Immunization of cross-bred cattle against Hyalomma anatolicum anatolicum by purified antigens

Extracts prepared from unfed larvae of Hyalomma anatolicum anatolicum were purified by immunoaffinity chromatography using anti-gut IgG as ligand. Affinity purified antigen (Aff-GHLAg) was used to immunize cross-bred (Bos taurus×B. indicus) calves of 6–7 months of age. Immunized calves rejected 70.6% larvae, 54.5% nymphs and 61.9% adults. No significant changes in the engorged weight of females was observed; however, significant decrease in the engorgement weight of larvae and nymphs was recorded. There was a significant decrease in the emerging nymphs (p<0.05) and adults (p<0.01) of the tick stages fed on immunized animals. SDS-PAGE analysis revealed three antigenic proteins of 100, 59.4 and 37kDa responsible for induction of resistance in the host.     

Comparative immunopotentiating properties of saponin and incomplete Freund's adjuvant coupled to affinity purified larval antigen of Hyalomma anatolicum anatolicum

Comparative immunostimulatory properties of saponin and incomplete Freund's adjuvant (IFA) were studied using affinity purified 39 kDa larval antigen of Hyalomma anatolicum anatolicum. Significant antibody response to 39 kDa antigen was detected in the sera of rabbits immunized with both 39 kDa plus saponin and 39 kDa plus IFA in comparison to their corresponding control animals. Insignificant differences were noted in the antibody response between the animals of two immunized groups. Upon challenge, the animals immunized with 39 kDa plus IFA rejected 76.2 +/- 9.7% of larvae and 45.8 +/- 4.1% of adults while in group of animals injected with 39 kDa plus saponin rejected 80.9 +/- 11.2% of larvae and 47.2 +/- 5.7% of adults.     

Transstadial transmission of Theileria annulata through common ixodid ticks infesting Indian cattle.

Transstadial transmission of Theileria annulata with Hyalomma anatolicum anatolicum, H. dromedarii and H. marginatum isaaci, and Haemaphysalis bispinosa, Rhipicephalus haemaphysaloides haemaphysaloides and Boophilus microplus was determined. It was found that the infection was successfully transmitted by H. a. anatolicum from larva to nymph and nymph to adult in all attempts. When larvae were fed on an infected calf the succeeding adults transmitted the infection when the intervening nymphs fed on a rabbit (non-susceptible host) but not when fed on a calf (susceptible host). Infective adult ticks transmitted the parasite during the first 24 h of feeding on a calf. When the feeding was interrupted after 24 h, and the tick transferred to another calf, the infection was transmitted to the latter as well. H. dromedarii successfully transmitted the infection from larva to nymph and from nymph to adult. Larvae of H. marginatum isaaci did not feed on calves but the infection was successfully transmitted from nymph to adult. Haemaphysalis bispinosa, Rhipicephalus h. haemaphysaloides and Boophilus microplus did not transmit Th. annulata from larva to nymph to adult.     

Experimental immunisation of crossbred cattle with glycoproteins isolated from the larvae of Hyalomma anatolicum anatolicum and Boophilus microplus

In India, control of tick infestations is a major problem facing the owners of highly productive crossbred cattle. To develop suitable immunoprotective measures against the multitick infestations, specific problem encountered by the Indian farmers, an attempt has been made to identify candidate protein molecules in the larvae of Hyalomma anatolicum anatolicum and Boophilus microplus. Employing two steps affinity chromatography method, glycoproteins of 34 and 29 kDa were isolated from H. a. anatolicum and B. microplus, respectively. The additive effect of the isolated glycoproteins was tested by the immunisation of crossbred male calves at a dose of 200 microg/animal of each glycoprotein, three divided dosages. Following challenge infestations by the stages of two species of ticks, a significant rejection of (73.6%; P < 0.01) of larvae and adults (75.0%; P < 0.01) of H. a. anatolicum and adults (89.8%; P < 0.01) of B. microplus were noted. The direct effect of immunisation on the stages of the challenged ticks (%DT) was calculated as 69%, 52% against larvae and adults of H. a. anatolicum and 60% against adults of B. microplus. A direct corelation between protection and anti-glycoprotein antibody titre was detected. The effect of the isolated glycoproteins on limiting the tick infestation is discussed in detail.     

Hyalomma anatolicum anatolicum: the role of humoral factors in the acquisition of host resistance

A significant degree of resistance to Hyalomma anatolicum anatolicum can be adoptively transferred to naive recipients with immune serum from rabbits repeatedly infected with adult H. a. anatolicum. Ticks fed on recipients of immune serum took longer to become engorged and showed a significant decrease (P less than 0.01) in engorgement weight and oviposition compared with ticks that fed on recipients of normal serum. A direct correlation between resistance and anti-saliva IgG antibodies was indicated by a progressive increase in the degree of resistance and IgG antibody titres following successive tick infestations. Challenge feeding sites on actively sensitised hosts and recipients of immune serum revealed significantly greater infiltration of basophils and eosinophils compared with feeding sites on recipients of normal serum. However, both the degree of resistance and the accompanying cutaneous basophil and eosinophil responses in recipients of immune serum were considerably weaker than those induced by active tick feeding, thus suggesting that nonhumoral (cell-mediated) mechanisms might also be involved in acquired host resistance to H. a. anatolicum.     

Laboratory rearing of <Emphasis Type="Italic">Theileria annulata</Emphasis>-free <Emphasis Type="Italic">Hyalomma anatolicum anatolicum</Emphasis> ticks

Hyalomma anatolicum anatolicum is a three-host tick which transmits Theileria annulata infection in Indian cattle. Laboratory rearing of ixodid ticks is an essential requirement of any laboratory engaged with research on ticks and tick borne diseases. The Entomology laboratory of Indian Veterinary Research Institute is fully equipped with all the facilities and skilled manpower to maintain a homogenous H. a. anatolicum population throughout the year. The continuous supply of eggs, larvae and adults of H. a. anatolicum is maintained to meet out the demand of different experiments viz., preparation of tick antigens for immunization of animals, experimental challenge, isolation of genomic DNA and RNA. Maintenance of a H. a. anatolicum colony free of T. annulata infection is imperative for the experimental challenge infestation on cross-bred (Bos indicus × B. taurus) calves, in order to prevent the transmission of T. annulata infection to the experimental animals. A system has been developed in the laboratory in which the larvae of H. a. anatolicum were fed on New Zealand white rabbits and the dropped fed nymphs molted to adults are fed on cross-bred calves free of T. annulata infection. This synthetic cycle prevents the transstadial transmission of T. annulata as the rabbits are unsusceptible to T. annulata infection and only the adults were fed on cross-bred animals. Moreover, absence of transovarial transmission of T. annulata prevents the chance of carry over infection to experimental animals in the next cycle. Declaration: The experiments have been conducted in accordance with the approved guidelines of Committee for the Purpose of Control and Supervision of Experimentation on Animals (CPCSEA). Besides, the institute animal ethics committee continuously monitored the animal experimentation.     

Effects of immunization of rabbits on establishment, survival, and reproductive biology of the tick Hyalomma anatolicum anatolicum.

Four antigenic preparations, viz. salivary gland antigen (SG Ag), whole tick extract antigen (WTE Ag) and 30,000-g supernatant fraction, and pellet of WTE Ag (TES Ag and TEP Ag, respectively), were made from partially fed adult female Hyalomma anatolicum anatolicum. Four groups of 5 rabbits each were immunized with the antigens, and a fifth group was kept as control. Following challenge with adult H. a. anatolicum, a significant decrease in engorgement weight and egg mass weight and an increase in engorgement period and preoviposition period were observed in WTE Ag-immunized rabbits. Similar results were observed with TES Ag and SG Ag, except that change in the engorgement period was insignificant. However, none of the tick parameter measurements showed significant changes with TEP Ag. None of the antigens produced significant changes in the percentage of engorgement or oviposition period of the challenged ticks. Thus, WTE Ag was the most effective in altering tick performances.     

The drop-off rhythm of Amblyomma triguttatum triguttatum Koch (Acari: Ixodidae) from laboratory hosts

The pattern of drop-off of Amblyomma triguttatum triguttatum Koch was studied by infesting rabbits and rats with larvae and nymphs, and calves with adult ticks maintained at a daily photoperiodic regime of 12 h light 12 h dark. Additional information was obtained by infesting rabbits with nymphs in or out of phase with the photoperiodic regime experienced by the host.The great majority of ticks detached during the photophase: only 9.7% of larvae and 11.3% of nymphs fed on rats, 12.0% of larvae and 15.8% of nymphs fed on rabbits, respectively, and 10.0% of female ticks detached during the scotophase. The rhythm was not substantially altered by feeding nymphs in or out of phase with the photoperiodic regime of the host. The drop-off rhythm seemed to be coupled with endogenous rhythms of the host.     

Evidence for the utility of the Bm86 antigen from Boophilus microplus in vaccination against other tick species

The Bm86 antigen, as originally identified in Boophilus microplus, is the basis of commercial tick vaccines against this tick species. The potential for using this antigen or homologues of the antigen in vaccination against other tick species has been assessed. We have conducted vaccine trials in cattle using the B. microplus-derived recombinant Bm86 vaccine (TickGARD) using pairs of vaccinated calves and control calves. These were infested with B. microplus and Boophilus decoloratus larvae simultaneously. For both species, the numbers of engorged female adult ticks, their weight and egg-laying capacity were all reduced, leading to a reduction in reproductive capacity of 74% for B. microplus and 70% for B. decoloratus. Hyalomma anatolicum anatolicum ticks were fed both as immatures as well as adults on vaccinated calves and non-vaccinated controls. There was an overall 50% reduction in the total weight of nymphs engorging on vaccinated calves, and a suggestion of a subsequent effect on feeding adults. For Hyalomma dromedarii there was a 95% reduction in the number of nymphs engorging and a further 55% reduction in weight of those ticks surviving. Rhipicephalus appendiculatus and Amblyomma variegatum ticks were fed simultaneously both as immatures and subsequently as adults. There was no evidence for a significant vaccination effect. Finally, the amino acid sequence of a Bm86 homologue found in H. a. anatolicum unequivocally demonstrated the conservation of this molecule in this species. Our strategy for the development of multivalent anti-tick vaccines is discussed in relation to these findings.     

The life cycle of Amblyomma neumanni Ribaga, 1902 (Acari: Ixodidae) in the laboratory

A colony of Amblyomma neumanni was started with engorged females collected from cattle in the province of Salta (24° 51S, 65° 33W), Argentina. The larvae and nymphs were fed on rabbits and the adults on calves. The non-parasitic stages were maintained in darkness at 27 ± 1°C and 83–86% RH. The life cycle (pre-feeding period not tested) had a mean duration of 205.7 days. The mean time (days) for the different phases of the cycle were as follows: feeding of females 8.8, pre-oviposition 23.8, oviposition 41.4, minimum egg incubation 76.1, feeding of larvae 8.5, pre-moult to nymphs 16.4, feeding of nymphs 7.9 and pre-moult to adults 22.8. The mean recovery rates of larvae, nymphs and females were 83.8, 85.6 and 89.3%, respectively. The nymphs moulting to females were heavier (8.1 ± 2.34 mg) than those moulting to males (6.0 ± 2.34 mg; p < 0.01), but their range of engorgement weight showed overlap (2.3–16.2 versus 2.2–12.8 mg, respectively). Two gynandromorphs were detected between the nymphs. A comparison of biological parameters of A. neumanni with other American Amblyomma species from mammals is presented. © Rapid Science Ltd. 1998     

Artificial immunization of rabbits with Hyalomma dromedarii tick-derived midgut antigen

New Zealand white rabbits were immunized with partly fed Hyalomma dromedarii tick-derived midgut concealed antigens (supernate and pellet fractions) and Freund's complete adjuvant (FCA). The rabbits received three inoculations subcutaneously on days 0, 14 day 21 at a dose rate of 1 mg antigen per animal. The effects of the immunity induced was determined by infesting the rabbits with adult H. dromedarii ticks. In immunized rabbits a significant reduction in tick yield, engorgement weight, oviposition period, egg mass weight and percentage of egg hatchability was found. The gut supernatant antigen fraction induced the best protection in terms of reduced feeding and reproductive performance of the ticks.     

Immunity against Boophilus annulatus induced by the Bm86 (Tick-GARD) vaccine

Friesian cattle were immunized with two inoculations of anti-tick Bm86 (Tick-GARD) vaccine and were challenged 30 or 90 d later with Boophilus annulatus larvae derived from 1.2 g of eggs. No nymphs or adult ticks were found on the immunized cattle during four weeks after challenge. Repeated infestations (2 to 4) with larvae on three other calves during a period of 160 and 390 d after the immunization did not result in development of nymphal and adult stages. In control, non-immunized cattle infested with corresponding batches of larvae 1380 to 4653 replete adult female ticks were collected. Larvae issued from Babesia bovis-infected female ticks transmitted the infection to Bm86-immunized cattle, but the progeny of B. bigemina-infected females did not. Since B. bigemina is transmitted exclusively by nymphal stages of Bo. annulatus these results support the observation that immunity induced by Bm86 affects the larval stage of this tick.     

Ixodes dammini: Induced skin lesions in guinea pigs and rabbits compared to erythema chronicum migrans in patients with lyme arthritis

Erythematous skin lesions occurred in rabbits 2 days after being fed upon by larvae or nymphs of the tick, Ixodes dammini. Similar lesions occurred in guinea pigs 7 days after a primary infestation with either larvae or nymphs. Host resistance to secondary feeding by larvae was demonstrated in guinea pigs and rabbits. Host resistance to secondary feeding by nymphs was seen in guinea pigs, but not in rabbits. Guinea pigs developed resistance to nymphs after being previously fed upon twice by larvae. All skin lesions in resistant guinea pigs contained large accumulations of basophils (49–76% of cells) with smaller (20–33%), but significant, numbers of eosinophils. These responses were characteristic of strong cutaneous basophil hypersensitivity reactions. Primary and secondary lesions in rabbits fed upon by larvae contained mostly mononuclear cells (46–52%) and moderate numbers (16–30%) of basophils and eosinophils. Primary and secondary lesions in rabbits fed upon by nymphs had few (3–11%) basophils and eosinophils and were dominated by mononuclear cells (73–86%). Thus, acquired resistance in guinea pigs and rabbits was associated with cutaneous basophil and eosinophil responses and the lack of resistance of rabbits to nymphs was associated with erythematous lesions dominated by mononuclear cells. The mononuclear nature of rabbit lesions induced by nymphal feeding was similar to that seen in erythema chronicum migrans in Lyme arthritis patients who are thought to have been fed upon by I. dammini nymphs. This study confirms the cutaneous basophil hypersensitivity characteristics of lesions in guinea pigs resistant to ticks and demonstrates a relationship between the mononuclear cell response of rabbits to nymphal I. dammini and the cellular response seen in patients with erythema chronicum migrans and Lyme arthritis.     

The feeding behaviour of larvae, nymphs and adults of Rhipicephalus appendiculatus.

The pre-feeding and feeding periods of larvae, nymphs and adults of Rhipicephalus appendiculatus on rabbits (Oryctolagus cuniculus) were investigated. Larvae and nymphs required at least 8-9 days after hatching and moulting respectively before they could attach and start feeding, while adults required at least 6-9 days. But longer periods of starvation improved the proportion of ticks that successfully fed. After attachment, there was always an initial period of slow feeding, which was followed by a phase of very rapid feeding before the engorged ticks detached from the host. Larvae detached 4-5 days after attachment, nymphs detached after 5-6 days and adults detached 7-9 days after attachment.     

The role of medium-sized mammals as reservoirs of Borrelia burgdorferi in southern New York

The ability of raccoons (Procyon lotor), striped skunks (Mephitis mephitis) and opossums (Didelphis virginiana) to serve as reservoirs of Borrelia burgdorferi, the spirochetal agent of Lyme disease, was compared with that of white-footed mice (Peromyscus leucopus). Twenty-eight (28) medium-sized mammals and 34 white-footed mice were captured in Westchester County, New York (USA) in summer 1986. Animals were caged over pans of water for 1 to 2 days to recover engorged tick larvae (Ixodes dammini) that detached from the hosts after feeding. With the exception of mice, numbers of engorged tick larvae recovered exceeded those counted during initial examinations of the hosts by 30% (opossums) to nearly 90% (raccoons). Newly-molted nymphal ticks derived from the engorged larvae were examined for the presence of spirochetes by darkfield microscopy. Percentage infection was 5% (n = 22) for ticks from skunks and 14% (n = 191) for ticks from raccoons. None of 24 nymphs from larvae that fed on opossums survived long enough for spirochete examination. By comparison, 40% (n = 72) of nymphs from larvae which fed on white-footed mice were infected. Of the individual hosts from which molted nymphs had fed as larvae, 67% of mice, 33% of skunks, and 55% of raccoons produced spirochete-positive ticks.     

Continuous cell lines from the tick Hyalomma anatolicum anatolicum.

The establishment of 5 continuous cell lines from embryonic tissues of the tick Hyalomma anatolicum anatolicum is reported. Each line comprises 2 or more cell types; they are maintained at 28 C and 32 C in L-15/H-Lac medium with 20% fetal calf serum, and have been cryopreserved successfully. Sustained and consistent growth was achieved only after 12-41 mo in culture.     

Expression of acquired immunity to immature stages of the tick Rhipicephalus evertsi evertsi by rabbits and guinea-pigs

Acquired immunity in guinea-pigs and rabbits to immature stages of the two-host tick Rhipicephalus evertsi evertsi Neumann was demonstrated. Repeated infestations of both hosts with larvae resulted in a significant reduction in the weight of later engorged nymphs. A sharp decline in the numbers of nymphs which successfully fed on both hosts was also observed. This study provides evidence for a gradual decrease in the mean weight of engorged nymphs towards the end of the detachment period suggesting that, in two-host ticks, the onset of nymphal feeding acts as an immune booster in a host already primed by the larval feed and that this results in a reduced feeding performance.     

The Effect of Male Ticks on the Feeding Performance of Immature Stages of Rhipicephalus Sanguineus and Amblyomma Americanum (Acari: Ixodidae)

A unique group of immunoglobulin-binding proteins (IGBPs), produced by ixodid male ticks during the latter half of their prolonged feeding period, improves the feeding performance of co-feeding females. As a follow-up to this observation, we investigated whether male tick feeding also affects the feeding of other developmental stages. Immature stages of Rhipicephalus sanguineus (Latreeille) and Amblyomma americanum (L.) were fed on rabbits in the presence or absence of conspecific males. The mean weight of larvae and nymphs of both species that fed around males and detached from the host on the first day of dropping was significantly higher than when the immature ticks fed on rabbits in the absence of males. However, larvae of both species and nymphs of R. sanguineus that fed slower and detached on the second day of dropping did not show significant differences in weight. A similar pattern was observed for A. americanum nymphs although, unlike R. sanguineus, the presence of males also influenced the feeding performance of the nymphs that fed slowly and detached on the second day of drop-off. The improved feeding performance demonstrated by immature ticks in the presence of males may be due to immunomodulatory saliva proteins, such as immunoglobulin-binding proteins (IGBPs) that are introduced into the co-feeding site. The results are considered in relation to the distribution of ixodid tick species on their natural hosts.     

Acquired immunity in rats against Angiostrongylus cantonensis infection

Acquired immunity against Angiostrongylus cantonensis was induced by immunizing rats with somatic antigens from fifth-stage larvae and adult worms and live third-stage larvae. Rats immunized twice had significantly fewer worms than rats immunized three times. Fewer worms were recovered from rats immunized with 200 live third-stage larvae than from any other groups. Rats immunized with somatic antigens had higher enzyme-linked immunosorbent assay (ELISA) antibody levels than rats immunized with live larvae. Rats immunized with live third-stage larvae of Angiostrongylus cantonensis were more strongly protected against challenge infections (62-92%) than rats immunized with antigens extracted from fifth-stage larvae (0-30%) and adult worms (11-24%).     

Immunomarking reveals food flow and feeding relationships in the eastern subterranean termite, Reticulitermes flavipes (Kollar)

Trophallaxis and feeding relationships in the eastern subterranean termite, Reticulitermes flavipes (Kollar), were examined using a novel marking technique, rabbit IgG protein coupled with an enzyme linked immunosorbent assay (ELISA) to detect the marker. Transfer experiments in small dishes evaluated the trophallactic transfer of the marker from donor workers fed IgG-treated paper to recipient workers or larvae. Worker donors rapidly acquired the marker, and 100% of donors tested positive within 24 h. Trophallactic transfer from donors to recipients was relatively inefficient, and 51 +/- 2% of recipient workers and 31 +/- 2% of recipient larvae tested positive at 72 h. Based on the mean optical density counts, approximately 27% of marker ingested by the donors was passed on to the recipient workers in the first 24 h, 14% to recipient larvae, and 26% to recipient soldiers. The ability of soldiers to feed independently of workers was examined in dish assays. Soldiers showed no significant uptake of the marker when isolated from the workers, and uptake increased significantly when workers were present. The distribution of the marker was further studied in larger colony fragments composed of workers, soldiers, nymphs, and larvae. Marker acquisition by the different castes/developmental stages was highly variable, with workers and nymphs acquiring the marker at a faster rate than soldiers and larvae. The results of this study contribute to our understanding of the foraging ecology and social behavior in R. flavipes. In addition, they may help design improved control programs for subterranean termites based on baits.