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Field trial of a combined vaccine against caprine contagious agalactia: Humoral immune response in lactating goats
Available online at www.sciencedirect.com
The Veterinary Journal The Veterinary Journal 174 (2007) 610–615 www.elsevier.com/locate/tvjl
Field trial of a combined vaccine against caprine contagious agalactia: Humoral immune response in lactating goats Christian de la Fe a,b,*, Patricia Assunc¸a˜o a, Pedro Saavedra a, Ana Ramı´rez a, Jose´ B. Poveda a a
Unidad de Epidemiologı´a y Medicina Preventiva, Facultad de Veterinaria, Universidad de Las Palmas, Trasmontan˜a s/n, 35416 Arucas, Las Palmas, Spain b Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30100 Murcia, Spain Accepted 25 October 2006
Abstract Two vaccines against Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (large colony type) were developed using inactivated strains selected in previous characterization studies. Formaldehyde and phenol were used as the inactivating agents for vaccines A and B, respectively. Aluminium hydroxide plus purified saponin (Quil-A) were added to both vaccines as adjuvant. The field trial was designed to evaluate the specific humoral immune response to the two mycoplasma species in lactating goats over a period of 7 months. The vaccines were tested on 120 goats randomly assigned to three groups of 40 animals each. Two groups received two injections of vaccine A or B respectively, and a third group remained in the herd as control. Antibody titres determined by ELISA indicated a significant difference between both vaccines and the control group over a 6-month period. Immunoblotting assays also revealed the production of antibodies against the two mycoplasma species. Further field trials are underway to evaluate the efficacy and protection conferred to the animals by these specific antibodies. 2006 Elsevier Ltd. All rights reserved. Keywords: Caprine contagious agalactia; Combined vaccine; Humoral immune response; Field trial
1. Introduction Contagious agalactia (CA) is among the most significant diseases affecting small ruminants, and is regarded by health authorities as endemic in most Mediterranean countries. In sheep and goats, Mycoplasma agalactiae (M. agalactiae), considered the ‘‘classic’’ agent of this disease, and Mycoplasma mycoides subsp. mycoides Large Colony type (Mmm LC) are the main causative agents of the syndrome (Nicholas, 1995; Bergonier et al., 1997). Prophylactic strategies to control CA have mainly involved the use of inactivated vaccines against M. agalac*
Corresponding author. Present address: Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30100 Murcia, Spain. Tel.: +34 968367259; fax: +34 968364147. E-mail address: [email protected] (C. de la Fe). 1090-0233/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2006.10.021
tiae. However, the real efficacy of this type of vaccine has been questioned, especially under field conditions (Nicholas, 1995; Bergonier et al., 1997). Moreover, results obtained using caprine CA (CCA) vaccines in experimental conditions seem not to be representative of their performance in the field, and so far there is little information in the literature on field trials designed to assess CCA vaccines. The reason that a number of vaccines fail may be attributed to the high degree of antigenic variability observed in field strains of M. agalactiae and Mmm LC (Solsona et al., 1996; Tola et al., 1996; de la Fe et al., 2006). In addition, goat herds in which commercial M. agalactiae vaccines have been ineffective indicate that, in addition to antigenic variability, there is also high aetiological variation (Villalba et al., 1991). Accordingly, the use of combined vaccines in CCA endemic areas has been proposed to control the disease (Consenti and Montagna, 1989).
C. de la Fe et al. / The Veterinary Journal 174 (2007) 610–615
Recent research efforts have explored the possibility of improving inactivated CCA vaccines. Thus, it has been reported that some adjuvants can enhance the Th1 and Th2 response shown by mice injected with an inactivated M. agalactiae vaccine (Avramidis et al., 2002). In a further study, the use of low phenol or formaldehyde concentrations was effective at inactivating all the causative mycoplasma species of CCA (de la Fe et al., 2004). These low levels of inactivating agents seem to improve antigen preservation. The present study was designed to evaluate the humoral immune response produced in lactating goats injected with two polyvalent vaccines against M. agalactiae and Mmm LC under field conditions. The vaccines we tested were prepared using killed field strains of both mycoplasma species selected according to their previously published protein and antigenic profiles (de la Fe et al., 2006). The vaccines differed in terms of the agents used to inactivate the mycoplasma strains. The idea was to try to overcome the aetiological variation of CCA and the high degree of antigenic variability of its causative microorganisms by minimizing the antigenic differences that could exist between the vaccine’s strains and field strains.
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Potential side effects of the vaccines were assessed by the intraperitoneal administration of 1 mL of each vaccine to 10 mice and by subcutaneous inoculation of 10 mL of the vaccines in two goats. 2.2. Herd The field trial was carried out on a CCA-free herd on a farm in Ingenio, Gran Canaria, Spain, over a 7-month period. The herd comprised 725 lactating Canary dairy goats managed under a semi-intensive production system. To confirm the herd was CCA free, before the trial 100 animals were randomly selected and checked for the presence of Mycoplasma spp. Milk samples and auricular swabs were cultured and blood samples were also taken for indirect ELISA and immunoblotting to detect specific antibodies against M. agalactiae and Mmm LC. We then selected 120 lactating goats, 2–3 years old, from the main herd for the trial. None of the management conditions applied to the herd before the trial was modified during the trial. No antibiotic therapy or other specific treatment against mycoplasma was administered before or during the trial. 2.3. Experimental design
2. Materials and methods 2.1. Strains and formulation of the vaccines The field strains used were M. agalactiae (L9, AGIN3, 9B) and Mmm LC (AG1, 153/93, IN3) isolated from local clinical cases of CCA. The vaccines were prepared using cells of these strains cultured in modified pH culture medium (Kirchhoff and Rosengarten, 1984) supplemented with 10% horse serum (BioWhitaker) at 37 C until the mid-log phase. The cells were then centrifuged at 20,000g for 30 min at 4 C and resuspended in saline (0.9% NaCl; pH 7.2); this process was repeated three times. Colony forming units (CFU)/mL (Albers and Fletcher, 1982) were determined and each 2 mL dose was prepared to contain more than 5 · 1010 cfu/ mL of each mycoplasma species. Washed cultures containing pooled mycoplasma were stored at 20 C. Aluminium hydroxide (25%, vol/vol, Superfos A/S) plus purified saponin (0.5 mg/dose, Quil-A, Superfos A/S) were added as adjuvant, and the vaccines were then incubated with constant rocking for 2 h at 37 C. Vaccines A and B were inactivated with formaldehyde (0.1%, vol/vol, Sigma–Aldrich, for 16 h at 37 C) or phenol (0.5%, vol/ vol, Sigma–Aldrich, for 16 h at 37 C), respectively, and stored at 4 C. Samples of the vaccine were checked for sterility and side effects. For sterility testing, 200 lL aliquots of each vaccine lot were inoculated on solid and in liquid pH culture media (Kirchhoff and Rosengarten, 1984) and incubated at 37 C. A general medium for microbiology studies (blood agar) was also inoculated. At the end of the incubation period, the culture media were examined microscopically for the presence of mycoplasma or other types of colonies.
One hundred and twenty lactating goats were randomly assigned to three groups of 40 goats. One group was vaccinated with vaccine A, another received vaccine B, and a third group (C) remained as controls. The goats received two subcutaneous injections (2 mL) on Day 0 and 25 of the experiment. The animals were monitored over a 7month period, and blood samples were withdrawn from all goats at 0, 25, 45, 100, 150, 180 and 210 days. Serum samples were then tested for IgG antibodies against M. agalactiae and Mmm LC by indirect ELISA. Subsequently, a pool of ELISA-positive sera from each group was analysed by immunoblotting to detect the presence of specific antibodies to these two mycoplasma species. 2.4. Clinical signs During the trial, all cases of mastitis, conjunctivitis, arthritis or abortion were examined for Mycoplasma spp. To perform the microbiological tests, specimens of corresponding tissue or fluid were plated on pH medium, as previously described (de la Fe et al., 2005). At the end of the trial, 100 milk samples were also analyzed for the presence of Mycoplasma spp. by PCR. DNA was extracted from milk samples as previously described (Tola et al., 1997b). Specific primers were used to identify mycoplasma species (Hotzel et al., 1996; Tola et al., 1997b; Peyraud et al., 2003). 2.5. ELISA test and statistical analysis Sera were immediately prepared from each blood sample and kept at 20 C at the Veterinary Faculty of Las Palmas University (ULPGC), Arucas, Spain. Anti-M. aga-
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lactiae and anti-Mmm LC antibody levels, expressed as optical densities (OD), were determined by a previously described ELISA procedure. This method showed 99% sensitivity and 78% specificity for M. agalactiae, and 100% sensitivity and 84% specificity for Mmm LC (Assunc¸a˜o et al., 2004). To analyse the results obtained statistically, we considered the effects of the treatment, observation day, the interaction of both these effects, the random effect of the experimental unit and the residual effect, which were all assumed to be normally distributed. The model was estimated by the method of maximum likelihood. The significance of the effects was tested by the corresponding F-test. When the treatment effect was found to be significant, Scheffe’s multiple comparisons were performed. The level of statistical significance was set at P < 0.05. All statistical tests were performed using SPSS software (version 11.5) for Windows.
The ELISA results are provided in Figs. 1 and 2. The results indicate a steady increase in the production of specific IgG antibodies against M. agalactiae and Mmm LC in both groups of vaccinated animals, while control animals preserved their initial antibody levels. In both groups, A
2.6. SDS-PAGE and immunoblotting The presence of specific antibodies against M. agalactiae and Mmm LC was determined in vaccinated and control animals by SDS-PAGE and immunoblotting. First the protein contents of each mycoplasma species were determined using a kit (Sigma, Microprotein-PR) according to the manufacturer’s instructions. Samples were mixed with lysis buffer (500 mM Tris/HCl pH 6.8, 4.6% (w/v) SDS, 20% (v/ v) glycerol, 10% (v/v) 2-mercaptoethanol and 0.004% bromphenol blue) and boiled for 5 min. Next, 16 lg protein of each species were separated by SDS-PAGE on a 1.5 mm thick, 4% stacking gel and 10% resolving gel. Electrophoresis was performed as previously described (Laemmli, 1970). Separated proteins were transferred to 0.45 lm nitrocellulose membranes at 70 V constant voltage for 90 min (Towbin et al., 1979). The membranes were blocked in PBS containing 1 M glycine, 1% (v/v) egg albumin and 5% skimmed milk for 2 h at room temperature (RT), and then washed three times in PBS for 15 min. The membranes were incubated with the pooled serum samples from vaccinated or control goats at 37 C for 2 h, washed three times in PBS at room temperature and then incubated with the appropriate dilution of peroxidase conjugated anti-rabbit IgG (Sigma, A0545) in dilution buffer (1:2000). After washing, the substrate comprising 30 mg 4-chloro-1-naphtol dissolved in 10 mL methanol plus 50 mL PBS and 30 lL H2O2 was added to the membranes which were then left for 5–15 min in the dark. The labelling reaction was stopped with distilled water. 3. Results Sterility tests were negative. None of the vaccinated mice died and the goats that received two doses of the vaccines showed no reaction after vaccination. All the microbiological and serological tests performed before the trial were also negative.
Fig. 1. Specific immune response to M. agalactiae recorded in vaccinated (groups A and B) and non-vaccinated (controls) lactating goats at 0, 25, 45, 100, 150, 180 and 210 days post-vaccination determined by indirect ELISA. The vertical axis indicates antibody levels expressed as optical densities (OD).
Fig. 2. Specific immune response to Mmm LC recorded in vaccinated (groups A and B) and non-vaccinated (controls) lactating goats at 0, 25, 45, 100, 150, 180 and 210 days post-vaccination determined by indirect ELISA. The vertical axis indicates antibody levels expressed as optical densities (OD).
Table 1 Statistical analysis of the serological results recorded in lactating goats Model effects
Vaccine Period Interaction Animal Residual effect Total effect
M. agalactiae
Mmm LC
Sum of squares
F-test (P-value)
Sum of squares
F-test (P-value)
11.912 12.931 6.806 0.378 1.810 33.837
<0.001 <0.001 <0.001
17.277 13.033 7.036 0.783 3.837 41.966
<0.001 <0.001 <0.001
Significance of the effects of the mathematical model.
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Table 2 Statistical comparisons among the different treatments (Scheffe’s test) M. agalactiae
Scheffe’s test
Mmm LC
A–B
A–CT
B–CT
A–B
A–CT
B–CT
NS
<0.001
<0.001
<0.001
<0.001
<0.001
NS <0.001 <0.001 <0.001 <0.001 <0.001 0.002
NS <0.001 <0.001 <0.001 <0.001 <0.001 <0.022
NS NS NS 0.018 0.003 <0.001 NS
NS <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
NS <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
Experiment day 0 NS 25 NS 45 NS 100 NS 150 0.056 180 NS 210 NS
A: vaccine A; B: vaccine B; CT: control group; NS: not statistically significant.
and B, serum IgG levels peaked in the second month (45 days), and thereafter remained stable until Day 180. On Day 210, similar antibody levels were recorded in the vaccinated and non-vaccinated animals. When the effects of treatment, observation day and interaction between these factors were examined (Table 1), significant differences between groups A and B also emerged from Days 100–180 of the experiment (Table 2). Low levels of Mmm LC antibodies were observed in animals receiving vaccine B. Positive sera taken from both groups of vaccinated animals between Day 25 and Day 180 were analysed by immunoblotting (Figs. 3 and 4). Vaccinated animal sera indicated the presence of different pro-
Fig. 4. Immunoblotting profile of M. agalactiae (A) and Mmm LC (B) whole cell proteins obtained using serum obtained from goats experimentally vaccinated with vaccine B (16 lL protein loaded per track) at 25 days (track 1), 45 days (track 2), 100 days (track 3), 150 days (track 4) and 180 days (track 5). Molecular weights are indicated in kDa and correspond to molecular weight markers (left) and to the antigens detected (right).
teins of molecular weights 42–50 kDa, or 60–75 kDa in each mycoplasma species. Moreover, M. agalactiae or Mmm LC rendered specific bands of 24–25 kDa and 15 kDa, respectively. These results confirm the production of specific antibodies against antigens common to these mycoplasma species, as well as antibodies specific to each species. Finally, only a few cases (n = 10) of clinical mastitis were detected in the herd during the trial. Staphylococcus spp. was identified as the causal agent. All mycoplasma analyses were negative. No other clinical sign associated with CCA was observed, and the absence of mycoplasma in milk samples was confirmed by PCR. 4. Discussion
Fig. 3. Immunoblotting profiles of M. agalactiae (A) and Mmm LC (B) whole cell protein contents obtained using serum collected from goats experimentally vaccinated with vaccine A (16 lL protein loaded per track) at 25 days (track 1), 45 days (track 2), 100 days (track 3), 150 days (track 4) and 180 days (track 5). Molecular weights are indicated in kDa and correspond to molecular weight markers (left) and to the antigens detected (right).
The efficacy of inactivated CCA vaccines has always raised many doubts, especially in field conditions (Fleury et al., 2002). However, the literature lacks information on field trials and most existing data have been obtained under experimental conditions. The situation is further complicated by the fact that many outbreaks in CCA endemic areas are of mixed aetiology (de la Fe et al., 2005). Despite this, most vaccination trials have tested monovalent M. agalactiae vaccines, which are ineffective against the other mycoplasma species involved in the syndrome (Villalba et al., 1991). The use of combined vaccines to control more than one mycoplasma species in endemic areas has been previously
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proposed (Consenti and Montagna, 1989) and these have been used in some areas of Portugal (Regalla, 1987). The present field trial is the first report of a study of the humoral immune response induced in goats in field conditions by a combined CCA vaccine containing M. agalactiae and Mmm LC strains selected according to their antigenic profiles determined by molecular procedures (de la Fe et al., 2006). The use of a pool of field strains could produce a more efficient vaccine since reference strains lack many of the antigens shown by field strains (Gummelt et al., 1996; de la Fe et al., 2006). Hence, the use of a pool of field strains could reduce the number of vaccines that fail because of the antigenic variability of M. agalactiae and Mmm LC (Tola et al., 1996; de la Fe et al., 2006). Both the vaccines tested here induced a specific humoral immune response against M. agalactiae and Mmm LC in lactating goats that lasted for 6 months. This finding is consistent with the results of published M. agalactiae vaccine trials (Regalla, 1987; Leo´n Vizcaı´no et al., 1995). Other authors have reported a shorter period of immunity (Buonavoglia et al., 1998). The use of purified saponin (Quil-A) improved the immunogenicity of the aluminium adjuvant, avoiding the use of oil components (Rurangirwa et al., 1987). With the combined vaccines, antibodies peaked at a similar time to that found when a monovalent vaccine is administered (Foggie et al., 1971; Buonavoglia et al., 1998). Tola et al. (1999), however, recorded the antibody peak at 3 months. The better serological results obtained here with the phenol-inactivated vaccine can be explained by the minimal damage produced by this agent to the immunogenic proteins of these mycoplasma, as previously described (Tola et al., 1999; de la Fe et al., 2004). Immunoblotting results confirmed the production of specific antibodies against both mycoplasma species, and indicated that several species may be included in a combined CCA vaccine. The main immunogenic proteins of M. agalactiae were between 40 and 90 kDa, in agreement with previous observations (Tola et al., 1997a). There is no similar information available for Mmm LC, although, for example, we detected specific antibodies against a 62 kDa protein. This band seems to be a common specific antigen to the two closely related mycoplasma, Mmm LC and M. mycoides subsp. capri (Monnerat et al., 1999). In summary, the results of our field trial indicate that a polyvalent vaccine against M. agalactiae and Mmm LC can induce a humoral immune response in lactating goats that persists for 6 months. To prepare this type of vaccine, preliminary characterization studies are needed to select the best strains. We are presently engaged in further studies designed to establish the protection level conferred by these specific antibodies. Acknowledgements The authors thank the Vet Med Nelo Pascual (ADS Ingenio) for his technical assistance. This study was sup-
ported by Project AGF99-0717-C02-02 of the Plan Nacional I+D, Ministerio de Educacio´n y Cultura, Spain. References Albers, A.C., Fletcher, R.D., 1982. Simple method for quantitation of viable mycoplasmas. Applied and Environmental Microbiology 43, 958–960. Assunc¸a˜o, P., De La Fe, C., Ramı´rez, A., Andrada, M., Poveda, J.B., 2004. Serological study of contagious agalactia in herds of goats in the Canary Islands. Veterinary Record 154, 684–687. Avramidis, N., Victoratos, P., Yiangou, M., Hadjipetrou-Kourounakis, L., 2002. Adjuvant regulation of cytokine profile and antibody isotype of immune responses to Mycoplasma agalactiae in mice. Veterinary Microbiology 88, 325–338. Bergonier, D., Berthelot, X., Poumarat, F., 1997. Contagious agalactia of small ruminants: current knowledge concerning epidemiology, diagnosis and control. Revue Scientifique et Technique (OIE) 16, 848–873. Buonavoglia, D., Fasanella, A., Sagazio, P., Tempesta, M., Iovane, G., Buonavoglia, C., 1998. Persistence of antibodies to Mycoplasma agalactiae in vaccinated sheep. Microbiologica 21, 209–212. Consenti, B., Montagna, C.O., 1989. Profilassi dell’agalassia contagiosa degli ovi-caprini. Obiettivi Veterinari 5, 31–33. de la Fe, C., Assunc¸ao, P., Ramirez, A.S., Poveda, J.B., 2004. Inactivation assays of mycoplasma species involved in contagious agalactia. Berliner und Mu¨nchener Tiera¨rztliche Wochenschrift 117, 1–5. de la Fe, C., Assunc¸a˜o, P., Antunes, T., Rosales, R.S., Poveda, J.B., 2005. Microbiological survey for Mycoplasma spp. in a contagious agalactia endemic area. The Veterinary Journal 170, 257–259. de la Fe, C., Assunc¸a˜o, P., Antunes, T., Rosales, R.S., Poveda, J.B., 2006. Protein and antigen variability among Mycoplasma mycoides subsp. mycoides (LC) and Mycoplasma agalactiae field strains by SDS-PAGE and Immunoblotting. The Veterinary Journal 171, 532–538. Fleury, B., Bergonier, D., Berthelot, X., Peterhans, E., Frey, J., Vilei, E.M., 2002. Characterization of P40, a cytadhesin of Mycoplasma agalactiae. Infection and Inmunity 70, 5612–5621. Foggie, A., Etheridge, J.R., Erdag, O., Arisoy, F., 1971. Contagious agalactia of sheep and goats studies on live and dead vaccines in lactating sheep. Journal of Comparative Pathology 81, 165–172. Gummelt, I., Hotzel, H., Runge, M., Kirchhoff, H., 1996. Taxonomic relationship between Mycoplasma bovis and Mycoplasma agalactiae. In: Frey, J., Sarris, K. (Eds.), COST 826 Agriculture and biotechnology, Mycoplasmas of Ruminants: Pathogenicity, Diagnostics, Epidemiology and Molecular Genetics, vol. I. Office for Official Publications of the European Communities, Brussels, Belgium, pp. 27–29. Hotzel, H., Sachse, K., Pfu¨tzner, H., 1996. A PCR scheme for differentiation of organisms belonging to the Mycoplasma mycoides cluster. Veterinary Microbiology 49, 31–43. Kirchhoff, H., Rosengarten, R., 1984. Isolation of a motile mycoplasma from fish. Journal of General Microbiology 130, 2439–2445. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 22, 680–685. Leo´n Vizcaı´no, L., Garrido Abella´n, F., Cubero Pablo, M.J., Perales, A., 1995. Immunoprophylaxis of caprine contagious agalaxia due to Mycoplasma agalactiae with an inactivated vaccine. Veterinary Record 137, 266–269. Monnerat, M.P., Thiaucourt, F., Poveda, J., Nicolet, J., Frey, J., 1999. Genetic and serological analysis of lipoprotein LppA in Mycoplasma mycoides subsp. mycoides LC and Mycoplasma mycoides subsp. capri.. Clinical and Diagnostic Laboratory Immunology 6, 224–230. Nicholas, R., 1995. Contagious agalactia. State Veterinary Journal 5, 13– 15. Peyraud, A., Woubit, S., Poveda, J.B., De La Fe, C., Mercier, P., Thiaucourt, F., 2003. A specific PCR for the detection of Mycoplasma putrefaciens. Molecular and Cellular Probes 17, 289–294. Regalla, J., 1987. Notes on the contagious agalactia syndrome in Portugal. In: Jones G.R. (Eds.), Agriculture: Contagious Agalactia and Other
C. de la Fe et al. / The Veterinary Journal 174 (2007) 610–615 Mycoplasmal Diseases of Small Ruminants, European Communities, Luxembourg, pp. 7–10. Rurangirwa, F.R., Mcguire, T.C., Kibor, Chema S., 1987. An inactivated vaccine for contagious caprine pleuropneumonia. Veterinary Record 121, 397–402. Solsona, M., Lambert, M., Poumarat, F., 1996. Genomic, protein homogeneity and antigenic variability of Mycoplasma agalactiae. Veterinary Microbiology 50, 45–58. Tola, S., Idini, G., Manunta, D., Casciano, I., Rocchigiani, A.M., Angioi, P.P., Leori, G., 1996. Comparison of Mycoplasma agalactiae isolates by pulsed field gel electrophoresis, SDS-PAGE and immunoblotting. FEMS Microbiology Letters 143, 259–265. Tola, S., Manunta, D., Cocco, M., Turrini, F., Rocchigiani, A.M., Idini, G., Angioi, P.P., Leori, G., 1997a. Characterization of membrane surface proteins of Mycoplasma agalactiae during natural infection. FEMS Microbiology Letters 154, 355–362.
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Tola, S., Angioi, P.P., Rocchigiani, A.M., Idini, G., Manunta, D., Galleri, G., Leori, G., 1997b. Detection of Mycoplasma agalactiae in sheep milk samples by polymerase chain reaction. Veterinary Microbiology 54, 17–22. Tola, S., Manunta, D., Rocca, S., Rocchigiani, A.M., Idini, G., Angioi, P.P., Leori, G., 1999. Experimental vaccination against Mycoplasma agalactiae using different inactivated vaccines. Vaccine 17, 2764–2768. Towbin, S.T., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrilamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America 76, 4350–4354. Villalba, E.J., Poveda, J.B., Garrido, A., Garrido, F., Ferna´ndez, A., 1991. Inmunizacio´n de cabras contra Mycoplasma mycoides subsp. mycoides (LC) en un caso de agalaxia persistente. Report of XIV Jornadas Cientı´ficas de la Sociedad Espan˜ola de Ovinotecnia y Caprinotecnia, pp. 377–381.
The Veterinary Journal The Veterinary Journal 174 (2007) 610–615 www.elsevier.com/locate/tvjl
Field trial of a combined vaccine against caprine contagious agalactia: Humoral immune response in lactating goats Christian de la Fe a,b,*, Patricia Assunc¸a˜o a, Pedro Saavedra a, Ana Ramı´rez a, Jose´ B. Poveda a a
Unidad de Epidemiologı´a y Medicina Preventiva, Facultad de Veterinaria, Universidad de Las Palmas, Trasmontan˜a s/n, 35416 Arucas, Las Palmas, Spain b Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30100 Murcia, Spain Accepted 25 October 2006
Abstract Two vaccines against Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (large colony type) were developed using inactivated strains selected in previous characterization studies. Formaldehyde and phenol were used as the inactivating agents for vaccines A and B, respectively. Aluminium hydroxide plus purified saponin (Quil-A) were added to both vaccines as adjuvant. The field trial was designed to evaluate the specific humoral immune response to the two mycoplasma species in lactating goats over a period of 7 months. The vaccines were tested on 120 goats randomly assigned to three groups of 40 animals each. Two groups received two injections of vaccine A or B respectively, and a third group remained in the herd as control. Antibody titres determined by ELISA indicated a significant difference between both vaccines and the control group over a 6-month period. Immunoblotting assays also revealed the production of antibodies against the two mycoplasma species. Further field trials are underway to evaluate the efficacy and protection conferred to the animals by these specific antibodies. 2006 Elsevier Ltd. All rights reserved. Keywords: Caprine contagious agalactia; Combined vaccine; Humoral immune response; Field trial
1. Introduction Contagious agalactia (CA) is among the most significant diseases affecting small ruminants, and is regarded by health authorities as endemic in most Mediterranean countries. In sheep and goats, Mycoplasma agalactiae (M. agalactiae), considered the ‘‘classic’’ agent of this disease, and Mycoplasma mycoides subsp. mycoides Large Colony type (Mmm LC) are the main causative agents of the syndrome (Nicholas, 1995; Bergonier et al., 1997). Prophylactic strategies to control CA have mainly involved the use of inactivated vaccines against M. agalac*
Corresponding author. Present address: Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30100 Murcia, Spain. Tel.: +34 968367259; fax: +34 968364147. E-mail address: [email protected] (C. de la Fe). 1090-0233/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2006.10.021
tiae. However, the real efficacy of this type of vaccine has been questioned, especially under field conditions (Nicholas, 1995; Bergonier et al., 1997). Moreover, results obtained using caprine CA (CCA) vaccines in experimental conditions seem not to be representative of their performance in the field, and so far there is little information in the literature on field trials designed to assess CCA vaccines. The reason that a number of vaccines fail may be attributed to the high degree of antigenic variability observed in field strains of M. agalactiae and Mmm LC (Solsona et al., 1996; Tola et al., 1996; de la Fe et al., 2006). In addition, goat herds in which commercial M. agalactiae vaccines have been ineffective indicate that, in addition to antigenic variability, there is also high aetiological variation (Villalba et al., 1991). Accordingly, the use of combined vaccines in CCA endemic areas has been proposed to control the disease (Consenti and Montagna, 1989).
C. de la Fe et al. / The Veterinary Journal 174 (2007) 610–615
Recent research efforts have explored the possibility of improving inactivated CCA vaccines. Thus, it has been reported that some adjuvants can enhance the Th1 and Th2 response shown by mice injected with an inactivated M. agalactiae vaccine (Avramidis et al., 2002). In a further study, the use of low phenol or formaldehyde concentrations was effective at inactivating all the causative mycoplasma species of CCA (de la Fe et al., 2004). These low levels of inactivating agents seem to improve antigen preservation. The present study was designed to evaluate the humoral immune response produced in lactating goats injected with two polyvalent vaccines against M. agalactiae and Mmm LC under field conditions. The vaccines we tested were prepared using killed field strains of both mycoplasma species selected according to their previously published protein and antigenic profiles (de la Fe et al., 2006). The vaccines differed in terms of the agents used to inactivate the mycoplasma strains. The idea was to try to overcome the aetiological variation of CCA and the high degree of antigenic variability of its causative microorganisms by minimizing the antigenic differences that could exist between the vaccine’s strains and field strains.
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Potential side effects of the vaccines were assessed by the intraperitoneal administration of 1 mL of each vaccine to 10 mice and by subcutaneous inoculation of 10 mL of the vaccines in two goats. 2.2. Herd The field trial was carried out on a CCA-free herd on a farm in Ingenio, Gran Canaria, Spain, over a 7-month period. The herd comprised 725 lactating Canary dairy goats managed under a semi-intensive production system. To confirm the herd was CCA free, before the trial 100 animals were randomly selected and checked for the presence of Mycoplasma spp. Milk samples and auricular swabs were cultured and blood samples were also taken for indirect ELISA and immunoblotting to detect specific antibodies against M. agalactiae and Mmm LC. We then selected 120 lactating goats, 2–3 years old, from the main herd for the trial. None of the management conditions applied to the herd before the trial was modified during the trial. No antibiotic therapy or other specific treatment against mycoplasma was administered before or during the trial. 2.3. Experimental design
2. Materials and methods 2.1. Strains and formulation of the vaccines The field strains used were M. agalactiae (L9, AGIN3, 9B) and Mmm LC (AG1, 153/93, IN3) isolated from local clinical cases of CCA. The vaccines were prepared using cells of these strains cultured in modified pH culture medium (Kirchhoff and Rosengarten, 1984) supplemented with 10% horse serum (BioWhitaker) at 37 C until the mid-log phase. The cells were then centrifuged at 20,000g for 30 min at 4 C and resuspended in saline (0.9% NaCl; pH 7.2); this process was repeated three times. Colony forming units (CFU)/mL (Albers and Fletcher, 1982) were determined and each 2 mL dose was prepared to contain more than 5 · 1010 cfu/ mL of each mycoplasma species. Washed cultures containing pooled mycoplasma were stored at 20 C. Aluminium hydroxide (25%, vol/vol, Superfos A/S) plus purified saponin (0.5 mg/dose, Quil-A, Superfos A/S) were added as adjuvant, and the vaccines were then incubated with constant rocking for 2 h at 37 C. Vaccines A and B were inactivated with formaldehyde (0.1%, vol/vol, Sigma–Aldrich, for 16 h at 37 C) or phenol (0.5%, vol/ vol, Sigma–Aldrich, for 16 h at 37 C), respectively, and stored at 4 C. Samples of the vaccine were checked for sterility and side effects. For sterility testing, 200 lL aliquots of each vaccine lot were inoculated on solid and in liquid pH culture media (Kirchhoff and Rosengarten, 1984) and incubated at 37 C. A general medium for microbiology studies (blood agar) was also inoculated. At the end of the incubation period, the culture media were examined microscopically for the presence of mycoplasma or other types of colonies.
One hundred and twenty lactating goats were randomly assigned to three groups of 40 goats. One group was vaccinated with vaccine A, another received vaccine B, and a third group (C) remained as controls. The goats received two subcutaneous injections (2 mL) on Day 0 and 25 of the experiment. The animals were monitored over a 7month period, and blood samples were withdrawn from all goats at 0, 25, 45, 100, 150, 180 and 210 days. Serum samples were then tested for IgG antibodies against M. agalactiae and Mmm LC by indirect ELISA. Subsequently, a pool of ELISA-positive sera from each group was analysed by immunoblotting to detect the presence of specific antibodies to these two mycoplasma species. 2.4. Clinical signs During the trial, all cases of mastitis, conjunctivitis, arthritis or abortion were examined for Mycoplasma spp. To perform the microbiological tests, specimens of corresponding tissue or fluid were plated on pH medium, as previously described (de la Fe et al., 2005). At the end of the trial, 100 milk samples were also analyzed for the presence of Mycoplasma spp. by PCR. DNA was extracted from milk samples as previously described (Tola et al., 1997b). Specific primers were used to identify mycoplasma species (Hotzel et al., 1996; Tola et al., 1997b; Peyraud et al., 2003). 2.5. ELISA test and statistical analysis Sera were immediately prepared from each blood sample and kept at 20 C at the Veterinary Faculty of Las Palmas University (ULPGC), Arucas, Spain. Anti-M. aga-
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lactiae and anti-Mmm LC antibody levels, expressed as optical densities (OD), were determined by a previously described ELISA procedure. This method showed 99% sensitivity and 78% specificity for M. agalactiae, and 100% sensitivity and 84% specificity for Mmm LC (Assunc¸a˜o et al., 2004). To analyse the results obtained statistically, we considered the effects of the treatment, observation day, the interaction of both these effects, the random effect of the experimental unit and the residual effect, which were all assumed to be normally distributed. The model was estimated by the method of maximum likelihood. The significance of the effects was tested by the corresponding F-test. When the treatment effect was found to be significant, Scheffe’s multiple comparisons were performed. The level of statistical significance was set at P < 0.05. All statistical tests were performed using SPSS software (version 11.5) for Windows.
The ELISA results are provided in Figs. 1 and 2. The results indicate a steady increase in the production of specific IgG antibodies against M. agalactiae and Mmm LC in both groups of vaccinated animals, while control animals preserved their initial antibody levels. In both groups, A
2.6. SDS-PAGE and immunoblotting The presence of specific antibodies against M. agalactiae and Mmm LC was determined in vaccinated and control animals by SDS-PAGE and immunoblotting. First the protein contents of each mycoplasma species were determined using a kit (Sigma, Microprotein-PR) according to the manufacturer’s instructions. Samples were mixed with lysis buffer (500 mM Tris/HCl pH 6.8, 4.6% (w/v) SDS, 20% (v/ v) glycerol, 10% (v/v) 2-mercaptoethanol and 0.004% bromphenol blue) and boiled for 5 min. Next, 16 lg protein of each species were separated by SDS-PAGE on a 1.5 mm thick, 4% stacking gel and 10% resolving gel. Electrophoresis was performed as previously described (Laemmli, 1970). Separated proteins were transferred to 0.45 lm nitrocellulose membranes at 70 V constant voltage for 90 min (Towbin et al., 1979). The membranes were blocked in PBS containing 1 M glycine, 1% (v/v) egg albumin and 5% skimmed milk for 2 h at room temperature (RT), and then washed three times in PBS for 15 min. The membranes were incubated with the pooled serum samples from vaccinated or control goats at 37 C for 2 h, washed three times in PBS at room temperature and then incubated with the appropriate dilution of peroxidase conjugated anti-rabbit IgG (Sigma, A0545) in dilution buffer (1:2000). After washing, the substrate comprising 30 mg 4-chloro-1-naphtol dissolved in 10 mL methanol plus 50 mL PBS and 30 lL H2O2 was added to the membranes which were then left for 5–15 min in the dark. The labelling reaction was stopped with distilled water. 3. Results Sterility tests were negative. None of the vaccinated mice died and the goats that received two doses of the vaccines showed no reaction after vaccination. All the microbiological and serological tests performed before the trial were also negative.
Fig. 1. Specific immune response to M. agalactiae recorded in vaccinated (groups A and B) and non-vaccinated (controls) lactating goats at 0, 25, 45, 100, 150, 180 and 210 days post-vaccination determined by indirect ELISA. The vertical axis indicates antibody levels expressed as optical densities (OD).
Fig. 2. Specific immune response to Mmm LC recorded in vaccinated (groups A and B) and non-vaccinated (controls) lactating goats at 0, 25, 45, 100, 150, 180 and 210 days post-vaccination determined by indirect ELISA. The vertical axis indicates antibody levels expressed as optical densities (OD).
Table 1 Statistical analysis of the serological results recorded in lactating goats Model effects
Vaccine Period Interaction Animal Residual effect Total effect
M. agalactiae
Mmm LC
Sum of squares
F-test (P-value)
Sum of squares
F-test (P-value)
11.912 12.931 6.806 0.378 1.810 33.837
<0.001 <0.001 <0.001
17.277 13.033 7.036 0.783 3.837 41.966
<0.001 <0.001 <0.001
Significance of the effects of the mathematical model.
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Table 2 Statistical comparisons among the different treatments (Scheffe’s test) M. agalactiae
Scheffe’s test
Mmm LC
A–B
A–CT
B–CT
A–B
A–CT
B–CT
NS
<0.001
<0.001
<0.001
<0.001
<0.001
NS <0.001 <0.001 <0.001 <0.001 <0.001 0.002
NS <0.001 <0.001 <0.001 <0.001 <0.001 <0.022
NS NS NS 0.018 0.003 <0.001 NS
NS <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
NS <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
Experiment day 0 NS 25 NS 45 NS 100 NS 150 0.056 180 NS 210 NS
A: vaccine A; B: vaccine B; CT: control group; NS: not statistically significant.
and B, serum IgG levels peaked in the second month (45 days), and thereafter remained stable until Day 180. On Day 210, similar antibody levels were recorded in the vaccinated and non-vaccinated animals. When the effects of treatment, observation day and interaction between these factors were examined (Table 1), significant differences between groups A and B also emerged from Days 100–180 of the experiment (Table 2). Low levels of Mmm LC antibodies were observed in animals receiving vaccine B. Positive sera taken from both groups of vaccinated animals between Day 25 and Day 180 were analysed by immunoblotting (Figs. 3 and 4). Vaccinated animal sera indicated the presence of different pro-
Fig. 4. Immunoblotting profile of M. agalactiae (A) and Mmm LC (B) whole cell proteins obtained using serum obtained from goats experimentally vaccinated with vaccine B (16 lL protein loaded per track) at 25 days (track 1), 45 days (track 2), 100 days (track 3), 150 days (track 4) and 180 days (track 5). Molecular weights are indicated in kDa and correspond to molecular weight markers (left) and to the antigens detected (right).
teins of molecular weights 42–50 kDa, or 60–75 kDa in each mycoplasma species. Moreover, M. agalactiae or Mmm LC rendered specific bands of 24–25 kDa and 15 kDa, respectively. These results confirm the production of specific antibodies against antigens common to these mycoplasma species, as well as antibodies specific to each species. Finally, only a few cases (n = 10) of clinical mastitis were detected in the herd during the trial. Staphylococcus spp. was identified as the causal agent. All mycoplasma analyses were negative. No other clinical sign associated with CCA was observed, and the absence of mycoplasma in milk samples was confirmed by PCR. 4. Discussion
Fig. 3. Immunoblotting profiles of M. agalactiae (A) and Mmm LC (B) whole cell protein contents obtained using serum collected from goats experimentally vaccinated with vaccine A (16 lL protein loaded per track) at 25 days (track 1), 45 days (track 2), 100 days (track 3), 150 days (track 4) and 180 days (track 5). Molecular weights are indicated in kDa and correspond to molecular weight markers (left) and to the antigens detected (right).
The efficacy of inactivated CCA vaccines has always raised many doubts, especially in field conditions (Fleury et al., 2002). However, the literature lacks information on field trials and most existing data have been obtained under experimental conditions. The situation is further complicated by the fact that many outbreaks in CCA endemic areas are of mixed aetiology (de la Fe et al., 2005). Despite this, most vaccination trials have tested monovalent M. agalactiae vaccines, which are ineffective against the other mycoplasma species involved in the syndrome (Villalba et al., 1991). The use of combined vaccines to control more than one mycoplasma species in endemic areas has been previously
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proposed (Consenti and Montagna, 1989) and these have been used in some areas of Portugal (Regalla, 1987). The present field trial is the first report of a study of the humoral immune response induced in goats in field conditions by a combined CCA vaccine containing M. agalactiae and Mmm LC strains selected according to their antigenic profiles determined by molecular procedures (de la Fe et al., 2006). The use of a pool of field strains could produce a more efficient vaccine since reference strains lack many of the antigens shown by field strains (Gummelt et al., 1996; de la Fe et al., 2006). Hence, the use of a pool of field strains could reduce the number of vaccines that fail because of the antigenic variability of M. agalactiae and Mmm LC (Tola et al., 1996; de la Fe et al., 2006). Both the vaccines tested here induced a specific humoral immune response against M. agalactiae and Mmm LC in lactating goats that lasted for 6 months. This finding is consistent with the results of published M. agalactiae vaccine trials (Regalla, 1987; Leo´n Vizcaı´no et al., 1995). Other authors have reported a shorter period of immunity (Buonavoglia et al., 1998). The use of purified saponin (Quil-A) improved the immunogenicity of the aluminium adjuvant, avoiding the use of oil components (Rurangirwa et al., 1987). With the combined vaccines, antibodies peaked at a similar time to that found when a monovalent vaccine is administered (Foggie et al., 1971; Buonavoglia et al., 1998). Tola et al. (1999), however, recorded the antibody peak at 3 months. The better serological results obtained here with the phenol-inactivated vaccine can be explained by the minimal damage produced by this agent to the immunogenic proteins of these mycoplasma, as previously described (Tola et al., 1999; de la Fe et al., 2004). Immunoblotting results confirmed the production of specific antibodies against both mycoplasma species, and indicated that several species may be included in a combined CCA vaccine. The main immunogenic proteins of M. agalactiae were between 40 and 90 kDa, in agreement with previous observations (Tola et al., 1997a). There is no similar information available for Mmm LC, although, for example, we detected specific antibodies against a 62 kDa protein. This band seems to be a common specific antigen to the two closely related mycoplasma, Mmm LC and M. mycoides subsp. capri (Monnerat et al., 1999). In summary, the results of our field trial indicate that a polyvalent vaccine against M. agalactiae and Mmm LC can induce a humoral immune response in lactating goats that persists for 6 months. To prepare this type of vaccine, preliminary characterization studies are needed to select the best strains. We are presently engaged in further studies designed to establish the protection level conferred by these specific antibodies. Acknowledgements The authors thank the Vet Med Nelo Pascual (ADS Ingenio) for his technical assistance. This study was sup-
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