Rabbits infested with Ixodes ricinus L. adults: effects of a treatment with cyclosporin A on the biology of ticks fed on naive and immune hosts

Rabbits have been infested 3 times with 10 female and 10 male Ixodes ricinus. Immunity which is induced when ticks feed on naive animals (1st infestation) perturbs feeding, oviposition and embryogenesis during reinfestations. Treatment of rabbits during a 3rd infestation (resistant animals) with cyclosporin A (CsA), an immunosuppressive agent which works on the cellular compartment (chiefly T helper cells), partially reversed the negative effects of the immunity on the biology of the ticks. Conversely, CsA may also directly affect the reproductive processes of ticks. Thus, the weight of the eggs laid and the egg conversion factor of ticks fed on naive treated hosts (1st infestation) were diminished. In addition, the preoviposition was prolonged, and finally failure in oviposition and hatching occurred more frequently.     

Performance of female Rhipicephalus sanguineus (Acari: Ixodidae) fed on dogs exposed to multiple infestations or immunization with tick salivary gland or midgut tissues

This investigation compared the effects of repeated infestations to immunization of dogs with tick salivary gland or midgut extracts on the feeding and fecundity performances of female Rhipicephalus sanguineus (Latrielle). In each immunized group, three tick-naive dogs were immunized three times with tick salivary gland or midgut extracts, and twice challenged at 21-d intervals by allowing 80 female and 40 male adult ticks to feed on each host. The repeated infestation group of three naive dogs was infested five times at 21-d intervals by the same numbers of ticks. The repeated infestation group showed a trend of reduced tick performance after the third infestation, but some of the tick performance parameters had recovered by the fifth infestation. Tick attachment was reduced by immunization with either tick salivary gland or midgut extract. Immunization with tick salivary gland extract had the greatest impact on the feeding period and engorgement weight of the female ticks. Immunization with tick midgut extract resulted in the greatest reduction of tick fecundity parameters, which included preoviposition, oviposition, and egg-incubation periods in addition to reduced egg production and egg viability. These results confirm that dogs can become resistant to R. sanguineus, and demonstrate that immunization with tick salivary gland or midgut extract has different effects on tick feeding and fecundity.     

Feeding efficiency of larval Hyalomma truncatum (Acari: Ixodidae) on hosts previously exposed to ticks.

The effect of guinea pig response to feeding larval Hyalomma truncatum Koch ticks was studied by measuring the percentage of ticks engorging and molting, their engorged weight, and hemoglobin content. Four guinea pigs were infested with 100, 200, 400, and 1,000 larvae, respectively, on three occasions at 21-d intervals, followed by a fourth infestation with 500 larvae. During the second, third, and fourth infestations, significantly fewer ticks engorged on each guinea pig than during the first infestation. The greatest reduction in percentage molting occurred during the fourth infestation on the animal with successive exposure to 400 larvae; only 24% of the ticks that fed molted. Ticks with the lowest mean weight and hemoglobin content also engorged on this animal during the fourth infestation. Guinea pigs exposed to 200 and 400 H. truncatum larvae elicited the greatest change in feeding efficiency during the fourth infestation. However, these hosts had no effect on a single subsequent fifth infestation with Amblyomma cajennense (F.) nymphs, as greater than 95% successfully engorged.     

Immunization of guinea-pigs and cattle against adult Rhipicephalus appendiculatus ticks using semipurified nymphal homogenates and adult gut homogenate.

Guinea-pigs inoculated with crude homogenate of unfed nymphs of the tick Rhipicephalus appendiculatus and with three semipurified fractions of the homogenate obtained by gel permeation chromatography, acquired a significant degree of immunity to infestation with adults of this tick. Fraction 2 induced the highest reduction (66%) in mean weight of engorged females followed by crude homogenate and fractions 1 and 3. Calves immunized with crude homogenates of unfed nymphs, fraction 2 of nymphal homogenate, and gut homogenate of unfed females also acquired immunity against adults of R. appendiculatus. The mean weight of engorged females fed on calves inoculated with nymphal fraction 2 was the lowest of all five groups of calves on which females fed. The reduction in weight (38%) was not significantly different from that observed for females fed on calves inoculated with crude nymphal homogenate (31%) or females from third infestation of adult ticks. No differences in the weight and hatchability of egg batches produced by engorged females collected from the five groups of calves were observed. Analysis of sera collected from the five groups of calves showed that the concentration of albumin, alpha-1, alpha-2 and beta-globulins fluctuated and no significant differences between the treated groups were observed. The levels of gamma-globulin increased in treated groups including the group inoculated with adjuvant only, but unlike previous reports no increase in gamma-globulin or a correlation between the level of gamma-globulin and the degree of resistance acquired were observed in calves exposed to repeated tick infestations. However, the increase in the concentration of gamma-globulin in calves inoculated with fraction 2 or crude nymphal homogenate was higher than that observed in the other groups.     

Acquired resistance to ticks. I. Passive transfer of resistance.

Guinea-pigs developed resistance to larvae of the ixodid tick, Dermacentor andersoni, after one infestation. Resistance was characterized by guinea-pigs allowing fewer larvae to engorge (5-15%) during a second exposure than during an initial infestation (70-90%). Larvae feeding on resistant hosts weighed less than larvae engorging on a host with no previous exposure to ticks. Evidence is presented which indicates that this resistance can be passively transferred with viable lymph node cells, but not with serum, from resistant guinea-pigs. Recipients of cells from such resistant animals allowed significantly fewer larvae to engorge than did controls previously unexposed to ticks. This was not so in recipients of immune serum. The protection provided by passive transfer of cells from a resistant donor was not as complete as the protection afforded by natural exposure.     

Efficacy and blood sera analysis of a long-acting formulation of moxidectin against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) on treated cattle

The therapeutic and persistent efficacy of a single subcutaneous injection of a long-acting formulation of moxidectin at a concentration of 1 mg/kg body weight was determined against Rhipicephalus (Boophilus) microplus (Canestrini), along with the concentration-time blood sera profile in treated cattle. The therapeutic efficacy against ticks of all parasitic stages on cattle at the time of treatment was >99.9%, and the mean tick number, index of fecundity, engorgement weight, and egg mass weight of ticks recovered from treated animals were all significantly lower than ticks from untreated animals. The index of fecundity, engorgement weight of females, and egg mass weight of ticks recovered from treated animals infested at weekly (7-d) intervals between 14 and 63 d posttreatment were significantly lower than for ticks on untreated animals, whereas the number of ticks per animal recovered from treated cattle remained lower than that of untreated cattle for up to 49 d posttreatment. The percentage control remained >99% at weekly intervals between 14 and 49 d posttreatment, which is the minimum level of efficacy considered acceptable for use in the United States Cattle Fever Tick Eradication Program. The serum concentration of moxidectin in treated cattle increased to 25.6 ppb (parts per billion) within 1 d after treatment, and peaked at 47.3 ppb at 8 d posttreatment. Moxidectin sera levels remained above the estimated 100% threshold level for elimination of feeding ticks (5-8 ppb) for 44-53 d after treatment. The label claim of 50 d of prevention against reinfestation for the long-acting moxidectin formulation used in the study was supported by the efficacy and sera concentration data obtained. Based on these results, cattle could be treated at 63-d intervals with minimal risk of viable ticks detaching from treated animals. This treatment interval would be 4.5-fold longer than the presently required treatment interval of 14 d, thus leading to approximately 75% reduction in gathering and handling costs of cattle incurred by producers.     

Induction of host resistance toRhipicephalus appendiculatus in rabbits: Effects of immunizing with detergent-solubilized tick tissue proteins

Resistance to the hard tick,Rhipicephalus appendiculatus, was induced in rabbits by immunizing them with tick tissue proteins extracted with a detergent, Triton X-100. There was 25% mortality in female ticks fed on immunized rabbits as compared with those fed on controls. Similarly, there was a 40% and 60% reduction in the engorged weight and the weight of egg batches, respectively, of ticks fed on immunized rabbits. Western blot analysis of detergent-solubilized tick tissue Western blot analysis of detergent-solubilized tick tissue proteins, carried out using immune sera, recognized a complex pattern of proteins. A strong reaction was observed with proteins with apparent molecular weights of 94000 and 40000 daltons.This work was carried out at the Institute of Zoology, University of Neuchâtel, Chantemerle 22, CH-2000 Neuchâtel, Switzerland     

Immunity against female Ixodes ricinus L.: effect on feeding and haemoglobin digestion

Rabbits have been infested 3 times with 10 females and 10 males Ixodes ricinus. Immunity which is acquired when ticks feed on naive rabbits (first infestation) perturbs tick feeding on reinfested animals (third infestation). Then ticks ingest less blood (p less than 0.001). Blood meal digestion in also altered. It was estimated by measuring haemoglobin concentration in ixodid midgut during 20 days after their drop off. After the first infestation this concentration decreases linearly with time (r2(1) = 46.14%, n1 = 63, p less than 0.001). After 3 infestations it is no longer correlated with time, indicating an impaired digestive process (r2(3) = 7.15%, n3 = 49, p greater than 0.05). This observation was corroborated by an analysis of multiple regression. Haemoglobin concentration of tick midgut only correlates with time after a first infestation (r2(1) = 45.25%). In ticks fed on immune animals this concentration is predicted with the quantity of midgut C3 and the weight of fed ticks and not with time (r2(3) = 60.99%).     

Identification of paramyosin as a potential protective antigen against Brugia malayi infection in jirds.

Vaccination of jirds with irradiated infective larvae of Brugia malayi has been reported to provide partial immunity to larval challenge. In the present study, we found that sera from vaccinated animals recognized larval antigens with apparent molecular weights of 97, 55-60, and 10 kDa that were not recognized by sera from infected animals. A B. malayi cDNA expression library in lambda gt11 was screened to identify clones that were preferentially recognized by sera from immunized animals. One of these clones (BM-5) was chosen for further study. BM-5 contains a 2.1 kb DNA insert and produces a fusion protein with a molecular weight of 185 kDa. Antibody, affinity-purified with the BM-5 fusion protein, binds to a 97 kDa native B. malayi antigen. Immunological studies and partial DNA sequence data confirm that BM-5 encodes paramyosin. Recombinant B. malayi paramyosin is strongly recognized by antibodies in sera from jirds that have been immunized either by injection with irradiated larvae or by chemotherapy-abbreviated infection. Most sera from infected jirds do not contain antibody to paramyosin. Additional studies are needed to determine whether paramyosin is actually protective in this filariasis model.     

Immunization of rabbits against nymphs of Amblyomma hebraeum and A. marmoreum (Acari: Ixodidae).

Himalayan rabbits immunized with homogenates prepared from nymphs of Amblyomma hebraeum Koch and A. marmoreum Koch ticks developed humoral and probably also cell-mediated immunity to their respective homogenates. Beta and gamma globulin levels and numbers of eosinophils and neutrophils increased significantly in inoculated rabbits. The recipient animals developed resistance to homospecific nymphal infestations. Cross resistance between the two species was not evaluated. Nymphs of both species that fed on inoculated rabbits demonstrated slightly shorter feeding periods, and their mean weights were significantly lower than nymphs that fed on Quil 'A' adjuvant-inoculated rabbits or on naive rabbits. Significantly higher proportions of nymphs from immunized animals failed to moult when compared with nymphs that fed on the two control groups. These parameters indicate that the inoculated rabbits had acquired protective immunity against nymphs of both ticks.     

Salivary gland extracts of partially fed Dermacentor reticulatus ticks decrease natural killer cell activity in vitro.

The salivary glands and saliva of ticks (Arachnida, Acari, Ixodida) play a vital role in blood feeding, including manipulation of the host's immune response to tick infestation. Furthermore, a diverse number of tick-borne pathogens are transmitted to vertebrate hosts via tick saliva. A factor synthesized in the salivary glands of feeding ticks potentiates the transmission of certain tick-borne viruses. We show that salivary gland extracts (SGE) derived from Dermacentor reticulatus female ticks fed for 6 days on laboratory mice (SGED6) induced a decrease in the natural killer (NK) activity of effector cells obtained from 16 healthy blood donors. The decreased activity ranged from 14 to 69% of NK activity observed with the respective untreated effector cells. Such a decrease was not observed after treatment of effector cells with SGE from unfed ticks. Ten-fold dilution of SGED6 significantly reduced the capacity to decrease NK activity and a further 10-fold dilution almost eliminated the effect. After addition of IFN-alpha 2, the SGED6-induced decrease in NK activity was restored to activity levels approaching those of untreated cells. The apparent reversibility of the inhibition indicates that the effect of SGED6 on NK activity was not due to cytotoxicity. The results demonstrate the presence of a factor(s) in the salivary gland products of feeding D. reticulatus female ticks that influences human NK activity in vitro. These data suggest a possible mechanism by which tick SGE potentiates the transmission of some tick-borne viruses through suppression of NK activity.     

Quantitative studies of host immunoglobulin G in the hemolymph of ticks (Acari)

A radioimmunoassay was used to measure the concentration of host immunoglobulin G (IgG) in the hemolymph of female hard and soft ticks. Hyalomma excavatum Koch, Rhipicephalus sanguineus Latreille, Ornithodoros tholozani (Laboulbene and Megnin), and O. moubata (Murry) were fed on rabbits immunized with ovalbumin; Argas persicus (Oken) was fed on chickens immunized with cytochrome 'C.' At 24 h after feeding, the concentration of antiovalbumin IgG in the hemolymph was 7 micrograms/ml for H. excavatum, 5 micrograms/ml for R. sanguineus, and 0.15 micrograms/ml for O. moubata; the percentage of intact IgG molecules was 30, 44, and 100%, respectively. Host IgG was not detected in the hemolymph of O. tholozani and A. persicus. There was no increase in the concentrations of host IgG in the hemolymph of the soft ticks during the first week following the blood meal. The potential contribution to the resistance of hosts against ticks by host antibodies that cross into the tick hemocoel is discussed.     

Stage-specific antigens of Nematospiroides dubius.

The results presented here demonstrate conclusively for the first time that Nematospiroides dubius exhibits stage specificity in the expression of cuticular antigens, and infer that the stage specific antigens demonstrable on L4 stages 6 days following infection account for the immunity generated by abbreviated infection regimes. L4 stages 6 days following infection appeared to possess a stage-specific surface molecule (MW 16,000) which either disappeared on maturity to adults or somehow became unavailable to the surface probes used in this study. A molecule of similar molecular size was recognized by sera raised in mice immunized either by the administration of irradiated larvae or by using an antihelminthic-abbreviated infection and also by sera from jirds which had recently expelled N. dubius. The immune derivation of these sera supports the hypothesis that the molecule having a MW of 16,000 is functionally immunogenic. A monoclonal antibody (29-1-D1) was used to demonstrate a further stage specific molecule (MW 65,000), this time on the surface of adult N. dubius. The application of biochemical techniques suggested that further heterogeneity existed in surface molecules resolving between 60,000-70,000 on SDS-PAGE and a hypothesis was formulated in an attempt to explain this phenomenon.     

Comparison of methods for introducing and producing artificial infection of ixodid ticks (Acari: Ixodidae) with Ehrlichia chaffeensis.

Only 29.5 +/- 8.91% of engorged Amblyomma americanum (L.) nymphs that we inoculated with Ehrlichia chaffeensis molted successfully to adults compared with 75.8 +/- 7.46% of engorged nymphs that were not inoculated. However, 65.4 +/- 6.02% of unfed nymphs of this species were exposed for 2 h to E. chaffeensis suspension introduced to them through glass capillaries gained weight. These nymphs were placed on rabbits, and approximately 50% of them completed their feeding and molted successfully to adults. Weight gained was higher (71.8 +/- 17.33% and 69.8 +/- 23.26%) for unfed A. americanum females that fed from capillaries for 2 and 24, h respectively, than for nymphs. Similar values were recorded for Dermacentor variabilis (Say) (61.0 +/- 16.23%) and Rhipicephalus sanguineus (Latreille) (59.0 +/- 18.62%) females after 24 h of capillary feeding. The amount of E. chaffeensis suspension taken in by females of A. americanum, D. variabilis, and R. sanguineus during 24 h of feeding was 11.2 +/- 3.56, 10.9 +/- 4.29 and 6.3 +/- 2.35 microliters, respectively. This volume is equivalent to approximately 12,969, 12,622, and 7,295 infected cells ingested by the species mentioned above. Positive correlation between the volume taken in by the ticks and the weight gained by the females was found, but the initial weight of the unfed females did not effect the weight they gained. The pathogen was found in the females of all 3 species by polymerase chain reaction procedures for at least 7 d, indicating that the capillary feeding method can be successfully used for infecting unfed ticks. The potential use of this method is discussed.     

Protein change of intestinal epithelial cells induced in vitro by <Emphasis Type="Italic">Trichinella spiralis</Emphasis> infective larvae

The aim of this study was to observe the protein changes of intestinal epithelial cells induced in vitro by Trichinella spiralis infective larvae and their excretory-secretory (ES) or surface antigens and identity the proteins related with invasion. HCT-8 cells were incubated for 2 h in the culture medium contained ES or surface antigens of infective larvae, and observed by Immunofluorescent test (IFT). The infective larvae were inoculated into culture of HCT-8 cells to incubate for 18 h, and the lysates of HCT-8 cells were analyzed by SDS-PAGE and Western blot. IFA showed that normal HCT-8 cells had positively reactions with sera of the infected mice and mice immunized with ES or surface antigens. However, after incubating with ES or surface antigens, HCT-8 cells had stronger positively reaction with the above sera. On Western blot, after cultured with infective larvae, additional seven protein bands (66, 61, 57, 45, 34, 21, and 17 kDa) of HCT-8 cells were recognized by sera of the infected or immunized mice, but three protein bands (48, 43, and 23 kDa) of HCT-8 cells were not recognized by the above sera, compared with normal HCT-8 cells. Our results showed that after cultured with infective larvae the protein components of HCT-8 cell changed, suggesting that additional seven proteins recognized by sera of the infected or immunized mice may be related with invasion of intestinal epithelial cells by infective larvae, these proteins might mediate or facilitate entry into the cells, while the three proteins not recognized by the above sera may be the specific mediators released from the cells which permit invasion.     

Alteration of the protein composition in the haemolymph of American cockroaches immunized with soluble proteins.

Previous in vivo experiments demonstrating the existence of humoral immunity in cockroaches prompted us to investigate the composition and number of proteins that are newly induced or increased in quantity in the haemolymph of immune cockroaches compared to saline-injected control animals. SDS-PAGE analysis showed that six different haemolymph proteins of molecular weights 220,000, 162,000, 115,000, 102,000, 95,000 and 45,000 were induced in response to immunization by honeybee toxoid. However, only the 115,000 and 102,000 MW proteins consistently increased in quantity in the haemolymph of females immunized with various other soluble protein antigens, such as phospholipase A2 toxoid and inactivated creatine phosphokinase. Other experiments showed that the 115,000 MW band was not induced in immunized male cockroaches. Therefore, only the 102,000 MW protein was consistently enhanced in all samples of immune haemolymph from animals injected with various antigens. In addition, this protein was found in precipitates recovered from assays where immune haemolymph proteins were precipitated by the antigen. Gel filtration chromatography showed that this induced haemolymph protein is large, and bears an apparent native molecular weight of 600,000 +/- 80,000. This suggests that the 600,000 MW protein may consist of multiple subunits of 102,000.     

A rickettsia-like organism showing positive immunofluorescence with antisera to Coxiella burnetii in Haemaphysalis inermis ticks

A rickettsia-like organism (RLO) was detected in the oocytes of Haemaphysalis inermis ticks. The RLOs were about 5 microns long bent rods with a definite inner structure. They were Gram-negative and could be visualized by Giemsa but not by Gimenez staining. Attempts to cultivate the RLO in chick embryo yolk sacs, various types of cell culture and tick body cavities were unsuccessful. The RLO displayed a bright immunofluorescence with antisera to Coxiella burnetii, but no immunofluorescence was obtained with antisera to representatives of typhus and spotted fever group rickettsiae, with the exception of very weak fluorescence with serum from a rabbit immunized with Rickettsia akari and one serum from Apodemus flavicollis immunized with Rickettsia conorii. These findings should be taken into consideration when studying the infestation of ticks with rickettsiae.     

Identification of potential allergens in white oak (Quercus alba) pollen by immunoblotting

Aqueous extracts of white oak pollen were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The nitrocellulose membranes were blocked with phosphate-buffered saline 15% nonfat dry milk, incubated with dilutions of sera from atopic or control subjects, and probed with a radiolabeled or peroxidase-labeled antihuman IgE. The IgE binding bands were detected by autoradiography or enzymatic reaction; 45 to 50 protein bands were observed in silver-stained gels. IgE from 30 of the 38 sera tested from oak-sensitive subjects bound to 23 bands with molecular weights (MWs) between 106 to 108 kd (band 1) and 13.2 to 15.2 kd (band 23). No band was recognized by sera of every patient. Band 5 (MW 74.0 to 77.9 kd) and band 21 (MW 16.2 to 17.7 kd) were recognized by 71% of the patients' sera. Multiple bands were recognized by 30% to 50% of the sera tested. All patients who were skin test positive to oak by prick testing had positive immunoblots. Of 12 patients positive by intradermal skin testing, only four patients had positive immunoblots. The average number of allergens recognized by a single patient was 6.6. The maximum number of allergens to which any individual reacted was 18; the minimum number was one. Extracts separated under nonreducing conditions resulted in aggregates that did not enter the polyacrylamide gel. Of the protein that did enter the gel, the higher MW species elicited banding patterns similar to patterns observed under reducing conditions, whereas lower MW IgE binding bands were lost. These data suggest that the extractable proteins of white oak pollen contain multiple proteins that are potentially allergenic.     

Immune response of lizards and rodents to larval Ixodes scapularis (Acari: Ixodidae).

The house mouse (laboratory strain), Mus musculus (L.), the cotton mouse, Peromyscus gossypinus (LeConte), the broad-headed skink, Eumeces laticeps (Schneider), and the guinea pig, Cavia porcellus (L.), were successively infested five times with larvae of the blacklegged tick, Ixodes scapularis Say. Tick feeding success, engorgement weight, and subsequent molting success were measured after each infestation. A greater percentage of ticks (P less than 0.05) fed on M. musculus and E. laticeps than on P. gossypinus or C. porcellus. P. gossypinus expressed a transitory partial resistance, measured in percentage of ticks feeding, during the third infestation but showed increased tolerance during the fourth and fifth infestations. Ticks fed on E. laticeps were heavier than those fed on any other host (P less than 0.05). Those fed on M. musculus were heavier than those fed on P. gossypinus, but the difference was not statistically significant. On C. porcellus, only 1.6% of larvae from the third infestation and none thereafter engorged; weights of larvae from first and second infestations were higher (P less than 0.05) than those fed on M. musculus and P. gossypinus and lower (P less than 0.05) than those fed on E. laticeps. A greater percentage of larvae from E. latticeps and M. musculus (P less than 0.05) molted to nymphs compared with those from P. gossypinus and C. porcellus. Molting success was the same for ticks fed on P. gossypinus and on C. porcellus during the first and second infestations. M. musculus, P. gossypinus, and E. laticeps expressed no resistance (measured as percentage feeding, engorgement weight, and molting) to feeding by I. scapularis larvae after five infestations. Host serum was tested using an ELISA for detection of antibodies against I. scapularis salivary gland antigens before and after each infestation. Antibodies were detected after the second infestation and thereafter in C. porcellus, the only species to show antibodies or to express and maintain an acquired resistance to tick feeding throughout five infestations.     

Campylobacter jejuni outer membrane proteins are antigenic for humans.

All Campylobacter jejuni strains have a major outer membrane protein (OMP) that migrates between a molecular weight of 41,000 (41K) and 45K and represents more than 50% of protein present, plus several more minor bands. Using 125I-radiolabeled C. jejuni cells in a radioimmunoprecipitation procedure to assess whether the OMPs were antigenic, we studied serum from rabbits immunized with C. jejuni cells, from humans convalescent after C. jejuni infection, and from appropriate controls. In this assay, the major OMP was the major antigen for both homologously and heterologously immunized rabbits and infected humans but not for controls. Minor bands at 29K and 50K were also antigenic. We tested human and animal sera in a Western blot procedure using anti-immunoglobulin A (IgA), anti-IgG, or anti-IgM conjugates. Homologous and heterologous immune rabbit serum, but not control serum, recognized a large number of membrane proteins between 15K and 91K, including the major OMP. Both Campylobacter spp.-infected and healthy humans showed IgA, IgG, and IgM responses to the major OMP, although the response was more pronounced in the former group. Sera from infected humans recognized several minor bands to a significantly greater extent than control sera did. Our data suggest that there is antigenic similarity between the OMPs of different C. jejuni strains and that some of these OMPs recognized by infected animals and humans have vaccinogenic potential.