Immunogenic potential of Rhipicephalus (Boophilus) microplus aquaporin 1 against Rhipicephalus sanguineus in domestic dogs / Potencial imunogênico da aquaporina 1 de Rhipicephalus (Boophilus) microplus contra o carrapato Rhipicephalus sanguineus em cães domésticos

Abstract This study evaluated a recombinant aquaporin 1 protein of Rhipicephalus (Boophilus) microplus (RmAQP1) as antigen in a vaccine against R. sanguineus. Five dogs were immunized with RmAQP1 (10 µg) + adjuvant (Montanide) (G1), and five were inoculated with adjuvant only (G2), three times. Twenty-one days after the last immunization, animals of both groups were challenged with R. sanguineus larvae, nymphs and adults, and their biotic potential was compared. Blood samples were collected before each immunization and every 28 days after the last immunization for 10 weeks. Serum antibody titers (IgG) were assessed by ELISA. We observed that: engorgement period of adult females from G1 was 12% shorter than G2; larvae from G1 had 8.7% longer engorgement period than G2 and weighed 7.2% less; nymphs from G1 had 4.5% shorter engorgement period than G2 and weighed 3.6% less; although the antibody titers increased following the second immunization, they rapidly decreased after the third immunization. Results indicated low immunoprotection of RmAQP1 against adult R. sanguineus ticks, and possible efficacy on larvae and nymphs fed on immunized dogs. Further studies should be performed for a full evaluation of the immunoprotection of RmAQP1 against R. sanguineus infestations in dogs.

Resumo Este estudo avaliou a proteína recombinante (aquaporina) do carrapato Rhipicephalus (Boophilus) microplus como antígeno em vacina contra Rhipicephalus sanguineus. Cinco cães foram imunizados com RmAQP1 (10 µg) + adjuvante (G1) e cinco foram inoculados apenas com adjuvante (G2), três vezes. 21 dias após a última imunização todos os animais foram desafiados com larvas, ninfas e adultos de R. sanguineus, e potencial biótico dos carrapatos foi comparado. Amostras de sangue foram coletadas antes de cada imunização e a cada 28 dias após a última imunização, durante 10 semanas. Títulos de anticorpos dos soros dos cães foram avaliados por ELISA. Resultados: o período de ingurgitamento das fêmeas do G1 foi 12% mais curto que o período de ingurgitamento de G2; o período de ingurgitamento das larvas do G1 8,7% foi mais longo e o peso 7,2% menor que no caso de G2; o período de ingurgitamento das ninfas do G1 4,5% foi mais curto e peso 3,6% menor que no caso do G2; aumento dos títulos de anticorpos do G1 após a segunda imunização e declínio após a terceira imunização. Os resultados indicaram baixo potencial de imunoproteção de RmAQP1 contra R. sanguineus adultos, e possível eficácia contra larvas e ninfas, na dose testada. Sugere-se desenvolver novos estudos para melhor avaliação da eficácia de RmAQP1 contra R. sanguineus em cães.

Evaluation of glycoproteins purified from adult and larval camel ticks (Hyalomma dromedarii) as a candidate vaccine

In order to identify antigens that can help prevent camel tick infestations, three major glycoproteins (GLPs) about 97, 66 and 40 kDa in size were purified from adult and larval Egyptian ticks, Hyalomma (H.) dromedarii, using a single-step purification method with Con-A sepharose. The purified GLPs were evaluated as vaccines against camel tick infestation in rabbits. The rabbits received three intramuscular inoculations of GLPs (20 microg/animal) on days 0, 14, and 28. In the immunoblot analysis, Sera from the immunized rabbits recognized the native GLPs and other proteins from larval and adult H. dromedarii ticks along with those from other tick species such as Rhipicephalus sanguineus but not Ornithodoros moubata. The effects of immunity induced by these GLPs were determined by exposing rabbits to adult H. dromedarii ticks. These results demonstrated that GLP immunization led to a slightly decreased reproductive index and significantly reduced rates of egg hatchability. These results demonstrated that immunization with the purified GLPs can provide protection against infestation by H. dromedarii and some other tick species. Further studies are needed to confirm the effectiveness of immunization with GLPs against other tick species.

Possible acquired resistance of dogs successively infested by Amblyomma cajennense (Fabricius, 1787) (Acari: Ixodidae) nymphs / Possibilidade de ocorrência de resistência adquirida em cães sucessivamente infestados por ninfas de Amblyomma cajennense (Fabricius, 1787) (Acari: Ixodidae)

The present study aimed to evaluate the occurrence of immune resistance in dogs successively infested with Amblyomma cajennense nymphs. Five animals were submitted to four consecutive infestations with A. cajennense nymphs, at fourteen-day intervals. For each infestation, 50 nymphs were used per animal and data on the parasitic and non-parasitic periods were recorded. The average recovering rate of engorged nymphs in the successive infestations were 52.0, 29.2, 9.6 and 12.8%, respectively, with a significant reduction (p < 0.05) of this parameter from the second infestation onwards. The modal drop-off day of engorged nymphs was Day 4 of parasitism in all infestations. The average mortality rates of nymphs seen on the first, second, third and fourth infestations were 3.6, 3.2, 2.0 and 2.8%, respectively, with no significant differences among them (p < 0.05). In addition, no significant differences were seen among the ecdysis rates for specimens recovered from successive parasitic challenges. The study results suggest that the acquired resistance of infested dogs had a negative effect on recovery rate of A. cajennense nymphs; however, it did not affect the other biological parameters evaluated.

O presente trabalho teve por objetivo avaliar a ocorrência de resistência imune em cães, frente a infestações sucessivas por ninfas de Amblyomma cajennense. Para tanto, cinco animais foram submetidos a quatro infestações consecutivas por ninfas de A. cajennense em intervalos de quatorze dias. Foram aplicadas 50 ninfas em cada animal por infestação e os dados referentes aos períodos parasitários e não parasitários, foram registrados. As taxas médias de recuperação de ninfas ingurgitadas, verificadas nas sucessivas infestações foram de 52,0, 29,2, 9,6 e 12,8%, sendo observada uma redução significativa (p < 0,05) nesse parâmetro a partir da segunda infestação. O dia modal de queda das ninfas ingurgitadas em todas as infestações foi o 4º dia de parasitimo. As taxas médias de mortalidade de ninfas observadas no primeiro, segundo, terceiro e quarto desafio parasitário foram de, respectivamente, 3,6, 3,2, 2,0 e 2,8%, não havendo diferença significativa entre elas (p < 0,05). Não observou-se diferença significativa (p < 0,05) entre as taxas de ecdise reportadas para os exemplares recuperados nos sucessivos desafios parasitários. Esses resultados sugerem que a resistência adquirida nos cães parasitados afetou negativamente a taxa de recuperação das ninfas de A. cajennense inoculadas nesses animais, contudo não apresentou nenhum efeito sobre os demais parâmetros biológicos avaliados.

Purification and characterization of two larval glycoproteins from the cattle tick, Boophilus annulatus

The present study was conducted to identify new target immunogenic molecules from the larval stage of the cattle tick, Boophilus annulatus (Acari: Ixodidae). Two specific larval glycoproteins (GLPs) were isolated by two-step affinity chromatography. The larval immunogens were first purified with CNBr-Sepharose coupled to rabbit anti-larval immunoglobulins, and the glycoproteins were then purified with Con-A Sepharose. These glycoproteins have molecular weights of approximately 32 and 15 kDa with isoelectric points between 6.8 and 7.2. Antibodies against the two GLPs, labeled I and II, were detected in the anti-whole tick, -whole larval, and -gut antigens through immunoblot analysis. These results suggest that these GLPs are good immunogens and can be useful in the vaccination of cattle against tick infestation.

Association of BoLA-DRB3.2 alleles with tick (Boophilus microplus) resistance in cattle

Losses caused by bovine tick burdens in tropical countries have a tremendous economic impact on production systems. Besides reducing production, this parasite can cause death in the most susceptible animals. The use of commercial acaricides has been the major method of control, but their misuse has led to tick resistance to many chemicals. More recently, vaccines have been used in some countries without solving the problem completely. An alternative could be the development of resistant animals and the use of genetic markers and candidate genes that could help with the enormous task of selecting resistant animals. The bovine lymphocyte antigen genes (BoLA) have been shown to be associated with some parasitic infestations and disease incidence. Thus, the objective of the present study was to determine the association of BoLA-DRB3.2 alleles with tick resistance in cattle. The study was conducted on 231 F2 (Gyr x Holstein) animals that were artificially infested with 10,000 tick larvae. Log of tick count +1 was used as the dependent variable in a mixed animal model with allele substitution effects in addition to fixed effects of year and season at tick count, sex of calves, age of animal at tick count, hair type (short-straight, short-curl, long-straight, and long-curl), coat color (white, >75% white, 50- 75% white, and 25-50% white), and additive genetic, permanent environmental and residual effects as random. Females showed fewer ticks than males. Animals with short-straight hair were more resistant to tick infestation than animals with long-curl hair, and animals with whiter coat color also had fewer ticks. An association between BoLA alleles and lower tick number was found for alleles DRB3.2 *18, *20 and *27 at the 5% significance level. Also, one allele (DRB3.2*16) showed an association at the 10% level. Allele *27 was the most frequent in the population (30.7%), followed by alleles *16 (10.8%), *20 (8.7%) and *18 (2.4%)...

Immunization effect of recombinant P27/30 protein expressed in Escherichia coli against the hard tick Haemaphysalis longicornis (Acari: Ixodidae) in rabbits

We investigated the induction of resistance to Haemaphysalis longicornis infestation in rabbits that had been immunized with recombinant H. longicornis P27/30 protein. The success of immunological control methods is dependent upon the use of potential key antigens as tick vaccine candidates. Previously, we cloned a gene encoding 27 kDa and 30 kDa proteins (P27/30) of H. longicornis, and identified P27/30 as a troponin I-like protein. In this study, rabbits that were immunized with recombinant P27/30 expressed in Escherichia coli showed the statistically significant longer feeding duration for larval and adult ticks (P< 0.05), low engorgement rates in larval ticks (64.4%), and an apparent reduction in egg weights, which suggest that H. longicornis P27/30 protein is a potential candidate antigen for a tick vaccine. These results demonstrated that the recombinant P27/30 protein might be a useful vaccine candidate antigen for biological control of H. longicornis.

Immunization of cattle against Hyalomma anatolicum anatolicum using larval antigens.

Development of immunity in cross-bred (Bos taurus x Bos indicus) calves against Hyalomma anatolicum anatolicum, vector of bovine tropical theileriosis, was studied using larval antigen (LS) in Freund's complete adjuvant (FCA). Calves immunized with LS + FCA showed significant rejection of larvae (57.25 +/- 6.8) and nymphs (45.75 +/- 5.16). Abnormally fed larvae (11.4 +/- 0.8) and nymphs (8.25 +/- 1.2) were also recovered from immunized calves. This abnormal feeding may possibly be attributed to their inability to gain access to the blood vessels owing to the host immunological reactions. Consequently, feeding of extravascular fluid leads to white colour of fed ticks. Sera from all immunized calves after a week of immunization were positive for anti-LS antibodies in ELISA. The investigation indicates that LS in FCA enhanced anti-tick immunity.

Imunidade a carrapatos Rhipicephalus sanguineus (Acarina: ixodidae) em cachorro-do-mato Cerdocyon thous (Linnaeus) e no cäo doméstico / Immunity ticks Rhipicephalus sanguineus (Latreille) in crab-eating-fox Cerdocyon thous (Linnaeus) and mongrel dogs

Algumas das relaçöes hospedeiro-parasita säo marcadas por desenvolvimento de resistência pelo hospedeiro, limitando o número de parasitas. Tal näo ocorre na relaçäo cäo doméstico x Rhipicephalus sanguineus (carrapato do cäo). Teria o cäo doméstico perdido aquela capacidade em seu processo de domesticaçäo? Esta hipótese foi testada em duas condiçöes, comparando-se a aquisiçäo de imunidade ao carrapato R. sanguineus pelo cäo doméstico em relaçäo ao cachorro-do-mato-Cerdocyon thous após três infestaçöes sucessivas por carrapatos adultos, ou através de infestaçäo desafio após imunizaçäo com um homogenato bruto de carrapatos da mesma espécie. A aquisiçäo ou näo de resistência foi avaliada a partir da análise do desempenho alimentar e reprodutivo das carrapatas durante e após as infestaçöes. Os resultados indicaram uma pequena diferença significativa entre o cäo doméstico e o cachorro-do-mato quanto ao desenvolvimento de resistência ao carrapato R. sanguineus, que, entretanto, näo foi considerada relevante na caracterizaçäo de uma imunidade efetiva