Effects of in utero retinoic acid exposure on mouse pelage hair follicle development

Abstract We investigated in vivo the histological and immunohistochemical responses of mouse hair pelage follicle morphogenesis to prenatal exposure to a potentially nonteratogenic dose of all- trans -retinoic acid (RA), as a basis studying the preventive effect of RA on adult mouse skin carcinogenesis. In pregnant mice, a single oral dose of RA at 30 mg kg⁻¹ body weight given on day 11.5 of gestation caused no RA-induced changes in the morphology or temporal expression patterns of keratins during pelage hair follicle morphogenesis. The only differential effect of RA was a statistically significant increase in the number of BrdU-positive nuclei in hair bulbs from RA exposed fetuses compared with nonexposed mice. The absence of adverse RA effects suggests that this experimental design may represent a valuable protocol for use in studies on the in vivo effects of this retinoid on different skin diseases.     

In vivo long-term effects of retinoic acid exposure in utero on induced hyperplastic epidermal foci in murine skin

Adult Naval Medical Research Institute (NMRI) mice, after prenatal exposure to retinoic acid (RA), were treated with a standard two-stage skin carcinogenesis regime to characterize hyperplastic epidermal foci that precede the appearance of cutaneous papillomas, and to investigate the in vivo long-term action of RA on adult mouse skin treated with DMBA (7,12 dimethyl benz[a]anthracene) and TPA (12-O-tetradecanoylphorbol 13-acetate). The results demonstrate that RA administered to pregnant mice had a long-term inhibitory action on the cell differentiation and development of hyperplastic lesions occurring prior to cancer on the adult skin of their offspring as well as a stimulatory effect on cell proliferation of these hyperplastic lesions.     

Mouse epidermal development: effects of retinoic acid exposure in utero

Epidermal morphogenesis was studied in vivo following prenatal exposure to retinoic acid (RA). In pregnant mice, a single oral dose of RA on day 11.5 of gestation failed to induce histological changes in fetal epidermal development except in epidermal thickness. Epidermal thickness increased from 16.5 days post-coitum (dpc) onwards, and temporal and spatial epidermal modifications in keratins K5 and K14 related to proliferative activity of keratinocytes were observed. An RA effect on cell proliferation was supported by a statistically significant increase in the number of epidermal S-phase cells, containing BrdU-incorporated DNA in RA-exposed mice compared with nonexposed animals. The prolonged in utero action of RA on epidermal proliferative activity in fetuses and newborns suggests a long-term RA effect that may play a role on the development and evolution of diseases in adult skin.     

Fermented Maesil (Prunus mume) with probiotics inhibits development of atopic dermatitis‐like skin lesions in NC/Nga mice

Maesil (Prunus mume Siebold & Zucc.), a potential source of free radical scavengers and inhibitor of pro‐inflammatory mediators, is used in traditional Korean medical preparations as a remedy for skin disorders as have probiotics. The action of a probiotic fermented Maesil preparation on the development of atopic dermatitis (AD)‐like skin lesions was determined in a NC/Nga mouse model as an initial step towards the development of a therapeutic feed supplement for use in dogs. Continuous ingestion of the experimental feed markedly inhibited the development of the AD‐like skin lesions, as evidenced by a marked decrease in skin signs and reduced inflammation within the skin lesions. Efficacy was confirmed by significant decreases in eosinophil ratio and serum IgE concentration, and a reduction in the number of Staphylococcus aureus recovered from the ear. Relative mRNA expression levels of IL‐4, interferon‐γ and tumour necrosis factor‐α in the spleens of the experimental animals were also decreased and there was an increased serum concentration of IL‐10 with a concurrent decreased IL‐4 concentration in comparison to a control group. Taken together, the results indicate that some component(s) of fermented Maesil have the ability to suppress the development of AD‐like skin lesions, possibly by stimulation of IL‐10. Beneficial effects of fermented Maesil may thus be expected in dogs with AD, although this and the nature of the active pathway remain to be explored.     

Ultrastructural,Sensory and Functional Anatomy of the Northern Elephant Seal (Mirounga angustirostris) Facial Vibrissae

Vibrissae (whiskers) play a key role in underwater orientation in foraging phocids through vibrotactile sensation processing. Our aim was to evaluate the structure of northern elephant seal (NES) vibrissae by means of light (LM) and transmission electron microscopy (TEM), in order to elucidate their function. Vibrissal follicles were processed using standardized laboratory methods and LM/TEM techniques. Individual follicular axonal numbers were counted and axonal diameter measured and averaged. NES have mystacial, rhinal, supraorbital and labial vibrissae. The vibrissal follicles are histologically subdivided into a ring, upper and lower cavernous sinuses (LCS). Each vibrissa is innervated by the deep vibrissal nerve. The average number of axons per large mystacial vibrissa is 1804 (±123), rhinal 985 (±241), supraorbital 1,064 (±204) and 374 (±65) in labial vibrissa. The entire vibrissal system carries an estimated 148 573 axons, and mystacial vibrissae alone have 125 323 axons. Axonal conduction velocity for each vibrissal type is 55.26 m/s for labial, 56.58 m/s for rhinal and 35.88 m/s for mystacial vibrissae. TEM and LM revealed a plethora of mechanoreceptors within the vibrissal follicles: Merkel cell‐neurite complexes, lanceolate and pilo‐Ruffini end organs. A vast number of sensory axons projecting from the entire vibrissal system indicate that the vibrissal sensory area takes up a large proportion of phocids’ somatosensory cortex. In conclusion, NES has highly sensitive and finely tuned vibrotactile vibrissal sense organs.     

Effects of pre-natal glucocorticoids on testicular development in sheep

We tested the hypothesis that acute pre-natal exposure to high levels of synthetic glucocorticoid (betamethasone) would alter fetal testicular development through actions on gonadal glucocorticoid receptors (GRs). Pregnant Merino ewes bearing singleton male fetuses (n = 24) were allocated randomly among four equal groups to be injected intramuscularly with saline or betamethasone (0.5 mg/kg) either on day 109 of gestation or on both day 109 and day 116 of gestation. Fetal testes were collected at post-mortem, 5 days after each treatment. The volume of interstitial tissue and the volume, length and diameter of the sex cords were measured, and Sertoli cells and gonocytes were counted. For cord volume and interstitial tissue volume, control testes demonstrated maturational changes as fetal age advanced from 109 to 116 days of gestation. For that period, the single injection of betamethasone significantly reduced Leydig cell proliferation (P < 0.05), but had no effect on Sertoli cell numbers. Immunohistochemistry was used to localize GR and proliferating cell nuclear antigen in testicular cells. GR immunoexpression in Leydig cells was higher in fetuses exposed to betamethasone at 109 days of gestation than in control fetuses. Sertoli cells showed low levels of GR. It was concluded that, during mid-gestation, a brief period of glucocorticoid treatment could affect testicular development in male sheep fetuses. The mechanism probably involves direct effects on Leydig cells, as these cells express extra-GR in response to the treatment. Sertoli cells seem to produce less GR than Leydig cells, perhaps explaining their lack of response to betamethasone. These outcomes may have important implications for future fertility in male offspring.     

不同冷冻保护剂和冷冻方法对小鼠耳皮肤成纤维细胞冻存效果的影响

通过比较不同冷冻保存方法和冷冻保护剂对小鼠耳皮肤成纤维细胞冷冻-解冻复苏后细胞存活率、48h贴壁率和细胞生长曲线的影响,筛选适宜的小鼠耳皮肤成纤维细胞冷冻保存方法和冷冻保护剂。结果表明,以100mL/L二甲基亚砜(DMSO)作为冷冻保护剂进行小鼠耳皮肤成纤维细胞冷冻保存时,方法3处理组解冻复苏后细胞存活率和培养48h细胞贴壁率均高于方法2处理组(P>0.05),并分别极显著高于方法1和方法4。采用方法3进行冷冻保存时,以100mL/L DMSO作为冷冻剂的细胞贴壁率显著高于100mL/L甘油(GL)组(P<0.05),且冷冻解冻后的细胞呈现正常的分裂增殖生长模式。因此,宜选择100mL/L胎牛血清(FBS)+100mL/L DMSO+DMEM作为冷冻保护液,采用方法3进行小鼠耳皮肤成纤维细胞的冷冻保存。     

The roles of vitamin A for cytoplasmic maturation of bovine oocytes

Vitamin A is one of the micronutrients which have been implicated in cattle reproduction. In cattle, ingested vitamin A, mainly as beta-carotene (BC) from forages and retinol ester from formula feed, is metabolized and transported to the oocytes and cumulus-granulosa cells in ovarian follicles through binding to various interacting molecules. The active form of vitamin A, retinoic acid (RA), functions as a regulator of gene expression in these targets. Early research showed the positive effects of vitamin A supplementation on bovine fertility in artificial insemination, and several studies on effects of vitamin A metabolites used in other artificial reproductive techniques (ART), including superovulation, ovum pick up, and in vitro maturation culture have provided evidence for the specific roles of vitamin A in oocyte cytoplasmic maturation (acquisition of developmental competence of oocytes during their meiotic maturation period for the embryonic development after fertilization). BC may enhance cytoplasmic maturation by its antioxidant properties which cannot be replaced by RA. Furthermore, RA may promote cytoplasmic maturation of bovine oocytes via its modulatory effects on the gene expression of gonadotrophin receptors, midkine, cyclooxygenase-2, and nitric oxide synthase in cumulus-granulosa cells.     

The effect of repeated tuberculin skin testing of cattle on immune responses and disease following experimental infection with Mycobacterium bovis

The comparative intradermal skin test, in which a delayed type hypersensitivity (DTH) response to purified protein derivative of tuberculin (PPD) from Mycobacterium bovis and M. avium is assessed and compared, may be used repeatedly on non-infected animals on farms where bovine tuberculosis (TB) has occurred. A skin test is known to affect subsequent skin tests in infected animals. The reported study was to determine whether repeated skin testing prior to infection with M. bovis might affect the development of the comparative skin test and IFNgamma response subsequent to exposure to virulent M. bovis. The comparative intradermal skin test was applied to one group of six calves five times at 8-week intervals. These and six control calves were subsequently inoculated intratracheally with a dose of M. bovis that produced mild disease. The development of the DTH reaction, IFNgamma, IL-10 and proliferative responses were compared in the two groups of animals. No differences in IFNgamma, IL-10 and proliferative responses were seen between the two groups of calves prior to challenge. After infection with M. bovis no differences in the development of the DTH and IFNgamma responses to PPD were noted as a consequence of the repeated skin testing prior to challenge. No differences between the groups were evident when ESAT-6 was used as antigen and IFNgamma was assayed, although two animals that responded to PPD did not respond with ESAT-6. However, there did appear to be subtle effects of repeated skin testing on the immune response post-challenge that did not affect the diagnostic tests. After challenge control animals showed greater proliferative responses than animals given repeated skin tests prior to challenge, indicating that the procedure did have consequences for immune responses following infection. In both groups a marked reduction in the intensity of the skin test and in the number of animals that would be recognized as reactors was evident when animals were tested 15 weeks post-infection compared to their responses 8 weeks earlier that could have consequences for diagnosis of TB. An antibody response was not evident as a result of repeat skin testing prior to infection but was seen in both groups of calves following skin testing performed 7 weeks after infection.     

新型中草药添加剂在母猪阶段的喂养试验

分两组从母猪产前30 d至断乳进行试验观察。考虑到猪场喂药、产仔、拌料方便,试验组选择53头母猪在产前30 d给怀孕母猪饲喂添加新型中草药制剂(主要含多喂康颗粒Ⅰ型、益母草、鱼腥草);对照组选择42头母猪,根据该场日常饲养状况进行饲养(产前10 d投喂抗生素进行预防性用药)。通过新型中草药制剂喂养试验说明,新型中草药添加剂在产前30 d投喂对提高健仔数、成活率、出生重和断乳重有重要作用,使用新型中草药新制剂作为预防用药与抗生素相比成本降低了,生态效益也得到改善。     

Role of the chancre in induction of immunity to tsetse-transmitted Trypanosoma (Nannomonas) congolense in goats

Local skin reactions (chancres) developed in goats at the sites of deposition, by tsetse flies, of metacyclics of Trypanosoma congolense. The chancres developed much faster and were more pronounced when ten infected tsetse were allowed to feed on a spot as compared to only one fly per spot. The initial host cellular reaction in the chancre was predominantly polymorphonuclear, followed at the peak of development of the chancre by a predominantly lymphoblastic and plasmacytic reaction. Trypanosomes were found in various stages of division as well as degeneration in chancre biopsies taken at various days post-infection (p.i.). Most of the trypanosomes recovered from the chancre tissue fluid were found to bear the same variable surface glycoprotein (VSG) epitopes as the corresponding metacyclics for as long as 13 days p.i., as revealed by indirect immunofluorescence using mouse anti-metacyclic VSG hyperimmune sera and monoclonal antibodies. Immunization of goats with metacyclic trypanosomes, by exposure to infected tsetse bites followed by treatment of the infected goats on day 13 p.i., gave rise to the development of protection to homologous tsetse-transmitted challenge, whilst immunization by intravenous inoculation of the metacyclics did not induce such protection. Chancre formation would thus appear to be vital for the induction of comprehensive immune recognition of the metacyclic variable antigen repertoire deposited in the skin by infected tsetse, and hence development of protective immunity.     

Immunological characteristics of experimental murine infection with Leishmania (Leishmania) amazonensis

The murine models of Leishmania infection are well-studied and suitable models for studying this disease, which, despite its incidence of nearly 2 million new cases worldwide per year and its prevalence of 12 million cases, has been a somewhat neglected disease. Data obtained using such models are important for a better understanding of the disease in humans due to similarities in physiology and the advantage provided by the uniform infection profile within each mouse strain. In this review, we focus on studies of experimental murine infection with Leishmania (Leishmania) amazonensis, a species that has been associated with infections exhibiting various clinical features in humans. Mainly, we point out and discuss reports on: the effects of variations of the inoculum (such as strain, site, and size) in the establishment and development of the infection; characteristics of the infection in distinct mouse strains; and, the effects and subversions of the infection on components of the host innate and adaptive immune responses. The results obtained in these studies show that L. (L.) amazonensis infection in mice presents some unique features and immunoregulatory mechanisms, making it an interesting model for obtaining further knowledge of potential drugs targets and immunotherapy in Leishmania infection.     

9-cis retinoic acid improves developmental competence and embryo quality during in vitro maturation of bovine oocytes through the inhibition of oocyte tumor necrosis factor-α gene expression

Retinoic acid (RA; all-trans RA and 9-cis RA) enhances embryo developmental competence and quality through multiple mechanisms affecting the oocyte and preimplantation embryo. Folliculogenesis and oocyte maturation are influenced by tumor necrosis factor-α (TNF-α) via inhibition of aromatase activity and estradiol secretion in granulosa cells. Retinoic acid inhibits TNF-α production in various cell lines. The aim of the present study was to determine whether oocyte TNF-α concentrations regulate developmental competence and embryo quality and if the beneficial effects of 9-cis RA are mediated through attenuation of oocyte TNF-α production. Bovine cumulus oocyte complexes collected from abattoir ovaries were matured in maturation medium in the absence (control) or presence of 5 nM 9-cis RA (RA), 100 ng/mL of recombinant bovine TNF-α (TNF), or 5 nM 9-cis RA + 100 ng/mL of recombinant bovine TNF-α (RA+TNF). Oocytes were subsequently collected for gene expression analysis or subjected to in vitro fertilization and culture. Apoptosis and gene expression were analyzed in d-8 blastocysts. Results indicated that 9-cis RA downregulated (P < 0.01) both basal and TNF-α-induced TNF-α mRNA in oocytes (1.0-fold in control, 0.4-fold in RA, 2.1-fold in TNF, and 0.7-fold in RA+TNF). The 9-cis RA increased (P < 0.001) blastocyst development rates (37.1 ± 6.9 vs. 23.6 ± 8.0%) and total cell number (138.4 ± 19.2 vs. 120.2 ± 24.5) and reduced (P < 0.001) the percentage of apoptotic cells (3.3 ± 2.0 vs. 5.6 ± 2.3%) compared with controls. Expression of caspase 3 (0.4- vs. 1.0-fold) and TNF-α (0.4- vs. 1.0-fold) mRNA was downregulated (P < 0.05) in RA-treated blastocysts compared with controls. Moreover, 9-cis RA rescued (P < 0.001) development rates (24.5 ± 11.1 vs. 15.6 ± 9.0%), increased total cell number (124.6 ± 36.5 vs. 106.9 ± 31.1), and reduced apoptosis (5.8 ± 2.0 vs. 8.1 ± 3.1%) in blastocysts exposed to TNF-α (TNF group). Caspase 3 (0.8-fold in RA+TNF vs. 2.2-fold in TNF) and TNF-α (0.3-fold in RA+TNF vs. 2.8-fold in TNF) mRNA expression was attenuated (P < 0.05) in TNF-α-treated blastocysts. In conclusion, the present study suggests that 9-cis RA exerts its beneficial roles on oocyte developmental competence and embryo quality by attenuating oocyte TNF-α mRNA expression.     

Direct embryotoxicity of chromium (III) exposure during preimplantation development

Chromium in its trivalent form (chromium (III)) is an essential component of a balanced diet, and its deficiency disturbs glucose and lipid metabolism in humans and animals. The prevailing view is that chromium (III) is notably less toxic than chromium (VI), which is genotoxic and carcinogenic. Thus, the biotransformation of environmental chromium (VI) to chromium (III) is a promising and environmentally friendly detoxification method. However, increasing evidence suggests that chromium (III) induces considerable cytotoxicity. However, the toxicity of chromium (III) to early embryos remains largely unknown. In the present study, we used in vitro fertilization (IVF) to produce mouse embryos and identified the direct embryotoxicity of chromium (III). On exposure to high concentrations of CrCl₃, blastocyst formation almost completely failed and a large proportion of embryos were arrested at the 2- to 4-cell stage. At low concentrations of CrCl₃, IVF embryos showed a significant decrease in blastocyst formation, reduced total cell numbers, aberrant lineage differentiation, increased oxidative stress, and apoptosis. We also found that chromium (III) exposure during the preimplantation stage, even at low concentrations, led to impaired post-implantation development. Thus, our study substantiates the direct embryotoxicity of chromium (III) during preimplantation development and prolonged impairment of development potential. The results further highlight the potential adverse effects of chromium (III) on public reproductive health with respect to increased environmental enrichment of and dietary supplementation with chromium (III) complexes.     

Experimental Infection of Brook Trout with Infectious Hematopoietic Necrosis Virus Types 1 and 2

Abstract

Fry of brook trout Salvelinus fontinalis became infected and diseased after immersion exposure to infectious hematopoietic necrosis virus (IHNV), but a long-lasting IHNV carrier state was not induced. Duplicate groups of 100 fish were immersed for 6 h in baths containing a type 1 (Round Butte, RB) or a type 2 (Rangen, RA) IHNV isolate at a high or low dose. Brook trout mortalities induced by immersion in a bath of the RB or RA IHNV isolate at 10² plaque-forming units (pfu) per milliliter were equivalent (1 and 0%), but fish were more susceptible to infection with RA IHNV. Only the single dead fish in the RB group was infected, but 24% of the RAexposed fish were infected 1 week after exposure. At a dose of 10⁶ pfu/mL, exposure to RB IHNV resulted in a higher mortality (35%) and prevalence of infection (89% of live fish sampled at 1 week postexposure), but no infectious virus was detectable by 5 weeks after exposure. In contrast, RA IHNV exposure at a dose of 10⁴ pfu/mL resulted in only 5% mortality, and live fish killed at 1 week postexposure had a 22% prevalence of infection, but infectious virus was not detectable by week 3. Although brook trout have been previously considered to be resistant to IHNV, this study has shown that brook trout become diseased and die after exposure to a high dose of one type I IHNV isolate and can be infected after immersion exposure to even a low dose of type 1 or type 2 IHNV.     

Development of a genetically modified nontoxigenic Pasteurella multocida toxin as a candidate for use in vaccines against progressive atrophic rhinitis in pigs

OBJECTIVE: To construct a genetically modified nontoxigenic Pasteurella multocida toxin (PMT) and examine its immunoprotective activity against challenge exposure with wild-type PMT in pigs. ANIMALS: 5 healthy pigs. PROCEDURE: A nontoxigenic PMT was created by replacing the serine at position 1164 with alanine (S1164A) and the cysteine at position 1165 with serine (C1165S). Toxic activity was determined by use of the guinea pig skin test and mouse lethality test. Three pigs were vaccinated twice with the modified PMT, and the remaining 2 pigs served as nonvaccinated control animals. Vaccinated and control pigs were challenge exposed with wild-type PMT. Pigs were euthanatized and necropsied on day 14 after challenge exposure. Turbinate atrophy was examined macroscopically and assigned a score. Serum anti-PMT antibodies were determined by use of an ELISA. RESULTS: The genetically modified PMT was characterized by a total lack of toxic activity. Pigs vaccinated with the modified PMT became seropositive; in contrast, control pigs remained seronegative. Necropsy revealed that the 2 control pigs had moderate and severe turbinate atrophy, respectively, whereas the 3 vaccinated pigs did not have any lesions in the turbinates or abnormalities in other organs. CONCLUSIONS AND CLINICAL RELEVANCE: Modification by use of S1164A and C1165S leads to a complete loss of toxic effects of PMT without impairment of the ability to induce protective immunity in pigs. Analysis of these results suggests that genetically modified PMT may represent a good candidate for use in developing a vaccine against progressive atrophic rhinitis in pigs.     

Evaluation of the Modifying Effect of Inhalation of Mainstream Cigarette Smoke on Mouse Bladder Carcinogenesis

Cigarette smoking is one of the major risk factors of bladder cancer in humans. To date, however, there is no experimental evidence for the effects of inhalation exposure to mainstream cigarette smoke on bladder carcinogenesis. The purpose of the present study was to evaluate the effect of inhalation of mainstream cigarette smoke on mouse bladder carcinogenesis using a cigarette smoke inhalation exposure system. Six-week-old male C57BL mice were administered 0.025% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in their drinking water for 8 weeks and then divided into 2 groups and exposed to 0 or 300 mg/m³ wet total particulate matter mainstream cigarette smoke for 2 h per day, five times per week, for 22 weeks. The incidences of bladder tumors (papilloma and urothelial carcinoma) tended to increase in the cigarette smoke-exposed group (25.0%) compared with the controls (15.8%), albeit without a statistically significant difference. We also evaluated mRNA expression levels of cytochrome P450 (cyp) enzymes and proliferating cell nuclear antigen (PCNA) in the bladder epithelium. Expression of cyp1a1 was significantly increased in the cigarette smoke-exposed group. Cigarette smoke exposure did not have a significant effect on the expression of cyp1a2, cyp 1b1, cyp 2a4, cyp 2b10, cyp 2e1, or PCNA. In conclusion, limited exposure to mainstream cigarette smoke for 22 weeks, caused a significant increase in cyp1a1 expression. This increase coupled with the nonsignificant increase in bladder tumors suggests that a longer period of exposure is required to clarify the effects of cigarette smoke on bladder carcinogenesis.     

The immunopathogenesis of flea allergy dermatitis in dogs, an experimental study

In this study, we investigated the development of clinical disease and immune responses in the development of an experimental model of flea allergy dermatitis. Dogs were randomly divided into four treatment groups and were infested with fleas on two different feeding schedules (continuous and episodic). Group 1 consisted of four non-exposed dogs (negative controls) and Group 2 consisted of six dogs exposed to fleas continually. Groups 3 and 4 consisted of 14 dogs each that were exposed to fleas on an episodic schedule (two consecutive days every other week for 12 weeks). Group 4 also received intraperitoneal injections of a low dose of lectin (ricin) with immunomodulatory properties. The purpose of Group 4 was to investigate the effects of ricin on enhancing the development of clinical signs, flea antigen-specific IgE levels and altering the number of CD4+ and CD8+ T cell subsets in peripheral blood. Clinical signs developed in all flea exposed dogs, however, the dermatology lesion scores were less and shorter in duration for continuously exposed dogs compared to episodic exposed dogs, independent of ricin treatment. Lesion development was concentrated in the flea triangle and consisted principally of erythema, followed by alopecia, excoriation, papules, and crusts. CD4+ and CD8+ lymphocyte subsets or IgE levels were not altered by ricin treatment. Flea antigen-specific IgE values were highest in dogs exposed to fleas on a continuous basis compared to those episodically exposed. A greater percentage of clinical responder dogs with negative flea-specific IgE titers or negative intradermal test (IDT) were present in the episodic exposure groups than in the continuous exposure group. IgE titers corresponded slightly better with clinical responders than the IDT. The agreement between the IgE titers and IDT was good (weighted K = 0.67). Histopathology of skin samples were consistent with a Type I hypersensitivity. In conclusion, we were able to develop a model of flea allergy dermatitis by experimentally exposing dogs to fleas on an episodic and continuous feeding schedule. In this study, continuously exposed dogs did not develop immunotolerance, and ricin did not enhance the development of FAD.