Joanne Gough

Immunization of cattle against Boophilus microplus using extracts derived from adult female ticks: Feeding and survival of the parasite on vaccinated cattle

Abstract Kemp D. H. , Agbede R. I. S. , Johnston L. A. Y. and Gough J. M. 1986. Immunization of cattle against Boophilus microplus using extracts derived from adult female ticks: feeding and survival of the parasite on vaccinated cattle. International Journal for Parasitology 16 : 115–120. Boophilus microplus adults and larvae were observed in chambers on three European breed cattle which had been vaccinated against the tick and on three unvaccinated controls. The moulting of larve on vaccinated cattle was delayed by up to 12 h but otherwise they were unaffected. On two of the three vaccinated cattle there was progressive death of tick females throughout feeding and up to 60% of females had a damaged gut. These females either failed to engorge, or if they did, many died before egg laying. Males also suffered gut damage. In contrast, the females which survived the first day on control cattle usually completed engorgement and neither females nor males showed damaged gut. No hypertensivity reaction or serious exudation was seen at the site of tick attachment on any animals. This was in contrast to resistance acquired following repeated tick infestations where hypersensitivity reactions caused rejection of attaching ticks, especially larvae, and where no gut damage occurred. Vaccination therefore produced effects which were additional to those that follow repeated infestations. Damage to tick gut was also demonstrated in vitro and a role for complement in this effect was suggested.

Vaccination againstBoophilus microplus: Localization of antigens on tick gut cells and their interaction with the host immune system

Cattle have been vaccinated againstBoophilus microplus with antigens derived from partially fed female ticks. The immune response of the host lyses the gut cells of adult ticks, causing a reduction in the number, weight and reproductive capacity of engorging ticks. This response is different from the immunity that cattle acquire after repeated tick infestation. Evidence is presented that the antigens used in vaccination are located on the plasma membrane of the gut cells and it is unlikely that these antigens are secreted into the host during feeding. Vaccination using such ‘concealed’ antigens may not encounter the mechanisms of immune evasion that parasites usually demonstrate.In-vitro assays suggest that vaccination immunity is not dependent on the need to stimulate cell-mediated responses. Immunoglobulin G alone, or with the aid of complement, is enough to damage tick gut.The normal function of the one protein antigen isolated so far is unknown but we speculate that it serves some vital function on the cell plasma membrane.

Localization of a low abundance membrane protein (Bm86) on the gut cells of the cattle tick Boophilus microplus by immunogold labeling

A preembedding immunogold technique was used to locate Bm86, an antigen from the gut digest cells of the cattle tick Boophilus microplus. Gut from partially engorged female ticks was everted to expose the cells, lightly fixed in 4% paraformaldehyde, and then incubated in rabbit antisera against a recombinant form of Bm86. Following incubation in a secondary antibody conjugated to 1-nm colloidal gold, Bm86 antigenic sites were visualized for both light and electron microscopy using silver enhancement. Bm86 was shown to be located predominantly on the microvilli of digest cells. Antiserum against a nonglycosylated Escherichia coli recombinant form of Bm86 was used to avoid cross-reactivity with carbohydrate epitopes of other digest cell proteins.

Exploring the midgut proteome of partially fed female cattle tick (Rhipicephalus (Boophilus) microplus).

The continued development of effective anti-tick vaccines remains the most promising prospect for the control of the cattle tick, Rhipicephalus (Boophilus) microplus. A vaccine based on midgut proteins could interfere with successful tick feeding and additionally interfere with midgut developmental stages of Babesia parasites, providing opportunities for the control of both the tick and the pathogens it transmits. Midgut proteins from partially fed adult female cattle ticks were analysed using a combination of 2-DE and gel-free LC-MS/MS. Analysis of the urea-soluble protein fraction resulted in the confident identification of 105 gut proteins, while the PBS-soluble fraction yielded an additional 37 R. microplus proteins. The results show an abundance of proteins involved in mitochondrial ATP synthesis, electron transport chain, protein synthesis, chaperone, antioxidant and protein folding and transport activities in midgut tissues of adult female ticks. Among the novel products identified were clathrin-adaptor protein, which is involved in the assembly of clathrin-coated vesicles, and membrane-associated trafficking proteins such as syntaxin 6 and surfeit 4. The observations allow the formulation of hypotheses regarding midgut physiology and will serve as a basis for future vaccine development and tick-host interaction research.

Peritrophins of adult dipteran ectoparasites and their evaluation as vaccine antigens

Several peritrophins of larvae of Lucilia cuprina (sheep blowfly) have demonstrated potential as vaccine antigens, and some have been characterised and cloned. These proteins are tightly associated with the peritrophic matrix, a chitinous tube or sac lining the lumen of the gut of most insects. The peritrophins require strong denaturants for their removal from peritrophic matrix. We now report the preliminary characterisation of peritrophins of the adult stage of L. cuprina and Haematobia irritans exigua (buffalo fly). Similar SDS-PAGE profiles were obtained for proteins extracted in SDS or urea from isolated adult peritrophic matrices of both species. Radioiodination of urea-extracted peritrophins improved sensitivity, indicating numerous proteins of 15-75 kDa. Direct radioiodination of L. cuprina peritrophic matrix preferentially labelled high molecular weight complexes and proteins of 80-90 kDa. Two-dimensional gel analyses of a urea extract of adult L. cuprina peritrophic matrix revealed that most proteins were moderately acidic. Antibodies produced against SDS-extracted peritrophins, or against sonicated peritrophic matrices of these two flies were crossreactive. The sera also appeared to recognise SDS-extracted components of Triton X-100 treated and washed adult peritrophic matrix of the mosquito, Aedes vigilax (Skause). This profile altered as the peritrophic matrix matured. In concordance with extracts from the adult L. cuprina and H.i. exigua peritrophic matrices, proteins in the 50-75 kDa region were immunodominant. The vaccine potential of the peritrophins of these Diptera were examined following vaccination of cattle and rabbits with adult H.i. exigua or L. cuprina peritrophins. When the adult life stages of H.i. exigua or two mosquitoes, A. vigilax and A. aegypti (Linnaeus), were fed on the sera or blood of vaccinated hosts, there were no detrimental effects to any life cycle stages of these Diptera.

Expression of angiotensin-converting enzyme-related carboxydipeptidases in the larvae of four species of fly

HieACE, a soluble 70 kDa protein related to the angiotensin-converting enzyme (ACE) has recently been identified, characterized and cloned from the adult buffalo fly (Haematobia irritans exigua). HieACE is enzymatically similar to the mammalian ACEs and its predicted amino acid sequence has 42% identity with the mammalian testicular ACEs. In adult H.i. exigua, HieACE expression is restricted to the compound ganglion and posterior midgut, and the maturing male reproductive system. Western blot analysis was used to investigate the expression of HieACE and its homologues in the larvae of H.i. exigua, Drosophila melanogaster, the sheep blowfly (Lucilia cuprina), the Old World screwworm fly (Chrysomya bezziana) and a secondary strike fly, Chrysomya rufifacies. Dipteran ACE homologues of 65-70 kDa were detected in all the larval instars investigated. Most of the immunoreactive proteins were concentrated in the soluble fraction. The first and second larval instars of L. cuprina and C. bezziana appeared to express two ACE homologues. These larvae were also found to secrete (or excrete) the ACE homologue in larval cultures. The presence of ACE-like enzymes in these larvae was confirmed by the measurement of carboxydipeptidase activity that was inhibited by the specific ACE inhibitor, captopril. The tissue distributions of the ACE homologues in the third instar larvae of H.i. exigua and L. cuprina were examined. As in adult H.i. exigua, HieACE was detected in the larval ganglion, but in contrast to the restricted distribution in the adult stage midgut, HieACE was found throughout the digestive system, and in the salivary glands of H.i. exigua larvae. The expression pattern in the gut of L. cuprina larvae was similar despite the differences in diet and habitat. The most striking difference from the adult stage H.i. exigua was the expression of HieACE and its L. cuprina homologues in the hindgut and Malpighian tubules of these larvae. These results suggest that the role(s) played by the dipteran ACE-like enzymes differ between the adult and larval stages.

Acid phosphatase in midgut digestive cells in partially fed females of the cattle tick Boophilus microplus

Enzyme cytochemistry was used to identify vesicles containing acid phosphatase in the midgut digestive cells of partially fed females of the cattle tick Boophilus microplus. The vesicles were elongated or tubular in shape and appeared to be involved with the digestion of host bloodmeal. In mature cells, they were sometimes in close contact with large endosomes, which contained host blood. The vesicles were identified as tubular lysosomes because their morphological and cytochemical characteristics were analogous to similar structures described in mammalian cells. This is the first report of such lysosomes in tick gut cells and suggests some parallels with the intracellular structures involved in the digestion process of mammalian cells. Acid phosphatase in tick gut was also assayed biochemically and was shown to be inhibited with 10 mM sodium fluoride. Cytochemistry showed that this inhibitor blocked activity within the cell and on the lumenal cell membrane.