Pia U. Olafson

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.

Widespread movement of invasive cattle fever ticks (Rhipicephalus microplus) in southern Texas leads to shared local infestations on cattle and deer

BackgroundRhipicephalus (Boophilus) microplus is a highly-invasive tick that transmits the cattle parasites (Babesia bovis and B. bigemina) that cause cattle fever. R. microplus and Babesia are endemic in Mexico and ticks persist in the United States inside a narrow tick eradication quarantine area (TEQA) along the Rio Grande. This containment area is threatened by unregulated movements of illegal cattle and wildlife like white-tailed deer (WTD; Odocoileus virginianus).MethodsUsing 11 microsatellite loci we genotyped 1,247 R. microplus from 63 Texas collections, including outbreak infestations from outside the TEQA. We used population genetic analyses to test hypotheses about ecological persistence, tick movement, and impacts of the eradication program in southern Texas. We tested acaricide resistance with larval packet tests (LPTs) on 47 collections.ResultsLPTs revealed acaricide resistance in 15/47 collections (32%); 11 were outside the TEQA and three were resistant to multiple acaricides. Some collections highly resistant to permethrin were found on cattle and WTD. Analysis of genetic differentiation over time at seven properties revealed local gene pools with very low levels of differentiation (FST 0.00-0.05), indicating persistence over timespans of up to 29 months. However, in one neighborhood differentiation varied greatly over a 12-month period (FST 0.03-0.13), suggesting recurring immigration from distinct sources as another persistence mechanism. Ticks collected from cattle and WTD at the same location are not differentiated (FST = 0), implicating ticks from WTD as a source of ticks on cattle (and vice versa) and emphasizing the importance of WTD to tick control strategies. We identified four major genetic groups (K = 4) using Bayesian population assignment, suggesting multiple introductions to Texas.ConclusionsTwo dispersal mechanisms give rise to new tick infestations: 1) frequent short-distance dispersal from the TEQA; and 2) rare long-distance, human-mediated dispersal from populations outside our study area, probably Mexico. The threat of cattle fever tick transport into Texas is increased by acaricide resistance and the ability of R. microplus to utilize WTD as an alternate host. Population genetic analyses may provide a powerful tool for tracking invasions in other parts of the world where these ticks are established.

R86Q, a Mutation in BmAChE3 Yielding a Rhipicephalus microplus Organophosphate-Insensitive Acetylcholinesterase

Abstract 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.

Multiple mutations in the para-sodium channel gene are associated with pyrethroid resistance in Rhipicephalus microplus from the United States and Mexico

BackgroundAcaricide resistant Rhipicephalus microplus populations have become a major problem for many cattle producing areas of the world. Pyrethroid resistance in arthropods is typically associated with mutations in domains I, II, III, and IV of voltage-gated sodium channel genes. In R. microplus, known resistance mutations include a domain II change (C190A) in populations from Australia, Africa, and South America and a domain III mutation (T2134A) that only occurs in Mexico and the U.S.MethodsWe investigated pyrethroid resistance in cattle fever ticks from Texas and Mexico by estimating resistance levels in field-collected ticks using larval packet discriminating dose (DD) assays and identifying single nucleotide polymorphisms (SNPs) in the para-sodium channel gene that associated with resistance. We then developed qPCR assays for three SNPs and screened a larger set of 1,488 R. microplus ticks, representing 77 field collections and four laboratory strains, for SNP frequency.ResultsWe detected resistance SNPs in 21 of 68 U.S. field collections and six of nine Mexico field collections. We expected to identify the domain III SNP (T2134A) at a high frequency; however, we only found it in three U.S. collections. A much more common SNP in the U.S. (detected in 19 of 21 field collections) was the C190A domain II mutation, which has never before been reported from North America. We also discovered a novel domain II SNP (T170C) in ten U.S. and two Mexico field collections. The T170C transition mutation has previously been associated with extreme levels of resistance (super-knockdown resistance) in insects. We found a significant correlation (r = 0.81) between the proportion of individuals in field collections that carried any two resistance SNPs and the percent survivorship of F1 larvae from these collections in DD assays. This relationship is accurately predicted by a simple linear regression model (R2 = 0.6635).ConclusionsThese findings demonstrate that multiple mutations in the para-sodium channel gene independently associate with pyrethroid resistance in R. microplus ticks, which is likely a consequence of human-induced selection.

Bm86 midgut protein sequence variation in South Texas cattle fever ticks

BackgroundCattle fever ticks, Rhipicephalus (Boophilus) microplus and R. (B.) annulatus, vector bovine and equine babesiosis, and have significantly expanded beyond the permanent quarantine zone established in South Texas. Currently, there are no vaccines approved for use within the United States for controlling these vectors. Vaccines developed in Australia and Cuba based on the midgut antigen Bm86 have variable efficacy against cattle fever ticks. A possible explanation for this variation in vaccine efficacy is amino acid sequence divergence between the recombinant Bm86 vaccine component and native Bm86 expressed in ticks from different geographical regions of the world.ResultsThere was 91.8% amino acid sequence identity in Bm86 among R. microplus and R. annulatus sequenced from South Texas infestations. When South Texas isolates were compared to the Australian Yeerongpilly and Cuban Camcord vaccine strains, there was 89.8% and 90.0% identity, respectively. Most of the sequence divergence was focused in one region of the protein, amino acids 206-298. Hydrophilicity profiles revealed that two short regions of Bm86 (amino acids 206-210 and 560-570) appear to be more hydrophilic in South Texas isolates compared to vaccine strains. Only one amino acid difference was found between South Texas and vaccine strains within two previously described B-cell epitopes. A total of 4 amino acid differences were observed within three peptides previously shown to induce protective immune responses in cattle.ConclusionsSequence differences between South Texas isolates and Yeerongpilly and Camcord strains are spread throughout the entire Bm86 sequence, suggesting that geographic variation does exist. Differences within previously described B-cell epitopes between South Texas isolates and vaccine strains are minimal; however, short regions of hydrophilic amino acids found unique to South Texas isolates suggest that additional unique surface exposed peptides could be targeted.

Analysis of expressed sequence tags from a significant livestock pest, the stable fly (Stomoxys calcitrans), identifies transcripts with a putative role in chemosensation and sex determination.

The stable fly, Stomoxys calcitrans L. (Diptera: Muscidae), is one of the most significant pests of livestock in the United States. The identification of targets for the development of novel control for this pest species, focusing on those molecules that play a role in successful feeding and reproduction, is critical to mitigating its impact on confined and rangeland livestock. A database was developed representing genes expressed at the immature and adult life stages of the stable fly, comprising data obtained from pyrosequencing both immature and adult stages and from small-scale sequencing of an antennal/maxillary palp-expressed sequence tag library. The full-length sequence and expression of 21 transcripts that may have a role in chemosensation is presented, including 13 odorant-binding proteins, 6 chemosensory proteins, and 2 odorant receptors. Transcripts with potential roles in sex determination and reproductive behaviors are identified, including evidence for the sex-specific expression of stable fly doublesex- and transformer-like transcripts. The current database will be a valuable tool for target identification and for comparative studies with other Diptera.

Genotyping Mutations in BmAChE3: A Survey of Organophosphate-Resistant and -Susceptible Strains of Rhipicephalus (Boophilus) microplus

ABSTRACT Mutations I48L, I54V, R86Q, V137I, I492M, and T548A were identified previously in BmAChE3, a gene encoding acetylcholinesterase, from the organophosphate (OP) acaricide-resistant San Román strain of Rhipicephalus (Boophilus) microplus. Recombinant BmAChE3 acetylcholinesterase containing the R86Q mutation was shown to exhibit nearly 20-fold reduction in the rate of phosphorylation by paraoxon relative to the wild-type sequence. In addition, the R86Q mutation was present in resistant laboratory strains at elevated frequency compared with OP-susceptible strains but was insufficient to alone generate the OP-resistant phenotype (J. Med. Entomol. 44:1013–1018). Here, we developed assays to genotype the remaining five mutations and evaluated frequency of all six BmAChE3 mutations in individual R. microplus ticks from laboratory and Mexican field-collected strains. We found a substantial number of individuals in known OP-susceptible strains that seemed to be homozygous for each of the mutations surveyed, the exception being I48L, which was infrequent in all strains, leading us to conclude that none of the mutations alone were responsible for generation of phenotypic resistance to OP acaricide.

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.

Survival and fate of Salmonella enterica serovar Montevideo in adult horn flies (Diptera: Muscidae).

ABSTRACT Contamination of cattle peripheral lymph nodes with Salmonella enterica is proposed to occur via a transdermal route of entry. If so, bacteria may be introduced to cattle by biting arthropods. Biting flies, such as horn flies (Haematobia irritans irritans (L.)) (Diptera: Muscidae), are intriguing candidates for transmitting Salmonella to cattle because they provide a route of entry when they breach the skin barrier during blood feeding. Using a green fluorescent protein-expressing strain of Salmonella Montevideo (S. Montevideo-GFP), the current study demonstrated that horn fly grooming subsequent to tactile exposure to the bacteria resulted in acquisition of the bacteria on mouthparts as well as microbial ingestion. Consumption of a bloodmeal containing ≈102, ≈104, or ≈106 S. Montevideo-GFP resulted in horn fly colonization for up to 72 h postingestion (PI). Epifluorescent microscopy indicated that the bacteria were not localized to the crop but were observed within the endoperitrophic space, suggesting that regurgitation is not a primary route of transmission. S. Montevideo-GFP were cultured from excreta of 100% of flies beginning 6–7 h PI of a medium or high dose meal and >12 h PI in excreta from 60% of flies fed the low-dose meal. Animal hides and manure pats are sources for horn flies to acquire the Salmonella and mechanically transmit them to an animal while feeding. Mean quantities of 5.65- 67.5 × 102 CFU per fly were cultured from fly excreta passed within 1 d after feeding, suggesting the excreta can provide an additional microbial source on the animals hide.

Serologically defined Rhipicephalus (Boophilus) microplus larval antigens in BmLF3, a partially pure Sephacryl S-300 fraction of crude larval proteins.

This report is designed to provide additional information regarding larval soluble proteins toward the planned development of a comprehensive database of Rhipicephalus (Boophilus) microplus proteins that elicit a humoral immune response in cattle as a result of natural ectoparasite infestation. Larval proteins of R. microplus are complex and the protein profile is not dominated by any major proteins. This report focuses upon an S-300 Sephacryl (molecular sieve) column fraction, fraction 3 (BmLF3). With the use of SDS-PAGE (without-2ME) and Western blotting with a composite pool of pre- and post-R. microplus larval infestation antiserum BmLF3 was found to contain 7 apparent common ixodid major antigens (207.3, 171.9, 98.0, 86.5, 65.7, 58.9, and 38.0 kDa), those potentially shared with other ixodid species, and 2 apparent R. microplus specific antigens evidenced by low-level antibody binding in crude BmLF3 (149.4 kDa) and HPLC peak 8 of BmLF3 (116.0 kDa). In addition, BmLF3 contains potent inhibitors of trypsin activity. However, these inhibitors of trypsin did not appear to elicit host antibodies as a result of natural ectoparasite exposure, as defined by Western blotting of reduced and denatured trypsin binding proteins purified by affinity chromatography.