Exhaled breath condensate hydrogen peroxide, pH and leukotriene B4 are associated with lower airway inflammation and airway cytology in the horse.

BACKGROUND: Exhaled breath condensate (EBC) analysis is a noninvasive method to assess the lower respiratory tract. In human subjects, EBC hydrogen peroxide (H2 O2 ), pH and leukotriene B4 (LTB4 ) are useful for detection and monitoring of inflammatory lung diseases, including asthma. OBJECTIVES: To determine associations between EBC biomarkers and cytological and endoscopic definitions of lower airway inflammation (LAI) while controlling for sampling and environmental variables. STUDY DESIGN: Prospective, cross-sectional study. METHODS: Clinical, endoscopic and airway cytological findings from 47 horses were compared with EBC pH and concentrations of H2 O2 and LTB4 by univariate and multivariable analyses. Dichotomous (presence/absence of airway inflammation) and continuous outcome variables (differential cell counts in tracheal aspirate and bronchoalveolar lavage fluid, BALF) were evaluated and potential effects of collection and methodological factors were included. RESULTS: EBC pH and H2 O2 concentrations were higher in horses with LAI and both were positively associated with the percentage of neutrophils in BALF (P<0.05). Mast cell percentage in BALF was negatively associated with EBC pH, and BALF eosinophil percentage was positively associated with EBC LTB4 (P<0.05). Ambient temperature, relative humidity and assay methodology significantly impacted some analytes. MAIN LIMITATIONS: LAI is challenging to categorise due to a variety of clinical and cytological phenotypes. Although the study was designed to overcome this limitation, numbers of horses were small in some categories. CONCLUSIONS: EBC pH and H2 O2 concentrations are altered by airway inflammation, suggesting a role for these biomarkers in the diagnosis and monitoring of airway disease. Environmental and methodological factors can influence these biomarkers and should be considered in the interpretation of results.

Equine pituitary pars intermedia dysfunction: current understanding and recommendations from the Australian and New Zealand Equine Endocrine Group.

The purpose of this article is to provide a review of the current knowledge and opinions about the epidemiology, clinical findings (including sequelae), diagnosis, treatment and monitoring of equine pituitary pars intermedia dysfunction, particularly in the Australian context. This information and the recommendations provided will assist practitioners in making informed decisions regarding the diagnosis and management of this disorder.

The effects of sample handling and N-phenylmaleimide on concentration of adrenocorticotrophic hormone in equine plasma.

REASONS FOR PERFORMING STUDY: Previous reports suggest that adrenocorticotrophic hormone (ACTH) degrades rapidly, limiting its use as a diagnostic test. OBJECTIVES: This study quantified effects of processing delays on ACTH concentrations and investigated the addition of N-phenylmaleimide (maleimide), a protease inhibitor, as a means of reducing ACTH degradation. STUDY DESIGN: Experimental study. METHODS: Venous blood was collected from 8 healthy horses and 8 horses with pituitary pars intermedia dysfunction (PPID) with a range of ACTH concentrations. Baseline ACTH concentrations were established immediately using a chemiluminescent assay. Plasma samples were then: 1) centrifuged immediately, 2) centrifuged immediately with the addition of maleimide, or 3) allowed to separate by gravity followed by the addition of maleimide, before all samples were stored at 22°C and analysed at 4, 8, 24 and 48 h post collection. A linear mixed effects model and Bland-Altman analyses were performed. Significance was set at P < 0.05. RESULTS: No significant effect of plasma treatment (P = 0.1) on change in ACTH concentration was identified. However, significant effects of horse health status (P < 0.001) and time (P < 0.001) on change in ACTH concentration were identified. No significant interactions were found. Significant decreases in ACTH concentration occurred in horses with PPID between 4 and 8 h after blood collection. In non-PPID horses, the decrease in ACTH concentration over time was not significant. Agreement with baseline values decreased over time and was greater for non-PPID horses than for PPID horses. CONCLUSIONS: Clinically useful results are still obtained if ACTH concentration is measured up to 48 h after sample collection. Allowing samples to separate by gravity rather than centrifugation did not have a significant effect on ACTH concentration, and the addition of maleimide was of no benefit.

Investigation of rhythms of secretion and repeatability of plasma adrenocorticotropic hormone concentrations in healthy horses and horses with pituitary pars intermedia dysfunction.

REASONS FOR PERFORMING STUDY: There is little published information on whether measurement of plasma ACTH concentration at a single timepoint is a repeatable indicator of pituitary pars intermedia dysfunction (PPID). OBJECTIVES: To determine whether ultradian or circadian fluctuations in ACTH production influence plasma ACTH concentration in normal horses and horses with PPID. STUDY DESIGN: Prospective observational study. METHODS: Plasma ACTH concentration in 8 non-PPID horses and 8 horses with PPID was measured at 08.00, 11.00, 14.00 and 17.00 h on 5 nonconsecutive days within a 3 week period. In addition, at 08.30 h on one day, 6 samples were collected from each horse at precisely 5 min intervals over a period of 25 min. Descriptive and graphical analysis was performed and a linear mixed effects model was fitted to assess the effect of time of day on ACTH concentration in non-PPID and PPID horses. RESULTS: Evidence of ultradian fluctuation in ACTH production was not identified in either non-PPID or PPID horses. Evidence for circadian fluctuation was identified in non-PPID horses; plasma ACTH concentrations were highest at 08.00 h and decreased through the day. There was no evidence of circadian fluctuation in PPID horses. In non-PPID horses, the magnitude of circadian changes in ACTH concentration was smaller than variations in concentration that occurred at random. Intrahorse variability of ACTH concentration was greater in PPID horses than in non-PPID horses. CONCLUSIONS: Ultradian and circadian fluctuations in ACTH concentration are unlikely to influence clinical decision making; however, variations of potential clinical relevance do occur in individual horses, for reasons that remain to be determined, and increase in magnitude with progression of PPID. Results of the current study indicate that when an ACTH concentration between 19 and 40 pg/ml is measured, further testing should be considered to increase the accuracy of PPID diagnosis.

Multicentric T-cell lymphoma presenting as inferior palpebral swelling in a Standardbred mare.

A 6-year-old Standardbred mare that presented with bilateral palpebral swelling and a left corneal ulcer was diagnosed with a multicentric T-cell lymphoma. Other clinical findings included submandibular lymphadenopathy and oedema, pharyngeal oedema and rhinitis. Prior to euthanasia the horse developed keratomalacia, the onset of which was coincidental with the use of high doses of topical and systemic corticosteroids. Although an unusual presentation, palpebral swelling should be considered as a clinical sign of lymphoma.

The application of exhaled breath gas and exhaled breath condensate analysis in the investigation of the lower respiratory tract in veterinary medicine: A review.

The analysis of biomarkers in exhaled breath (EB) and exhaled breath condensate (EBC) may allow non-invasive and repeatable assessment of respiratory health and disease in mammals. Compared to human medicine, however, research data from EB and EBC analysis in veterinary medicine are limited and more patient variables influencing concentrations of EB/EBC analytes may be present. In addition, variations in methodologies between studies may influence results. A comparison of the approaches used in veterinary research by different groups may aid in the identification of potentially reliable and repeatable biomarkers suitable for further investigation. To date, changes in acid-base status and increased concentrations of inflammatory mediators have been the main findings in studies of pulmonary disease states in animals. Whilst these biomarkers are unlikely to represent specific and sensitive diagnostic parameters, they do have potential application in monitoring disease progression and treatment response.

Long-term management of sabulous cystitis in five horses.

Five horses with sabulous cystitis were managed for up to three years. They were treated by emptying the bladder through a urinary catheter and saline lavage with cytoscopic guidance to remove residual sabulous material. The cystitis was treated with antimicrobial and anti-inflammatory medications, and bethanechol chloride was also administered. Frequent catheterisation and emptying of the bladder was an alternative to regular cystoscopic examination with saline lavage but it resulted in the development of a urethral stricture in one case. Four of the horses returned to work and one was retired owing to persistent incontinence.

Results of surgical treatment of colic in miniature breed horses: 11 cases.

OBJECTIVE: To report on the outcome of surgical treatment of acute abdominal crises in miniature breed horses. DESIGN: Retrospective case series of miniature horses presented to the University Veterinary Centre, Camden with an acute abdominal crisis. METHODS: Hospital records of all miniature horses that underwent ventral midline laparotomy for acute abdominal crisis between 1997 and 2001 were reviewed. The signalment, history, clinical signs, results of ancillary diagnostic procedures, location and type of intestinal lesion, treatment and outcome were retrieved from each case record. Long-term survival was determined by telephone interview of owners. RESULTS: Eleven miniature horses including five females and six males underwent ventral midline laparotomies for acute abdominal crisis during the study period. Ages ranged between 1 month and 19 years. Surgical findings included faecalith obstruction (seven horses), enterolith (one horse), strangulating lipoma of the descending colon (one horse), jejunal infarction (one horse), and caecal infarction (one horse). Long-term survival rate (minimum 12 months post surgery) was 55%. Six of eight horses with simple intraluminal obstructions survived, while the three horses with gastrointestinal lesions associated with vascular compromise were euthanased either at surgery (caecal infarction), or postoperatively, due to complications (strangulating lipoma of the descending colon, jejunal infarction). Postoperative complications in this study included impaction of the descending colon (two horses), diarrhoea (two horses), peritonitis (one horse), hyperlipaemia (two horses), incisional infection (two horses) and abdominal adhesions (one horse). Hyperlipidaemia was present in five of seven horses in which serum triglycerides were measured at presentation. CONCLUSIONS: Simple intraluminal obstructions of the large intestine were frequently encountered during exploratory laparotomy in miniature horses presented for acute abdominal crises, and their surgical treatment was associated with a good prognosis. In contrast, this study suggested that abdominal pain associated with vascular compromise of gastrointestinal tissues in miniature horses was associated with a poorer prognosis, consistent with reports in other horse breeds. Possible contributing factors to faecalith formation, including poor quality roughage, dental disease, and inadequate water consumption, should be recognised and avoided in miniature horses. Serum triglyceride concentrations should be measured in miniature horses presented for acute abdominal pain. If elevated, nutritional supplementation should be provided.

Comparison of tracheal aspirates and bronchoalveolar lavage in racehorses. 1. Evaluation of cytological stains and the percentage of mast cells and eosinophils.

OBJECTIVE: To compare a fast Romanowsky cytological stain (Diff-Quik) and Leishman's stain for the detection of mast cells in samples from the lower airways of racehorses, and to compare the proportion of mast cells and eosinophils in the total inflammatory cells in tracheal aspirate (TA) with those in paired bronchoalveolar lavage (BAL) samples. DESIGN: Retrospective case series of 48 young Thoroughbred and Standardbred racehorses. PROCEDURE: Fifty-one paired TA and BAL samples were collected after treadmill exercise from 48 horses with poor racing performance. Two slides were prepared from each sample; one was stained with Diff-Quik stain and the other with Leishman's stain. Differential cell counts of eosinophils and mast cells were recorded from each slide. Comparison of the suitability of the stains for the detection of mast cells, and comparisons of eosinophil and mast cell percentages in TA and BAL samples were analysed using the non-parametric Wilcoxon matched pairs test. RESULTS: Percentages of mast cells were significantly higher in Leishman than in Diff-Quik stained slides in both TA (P = 0.03) and BAL samples (P < 0.0001). Mast cell percentages were significantly higher in BAL than in TA samples using Leishman's stain (P < 0.0001). There was no significant difference in eosinophil percentages between TA and BAL samples (P = 0.07). CONCLUSIONS: Fast Romanowsky type stains (for example Diff-Quik) are not appropriate for the detection of mast cells in samples from the equine lower respiratory tract. Therefore, a metachromatic stain that reliably identifies mast cells (for example Leishman's) should be used if evaluation of mast cells in lower respiratory tract is undertaken. Mast cells are predominantly found in the distal small airways and alveoli sampled with a BAL. In contrast, eosinophils appear to be evenly distributed in the lower respiratory tract. However, high percentages of eosinophils are occasionally found only in TA samples. We recommend that both a TA and BAL be used for the evaluation of eosinophils and mast cells within the equine lower respiratory tract.

Acute clinical disease in cats following infection with a pathogenic strain of Bartonella henselae (LSU16).

Bartonella henselae is the causative agent of human cat scratch disease as well as several serious sequelae of infections, including bacillary angiomatosis and bacillary peliosis. Conflicting reports describe the pathogenesis of B. henselae in the cat. In this study, we characterized a strain of B. henselae termed LSU16. This strain was isolated on rabbit blood agar from a naturally infected 10-month-old female cat during a recurrent episode of bacteremia. The bacterial species was confirmed by PCR-restriction fragment length polymorphism analysis. Nine cats were infected intradermally with 5 x 10(7) CFU of LSU16, and clinical signs, antibody responses, and bacteremia were monitored. All nine cats developed raised, erythematous areas at the site of inoculation within 72 h postinoculation; the swelling peaked at 14 days postinfection and was not palpable by 28 days postinfection. Fever developed in all nine cats between 6 and 16 days postinfection and lasted for 1 to 8 days. Between 6 and 16 days postinfection, all nine cats experienced lethargy which persisted 5 to 18 days. Seven of nine cats were bacteremic by day 7, and all nine cats had become bacteremic by 14 days postinfection. Bacteremia peaked at 14 to 28 days postinfection in all cats. In six of the nine infected cats, bacterial numbers reached nondetectable levels during the 7th week postinfection; however, a single animal maintained bacteremia to 18 weeks postinfection. All nine cats developed strong antibody responses to B. henselae, as determined by Western blot analysis and enzyme-linked immunosorbent assay. Subsequently, three naive cats were injected intradermally with blood from cats infected with LSU16 from a pure culture, and five naive cats were injected with feces from fleas which had been feeding on cats infected with a pure culture of LSU16. These cats developed signs similar to those described in the previous experiment and were euthanized at 5 weeks postinfection. We conclude that B. henselae LSU16 is a virulent strain of B. henselae in cats and propose that the virulence of B. henselae in cats is strain dependent.

Identification of a Vibrio cholerae ToxR-activated gene (tagD) that is physically linked to the toxin-coregulated pilus (tcp) gene cluster.

The toxin-coregulated pilus (TCP)-encoding gene cluster (tcp) specifies a type-IV pilus that is a major colonization determinant of Vibrio cholerae. We have identified a gene 200 bp upstream from the tcp cluster that requires ToxR for expression. We have designated this gene tagD (ToxR-activated gene) and have shown that tagD is encoded on a 600-nt transcript. The deduced tagD product is a 164-amino-acid polypeptide (20 kDa). Interestingly, TagD shares a high degree of similarity to a protein of Streptococcus sanguis 12 that is thought to play a role in fimbriae synthesis or assembly. The high degree of similarity between tagD and the Ss 12 protein provides preliminary evidence that tagD represents an additional member of the tcp cluster.

The Vibrio cholerae acfB colonization determinant encodes an inner membrane protein that is related to a family of signal-transducing proteins.

Vibrio cholerae accessory colonization factor genes (acfA, B, C, and D) are required for efficient intestinal colonization. Expression of acf genes is under the control of a regulatory cascade that also directs the synthesis of cholera toxin and proteins involved in the biogenesis of the toxin-coregulated pilus. The gene for acfB was cloned by using an acfB::TnphoA fusion junction to probe a V. cholerae O395 bacteriophage lambda library. DNA sequence analysis revealed that acfB is predicted to encode a 626-amino-acid protein related to the V. cholerae HlyB and TcpI proteins. These three Vibrio proteins have amino acid sequence similarity in a region highly conserved among bacterial methyl-accepting chemotaxis proteins. Analysis of the predicted AcfB amino acid sequence suggests that this colonization determinant possesses a membrane topology and domain organization similar to those of methyl-accepting chemotaxis proteins. Heterologous expression of acfB in Escherichia coli generates four polypeptide species with apparent molecular masses of 34, 35, 74, and 75 kDa. The 74- and 75-kDa proteins appear to represent modified forms of the full-length AcfB protein. The 34- and 35-kDa polypeptide species most likely correspond to a C-terminal 274-amino-acid polypeptide that results from internal translation initiation of acfB mRNA. Localization studies with AcfB-PhoA hybrid proteins indicate that AcfB resides in the V. cholerae inner membrane. V. cholerae acfB::TnphoA mutants display an altered motility phenotype in semisolid agar. The relationship between AcfB and Vibrio motility and the amino acid similarities between AcfB and chemotaxis signal-transducing proteins suggest that AcfB may interact with the V. cholerae chemotaxis machinery. The data presented in this report provide preliminary evidence that acfB encodes an environmental sensor/signal-transducing protein involved in V. cholerae colonization.