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.     

Basophils in skin reactions of mast cell-deficient mice infested with Dermacentor variabilis

Acquired resistance to ticks in guinea pigs has been found to be associated with basophil-rich skin reactions. Mice, which are generally believed to possess few, if any basophils, also acquire resistance following repeated tick infestations and this has been found to be associated with increased numbers of dermal mast cells. Mast cell-deficient W/Wv mice have, however, also been shown capable of acquiring resistance after two infestations with Dermacentor variabilis larvae. In the studies described here, we have examined, with the electron microscope, skin reactions in W/Wv and +/+ mice undergoing their third infestation with Dermacentor variabilis. Basophils, along with neutrophils and eosinophils, were identified using established criteria. The possibility that basophils contribute to various pathogenetic mechanisms in these and other strains of mice is discussed.     

Ixodes holocyclus: Kinetics of cutaneous basophil responses in naive,and actively and passively sensitized guinea pigs

In guinea pigs, macroscopic cutaneous reactions to initial (primary) Ixodes holocyclus feeding were first apparent at 96 hr post-tick attachment, peaked at 7 days (5 mm), and were gone by Day 14. Microscopic analyses of these primary tick feeding sites at 12, 24, 48, 72, and 96 hr post-attachment revealed the dominance of mononuclear cells (63–94% of the infiltrate) at all times. Neutrophil levels were high initially (34% of the infiltrate), but quickly subsided to 6–15% of the cellular response. Eosinophils were essentially absent from primary sites, comprising only 1–3% of the infiltrate. Basophils were absent initially, but accumulated in small but significant numbers (12% of the infiltrate) at the epidermal-dermal border by 96 hr post-tick attachment, 3 days prior to maximum erythematous skin reactions. In sensitized and challenged (secondary) animals, erythematous reactions in response to secondary tick feeding were apparent as early as 18 hr post-tick attachment with peak responses at 48 hr (3–4 mm). Cutaneous leukocyte responses to challenge feedings were quantitated at 12, 24, 48, and 72 hr posttick attachment and consisted initially (12–24 hr) of a strong mononuclear cell response (72–79% of the infiltrate) that was replaced by a dominant basophil response at 48 and 72 hr. Strong cutaneous basophil responses coincided with a significant level of tick rejection. Eosinophils and neutrophils were virtually absent from these secondary responses. Other ixodid tick-guinea pig systems have a relatively stronger eosinophil response, and both basophils and eosinophils participate in immune resistance in these systems. This suggests that basophils alone mediate resistance in the I. holocyclus system. Recipients of immune serum or immune cells expressed significant cutaneous basophil responses 72 hr post-tick challenge (23 and 39% of the infiltrate, respectively). Eosinophil and neutrophil responses were weak (1–6%), whereas mononuclear cells were dominant (54–72%). Recipients of immune lymph node cells expressed a cutaneous basophilia approximately two-thirds (69%) of that observed in actively sensitized and challenged hosts, whereas immune serum recipients recruited less than one-third (31%) as many basophils. In summary, these results indicate that T-cell- and antibody-dependent mechanisms recruit infiltrates of basophils that are responsible for acquired immune resistance to I. holocyclus in guinea pigs.     

Rhipicephalus appendiculatus: Larval feeding sites in guinea pigs actively sensitized and receiving immune serum

Histological analyses of larval Rhipicephalus appendiculatus feeding sites in naive and actively sensitized guinea pigs were made at 6, 24, 48, 72, and 96 hr post-tick attachment. As primary feedings progressed the cavity at the entrance of the ticks mouthparts into the uppermost dermis, and the surrounding cellular infiltrate (lesion) both increased. Early (6 hr) lesions were dominated by eosinophils (46% of the infiltrate), neutrophils predominated at 24 (55%) and 48 hr (68%), eosinophils again predominated at 72 hr (44%), and finally basophils were dominant at 96 hr (78%). At sites of secondary feedings in animals expressing acquired resistance, lesion size increased as tick feeding progressed and at each observation time was at least twice that observed in primary feedings. Dermal cavities at the site of entrance of the ticks mouthparts were occasional in occurrence and were reduced in size indicating altered tick feeding. Basophils were dominant at all observation times ranging from 61 to 91% of the infiltrate. The second cell type of significance was the eosinophil, ranging in abundance from 7 to 21%. Recipients of immune serum had a smaller cellular infiltrate around feeding ticks, but basophils were also dominant. Basophils appear to be the principal host cell involved in acquired resistance to tick feeding as indicated by the profound cutaneous basophil reaction that characterized the immune response to larval ticks both in actively and passively sensitized hosts. The finding of significant eosinophil accumulations at tick feeding sites of both hosts indicates that these cells may also contribute to acquired resistance.     

Histological study of the attachment sites of adult Rhipicephalus appendiculatus on rabbits and cattle

Biopsies of tick attachment sites on ears of five rabbits and four calves (Bos taurus) were taken at first and third infestations and were examined by histochemistry, light microscopy and electron microscopy. Resistance was expressed by all hosts by reduction of tick engorgement weights. Attachment sites at first infestations on rabbits and cattle were similar and were characterized by an acute inflammatory abscess with a preponderance of neutrophils and macrophages infiltrating the site. Attachment sites at third infestations on rabbits and cattle were similar and were characterized by infiltration of neutrophils and macrophages and a 2- to 5-fold increase in the proportions of eosinophils and basophils. Lymphoblasts and plasmacytes were found in third infestation sites. In rabbits there was much necrosis and in cattle there were large intraepidermal pustules in third infestations. The cement attachment cone of the ticks was of lipoprotein and contained aminopeptidase. Salivary glycoproteins and esterases were detected in the attachment sites.     

Reactivity of rabbit antiserum to guinea pig eosinophils.

Although the eosinophil has been recognized as a distinctive cell type for almost 100 years, the major functions of these cells remain unknown. As an approach to defining these functions we have treated guinea pigs with rabbit antiserum to eosinophils (AES) in an attempt to ablate these cells from tissues. Rabbits were immunized thrice with purified eosinophils and the antisera were absorbed with peripheral blood cells from guinea pigs made eosinopenic with methyprednisolone to remove antibodies reactive with serum proteins and erythrocytes. The resulting sera reacted strongly with eosinophils in cytotoxicity tests and had weak or no reactivity with neutrophils. However, absorption of AES with purified neutrophils removed antieosinophil activity. Intraperitoneal injection of potent AES into guinea pigs resulted in complete absence of eosinophils from the peripheral blood and from the peritoneal cavity with only transient or no reduction in circulating neutrophils. Eosinophils were also reduced in bone marrow, spleen, and intestine. The ability of neutrophils to absorb AES activity in spite of weak reactivity in cytotoxicity tests may reflect a quantitative difference in antigenic determinants between eosinophils and neutrophils.     

Xenopsylla cheopis: cellular expression of hypersensitivity in guinea pigs

Guinea pigs multiply exposed to Xenopsylla cheopis adult fleas exhibited marked blood basophil responses to challenge infestation with only minor changes in blood eosinophil levels. Dermal responses to flea feeding were marked by dominant neutrophil (52% of the infiltrate) and eosinophil (32%) accumulations 24 hr after primary feeding, with a weak basophil response (11%). However, after challenge feeding 14 days later, eosinophils dominated, representing 59% of the infiltrate with basophils comprising 30% of the cellular response; neutrophils were rare (7%). Mast cells did not exhibit any increases in density during either the primary or secondary infestation, representing 4-7% of the infiltrate. These results demonstrate that flea feeding induces systemic and local basophil responses as demonstrated for all hematophagous arthropods examined so far. Flea feeding success was not adversely affected by feeding on homologously hypersensitized guinea pigs or guinea pigs sensitized by Ornithodoros parkeri feeding. However, basophil responses at flea feeding sites in heterologous (tick) sensitized animals were more basophilic (26 +/- 4 cells/0.03-mm2 area) than feeding sites in homologous (flea) sensitized hosts (9 +/- 6 cells). Furthermore, primary tick feeding sites become erythematous and indurated after flea feeding on the opposite flank, and were marked histologically by strong basophil abscess (276 +/- 56 cells/0.03-mm2 area); primary flea feeding sites were not activated by tick challenge feeding. These cross-generic challenge feeding experiments suggest antigen cross-reactivity, resulting in activation of feeding sites of a heterologous arthropod.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rejection of ticks from guinea pigs by anti-hapten-antibody-mediated degranulation of basophils at cutaneous basophil hypersensitivity sites: role of mediators other than histamine

Previous studies have established that recruitment of basophils to sites of tick feeding in guinea pigs is required to effect immune resistance. In the current study, actively sensitized guinea pigs treated three times daily with H-1 (mepyramine) and H-2 (cimetidine) histamine receptor antagonists, during the challenge tick infestation period, expressed normal resistance to Amblyomma americanum larvae. Similarly, naive guinea pigs treated with anti-histamines four times daily, beginning 7 days before transfer of immune serum and tick challenge and continuing through the tick infestation period, also expressed normal antibody-mediated resistance to A. americanum. These results indicated that histamine was not an important basophil mediator of the resistance response. Ticks allowed to feed on tissue rich in basophils that were induced by sensitization and subsequent local challenge with non-tick protein antigen, keyhole limpet hemocyanin (KLH), expressed normal yield. Ticks that fed on similar tissue rich in basophils induced by sensitization and challenge with KLH, in which the basophils expressed anti-picryl specificity due to systemic passive transfer of anti-picryl antibodies, were rejected when basophils were induced to degranulate by i.v. challenge with picryl antigen at 6 hr (29% rejection), 12 hr (18% rejection), 24 hr (22% rejection), and 48 hr (37% rejection) post-tick attachment. However, basophil degranulation at 18, 72 and 96 hr post-tick attachment had no adverse effect on tick feeding. These hosts were protected from systemic anaphylaxis by treatment with the anti-histamine mepyramine. Release of histamine occurred at tick feeding sites, but vasoactive effects were blocked by mepyramine treatment as evidenced by a lack of increased vascular permeability (bluing) at these sites compared with non-tick-infested tissues, or to cutaneous basophil hypersensitivity (CBH) sites of animals not protected with mepyramine. These results indicate that local recruitment and subsequent degranulation of basophils via immune mechanisms dependent on non-tick antigens can lead to tick rejection, and that basophil-derived mediators other than histamine are involved in this immune resistance response to A. americanum ticks. The identity of the crucial basophil mediator(s) is not known. The significant susceptibility of ticks to basophil-mediator release at 6 to 12 hr and 24 to 48 hr post-attachment coincides with the tick attaching and fast-feeding phases, respectively, suggesting that these phases of tick parasitism are particularly susceptible to the effect of basophil mediators other than histamine.     

Leukocyte differential analysis in multiple laboratory species by a laser multi-angle polarized light scattering separation method.

Leukocyte differential analysis was performed in various species, particularly laboratory animals, by the laser multi-angle polarized light scattering separation method. Venous blood specimens were drawn from the following subjects: healthy adult men and women ("humans"); cynomolgus monkeys ("monkeys"); common marmosets ("marmosets"); beagle dogs ("dogs"); miniature potbelly pigs ("swine"); Japanese white rabbits ("rabbits"); Hartley guinea pigs ("guinea pigs"); and Sprague-Dawley rats ("rats"). 90 degrees/10 degrees scatter plot: Basophils and mononuclear-polymorphonuclear cells were separated in all subjects, but individual 10 degrees and 90 degrees scatter plots overlapped in dogs and guinea pigs, respectively. 90 degrees depolarized /90 degrees scatter plot: Neutrophils and eosinophils were clearly separated in human, monkey, guinea pig, swine and rat subjects. The eosinophil cluster was not clearly plotted in marmoset, dog, or rabbit. 0 degree/10 degrees scatter plot: Regarding this plot for monocytes and lymphocytes, cells were plotted in the following order in all subjects: lymphocytes < basophils < or = monocytes in the 0 degree (size) scatter; and lymphocytes [symbol: see text] monocytes < or = basophils in the 10 degrees (complexity) scatter. Compared to other species, the rat scatter showed a tendency to overlapping plots in both the 0 degree and 10 degrees scatters in the monocyte and lymphocyte clusters. In both dog and guinea pig, the monocyte and neutrophil plots overlapped in the 0 degree and 10 degrees scatters. Basobox: In the human and rabbit subjects, the basophil cluster was plotted within the established basobox, but no clear cell cluster was plotted in the other subjects. As a result of comparing the percentage values for leukocytes in various species obtained by using the CD3500 apparatus versus the corresponding values obtained manually, good correspondence was found in the monkey, and eosinophils in the marmoset were lower with CD3500 than manually. In the rabbit, the mean measured value for basophils matched in the manual and CD3500 findings. In the guinea pig, the CD3500 values were lower than the manual values for lymphocytes, but higher for monocytes and neutrophils. The above findings suggest that the laser multi-angle polarized light scattering separation method is indeed capable of analyzing leukocytes from various species based on cell size and cell complexity, i.e., the presence or absence of nuclei, granules and cell enclosures.     

Orally administrated Lactobacillus gasseri TMC0356 and Lactobacillus GG alleviated nasal blockage of guinea pig with allergic rhinitis

Lactobacillus GG (LGG) and L. gasseri TMC0356 (TMC0356) were investigated for their ability to alleviate nasal blockage associated with allergic rhinitis using a guinea pig model. The increases in sRaw at 10 min and 5 hr after the exposure of the nasal mucosa to OVA were significantly alleviated in the guinea pigs orally administrated with LGG and TMC0356 compared with those of the control (P<0.05 and P<0.01). The total numbers of leukocytes, particularly eosinophils and neutrophils from the nasal cavity lavage fluid, and the OVA-specific IgE concentration in the serum were also decreased in the guinea pigs orally administrated with LGG and TMC0356, although the decreases were not statistically significant. These results suggest that LGG and TMC0356 can alleviate antigen-induced nasal blockage in earlyphase and late-phase inflammatory responses associated with allergic rhinitis.     

Histopathology of Tick-Bite Lesions in Naturally Infested Capybaras (Hydrochoerus hydrochaeris) in Brazil

In the present work features of tick-bite lesions were evaluated in capybaras naturally infested with Amblyomma cajennense and Amblyomma dubitatum ticks. Gross appearance of tick bite site was characterized by a mild swelling and erythema. Microscopic examination revealed the cement cone, a tube-like homogenous eosinophilic mass penetrating deep into the dermis. This structure was surrounded in the dermis by a cellular infiltrate and free eosinophilic granules and was associated to edema of variable intensity. Necrosis was a common feature deep in the dermis particularly at the far end of the eosinophilic tube. Hyperplasia, cellular edema and occasionally necrosis of keratinocytes could be seen at both sides of the ruptured epidermis. Cellular infiltrate was constituted overwhelmingly by polymorphonuclear leukocytes with eosinophilic granules. In capybaras cells with such features can be either eosinophils or heterophils (pseudoeosinophils), the latter being the equivalent of neutrophils of other mammals. Ultrastructural analysis of the cellular infiltrate revealed the predominance of heterophils over eosinophils. Mononuclear cells and mast cells and, in lesser numbers, basophils were also seen at skin attachment sites. The presence of heterophils in the reaction of capybaras against Amblyomma ticks is an outstanding feature but its role in the reaction to the tick is not known. It is however speculated that capybara heterophils might be associated with a more permissive environment for tick feeding and pathogen transmission as already shown for the equivalent cell type, the neutrophil, in the reaction of the dog against the Rhipicephalus sanguineus tick.     

Schistosoma mansoni: the cutaneous response to cercarial challenge in naive guinea pigs and guinea pigs vaccinated with highly irradiated cercariae

Schistosoma mansoni: the cutaneous response to cercarial challenge in naive guinea pigs and guinea pigs vaccinated with highly irradiated cercariae. International Journal for Parasitology16: 491–510. Naive guinea pigs and guinea pigs vaccinated 4 weeks previously with highly irradiated cercariae were challenged percutaneously with normal cercariae. Skin samples from the challenge site were then harvested at varying times to provide histological, quantitative and ultrastructural data on the respective cellular responses to cercarial invasion. The primary cutaneous reaction was characterised by neutrophils; these cells reached peak numbers (16% of total cells) by 18 h. Eosinophils and basophils made only a small contribution to the infiltrate (2.9 and 5.7% respectively). Some basophils showed evidence of anaphylactic degranulation, others seemed to be damaged, but most appeared normal. Mononuclear cells of varied morphology were present at all times, but activated fibroblasts were prominent, and collagen deposition increased with time. Degranulating mast cells were recognised at 24 and 48 h. Dead schistosomula were never seen in naive-challenged skin, although one or two of the observed larvae showed minor tegumental abnormalities. In vaccinated guinea pigs, the cutaneous cellular response to challenge was significantly enhanced, with basophils dominating the reaction (33% of total cells at 24 h). Many basophils were already degranulating by the anaphylactic mechanism at 3 h post challenge, and free basophil granules were seen frequently. Both intact cells and free granules congregated in close proximity to invading larvae. Eosinophils were also present in greater numbers at secondary reaction sites, but they never exceeded 6% of the total infiltrate. Mononuclear cells believed to be immature eosinophils were prominent from 3 h. For the first time, the mechanism of eosinophil attachment and degranulation onto a multicellular target, described previously only from in vitro investigations, was recognized in vivo. Neutrophil numbers matched those recorded in naive-challenged skin at 3 and 6 h, but declined thereafter, while mast cells were seen degranulating at these early times. Mononuclear cells again presented a variety of morphological appearances; of particular note were large cells that had phagocytosed debris and were presumed to be macrophages, and small rounded cells with scant cytoplasm and few organelles, that may have been lymphocytes. By 12 h, large areas of the dermis had become severely disorganised and numerous, free basophil granules were distributed amongst the other cellular constituents. Dead schistosomula, denuded of tegument, were clearly recognised at 6 h, and such individuals invariably had neutrophils attached to their exposed muscle layers. Since dead schistosomula were not identified in naive-challenged guinea pig skin, it is concluded that a percentage of the challenge larvae, however small, is preferentially killed in the skin of the vaccinated animals.     

The Use of a 4% (w/w) Deltamethrin Collar (Scalibor® ProtectorBand) in the Extended Control of Ticks on Dogs

Deltamethrin (4%, w/w) formulated in a polymer matrix system (Scalibor ProtectorBand, Intervet International BV) was evaluated for the prevention of ticks on dogs. Controlled laboratory trials were conducted, wherein dogs wearing anti-tick collars were compared with control dogs and artificially infested by adult Ixodes ricinus and/or Rhipicephalus sanguineus ticks. The activity of the collars increased from 24 to 48 h post-treatment (pt) wherein tick numbers were reduced to 78.7% for I. ricinus and to 77% for R. sanguineus. The proportion of ticks controlled after the second infestation on D7 pt increased to 99% for I. ricinus and 99.5% for R. sanguineus. For I. ricinus adult ticks, an efficacy of 95.4% was reached over a period of 5.5 months and approximated 90% for R. sanguineus. Hence, the efficacy of the deltamethrin-impregnated dog collar was >90% for 6 months against both tick species. There was no significant difference between the efficacy of the collar against either R. sanguineus or I. ricinus. A subsequent field study wherein 82 dogs were wearing the collar and monitored for tick infestation indicated that the duration of efficacy was between 5 and 6 months.     

Rhipicephalus sanguineus: sequential histopathology at the host-arthropod interface

The reaction beneath the mouth parts of an adult, female Rhipicephalus sanguineus attached to a dog develops progressively over 4–5 days. The cement substance is confined to the external surface of the epidermis and does not form any organized structure in the dermis. The hypostome is imbedded in the cement substance and does not penetrate the epidermis. The cheliceral shafts are at the epidermal-dermal junction and do not extend into the dermis to any degree. Thus, it is the adhesive quality of the cement for the dog's skin that holds this tick firmly attached to its host.Edema of the epidermis appears 24 hr after attachment of the tick; the dermal infiltrate becomes prominent from 24 hr after attachment onward and initially consists of polymorphonuclear leukocytes. This cell type predominates until the tick has detached. Rupture of lymphatics occurs and infiltrated cells can be found entering these open channels. Degranulation of mast cells is associated with polymorphonuclear leukocytic infiltration. A cavity develops in the dermis progressively over the period of tick attachment and feeding. This cavity appears during the first 24 hr and its full development depends upon leukocyte infiltration and the feeding activity of the tick. During the healing phase, macrophages, lymphocytes and fibroblasts gradually replace the polymorphonuclear leukocytes. The dermis is still hypercellular in the area of former attachment 2 wk after a female tick has detached fully engorged. The dog does not appear to develop resistance to the tick's engorgment even after 2 yr of intermittent exposure, nor does the host's reaction hinder the tick's engorgment. A dog never before exposed to arthropods of any kind reacted, histologically, to tick exposure with a very similar infiltration of polymorphonuclear leukocytes as seen in dogs repeatedly exposed. It is suggested that the inflammatory response by the host to the feeding tick may play an important role in the development and spread of infectious agents transmitted by this pool feeding arthropod.     

Characterization of a localized basophil hypersensitivity lesion in guinea pig conjunctiva

Basophils accumulate in response to antigen challenge in cutaneous basophil hypersensitivity (CBH) reactions. Two ocular diseases, vernal conjunctivitis and contact-lens-associated conjunctivitis, are also characterized by this histopathology. We have refined a model previously developed in guinea pig conjunctiva by precisely defining the site of antigen injection and correlating the site with the clinical and histologic changes. Guinea pigs were primed by an intradermal injection of keyhole limpet hemocyanin (KLH) in the flank and challenged (Day 6) by injection of a small bolus of KLH just under the conjunctival epithelium. Twenty-four hours later histologic examination showed a perivascular infiltrate of inflammatory cells containing large numbers of basophils. Eosinophils, neutrophils, macrophages, and mast cells were also seen. Serial sections of the reaction site showed discrete boundaries. At all sites examined in antigen-challenged tissues, there were significantly more basophils than in control-injected conjunctiva. Insertion of a sterile needle or injection of PBS or KLH into normal conjunctiva induced a significant increase in neutrophils and some macrophages. Injection of graded doses of antigen into the conjunctiva of primed animals, resulted in a dose-dependent increase in basophils up to 50 micrograms KLH (optimal dose).     

Acquired resistance to ixodid ticks induced by tick cement antigen

Antisera from guinea pigs made resistant to infestation with an ixodid tick of east and central Africa,Rhipicephalus appendiculatus, were used to identify the tick antigens they recognized by immunoblotting. Most of the antigens were found in tick salivary glands and in tick attachment cement. Antisera fromR. appendiculatus-resistant guinea pigs also recognized some salivarygland antigens in ticks of other species (R. pulchellus, R. evertsi, Amblyomma variegatum andA. gemma). Antibodies against the most strongly recognizedR. appendiculatus antigen, a 20-kDa molecule, were only poorly reactive with similar-sized molecules in the other ticks. A 94-kDa antigen, which appeared to have broader cross-reactivity, was purified fromR. appendiculatus attachment cement, and a monospecific rabbit serum was raised against it. This antiserum clearly recognized a molecule of similar molecular weight inR. pulchellus andR. evertsi. Intravenous inoculation of rabbits with the purified molecule elicited delayed-type hypersensitivity to the antigen. The hypersensitive rabbits demonstrated resistance to feeding ofR. appendiculatus ticks but slight enhanced feeding ofR. pulchellus ticks. These results are discussed with respect to their relevance for artificial induction of tick-feeding resistance.     

First report of amitraz and cypermethrin resistance in Rhipicephalus sanguineus sensu lato infesting dogs in Mexico

Engorged female Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) were collected from dogs in the state of Yucatán, Mexico. Fourteen tick populations were collected from dogs at seven veterinary clinics, four residential homes and three cattle farms. The larval immersion test was used in the progeny of collected adult females to test susceptibility to amitraz and cypermethrin. Dose–mortality regressions, 50% lethal concentrations (LC₅₀), confidence intervals and slope were estimated by probit analysis. For amitraz, 12 tick populations (85.7%) were classified as resistant and low inter‐population variation in the phenotypic level of resistance was evident [resistance ratios (RRs) at LC₅₀: 1.0–13.0]. For cypermethrin, 12 tick populations (85.7%) were classified as resistant and substantial inter‐population variation in the phenotypic level of resistance was evident (RRs at LC₅₀: 1.0–104.0). Thus, amitraz resistance in R. sanguineus s.l. is common, but generally occurs at low levels; however, alarmingly high levels of cypermethrin resistance are present in R. sanguineus s.l. populations in dogs in Yucatán, Mexico. The intensive use of both acaricides to control ectoparasites on dogs is likely to lead to more serious resistance problems that may cause high levels of control failure in the future.     

Repeated antigen challenge induces airway hyperresponsiveness with tissue eosinophilia in guinea pigs

To test the hypothesis that the development of airway hyperresponsiveness (AHR) lasting greater than or equal to 3 days after the last antigenic exposure required repeated mediator release, we compared dose-response changes in lung resistance (RL) to acetylcholine (ACh) in animals sensitized with 1% ovalbumin (OA), 4% Bordatella pertussis aerosol and subsequently challenged with 0.5% OA aerosol twice weekly for 4-6 wk vs. animals receiving saline aerosol instead of OA. Despite antihistamine pretreatment, each OA challenge produced cyanosis and inspiratory indrawing. Blood gas analysis in six guinea pigs revealed an immediate fall in arterial PO2 (PaO2) from 104.3 +/- 4.9 to 35.4 +/- 2.2 Torr after a 1-min exposure to aerosolized OA. ACh dose-response measurements of RL 3 days after the last OA challenge demonstrated a leftward shift and an increased magnitude of response. These differences were less marked at 7 days, and by 14 days after the last OA challenge, ACh dose-response curves were not different from those of control guinea pigs. Sensitization without repeated antigen challenge did not cause hyperresponsiveness. Morphometric analysis showed significantly increased numbers of eosinophils in the epithelium of airways in hyperresponsive guinea pigs, without neutrophil infiltration or alterations in epithelium and airway wall areas. We conclude that repeated antigenic challenge, but not sensitization alone, causes prolonged AHR in guinea pigs, which is associated with tissue eosinophilia.     

Neurokinins and inflammatory cell iNOS expression in guinea pigs with chronic allergic airway inflammation

In the present study we evaluated the role of neurokinins in the modulation of inducible nitric oxide synthase (iNOS) inflammatory cell expression in guinea pigs with chronic allergic airway inflammation. In addition, we studied the acute effects of nitric oxide inhibition on this response. Animals were anesthetized and pretreated with capsaicin (50 mg/kg sc) or vehicle 10 days before receiving aerosolized ovalbumin or normal saline twice weekly for 4 wk. Animals were then anesthetized, mechanically ventilated, given normal saline or N(G)-nitro-l-arginine methyl ester (l-NAME, 50 mg/kg ic), and challenged with ovalbumin. Prechallenge exhaled NO increased in ovalbumin-exposed guinea pigs (P < 0.05 compared with controls), and capsaicin reduced this response (P < 0.001). Compared with animals inhaled with normal saline, ovalbumin-exposed animals presented increases in respiratory system resistance and elastance and numbers of total mononuclear cells and eosinophils, including those expressing iNOS (P < 0.001). Capsaicin reduced all these responses (P < 0.05) except for iNOS expression in eosinophils. Treatment with l-NAME increased postantigen challenge elastance and restored both resistance and elastance previously attenuated by capsaicin treatment. Isolated l-NAME administration also reduced total eosinophils and mononuclear cells, as well as those cells expressing iNOS (P < 0.05 compared with ovalbumin alone). Because l-NAME treatment restored lung mechanical alterations previously attenuated by capsaicin, NO and neurokinins may interact in controlling airway tone. In this experimental model, NO and neurokinins modulate eosinophil and lymphocyte infiltration in the airways.