Molecular Detection of Rickettsia Species Within Ticks (Acari: Ixodidae) Collected from Arkansas United States.

Rocky Mountain spotted fever (RMSF), caused by the etiological agent Rickettsia rickettsii, is the most severe and frequently reported rickettsial illness in the United States, and is commonly diagnosed throughout the southeast. With the discoveries of Rickettsia parkeri and other spotted fever group rickettsiae (SFGR) in ticks, it remains inconclusive if the cases reported as RMSF are truly caused by R. rickettsii or other SFGR. Arkansas reports one of the highest incidence rates of RMSF in the country; consequently, to identify the rickettsiae in Arkansas, 1,731 ticks, 250 white-tailed deer, and 189 canines were screened by polymerase chain reaction (PCR) for the rickettsial genes gltA, rompB, and ompA. None of the white-tailed deer were positive, while two of the canines (1.1%) and 502 (29.0%) of the ticks were PCR positive. Five different tick species were PCR positive: 244 (37%) Amblyomma americanum L., 130 (38%) Ixodes scapularis Say, 65 (39%) Amblyomma maculatum (Koch), 30 (9%) Rhipicephalus sanguineus Latreille, 7 (4%) Dermacentor variabilis Say, and 26 (44%) unidentified Amblyomma ticks. None of the sequenced products were homologous to R. rickettsii. The most common Rickettsia via rompB amplification was Rickettsia montanensis and nonpathogenic Candidatus Rickettsia amblyommii, whereas with ompA amplification the most common Rickettsia was Ca. R. amblyommii. Many tick specimens collected in northwest Arkansas were PCR positive and these were commonly A. americanum harboring Ca. R. amblyommii, a currently nonpathogenic Rickettsia. Data reported here indicate that pathogenic R. rickettsii was absent from these ticks and suggest by extension that other SFGR are likely the causative agents for Arkansas diagnosed RMSF cases.

Population genetics of Amblyomma americanum (Acari: Ixodidae) collected from Arkansas.

Lone star ticks, Amblyomma americanum L. (Acari: Ixodidae),infest multiple hosts such as birds, and mammals of various sizes (rodents to white-tailed deer) and can harbor human pathogens such as Borrelia lonestari and Ehrlichiosis chaffeensis. The population structure of 251 A. americanum ticks, collected from canines and two white-tailed deer in six Arkansas ecoregions, was examined using DNA sequences of a 247-bp region of the mitochondrial DNA ribosomal RNA 16S gene. Of the 247 nucleotide characters, 26 were variable. Thirty-three haplotypes were identified of which 25 haplotypes occurred once (10%). The most common haplotype was Aa25, occurring in 60% of the samples and found in all six ecoregions. The excess of low frequency haplotypes combined with the overall negative Tajima's D and Fu and Li statistics suggests population expansion. Phylogenetic relationships of the 33 A. americanum haplotypes were constructed with other Amblyomma species and identified A. americanum as a monophyletic species with two groups. The patterns of high nucleotide and haplotype diversity found in this study suggests that the A. americanum population is expanding perhaps due to its ability to survive in a variety of habitats and feed on multiple hosts. Given the gene flow in Arkansas, the spread of acaricide resistance and pathogens may be rapid.

Phylogenetics and population genetics of the louse fly, Lipoptena mazamae, from Arkansas, USA.

Louse flies, also known as deer keds (Lipoptena mazamae Rondani), infest cervids such as white-tailed deer, Odocoileus virginianus and vector pathogens such as Anaplasma and Bartonella schoenbuchensis to cattle and humans, respectively. The population genetic structure of 30 L. mazamae collected from white-tailed deer in four regions of Arkansas, U.S.A., designated by county boundaries, was examined using DNA sequences of a 259-bp region of the mitochondrial DNA rRNA 16S gene. Of the 259 nucleotide characters, 33 were variable and 6 haplotypes were identified. Two haplotypes occurred only once (haplotype 3 and 4), whereas two other haplotypes occurred in 43% (haplotype 1 in two regions) and 40% (haplotype 6 in three regions) of the samples. Phylogenetic relationships of the six L. mazamae haplotypes were constructed with other Hippoboscid and Glossinid samples and two clades resulted. Clade 1 was located in the north and western Ozarks whereas clade 2 was found in the northern and eastern Ozarks. Results from the present study indicate that Lipoptena may be a polyphyletic genus; consequently, more research into genetic variation within this genus is necessary.

Effect of aggregation of horn fly populations within cattle herds and consequences for sampling to obtain unbiased estimates of abundance.

Reanalysis of counts of horn fly, Hematobia irritans (L.), obtained from a variety of cattle herds indicated that aggregation of the flies within herds decreased as mean fly density increased. Aggregation was also related to the proportion of fly-resistant and fly-susceptible cattle in a herd. Herds were grouped according to their degree of horn fly aggregation. Low aggregation herds included larger framed Angus, Horned Hereford, Polled Hereford, and Red Poll breeds. Moderate aggregation occurred with Brahman, Charolais, small-framed Angus, mixed cows, and Hereford x Charolais cross. High aggregation occurred with Chianina and mixed herds. Relationships between the sample means and variances varied among aggregation groups. A resampling approach was used to determine the influence of random sampling of a herd on the proportion of horn fly population estimates within fixed percentages of the true mean. The proportion of sample means within +/- 5, 10, 15, and 20% of the true means varied with the proportion of the herd sampled, the mean and variance of fly density, and herd size. Recommendations for obtaining sample size to estimate fly density within a fixed percentage of the true mean are given.

Feasibility of controlling Ixodes scapularis ticks (Acari: Ixodidae), the vector of Lyme disease, by parasitoid augmentation.

A theoretical analysis of the feasibility of controlling tick populations (Ixodidae) by the release of reared Ixodiphagus parasitoids in tick ecosystems yielded promising results. The analysis suggested that if reasonable progress could be made in mass-rearing the parasitoids, it would be possible to control the blacklegged tick, Ixodes scapularis (Say), the vector of Lyme disease, by this biological control procedure. Lyme disease has become the most important vector-borne disease in the United States. In a field-release experiment conducted in Africa by members of the International Center for Insect Physiology and Ecology, effective control of Amblyomma variegatum (F.) was obtained by the release of Ixodiphagus parasitoids in tick habitats. Encouraging theoretical results along with the encouraging results of a field-release experiment indicate the need for civil and political leaders in countries where ticks are a major problem to sponsor strong and well-coordinated research initiatives focused on the development of this new method of dealing with tick problems.

Reservoir competence of the lesser mealworm (Coleoptera: Tenebrionidae) for Salmonella typhimurium (Eubacteriales: Enterobacteriaceae).

The reservoir competence of the lesser mealworm, Alphitobius diaperinus (Panzer) is reported for Salmonella typhimurium (Loeffler) relative to broiler chicken production. Salmonella typhimurium was isolated from feces of the adult lesser mealworm at least 28 d after feeding for 24 h on 1 g of chicken feed inoculated with 3 x 10(8) bacteria/ml. All larvae fed S. typhimurium ceased voiding the bacteria in their feces before pupal molt, except one. One beetle continued to void S. typhimurium after it emerged as an adult, providing evidence that transstadial transmission of S. typhimurium may occur. The bacteria were found both on the external body surface and inside the body of surface-sterilized adults and larvae during 16 d of exposure. Salmonella-positive cloacal swabs were obtained from 1-day-old broiler chicks within 24 h after eating one infected lesser mealworm adult or larva.

Individual variation within breeds of beef cattle in resistance to horn fly (Diptera: Muscidae).

Weekly estimates of populations of horn fly, Haematobia irritans (L.), were made on individual purebred Charolais, Chianina, Hereford, Polled Hereford, and Red Poll cows in 1988, 1989, and 1990 with Angus included in 1989 and 1990. During the study period, 94 of approximately 200 individual cows were classified as either fly resistant or fly susceptible. In general, individuals retained the same classification over the 3-yr study period. The mean number of flies on individual susceptible cows in all breeds was at least twice the number of flies on resistant cows. Susceptible Chianina cows averaged > 4.5 times as many flies as resistant cows of the same breed. Individual cows within all breeds classified as fly resistant ranged in age from 2 to 12 yr, whereas fly-susceptible cows ranged in age from 2 to 14 yr; therefore, cow age did not influence the abundance of horn flies on individual cows within breeds. Two-year-old fly-resistant cows were identified accurately according to their horn fly-population density, showing the potential of host resistance in horn fly-population management.

Reservoir competence of Alphitobius diaperinus (Coleoptera:Tenebrionidae) for Escherichia coli (Eubacteriales:Enterobacteriaceae).

Larval and adult lesser mealworms, Alphitobius diaperinus (Panzer), were found to harbor a Congo red-binding strain of Escherichia coli (Migula) Castellani & Chalmers both on the external surface of their body and internally for 12 d. Thereafter, E. coli was not detected, even though the beetles were exposed continuously to a food source inoculated with the bacteria. Lesser mealworm larvae and adults discharge E. coli bacteria in their feces for up to 6 and 10 d, respectively. However, bacteria were no longer detected in their feces after larvae underwent a single molt to the next larval stage. This indicated there was no transstadial transmission of this strain of E. coli. Consumed infected larvae were found to cause more 1-d-old chicks to have positive cloacal swabs for Congo red-binding E. coli than consumed infected adults. The data indicated that the lesser mealworm may play a role in the direct transmission of E. coli and contribute to the spread of this bacteria in broiler production systems. This may be achieved by beetles being directly consumed by chickens or indirectly by spread of the bacteria throughout the broiler house by lesser mealworm feces.

Distribution of horn flies on individual cows as a percentage of the total horn fly population.

Twenty-three mixed-breed herd cows were phenotyped for their ability to serve as a suitable host for Haematobia irritans, the horn fly. Based upon consistent observations within the lower quartile or upper quartile of individual fly counts, four cows were phenotyped as low carriers and five cows were phenotyped as high carriers of horn flies. The cows designated as low carriers consistently carried levels of flies below the economic threshold. However, during a period of fly population explosion, low carriers harbored flies well above the economic threshold. Although the number of flies counted on these low carrying cattle increased as the population increased, the relative percentage of the population that they carried changed very little. A hypothesis is proposed to explain this observation, and future studies are suggested.

Estimates of repeatability and heritability of horn fly resistance in beef cattle.

Horn fly population density on 215 beef cows representing seven breed groups and 51 sires was used to obtain estimates of repeatability (rXX) and heritability (h2) for resistance to the horn fly (Haematobia irritans [L.] Diptera: Muscidae). Total horn fly densities were determined weekly on each cow beginning in May and ending in late October or early November of 1988, 1989, and 1990. No insecticides were used on cattle in this study. Estimates of h2 for horn fly resistance (low horn fly number per cow) were obtained by the paternal half-sib method (4 sigma 2S) and as twice the intrasire regression of offspring on dam (2bDD/S). Variance component estimates were obtained using a completely nested ANOVA that included overall mean, breed, sire/breed, cow/sire, and residual error. One hundred twenty-six daughter-dam pairs were available for regression analysis. In a preliminary analysis, the within-breed regression was nonsignificant (P greater than .05), implying that the regression was the same for all breeds; therefore, breed was deleted from the model. The regression model included an overall mean, year, and the intrasire regression of daughter on dam (bDD/S). The estimate of rXX was .47 +/- .02. Estimates of h2 were .78 +/- .16 and .59 +/- .10 from the 4 sigma 2S and 2bDD/S methods, respectively. Similar estimates of rXX and h2 were obtained when each observation of horn fly number per cow (x) was transformed to both log10 (x) and square root of x. These estimates suggest the possibility of selection procedures as an environmentally safe alternative to the use of chemical control.

Plasma characteristics of beef cattle classified as resistant or susceptible to horn flies.

Horn flies inflict economic losses on cattle producers. Therefore, the objective of this study was to identify a serological marker for cows that are innately resistant to horn flies. Plasma characteristics (optical density, cortisol, and protein pattern) were studied in beef cattle classified (cow-type) as horn fly resistant or susceptible. Cows (n = 30) of five breed groups were used in this study. Cows were bled via jugular venipuncture in May (the beginning of the horn fly season). Plasma optical density at wavelengths 200 and 464 nm was different (P < .05) between resistant and susceptible cattle. Cow-type also affected (P < .05) area percentage for proteins with running molecular weights (M(r)) of 74,000 and 54,000 daltons. Breed group affected (P < .05) optical density at wavelengths 200, 280, 320, and 464 nm and concentration of cortisol in plasma. When the ratio of area percentage for protein bands 7 and 9 (M(r) 74,000 and 54,000, respectively) was determined, cows could be categorized as horn fly resistant or susceptible. These data suggest that a serological marker for horn fly resistant cattle has been identified; however, the marker will need to be tested on a larger population of cattle.

Relationship of horn fly to face fly infestation in beef cattle.

Horn fly and face fly counts (n = 394) taken on 194 beef cows representing seven breed groups were used to determine the effects of horn fly and face fly counts. Breed groups included were Angus (ANI and ANII), Chianina (CA), Charolais (CH), Hereford (HH), Polled Hereford (PH), and Red Poll (RP). The breed group designated ANI consisted of small-framed cows. Total horn fly and total face fly counts were determined weekly on each cow beginning in May and ending in late October or early November in a 3-yr (1988-90) study. Face flies were not counted on the ANI and ANII breed groups in 1988. All fly counts were taken when cows were grazing Ozark upland native grass pastures with only containment fences separating breeding groups. No insecticides were used in the study. Data for analysis were the mean annual horn fly and face fly counts (averaged across weeks), spring weight and fall weights, gain/day between spring and fall weights, and skin surface area in the spring (SSAS) and fall (SSAF) for each cow. Relationships among measurements were examined by correlation and regression procedures. Horn fly count was correlated (P < .05) with face fly count, spring weight, gain/day, and SSAS (.23, .11, -.25, and .12, respectively). Correlations of horn fly count with fall weight and SSAF were non-significant. Horn fly count, breed, and the breed x horn fly count interaction were significant (P < .05) for the face fly regression.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies with equine infectious anemia virus: transmission attempts by mosquitoes and survival of virus on vector mouthparts and hypodermic needles, and in mosquito tissue culture.

Biological and mechanical transmission trials with Psorophora columbiae (Dyar and Knab) and Aedes sollicitans (Walker) and ponies acutely infected with equine infectious anemia virus (EIAV) were negative. The EIAV antigen was detected by radioimmunoassay in Ae sollicitans immediately after the mosquitoes had fed on an acutely ill pony, but not 14 days after feeding. Psorophora columbiae mosquitoes had detectable EIAV antigen as determined by radioimmunoassay 24 hours after they fed on an acutely ill pony; this antigen was not detected again until 6 days after feeding and was still detected 14 days after feeding. The EIAV was detected on hypodermic needles held at 25 C for 96 hours, but was not detected 120 hours after the needles were dipped in solutions of EIAV. The virus was detected on the mouthparts of mosquitoes for 1 hour after they had fed on an EIAV-rich medium, but was not detected 4 hours after feeding. Culex pipiens quinquefasciatus Say ovarian cells maintained the infectivity of EIAV for 10 weekly passages, but no evidence for virus multiplication was obtained.

Apparent propagation of the equine infectious anemia virus in a mosquito (Culex pipiens quinquefasciatus Say) ovarian cell line.

A tissue culture of Culex pipiens quinquefasciatus Say ovarian cells appeared to support the growth of equine infectious anemia (EIA) virus. Shetland ponies inoculated with 2nd, 7th, 9th, and 11th passages of mediums harvested from infected tissue culture had clinical signs of the disease and became EIA positive on 11, 19, 23, and 43 days after inoculation, respectively.

Interactive response of the horn fly (Diptera: Muscidae) and selected breeds of beef cattle.

Statistically significant differences were observed in the population density of the horn fly, Haematobia irritans irritans (L.), on different breeds of beef cattle. The European breed Chianina had a population density of horn flies generally less than or equal to 50% than that of the British cattle breeds (Angus, Hereford, Polled Hereford, and Red Poll) and another European breed (Charolais). Generally, no significant difference existed among numbers of horn flies on Hereford, Polled Hereford, and Red Poll cows in 1988 or among Angus, Hereford, Polled Hereford, and Red Poll cows in 1989. Factors other than color appeared to be involved in the selective process between the horn fly and its host. Population densities on two white European breeds (Charolais and Chianina) were significantly different on all weekly intervals except for 4 wk in both 1988 and 1989. No significant difference existed among Charolais and British breeds except during 4 wk in 1988 and 3 wk in 1989. When weaning weights of all calves were adjusted for the effects of age to 205 d, sex of calf, and age of dam, the indirect effect of the horn fly on weaning weight showed a significant linear regression. Each 100 flies per cow caused a reduction of 8.1 kg in calf weaning weight. Cows within each breed with low numbers of horn flies weaned significantly heavier calves than cows with higher numbers of horn flies.

Horn fly (Diptera: Muscidae) insecticide resistance in Kentucky and Arkansas.

The effect of previous insecticide use patterns for horn fly control on the susceptibility spectrum of horn fly (Haematobia irritans [L.]) populations from Kentucky and Arkansas is described. Populations of horn flies from both states were tested with three pyrethroids (cyhalothrin, cypermethrin, and permethrin), three organophosphates (diazinon, pirimiphos methyl, and tetrachlorvinphos), and a chlorinated hydrocarbon (methoxychlor). Dose-mortality data indicated insecticide resistance in Arkansas and Kentucky. Two permethrin-resistant horn fly populations in Kentucky that did not have a history of exposure to methoxychlor were cross-resistant to this chlorinated hydrocarbon. Horn fly populations from both states with a history of at least three consecutive years of exposure to various pyrethroid ear tags were subsequently exposed to cattle tagged with cyhalothrin-impregnated ear tags for 15-16 wk. Such exposure resulted in a decrease in susceptibility to this pyrethroid (ranging from approximately 30 to greater than 100-fold) when compared with levels before treatment. Horn fly populations from Arkansas resistant to cyhalothrin (as a result of exposure to cyhalothrin ear tags) were cross-resistant to pirimiphos methyl. Seasonal exposure of an Arkansas and Kentucky horn fly population to cattle with ear tags impregnated with pirimiphos methyl resulted in a significant decrease in susceptibility to this organophosphate.

Efficacy of Brahman breeding in the management of insecticide-resistant horn flies (Diptera: Muscidae) on beef cattle.

The efficacy of Brahman breeding used as an alternative tactic to manage insecticide-resistant populations of adult horn flies, Haematobia irritans irritans (L.), was determined. Concentration-mortality bioassays done at Booneville and Hope, AR, in 1988 and 1989, respectively, showed that horn fly populations were resistant to diazinon, pirimiphos methyl, tetrachlorvinphos, and methoxychlor. Data showed loss of field efficacy for coumaphos and delnav. Mean horn fly counts on Braham cows were significantly lower than on Angus cows for all sampling dates in 1989 and 1990. Mean fly counts on Brahman x Angus cows were approximately intermediate to the two purebred mean fly counts. Brahman breeding caused significant reductions in the number of organophosphate-resistant horn flies, which had been equal to or greater than that obtained from continued spraying with organophosphate insecticides. The Brahman x Hereford cows, which have one-eighth greater Brahman breeding than the Brangus cows, had fewer horn flies on 48 of 56 sampling dates in 1988-1990 and significantly fewer flies on 37 sampling dates. The effectiveness of Brahman breeding in causing lower numbers of insecticide-resistant horn flies significantly increased as the percentage of Brahman breeding increased.

Evaluation of alternative tactics for management of insecticide-resistant horn flies (Diptera: Muscidae).

A 3-yr study was conducted to determine the efficacy of tactics that could be used to manage populations of insecticide-resistant horn flies, Hematobia irritans irritans (L.). Insecticide spray, spot-on or pour-on formulations and two IGRs in bolus formulation, 1.3- and 3.2-ha pasture rotations on different rotation schedules, 0-50% Brahman breeding, selected fly-resistant cows, and a mechanical trap were evaluated singly and in combination. Concentration-mortality tests indicated that horn flies collected from cows used in the current study were significantly less susceptible to diazinon, coumaphos, and methoxychlor than horn flies from cows at the same locations previously used to determine baseline susceptibility. During the 3-yr study at the Southeast Research and Extension Center (SEREC), the IGR-bolus significantly reduced (P < 0.05) horn fly numbers on both the continuous and rotational graze regimens, resulting in significantly (P < 0.05) greater calf weaning weights (average of 24 kg). Horn fly numbers were significantly greater on untreated cows during the 3-yr study at the Southwest Research and Extension Center (SWREC) compared with the mean fly numbers on cows that received fly-management treatments. All tactics and tactic-combinations used at SWREC on cattle having no Brahman breeding failed to significantly reduce insecticide-resistant horn fly numbers. However, the combination of Brahman breeding with the IGR-Bolus and mechanical trap significantly reduced horn fly numbers and resulted in significant increases in calf weaning weight. In addition, mean horn fly numbers decreased significantly as the percentage Brahman breeding increased with 50% Brahman breeding reducing horn fly numbers by 140 flies per cow. No significant difference was found between the mean fly numbers on the fly-resistant purebred group and the cows that had no Brahman breeding but received the IGR-Bolus or used the mechanical trap. The use of synergized zeta-cypermethrin pour-on treatment successfully complimented the use of IGR-bolus and mechanical traps in reducing insecticide-resistant horn fly numbers. Neither 1.3- nor 3.2-ha size paddocks and stocking rates used in the rotation graze regimens at SEREC and SWREC, respectively, significantly reduced horn fly numbers when compared with continuously grazed paddocks. Data indicated the importance of using tactics that reduce horn fly numbers to approximately 150 horn flies per cow. These data demonstrated the efficacy of using tactic combinations to manage insecticide-resistant horn fly populations.

Isolation of infectious bursal disease virus from the lesser mealworm, Alphitobius diaperinus (Panzer).

Infectious bursal disease virus (IBDV) was isolated from adult lesser mealworms, Alphitobius diaperinus (Panzer), up to 14 d after exposure, but isolation of the virus was erratic over this period of time. The virus was undetected after 24 h in beetle larvae. Virus was isolated from the adult beetle's mouth parts, foregut, midgut, hindgut, and blood 24 h after they fed on feed inoculated with IBDV. Ten days after exposure, virus was isolated from the foregut but not the blood, mouth parts, or remaining digestive tract of the adult beetles. The adult lesser mealworm is capable of serving as a reservoir for IBDV, rather than a fomite, between broiler growouts.