Purification, characterization, and pathogenicity of Moraxella bovis pili.

Pilins composed of the alpha or beta pilins of Moraxella bovis strain Epp63 were purified, subjected to chemical or enzymatic cleavage, and the resulting fragments sequenced by automated Edman degradation. alpha Pilin was found to be a 155-amino-acid polypeptide with a single intramolecular disulfide bridge. The beta pilin amino acid sequence substantiated the previously reported structure derived from the beta pilin gene DNA sequence, and indicated that the alpha and beta pilins of this strain are approximately 70% homologous. DNA hybridization studies of genomic DNA from the alpha- and beta-piliated variants of strain Epp63 indicated that the expression of the two pilin types was governed by an oscillating mechanism of chromosomal rearrangement. The alpha and beta pili were evaluated serologically and found to exhibit approximately 50% shared antigenicity, indicating that regions of conserved and heterologous sequence specify both type-specific and crossreacting epitopes. The pathogenicity of the alpha- and beta-piliated variants was studied by ocular inoculation of calves eyes; beta-piliated organisms were significantly more infectious than alpha-piliated organisms, indicating that beta pili confer, or are associated with, a relative advantage during the first stages of ocular infection. Preliminary analysis of other M. bovis strains suggests that each strain produces two types of pilin, and that this property may be characteristic of the species.

Determinants of the antitumor effect of radiolabeled monoclonal antibodies.

The murine B-cell lymphoma 38C13 model was used to study the radiobiological effect of 131I-monoclonal antibody (MAB) therapy compared with dose equivalent external beam irradiation. Continuous exponentially decreasing low dose rate (LDR) gamma-irradiation, and multiply fractionated (MF) X-irradiation were compared with dose equivalent 131I-MAB. The relative therapeutic efficacy of radioimmunotherapy, and the relative contribution of (a) low dose rate; (b) whole body irradiation; and (c) microdosimetry to the overall effect were determined. Groups of mice with or without B-cell lymphoma were treated with either (a) 131I-anti-idiotype MAB; (b) 131I-isotype-matched irrelevant control MAB; (c) 5-15 Gy 250 kV X-irradiation given as a single fraction; (d) 2.5-30 Gy 250 kV X-irradiation given in 10 fractions/2 weeks; or by (e) continuous exponentially decreasing gamma-irradiation via a 137Cs source, which simulated the effective t1/2 of the 131I-MAB. In tumor-free mice the LD50/30 was approximately 10 Gy for MF and LDR external irradiation, and 11-12 Gy for 131I-MAB. However, the effect of these modes of irradiation on tumor size differed significantly. The cumulative percentage of tumor reduction averaged over 12 days was 0.635 +/- 0.055%/Gy for MF, and 1.36 +/- 0.061%/Gy for LDR external irradiation (a relative efficacy factor of 1.63 for LDR irradiation; P = 0.01). Assuming homogeneous body distribution, the tumor reduction effect over 12 days for 131I-MAB was 2.064 +/- 0.133%/Gy for specific, and 1.742 +/- 0.1%/Gy for nonspecific isotype-matched irrelevant 131I-MAB (P = 0.02). When 131I-MAB was compared to LDR external irradiation, the relative efficacy factor was 1.99 (P less than 0.001). In summary, there was a dose rate effect on tumor response, which may in part explain the efficacy of radioimmunotherapy. The additional effect of 131I-MAB on tumor response was only partially explained by the cumulative concentration ratio of 131I-MAB tumor/131I-MAB whole body, which was on average 1.7. This relatively low concentration ratio was partly due to tumor-mediated dehalogenation. Thus, the overall tumor response was a function of the total dose, dose rate, and both the specific and nonspecific distribution of 131I-MAB.

The canine as an animal model of human aging and dementia.

The aged canine displays many features that make it an excellent model for studying the progression of pathology in brain aging and linking these findings to learning, memory and other cognitive functions. Canines develop extensive beta-amyloid deposition within neurons and their synaptic fields, which appears to give rise to senile plaques. These plaques are primarily of the early diffuse subtype. Aged canines also exhibit accumulations of lipofuscin, cerebral vascular changes, dilation of the ventricles, and cytoskeletal changes. Neurofibrillary tangles (NFTs) are not present in the aged canine. Thus, the aged canine brain provides a suitable model for studying early degeneration normally considered to be pre-Alzheimer's. This supposition is also supported by behavioral data. We have found that the extent of beta-amyloid deposition correlates with a decline in select measures of cognitive function. These data provide the first evidence of a correlation between beta-amyloid accumulation and cognitive decline in the absence of NFTs. We summarize four lines of evidence that support using the aged canine as a model of human aging: (a) Aged canines develop aspects of neuropathology similar to that observed in aged humans; (b) Veterinarians have observed that many canines exhibit a clinical syndrome of age-related cognitive dysfunction; (c) Aged canines are deficient on a variety of neuropsychological tests of cognitive function; (d) The level of beta-amyloid accumulation correlates with cognitive dysfunction in the canine. These data indicate that the aged canine is a particularly useful model for studying age-related cognitive dysfunction (ARCD), early neuronal changes associated with aging, and the initial stages of senile plaque formation.

The effect of unlabelled monoclonal antibody (mAb) on the biodistribution of 131I-anti-idiotype mAb in murine B cell lymphoma.

The 38C13 murine B cell lymphoma model was used to study the effect of the preinjection of unlabelled anti-idiotype monoclonal antibody (mAb) on the subsequent biodistribution of 131I-anti-idiotype mAb. Mice with established tumors received 0-500 micrograms of unlabelled anti-idiotype mAb 24 h prior to the administration of 131I-anti-idiotype (specific), or both 125I-anti-idiotype and 131I-isotype-matched irrelevant control (nonspecific) mAb. Mice were counted daily in a gamma counter and sacrificed at 2-144 h following injection. Mice were dissected and the weight and activity of the animals and organs were measured. Mice were bled periodically and circulating idiotype levels were measured using an ELISA assay. Five hundred micrograms of unlabelled anti-idiotype mAb increased the retention time of the specific but not the nonspecific mAb in all organs and tumor. Following pretreatment with unlabelled mAb, the cumulative tumor/whole body and tumor/normal organ ratios became similar to those of the nonspecific mAb, with concentration ratios (specific/nonspecific mAb) of approximately 1, which persisted until 96 h post injection when circulating idiotype reappears in antigen excess. In the absence of unlabelled mAb there was less retention in tumor and normal tissue. This is presumed to be due in part to decreased levels of circulating 131I-mAb secondary to rapid plasma clearance of antigen-antibody complexes and tumor cell mediated dehalogenation, which results when the specific mAb specifically binds the targeted antigen. Thus, the addition of unlabelled mAb increased the retention by decreasing the specific behavior of the anti-idiotypic antibody.

Spatial learning and memory as a function of age in the dog.

Spatial learning and memory were studied in dogs of varying ages and sources. Compared to young dogs, a significantly higher proportion of aged dogs could not acquire a spatial delayed nonmatching-to-sample task. A regression analysis revealed a significant age effect during acquisition. Spatial memory was studied by comparing performance at delay interval of 20, 70, and 110 s. At short delays aged and young dogs were similar; at longer delays, errors increased to a greater extent in old than in young dogs; however this was not statistically significant. It was possible to identify 2 groups of aged animals, age-impaired and age-unimpaired. Several of the dogs were also tested on an object recognition memory task, which was more difficult to learn than the spatial task. The possibility that these findings are confounded by breed differences is considered. Overall, the present results provide further evidence of the value of a canine model of aging.

DNA damage and apoptosis in the aged canine brain: relationship to Abeta deposition in the absence of neuritic pathology.

1. In addition to beta-amyloid (Abeta) deposition and cytoskeletal neuropathology, both the Alzheimer's disease (AD) and Down's syndrome (DS) human brain exhibit marked evidence of DNA damage, however, it is difficult to separate events that occur in conjunction with neurofibrillary pathology versus Abeta pathology in these systems. 2. In contrast, the aged canine brain exhibits the accumulation of Abeta into diffuse deposits similar to those found in early AD and DS in the absence of neurofibrillary pathology. Furthermore, Abeta deposition in canine brain is correlated with cognitive deficits. 3. In order to test the hypothesis that TUNEL labeling for DNA damage in AD is not simply a consequence of agonal artifacts, postmortem artifacts, or neurofibrillary pathology, and may be directly related to Abeta deposition, we examined Abeta immunoreactivity, PHF-1 immunoreactivity, and TUNEL labeling in this animal model. 4. These experiments reveal a relationship between the amount of DNA damage detected by TUNEL labeling and levels of Abeta deposition. Further, in animals with no TUNEL labeling, we detected no Abeta immunoreactivity. 5. These data support the hypothesis that TUNEL labeling in AD ans DS is not a consequence of agonal artifact, postmortem artifact, or tau pathology, and may be directly related to Abeta deposition and perhaps AD pathogenesis.

The effects of L-deprenyl on spatial short term memory in young and aged dogs.

1. Young and aged dogs were tested on a spatial memory task using a delayed non matching to sample technique. Dogs were tested with 20, 70 and 110 second delay intervals. Animals were pretrained to a stable level of performance prior to treatment. 2. During treatment periods, dogs were orally administered a placebo or I-deprenyl in doses of 0.5 and 1.0 mg/kg in a repeated measures design. 3. Young dogs did not show any significant effects of I-deprenyl, however the sample size was limited. 4. L-deprenyl administration improved spatial memory in aged dogs. 5. The optimal dose or length of treatment time of I-deprenyl varied among individual dogs.

Treatment with L-deprenyl prolongs life in elderly dogs.

Eighty two beagle dogs ranging in age from 2.8 to 16.4 years and in weight from 6.3 to 15.8 kg were allotted to 41 pairs and administered placebo or 1 mg/kg L-deprenyl orally once daily for 2 years and 10 weeks. When survivorship for all dogs in the study was analyzed there was no significant difference between the L-deprenyl and placebo treated groups, most likely due to the (expected) survival of virtually all young dogs in both groups for the duration of the study. To assess whether L-deprenyl treatment begun in later life might enhance canine longevity in a fashion similar to that documented in rodents we also examined survival in a subset of elderly dogs who were between the ages of 10 and 15 yrs at the start of tablet administration and who received tablets for at least 6 months. In this subset, dogs in the L-deprenyl group survived longer (p < 0.05) than dogs in the placebo group. Twelve of 15 (80%) dogs in the L-deprenyl group survived to the conclusion of the study, in contrast to only 7 of 18 (39%) of the dogs who received placebo (P=0.017). Furthermore, by the time the first L-deprenyl treated dog died on day 427, 5 placebo treated dogs had already succumbed, the first on day 295. Specifically with respect to dogs, the findings reported herein suggest daily oral administration of 1 mg/kg L-deprenyl prolongs life when begun in relatively healthy dogs 10-15 years of age and maintained for the duration of the individual's life, but in any event for no less than six months.

Open field activity and human interaction as a function of age and breed in dogs.

Open field (OF) activity was studied in kennel reared purebred beagles from two separate colonies (2-13 years in age) and pound source mixed breed dogs (9 months to 10 years in age). Dogs were observed for 10 min sessions and records were taken of: locomotion, urination, sniffing, grooming, rearing, vocalizing, jumping frequencies and inactivity (16). Since dogs are uniquely social towards people, we also measured human interaction (HI), which recorded the same behaviors as during OF when a person was present in the room. Measures of exploratory behavior decreased as a function of age in pound source dogs in the OF test, but not in beagles from either colony. No breed differences were found between the young dogs. In the HI test, age effects were found in beagles but not pound source dogs. OF activity correlated with tests of cognitive function, but differences were found between the three groups. These findings indicate that OF activity is age-sensitive in dogs, but that breed and test conditions are also essential factors.

Effects of chronic oral administration of L-deprenyl in the dog.

Dogs were administered capsules containing L-deprenyl daily over 3 weeks at dose levels of 0, 0.1, 0.5, and 1.0 mg/kg. Spontaneous behavior was measured using a canine open field test, and was not significantly affected by L-deprenyl. Plasma levels of amphetamine showed a clear dose-dependent elevation 2 h and was not significantly affected by L-deprenyl. Plasma levels of amphetamine showed a clear dose-dependent elevation 2 h following treatment, but were markedly lower after 24 h, and were undetectable 5 days following the last treatment. Plasma levels of phenylethylamine were increased, but were highly variable. Animals sacrificed 1 day following the last treatment showed a dose-dependent inhibition of monoamine oxidase B in the brain, liver, and kidney, whereas monoamine oxidase A was unaffected in these tissues. L-Deprenyl also caused an increase in phenylethylamine in the striatum and hypothalamus, but not in the neocortex. Brain levels of DA, DOPAC, 3-MT, HVA, 5-HT, and 5-HIAA were unaffected. The pharmacological profile for the dog is distinct from that of other species in that long-term treatment did not produce any significant inhibition of MAO-A activity. The absence of an effect on biogenic amines or metabolites suggests that the metabolism of dopamine is mediated at least in part through pathways other than MAO-B in the normal adult dog.

Infection rates, disease frequency, pilin gene rearrangement, and pilin expression in calves inoculated with Moraxella bovis pilin-specific isogenic variants.

Pili have been implicated as virulence factors that result in increased infectivity of Moraxella bovis, the causative agent of infectious bovine keratoconjunctivitis (IBK). Healthy calves' eyes were inoculated with I- or Q-piliate or nonpiliate M bovis Epp63 to compare the pathogenicity of these isogenic variants. Pathogenicity was determined by the rate of persistent M bovis infection and the prevalence of clinical IBK. Inoculation with M bovis expressing the Q pili resulted in the highest frequency of infection and IBK, whereas I-piliate M bovis elicited a lower rate and nonpiliate M bovis did not result in infection. In vivo pilin gene rearrangement and pilin-type switching were evaluated by DNA hybridization and immunoblot. Gene rearrangement and type switching varied dependently, and were observed only in eyes inoculated with Q-piliate M bovis. This study suggests that Q pili are specific for colonization of bovine corneal epithelium, whereas I pili enable maintenance of an established infection.

Diagnostic value of pericardial fluid analysis in the dog.

The physical, chemical, and cytologic characteristics of 50 pericardial effusions were reviewed to determine their value to the clinician for distinguishing a variety of pericardial disorders in the dog. Pericardial fluid analysis allowed identification of chylous and bacterial pericardial effusions. Overlap in the ranges of RBC counts, nucleated cell counts, and protein concentrations between dogs with neoplastic and nonneoplastic disorders precluded identification of the cause of the effusion. Of 19 neoplastic effusions, 74% were not detected on the basis of cytologic findings and 13% of 31 nonneoplastic effusates were falsely reported as positive or suspect for a neoplasm. It was concluded that pericardial fluid analysis, including cytologic examination, did not reliably distinguish neoplastic from nonneoplastic disorders.

Prevalence of behavioral changes associated with age-related cognitive impairment in dogs.

OBJECTIVE: To determine the prevalence of age-related behavioral changes, namely impairment, in a randomly chosen population of dogs. DESIGN: Age-stratified cohort study. ANIMALS: 97 spayed female and 83 castrated male dogs that were 11 to 16 years old. PROCEDURE: Data on possible impairment in 4 behavioral categories (ie, orientation in the home and yard, social interaction, house training, and sleep-wake cycle) linked to cognitive dysfunction were obtained from dog owners, using a structured telephone interview. Hospital records of dogs had been screened to exclude dogs with dysfunction in organ systems that may cause behavioral changes. Dogs with behavioral impairment were those with > or = 2 signs of dysfunction within a category. Dogs with impairment in 1 category were considered mildly impaired and those with impairment in > or =2 categories were considered severely impaired. RESULTS: Age by sex interactions for dogs with impairment in any category were not significant, and, therefore, data on castrated males and spayed females were pooled for analyses across ages. The prevalence of age-related progressive impairment was significant in all categories. The percentage of 11- to 12-year-old dogs with impairment in > or = 1 category was 28% (22/80), of which 10% (8/80) had impairment in > or = 2 behavioral categories. Of 15- to 16-year-old dogs, 68% (23/34) had impairment in > or =1 category, of which 35% (12/34) had impairments in > or = 2 categories. There were no significant effects of body weight on the prevalence of signs of dysfunction in the behavioral categories. CONCLUSIONS AND CLINICAL RELEVANCE: Data collected provide estimates of the prevalence of various degrees of age-related behavioral changes associated with cognitive dysfunction in dogs. Age-related behavioral changes may be useful indicators for medical intervention for dogs with signs of cognitive impairment.

Predicting behavioral changes associated with age-related cognitive impairment in dogs.

OBJECTIVE: To monitor the progression of age-related behavioral changes in dogs during a period of 6 to 18 months and to determine whether signs of dysfunction in any of 4 behavioral categories can be used to predict further impairment. DESIGN: Age-stratified cohort study. ANIMALS: 63 spayed female and 47 castrated male dogs 11 to 14 years of age. PROCEDURE: Data were collected from randomly selected dog owners who were interviewed by telephone twice at a 12- to 18-month interval; data were included if the dog had lived > or = 6 months between interviews. The interview focused on signs of impairment in the following behavioral categories: orientation in the home and yard, social interactions with human family members, house training, and the sleep-wake cycle. Dogs were determined to have impairment in 0 behavioral categories (on the basis of < or = 1 sign for each category), impairment in 1 category (> or = 2 signs of dysfunction in that category), or impairment in > or = 2 categories. RESULTS: Between interviews, 22% (16/73) of dogs that did not have impairment in a category at the time of the first interview developed impairment in that category by the time of the second interview. Forty-eight percent (13/27) of dogs that had impairment in 1 category at the time of the first interview developed impairment in > or = 2 categories by the time of the second interview and were significantly more likely to develop impairment in > or = 2 categories, compared with dogs that initially had impairment in 0 categories. Dogs with 1 sign of dysfunction in orientation were significantly more likely to develop impairment in that category, compared with dogs that had 0 signs of dysfunction in orientation. CONCLUSIONS AND CLINICAL RELEVANCE: Age-related behavioral changes in dogs are progressive. Clinicians should consider trying to predict which dogs are most likely to become progressively impaired during the subsequent 6 to 18 months.

Geriatric behavioral problems.

Behavior problems in older pets may be due to many of the same causes as in younger pets. However, the effects of aging on the pet's body may cause a dramatic decline or deterioration in organ and sensory function, which may have a profound impact on the pet's behavior. A decline in cognitive function may also afflict older pets, in many instances due to the occurrence of Alzheimer's disease brain pathology, especially beta amyloid plaque formation. Correlation of geriatric behavior problems, therefore, entails first diagnosing and treating any underlying medical problems. Behavior modification techniques and drug therapy may then be required, with modifications and adjustments made to suit the specific needs of the older pet.

Management of canine pituitary-dependent hyperadrenocorticism with l-deprenyl (Anipryl).

In this chapter we have discussed the pathogenesis of canine PDH focusing on its relationship to aging, dopamine deficiency, and neurodegenerative disease. We have outlined the successful management of canine PDH patients with l-deprenyl, a selective MAO-B inhibitor. Treatment with l-deprenyl results in clinical and endocrinologic improvement (partial to complete) in approximately 83% of dogs, with improvement noted within the first 1 to 2 months of therapy. The safety profile of l-deprenyl is excellent, especially in light of the fact that the majority of patients are elderly. l-Deprenyl is a safe and effective first-line therapy for the medical management of uncomplicated cases of canine PDH.