P-gp substrate-induced neurotoxicity in an Abcb1a knock-in/Abcb1b knock-out mouse model with a mutated canine ABCB1 targeted insertion.

Certain dog breeds, especially Collies, are observed to exhibit neurotoxicity to avermectin drugs, which are P-glycoprotein (P-gp) substrates. This neurotoxicity is due to an ABCB1 gene mutation (ABCB1-1Δ) that results in non-functional P-gp expression. A developed Abcb1a knock-in/Abcb1b knock-out mouse model expressing the ABCB1-1Δ canine gene was previously reported and mice exhibited sensitivity upon ivermectin administration. Here, model and wild-type mice were administered P-gp substrates doramectin, moxidectin, and digoxin. While knock-in/knock-out mice exhibited ataxia, lethargy and tremor, wild-type mice remained unaffected. In addition, no neurotoxic clinical signs were observed in either mouse type administered domperidone, a P-gp substrate with no reported neurotoxicity in ABCB1-1Δ Collies. Overall, neurotoxic signs displayed by model mice closely paralleled those observed in ivermectin-sensitive Collies. This model can be used to identify toxic P-gp substrates with altered safety in dog populations and may reduce dog use in safety studies that are part of the drug approval process.

Inactivation of Cryptosporidium parvum oocysts in fresh apple cider by UV irradiation.

This study evaluated the efficacy of UV irradiation on the inactivation of Cryptosporidium parvum oocysts in fresh apple cider. Cider was inoculated with oocysts and exposed to 14.32 mJ of UV irradiation/cm(2). Oocyst viability was assessed with the gamma interferon gene knockout (GKO) mouse and infant BALB/cByJ mouse models. All GKO mice challenged with UV-treated cider demonstrated no morbidity or mortality, and infant BALB/c mice challenged with treated cider were negative for the presence of C. parvum. In contrast, the GKO mice challenged with non-UV-treated inoculated cider died and the parasite was detected in the ileums of all challenged infant mice. This study shows that UV irradiation can be used to inactivate C. parvum in fresh apple cider.

New Zealand white rabbit as a nonsurgical experimental model for Salmonella enterica gastroenteritis.

Rabbits orally challenged with Salmonella enterica developed a dose-dependent diarrheal disease comparable to human salmonellosis. Viable Salmonella organisms recovered from the intestine and deep tissues indicate local and systemic infections. Therefore, results show that the rabbit can be used as a model for diarrheal disease and sequelae associated with salmonellosis.

Attempts to establish experimental Cyclospora cayetanensis infection in laboratory animals.

Attempts were made to develop an animal model for Cyclospora cayetanensis to identify a practical laboratory host for studying human cyclosporiasis. Oocysts collected from stool of infected humans in the United States, Haiti, Guatemala, Peru, and Nepal were held in potassium dichromate solution to allow development of sporozoites. The following animal types were inoculated: 9 strains of mice, including adult and neonatal immunocompetent and immune-deficient inbred and outbred strains, rats, sandrats, chickens, ducks, rabbits, jirds, hamsters, ferrets, pigs, dogs, owl monkeys, rhesus monkeys, and cynomolgus monkeys. Most animals were inoculated by gavage, although some of the primates were fed oocysts on food items. The animals were examined for signs of infection, particularly diarrhea, and stool samples were examined for 4-6 wk after inoculation. None of the animals developed patent infections or signs of infection. We conclude that none of the animals tested is susceptible to infection with C. cayetanensis.

The effects of an alpha hydroxy acid (glycolic acid) on hairless guinea pig skin permeability.

The barrier integrity of hairless guinea pig skin after treatment with an alpha hydroxy acid was assessed through in vivo topical application of an oil-in-water emulsion containing 5 or 10% glycolic acid at pH 3.0. The control was a commercial moisturizing lotion, pH 7.8. A dosing regimen for the glycolic acid formulations that was tolerated by the hairless guinea pigs and significantly decreased stratum corneum turnover time was determined using the dansyl chloride staining technique. Once-daily dosing of hairless guinea pig skin for 3 weeks with the glycolic acid formulations resulted in approximately a 36-39% decrease in stratum corneum turnover time compared with the control lotion. After this treatment, hairless guinea pigs were sacrificed for the in vitro measurement of the percutaneous absorption of [14C]hydroquinone and [14C]musk xylol. No significant differences in the 24-hour absorption of either test compound were found for skin treated with the control lotion or the glycolic acid formulations. There were also no significant differences found in the absorption of [3H]water through skin from the different treatment groups. Although no increase in skin penetration occurred after treatment with the glycolic acid formulations, histology revealed approximately a twofold increase in epidermal thickness. Also the number of nucleated cell layers nearly doubled in skin treated with 5% and 10% glycolic acid compared with the control lotion and untreated skin. These studies demonstrate that substantial changes in the structure of hairless guinea pig epidermis can occur without significant effect on skin permeability of two model compounds.

Histopathological evaluation of liver, pancreas, spleen, and heart from iron-overloaded Sprague-Dawley rats.

The effects of increasing dietary levels of Fe on the histopathology of liver, pancreas, spleen, and heart were examined in a rat model for iron overload. Sprague-Dawley rats were fed diets containing 35, 350, 3,500, or 20,000 micrograms Fe/g, and, after 12 wk, there was a direct correlation between increased liver nonheme Fe and lipid peroxidation measured by the lipid-conjugated diene assay. Histopathological examination of tissues revealed the following: (a) hepatocellular hemosiderosis in all groups of rats, with a dose-related accumulation of cytoplasmic Fe-positive material predominantly in hepatocytes located in the periportal region (Zone 1), (b) myocardial degeneration and necrosis (cardiomyopathy) with hemosiderin in interstitial macrophages or in myocardial fibers of animals with heart damage, (c) splenic lymphoid atrophy affecting the marginal zone of the white pulp and hemosiderin deposition in the sinusoidal macrophages, and (d) pancreatic atrophy with loss of both the endocrine and exocrine pancreatic tissue in those animals receiving 3,500 and 20,000 micrograms Fe/g of diet. The toxic effects of Fe overload in this rat model include cellular apoptosis or necrosis in heart, spleen, and pancreas and, when coupled with the findings on lipid peroxidation, suggests that oxidative stress is involved in the pathogenesis of the lesions.

Epidemiology of Potomac horse fever: an investigation into the possible role of non-equine mammals.

A serological study of antibodies to Ehrlichia risticii was carried out on 10 species of wild and domestic mammals found on or near 21 horse farms in an area of the USA in which Potomac horse fever is endemic. No antibodies were found in 133 peridomestic rodents (Norway rats and house mice), nor in 108 wild rodents (white-footed mice and meadow voles) captured on farms. Three of the six domestic animal species examined, cats, pigs and a goat, showed serological evidence of exposure to E risticii. Seropositive animals were detected on three of the 21 premises. The eight seropositive cats (of 48 cats tested) were on two farms, and the three seropositive pigs (of 14 tested) were all on one farm which lay some 3 km from where the one seropositive goat (of three tested) was found. None of the 79 dogs, 75 cattle and seven sheep tested had antibodies to E risticii. The significance of these findings is discussed in the light of current understanding of the transmission of Potomac horse fever and of the epidemiology of other related ehrlichial diseases.

Disease features in horses with induced equine monocytic ehrlichiosis (Potomac horse fever).

Fifty-five horses were inoculated IV and/or SC with materials containing Ehrlichia risticii, ie, infected whole blood, buffy coat cells, or cell culture, to study clinical and hematologic features of equine monocytic ehrlichiosis (Potomac horse fever). Major clinical and hematologic features of induced E risticii infection were biphasic increase in rectal temperature with peak increases of 38.9 C and 39.3 C on postinoculation days (PID) 5 and 12, respectively; depression; anorexia; decreased WBC count (maximal decrease of 47% on PID 12); and diarrhea from PID 14 to PID 18. Increased WBC count was an inconsistent feature, with a maximal increase of 51.5% on PID 20. During times of decreased and increased WBC counts, lymphocyte/neutrophil ratios remained fairly constant. However, not all horses had all clinical and hematologic features, and these features were present in different degrees among horses. Increased rectal temperature, depression, anorexia, and decreased WBC count were more consistent features, whereas diarrhea developed in 73% of the horses. Of 55 horses, 39 (71%) had all clinical and hematologic features of the disease (classic disease), whereas 16 (29%) horses did not have greater than or equal to 1 of these features (nonclassic disease). The E risticii titer in the blood (ehrlichemia) was maximum during the peak increase in rectal temperature. In 55 horses, mortality was 9%. Significant differences (P greater than 0.5) in clinical and hematologic features were not detected between horses that survived and those that died of E risticii infection.

Attempted transmission of Ehrlichia risticii by field-captured Dermacentor variabilis (Acari: Ixodidae).

The capability of field-collected American dog ticks, Dermacentor variabilis, to infect horses with Ehrlichia risticii, causative agent of Potomac horse fever (PHF), was examined by allowing adult ticks collected from horse farms with a history of PHF to feed on susceptible horses. More than 500 male and female ticks attached and fed on 3 test horses; however, no clinical or serologic evidence of PHF was observed in treated or control horses. All horses were challenge exposed with E risticii-infective blood by inoculation at 60 to 65 days after ticks fed, and all developed clinical PHF with subsequent seroconversion. The data, therefore, indicated that adult D variabilis, a common parasite of horses on Maryland premises where PHF is enzootic, may not serve as a vector of E risticii.

Effect of a single therapeutic dose of levamisole on bovine viral diarrhea virus infection in calves.

The effect of a single therapeutic dose of levamisole in calves experimentally infected with bovine viral diarrhea virus (BVDV) was evaluated in 2 separate double-blind experiments. The infection was mild and there was no difference in severity of infection or speed of recovery between levamisole-treated and 0.9% Nacl solution-treated (control) calves. There were no significant differences between drug-treated and control calves in respect to total white blood cell, lymphocyte, and neutrophil counts, and the viral recovery data from these calves were comparable. The differences in blastogenic response of lymphocytes to the mitogen phytohemagglutinin and serum antibody titers of the 2 groups of calves were also insignificant. An increased lymphocytic hyperplasia was observed in a larger number of lymphoid tissues of drug-treated calves than in those of control calves. It appeared that a single therapeutic dose of levamisole was ineffective in altering the response of calves infected with BVDV.

Experimental reproduction of Potomac horse fever in horses with a newly isolated Ehrlichia organism.

Potomac horse fever, a recently recognized disease of equines, characterized by high fever, leukopenia, and a profuse diarrhea, was studied for its etiology. An Ehrlichia organism was isolated in equine macrophage-fibroblast cell cultures and mouse macrophage cell cultures from the mononuclear cells of blood of infected horses. The agent was continuously propagated in mouse macrophage cell cultures. The organism multiplied in the cytoplasm of mouse macrophage cells and was identified by Giemsa staining, acridine orange staining, and by indirect immunofluorescence with convalescent sera from infected horses. The disease was experimentally reproduced in horses inoculated with Ehrlichia-infected cell culture material. The Ehrlichia organism was reisolated from the blood of these infected horses during the course of the disease. Antibody against the organism was detected in the sera of experimentally infected horses. This study confirmed that the new Ehrlichia organism is the etiological agent of Potomac horse fever.

Toxicity and residue studies in dairy animals with firemaster FF-1 (polybrominated biphenyls).

Two studies (one in Holstein calves and one in Holstein cows) were conducted to determine potential toxicity and residue levels following oral ingestion of polybrominated biphenyls (PBB). The material was FireMaster FF-1. Administration was by gelatin capsules. Doses in calves were 0.1, 1.0, 10, or 100 mg/kg body weight, while doses in cows were equivalent to 0.01, 0.1, 1.0, or 10 ppm in the diet. The calves were sacrificed after 2, 4, 6, or 12 weeks. The cows were fed 158 or 228 days, and were then in a recovery period for 182 or 112 days. In the calf study, signs of toxicity were observed only in animals fed 100 mg/kg-day. Administration of 10 mg/kg-day or less for up to 12 weeks caused no overt signs of toxicity. Histologic studies were conducted upon selected organs and tissues taken at time of sacrifice. The only treatment-induced lesions among animals fed 0.1 mg/kg-day were minimal lesions in the kidney and skin in the one calf fed at this level for 12 weeks. Treatment-induced lesions were present in the kidneys, skin, and/or liver from some animals fed levels of 1.0 mg/kg-day and above. The relative severity of these lesions was related to the level and length of exposure. Treatment-associated changes were observed in the testes of all males in this study. The hypospermatogenesis observed was consistent with the age of the animals due to prepuberal development of the testes. No clinical signs of toxicity or histologic changes attributed to PBB were observed in the cows. Two cows were pregnant at the initiation of the study and give birth to normal, healthy calves during the study. These calves grew normally and appeared healthy when sacrificed at about 6 months of age. Residue levels were quantitated as the hexabromobiphenyl isomer (BP-6) which is the major isomer present in the PBB mixture. Tissue residue levels in calves increased with dose and duration of administration of PBB with highest levels being found in the fat. At 100 mg/kg the levels in fat were about 6000 to 6300 ppm after 6 to 12 weeks. Residue levels of BP-6 in milk and fat of the cows also increased with dose and duration of administration. Maximum levels in milk were about 1/3 the levels in the diet. In general, the levels plateaued after about 4 to 6 weeks and did not go appreciably higher even though administration of the FireMaster FF-1 was continued. Maximum levels in fat were on the order of approximately 4.5 times the levels in the diet, except at the lowest dose level. Residues decreased in milk after administration of PBB was discontinued, but detectable levels were still present 6 months later. Residues were found in the calves born of treated cows indicating passage of the PBBs through the placental barrier and/or ingestion through the milk.