Gabaergic inhibition of the pituitary release of adrenocorticotropin and α-melanotropin is impaired in dogs with hepatic encephalopathy

γ-Aminobutyric acid (GABA) is the principal depressant neurotransmitter system, but its possible role in the regulation of the hypothalamic-pituitary-adrenocortical (HPA) axis has not yet been investigated in the dog. Moreover, GABA is one of the factors underlying the syndrome of hepatic encephalopathy (HE), and in dogs with HE, the regulation of the HPA axis is deranged. We have therefore investigated the role of the GABA system in the regulation of the HPC system in 10 healthy dogs and 10 dogs with HE due to congenital portosystemic shunts. The effect of an intravenous injection of the GABA antagonist bicuculline on the release of adrenocorticotropin (ACTH), α-melanotropin (MSH), and cortisol was measured in plasma. In healthy dogs, a dose of 1.0 mg/kg caused a marked release of ACTH, MSH, and cortisol, but doses of 0.001 to 0.5 mg/kg produced an inconsistent or no response. The high release of MSH after bicuculline administration indicated that the effect of GABA was predominantly in the neurointermediate lobe of the pituitary. In order to investigate whether the effect of GABA was exerted in the pituitary or at a suprapituitary level, the effect of incubation with GABA on basal and corticotropin-releasing hormone-induced ACTH release was measured in primary cultures of anterior and neurointermediate lobe cells from healthy dogs, and no response was observed. We conclude that in healthy dogs, GABA inhibits the release of ACTH and MSH from the neurointermediate lobe of the pituitary at a suprapituitary level. In dogs with HE, 1.0 mg/kg of bicuculline caused virtually no stimulation of the secretion of ACTH, MSH, or cortisol, indicating deranged GABAergic neurotransmission in HE. This may be explained by an increased GABA tone that prevents the effect of the antagonist. Such a high GABA tone associated with HE has been documented in several other species. Dogs with HE had significantly increased basal levels of ACTH, MSH, and cortisol in plasma, and their cortisol:creatinine ratios in 24-hr urine samples (63 ± 14 · 10⁻⁶ were higher than those of healthy dogs (9 ± 2 · 10⁻⁶). An increased basal HPA activity in dogs with HE is not in agreement with augmented GABAergic inhibition, but this contradiction may be explained by the predominance of effects of dopaminergic disinhibition that has been reported in such dogs.     

Effects of trilostane on the pituitary-adrenocortical and renin–aldosterone axis in dogs with pituitary-dependent hypercortisolism

The medical records of 63 dogs with pituitary-dependent hypercortisolism (PDH) before and during treatment with trilostane were reviewed retrospectively. The correct trilostane dosage in dogs with PDH was based on the resolution of clinical signs and the results of an adrenocorticotropic hormone (ACTH) stimulation test. The mean (±SD) dose rate of trilostane to achieve good clinical control was 2.8 ± 1.0 mg/kg bodyweight. Trilostane treatment resulted in a significant decline in basal plasma cortisol concentrations. The median plasma ACTH concentration (39 pmol/L, range 7–132 pmol/L; n = 60) at the optimal trilostane dosage time was significantly higher (P < 0.001) than before treatment (13 pmol/L, range 2–102 pmol/L). These values did not overlap with plasma ACTH concentrations (range 212–307 pmol/L) of five PDH dogs with trilostane-induced hypocortisolism.The median cortisol/ACTH ratio in well-controlled dogs (0.23, range 0.03–2.5; n = 46) was significantly lower (P < 0.001) than before treatment (2.59, range 0.27–13.25). Trilostane treatment resulted in an insignificant decrease in plasma aldosterone concentration (PAC), but the median plasma renin activity (PRA) at the time the trilostane dosage was considered optimal (265 fmol/L/s, range 70–3280 fmol/L/s; n = 18) was significantly higher (P < 0.001) than prior to treatment (115 fmol/L/s, range 15–1330 fmol/L/s). Similarly, the median PAC/PRA ratio during trilostane treatment (0.16, range 0.003–0.92; n = 17) was significantly lower (P < 0.001) than before treatment (median 0.44, range 0.04–1.33). Trilostane affected both the hypothalamic-pituitary-adrenocortical and the renin–aldosterone axes. The results also suggested that basal plasma ACTH concentration may be used to detect trilostane overdosage.     

Survey of bacterial microorganisms in the conjunctival sac of clinically normal dogs and dogs with ulcerative keratitis in Fortaleza, Ceará, Brazil

OBJECTIVE: The ocular microflora in dogs has not been established in north-east Brazil. Thus, the main aim of this research was to determine the bacterial microorganisms in the conjunctival sac of clinically normal dogs and dogs with ulcerative keratitis in Fortaleza, Ceará, Brazil. ANIMALS STUDIED: This study included 60 healthy dogs, 15 dogs with unilateral corneal ulcer, and three dogs with bilateral corneal ulcers. Procedure Samples were taken by a calibrated platinum loop (1 microL) placed directly onto the conjunctival sac and on sterile blood agar. The clinical specimens were incubated at 37 degrees C in an atmosphere of 5% CO2 for 48 h. RESULTS: Of the 120 samples from healthy dogs, only 47 (39%) had positive culture for bacteria, while all of the specimens from eyes with corneal ulcer were positive for bacterial growth. The group of dogs with corneal ulcer had a higher (P < 0.05) number of colony-forming units (CFU) per plate than the group of healthy animals. Of the 59 isolates from healthy eyes, only nine (15.3%) had more than 50 CFU per plate, while in the group of dogs with corneal ulcer, 23 (62.2%) of the 37 isolates presented more than 50 CFU per plate. In both groups Gram-positive bacteria (86.5%) predominated over Gram-negative (13.5%). Staphylococcus spp. was the most frequently isolated genus and S. intermedius predominated in both groups. CONCLUSION: The results of our study are directly applicable to initiate rational, preventive and therapeutic measures with greater accuracy in dogs with corneal ulcer.     

A Comparison of the Concentrations of C-reactive Protein and α1-Acid Glycoprotein in the Serum of Young and Adult Dogs with Acute Inflammation

The concentrations of C-reactive protein (CRP) and ₁-acid glycoprotein (AAG) were evaluated in 1-, 3- and 18-month-old dogs (four of each age) that had been inoculated with turpentine oil. The CRP and AAG in 3-month-old and younger dogs subjected to surgery or inoculated with either Staphylococcus aureus or a viral vaccine were also evaluated. The average CRP concentration in the sera peaked 2 days after inoculation of turpentine oil. The peak CRP concentrations in 3- and 18-month-old dogs were significantly (p<0.05) greater than those in 1-month-old dogs. The average AAG concentration in the sera peaked 4 days after inoculation of turpentine oil. No significant difference was found in AAG concentrations between any of the age groups. When experimentally inoculated with S. aureus or subjected to oophorohysterectomy, the CRP and AAG concentrations increased in 3-month-old dogs, but they increased little in 1-month-old dogs. The CRP and AAG in dogs inoculated with the viral vaccine did not increase. In dogs with fractures or subjected to percutaneous gastrostomy, the CRP and AAG concentrations correlated with the condition of dogs.     

Effect of β-mannanase on the digestibility of diets with different protein sources in dogs determined by different methodologies

This experiment aimed at evaluating the effects of including the enzyme, β-mannanase, in dog (Canis lupus familiaris) diets based on either poultry (Gallus gallus domesticus) by-product meal (PBM) or soybean [Glycine max (L.) Merr.] Meal (SBM). The second objective was to evaluate 3 methods for determining energy and nutrient digestibility values in diets fed to dogs: total fecal collection (TFC) and use of aia or crude fiber (CF) as a marker. Eight dogs were allotted to a replicated latin square (4 by 4) design. There were 2 diets based on PBM as the major protein source and 2 diets based on SBM as the major protein source. Within each protein source, 1 diet contained no β-mannanase and 1 diet contained 0.01% β-mannanase. Diets were fed for an adaptation period of 5 d followed by 5 d of TFC. Fecal score (1 = watery feces to 5 = dry, hard pellets), pH, DM, and fecal volume were determined. The apparent total tract digestibility (ATTD) of DM, OM, CP, ether extract (EE), N-free extract (NFE), and GE, and ME content were calculated using the methods of TFC, AIA, and CF. Data were analyzed as a 2 by 2 by 3 split-split-plot design (β-mannanase, protein source, and digestibility calculation procedure). There were interactions between protein source and β-mannanase (P < 0.05). Supplementation of β-mannanase increased ATTD of nutrients and energy and ME (+ 195.3 kcal/kg) and also reduced fecal production in the diet with SBM, but not in the diet that contained PBM. There was an interaction between digestibility calculation procedure and protein source (P < 0.05). The use of AIA overestimated ATTD of the diets containing PBM, but digestibility values estimated based on TFC and CF were not different. Dogs fed diets containing SBM produced more feces with greater moisture content and lower pH compared with dogs fed the PBM diet (P < 0.05). Addition of 0.01% β-mannanase increased (P < 0.05) the digestibility and ME content of the diets containing SBM, but did not improve (P > 0.05) fecal texture. Results indicated that values for ATTD of energy and nutrients in diets containing sbm are not different if they are calculated based on TFC, AIA, or CF, but use of AIA may result in an overestimation of values for ATTD of energy and nutrients in diets containing PBM.     

A review of the off-label use of selamectin (Stronghold?/Revolution?) in dogs and cats

Since its introduction approximately seven years ago, selamectin (Stronghold^®/Revolution^®, Pfizer Inc.) has been used off-label to treat a number of ecto- and endoparasite conditions in dogs and cats. It has been used as a successful prophylactic against Dirofilaria repens and as a treatment for Aelurostrongylus abstrusus in cats. It has also been used to treat notoedric mange, infestation with the nasal mite Pneumonyssoides caninum, Cheyletiella spp. and Neotrombicula autumnalis infestations and larval Cordylobia anthropophaga infection. However, to date attempts to treat generalised canine demodicosis have not been successful. In all cases, treatment was apparently well tolerated by the host.     

Trilostane-induced inhibition of cortisol secretion results in reduced negative feedback at the hypothalamic–pituitary axis

Cushing's disease caused by pituitary corticotroph adenoma in dogs is usually treated by medical treatment, and the efficacy of this treatment has been reported. However, controversy remains as to whether reduced negative feedback through the inhibition of cortisol secretion, similar to Nelson's syndrome, may appear as an adverse effect. The purpose of this study was to investigate the effect of reduced negative feedback through the inhibition of cortisol secretion by daily trilostane administration on the pituitary–adrenal axis in clinically normal dogs. Dogs were administered 5 mg/kg trilostane twice a day every day for 8 weeks (n = 8) or 16 weeks (n = 3). After the initiation of trilostane administration, plasma adrenocorticotropic hormone (ACTH) concentrations were increased remarkably. As assessed by magnetic resonance imaging (MRI) during administration, the pituitary became enlarged. After trilostane administration, the cytoplasmic areas of the pituitary corticotrophs were increased and the ratio of pituitary corticotrophs to all cells in the anterior lobe was greater in the trilostane-treated dogs than that in untreated animals. In addition, histological examinations revealed bilateral adrenal cortical hyperplasia. Using real-time PCR quantification, the expression of proopiomelanocortin (POMC) mRNA in the pituitary and ACTH receptor (ACTH-R) mRNA in the adrenal gland was greater in the dogs treated with trilostane than in untreated dogs. These results indicate that reduced negative feedback induced hyperfunction of the pituitary corticotrophs and pituitary enlargement in healthy dogs. These changes suggest that the inhibition of cortisol secretion by trilostane may increase the risk for accelerating the growth of corticotroph adenomas in dogs with Cushing's disease.     

Investigation of serum concentrations and immunohistochemical localization of α1-acid glycoprotein in tumor dogs

The purpose of this study was to measure the dynamics of serum α1-acid glycoprotein (AGP) concentration in dogs with various tumors, and to investigate the localization of AGP in some tissues using immunohistochemical staining. Sera were obtained from 171 dogs bearing tumors of various types. Serum AGP concentration was measured by single radial immunodiffusion. Tumors occurring in the liver and spleen were also investigated immunohistochemically using anti-canine AGP antibody. Mean serum AGP levels were 749 ± 602 mg/L in dogs with carcinoma (n = 39), 1,014 ± 971 mg/L with sarcoma (n = 18), and 887 ± 935 mg/L with round cell tumors (n = 46), all significantly higher than serum AGP level in healthy dogs (n = 137, 364 ± 106 mg/L). Mean serum AGP levels were significantly higher than in healthy dogs in complex mammary gland carcinoma (n = 5, 876 ± 721 mg/L), malignant melanoma (n = 7, 1,010 ± 821 mg/L), and hepatocellular carcinoma (n = 5, 936 ± 741 mg/L) among carcinomas, hemangiosarcoma (n = 5, 1,740 ± 1,323 mg/L) among sarcomas, and lymphoma (n = 19, 1,072 ± 965 mg/L) and histiocytic tumor (n = 6, 1,800 ± 1,387 mg/L) among round cell tumors. In an immunohistochemical investigation of AGP localization, both weak and strong staining for anti-AGP antibody were seen in hepatic tissue in dogs with primary non-tumorous lesions originating in the spleen (hematoma) and elevated serum AGP, but all tumor tissue in the spleen was negative. Among dogs with primary tumor lesions of the spleen (hemangiosarcoma) and elevated serum AGP levels, both weak, moderate and strong staining for anti-AGP antibody were seen in hepatic tissue, while strong positive staining was apparent in all tumorous tissue from the spleen. In primary tumor lesions in the liver (hepatocellular carcinoma), both moderate and strong staining for anti-AGP antibody were seen in normal hepatic tissue, and both weak, moderate and strong staining were seen in tumor tissues of the liver. AGP levels thus appear to be elevated in dogs with carcinomas, sarcomas, and round cell tumors. With some of these malignant tumors, localization of AGP in tumor tissue was seen.     

Topical KINOSTAT™ ameliorates the clinical development and progression of cataracts in dogs with diabetes mellitus

Objective To determine whether topical administration of the aldose reductase inhibitor Kinostat™ can ameliorate the onset or progression of cataracts in dogs with naturally occurring diabetes mellitus (DM). Materials and Methods A randomized, prospective, double‐masked placebo control pilot study was conducted with 40 dogs newly diagnosed with DM with no or minimal lens changes. Twenty‐eight dogs received Kinostat™ and 12 dogs received placebo. Procedures Owners administered the agent into both eyes three times daily for 1 year and compliance was monitored with log sheets. Complete ophthalmic examinations were performed on dilated eyes at the time of enrollment and 1, 2, 3, 6, and 12 months into treatment. Cataract severity was assessed on a scale of 0–3. At 12 months, full bloodwork, including HbA1C and blood Kinostat^TM levels were performed. Results After 12 months of treatment, the cataract score in the placebo group significantly increased with seven dogs (14 eyes) developing mature cataracts, two dogs (4 eyes) developing cortical opacities, and one dog (2 eyes) developing equatorial vacuoles with mild punctate cortical opacities. In contrast, the cataract score in the Kinostat^TM treated dogs was significantly less with seven developing anterior equatorial vacuoles, two developing incipient anterior cortical cataracts, and four developing mature cataracts. In fact, the cataract scores of the Kinostat^TM group at 12 months did not significantly increase from the score at the time of enrollment. The HbA1C values between the two groups after 12 months of treatment were similar, and no blood levels of Kinostat^TM were found in any enrolled dog. Conclusion The onset and/or progression of cataracts in dogs with DM can be significantly delayed by topical administration of Kinostat™.     

A comparative study of the seroprevalence of brucellosis in commercial and small-scale mixed dairy–beef cattle enterprises of Lusaka province and Chibombo district,Zambia

A cross-sectional study was conducted between January 2007 and February 2008 to estimate seroprevalence of brucellosis and identify risk factors associated with Brucella infections in commercial cattle in three districts of Lusaka province (Chongwe, Luangwa, and Kafue; n = 849) and in one rural district from the Central province (n = 48). A total of 897 serum samples were randomly collected from 55 farms along with animal-level data such as sex, age, and parity. Sera were screened for presence of anti-Brucella antibodies using the Rose Bengal test, and positive samples were confirmed using competitive enzyme-linked immunosorbent assay. At the animal level, seroprevalence was estimated at 7.9% (95% CI = 4.4–11.4%) in the Lusaka province and 18.7% (95% CI = 7.5–29.9%) for Chibombo district. Brucellosis seroprevalence varied according to district, with Chongwe district recording the highest compared to other districts. Seroprevalence also varied according to sex with bulls (n = 96) having higher seroprevalence (12.5%; 95% CI = 3.8–21.1%) compared to females (8.1%; 95% CI = 4.6–11.6). Similarly, seroprevalence varied according to age groups, with the age category 1–4 years recording the highest (10.7%). The study recorded relatively low Brucella seroprevalence in commercial farms in Lusaka, compared to the traditional small-scale farms. We suggest that testing and stamping out of infected animals is likely to improve the situation and significantly reduce the public health risk associated with Brucella infections in animals.     

Cryptic Leishmaniosis by Leishmania infantum, a feature of canines only? A study of natural infection in wild rabbits, humans and dogs in southeastern Spain

An epidemiological study was carried out to investigate asymptomatic Leishmania infantum infection by PCR and ELISA in wild rabbits, humans and domestic dogs in southeastern Spain. Seroprevalence was 0% (0/36) in rabbits, 2% (13/657) in humans and 7% (14/208) in dogs. The prevalence of PCR-positives was 0.6% (1/162) in rabbits tested in a wide range of tissue samples, 2% (8/392) in humans analysed in blood samples and 10% (20/193) and 67% (29/43) in dogs analysed in blood and lymphoid tissue samples, respectively. Results suggest that wild rabbits have a very low risk of becoming chronically infected with L. infantum, and provide further evidence that cryptic L. infantum infection is widespread in the domestic dog population and is also present in a comparatively smaller proportion of healthy humans. The epidemiological and clinical implications of these findings are discussed.     

The immunohistochemical evaluation of selected markers in the left atrium of dogs with end-stage dilated cardiomyopathy and myxomatous mitral valve disease – a preliminary study

Background

Dilated cardiomyopathy (DCM) and myxomatous mitral valve disease (MMVD) are the most common diseases noted in dogs. Although their pathogenesis varies, both include a significant enlargement of the left atrium.The study was carried out on left atrial specimens obtained from 56 dogs, including those from 34 dogs with clinically diagnosed MMVD, 15 dogs with DCM and 7 dogs without heart disease (control group). Dogs in the MMVD and the DCM groups presented with left atrial enlargement and stage D heart failure. The specimens underwent immunohistochemical examination using desmin, vimentin, periostin and caspase-3 antibodies.

Results

There were alterations in the expression of the studied proteins in the study groups compared to the control group. The changes included: irregularity of desmin cross-striation and desmosomes, a higher amount of vimentin-positive cells, a change in the periostin expression pattern from cytoplasmic to extracellular, and a lower expression of caspase-3. The alterations were more pronounced in the DCM group than in the MMVD group.

Conclusions

During heart failure, the pattern of desmin, vimentin, periostin and caspase-3 expression alters in the left atrium, regardless of the cause. The changes are more pronounced in dogs with DCM than in dogs with MMVD and similar left atrial enlargement, suggesting that volume overload may not be the only cause of myocardial changes in DCM.

     

Circadian and circannual rhythms of cortisol,ACTH, and α-melanocyte-stimulating hormone in healthy horses

Cosinor analysis was used to evaluate whether pituitary and adrenal hormones exhibit circadian rhythmicity in horses. The effect of season and animal age on their respective rhythms was also determined. In addition, the usefulness of evaluating cortisol rhythmicity for the diagnosis of pituitary pars intermedia dysfunction (PPID) was assessed. Serum cortisol concentrations (P < 0.01), but not plasma ACTH or α-melanocyte-stimulating hormone (α-MSH), showed a significant circadian periodicity in horses. An effect of season on hormone concentration was observed with plasma ACTH and α-MSH concentration greater in the fall and cortisol concentration greater in the spring (P < 0.001). Age did not affect cortisol rhythm, but it did blunt the variation in cortisol concentration in horses, similar to what has been previously reported to occur in aged people and dogs. In addition, our results suggest that clinically and diagnostically normal, non–PPID-affected horses commonly have a loss of cortisol diurnal rhythm. Therefore, measurement of circadian rhythm is not an appropriate diagnostic test for PPID.     

Effect of age of donor on the responsiveness of dispersed and cultured chicken anterior pituitary cells to GnRH‐I

1. The aim of this study was to devise a method to prepare and culture anterior pituitary cells from juvenile and adult chickens in order to investigate mechanisms controlling gonadotrophin‐releasing hormone‐I (GnRH‐I) ‐induced luteinising hormone (LH) release in vitro.

2. The optimum culture medium for maintaining gonadotroph responsiveness to GnRH‐I was bicarbonate‐buffered and phenol red‐free Medium 199 supplemented with 10% foetal calf serum.

3. Cultured pituitary cells from juvenile chickens were more responsive to GnRH‐I than cells from adult cockerels, while no LH was released in response to GnRH‐I from pituitary cells from laying hens.

4. Cultured pituitary cells from adult chickens of both sexes released LH in response to 12‐O‐tetradecanoyl‐13‐phorbol acetate (TPA), an activator of an enzyme involved in intracellular signalling, protein kinase C.

5. It is concluded that freshly‐dispersed and cultured gonadotrophs from adult chickens do not regain their responsiveness to GnRH‐I as well as freshly‐dispersed and cultured gonadotrophs from juvenile chickens. It appears that the stimulus‐secretion coupling pathway between the GnRH‐receptor and the activation of protein kinase C in gonadotrophs from adult chickens is more easily disrupted by dispersion and culture than in gonadotrophs from juvenile chickens.     

Toxocara eggs shed by dogs and cats and their molecular and morphometric species-specific identification: is the finding of T. cati eggs shed by dogs of epidemiological relevance?

Intestinal infections with Toxocara cati and Toxocara canis in their definitive host (felids and canids, respectively) are diagnosed by egg identification in faeces using coproscopical techniques. The Toxocara species is assumed to comply with the species from which the examined faeces were obtained, i.e. T. cati in cats and T. canis in dogs. We isolated and measured Toxocara eggs from faecal samples of 36 cats and 35 dogs from Switzerland and identified the Toxocara species by PCR. Amongst the isolates originating from dogs, 24 (68.5%) were determined as T. canis and 11 (31.5%) as T. cati. In all samples originating from cats, only T. cati was identified. Based on PCR identification, eggs of T. canis (n=241) and T. cati (n=442) were measured, revealing statistically significant different (p<0.001) mean sizes of 62.3 by 72.7 μm for T. cati and 74.8 by 86.0 μm for T. canis eggs. Considering that coprophagy is not unusual for dogs, a considerable percentage of Toxocara infections coproscopically diagnosed in dogs, as well as assumptions on anthelminthic resistance in regularly treated dogs, might in fact relate to intestinal passages of eggs following the uptake of other animals' faeces.     

A pilot study of 4CYTE™ Epiitalis® Forte,a novel nutraceutical,in the management of naturally occurring osteoarthritis in dogs

The primary goal of this pilot study was to assess, the efficacy of a new nutraceutical, 4CYTE™ Epiitalis® Forte, containing, as a standalone, a proprietary plant oil extract, Epiitalis, in dogs presenting with signs of osteoarthritis (OA). Fifty dogs aged 9.2 (±3.2) years with signs of naturally occurring OA were included in this report. They were free of other comorbidities and were not on any medications except for those utilised for managing their OA. In these dogs, the current treatments were continued to avoid any sudden changes in their disease management. The effects of the 4CYTE Epiitalis Forte were assessed both at the beginning and at the end of a 1 month-long treatment period. The evaluation consisted of an objective lameness assessment (TPI%[total pressure index]) using a gait analysis (GAITRite® Portable Walkway System) and a subjective quality-of-life questionnaire, the Helsinki Chronic Pain Index (HCPI). Additional exploratory objective measurements included the Symmetry Index (SI) and the fore/hind limb ratio (T/P TPI%). Of dogs, 74% (34/46) registered a numerical improvement in TPI% in their worse limb. In addition, of the 93.5% of the dogs that demonstrated improvement in their HCPI scores by at least 5% on the quality-of-life questionnaire, 79% demonstrated improvements in gait based on TPI%. Finally, there were improvements measured in both exploratory objective endpoints SI and T/P TPI%. These encouraging results will be used to develop a protocol for a follow-up placebo-controlled randomised study to confirm the efficacy of this new nutraceutical for dogs suffering from OA.