Survey of Borreliae in ticks, canines, and white-tailed deer from Arkansas, U.S.A.

BACKGROUND: In the Eastern and Upper Midwestern regions of North America, Ixodes scapularis (L.) is the most abundant tick species encountered by humans and the primary vector of B. burgdorferi, whereas in the southeastern region Amblyomma americanum (Say) is the most abundant tick species encountered by humans but cannot transmit B. burgdorferi. Surveys of Borreliae in ticks have been conducted in the southeastern United States and often these surveys identify B. lonestari as the primary Borrelia species, surveys have not included Arkansas ticks, canines, or white-tailed deer and B. lonestari is not considered pathogenic. The objective of this study was to identify Borrelia species within Arkansas by screening ticks (n=2123), canines (n=173), and white-tailed deer (n=228) to determine the identity and locations of Borreliae endemic to Arkansas using PCR amplification of the flagellin (flaB) gene. METHODS: Field collected ticks from canines and from hunter-killed white-tailed were identified to species and life stage. After which, ticks and their hosts were screened for the presence of Borrelia using PCR to amplify the flaB gene. A subset of the positive samples was confirmed with bidirectional sequencing. RESULTS: In total 53 (21.2%) white-tailed deer, ten (6%) canines, and 583 (27.5%) Ixodid ticks (252 Ixodes scapularis, 161 A. americanum, 88 Rhipicephalus sanguineus, 50 Amblyomma maculatum, 19 Dermacentor variabilis, and 13 unidentified Amblyomma species) produced a Borrelia flaB amplicon. Of the positive ticks, 324 (22.7%) were collected from canines (151 A. americanum, 78 R. sanguineus, 43 I. scapularis, 26 A. maculatum, 18 D. variabilis, and 8 Amblyomma species) and 259 (37.2%) were collected from white-tailed deer (209 I. scapularis, 24 A. maculatum, 10 A. americanum, 10 R. sanguineus, 1 D. variabilis, and 5 Amblyomma species). None of the larvae were PCR positive. A majority of the flaB amplicons were homologous with B. lonestari sequences: 281 of the 296 sequenced ticks, 3 canines, and 27 deer. Only 22 deer, 7 canines, and 15 tick flaB amplicons (12 I. scapularis, 2 A. maculatum, and 1 Amblyomma species) were homologous with B. burgdorferi sequences. CONCLUSIONS: Data from this study identified multiple Borreliae genotypes in Arkansas ticks, canines and deer including B. burgdorferi and B. lonestari; however, B. lonestari was significantly more prevalent in the tick population than B. burgdorferi. Results from this study suggest that the majority of tick-borne diseases in Arkansas are not B. burgdorferi.

Genetic diversity of Aedes vexans (Diptera, Culicidae) from New Orleans: pre- and post-Katrina.

The floodwater mosquito Aedes vexans, a potential vector of West Nile virus, has a worldwide distribution that includes the continental United States and southern Canada. In order to determine the effect that Hurricane Katrina had on the temporal genetic variation of Ae. vexans from New Orleans, we compared genetic diversity of a portion of the mtDNA ND5 gene of mosquito specimens collected during 2005 (n = 99) with specimens from 2006 (n = 103), after the hurricane. Average haplotype diversity (Hd) was high (>0.88) in 2005 and 2006 for both the parishes studied. It does not appear that Hurricane Katrina had any impact on genetic diversity, and despite the intense efforts to control mosquitoes in New Orleans, Ae. vexans has not undergone a population bottleneck. A bottleneck effect may be lacking because this species breeds outside the city and the adults migrate into the city.

Rickettsiae in Gulf Coast ticks, Arkansas, USA.

To determine the cause of spotted fever cases in the southern United States, we screened Gulf Coast ticks (Amblyomma maculatum) collected in Arkansas for rickettsiae. Of the screened ticks, 30% had PCR amplicons consistent with Rickettsia parkeri or Candidatus Rickettsia amblyommii.

Male-produced aggregation pheromone of the lesser mealworm beetle, Alphitobius diaperinus.

The lesser mealworm beetle, Alphitobius diaperinus (Panzer), is a widespread serious pest in poultry production facilities and is difficult to control by conventional means. Although pheromone-based tools have become useful in the management of other beetle pests, no pheromone was known for A. diaperinus, and this study sought to develop basic pheromone information. Volatiles were collected in the laboratory from groups of male and female A. diaperinus maintained on poultry food (chick starter mash). Gas chromatographic-mass spectrometric analysis of volatiles collected from feeding males and females revealed five male-specific compounds that were identified as (R)-(+)-limonene, (E)-beta-ocimene, (S)-(+)-linalool, (R)-(+)-daucene, and 2-nonanone. Emission of these began 1-2 weeks after adult emergence and could continue for at least 1 year, ceasing and resuming in response to changes in food availability and quality and other factors. No female-specific compounds were discovered. A synthetic blend of the five male compounds was attractive to both sexes in poultry production facilities in Illinois and Arkansas, indicating that the blend functions as an aggregation pheromone, but it is not yet known whether all five compounds are required for activity. A new pitfall trap is described for field use.

Mitochondrial and ribosomal internal transcribed spacer 1 diversity of Cimex lectularius (Hemiptera: Cimicidae).

Understanding genetic variation among populations of medically significant pest insects is important in studying insecticide resistance and insect dispersal. The bed bug, Cimex lectularius L. (Hemiptera: Cimicidae), is widespread hematophagus insect pest around the world, including North America, and it has recently been identified as an emerging resurgent pest. To date, no studies have been conducted on genetic variation of this species. For this study, 136 adult bed bugs representing 22 sampled populations from nine U.S. states, Canada, and Australia were subjected to genetic analysis using polymerase chain reaction (PCR) to amplify and sequence a region of the mitochondrial DNA (mtDNA) 16S rRNA gene and a portion of the nuclear rRNA internal transcribed spacer (ITS) 1 region. For the 397-bp 16S marker, a 12 nucleotide sites in total were polymorphic, and 19 unique haplotypes were observed. Heterozygosity was observed within many of the sampled populations for the mtDNA marker. This suggests that bed bug populations did not undergo a genetic bottleneck as one would expect from insecticide control during the 1940s and 1950s, but instead, that populations may have been maintained on other hosts such as birds and bats. In contrast to the high amount of heterozygosity observed with the mitochondrial DNA marker, no genetic variation in the 589-bp nuclear rRNA marker was observed. This suggests increased gene flow of previously isolated bed bug populations in the United States, and given the absence of barriers to gene flow, the spread of insecticide resistance may be rapid.

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.

Reservoir competence of lesser mealworm (Coleoptera: Tenebrionidae) for Campylobacter jejuni (Campylobacterales: Campylobacteraceae).

The lesser mealworm, Alphitobius diaperinus (Panzer), is a carrier of Campylobacter spp. in poultry facilities; however, the beetle's importance in the epidemiology of campylobacteriosis is not known. A series of laboratory experiments were designed to test the vector and reservoir competence of the lesser mealworm for Campylobacter jejuni. In the first experiment, C. jejuni was swabbed onto the outer surface of adult and larval beetles to determine how long bacteria can survive on the beetles' exterior. Next, adult and larval mealworms were allowed to drink from a solution containing C. jejuni and the duration of internal carriage was monitored. For the third experiment, beetles drank from a Campylobacter suspension and the duration of fecal shedding of bacteria was determined. In the last experiment, 3-d-old chickens were fed either one or 10 infected beetles, and cloacal swabs were tested periodically for Campylobacter. C. jejuni was detected on the exterior of larval beetles for 12 h, from the interior of larvae for 72 h, and from the feces of larvae for 12 h after exposure. Ninety percent of the birds that consumed a single adult or larval beetles became Campylobacter-positive, whereas 100% of the birds that consumed 10 adults or larvae became positive. These experiments demonstrated that the lesser mealworm could acquire and harbor Campylobacter from an environmental source. We found that the lesser mealworm was capable of passing viable bacteria to chickens that consumed the beetle. The beetle should be included in attempts to maintain Campylobacter-free poultry facilities.

Molecular assay for the detection of Cochlosoma anatis in house flies and turkey specimens by polymerase chain reaction.

A 1520 bp region of Cochlosoma anatis mtDNA 16S gene was subjected to DNA sequencing and a 466 bp portion was compared with other protozoan 16S sequences to develop PCR primers specific for C. anatis. This PCR diagnostic method allowed identification of C. anatis from house flies, Musca domestica L., turkey gut, and fecal samples within 6 h after field-collected samples reached the laboratory. House flies detected carrying C. anatis using the diagnostic 374 bp amplicons represented the first record of this protozoan in house flies.