Differential protein expression in ovaries of uninfected and Babesia-infected southern cattle ticks, Rhipicephalus (Boophilus) microplus

We used gel electrophoresis and mass spectrometry to investigate differences in protein expression in ovarian tissues from Babesia bovis-infected and uninfected southern cattle tick, Rhipicephalus (Boophilus) microplus. Soluble and membrane proteins were extracted from ovaries of adult female ticks, and analyzed by isoelectric focusing (IEF) and one-dimensional or two-dimensional (2-D) gel electrophoresis. Protein patterns were analyzed for differences in expression between infected and uninfected ticks. 2-D separation of proteins revealed a number of proteins that appeared to be up- or down-regulated in response to infection with Babesia, in particular membrane/membrane-associated proteins and proteins in a low molecular mass range between 6 and 36 kDa. A selection of differentially expressed proteins was subjected to analysis by capillary-HPLC-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). Among the ovarian proteins that were up-regulated in infected ticks were calreticulin, two myosin subunits, an endoplasmic reticulum protein, a peptidyl-prolyl cis–trans isomerase (PPIase), a cytochrome c oxidase subunit, a glutamine synthetase, and a family of Kunitz-type serine protease inhibitors. Among the down-regulated ovarian proteins were another PPIase, a hemoglobin subunit, and a lysozyme. This study is part of an ongoing effort to establish a proteome database that can be utilized to investigate specific proteins involved in successful pathogen transmission.     

Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba) skin biopsies

Background  

Odontocete cetaceans occupy the top position of the marine food-web and are particularly sensitive to the bioaccumulation of lipophilic contaminants. The effects of environmental pollution on these species are highly debated and various ecotoxicological studies have addressed the impact of xenobiotic compounds on marine mammals, raising conservational concerns. Despite its sensitivity, quantitative real-time PCR (qRT-PCR) has never been used to quantify gene induction caused by exposure of cetaceans to contaminants. A limitation for the application of qRT-PCR is the need for appropriate reference genes which allow the correct quantification of gene expression. A systematic evaluation of potential reference genes in cetacean skin biopsies is presented, in order to validate future qRT-PCR studies aiming at using the expression of selected genes as non-lethal biomarkers.     

Selection of optimal reference genes for quantitative RT-PCR studies of boar spermatozoa cryopreservation

Reference genes can be used to normalize mRNA levels across different samples for the exact comparison of the mRNA expression level. It is important to select reference genes with high quality for the accurate interpretation of qRT-PCR data. Although several studies have attempted to validate reference genes in pigs, no validation studies have been performed on spermatozoa samples frozen with different cryoprotectants. In this study, 11 commonly used reference genes (ACTB, B2M, GAPDH, HPRT1, RPL4, SDHA, YWHAZ, PPIA, PGK1, S18, and BLM) were investigated in boar spermatozoa frozen with six different cryoprotectants using qRT-PCR. The expression stability of these reference genes in different samples was evaluated using geNorm (qbase^plus software), NormFinder, and BestKeeper. The geNorm results revealed that PGK1, ACTB, and RPL4 exhibit high expression stability in all of the samples, and the NormFinder results indicated that GAPDH is the most stable gene. Furthermore, the BestKeeper results indicated that the three most stable genes are PPIA, GAPDH, and RPL4 and that S18, B2M and BLM are the three least stable genes. There are a number of differences in the ranking order of the reference genes obtained using the different algorithms. In conclusion, GAPDH, RPL4, and PPIA were the three most stable genes in frozen boar spermatozoa, as determined based on the cycle threshold coefficient of variation (Ct CV%) and the comprehensive ranking order, and this finding is consistent with the BestKeeper results     

Two initial vaccinations with the Bm86-based Gavacplus vaccine against Rhipicephalus (Boophilus) microplus induce similar reproductive suppression to three initial vaccinations under production conditions

Background

The cattle tick, Rhipicephalus (Boophilus) microplus, affects livestock production in many regions of the world. Up to now, the widespread use of chemical acaricides has led to the selection of acaricide-resistant ticks and to environmental contamination. Gavac^plus is a subunit vaccine based on the recombinant Bm86 tick antigen expressed in yeast, capable to control infestations of R. microplus under controlled and production conditions. The vaccine constitutes the core element of broad control programs against this ectoparasite, in which acquired immunity in cattle to Bm86 is combined with a rational use of acaricides. At present, the conventional vaccine scheme consists of three doses that should be administered at weeks 0, 4 and 7, followed by a booster every six months.

Results

In this study we assayed a reduction in the number of the initial doses of Gavac^plus, evaluated the time course and the level of bovine anti-Bm86 antibodies elicited, and analyzed the vaccine effect on ticks engorging on immunized cattle under production conditions. Following three different immunization schemes, the bovines developed a strong and specific immune response characterized by elevated anti-Bm86 IgG titers. A reduction in the weight of engorging female ticks, in the weight of the eggs laid and also in R. microplus viable eggs percentage was obtained by using only two doses of Gavac^plus administered at weeks 0 and 4, followed by a booster six months later. This reduction did not differ from the results obtained on ticks engorging on cattle immunized at weeks 0, 4 and 7. It was also demonstrated that anti-Bm86 antibody titers over 1:640, measured in bovines immunized at weeks 0 and 4, were sufficient to affect weight and reproductive potential of female ticks as compared with ticks engorging on unvaccinated animals. In addition, no statistically significant differences were detected in the average weight of eggs laid by ticks engorged on immunized cattle that showed anti-Bm86 specific titers in the range of 1:640 to 1:81920.

Conclusion

The administration of two initial doses of Gavac^plus containing 100 μg of Bm86 antigen to non-immunized cattle under production conditions is sufficient to affect the weight and the reproductive capacity of R. microplus engorging females. According to these results, cattle herds' manipulation and vaccine costs could be potentially reduced with a positive impact on the implementation of integrated control programs against R. microplus.     

Selection of optimal reference genes for normalization in quantitative RT-PCR

Background  

Normalization in real-time qRT-PCR is necessary to compensate for experimental variation. A popular normalization strategy employs reference gene(s), which may introduce additional variability into normalized expression levels due to innate variation (between tissues, individuals, etc). To minimize this innate variability, multiple reference genes are used. Current methods of selecting reference genes make an assumption of independence in their innate variation. This assumption is not always justified, which may lead to selecting a suboptimal set of reference genes.     

Comparative studies on the invasion of cattle ticks (Rhipicephalus (Boophilus) microplus) and sheep blowflies (Lucilia cuprina) by Metarhizium anisopliae (Sorokin)

Microscopic investigations over time were carried out to study and compare the pathogenesis of invasion of ticks and blowflies by Metarhizium anisopliae. The scanning electron microscope and stereo light microscope were used to observe and record processes on the arthropods' surfaces and the compound light microscope was used to observe and record processes within the body cavities. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle, particularly the outer endocuticle. Although large numbers of conidia adhered to the surface of blowflies, no conidia were seen to germinate on external surfaces. A single germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggests that fungal invasion of the blowfly is primarily through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on tick cuticles, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16.     

Evidence for the utility of the Bm86 antigen from Boophilus microplus in vaccination against other tick species

The Bm86 antigen, as originally identified in Boophilus microplus, is the basis of commercial tick vaccines against this tick species. The potential for using this antigen or homologues of the antigen in vaccination against other tick species has been assessed. We have conducted vaccine trials in cattle using the B. microplus-derived recombinant Bm86 vaccine (TickGARD) using pairs of vaccinated calves and control calves. These were infested with B. microplus and Boophilus decoloratus larvae simultaneously. For both species, the numbers of engorged female adult ticks, their weight and egg-laying capacity were all reduced, leading to a reduction in reproductive capacity of 74% for B. microplus and 70% for B. decoloratus. Hyalomma anatolicum anatolicum ticks were fed both as immatures as well as adults on vaccinated calves and non-vaccinated controls. There was an overall 50% reduction in the total weight of nymphs engorging on vaccinated calves, and a suggestion of a subsequent effect on feeding adults. For Hyalomma dromedarii there was a 95% reduction in the number of nymphs engorging and a further 55% reduction in weight of those ticks surviving. Rhipicephalus appendiculatus and Amblyomma variegatum ticks were fed simultaneously both as immatures and subsequently as adults. There was no evidence for a significant vaccination effect. Finally, the amino acid sequence of a Bm86 homologue found in H. a. anatolicum unequivocally demonstrated the conservation of this molecule in this species. Our strategy for the development of multivalent anti-tick vaccines is discussed in relation to these findings.     

Evaluation of coffee reference genes for relative expression studies by quantitative real-time RT-PCR

Accuracy in quantitative real-time polymerase chain reaction (qPCR) requires the use of stable endogenous controls. Normalization with multiple reference genes is the gold standard, but their identification is a laborious task, especially in species with limited sequence information. Coffee (Coffea ssp.) is an important agricultural commodity and, due to its economic relevance, is the subject of increasing research in genetics and biotechnology, in which gene expression analysis is one of the most important fields. Notwithstanding, relatively few works have focused on the analysis of gene expression in coffee. Moreover, most of these works have used less accurate techniques such as northern blot assays instead of more accurate techniques (e.g., qPCR) that have already been extensively used in other plant species. Aiming to boost the use of qPCR in studies of gene expression in coffee, we uncovered reference genes to be used in a number of different experimental conditions. Using two distinct algorithms implemented by geNorm and Norm Finder, we evaluated a total of eight candidate reference genes (psaB, PP2A, AP47, S24, GAPDH, rpl39, UBQ10, and UBI9) in four different experimental sets (control versus drought-stressed leaves, control versus drought-stressed roots, leaves of three different coffee cultivars, and four different coffee organs). The most suitable combination of reference genes was indicated in each experimental set for use as internal control for reliable qPCR data normalization. This study also provides useful guidelines for reference gene selection for researchers working with coffee plant samples under conditions other than those tested here. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Resistance of the cattle tick Rhipicephalus (Boophilus) microplus to fluazuron in Argentina

The aim of this work is to report the presence of resistance to fluazuron in a population of Rhipicephalus microplus in Argentina. The evidence was obtained from field and in vitro trials. In the field trial, cattle infested with ticks was treated with two commercial formulations of fluazuron. The in vitro trial (adult immersion test, AIT) was performed by using technical grade fluazuron. In the field trial, there were no significant differences between the treated and control groups between days 2 and 34 post-treatment. The only exceptions (treated group I in day 14 post-treatment, treated group II in days 23 and 29 post-treatment) had a significantly lower tick load than the untreated group, but the efficacy was not higher than 70%. Viable engorged females were collected on both groups of treated bovines in all counts, and the production of viable larvae was not precluded with the application of the two commercial formulations of fluazuron evaluated in this study. The results obtained with the in vitro assay (AIT) also indicate that the R. microplus population tested in this work has a higher level of resistance to fluazuron than another susceptible field strain. The integrated analysis of the field and in vitro trials clearly reveals the emergence of resistance to fluazuron in a R. microplus population from Argentina. This diagnosis of resistance does not imply that the fluazuron has lost its functionality at a regional scale, but it highlights the need to establish control strategies that minimize the use of this drug in order to preserve its functionality as an acaricide.

     

Immunity against Boophilus annulatus induced by the Bm86 (Tick-GARD) vaccine

Friesian cattle were immunized with two inoculations of anti-tick Bm86 (Tick-GARD) vaccine and were challenged 30 or 90 d later with Boophilus annulatus larvae derived from 1.2 g of eggs. No nymphs or adult ticks were found on the immunized cattle during four weeks after challenge. Repeated infestations (2 to 4) with larvae on three other calves during a period of 160 and 390 d after the immunization did not result in development of nymphal and adult stages. In control, non-immunized cattle infested with corresponding batches of larvae 1380 to 4653 replete adult female ticks were collected. Larvae issued from Babesia bovis-infected female ticks transmitted the infection to Bm86-immunized cattle, but the progeny of B. bigemina-infected females did not. Since B. bigemina is transmitted exclusively by nymphal stages of Bo. annulatus these results support the observation that immunity induced by Bm86 affects the larval stage of this tick.     

Esterase profile of Rhipicephalus (Boophilus) microplus populations collected from Northern India exhibiting varied susceptibility to deltamethrin

Rhipicephalus (Boophilus) microplus is an economically important ectoparasite of cattle. Chemical acaricides remain the most practical method for control of these pests. During past two decades there have been increasing reports of resistance development against synthetic pyrethroids in tick populations of this species throughout the world. A study was conducted to determine the level of susceptibility of R. (B.) microplus to deltamethrin collected from different geographical locations of northern India. LPT bioassay results revealed LC(50) values of deltamethrin ranging from 0.035 to 0.00037?% A.I. Esterase profile of the tick larval extracts using native PAGE, revealed 5 bands of esterase activity designated EST-5 to EST-1A. Inhibitory tests recognized EST-1, EST-2 and EST-3 as Acetylcholinesterases (AchEs), EST-4 and EST-5 as Carboxylesterases (CaEs). The band intensity varied between tick populations of various locations, being more intense in case of the resistant populations. An extra band of esterase activity (EST-1A) was obtained in larval extracts of ticks from 3 locations. This increased esterase activity may be involved in the resistance development in these tick populations. Acaricide resistance is a multi-factorial phenomenon, thus other causes of increased resistance like sodium channel mutation and reduced drug penetration (e.g. cuticle thickening) and behavioural changes (e.g. avoiding the pesticides) are to be tested in future in order to confirm the basic cause of the resistance development in these acaricide resistant tick populations.     

A Rhipicephalus (Boophilus) microplus cathepsin with dual peptidase and antimicrobial activity

The cattle tick, Rhipicephalus (Boophilus) microplus, is a haematophagous arthropod responsible for considerable losses in the livestock industry. Immunological control with vaccines is a promising alternative to replace chemical acaricides. Due to their importance in parasite physiology, cysteine endopeptidases are potential targets. In a previous study, native Vitellin Degrading Cysteine Endopeptidase (VTDCE) was successfully tested as a vaccine antigen for bovines against R. microplus. In this work, nucleotide and amino acid VTDCE sequences were obtained from cDNA databanks, based on data from Edman sequencing and mass spectrometry. Subsequently, cloning and expression, purification, immunological and biochemical characterisation of the recombinant protein were performed to determine the biological importance of VTDCE. By Western blot, polyclonal antibodies produced against recombinant VTDCE recognised native VTDCE. Interestingly, molecular analysis showed that the VTDCE sequence has similarity to antimicrobial peptides. Indeed, experimental results revealed that VTDCE has an antimicrobial activity which is independent of endopeptidase activity. We believe that this is the first known study to show that an arthropod enzyme has antimicrobial activity.