ANIMAL BIODIVERSITY AND EMERGING DISEASES
Anaplasma marginale Yucatan (Mexico) Strain Assessment of Low Virulence and Potential Use as a Live Vaccine ´ Sergio D. Rodr´ıguez Camarillo,a Miguel Angel Garc´ıa Ortiz,a Edmundo E. Rojas Ram´ırez,a Germinal J. Canto´ Alarcon, ´ b,c a Jesus ´ F. Preciado de la Torre, Rodrigo Rosario Cruz,a Juan A. Ramos Aragon, ´ a and Ramon ´ Aboytes Torresd a
Centro de Investigaciones Disciplinarias (CENID) en Parasitolog´ıa Veterinaria, Instituto Nacional de Investigaciones Forestales, Agr´ıcolas y Pecuarias (INIFAP), Secretar´ıa de Agricultura, Ganader´ıa, Desarrollo Rural, Pesca y Alimentacio´ (SAGARPA), Jiutepec, Morelos, M´excio b
´ CENID Fisiolog´ıa y Mejoramiento Animal, INIFAP, SAGARPA, Ajuchitlan, Quer´etaro, M´exico c
´ Facultad de Ciencias Naturales, Universidad Autonoma de Quer´etaro, Quer´etaro, Quer´etaro, M´exico d
Private Practice
Anaplasma marginale Yucatan strain was found to have low virulence in cattle. We studied the virulence of this isolate by experimental inoculation of 113 susceptible cattle at increasing doses, after which only one animal required treatment for clinical disease. Subsequently, 104 cattle received a live vaccine of this strain by inoculation, which induced immunoprotection after heterologous challenged exposure with a different A. marginale isolate. In this study 14% of the immunized cattle required treatment as compared with the control nonimmunized cattle, in which 56% required treatment. The A. marginale vaccine strains used for the immunization studies had MSP1a variable regions that were different from those used for the challenge exposure. Key words: Anaplasma marginale; cattle; low-virulence Mexican strain; immunoprotection
Introduction Both inactivated and attenuated vaccines have been tested for the control of bovine anaplasmosis. The efficacy of live vaccines using low-virulence/attenuated strains depends upon the capacity of the vaccine strain to in-
´ Address for correspondence: Miguel Angel Garc´ıa Ortiz, Carr. Cuernavaca-Cuautla No. 8534, Col. Progreso, Jiutepec, Morelos 62550, M´exico. Voice: +52-777-3192850, ext. 127; fax: +52-7777-3192850, ext. 129.
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duce protection.1 A live vaccine tested in Mexico in 1975 using a Florida strain of Anaplasma marginale did not induce protection.2 The use of Mexican strains as inactivated vaccines has shown promise by affording 80–100% protection against homologous challenge.3 Recently, we characterized a Mexican strain of A. marginale for its virulence and molecular markers and tested the use of this strain as live vaccine. An effective live vaccine may provide an alternative for protection of cattle in endemic areas of Mexico against field challenge with heterologous strains of A. marginale.
Animal Biodiversity and Emerging Diseases: Ann. N.Y. Acad. Sci. 1149: 98–102 (2008). C 2008 New York Academy of Sciences. doi: 10.1196/annals.1428.067
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Rodr´ıguez Camarillo et al.: Anaplasma marginale Yucatan (Mexico) Strain TABLE 1. Evaluation of Virulence and Protection Trial
Strain
1
3
Yucatane M´exico Yucatan M´exico Morelos Yucatan
4e
Control Yucatan
2
Control Yucatan Control Yucatan Control Yucatan Yucatan Control
5 6 7f
Dose of IEa 106 108
104 106 108 1010 106 108 1010 108 108 108 108
N 4 4 5 5 5 7 5 7 7 7 6 6 6 5 10 5 24 24 26
Days observedb
% Treated post vaccinec
Challenge strain
% Treated post challenged
65
0 0 0 80 60 0 0 0 0 na 17 0 0 na 0 na 0 na 0 0 na
na na na na na Mor
na na na na na 86 40 43 14 86 17 33 17 100 0 40 4 42 0 0 63
46
56
64
74 40 85
8
Mor
Chis Ags PV
a
Infected erythrocytes. Period during which animals were monitored for vaccine reactions. c Animals receiving treatment on account of vaccine reactions. d Animals receiving treatment on account of challenge reactions. e Yucatan strain was previously cryopreserved for trials 4 through 7. f Field trial: All vaccinations were performed while animals were kept at a ranch in Queretaro State under Anaplasma-free conditions, where they remained for 85 days after vaccination. At the end of this period, animals were transported to Playa Vicente, Veracruz, where they remained for an additional 118 days. na = not applicable. b
Materials and Methods Trials Seven trials were conducted to evaluate virulence and immunity induced by the Yucatan strain of A. marginale (Table 1).
strains used for heterologous challenge were selected because they had different MSP1a variable regions.4 The challenge exposure dose for all trials was 108 infected erythrocytes (IEs).
Bovines Organisms A Yucatan strain, isolated from an asymptomatic carrier, was maintained by passage in splenectomized cattle and by preservation in liquid nitrogen. Morelos (Mor), Chiapas (Chis), Aguascalientes (Ags), and Playa Vicente (PV)
Eighteen-month-old steers or heifers found to be negative for TB and brucellosis, and ELISA- and PCR-negative for anaplasmosis, were used for these experiments. The protocol for use of experimental cattle was approved by the INIFAP Animal Welfare Committee.
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Clinical Evaluation Rectal temperature, packed cell volume (PCV), and percent of infected erythrocytes (PIEs) in Giemsa-stained blood smears were determined and used to monitor cattle infections. Blood and serum samples were assayed by msp5 and msp1α variable region PCR5 and indirect ELISA was used to determine antibody levels. Clinical evaluations were done on cattle from inoculation until complete recovery after both vaccination and challenge exposure. When indicated by the appearance of clinical disease, cattle were treated for 3 consecutive days with oxytetracycline (20 mg/kg).
Results During all trials, most animals inoculated with Yucatan vaccine strain developed increasing A. marginale antibody titers after challenge exposure. All control animals remained negative during vaccination periods, but became seropositive after challenge exposure. PCRpositive reactions were consistent with vaccination and challenge exposure in both vaccinated and control cattle. Trials 1 and 2 Clinical signs were not observed in cattle inoculated with Yucatan strain in Trials 1 and 2 (Table 1). All animals inoculated with Mexico strain in Trial 1 demonstrated reduction in PCVs, fever, general depression, and weight loss. In Trial 2, 80% and 60% of animals inoculated with Mexico and Morelos strains, respectively, required antibiotic treatment. In addition, one cow inoculated with the Morelos strain died. In Trials 1 and 2, PIE values for animals inoculated with Mexico (>23%), or Morelos (17%) strains were higher (P < 0.05) than in those inoculated with Yucatan strain (9% and 5%, respectively). The mean maximum percent reduction of PCVs (64% and 72%) observed in cattle inoculated with Morelos and Mexico
strains were higher (P < 0.05) than the percent reduction of PCV of 35–50% observed in cattle inoculated with Yucatan strain. Trials 3 and 4 Vaccination of cattle with fresh or rapidly thawed frozen infected blood samples induced clinical disease in only one group (Trial 4; dose 106 ), and most of the cattle in both trials were identified as positive by microscopic observation of stained blood smears. In Trial 3 and Trial 4, 86% and 100% controls at challenge exposure required antibiotic treatment. Likewise in Trial 3, 86% of vaccinates in the 104 dose group and 14–43% of vaccinated cattle (doses 106 –1010 ) required antibiotic treatment. PIE values for vaccinates (106 –1010 ) ranged from
