultivo. Todas los pollos vacunados y no vacunados en contacto resultaron infectados con Mg. El intervalo de confianza del 95% de la ratio de reproducción, R, una medida de la transmisión definida como la media del número de casos secundarios causados por un solo individuo infectado, fue del 4.48 hasta el ∞ en ambos grupos. Aún así, el logaritmo del área bajo la curva (AUC) en el grupo vacunado fue de 0.51‘′o menor (P=0.02) que en el grupo no vacunado, lo que indica que hay un efecto de la vacunación en los niveles de excreción potencial de Mg. No obstante, los resultados de este estudio indican que el uso de esta vacuna inactivada de Mg no reduce la transmisión horizontal de Mg entre gallinas ponedoras.
Introduction
Vaccination is one of the tools used to control Mycoplasma gallisepticum infections. However, although it has been well documented that vaccination may protect against airsacculitis, egg production losses and vertical transmission of M. gallisepticum (Carpenter et al., 1981; Glisson & Kleven, 1985; Sasipreeyajan et al., 1987; Evans et al., 1992), little is known about the ability of vaccination to reduce horizontal transmission. In this study we quantified experimentally the effect of an inactivated vaccine on the horizontal transmission of M. gallisepticum between layer hens. Moreover, by means of quantitative-polymerase chain reaction (Q-PCR) we compared the difference in the potential levels of shedding of M. gallisepticum between vaccinated and non-vaccinated M. gallisepticum-challenged birds.
Materials and Methods
Experimental design
The experimental design was as described in detail by Feberwee et al. (2005b). Briefly, the experiment consisted of a non-vaccinated group, a vaccinated group and a control group, each housed in a separate room. The first two groups each consisted of 20 specific pathogen free White Layers 23 weeks of age, housed in pairs in 10 cages. The cages were a distance of 178 cm from each other. The control group consisted of five birds from the same source housed individually in cages. This group was included to examine the specificity of the test methods. Birds were vaccinated intramuscularly at 16 and 20 weeks of age with a commercially available inactivated vaccine. They were then given 2 weeks of acclimatization time before starting the experiment. After this period one chicken per pair of the non-vaccinated group and the vaccinated group was removed and housed in a negative pressure isolator. These birds were then inoculated intratracheally with 1.0 ml containing 104 colony forming units (CFU) of field strain M. gallisepticum F1999. It has been documented previously that this strain does not induce noticeable clinical signs in specific pathogen free layers (Feberwee et al., 2005b). Twenty-four hours after challenge the infected chickens were reunited with their contact mates. This moment of contact was defined as day 0.
Monitoring, handling and testing
The mycoplasma-free status of the birds was confirmed by serology and PCR before the start of the experiment. Detection of infection was made by Q-PCR, serology (rapid plate agglutination [RPA] and haemagglutination inhibition [HI] test; a positive reaction at dilution ≥1:8 in both tests was regarded as positive) and culture as described previously (Feberwee et al., 2005a b; Mekkes & Feberwee, 2005). At days 2, 3, 4, 5, 6, 7, 9, 11, 14, 21, 25, 28, 31 and 35 after contact, tracheal samples were taken from all chickens and tested for the concentration of M. gallisepticum bacteria by Q-PCR, a method able to predict the number of CFU equivalents in tracheal swabs (Mekkes & Feberwee, 2005). At days 5, 14 and 28 culture was also performed using the tracheal swab from the same five inoculated chickens of both groups. Serology was performed on blood samples taken at days 7, 14, 21, 28 and 35 after inoculation.
Sampling of birds was always performed starting with the negative control group first, then vaccinated chickens and finally to the non-vaccinated group. Within groups the order of sampling of pairs was changed daily in order to prevent the occurrence of systemic errors.
Analysis of data
A measure for the transmission is the reproduction ratio (R), which is defined as the average number of secondary cases caused by one infectious individual. This implies that an infection will fade out in a population when R < 1, but will most probably continue to spread when R > 1. The transmission of M. gallisepticum in the present study was quantified as described earlier (Feberwee et al., 2005b). We compared transmission in the vaccinated group with that in the non-vaccinated group and calculated the 95% confidence interval of R by a maximum likelihood estimator in order to determine whether vaccination results in R < 1.
The effect of vaccination on the potential shedding levels of M. gallisepticum was assessed by calculating the area under the curve (AUC) in both vaccinated and non-vaccinated birds. For the inoculated birds it was performed from days 2 to 31, and for the in-contact birds from days 6 to 35—the start days being when the organism was detected in the inoculated and in-contact birds, respectively. In the statistical model the logarithm of the AUC was analysed as the response, and the independent factors were vaccination (yes/no) and the infection route (inoculated/in-contact) and the interaction between the factors. A linear mixed-effects model with the chicken pair as the random variable was performed in the statistical program R version 2.0.1. (R Development Core Team, 2003).
Results and Discussion
In the negative control group, Q-PCR and serology yielded negative results in the M. gallisepticum RPA and Q-PCR tests 35 days after inoculation, underlining the specificity of the used methods. All vaccinated birds developed an antibody response: before inoculation, all samples were positive in both the RPA and HI tests, 80% of the samples showing titres ≥1:8 in both tests (Table 1). Serology could therefore not be used to detect infection in vaccinated birds. The Q-PCR demonstrated M. gallisepticum in all inoculated and in-contact birds of both the non-vaccinated and the vaccinated groups. However, the vaccinated inoculated birds appeared to clear M. gallisepticum more rapidly from the trachea than the non-vaccinated inoculated birds (Table 1). In both challenged groups, the inoculated and in-contact birds were positive for the first time at days 2 and 6, respectively. At 11 and 14 days after exposure all contact birds (in both vaccinated and non-vaccinated groups) were positive in the Q-PCR (Table 1). In 10 of the non-vaccinated inoculated birds and in nine of the in-contact non-vaccinated birds, infection was also confirmed by a positive serological result. Viable mycoplasmas were detected by culture at days 5, 14 and 28 also in both groups (Table 1).
Table 1. Q-PCR and serology results of the non-vaccinated and vaccinated groups
As all non-vaccinated and vaccinated birds became infected, the 95% confidence interval of R ranged from 4.48 to ∞ in both groups. However, the possibility cannot be completely excluded that the frequent swabbing of the trachea of the birds had a negative effect on the integrity of the tracheal mucosa and thus influenced the calculated R value.
The potential shedding patterns of infected and in-contact animals, based on Q-PCR (Mekkes & Feberwee, 2005), were similar (Figure 1). However, the analysis of the M. gallisepticum levels showed that the logarithm of the AUC in the vaccinated pairs was 0.51 lower (P=0.02) than in the non-vaccinated group, indicating a possible effect of vaccination on shedding.
Published online:
18 January 2007Figure 1. Potential shedding patterns (mean+standard error of the mean) as indicated by Q-PCR of the non-vaccinated (nv) and vaccinated (v) inoculated and in-contact exposed birds, after inoculation of 104 CFU M. gallisepticum (Mg) F1999. The log (AUC) in the vaccinated pairs was 0.51 lower (P = 0.020) than in the non-vaccinated pairs.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2006/cavp20.v035.i01/03079450500465700/20160222/images/medium/cavp_a_146553_o_fig001.gif"})
Figure 1. Potential shedding patterns (mean+standard error of the mean) as indicated by Q-PCR of the non-vaccinated (nv) and vaccinated (v) inoculated and in-contact exposed birds, after inoculation of 104 CFU M. gallisepticum (Mg) F1999. The log (AUC) in the vaccinated pairs was 0.51 lower (P = 0.020) than in the non-vaccinated pairs.
The results of the estimation of R indicate that vaccination with an inactivated M. gallisepticum vaccine at 16 and 20 weeks of age does not reduce the horizontal transmission of M. gallisepticum between laying hens in the present model. Moreover, because R significantly exceeds 1 we found no indication that vaccination may help to halt the spread of M. gallisepticum.
Although this is first report where the horizontal transmission of M. gallisepticum among vaccinated birds has been quantified, the indication of a reduction in M. gallisepticum shedding is in agreement with earlier studies (Hildebrand et al., 1983; Yoder et al., 1984; Kleven, 1985; Talkington & Kleven, 1985; Whithear, 1996). This finding prompted several authors to hypothesize that vaccination might have a long-term effect, eventually stopping horizontal transmission (Talkington & Kleven, 1985; De Nie & Goren, 1989). However, this does not seem very probable considering the small amount of the reduction induced by the vaccination. Also, it is questionable whether under field conditions, especially in high prevalence areas, exposure doses are lower than 1500 CFU—a dose that was demonstrated as infective by Talkington & Kleven (1985) in chickens vaccinated twice with an inactivated vaccine.
| Inoculated (non-vaccinated) | In-contact (non-vaccinated) | Inoculated (vaccinated) | In-contact (vaccinated) | |||||
|---|---|---|---|---|---|---|---|---|
| Q-PCRa | Serologyb | Q-PCR | Serology | Q-PCR | Serology | Q-PCR | Serology | |
| Day –1e | 0 | 0 | 0 | 0 | 0 | 7c | 0 | 9d |
| Day 2f | 3 | – | 0 | – | 4 | – | 0 | – |
| Day 3 | 10 | – | 0 | – | 10 | – | 0 | – |
| Day 4 | 10 | – | 0 | – | 10 | – | 0 | – |
| Day 5 | 10g | – | 0 | – | 10c | – | 0 | – |
| Day 6 | 10 | – | 2 | – | 10 | – | 1 | – |
| Day 7 | 10 | 8 | 3 | 0 | 10 | 9h | 3 | 10 |
| Day 9 | 10 | – | 6 | – | 10 | – | 7 | – |
| Day 11 | 10 | – | 10 | – | 10 | – | 9 | – |
| Day 14 | 10i | 10 | 10 | 0 | 10i | 7j | 10 | 9k |
| Day 21 | 10 | 10 | 10 | 6 | 10 | 10 | 10 | 10 |
| Day 25 | 10 | – | 10 | – | 10 | – | 10 | – |
| Day 28 | 9l | 10 | 10 | 9 | 8m | 10 | 10 | 10 |
| Day 31 | 10 | – | 10 | – | 6 | – | 10 | – |
| Day 35 | 9 | 10 | 10 | 8 | 7 | 10 | 10 | 10 |
| –, not done. | ||||||||
| aNumber of birds positive in the Q-PCR test | ||||||||
| bNumber of birds with a titre of 1:8 or higher in both the RPA and HI tests. | ||||||||
| cAll samples showed an RPA and HI titre due to vaccination; lowest titres were the RPA titre of 1:2 combined with an HI titre of 1:16. | ||||||||
| dAll samples showed a positive RPA and HI titre due to vaccination; one bird showed an RPA titre of 1:2 in combination with an HI titre of 1:8. | ||||||||
| eDay before inoculation. | ||||||||
| fTwo days after inoculation. | ||||||||
| gFive birds were mycoplasma culture-positive. | ||||||||
| hOne sample was not tested as the serum was coagulated. | ||||||||
| iFive birds were mycoplasma culture-positive. | ||||||||
| jThree samples were not tested in the HI test (not enough serum). All three had an RPA titre ≥1:32. | ||||||||
| kOne sample was not tested in the HI test (not enough serum). The RPA titre was 1:16. | ||||||||
| lFour birds were mycoplasma culture-positive, one inoculated bird was Q-PCR and culture-negative. | ||||||||
| mFive birds were mycoplasma culture-negative. One of five birds was negative in the Q-PCR. The Q-PCR counted low M. gallisepticum concentrations (101 to 102 CFU equivalents). | ||||||||
Acknowledgments
This research was supported by a grant from the National Board for Poultry and Eggs (PPE) of The Netherlands. The authors thank Wil Landman for critically reading the manuscript.
References
- Carpenter, T.E., Mallinson, E.T., Miller, K.F., Gentry, R.F. and Schwartz, L.D. 1981. Vaccination with F strain Mycoplasma gallisepticum to reduce production losses in layer chickens. Avian Diseases, 25: 404–409. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- De Nie , S. & Goren E. (1989) . Enting ter preventie van eiproductie-verliezen door Mycoplasma gallisepticum . Dier en Arts , 79 – 81 . [Google Scholar]
- Evans, R.D., Hafez, Y.S. and Orthel, F.W. 1992. Mycoplasma gallisepticum vaccination-challenge: an egg-production model. Avian Diseases, 36: 956–963. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Feberwee, A., Mekkes, D.R., De Wit, J.J., Hartman, E.G. and Pijpers, A. 2005a. Comparison of culture, PCR and different serological tests for detection of Mycoplasma gallisepticum and Mycoplasma synoviae infections. Avian Diseases, 49: 260–268. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Feberwee, A., Mekkes, D.R., Klinkenberg, D., Vernooij, J.C.M., Gielkens, A.L.J. and Stegeman, J.A. 2005b. An experimental model to quantify horizontal transmission of Mycoplasma gallisepticum. Avian Pathology, 34: 355–361. [Taylor & Francis Online], [Web of Science ®], [Google Scholar]
- Glisson, J.R. and Kleven, S.H. 1985. Mycoplasma gallisepticum vaccination: further studies on egg transmission and egg production. Avian Diseases, 29: 408–415. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Hildebrand, D.G., Page, D.E. and Berg, J.R. 1983. Mycoplasma gallisepticum (MG)—laboratory and field studies evaluating the safety and efficacy of an inactivated MG bacterin. Avian Diseases, 27: 792–802. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Kleven, S.H. 1985. Tracheal populations of Mycoplasma gallisepticum after challenge of bacterin-vaccinated chickens. Avian Diseases, 29: 1012–1017. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Mekkes, D.R. and Feberwee, A. 2005. Real-time PCR for the qualitative and quantitative detection of Mycoplasma gallisepticum. Avian Pathology, 34: 348–354. [Taylor & Francis Online], [Web of Science ®], [Google Scholar]
- R Development Core Team . (2003) . R: A Language and Environment for Statistical Computing . Vienna , , Austria : R Foundation for Statistical Computing . [ http://www.R-project.org ]. [Google Scholar]
- Sasipreeyajan, J., Halvorson, D.A. and Newman, J.A. 1987. Effect of Mycoplasma gallisepticum bacterin on egg transmission and egg production. Avian Diseases, 31: 776–781. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Talkington, F.D. and Kleven, S.H. 1985. Evaluation of protection against colonization of the chicken trachea following administration of Mycoplasma gallisepticum bacterin. Avian Diseases, 29: 998–1003. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Whithear, K.G. 1996. Control of avian mycoplasmosis by vaccination. Revue scientifique et technique, 15: 1527–1553. [PubMed], [Web of Science ®], [Google Scholar]
- Yoder, H.W., Hopkins, S.R. and Mitchell, B.W. 1984. Evaluation of inactivated Mycoplasma gallisepticum oil–emulsion bacterins for protection against airsacculitis in broilers. Avian Diseases, 28: 224–234. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]