Claudio Bellino

In vitro evaluation of canine hemostasis following dilution with hydroxyethyl starch (130/0.4) via thromboelastometry.

OBJECTIVE To assess the effects of in vitro hemodilution of canine blood with a low molecular weight hy-droxyethyl starch (HES 130/0.4) by means of thromboelastometry (TEM). DESIGN In vitro experimental study. SETTING University Teaching Hospital. ANIMALS Ten healthy adult staff-owned dogs were sampled for the purposes of the study. Samples were also collected from 45 clinically normal dogs to establish thromboelastometric reference intervals. INTERVENTIONS For each dog whole blood was collected by jugular venipuncture and placed into tubes containing 3.8% trisodium citrate and stored at 37°C. Two standard dilutions (1:4 and 1:10) from canine blood specimens were prepared with HES 130/0.4 and saline 0.9%. MEASUREMENTS AND MAIN RESULTS The effects of dilution were studied by TEM. No statistically significant differences were observed between the TEM values of the control samples and the samples diluted with saline solution (ratio 1:10 and 1:4). In contrast, hypocoagulability was observed in the samples treated with HES 130/0.4, with more severe effects at 1:4 dilution than at 1:10 dilution. The 1:4 dilution with HES 130/0.4 produced marked alterations: CT (P = 0.035) and CFT (P = 0.0007) on the ex-TEM profile, CT (P = 0.0005) and ML (P = 0.0017) on the fib-TEM profile and CFT (P = 0.0043) on the in-TEM, were all significantly increased (P < 0.05), whereas MCF (P = 0.0042) and alpha angle (P = 0.002) on the in-TEM and MCF (P = 0.011) and alpha angle (P = 0.001) ex-TEM profiles and MCF (P = 0.0001) on the fib-TEM profile were significantly decreased (P < 0.05). CONCLUSIONS Dilution of canine blood with HES 130/0.4 results in a thromboelastometric pattern consistent with hypocoagulation and this effect appears to result from a dose-dependent alteration in fibrinogen concentration and inhibition of platelet function. As this is an in vitro study, further in vivo investigations are necessary to confirm the results.

Transanal pull-through rectal amputation for treatment of colorectal carcinoma in 11 dogs.

OBJECTIVE To evaluate outcome after transanal rectal pull-through amputation of single colorectal adenocarcinoma and in situ carcinoma (Tis) in dogs. STUDY DESIGN Retrospective case series. ANIMALS Dogs (n=11) with colorectal cancer. METHODS Full-thickness colorectal amputation by either simple transanal (7 dogs) or combined abdominal-transanal (4) pull-through technique. RESULTS Adenocarcinoma (8) and Tis (2) were removed with 3-6 cm of grossly normal tissue, cranial and caudal to the tumor, or in 1 Tis with 2 cm grossly normal tissue, cranial and caudal. Two dogs that had a combined abdominal-transanal approach died within 4 days. In the other dogs, postoperative complications included short-term tenesmus (6 dogs), rectal bleeding (11), rectal stricture (3), and long-term fecal incontinence (1). Postoperative recurrence and metastatic rates for adenocarcinoma were 18.2% and 0%, respectively. Median disease-free interval and survival time were not reached. Mean disease-free and overall survival times were 44.3 and 44.6 months (range, 0-75 months), respectively. CONCLUSION En bloc excision of colorectal Tis and adenocarcinoma may be followed by a long survival. Complications of the transanal approach are usually moderate and self-limiting, but complications are more common and severe when more extensive resections are performed through a combined abdominal-transanal approach. CLINICAL RELEVANCE Transanal rectal pull-through amputation is suitable for en bloc resection of colorectal neoplasia. A combined abdominal-transanal approach should be reserved for tumors extending from the mid-cranial region of the rectum to the descending colon.

Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood

The use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors in the quantification of gene expression. The most suitable reference genes in sheep have been identified for a restricted range of tissues, but no specific data on whole blood are available. The aim of this study was to identify a set of reference genes for normalizing qRT-PCR from ovine whole blood. We designed 11 PCR assays for commonly employed reference genes belonging to various functional classes and then determined their expression stability in whole blood samples from control and disease-stressed sheep. SDHA and YWHAZ were considered the most suitable internal controls as they were stably expressed regardless of disease status according to both geNorm and NormFinder software; furthermore, geNorm indicated SDHA/HPRT, YWHAZ/GAPDH and SDHA/YWHAZ as the best reference gene combinations in control, disease-stressed and combined sheep groups, respectively. Our study provides a validated panel of optimal control genes which may be useful for the identification of genes differentially expressed by qRT-PCR in a readily accessible tissue, with potential for discovering new physiological and disease markers and as a tool to improve production traits (e.g., by identifying expression Quantitative Trait Loci). An additional outcome of the study is a set of intron-spanning primer sequences suitable for gene expression experiments employing SYBR Green chemistry on other ovine tissues and cells.

Clinical Evaluation and Endoscopic Classification of Bronchomalacia in Dogs

BACKGROUND Little information is available about the association between bronchomalacia and historical or clinicopathologic data. Also, studies applying an endoscopic classification scheme that differentiates between static and dynamic bronchial collapse and based on a scoring system are lacking. OBJECTIVES To describe the clinical presentation of bronchomalacia in dogs, to classify endoscopic findings, and to evaluate associations among historical, clinicopathologic data, and endoscopic findings. ANIMALS Fifty-nine client-owned dogs with an endoscopic diagnosis of bronchomalacia. METHODS In this retrospective study, medical records were analyzed and video documentation was reviewed to assign a score to endoscopic findings. Univariate analysis was performed on categorical variables organized in contingency tables, and a stepwise logistic regression model was used for multivariate analysis. RESULTS Of the 59 dogs included in the study, 2 were affected by static bronchial collapse (SBC), 35 by dynamic bronchial collapse (DBC), and 22 by both SBC and DBC. The association between SBC and DBC was more frequently seen in the dogs with higher body weight, pulmonary hypertension, a bronchial type of radiographic pattern, and nodularity at endoscopic examination. Thirty-one dogs were presented with tracheomalacia and bronchomalacia; an association emerged between these concurrent disorders in dogs living indoors. Multivariate analysis of the endoscopic scores showed a correlation between DBC severity and cough duration. CONCLUSION AND CLINICAL IMPORTANCE Results of this study provide evidence for 2 different types of bronchial collapse. Endoscopic scoring scheme has proved to be promising in the bronchomalacia classification, although further evaluation of its applicability in larger canine populations is needed.

Neurological signs associated with aflatoxicosis in Piedmontese calves

MYCOTOXINS are secondary metabolites produced by fungi, which may be developed under particular storage and environmental conditions. The principal mycotoxins affecting agriculture and livestock are aflatoxins, ochratoxins, trichothecenes, zearalenone and fumonisins; these agents may significantly affect public health and economies worldwide (Moss 1991). The toxicity of mycotoxins varies according to the livestock species affected. Ruminants are among the most mycotoxin-tolerant species, as the agents are partly degraded by the rumen microbiota (Hussein and Jeffrey 2001, Yiannikouris and Jouany 2002). In bovids, natural intoxication by fumonisin is rare, and toxicological studies on this mycotoxin have been mostly experimental (Osweiler and others 1993, Mathur and others 2001). Aflatoxins are among the most widely diffuse mycotoxins in foods and feedstuffs. In bovids, the prolonged consumption of contaminated feed has a negative impact on both production and reproduction (Dekman and Green 1992, Pier 1992, Hussein and Jeffrey 2001). By binding to macromolecules such as nucleic acids and nucleoproteins, aflatoxins cause a number of toxic effects such as mutagenesis, carcinogenesis, teratogenesis, reduced protein synthesis and immunosuppression (Hussein and Jeffrey 2001). The liver is the principal target organ of aflatoxicosis, and chronic intoxication induces lesions that are characterised by proliferation and fibrosis of the bile ducts accompanied by hepatocyte megalocytosis (Marcato 2002). This short communication describes the clinical, laboratory, macroscopic and histopathological findings relating to an outbreak of hepatic encephalopathy consequent to accidental intoxication by aflatoxin in Piedmontese calves. The affected farm housed 75 Piedmontese cows that were kept for the production of beef calves. Animals kept for reproduction were fed hay, while the beef calves were fed home-produced cornmeal for approximately four months. A clinical examination carried out on site identified six calves, aged six to 15 months, showing varying degrees of clinical signs; neurological signs of varying intensity had appeared 36 to 48 hours previously. Two animals (calves 1 and 2) were in lateral recumbency; physical examination revealed hypothermia and weight loss. Neurological examination showed a comatose mental status, characterised by the absence of response to any stimulus, including pain sensation. Generalised spinal hyporeflexia was also present, as well as dilated pupils and the absence of direct and consensual pupillary light reflexes. One of these animals showed myoclonus in the right forelimb. Blood samples and cerebro spinal fluid were collected from these two animals, which were euthanased and underwent postmortem examination. The remaining four animals showed signs of weight loss, dysorexia, ruffled coats and reduced ruminal activity. Neurological examination of each animal revealed a depressed mental status and compulsive behaviour characterised by anteropulsion and circling in large circles (Fig 1); hyperexcitability was also observed on occasion. The gait was slightly ataxic in all four animals, while the postural reactions were altered in one calf and slightly diminished in the other three. Spinal reflexes were normal, and evaluation of the cranial nerve reflexes revealed the absence of or reduction in the menace response of all four animals. Both direct and consensual pupillary light reflexes were normal. All four animals had a tendency to walk into objects, suggesting amaurosis. A blood sample was taken from one calf (calf 3) for analysis. The clinical signs were compatible with an intracranial prosencephalic localisation. On the basis of the history and clinical signs, differential diagnoses included inflammatory, toxicometabolic or degenerative pathologies. Haematology, blood chemistry analysis and electrophoresis of serum proteins was performed on the blood samples, and while waiting for the results of the diagnostic tests, the animals were administered a high dose (10 mg/kg) of intravenous vitamin B1. However, all four calves deteriorated and died within 48 hours. Calf 3 underwent postmortem examination. As the animals affected were only those that had consumed cornmeal, feeding with the cornmeal was discontinued. Hepatic, renal, encephalic and cervical spinal cord samples were taken during the postmortem examinations and fixed in 10 per cent buffered formalin. After embedding in paraffin, standard histological sections were stained with haematoxylin and eosin. The following areas of the central nervous system (CNS) were analysed: cervical spinal cord, medulla oblongata, pons, cerebellum, midbrain, thalamus, hippocampus, basal nuclei and cerebral cortex. Veterinary Record (2007) 160, 698-700

Development of a diagnostic diagram for rapid field assessment of acidosis severity in diarrheic calves.

OBJECTIVE To develop a diagnostic diagram for rapid field assessment of acidosis severity in diarrheic calves. DESIGN Prospective cross-sectional study. ANIMALS 148 Piedmontese calves (38 calves in preliminary experiments; 83 diarrheic calves and 27 healthy control calves in the primary experiment). PROCEDURES Physical examination was performed and a standard data collection form was completed for each calf. Blood samples were obtained and submitted for evaluation of acid-base balance, performance of a CBC, and measurement of electrolyte and total protein concentrations. RESULTS Severe metabolic acidosis (extracellular base excess more negative than -10 mmol/L) was associated with abnormal mental status, delayed or absent suckle reflex, abnormal posture or gait, enophthalmos, and cold oral mucosal membranes. Clinical signs associated with severe metabolic acidosis were arranged into a grid to create a diagnostic diagram. Sensitivity and specificity of the diagnostic diagram for the prediction of severe metabolic acidosis were 88% and 79%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Use of the diagnostic diagram may aid differentiation between severe and nonsevere acidosis patterns as determined on the basis of clinical signs.

Helcococcus kunzii Isolated from a Sow with Purulent Urocystitis

ABSTRACT Helcococcus kunzii has never been reported in veterinary medicine. The isolation of H. kunzii from a sow with purulent urocystitis is described, suggesting this organisms potential pathogenic role in swine.

Analysis of cerebrospinal fluid from 20 calves after storage for 24 hours

Samples of CSF collected from 20 normal healthy calves were analysed either immediately or after having been stored for 24 hours at 4°C in the presence of 11 per cent autologous serum. There were no significant differences between the total and differential cells counts of the fresh and stored samples, but there was a positive linear correlation between them. There were some morphological changes to the nuclei of the mononuclear cells in the stored samples.

Diprosopia/dicephalia in calves in northern Italy: clinical and aetio-pathological features.

With 1 table and 4 figures

Characterization of the upper and lower respiratory tract microbiota in Piedmontese calves

BackgroundThe microbiota of the bovine upper respiratory tract has been recently characterized, but no data for the lower respiratory tract are available. A major health problem in bovine medicine is infectious bronchopneumonia, the most common respiratory syndrome affecting cattle. With this study, we used 16S rRNA gene sequencing to characterize and compare the microbial community composition of the upper and lower respiratory tracts in calves.ResultsThe microbiota of the upper (nasal swab [NS]) and the lower (trans-tracheal aspiration [TTA]) respiratory tracts of 19 post-weaned Piedmontese calves with (8/19) and without (11/19) clinical signs of respiratory disease, coming from six different farms, was characterized by 16S rRNA gene metabarcoding. A total of 29 phyla (29 in NS, 21 in TTA) and 305 genera (289 in NS, 182 in TTA) were identified. Mycoplasma (60.8%) was the most abundant genus identified in both the NS (27.3%) and TTA (76.7%) samples, followed by Moraxella (16.6%) in the NS and Pasteurella (7.3%) in the TTA samples. Pasteurella multocida (7.3% of total operational taxonomic units [OTUs]) was the most abundant species in the TTA and Psychrobacter sanguinis (1.1% of total OTUs) in the NS samples. Statistically significant differences between the NS and the TTA samples were found for both alpha (Shannon index, observed species, Chao1 index, and Simpson index; P = 0.001) and beta (Adonis; P = 0.001) diversity. Comparison of the NS and TTA samples by farm origin and clinical signs revealed no statistical difference (P > 0.05), except for farm origin for the NS samples when compared by the unweighted UniFrac metric (P = 0.05).ConclusionsUsing 16S rRNA gene sequencing, we characterized the microbiota of the upper and lower respiratory tracts of calves, both healthy individuals and those with clinical signs of respiratory disease. Our results suggest that environmental factors may influence the composition of the upper airway microbiota in cattle. While the two microbial communities (upper and lower airways) differed in microbial composition, they shared several OTUs, suggesting that the lung microbiota may be a self-sustaining, more homogeneous ecosystem, influenced by the upper respiratory tract microbiota.