A new method for in vitro feeding of Rhipicephalus australis (formerly Rhipicephalus microplus) larvae: a valuable tool for tick vaccine development
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Rhipicephalus microplus is a hard tick that has a major impact on cattle health in tropical and subtropical regions because it feeds on cattle and is implicated in the transmission of pathogens that cause diseases such as bovine anaplasmosis and babesiosis. Presently, acaricides are used to control tick infestation but this is becoming increasingly less effective due to the emergence of tick strains that are resistant to one or more classes of acaricides. Anti-tick vaccines are a promising alternative to control tick infestation in cattle. The life-cycle and host preference of R. microplus, however, makes vaccine research in cattle costly and would therefore greatly benefit from an in vitro screening system. To this aim, a stacked 24-well in vitro feeding system was designed in which the blood meal was administered in a chamber on top of the compartment containing the ticks, exploiting their anti-gravitational tendency. Both compartments were separated by a special feeding membrane, which was made by applying a silicone mixture to a gold beater's skin (baudruche membrane) with a paint roller to create a slightly uneven surface of 17–40 μm variable thickness. To further stimulate feeding, the membrane was treated with bovine hair extract and the unit was placed at 37 °C with 90% RH and 5% CO2. Using this set-up with Rhipicephalus australis (formerly Rhipicephalus microplus), a larval engorgement rate of up to 71% could be achieved. The larvae could successfully feed on blood, but also on serum. The latter allows easy screening of the effect of sera that are raised against tick proteins on feeding. As an example, serum from cattle that were vaccinated with the Bm86 midgut protein of R. microplus significantly reduced larval engorgement rates by 42%. The in vitro feeding system's high throughput design and its ability to measure statistically significant anti-tick effects in sera from immunized cattle enables screening of multiple vaccine candidates in a cost-effective manner.Keywords:
Rhipicephalus microplus
Rhipicephalus
Tick infestation
Parasitology
Blood meal
Veterinary parasitology
Rhipicephalus sanguineus
The vaccination represents optimum method evaluated with effective cost to prevent economic losses and to increase the duration and quality of life of the production animals. . Diverse vaccines are produced from the intestinal protein Bm86 of the Rhipicephalus. (B.) microplus. The knowledge of the conservation of the gene bm86 is very important to evaluate the vaccine efficiency and the possibility of reaction crossed between different species of ticks. Samples of R. (B.) microplus come from different localities had been sequenced. The analyses of multiple alignments of the sequences had been made through the BioEdit program 7.0.5.3 version and the verification of polymorphism for visual inspection. In this work the alignment of all was become fulfilled sequences using itself BLAST in the search for similarity. Similarity was observed enters the sequenced fragments of R. (B.) microplus with the sequence of the protein Rs86 de Rhipicephalus sanguineus and with protein HA98 of the tick Hyalomma anatolicum anatolicum. The results give molecular support to synthetic the vaccine use based in the gene bm86 (SBm7462®) to be used in different species of ticks.
Rhipicephalus sanguineus
Rhipicephalus microplus
Rhipicephalus
Hyalomma
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The vaccination represents optimum method evaluated with effective cost to prevent economic losses and to increase the duration and quality of life of the production animals. . Diverse vaccines are produced from the intestinal protein Bm86 of the Rhipicephalus. (B.) microplus. The knowledge of the conservation of the gene bm86 is very important to evaluate the vaccine efficiency and the possibility of reaction crossed between different species of ticks. Samples of R. (B.) microplus come from different localities had been sequenced. The analyses of multiple alignments of the sequences had been made through the BioEdit program 7.0.5.3 version and the verification of polymorphism for visual inspection. In this work the alignment of all was become fulfilled sequences using itself BLAST in the search for similarity. Similarity was observed enters the sequenced fragments of R. (B.) microplus with the sequence of the protein Rs86 de Rhipicephalus sanguineus and with protein HA98 of the tick Hyalomma anatolicum anatolicum. The results give molecular support to synthetic the vaccine use based in the gene bm86 (SBm7462 ® ) to be used in different species of ticks.
Rhipicephalus sanguineus
Rhipicephalus microplus
Rhipicephalus
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The objective of this article was to discuss some aspects of ticks associated with domestic animals in the State of Pernambuco, northeastern Brazil, based on a literature review and present new data obtained from recent tick collections carried out in this northeastern Brazilian state. From August 2007 to June 2008, 1,405 ticks were collected and five species were identified: Amblyomma cajennense (Fabricius, 1787), Amblyomma ovale Koch, 1844, Dermacentor nitens Neumann, 1897, Rhipicephalus (Boophilus) microplus (Canestrini, 1887), and Rhipicephalus sanguineus (Latreille, 1806). Dogs from urban areas were found exclusively infested by R. sanguineus, whereas dogs from rural areas were found infested by A. cajennense, A. ovale, R. (B.) microplus, and R. sanguineus. The only tick species found on cattle and goats was R. (B.) microplus. Horses were found infested by D. nitens and at a lesser extent by A. cajennense and R. (B.) microplus. The only tick species found on donkeys was D. nitens. Tis study confirms the presence of A. cajennense in Pernambuco and describes for the first time the presence of A. ovale in this state. The medical and veterinary relevance and control of ticks associated with domestic animals in Pernambuco are also briefly discussed.
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This study was conducted during 3 years period (2002-2004). Tick sampling was carried out randomly from domestic animals during seasonal activity of ticks from different parts of Iran. 2170 ticks from 151 cattle, 629 sheep, 336 goats and 33 camels were collected. The occurance of tick infestation in cattle, sheep, goats and camels was 60%, 71.4%, 53% and 46 % respectively. Sampled ticks of Rhipicephalus species have been identified as; Rhipiephalus sanguineus (37.9%) Rhipiephalus bursa (49.8%) and Rhipicephalus turanicus (12.23%). All three species adopted in ecological zone II but Rhipicephalus sanguineus was the main tick species found in four zones ofIran. The comprative tick yield obtained from animals showed that Rhipicephalus bursa was the most aboundant in zone II but Rhipicephalus sanguineus was the rarest species in zone IV. The results described here suggest that livestock had almost different pattern of tick species in any localities of Iran.
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Abstract Background Tick control is mostly hampered by the rise of acaricide-resistant tick populations. Significant efforts have focused on developing alternative control methods, including cross-species protective and/or cocktail-based anti-tick vaccines, to achieve protection against various tick species. Methods In this study, full-length open reading frames encoding subolesin (SUB) from Rhipicephalus microplus and ferritin 2 (FER2) from Hyalomma anatolicum as well as the partial 60S acidic ribosomal protein (P0) from R. microplus were cloned, expressed in Escherichia coli and used as vaccine antigens against Rhipicephalus sanguineus sensu lato ( R. sanguineus s.l.) infestation in rabbits. Results In silico analyses revealed that the SUB, P0 and FER2 proteins were antigenic and displayed limited similarity to the host's homologous proteins. The proteins shared identities of 97.5%, 100% and 89.5% with their SUB, P0 and FER2 R. sanguineus s.l. orthologous sequences, respectively. Antibodies against each recombinant protein cross-recognized the native proteins in the different tissues and developmental stages of R. sanguineus s.l. Overall efficacy of the SUB, FER2 and cocktail (SUB+FER2+P0) vaccines against R. sanguineus s.l. infestation was 86.3%, 95.9% and 90.9%, respectively. Conclusions Both mono-antigen and the cocktail anti-tick vaccines affected the biological parameters of R. sanguineus s.l. infestation in the rabbit model, which could be extrapolated to its infested host under natural conditions. These findings support the possibility of using mono-antigenic and cocktail-based vaccines for large-scale anti-tick vaccine development against multiple tick species. Graphical Abstract
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Rhipicephalus microplus
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Rhipicephalus microplus is a hard tick species that has a high impact on cattle health and production in tropical and subtropical regions. Recently, ribosomal DNA and morphological analysis resulted in the reinstatement of R. australis as a separate species from R. microplus. Both feed on cattle and can transmit bovine pathogens such as Anaplasma and Babesia species. The current treatment with acaricides is becoming increasingly less effective due to the emergence of resistant tick strains. A promising alternative can be found in the form of anti-tick vaccines. The available commercial vaccines can be used to control tick infestation, but the lack of a knockdown effect (> 90% reduction in tick numbers as seen with effective acaricides) hampers its widespread use, hence higher efficacious vaccines are needed. Instead of searching for new protective antigens, we investigated the efficacy of vaccines that contain more than one (partially) protective antigen. For screening vaccine formulations, a previously developed in vitro feeding assay was used in which R. australis larvae are fed sera that were raised against the candidate vaccine antigens. In the present study, the efficacy of the Bm86 midgut antigen and the cytosolic Subolesin (SUB) antigen were evaluated in vitro.Antiserum against recombinant Bm86 (rBm86) partially inhibited larval engorgement, whereas antiserum against recombinant SUB (rSUB) did not have any effect on feeding of larvae. Importantly, when larvae were fed a combination of antiserum against rBm86 and rSUB, a synergistic effect on significantly reducing larval infestations was found. Immunohistochemical analysis revealed that the rBm86 antiserum reacted with gut epithelium of R. australis larvae, whereas the antiserum against rSUB stained salivary glands and rectal sac epithelium.Combining anti-Bm86 and anti-subolesin antibodies synergistically reduced R. australis larval feeding in vitro. Rhipicephalus australis is a one host tick, meaning that the larvae develop to nymphs and subsequently adults on the same host. Hence, this protective effect could be even more pronounced when larvae are used for infestation of vaccinated cattle, as the antibodies could then affect all three developmental stages. This will be tested in future in vivo experiments.
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Tick infestation
Rhipicephalus
Parasitology
Tick-borne disease
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Ticks and tick-borne diseases are considered a major challenge for human and animal health in tropical, sub-tropical, and temperate regions of the world. However, only scarce information is available on the characterization of tick species infesting dogs in Pakistan. In this study, we present a comprehensive report on the epidemiological and phylogenetic aspects of ticks infesting dogs in Pakistan using the mitochondrial markers i.e. Cytochrome c oxidase subunit 1 (cox1) and 16S ribosomal RNA (16S rRNA) nucleotide sequences. A total of 300 dogs were examined and 1150 ixodid ticks were collected across central Khyber Pakhtunkhwa, Pakistan. The morpho-molecular characterization of hard ticks revealed the presence of two ixodid tick genera on dogs, i.e., Hyalomma and Rhipicephalus, including six tick species viz. Hyalomma dromedarii (15.9%), Hyalomma excavatum (3%), Rhipicephalus sanguineus s.l. (41.3%), Rhipicephalus turanicus s.s. (28.7%), Rhipicephalus haemaphysaloides (10.2%), and Rhipicephalus microplus (2%). The total prevalence of tick infestation in dogs was 61%. The district with the highest tick prevalence rate in dogs was Mardan (14.7%), followed by Peshawar (13%), Swabi (12%), Charsadda (11%), and Malakand (10.3%), respectively. Risk factors analysis indicated that some demographic and host management-associated factors such as host age, breed, exposure to acaricides treatment, and previous tick infestation history were associated with a higher risk of tick infestation on dogs. This is the first molecular report confirming the infestation of Hyalomma and Rhipicephalus tick species in the dog population from the study area. The present study also reported a new tick–host association between Hy. excavatum, Hy. dromedarii, and dogs. Phylogenetic analysis revealed that cox1 partial nucleotide sequences of Hy. excavatum in our dataset were 100% identical to similar tick specimens identified in Turkey, and those of Hy. dromedarii were identical to tick specimens from Iran. Whereas, Rh. haemaphysaloides and Rh. microplus’ cox1 partial nucleotide sequences were identical to sequences previously published from Pakistan. Rhipicephalus turanicus s.s. ‘s cox1 isolates from the present study were 99.8–100% identical to Pakistani-reported isolates, and those of Rh. sanguineus s.l. were 100% identical to Chinese specimens. Results on the genetic characterization of ticks were further confirmed by 16S rRNA partial nucleotide sequences analysis, which revealed 100% identity between the tick isolates of this study and those of Hy. excavatum reported from Turkey; Hy. dromedarii specimens reported from Senegal; Rh. haemaphysaloides, Rh. microplus, and Rh. turanicus s.s., previously published from Pakistan, and Rh. sanguineus s.l., published from China. Furthermore, phylogenetic analysis showed that the Rh. sanguineus s.l. isolates of this study clustered with specimens of the tropical lineage with 7.7–10% nucleotide divergence from the specimens of the temperate lineage. Further molecular works need to be performed throughout Pakistan to present a more detailed map of tick distribution with information about dog host associations, biological characteristics, and pathogen competence.
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Hyalomma
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