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.

Humoral immune response of dogs immunized with salivary gland, midgut, or repeated infestations with Rhipicephalus sanguineus.

The antibody (Ab) responses of dogs immunized with adult tick salivary gland (TSG), midgut (TMG), or repeated infestations of Rhipicephalus sanguineus were monitored to determine if there is an association between Ab production and R. sanguineus performance. Tick-naïve dogs were immunized with TSG or TMG and subjected to two challenge infestations. The control group was infested five times at 21-day intervals. The ELISA technique was used to measure Ab levels in sera from these dogs, which expressed different forms of resistance against R. sanguineus. In dogs immunized with TSG or TMG, similar Ab levels were detected against TMG, TSG, muscle, synganglion, and reproductive organs. However, these sera had different Ab levels against egg mass, unfed larvae, fed larvae, and nymph antigens. Ab levels to muscle, nerve, and reproductive antigens were lower than those observed when TMG or TSG antigens were used. Sera from dogs immunized with TMG or TSG responded to most tick stages or tissue antigens, whereas repeated infestation sera showed the lowest response among the three groups.

Evidence and mechanisms of immunosuppression in tick infestations.

The abundance and ubiquity of ticks from ancient times long ago suggested that they have eluded host immunity. In the last 15 years, several authors have demonstrated suppression of the Th1 responses (cell-mediated immunity), and sometimes the Th2 responses (humoral immunity), subsequent to tick infestations in laboratory and natural models. Although the mechanisms to produce suppression are not well-defined yet, evidences for antigenic competition, lymphocyte cytotoxicity, presence of immuno-inhibiting substances in the saliva, and existence of modulators of cytokines in salivary extracts have been reported. Management of tick-induced immunosuppression is essential to replace tick control by acaricide application with more environmentally sound vaccination.

Comparative immunizing power of infections, salivary extracts, and intestinal extracts of Hyalomma marginatum marginatum in cattle.

Tick concealed antigens have been successful in producing immunity that inhibits tick fertility, but require periodic revaccination and are little effective in preventing tick feeding, which is critical to stop pathogen transmission. Tick natural salivary antigens also induce important immunity, but revaccination may be unnecessary in enzootic areas. In addition these antigens may inhibit tick feeding. We immunized groups of three tick-naive calves with four prior infestations with Hyalomma marginatum marginatum, a salivary extract (SE), or an intestinal extract (IE) of the ticks. The calves were challenged with 100 pairs of homologous ticks and characteristics representing tick feeding or fertility were recorded and compared between groups. The percentage of attachment was inhibited by 46% by the infestation-generated immunity, 47% by the SE-generated immunity, and 0% by the IE-generated immunity. The percentage of engorgement was reduced 40% by the infestations, 57% by the SE, and 29% by the IE. The length of feeding was prolonged 92% by the infestations, shortened 44% by the SE, and not affected by the IE. The weight of the engorged females was decreased 67% by the infestations, 64% by the SE, and 31% by the IE. The percentage of engorged ticks that oviposited was inhibited 52% by the infestations, 27% by the SE, and 63% by the IE. The preoviposition period was prolonged 160% by the infestations, 80% by the SE, and 140% by the IE. The egg weight was reduced 60% by the infestations, 60% by the SE, and 66% by the IE. Taking into account mortality before oviposition, fertility was inhibited 88.2% by the infestations, 87.5% by SE, and 91.4% by the IE. The effect of IE immunization on tick feeding was not significant statistically.

Relationships and influences between Boophilus microplus characteristics in tick-naive or repeatedly infested cattle.

Six tick-naive male Hereford calves were infested once a month for 6 months with 18,000 Boophilus microplus larvae on the back and with 400 larvae in a cloth bag glued on the lumbar region. Working with the bag ticks, 12 tick characteristics were recorded for each infestation. Each tick attribute was analyzed for significant differences with those of the first infestation (analysis of variance), and for similarity (clustering), degree of relationship (correlation), and concomitant variation (regression) against all the other attributes during the first, third, and sixth infestations. Some attributes were affected maximally by host immunity about the third infestation but recovered later (length of feeding, detachment weight, egg weight, start of oviposition, fertility efficiency index), whereas others continued to be affected until the last infestation (length of oviposition, corpse weight, start of hatching, feeding efficiency index). All analyses showed that weight at detachment and egg weight were closely related, and corpse weight was partially related to these two. All other natural characteristics were largely independent. Length of feeding showed no significant relation with weight at detachment nor length of oviposition with egg weight. These findings suggest that different tick functions are independently affected by host immunity and recommends against estimating general anti-tick resistance by the evaluation of only a few tick characteristics.

A review on vaccination against protozoa and arthropods of veterinary importance.

The recent advances in immunology and biotechnology have stimulated much research on the control of parasitic diseases through vaccination. This is a review of the state of the art regarding important protozoan and arthropod veterinary parasites. A live oocyst vaccine for avian coccidiosis is still in use but much work has been done on the identification, cloning, and assay of protective antigens. The sporozoites of Eimeria tenella have been the preferred subject and at least four recombinant antigens have already been tested with partial success. Premunization against babesiosis is still widely used in Latin America as is a live vaccine with attenuated parasites in Australia. At least three Babesia bovis and three Babesia bigemina antigens that generate partial protection have been produced as recombinant proteins. A vaccine against canine babesiosis is being commercialized in France. Infection-treatment is still used to vaccinate against Theileria parva and a schizont vaccine against Theileria annulata. Recombinant sporozoite antigens have been assayed with partial success against both species but the identification and administration of protective schizont antigens, regarded as the most important, still requires considerable work. The immunological control of African trypanosomoses is still impaired by the antigenic variation that the parasites experience during the infection. Although some possibilities exist, most specialists are pessimistic about the promise of developing a vaccine in the near future. Control of Boophilus ticks with an occult tick intestine recombinant antigen seems to have potential in inhibiting reproduction of the tick but salivary antigens appear to be more effective at inhibiting feeding and pathogen transmission. Vaccination with a Hypoderma protein, recently cloned, has induced 90% protection against subsequent infestations. It is very likely that effective vaccines against veterinary parasites will become available in the near future.

Inhibition and recovery of tick functions in cattle repeatedly infested with Boophilus microplus.

Six tick-naive male Hereford calves were infested with about 18,000 Boophilus microplus larvae, 400 in a cloth bag, once a month for 6 mo. Working with the bag ticks, 12 tick characteristics were recorded for each infestation. Manifestations of resistance to some attributes appeared by the second infestation, peaked by the third (detachment weight, egg weight, fertility index) or fourth (feeding length, start of oviposition), and vanished by the fifth or sixth. Resistance to corpse weight, oviposition length, start of eclosion, feeding index, and survival developed more slowly but continued until the last infestation. Lymphocyte transformation after the fifth infestation was normal for concanavalin A and augmented in only 2 calves for phytohemagglutinin. In each infestation and at the end of all infestations some calves were significantly more resistant to some tick characteristics. There was no relationship between natural resistance and the ability to mount an acquired resistance. We concluded that B. microplus can depress or evade host's immunity, that resistance to different tick functions occurs independently, that natural and acquired resistance are unrelated, and that there exist genetic differences in natural or acquired resistance to the tick among seemingly homogeneous animals.

Immunity to Toxocara vitulorum repeated infections in a rabbit model.

Eleven rabbits were infected with 10 embryonated eggs of Toxocara vitulorum per g body weight on Days 0, 35 and 72. Embryonated eggs and larvae were enumerated in feces on Days 1-3 after each infection. Two rabbits were killed and larvae were enumerated in small intestine, liver, lungs, skeletal muscles, heart, kidney, brain, eye, uterus, and mammary glands on Days 5, 15, 30, 65 and 101. Serum was obtained on Days 0, 5, 15, 30, 42, 50, 65, 78, 86 and 101 to perform enzyme-linked immunosorbent assays (ELISAs) and Western blots against an extract of embryonated eggs. Between 4 and 10% of the administered parasites, almost all embryonated eggs, were found in the feces after the first or second infection, but 32% (27% of them larvae) after the third. Yields of tissue parasites were 4.1% of the administered dose on Day 5, 2% on Day 15, and 0.8% on Day 30 of the first infection, 0.1% on Day 30 of the second infection, and 0.06% on Day 30 of the third. Larvae were found only in liver, lungs and muscle, including heart. Larva content declined steadily in liver and lungs from Day 5 to 30 of the first infection, was absent in the liver at Day 30 of the second, and in both organs at Day 30 of the third. Muscle larva content increased from Day 15 to 30 of the first infection, and persisted throughout the third infection. Production of IgM antibody was minimal, IgG and the sum of IgMGA antibodies increased slightly or moderately after the first and second infections, but dramatically after the third. Western blots revealed the first antigens (12) by Day 15 of the first infection. Their total number increased with time and number of infections, but some antigens disappeared, whereas new antigens appeared in the course of the observations. Four antigens (32,500-41,000 mol.wt.) may be related to protection. Comparison of the Western blot patterns of two rabbits showed differences in the antigens, recognizable for each rabbit.

Evidence of immunosuppression by Demodex canis.

Three clinically normal beagles, 3 beagles with localized demodectic mange (LDM), and 3 beagles with generalized demodectic mange (GDM) were investigated simultaneously 1-3 and 4-6 weeks from the appearance of the clinical signs. Blood clinical examination and reactivity of peripheral lymphocytes to Con A and PHA were investigated in the first instance, and reactivity to Con A, PHA, and LPS in the second. Eight aliquots were used in each blastogenesis assay for each dog. All dogs were negative for rheumatoid factor. The results of blastogenesis showed that many observations were distributed non-normally, and that not all dogs in each group responded homogeneously. Comparison of blastogenesis results between dogs demands careful statistical analysis. Responses to mitogens were normal in all dogs at 1-3 weeks except for the LDM dogs that showed an increased response to PHA. Only the response to Con A was moderately inhibited in the LDM dogs at 4-6 weeks. All responses were severely depressed in the GDM dogs at 4-6 weeks. This means that immunosuppression follows rather than precedes the clinical manifestations of GDM, and implies that the phenomenon is induced by the parasite or the host's reaction to it.

Effect of host sex and litter on the population dynamics of Echinococcus granulosus in dogs.

Twenty-one Beagle dogs consisting of 10 males and 11 females and belonging to 3 litters were infected with 60,000 E. granulosus protoscolices each. They were killed on day 40, the parasites from their intestines recovered, and the number of worms, average number of proglottides per worm, average length per worm, percentage of worms with a uterine cavity, and percentage of egg-bearing worms were determined for each dog and analyzed per sex and litter. On average, the dogs had 1,253 +/- 339 worms (means +/- standard error) with 2.42 +/- 0.1 proglottides, were 1.59 +/- 0.07 mm long, and 25.6 +/- 4.8% of the worms presented a uterine cavity and 1.2 +/- 0.6% bore eggs. The number of worms exhibited a bimodal distribution with 19 dogs having less than or equal to 2,565 worms and 2 greater than or equal to 5,520 worms. Average number of proglottides also showed a bimodal distribution with 7 dogs having less than or equal to 2.1 proglottides per worm and 14 dogs having greater than or equal to 2.4 proglottides per worm. The parasites were significantly more numerous in females than in the males (1,964 +/- 573 vs. 681 +/- 202), had more proglottides (2.67 +/- 0.08 vs. 2.15 +/- 0.16), and were longer (1.72 +/- 0.07 vs. 1.44 +/- 0.11 mm). The percentages of parasites with a uterine cavity (27.8 +/- 5.9 vs. 23.2 +/- 8.1) or bearing eggs (1.0 +/- 0.5 vs. 1.5 +/- 1.8) were comparable in females and males.(ABSTRACT TRUNCATED AT 250 WORDS)

Biology and pathophysiology of Toxocara vitulorum infections in a rabbit model.

Ten female New Zealand rabbits were infected via stomach intubation with eggs of Toxocara vitulorum at a dosage of 10 embryonated eggs per gram of body weight on Days 0, 35 and 72. Ten or 4% of the administered parasites passed in the feces during the 3 days following the first or second infection, but 32% after the third infection. Many larvae were passed in the third infection, but not in the first or second. Tissue parasite yields were 4.1% on Day 5, 2% on Day 15, 0.8% on Day 30, 0.1% on Day 65 and 0.06% on Day 101. Five hundred and ninety-three larvae were recovered from liver, 243 from lungs and 0 from muscles on Day 5; 282 from liver, 138 from lungs and 21 from muscles on Day 15; 151 from liver, 21 from lungs and 50 from muscles on Day 30; 0 from liver, 26 from lungs and 15 from muscles on Day 65; 0 from liver, 0 from lungs and 9 from muscles on Day 101. No larvae were found in other tissues. The size of the muscle larvae at 30, 65 and 101 days indicated that the parasites did not develop beyond the infective stage and suggested that they were probably hypobiotic organisms. Erythrocytes, packed cell volume and monocytes decreased, but eosinophils and basophils increased, after each infection. Serum enzyme levels indicated that liver damage occurred only after the first infection, but muscle injury occurred after each infection and was increasingly more precocious after each infection.

Antigens of Amblyomma americanum ticks recognized by repeatedly infested sheep.

Sera were taken from 3 sheep that had been infested 5 times with Amblyomma americanum and that exhibited manifestations of humoral depression to homologous antigens and anti-tick resistance. Proteins extracted from the intestine or salivary glands of unfed ticks or salivary glands from partially (3-day) fed ticks were analyzed by polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE. Antigens recognized by the sheep in the same materials before and after each infestation were analyzed by western blots. The sheep responded to 44 antigens. Nine to 23 antigens were recognized by the preinfestation sera and the sera of 2 gnotobiotic sheep. Four antigens (34,000, 36,500, 38,000, and 115,000 MW) were revealed conspicuously by the serum of the first infestation but very weakly or not at all by the sera of the third infestation onward. Two antigens (35,500 and 29,000 MW) from fed salivary glands were revealed only by sera taken after manifestations of resistance had appeared. These antigens may be responsible for anti-tick protection. The 29,900 MW antigen was present also in salivary extracts of Boophilus microplus.

Manifestations of immunity in sheep repeatedly infested with Amblyomma americanum ticks.

Three sheep were infested 4 times with 100 Amblyomma americanum tick pairs and kept indoors until the natural termination of the infestations. Characteristics of the tick populations that show efficiency of feeding, fertility, and offspring development, and ELISA antibodies to tick salivary gland extracts were studied at each infestation. On average, female ticks fed on tick-naive sheep detached at 12.1 +/- 0.2 (mean +/- standard error) days, weighed 492 +/- 16.8 mg, engorged 40.7 mg per day, and 35% survived to detachment during the first infestation. During the fourth infestation, they detached at 17.3 +/- 0.8 days, weighed 321 +/- 14.4 mg, engorged 18.8 mg per day on average, and 23% survived to detachment. On average, oviposition of female ticks fed on tick-naive sheep started at 11.8 +/- 0.6 days of detachment, the egg mass weighed 236 +/- 13.2 mg, 43% of the female weight turned into eggs, and 89% of the ticks that detached survived to oviposition during the first infestation. During the fourth infestation, oviposition started at 15.1 +/- 0.5 days, the eggs weighed 103 +/- 9.9 mg, 13% of the tick weight became eggs, and 67% of the ticks survived to oviposition. On average, eclosion started at 35.4 +/- 0.9 days of oviposition and 83% of the egg batches hatched in the first infestation. During the fourth infestation, eclosion started on day 34.9 +/- 0.7, and 47% of the egg batches hatched. Anti-tick resistance was expressed as an inhibition of feeding, fertility, and offspring development.(ABSTRACT TRUNCATED AT 250 WORDS)

IgG response in guinea pigs to Trichinella spiralis infection.

An enzyme-linked immunosorbent assay (ELISA) using excretory-secretory antigens was developed to study the dynamics of the IgG antibody response to varying levels of Trichinella spiralis infection in the guinea pig. Four groups of four Hartley guinea pigs each were infected with 1250, 250, 50 or 10 T. spiralis infective muscle larvae. They were bled every 15 days for 6 months and the IgG antibody response determined by ELISA. The time of seroconversion was dose dependent as the larger the dose, the earlier the response occurred. Significant differences in antibody response between the dose groups were evident at 30 days post-infection (P less than 0.05). Beyond 60 days post-infection, the response was similar in the four groups. The antibody response in the groups infected with 250 and 50 infective larvae was similar, but was significantly different from that of the high (1250) and low (10) dose groups from 30 days post-infection (P less than 0.01). Once seroconversion occurred, the antibody titer rose to the same level, irrespective of the initial dose. To compare the antibody response according to muscle larvae recovered, the guinea pigs were grouped into four categories: less than 10 larvae; 10-25 larvae, 50-80 larvae, greater than 100 larvae. A significant positive correlation (P less than 0.05) was observed at 60 days post-infection when these groups were compared.

A critical look at the importance, prevalence and control of toxocariasis and the possibilities of immunological control.

The visceral infection of humans with Toxocara canis is particularly prevalent in children and may cause a variety of symptoms that commonly persist for 6-24 months. The ocular infection usually causes permanent loss of visual acuity. Human infection is acquired by ingestion of embryonated T. canis eggs with contaminated dirt. Review of recent reports indicates that patent T. canis infection is widely prevalent in the general population of dogs all over the world (3-81%) and results in a substantial contamination of the ground (0.3-87%). The results of sensitive and specific serological tests suggest that about 7% of the clinically healthy human population of the United States, about 5% of that of Canada, and about 4% of that in Great Britain is infected with the parasite. Control of transmission of the parasite to man is often attempted by eliminating the infection in dogs, reducing the population of dogs and the environmental contamination with their feces, and educating the public about the zoonotic potential of toxocariasis. The evidence reviewed indicates that these methods are only marginally effective. Because T. canis relies on congenital and lactogenic transmission to persist in nature, only a procedure that effects the sustained killing of the reservoir larvae in the tissues of the bitch, or of newly-acquired parasites, is expected to be successful. Research with mice, rabbits and dogs demonstrated that prior infections of the host induce the development of protective immunity to reinfections. This procedure, however, leaves remnant populations of larvae from the immunizing infections that are resistant to anthelmintics and to the effect of prior irradiation. Hyperimmunization with partially-purified extracts of T. canis larvae induced 37% resistance to a challenge in mice when the extract was administered alone, and 76% resistance when administered with lipopolysaccharide adjuvant. Production of complete resistance, however, will probably require the prior control of the immunosuppression induced by the parasite. T. canis infections inhibit the production of homologous protective immunity and antibody responses to heterologous antigens, probably by interfering with the activity of helper T-cells, competing with protective antigens, and suppressing antibody synthesis. The evidence indicates, however, that an anti-T. canis vaccine to eliminate the parasite in dogs is feasible.

Limited efficacy of levamisole against adults of Dirofilaria immitis in a dog.

A dog with chronic dirofilariasis (Dirofilaria immitis) was given 15 doses of 12 or 24 mg of levamisole/kg of body weight in 6 treatments within 96 days. Concentration of microfilariae in the blood was determined before and after treatment, and Aedes triseriatus mosquitoes were fed on the dog 1 to 5 days after each treatment and examined for D immitis larvae. Several adult worms were recovered from the dog 160 days after the end of the treatments.

Anthelmintic effect of levamisole hydrochloride or ivermectin on tissue toxocariasis of mice.

Paranatal transmission of Toxocara canis infection could be prevented in pups if an effective drug were administered to pregnant bitches. This drug also could eliminate the larvae in dogs that have been experimentally infected repeatedly to produce protective immunity. For these reasons, we assayed the effect of 2 doses of levamisole hydrochloride or ivermectin on T canis larvae. Mice (5 groups) were infected with 1,000 infective T canis larvae and then treated with 2 different dosages of levamisole hydrochloride (6 mg/kg or 12 mg/kg, given subcutaneously), 2 different dosages of ivermectin (0.2 mg/kg or 0.4 mg/kg, given IM) or 0.15M NaCl (given subcutaneously) once a day from days 15 to 28 of infection. On day 33 of infection, the parasites in liver, lungs, brain, and carcass were obtained and compared between groups. The smaller dosage of levamisole hydrochloride (6 mg/kg) significantly decreased only carcass parasitism to 17% of that in the controls, but did not affect significantly the total parasite load. The larger dosage of levamisole hydrochloride (12 mg/kg) decreased the infection in all organs, but particularly in carcass and brain; total parasitism was only 36% of that in the controls. The smaller dosage of ivermectin (0.2 mg/kg) significantly increased the number of larvae in the lungs to 550% of that in the controls, but it did not significantly affect the total parasite load. The larger dosage of ivermectin (0.4 mg/kg) significantly decreased only brain parasitism, but liver and total parasitism were decreased to 40% and 57%, respectively, compared with that in the controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of irradiation on the biology of the infective larvae of Toxocara canis in the mouse.

Mice were infected with either 2,000 normal or irradiated embryonated eggs of Toxocara canis and the number of larvae in their livers, lungs, brains, and carcasses investigated at 5, 20, and 33 days of infection. Mortality of mice infected with normal eggs was 33% between day 4 and 8 postinfection but there was no mortality among mice infected with irradiated eggs. Irradiation with 60, 90, or 150 kr of X-rays inhibited the migration of larvae from the livers and lungs and their accumulation in brain and carcass in proportion to the irradiation dose. By day 33 of infection, the ratio of larvae in liver and lungs to larvae in brain and carcass was 0.16 in normal mice, 0.42 in 60-kr mice, 0.98 in 90-kr mice, and 23.3 in 150-kr mice. Irradiated larvae, particularly those migrating through the peritoneal cavity, died faster than normal larvae until day 20. Irradiation favored survival after day 20. By days 20 and 33 postinfection the total parasite load was 29% and 8%, respectively, of the administered dose in control mice, 18% and 12% in 60-kr mice, 8% and 4% in 90-kr mice, and 0.9% and 0.3% in 150-kr mice. Irradiation of infective T. canis larvae, then, reduces their pathogenicity, inhibits their migration from liver and lungs, kills some of the parasites during the first 3 weeks of infection, but favors their late survival in the host.

Identification by transfer blot of antigens reactive in the enzyme-linked immunosorbent assay (ELISA) in rabbits immunized and a calf infected with Cryptosporidium sp.

Soluble and particulate fractions of Cryptosporidium sp. oocysts from cattle were obtained by homogenization and sonication. Electrophoresis of the soluble fraction in polyacrylamide gels with sodium dodecyl sulfate and silver staining revealed the presence of 41 bands. Enzyme-linked immunosorbent assay (ELISA) of sera from rabbits immunized with either fraction and from a calf 40 days after infection showed that the animals produced specific antibodies. Enzyme-linked immunoelectrotransfer blot tests revealed the presence of five antigens with the rabbit sera and nine with the calf serum. ELISA proved to be an appropriate test for diagnosis of cryptosporidiosis. Selection of reactive antigens may improve the quality of diagnosis and/or reveal the presence of protective materials in the parasite.