Local immune response to larvae of Rhipicephalus microplus in Santa Gertrudis cattle.

This study investigated the local immune response at larval attachment sites in Santa Gertrudis cattle with low and high levels of tick resistance. Skin samples with tick larvae attached were collected from Santa Gertrudis cattle at the end of a period of 25 weekly infestations, when the animals manifested highly divergent tick-resistant phenotypes. There was a tendency for more CD3+ , CD4+ , CD8+ , CD25+ , γδ T cells and neutrophils to concentrate at larval tick attachment site in susceptible cattle than in resistant cattle but the differences were significant only for γδ T cells and CD4+ cells. Most of the cattle developed intra-epidermal vesicles at the larval attachment site but the predominant cell within or around the vesicles was the neutrophil in susceptible animals and eosinophil in the resistant animals. The monoclonal antibodies (mAbs) specific for CD45 and CD45 RO antigens reacted with skin leucocytes from a higher number of susceptible cattle than resistant cattle. Our data suggest that some of the cellular responses mounted at larval attachment site are not involved in tick protection. The mAbs specific for CD45 and CD45 RO directly, or a test for CD45 genotype might be developed as markers of tick susceptibility or resistance.

Peripheral cellular and humoral responses to infestation with the cattle tick Rhipicephalus microplus in Santa Gertrudis cattle.

Resistance to cattle tick infestation in single-host ticks is primarily manifested against the larval stage and results in the immature tick failing to attach successfully and obtain a meal. This study was conducted to identify immune responses that characterize the tick-resistant phenotype in cattle. Thirty-five tick-naïve Santa Gertrudis heifers were used in this study, thirty of which were artificially infested for thirteen weeks with tick larvae while five animals remained at a tick-free quarantine property to serve as a control group. Following thirteen weeks of tick infestation, the animals in this trial exhibited highly divergent tick-resistant phenotypes. Blood samples collected throughout the trial were used to measure peripheral immune parameters: haematology, the percentage of cellular subsets comprising the peripheral blood mononuclear cell (PBMC) population, tick-specific IgG1 and IgG2 antibody titres, IgG1 avidity for tick antigens and the ability of PBMC to recognize and proliferate in response to stimulation with tick antigens in vitro. The tick-susceptible cattle developed significantly higher tick-specific IgG1 antibody titres compared to the tick-resistant animals. These results suggest that the heightened antibody response either does not play a role in resistance or might contribute to increased susceptibility to infestation.

Strategies for new and improved vaccines against ticks and tick-borne diseases.

Ticks infest a variety of animal species and transmit pathogens causing disease in both humans and animals worldwide. Tick-host-pathogen interactions have evolved through dynamic processes that accommodated the genetic traits of the hosts, pathogens transmitted and the vector tick species that mediate their development and survival. New approaches for tick control are dependent on defining molecular interactions between hosts, ticks and pathogens to allow for discovery of key molecules that could be tested in vaccines or new generation therapeutics for intervention of tick-pathogen cycles. Currently, tick vaccines constitute an effective and environmentally sound approach for the control of ticks and the transmission of the associated tick-borne diseases. New candidate protective antigens will most likely be identified by focusing on proteins with relevant biological function in the feeding, reproduction, development, immune response, subversion of host immunity of the tick vector and/or molecules vital for pathogen infection and transmission. This review addresses different approaches and strategies used for the discovery of protective antigens, including focusing on relevant tick biological functions and proteins, reverse genetics, vaccinomics and tick protein evolution and interactomics. New and improved tick vaccines will most likely contain multiple antigens to control tick infestations and pathogen infection and transmission.

Specific detection of the Old World screwworm fly, Chrysomya bezziana, in bulk fly trap catches using real-time PCR.

The Old World screwworm fly (OWS), Chrysomya bezziana Villeneuve (Diptera: Calliphoridae), is a myiasis-causing blowfly of major concern for both animals and humans. Surveillance traps are used in several countries for early detection of incursions and to monitor control strategies. Examination of surveillance trap catches is time-consuming and is complicated by the presence of morphologically similar flies that are difficult to differentiate from Ch. bezziana, especially when the condition of specimens is poor. A molecular-based method to confirm or refute the presence of Ch. bezziana in trap catches would greatly simplify monitoring programmes. A species-specific real-time polymerase chain reaction (PCR) assay was designed to target the ribosomal DNA internal transcribed spacer 1 (rDNA ITS1) of Ch. bezziana. The assay uses both species-specific primers and an OWS-specific Taqman((R)) MGB probe. Specificity was confirmed against morphologically similar and related Chrysomya and Cochliomyia species. An optimal extraction protocol was developed to process trap catches of up to 1000 flies and the assay is sensitive enough to detect one Ch. bezziana in a sample of 1000 non-target species. Blind testing of 29 trap catches from Australia and Malaysia detected Ch. bezziana with 100% accuracy. The probability of detecting OWS in a trap catch of 50 000 flies when the OWS population prevalence is low (one in 1000 flies) is 63.6% for one extraction. For three extractions (3000 flies), the probability of detection increases to 95.5%. The real-time PCR assay, used in conjunction with morphology, will greatly increase screening capabilities in surveillance areas where OWS prevalence is low.

Real-time polymerase chain reaction (PCR) assays for the specific detection and quantification of seven Eimeria species that cause coccidiosis in chickens.

Coccidiosis of chickens is an economically important disease caused by infection with species of Eimeria. The oocysts of some of the seven recognized species are difficult to distinguish morphologically and for this reason diagnostic laboratories are increasingly utilizing DNA-based technologies for the specific identification of Eimeria. The real-time PCR provides both sensitivity and speed for the analysis of DNA samples, and the approach has the capability of quantifying DNA. Together with a protocol for the extraction of DNA directly from faecal samples, real-time PCR assays have been established for the detection and quantification of seven species of Eimeria that infect chickens in Australia. The assays target one genetic marker, the second internal transcribed spacer of nuclear ribosomal DNA (ITS-2), use TaqMan MGB technology with species-specific probes, and can be multiplexed in pairs such that the seven species of Eimeria can be screened in four reaction tubes. A test screen of commercial flocks identified more Eimeria-infected chickens than were detected by coproscopic examination for oocysts. These molecular assays can also be used for the quality control of mixed-species vaccines. The ability to multiplex the assays makes them particularly practical for screening samples from chickens with mixed-species infections where the relative abundance of each Eimeria species present is required.

A review of reverse vaccinology approaches for the development of vaccines against ticks and tick borne diseases.

The field of reverse vaccinology developed as an outcome of the genome sequence revolution. Following the introduction of live vaccinations in the western world by Edward Jenner in 1798 and the coining of the phrase 'vaccine', in 1881 Pasteur developed a rational design for vaccines. Pasteur proposed that in order to make a vaccine that one should 'isolate, inactivate and inject the microorganism' and these basic rules of vaccinology were largely followed for the next 100 years leading to the elimination of several highly infectious diseases. However, new technologies were needed to conquer many pathogens which could not be eliminated using these traditional technologies. Thus increasingly, computers were used to mine genome sequences to rationally design recombinant vaccines. Several vaccines for bacterial and viral diseases (i.e. meningococcus and HIV) have been developed, however the on-going challenge for parasite vaccines has been due to their comparatively larger genomes. Understanding the immune response is important in reverse vaccinology studies as this knowledge will influence how the genome mining is to be conducted. Vaccine candidates for anaplasmosis, cowdriosis, theileriosis, leishmaniasis, malaria, schistosomiasis, and the cattle tick have been identified using reverse vaccinology approaches. Some challenges for parasite vaccine development include the ability to address antigenic variability as well the understanding of the complex interplay between antibody, mucosal and/or T cell immune responses. To understand the complex parasite interactions with the livestock host, there is the limitation where algorithms for epitope mining using the human genome cannot directly be adapted for bovine, for example the prediction of peptide binding to major histocompatibility complex motifs. As the number of genomes for both hosts and parasites increase, the development of new algorithms for pan-genomic mining will continue to impact the future of parasite and ricketsial (and other tick borne pathogens) disease vaccine development.

Evaluation and histological examination of a Campylobacter fetus subsp. venerealis small animal infection model.

Bovine genital campylobacteriosis (BGC), caused by Campylobacter fetus subsp. venerealis, is associated with production losses in cattle worldwide. This study aimed to develop a reliable BGC guinea pig model to facilitate future studies of pathogenicity, abortion mechanisms and vaccine efficacy. Seven groups of five pregnant guinea pigs (1 control per group) were inoculated with one of three strains via intra-peritoneal (IP) or intra-vaginal routes. Samples were examined using culture, PCR and histology. Abortions ranged from 0% to 100% and re-isolation of causative bacteria from sampled sites varied with strain, dose of bacteria and time to abortion. Histology indicated metritis and placentitis, suggesting that the bacteria induce inflammation, placental detachment and subsequent abortion. Variation of virulence between strains was observed and determined by culture and abortion rates. IP administration of C. fetus subsp. venerealis to pregnant guinea pigs is a promising small animal model for the investigation of BGC abortion.

Immuno-fluorescence staining patterns of leukocyte subsets in the skin of taurine and indicine cattle.

The immuno-staining patterns of skin leukocytes were investigated in three breeds of cattle: Holstein-Friesian, Brahman and Santa Gertrudis of similar age before and after tick infestation. The antibodies specific for CD45 and CD45RO reacted with cells in the skin of all Holstein-Friesian cattle but did not react with cells in the skin of any Brahman cattle. The same antibodies reacted with cells from the skin of four (CD45) and seven (CD45RO) of twelve Santa Gertrudis cattle. The antibodies specific for T cells and γδ subset of T cells recognized cells from all three breeds of cattle. The antibody specific for MHC class II molecules labelled cells of mostly irregular shape, presumably dermal dendritic cells and/or macrophages and Langerhans cells. The antibody specific for granulocytes (mAb CH138) reacted with cells only in sections cut from skin with lesions. The antibody specific for CD25(+) cells labelled regularly shaped cells that showed a wide range of intensities of staining.

Gene expression evidence for off-target effects caused by RNA interference-mediated gene silencing of Ubiquitin-63E in the cattle tick Rhipicephalus microplus.

Knowledge of cattle tick (Rhipicephalus (Boophilus) microplus; Acari: Ixodidae) molecular and cellular pathways has been hampered by the lack of an annotated genome. In addition, most of the tick expressed sequence tags (ESTs) available to date consist of ∼50% unassigned sequences without predicted functions. The most common approach to address this has been the application of RNA interference (RNAi) methods to investigate genes and their pathways. This approach has been widely adopted in tick research despite minimal knowledge of the tick RNAi pathway and double-stranded RNA (dsRNA) uptake mechanisms. A strong knockdown phenotype of adult female ticks had previously been observed using a 594 bp dsRNA targeting the cattle tick homologue for the Drosophila Ubiquitin-63E gene leading to nil or deformed eggs. A NimbleGen cattle tick custom microarray based on the BmiGI.V2 database of R. microplus ESTs was used to evaluate the expression of mRNAs harvested from ticks treated with the tick Ubiquitin-63E 594 bp dsRNA compared with controls. A total of 144 ESTs including TC6372 (Ubiquitin-63E) were down-regulated with 136 ESTs up-regulated following treatment. The results obtained substantiated the knockdown phenotype with ESTs identified as being associated with ubiquitin proteolysis as well as oogenesis, embryogenesis, fatty acid synthesis and stress responses. A bioinformatics analysis was undertaken to predict off-target effects (OTE) resulting from the in silico dicing of the 594 bp Ubiquitin-63E dsRNA which identified 10 down-regulated ESTs (including TC6372) within the list of differentially expressed probes on the microarrays. Subsequent knockdown experiments utilising 196 and 109 bp dsRNAs, and a cocktail of short hairpin RNAs (shRNA) targeting Ubiquitin-63E, demonstrated similar phenotypes for the dsRNAs but nil effect following shRNA treatment. Quantitative reverse transcriptase PCR analysis confirmed differential expression of TC6372 and selected ESTs. Our study demonstrated the minimisation of predicted OTEs in the shorter dsRNA treatments (∼100-200 bp) and the usefulness of microarrays to study knockdown phenotypes.

Local immune response against larvae of Rhipicephalus (Boophilus) microplus in Bos taurus indicus and Bos taurus taurus cattle.

Bos taurus indicus cattle are less susceptible to infestation with Rhipicephalus (Boophilus) microplus than Bos taurus taurus cattle but the immunological basis of this difference is not understood. We compared the dynamics of leukocyte infiltrations (T cell subsets, B cells, major histocompatibility complex (MHC) class II-expressing cells, granulocytes) in the skin near the mouthparts of larvae of R. microplus in B. t. indicus and B. t. taurus cattle. Previously naïve cattle were infested with 50,000 larvae (B. t. indicus) or 10,000 larvae (B. t. taurus) weekly for 6 weeks. One week after the last infestation all of the animals were infested with 20,000 larvae of R. microplus. Skin punch biopsies were taken from all animals on the day before the primary infestation and from sites of larval attachment on the day after the first, second, fourth and final infestations. Infiltrations with CD3(+), CD4(+), CD8(+) and gammadelta T cells followed the same pattern in both breeds, showing relatively little change during the first four weekly infestations, followed by substantial increases at 7 weeks post-primary infestation. There was a tendency for more of all cell types except granulocytes to be observed in the skin of B. t. indicus cattle but the differences between the two breeds were consistently significant only for gammadelta T cells. Granulocyte infiltrations increased more rapidly from the day after infestation and were higher in B. t. taurus cattle than in B. t. indicus. Granulocytes and MHC class II-expressing cells infiltrated the areas closest to the mouthparts of larvae. A large volume of granulocyte antigens was seen in the gut of attached, feeding larvae.