Salivary gland thrombostasin isoforms differentially regulate blood uptake of horn flies fed on control- and thrombostasin-vaccinated cattle.

Thrombostasin (TS) is an anticlotting protein found in saliva of Haematobia irritans (horn flies). The polymorphic nature of the ts gene was first associated with success of horn flies blood feeding on a laboratory host, New Zealand White rabbits. In this study, we report results of similar studies testing blood uptake of horn flies feeding on a natural host, cattle. These studies confirmed the association of ts genotype with blood uptake of horn flies and showed that it was host species specific. In contrast to rabbits, blood uptake volumes of homozygous ts10 horn flies were lower than those of other ts genotypes when fed on control (ovalbumin-vaccinated) cattle. Cattle vaccinated with recombinant protein isoforms, rTS9 or rTB8, resisted horn fly feeding by yielding lower blood volumes compared with flies feeding on control cattle. The specific impact of vaccination, however, varied by ts genotype of flies. Cattle vaccinated with isoform rTS9 resisted flies of ts2, ts9, and tb8 genotype. Vaccination with isoform rTB8 produced resistance to ts8, ts9, and tb8 genotype flies. Horn flies of genotype ts10 were not affected by vaccination with either TS isoform and fed as well on rTS9- and rTB8-vaccinated as on control-vaccinated cattle. These experimental results confirm the efficacy of vaccines targeting horn fly salivary proteins and provide new insight into the dynamics of horn fly-cattle interactions in nature.

Salivary gland thrombostasin isoforms differentially regulate blood uptake of horn flies fed on New Zealand White rabbits.

Thrombostasin (TS) is a previously characterized anticlotting protein with multiple isoforms found in the saliva of horn flies. In this report, the effect of TS isoforms on blood feeding was assessed using individual flies that carried corresponding ts allelles. Laboratory studies of horn fly blood feeding were conducted using colony-reared flies fed on New Zealand White (NZW) rabbits. After timed 20-min feeding periods, each fly was characterized for gender, blood volume uptake, and ts genotype. The results showed that mean blood volumes obtained by individual flies were not related to fly gender but were correlated to the ts genotype(s) carried by each fly. A fly having one or both ts alleles coding for the TS9 isoform took less blood than those possessing one or both ts alleles coding for the TS10 isoform. These results confirm the significant role that TS plays in horn fly feeding and highlight the differential impact of TS protein isoforms that vary by as few as three amino acids.

Chromosomal localization of two antihemostatic salivary factors in Simulium vittatum (Diptera: Simuliidae).

The chromosomal locations of two genes encoding the salivary protein products Simulidin (SVAT) and Simulium vittatum erythyma protein (SVEP) were identified using high-resolution cytogenetic mapping. Chromosomal subsection levels were determined from larval and adult salivary gland polytene chromosomes and adult Malphigian tubule chromosomes. Syntenic relationships occurred for both loci from a wild population of Simulium vittatum IIIL-1 collected in central Alabama and the colonized IS-7 S. vittatum sibling. cSVAT mapped to the short arm of chromosomes III, IIIS-72a4.5 and cSVEP mapped to the long arm of chromosome III, IIIL-96b1. cSVAT sits proximal to a common IIIS-2 paracentric inversion, which occurs predominately as the standard sequence in the IIIL-1 sibling and as the inverted sequence in the IS-7 sibling. cSVEP sits close to the differentiated X chromosomes in the IIIL-1 sibling.

Short report: impact of ivermectin community-level treatments on elimination of adult Onchocerca volvulus when individuals receive multiple treatments per year.

A reanalysis of several published reports indicates that when community-wide biannual treatment disrupts transmission of new infection, a profound macrofilaricidal effect of ivermectin (Mectizan) occurs that is accelerated for individuals treated 4 times a year (4x/yr). The effect is particularly obvious on adult male worms and suggests that this gender is susceptible to repetitive treatment after transmission has been blocked or greatly reduced as a result of community treatment.

The effects of long-term community level treatment with ivermectin (Mectizan) on adult Onchocerca volvulus in Latin America.

The objective of this study was to examine nodules from Mexico, Guatemala, and Ecuador collected over a one-year period (2001) to determine the effects of semi-annual ivermectin treatments on Onchocerca volvulus macrofilarial populations. Nodules were sectioned, stained with hematoxylin and eosin, and histologic findings were compared between countries and with historical data prior to the introduction of ivermectin into the region. Nodules from Ecuador had 10 times more dead or moribund worms than the historical control (66.6% versus 6.5%); nodules from patients from Mexico and Guatemala did not differ from the control. More than 80% of the female worms in each country were uninseminated and producing unfertilized oocytes. Nodules containing males differed in each country from the historical control (P < 0.0001), with presence of males ranging from 19.7% in Mexico to 13.6% in Ecuador versus 73% in the control. Nodules with females producing active microfilariae ranged from 7.8% (Mexico) to 2.7% (Ecuador) versus 60% in the historical control (P < 0.0001). Nodules from Ecuador and Mexico were significantly smaller in size than those from Guatemala or historical controls (P < 0.0005). These results depict a deteriorating condition of adult O. volvulus populations in Mexico, Guatemala and Ecuador, indicating that semi-annual ivermectin treatment of >/=6 years has had a profound effect on survival and reproduction of this species.

Processing of pro-thrombostasin by a recombinant subtilisin-like proprotein convertase derived from the salivary glands of horn flies (Haematobia irritans).

Thrombostasin (TS) is a thrombin inhibitor found in the salivary glands of horn flies (Haematobia irritans). It is produced as an inactive form with a 76-amino acid propeptide in the N-terminus preceding the mature TS. A minimal recognition sequence by subtilisin-like proprotein convertases, Arg-Xaa-Xaa-Arg, is localized C-terminal to the propeptide. This study demonstrated that a gene cloned from the salivary glands of the horn fly encodes a new convertase, subsequently named horn fly proprotein convertase (HFPC), and that the recombinant HFPC expressed in insect HighFive cell culture specifically cleaves recombinant pro-thrombostasin, produced in E. coli, at the expected site. The relative cleavage efficiency of rHFPC was compared with that of recombinant human furin, a commercially available proprotein convertase. The result indicated that this newly identified proprotein convertase is of importance for the proteolytic maturation of thrombostasin, a protein secreted in horn fly saliva and used by the insect to counteract its host's haemostatic response.

Identification of reptilian and amphibian blood meals from mosquitoes in an eastern equine encephalomyelitis virus focus in central Alabama.

Uranotaenia sapphirina, Culex erraticus, and Cx. peccator were collected in an enzootic eastern equine encephalomyelitis (EEE) virus focus in central Alabama (Tuskegee National Forest) from 2001 to 2003 and analyzed for virus as well as host selection. EEE virus was detected in each species every year except 2003, when pools of Cx. peccator were negative. Most (97%) of the 130 Cx. peccator blood meals identified were from ectothermic hosts; 3% were from birds. Among blood meals from reptiles (approximately 75% of the total), 81% were from Agkistrodon piscivorus (cottonmouth); all amphibian blood meals (approximately 25%) were from Rana spp. with > 50% taken from the bullfrog R. catesbeiana. Host identifications were made from 131 of 197 Cx. erraticus, but only 3 (2%) were derived from ectothermic species. Identification of Ur. sapphirina blood meals proved difficult and only 2 of 35 hosts were determined. Both were from R. catesbeiana. Ectothermic species are possible EEE virus reservoirs in the southeastern United States where species such as Cx. peccator and Ur. sapphirina occur with large, diverse reptilian, amphibian, and avian populations such as those at the Tuskegee site.

Evaluation of a recombinant salivary gland protein (thrombostasin) as a vaccine candidate to disrupt blood-feeding by horn flies.

The potential for controlling blood-feeding by the cattle pest, Haematobia irritans irritans (horn fly), was tested by vaccination against thrombostasin (TS), an inhibitor of mammalian thrombin that is released into skin during horn fly blood-feeding. The increase in blood meal size that occurred for flies feeding on sensitized non-vaccinated hosts was blocked and egg development in female flies was delayed when horn flies fed on rabbits and cattle immunized with recombinant TS. This demonstration of the impact of disrupting TS action by vaccination provides a novel approach toward control of this veterinary pest and offers a paradigm for limiting blood-feeding in other medically-important insect species.