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Phospholipase A2 (PLA2) activity levels in tick [Amblyomma americanum (L.)] salivary glands and saliva were examined during tick feeding by using 14C-phosphatidylcholine as the substrate. Saliva produced by stimulating female ticks to salivate with dopamine contains PLA2 (ts-PLA2) activity. The ts-PLA2 activity level in saliva did not change significantly during tick feeding except for a decrease in the last rapid feeding phase (> 200 mg) and in replete ticks. Phospholipase A2 activity was higher in salivary glands of fed than unfed ticks, both in males and females; activity increased during tick feeding correlating with salivary secretory rates during tick feeding suggesting that much of the PLA2 activity measured in whole salivary glands is synthesized for subsequent secretion. During the time course of in vitro salivation, the first 10 microliters of saliva contained higher ts-PLA2 activity than saliva secreted thereafter. Phospolipase A2 was identified in the saliva of artificially fed ticks indicating that ts-PLA2 is a physiological component of tick saliva.

Hemolytic activity was identified in the saliva of Amblyomma americanum (L.) when red blood cells from sheep were incubated with tick saliva in the presence of phosphatidylcholine and sodium deoxycholate. The hemolytic activity was destroyed by boiling or treating with trypsin. The hemolytic activity in tick saliva was calcium-dependent, and inhibited by a phospholipase A2 inhibitor oleyloxyethyl phosphorylcholine. Phosphatidylserine could replace phosphatidylcholine in the hemolytic assays but phosphatidylethanolamine and phosphatidylinositol were ineffective. Size exclusion chromatography of tick saliva revealed one peak of hemolytic activity, which correlated with the activity of tick salivary phospholipase A2, both having a molecular weight approximately 55,000 daltons. These results suggest that the hemolytic activity in tick saliva results from salivary phospholipase A2. The hemolytic activity in tick saliva may play a role in lysing host red blood cells, thus facilitating the tick digestive process.

The polymerase chain reaction (PCR) was used to detect Anaplasma marginale in secretagogue-induced oral secretions of male and female Dermacentor andersoni Stiles exposed as nymphs or adults by feeding on infected calves. A 409-bp DNA fragment derived from the A. marginale (Florida isolate) msp1 beta gene was amplified with oligonucleotide primers BAP-2 (5'-GTATGGCACGTAGTCTTGGGATCA-3') and AL34S (5'-CAGCAGCAGCAAGACCTTCA-3'). The target DNA was amplified in oral secretions of female ticks exposed to A. marginale as adults and stimulated to secrete by injection of dopamine. Conversely, A. marginale was detected in saliva from prefed female ticks exposed as nymphs only after stimulation with a combination of dopamine, gamma-aminobutyric acid, pilocarpine, and theophylline. Saliva from ticks exposed as nymphs and stimulated with ergot alkaloids did not contain the A. marginale target DNA. Saliva collected after 11 d of feeding from dopamine-stimulated male ticks contained A. marginale DNA. The results indicate that A. marginale is present in tick saliva and suggest that the parasite can be transmitted to cattle via saliva of feeding ixodid ticks. The variable appearance of A. marginale in saliva, regardless of the method used to induce salivation, suggests that transmission of A. marginale may be affected by the physiological state of the tick.

The abilities of various effectors of signal pathways in tick salivary glands to stimulate oral secretions in partially fed Amblyomma americanum (L.) female ticks were compared. Pilocarpine stimulated secretion the most rapidly following tick stimulation, but its effectiveness declined with time and subsequent injections. Secretion rates induced by dopamine and theophylline increased with time of collection and additional injections and were as effective as pilocarpine after 60 min. Activators of protein kinase C, phorbol 12-myristate 13-acetate (PMA), and 1-oleoyl-2-acetyl-sn-glycerol inhibited dopamine, and theophylline stimulated secretion. gamma-amino-butyric acid either by itself or in combination with other drugs had no effect on volume of oral secretions. Peptides were visible in the oral secretion stimulated by the various drugs after separation and staining in SDS-polyacrylamide gels. More peptides were present in the oral secretion obtained from the smallest partially fed (slow feeding) ticks. In some ticks, proteins were observed in oral secretions obtained from the same tick at one collection time but not another in response to the same pharmacological agent.

Females of the lone star tick, Amblyomma americanum (L.), and the American dog tick, Dermacentor variabilis (Say), were fed on ivermectin (Ivomec)-treated and untreated bovines to determine the effect of the acaricide on volume of blood ingested and to compare the weight differences between the treatment and control groups at various time intervals after attachment. Adult females from each genus were collected from Bos tarus hosts and subjected to hematin assays on three collection dates to estimate the volume of blood ingested. Before feeding, lone star ticks contained an average of 2.0 microliters of blood and had an average weight of 5.2 mg. Unengorged American dog tick females had an average blood volume of 3.3 microliters and a mean weight of 5.8 mg. Ticks of both species reacted to ivermectin by expressing lower mean weights, and they consumed smaller quantities of blood. Lone star tick females were significantly affected in terms of amount of blood consumed and body weight changes when compared with control ticks. After feeding on treated cattle, lone star tick females contained smaller quantities of blood than pretreatment females, but there were no significant differences observed until day 12 between the control and the treated groups. American dog tick females on treated hosts had measurable quantities of blood that were significantly different among the experimental groups.