Evaluation of methods for the isolation of high quality RNA from bovine and cervine hide biopsies.

Molecular investigations of the ruminant response to ectoparasites at the parasite-host interface are critically dependent upon the quality of RNA. The complexity of ruminant skin decreases the capacity to obtain high quality RNA from biopsy samples, which directly affects the reliability of data produced by gene expression experiments. Two methods for isolating total RNA from skin were compared and the use of 4M guanidinium isothiocyanate (GITC) during frozen storage of the specimens was evaluated. In addition, the best procedure for RNA isolation from bovine skin punch biopsies was also tested on white-tailed deer skin biopsies. Skin biopsy punches were collected and frozen prior to pulverization for RNA isolation. Total RNA quantity and integrity were determined by spectrophotometry and capillary electrophoresis technology, respectively. Significantly increased total RNA yield (P < 0.05) and higher integrity (P < 0.05) were obtained with a TRI Reagent® isolation method. Freezing and subsequent storage of bovine skin punch biopsies in 4 M GITC did not affect the amount or integrity of total RNA recovered by either RNA isolation method. However, quantity and integrity of total RNA extracted with the TRI Reagent method were again significantly higher than with the alternate technique, confirming it as the superior method. The TRI Reagent isolation method also yielded high quality total RNA from white-tailed deer skin punch biopsies, suggesting the usefulness of this method for obtaining RNA of a quality suitable for gene expression studies in other ruminant species.

Sequence polymorphism in acetylcholinesterase transcripts and genotyping survey of BmAchE1 in laboratory and Mexican strains of Rhipicephalus (Boophilus) microplus.

ABSTRACT Acetylcholinesterase cDNAs, BmAChE1, BmAChE2, and BmAChE3 of Rhipicephalus (Boophilus) microplus (Canestrini) were sequenced and found to exhibit significant polymorphism. A portion of the predicted amino acid substitutions in BmAChE1, BmAChE2, and BmAChE3 were found predominantly in organophosphate-resistant strains, but most did not correlate with resistant status. Multiple transcripts were observed from individual ticks, suggesting possible gene duplication or alternative splicing to produce more than two transcripts per individual. BmAChE1 transcript polymorphisms associating with organophosphate-resistant status in laboratory strains were surveyed in laboratory and Mexican strains of R. microplus by sequencing BmAChE1 genomic DNA. Quantitative real-time polymerase chain reaction was used to determine copy numbers of BmAChE1 (eight copies/haploid genome), BmAChE2 (16 copies/haploid genome), and BmAChE3 (four copies/haploid genome). Presence of at least three highly polymorphic amplified genes expressing AChE in tick synganglion suggested that ticks maintain a large and diverse assortment of AChE alleles available for rapid recombination and selection, which potentially reduces fitness costs associated with individual mutations. Elevated copy numbers for each of the BmAChEs may also explain previous failures to identify mutations resulting in insensitivity to organophosphates. It is clear that development of phenotypic resistance to organophosphates is highly complex and may be multigenic in character.

Multiple transcripts encode glucose 6-phosphate dehydrogenase in the southern cattle tick, Rhipicephalus (Boophilus) microplus.

Glucose 6-phosphate dehydrogenase (G6PDH) is an enzyme that plays a critical role in the production of NADPH. Here we describe the identification of four transcripts (G6PDH-A, -B, -C, and -D) that putatively encode the enzyme in the southern cattle tick, Rhipicephalus (Boophilus) microplus. The genomic DNA that is spliced to produce G6PDH-A and -B is 8,600-9,000 bases in length and comprises 12 exons. Comparison of the R. microplus G6PDH gene structure with those available from insects and mammals revealed that the tick gene is most like that of humans. Detection of the four transcripts was evaluated by quantitative RT-PCR using template from larvae, unfed adult females and males, salivary gland tissues from 2- to 3-day-fed adult females and males, and salivary gland tissue of 4- to 5-day-fed adult females. The G6PDH-A and -C transcripts were present in all templates, and both displayed induced expression in salivary gland tissue of fed, adult females but not matched males. The G6PDH-D transcript was detected only in unfed adults and in larvae, a stage in which it was most abundant relative to the other three transcripts. The G6PDH-B transcript, while detectable in all templates, was of low copy number suggesting it is a rare transcript. Induced expression of G6PDH-A and G6PDH-C in fed females may play a role in the tolerance of oxidative stress that is induced upon feeding, and the transcript abundance in fed females may be a function of bloodmeal volume and the time adult females spend on the host relative to adult males.

Baculovirus expression, biochemical characterization and organophosphate sensitivity of rBmAChE1, rBmAChE2, and rBmAChE3 of Rhipicephalus (Boophilus) microplus.

Rhipicephalus (Boophilus) microplus cDNAs, BmAChE1, BmAChE2, and BmAChE3, were previously identified as presumptively encoding acetylcholinesterases (AChEs), but biochemical identity was confirmed only for recombinant BmAChE3. In the present study, four recombinant BmAChE1 constructs and single recombinant constructs of BmAChE2 and BmAChE3 were expressed in baculovirus. Biochemical characterization of the recombinant proteins supports classification of rBmAChE1, rBmAChE2, and rBmAChE3 as AChEs (E.C.3.1.1.7), as evidenced by (i) substrate preference for acetylthiocholine, (ii) inhibition by eserine, BW284c51, and the organophosphates (OPs) malaoxon and paraoxon, (iii) insensitivity to iso-OMPA, and (iv) rapid hydrolysis of acetyl-beta-methyl-thiocholine. Unlike reports for insect AChEs, we did not observe substrate inhibition of activity at acetylthiocholine concentrations as high as 40 mM, however, product inhibition was apparent at 10-100 microM choline in agreement with properties reported for the catalytic domain of Anopheles gambiae acetylcholinesterase-1. Substrate affinity and V(max) values were highest for rBmAChE1 proteins, and one rBmAChE1 enzyme (Tx11, derived from the OP-resistant strain Tuxpan), was insensitive to paraoxon and exhibited a greatly reduced V(max) near that of rBmAChE2. To date, recombinant BmAChE1 and BmAChE3 enzymes with reduced sensitivity to OP-inhibition have been cloned and expressed from OP-resistant strains. The presence of at least three genes expressing AChEs in R. (B.) microplus, at least two of which contain mutations expressed as OP-insensitive enzymes, strongly suggests that phenotypic resistance to OPs may be complex and multigenic in character.

R86Q, a mutation in BmAChE3 yielding a Rhipicephalus microplus organophosphate-insensitive acetylcholinesterase.

Mutations were identified in the cDNA sequence encoding the acetylcholinesterase BmAChE3 in strains of Rhipicephalus (Boophilus) microplus (Canestrini) resistant or susceptible to organophosphate (OP) acaricide. The mutation that occurred most frequently in the OP-resistant San Román strain resulted in a substitution of glutamine (Q) for arginine (R) at position 86 in BmAChE3 (position 66 in mature BmAChE). Clones containing the mutant and wild-type cDNA sequences were expressed in the baculovirus system. Enzyme kinetics of recombinant BmAChE3 containing or lacking the R86Q mutation demonstrated that the R86Q mutation increased substrate affinity and conferred insensitivity to paraoxon inhibition. This is the first demonstration of a mutation in a gene encoding an ixodid acetylcholinesterase resulting in OP insensitivity. A restriction fragment length polymorphism assay was developed and used to diagnose the frequency of the R86Q mutation in BmAChE3 genomic DNA from seven laboratory-colonized strains. Use of the R86Q diagnostic assay detected an increased frequency of the R86Q mutation in OP-resistant tick strains compared with that of OP-susceptible strains; however, the R86Q mutation was also present in OP-susceptible strains at unexpectedly high frequency. Because the R86Q mutation generates an OP-resistant enzyme in vitro and it is present at an elevated frequency in laboratory strains selected for OP resistance, we conclude that the data are consistent with a potential role for BmAChE3 in development of OP resistance; however, because the R86Q mutation has a high frequency in susceptible strains, the R86Q mutation alone is insufficient to generate the OP-resistant phenotype at the organismal level. There are likely to be additional mutations in BmAChE3, mutations in additional acetylcholinesterase genes, or additional resistance mechanisms (e.g., oxidative metabolism) that contribute to expression of the OP-resistant phenotype.

Association of the bovine leukocyte antigen major histocompatibility complex class II DRB3*4401 allele with host resistance to the Lone Star tick, Amblyomma americanum.

The MHC of cattle, known as the bovine leukocyte antigen (BoLA) complex, plays an integral role in disease and parasite susceptibility, and immune responsiveness of the host. While susceptibility to tick infestation in cattle is believed to be heritable, genes that may be responsible for the manifestation of this phenotype remain elusive. In an effort to analyze the role that genes within the BoLA complex may play in host resistance to ticks, we have evaluated components of this system within a herd of cattle established at our laboratory that has been phenotyped for ectoparasite susceptibility. Of three microsatellite loci within the BoLA complex analyzed, alleles of two microsatellite loci within the BoLA class IIa cluster (DRB1-118 and DRB3-174) associated with the tick-resistant phenotype, prompting further investigation of gene sequences within the DRB3 region. DRB3 is a class IIa gene, the second exon of which is highly polymorphic since it encodes the antigen recognition site of the DR class II molecule. Analysis of the second exon of the DRB3 gene from the phenotyped calves in our herd revealed a significant association between the DRB3*4401 allele and the tick-resistant phenotype. To our knowledge, this is the first report of a putative association between a class IIa DRB3 sequence and host resistance to the Lone Star tick. Elucidation of the mechanism involved in tick resistance will contribute to improving breeding schemes for parasite resistance, which will be beneficial to the cattle industry.

Thrombostasin isoform frequency in a Central Texas field population of the horn fly, Haematobia irritans.

Thrombostasin is an anti-thrombin factor that plays a role in successful feeding of the horn fly, Haematobia irritans. It has been isolated and characterized from saliva, and polymorphisms in the gene coding sequence have been previously reported. In the present study, the thrombostasin gene was analyzed from 60, field-collected flies from Camp Stanley, Texas and the allele and genotype frequencies were compared with previously published data for an Alabama field collection and a Texas in vitro colony-reared collection. Significant differences in genotype frequency and extent of genotypic diversity observed between the Alabama and Camp Stanley field collections may be attributable to host genetic differences. In addition, bull calves that were phenotyped as either high- or low-carriers were parasitized by horn flies that displayed a significantly different genotype distribution, supporting a possible explanation for horn fly host selection behavior, as evidenced by thrombostasin sequence analysis of 95 additional horn flies collected from respective hosts.

Baculovirus expression of BmAChE3, a cDNA encoding an acetylcholinesterase of Boophilus microplus (Acari: Ixodidae).

The complete cDNA sequence encoding a Boophilus microplus (Canestrini) (Acari: Ixodidae) acetylcholinesterase (AChE3) was expressed in the baculovirus system. The recombinant AChE3 protein (rBmAChE3) was secreted as a soluble form into the cell culture medium and was identified as a functional AChE by substrate specificity and by inhibition with the AChE-specific inhibitors eserine sulfate and BW284c51. Inhibition kinetics of rBmAChE3, in the presence of the organophosphate paraoxon, revealed sensitivity comparable with that of adult, organophosphate-susceptible neural AChE. To our knowledge, this is the first report of the cloning and successful expression of a functional ixodid AChE.

Cloning and sequence analysis of a cDNA encoding Pso o II, a mite group II allergen of the sheep scab mite (Acari: Psoroptidae).

Psoroptes ovis (Hering), the sheep scab mite, is responsible for psoroptic scabies of cattle and sheep. Reverse translation of 30 N-terminal amino acids of the major P. ovis allergen, previously chosen as a candidate immunogen and identified as a 16 kDa protein yielded a degenerate sequence used to design oligodeoxynucleotide polymerase chain reaction (PCR) primers. Use of the PCR primers with a P. ovis cDNA library succeeded in amplification of a 90 bp cDNA gene fragment that was cloned, sequenced, and used to select unique sequencing/PCR primers. Primer walking generated overlapping subclones which yielded the 588 nucleotide consensus sequence of the cDNA encoding the 143 amino acid P. ovis allergen precursor. Nucleotide and translated sequences of the cDNA were compared with sequences in GenBank and found to be homologous to mite group II allergens Lep d II (formerly Lep d I) of Lepidoglyphus destructor Schrank, DerfII of Dermatophagoides farinae Hughes, Der p II of Derrmatophagoides pteronyssinus (Trouessart), Tyr p II of Tyrophagus putrescentiae (Schrank), Eur m II of Euroglyphus maynei (Cooreman) and Gly d II of Glycophagus domesticus (De Geer). The mature P. ovis allergen is composed of 126 amino acids with a calculated molecular mass of 13,468 Da, three disulfide bonds, and pI of 6.06 with one potential o-glycosylation site at Thr116. We designate the P. ovis 16 kDa protein as Pso o II in conformity with nomenclature for mite group II allergens.

Molecular and biochemical diagnosis of esterase-mediated pyrethroid resistance in a Mexican strain of Boophilus microplus (Acari: Ixodidae).

We examined pyrethroid resistant Mexican strains of Boophilus microplus using biochemical and molecular tests to determine the mechanisms conferring resistance. Permethrin hydrolysis assays and esterase activity gels indicated enhanced esterase-mediated metabolic detoxification in the Cz strain, while one other pyrethroid resistant strain, SF, and two pyrethroid susceptible strains had lower levels of permethrin hydrolysis. Results from assays using a PCR-based test to detect a pyrethroid target site resistance-associated mutation in the tick sodium channel gene found only low levels of mutations in the Cz strain, while the SF strain had a high level of the mutated sodium channel alleles. A specific esterase, designated CzEst9, believed to be responsible for the esterase-mediated pyrethroid resistance in the Cz strain was purified, and the gene encoding CzEst9 cloned.

Comparative inhibition kinetics for acetylcholinesterases extracted from organophosphate resistant and susceptible strains of Boophilus microplus (Acari: Ixodidae).

In this study, acetylcholinesterases (AChEs) were extracted from two Mexican Boophilus microplus strains that demonstrated resistance to the organophosphate (OP) acaricide, coumaphos, in bioassay. The rate of inhibition of the extracted AChEs by the diethyl-OP paraoxon was determined for two resistant strains and two susceptible strains of B. microplus. The time to inhibition of 50% AChE activity was approximately two-fold greater for the resistant strains. Kinetic analysis of the interaction of the resistant AChEs with paraoxon revealed reduced bimolecular reaction constants (ki). Apparent conformational changes in the AChE of the resistant strains were reflected in reduced Km and Vmax values. The bimolecular reaction constants (ki) of the resistant strains were most affected by a slower rate of enzyme phosphorylation (k2).