Identification of a calcitonin-like diuretic hormone that functions as an intrinsic modulator of the American lobster, Homarus americanus, cardiac neuromuscular system.

In insects, a family of peptides with sequence homology to the vertebrate calcitonins has been implicated in the control of diuresis, a process that includes mixing of the hemolymph. Here, we show that a member of the insect calcitonin-like diuretic hormone (CLDH) family is present in the American lobster, Homarus americanus, serving, at least in part, as a powerful modulator of cardiac output. Specifically, during an ongoing EST project, a transcript encoding a putative H. americanus CLDH precursor was identified; a full-length cDNA was subsequently cloned. In silico analyses of the deduced prepro-hormone predicted the mature structure of the encoded CLDH to be GLDLGLGRGFSGSQAAKHLMGLAAANFAGGPamide (Homam-CLDH), which is identical to a known Tribolium castaneum peptide. RT-PCR tissue profiling suggests that Homam-CLDH is broadly distributed within the lobster nervous system, including the cardiac ganglion (CG), which controls the movement of the neurogenic heart. RT-PCR analysis conducted on pacemaker neuron- and motor neuron-specific cDNAs suggests that the motor neurons are the source of the CLDH message in the CG. Perfusion of Homam-CLDH through the isolated lobster heart produced dose-dependent increases in both contraction frequency and amplitude and a dose-dependent decrease in contraction duration, with threshold concentrations for all parameters in the range 10(-11) to 10(-10) mol l(-1) or less, among the lowest for any peptide on this system. This report is the first documentation of a decapod CLDH, the first demonstration of CLDH bioactivity outside the Insecta, and the first detection of an intrinsic neuropeptide transcript in the crustacean CG.

The peptide hormone pQDLDHVFLRFamide (crustacean myosuppressin) modulates the Homarus americanus cardiac neuromuscular system at multiple sites.

pQDLDHVFLRFamide is a highly conserved crustacean neuropeptide with a structure that places it within the myosuppressin subfamily of the FMRFamide-like peptides. Despite its apparent ubiquitous conservation in decapod crustaceans, the paracrine and/or endocrine roles played by pQDLDHVFLRFamide remain largely unknown. We have examined the actions of this peptide on the cardiac neuromuscular system of the American lobster Homarus americanus using four preparations: the intact animal, the heart in vitro, the isolated cardiac ganglion (CG), and a stimulated heart muscle preparation. In the intact animal, injection of myosuppressin caused a decrease in heartbeat frequency. Perfusion of the in vitro heart with pQDLDHVFLRFamide elicited a decrease in the frequency and an increase in the amplitude of heart contractions. In the isolated CG, myosuppressin induced a hyperpolarization of the resting membrane potential of cardiac motor neurons and a decrease in the cycle frequency of their bursting. In the stimulated heart muscle preparation, pQDLDHVFLRFamide increased the amplitude of the induced contractions, suggesting that myosuppressin modulates not only the CG, but also peripheral sites. For at least the in vitro heart and the isolated CG, the effects of myosuppressin were dose-dependent (10(-9) to 10(-6) mol l(-1) tested), with threshold concentrations (10(-8)-10(-7) mol l(-1)) consistent with the peptide serving as a circulating hormone. Although cycle frequency, a parameter directly determined by the CG, consistently decreased when pQDLDHVFLRFamide was applied to all preparation types, the magnitudes of this decrease differed, suggesting the possibility that, because myosuppressin modulates the CG and the periphery, it also alters peripheral feedback to the CG.

Anti-lipopolysaccharide factors in the American lobster Homarus americanus: molecular characterization and transcriptional response to Vibrio fluvialis challenge.

Two partial mRNA sequences predicted to encode anti-lipopolysaccharide factors (ALFs) were identified among expressed sequence tags generated from the American lobster Homarus americanus and complete cDNA sequences were obtained from library clones. Comparison of the translated amino acid sequences to those publicly available confirmed similarity to arthropod anti-lipopolysaccharide factors. Both protein sequences, designated ALFHa-1 and ALFHa-2, contained an N-terminal signal peptide and two half-cysteines participating in a disulfide bridge, features conserved in other ALFs. Predicted secondary structures were similar to that described for the ALF from the horseshoe crab Limulus polyphemus. As part of an exploratory study of immunity in H. americanus, lobsters were injected with the bacterium Vibrio fluvialis and gill, hematopoietic, and hepatopancreas tissues were sampled for analysis of gene expression of ALFHa-1 and ALFHa-2 by quantitative PCR. The relative abundance of ALFHa-2 mRNA was not significantly affected by Vibrio injection in any of the three tissues tested. In contrast, ALFHa-1 mRNA levels in gills were increased by the treatment some 17-fold. Our results support a molecularly specific regulation of antimicrobial proteins in response to bacterial infection in H. americanus.

Effects of racing and training on serum thyroid hormone concentrations in racing Greyhounds.

OBJECTIVES: To determine the effects of racing and training on serum thyroxine (T4), triiodothyronine (T3), and thyroid stimulating hormone (TSH) concentrations in Greyhounds. ANIMALS: 9 adult racing Greyhounds. PROCEDURE: Serum thyroid hormone concentrations were measured before and 5 minutes after a race in dogs trained to race 500 m twice weekly for 6 months. Resting concentrations were measured again when these dogs had been neutered and had not raced for 3 months. Postrace concentrations were adjusted relative to albumin concentration to allow for effects of hemoconcentration. Thyroid hormone concentrations were then compared with those of clinically normal dogs of non-Greyhound breeds. RESULTS: When adjusted for hemoconcentration, total T4 concentrations increased significantly after racing and TSH concentrations decreased; however, there was no evidence of a change in free T4 or total or free T3 concentrations. Resting total T4 concentrations increased significantly when dogs had been neutered and were not in training. There was no evidence that training and neutering affected resting TSH, total or free T3, or free T4 concentrations. Resting concentrations of T3, TSH, and autoantibodies against T4, T3, and thyroglobulin were similar to those found in other breeds; however, resting free and total T4 concentrations were lower than those found in other breeds. CONCLUSIONS AND CLINICAL RELEVANCE: Except for total T4, thyroid hormone concentrations in Greyhounds are affected little by sprint racing and training. Greyhounds with low resting total and free T4 concentrations may not be hypothyroid.

A retrospective study of canine and feline cutaneous vasculitis.

Twenty-one cases of cutaneous vasculitis in small animals (dogs and cats) were reviewed, and cases were divided by clinical signs into five groups. An attempt was made to correlate clinical types of vasculitis with histological inflammatory patterns, response to therapeutic drugs and prognosis. Greater than 50% of the cases were idiopathic, whereas five were induced by rabies vaccine, two were associated with hypersensitivity to beef, one was associated with lymphosarcoma and two were associated with the administration of oral drugs (ivermectin and itraconazole). Only the cases of rabies vaccine-induced vasculitis in dogs had a consistent histological inflammatory pattern (mononuclear/nonleukocytoclastic) and were responsive to combination therapy with prednisone and pentoxifylline, or to prednisone alone. Most cases with neutrophilic or neutrophilic/eosinophilic inflammatory patterns histologically did not respond to pentoxifylline, but responded to sulfone/sulfonamide drugs, prednisone, or a combination of the two.

Clinical efficacy of selamectin for the control of fleas on dogs and cats.

The topical endectocide selamectin (Revolution, Pfizer Animal Health) was evaluated in seven veterinary dermatology specialty clinics for its ability to control fleas on 75 dogs and 46 cats from single- and multiple-animal households. All animals were treated on days 0, 30, and 60 with a minimum unit dose of 6 mg/kg of selamectin(h) applied to the skin in a single spot at the base of the neck in front of the scapulae. The product was applied according to label instructions, and the use of other topical or environmental flea control products was prohibited during the study. Efficacy was assessed by percentage reductions in geometric mean flea comb counts. The reductions in flea numbers for dogs and cats combined were 90.6%, 97.0%, and 98.0% on days 30, 60, and 90, respectively, compared with day 0. This study demonstrates that selamectin, applied at 30-day intervals to dogs and cats, effectively controls flea infestations without other flea control products in single- and multiple-animal households.

Cutaneous vasculitis and vasculopathy.

Although uncommon to rare in occurrence, cutaneous vasculitides and vasculopathies are becoming better recognized entities in veterinary medicine. The heterogeneity in their clinical presentations can be confusing, and the diagnostic evaluation of suspected cases can be clinically and financially challenging. The most important step is confirming a vasculitic/vasculopathic process histologically and then searching for an underlying cause. As many cases are diagnosed as idiopathic by exclusion, a familiarity with treatment options is important and may be guided in some cases by the histological inflammatory pattern. Because of the frequent temporal association with drug administration, any case of vasculitis requires a thorough drug history and physical examination for systemic complications. Familiarity with breed-associated vasculopathies is also key in properly diagnosing and managing these problems.

Nodules and draining tracts.

Many potential causes exist of nodules and draining tracts in cats. Treatment should not be undertaken until a definitive diagnosis is made. This may involve biopsies for histopathologic evaluation, sample collection and submittal for culture and sensitivity, cytologic evaluation of impression smears or needle aspirates, and serologic testing.

Serum thyroid hormone concentrations and thyrotropin responsiveness in dogs with generalized dermatologic disease.

Fifty-eight dogs with generalized dermatologic disease that had not been given glucocorticoids systemically or topically within 6 weeks of entering the study were evaluated for thyroid function by use of the thyrotropin-response test. Dogs were classified as euthyroid or hypothyroid on the basis of test results and response to thyroid hormone replacement therapy. Baseline serum thyroxine (T4), free T4 (fT4), and triiodothyronine (T3) concentrations were evaluated in the 58 dogs. Serum T4, fT4, and T3 concentrations were evaluated in 200 healthy dogs to establish normal values. Hormone concentrations were considered low if they were less than the mean -2 SD of the values for control dogs. Specificity of T4 and fT4 concentrations was 100% in predicting hypothyroidism; none of the euthyroid dogs with generalized skin disease had baseline serum T4 or fT4 concentration in the low range. Sensitivity was better for fT4 (89%) than for T4 (44%) concentration. Significant difference was not observed in serum T4 and fT4 concentrations between euthyroid dogs with generalized skin disease and healthy control dogs without skin disease. Serum T3 concentration was not accurate in predicting thyroid function; most of the euthyroid and hypothyroid dogs with skin disease had serum T3 concentration within the normal range.

Systemic toxicosis associated with azathioprine administration in domestic cats.

Five cats were treated with an azathioprine suspension (2.2 mg/kg of body weight on alternate days) and 2 cats were given vehicle (controls) for 9 weeks. Complete blood and platelet counts and serum biochemistry variables were monitored weekly. Bone marrow aspirates were evaluated every 3 weeks, and core bone marrow biopsy was performed at the end of the study. Profound neutropenia (less than 600 cells/microliters) was observed in all treated cats, and 1 cat developed pancytopenia. Treatment was discontinued if the WBC count was less than 3,000 cells/microliters. Four weeks after discontinuation of azathioprine, 1 treated cat again was given azathioprine at a lower dosage (1.1 mg of azathioprine/kg on alternate days) and neutropenia recurred within 2 weeks. During treatment, 3 cats developed thrombocytosis, and 2 developed thrombocytopenia. In 4 of 5 cats, neutropenia and thrombocytopenia resolved when azathioprine was discontinued. Bone marrow cytologic examination during treatment revealed reduction of the neutrophil line, with relative increase in monocytes. Core bone marrow biopsy at the completion of the study revealed hypocellular marrow with marked decrease in the myeloid series in cats given azathioprine. One of the cats that was treated with azathioprine had a hypercellular marrow with increased numbers of mature granulocytes and precursors; however, azathioprine had been discontinued 3 weeks prior to biopsy. Alterations in serum biochemical variables were not associated with azathioprine. Two cats that were treated with azathioprine developed respiratory tract infections, and 1 of them was euthanatized during the study.

Comparison of serum cortisol concentration before and after intradermal testing in sedated and nonsedated dogs.

Serum cortisol concentration was evaluated in 71 dogs before and after a stressful procedure was performed. Thirty dogs were skin tested with sedation (group S), 21 dogs were skin tested without sedation (group NS), and 20 dogs had other dermatologic procedures performed (group C). Group-S dogs had significant (P less than 0.001) decrease in serum cortisol concentration after skin testing, compared with baseline values. In contrast, dogs of groups NS and C had significant (P less than 0.001) increase in poststress serum cortisol concentration. Mean cortisol concentration after stress was significantly lower for dogs of group S, compared with that for dogs of the other 2 groups. The second part of the analysis consisted of determining the number of false-negative skin test results for dogs of groups S and NS and comparing these with serum cortisol concentration. Difference in the number of suspected atopic dogs with negative skin test results (false-negative) was not evident between groups S and NS. Also, difference was not apparent between cortisol concentration in dogs that had positive or false-negative skin test results in either group. This finding indicates that high serum cortisol concentration does not affect results of skin testing in suspected atopic dogs.

Current diagnostic techniques for evaluating thyroid function in the dog.

Hypothyroidism is a common endocrinopathy in dogs. There are a wide variety of diagnostic tests to evaluate thyroid function. The measurement of basal thyroid hormone concentrations is used widely as an indicator of thyroid function; however, there are many factors that may affect basal hormone concentrations. The thyrotropic stimulation test is not affected by many of these factors, and often it is preferred for this reason. Current diagnostic procedures for evaluating thyroid function for the veterinary practitioner are reviewed.

Effects of sedation on intradermal skin testing in flea-allergic dogs.

Effects of 4 commonly used sedatives on the wheal-and-flare response to histamine and flea antigen were evaluated in 8 flea-allergic Beagles. Skin testing was performed in 12 separate occasions, 3 to 4 days apart. Twelve intradermal injections were given during each skin test: 5 doubling dilutions of histamine phosphate, 6 doubling dilutions of flea antigen, and a phosphate-buffered saline solution (negative control). Of the 12 intradermal skin tests, 8 were control tests performed on nonsedated dogs. The remaining 4 tests were performed on dogs sedated with xylazine, ketamine and valium combination, acepromazine, or oxymorphone. Oxymorphone had the most profound effect on skin test results, significantly (P less than 0.05) decreasing skin responsiveness in 8 of 11 test sites (by objective evaluation) and in 5 of 11 test sites (by subjective evaluation). Xylazine sedation enhanced skin test results in 4 of 11 test sites (by objective evaluation) and in 1 of 11 test sites (by subjective evaluation). In non instance did xylazine significantly decrease skin responsiveness to histamine or flea antigen. Xylazine is the recommended sedative in dogs when sedation is necessary for intradermal skin testing.

Comparison of two doses of aqueous bovine thyrotropin for thyroid function testing in dogs.

Thyroid function was evaluated in 20 healthy dogs by thyrotropin (TSH) response testing. Two dose regimens were used: 5 IU of TSH given IV and 1 IU of TSH given IV. Blood samples were collected prior to and at 4 and 6 hours after TSH administration. Serum was obtained and analyzed for total 3,5,3'-tri-iodothyronine and thyroxine (T4) concentrations by radioimmunoassay. All dogs were classified as euthyroid on the basis of response to 5 IU of TSH at 4 and 6 hours. The 1-IU dose of TSH failed to induce adequate increase in T4 concentration in 7 dogs at 4 and 6 hours when the criteria for normal response were post-TSH serum concentration T4 greater than or equal to 3.0 micrograms/dl and serum T4 increase by greater than or equal to 100% over baseline serum T4 concentration. One IU of TSH induced increase in serum T4 concentration over baseline; however, the increase was significantly (P less than 0.05) less than that in response to a 5-IU dose at 6 hours after administration of TSH.

Prevalence of antithyroglobulin antibodies detected by enzyme-linked immunosorbent assay of canine serum.

Antithyroglobulin antibody (ATA) values were higher in dogs with low total serum thyroxine (T4) and triiodothyronine (T3) values than in dogs with T3 and T4 values within reference value limits. In the population studied, Doberman Pinschers were predisposed to the development of ATA; there was no sex predilection for development of ATA. Antithyroglobulin antibodies and thyroid status were evaluated in 2 groups of healthy dogs (n = 30) and in 470 canine serum samples submitted for T3 and T4 value determination. Antithyroglobulin antibodies were evaluated by ELISA, and thyroid status was evaluated by measurement of total serum T3 and T4 by radioimmunoassay.