Abstract In this study, we report for the first time the successful infestation of rabbits with just-molted, unfed adults of Rhipicephalus microplus. Six New Zealand White rabbits were experimentally infested with 20 female and 20 male unfed adult ticks released into plastic chambers fixed on the shaved backs of each host. The attachment and feeding processes were successful. The biological characteristics of the ticks and the occurrence of adverse events in the tick-attachment area were studied. The average engorgement period was 10.7 days, and 33.3% of the engorged females completed the parasitic phase. The average weight of the recovered engorged females was 149.8 mg, with an average egg mass weight of 70.9 mg, a conversion efficiency index of 47.3%, and a hatching percentage of 88.31%. The adverse reactions found in the tick-attachment area were the usual inflammatory responses of the organism to infestation by these ectoparasites; however, it did not prevent the ticks from feeding and completing their life cycle. These data indicate that the infestation of rabbits with just-molted, unfed adult ticks could be a valuable, alternative animal model for rapid and economical evaluation of vaccine candidates and new molecules with acaricidal activity against Rhipicephalus microplus.

Resumo Neste estudo, relata-se, pela primeira vez, o sucesso da infestação de coelhos por carrapatos adultos recém-mudados e não alimentados. Seis coelhos brancos da Nova Zelândia foram infestados artificialmente com 20 carrapatos adultos fêmeas e 20 machos adultos não alimentados no interior de câmaras de plástico fixadas no dorso de cada hospedeiro. Os processos de fixação e alimentação dos carrapatos foram bem sucedidos. Foram estudadas as características biológicas dos carrapatos e a ocorrência de eventos adversos na área de fixação do ectoparasito. O período médio de ingurgitamento foi de 10,7 dias, e 33,3% das fêmeas ingurgitadas completaram a fase parasitária. O peso médio das fêmeas ingurgitadas recuperadas foi de 149,8 mg, com peso médio da massa de ovos de 70,9 mg, índice de produção de ovos de 47,3% e porcentagem de incubação de 88,31%. Uma resposta inflamatória usual do organismo à infestação por esses ectoparasitas foi a única reação adversa encontrada na área de fixação dos carrapatos, o que não impediu que eles se alimentassem e completassem seu ciclo de vida. Esses dados apontam que a infestação de coelhos com carrapatos adultos recém-mudados e não alimentados pode ser um modelo animal alternativo e útil para a avaliação rápida e econômica de candidatos a vacinas e novas moléculas com atividade acaricida contra Rhipicephalus microplus.

RESUMEN El candidato vacunal E2-CD154 es una vacuna de subunidad cuya seguridad y capacidad protectora en crías frente a la peste porcina clásica ha sido demostrada en estudios previos. Esos hallazgos se confirman en el presente estudio de fase III realizado en dos unidades de producción porcina en la provincia de Pinar del Rio con un número mayor de animales. Todos los animales en ambas granjas recibieron dos dosis de E2-CD154 los días 0 y 21. El estudio se extendió por 60 semanas, la vacuna fue bien tolerada en las crías vacunadas entre los 15 y 28 días de edad, no se documentaron efectos adversos locales o sistémicos. Las cerdas gestantes inmunizadas fueron capaces de trasmitir altos títulos de anticuerpos a sus crías (Unidad A, media geométrica = 1:1295, valor mínimo 1:100 y Unidad B, media geométrica = 1:474, valor mínimo 1:150), muy por encima del umbral de protección (1:50). Estos elevados títulos de anticuerpos neutralizantes derivados de la madre no interfirieron con la inmunogenicidad del candidato vacunal en estudio. Todas las crías vacunadas estudiadas al azar (más del 10% de las 2804) desarrollaron títulos de anticuerpos neutralizantes mayores de 1:400 en los cuatro muestreos realizados (nueve, 21, 41, y 44 semanas) en ambas granjas. Los resultados de este estudio confirman la seguridad, inmunogenicidad y robustez del candidato vacunal en esta sensible categoría en condiciones de campo.

ABSTRACT E2-CD154 is a subunit vaccine candidate that has been proven to be safe and to protect piglets from classical swine fever (CSF). In this study, those previous findings were confirmed and extended to a larger number of animals in a phase III clinical trial conducted on two production farms in Pinar del Río province. All animals in both farms were vaccinated with two doses of E2-CD154 on days 0 and 21. The study extended up to 60 weeks. The vaccine was well tolerated in piglets between 15 and 28 days of life, with neither local nor systemic side effects documented. Immunized pregnant sows were capable of transmitting high levels of maternally-derived neutralizing antibodies (MDNAs) to their offspring (Unit A, geometric mean titer = 1:1295, minimum value 1:100 and Unit B geometric mean titer = 1:474, minimum value 1:150), well above the protection threshold (1:50). These high MDNA titers in the piglets did not interfere with the immunogenicity of the candidate. All vaccinated piglets evaluated at random (more than 10% of 2804 vaccinated) developed protective neutralizing antibody titers higher than 1:400 at the four time points analyzed (nine, 21, 41, and 44 weeks) in both farms. The results of this study confirm the safety, immunogenicity and robustness of this vaccine candidate in this sensitive pig category in the field.

ABSTRACT Introduction. Porvac( is a subunit vaccine against Classical Swine Fever (CSF), whose active principle is the chimeric protein between the E2 antigen and the molecular adjuvant CD154. Its ability to prevent transplacental transmission and to protect 7 days after one dose was demonstrated. This paper summarizes the safety, immunogenicity and efficacy studies of this vaccine at different ages and handling conditions. Methods. The vaccine was produced under GMP conditions. Dubroc x Yorkshire crossbreed swine were used in all experiments. The general immunization schedule consists in two immunizations on days 0 and 21 by the intramuscular route in the neck. The neutralizing antibody response was measured by the NPLA assay. Viral challenge experiments were conducted in controlled areas. Results and Discussion. Porvac( was safe and well tolerated in piglets and pregnant sows. No local or systemic reactions were documented. The number of offspring, their weight and survival in vaccinated sows was similar to unvaccinated controls. Different immunization schedules with Porvac( induce protective neutralizing antibodies (NAb). The titers of maternal derived antibodies (MDA) in the offspring of Porvac( or Attenuated Life vaccine and Porvac( vaccinated sows were higher than 1:200 up to at least 8 weeks of life. The MDA were able to protect piglets from a lethal viral challenge. Those MDA did not interfere with Porvac( vaccination of the piglets 15 days after birth or more. Finally, Porvac( induced NAb titers persisted during 9 months above 1:2000 and were still protective against the viral challenge. Conclusions. The results demonstrate the capacity of Porvac( to be safe and to induce robust and long term immunity through all the life of the animals, without immunological windows.

RESUMEN Introducción. Porvac( es una vacuna de subunidad contra la peste porcina clásica, cuyo principio activo es la proteína quimérica entre el antígeno E2 y el adyuvante molecular CD154. Se ha demostrado su capacidad para evitar la transmisión transplacentaria y proteger 7 días después de una dosis. El presente trabajo ha resumido los estudios de seguridad y eficacia de esta vacuna en diferentes edades y condiciones de manejo. Métodos. La vacuna se produjo en condiciones de BPM. Para los ensayos clínicos se emplearon cerdos Dubroc x Yorkshire. Por lo general, se empleó un esquema de 2 dosis los días 0 y 21 y se administraron 2 mL por vía intramuscular. La respuesta de anticuerpos neutralizantes se midió por NPLA. Los ensayos de desafío viral se realizaron en áreas controladas. Resultados. Se demostró la seguridad de Porvac( en crías y en cerdas gestantes. No se observaron reacciones locales ni sistémicas. El número de crías, su peso y sobrevida fue similar en las cerdas vacunadas y sus controles. Se evidenció la capacidad de Porvac( al inducir títulos de anticuerpos neutralizantes (AcN) protectores con diferentes esquemas. Los títulos de AcN calostrales (AcNC) en las crías de madres vacunadas con Porvac® o con VVA y Porvac( fueron superiores a 1:200 hasta al menos 8 semanas. Los AcNC fueron suficientes para proteger a las crías frente al reto viral letal. Se demostró que los AcNC no interferían en la vacunación con Porvac( en crías a partir de 15 días de nacidas. Finamente, los títulos de AcN inducidos por Porvac( persistieron durante 9 meses por encima de 1:2000 y protegieron frente al retoviral. Conclusiones . Los resultados evidenciaron la seguridad de Porvac( y su capacidad para inducir una inmunidad robusta y sostenida en los animales desde su nacimiento hasta la muerte, sin ventanas inmunológicas durante todo el ciclo productivo.