Antonio Borrelli

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.

Serum Values of Cardiac Troponin-T in Normal and Cardiomyopathic Dogs

Together with tropomyosin, the troponin complex participates in the contraction of skeletal and cardiac muscles. Troponin is composed of three subunits, namely troponin C (TnC), troponin I (TnI), and troponin T (TnT), which binds tropomyosin. There are three different isoforms of TnT and TnI, two of which are specific for skeletal muscle, while the third is specific for the myocardium (cTnT and cTnI). As the homology between troponins is about 95% among mammals, commercial diagnostic kits designed for use in humans also provide excellent results in other animals. (O’Brien et al., 1997). In human medicine, the measurement of cTnT or cTnI levels is now used together with measurement of the levels of creatine kinase (CK) and lactate dehydrogenase (LDH) and their isozymes for assessment of myocardial damage. For diagnostic purposes, one advantage is that these proteins are normally absent in blood in subjects without myocardial damage. Even minimal increases in the serum levels of these proteins are indicative of myocardial damage and, in the case of cTnT, it is detectable in serum from 2 h to 14 days following the onset of myocardial damage. Moreover, in contrast to the isozymes of CK and LDH, the results are not influenced by use of different clinical samples (hemolytic, lipemic, bile samples). The degree of increase in the level of cTnT is relatively proportional to the degree of myocardial damage, with prognostic implications as well. At the same time, the extreme sensitivity of this technique also allows for its use in other nonischemic pathologies including chronic cardiac insufficiency, pulmonary emboli, acute myocarditis, systemic hypertension, arrhythmias, and toxicity from doxorubicin (De Francesco, 2002). In veterinary medicine, O’Brien et al. (1997) have demonstrated the utility of measuring the levels of cTnT in the diagnosis of experimentally induced myocardial damage.

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.

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.

Late onset and rapid progression of cerebellar abiotrophy in a domestic shorthair cat.

A two-year-old male domestic shorthair cat was seen because of sudden onset of incoordination and tremors that had rapidly worsened over five days. Neurological examination revealed severe cerebellar ataxia, intention tremors and bilaterally decreased menace response. Blood work evaluation included a complete cell blood count (CBC), serum biochemistry profile, urinalysis, faecal flotation, cerebrospinal fluid (CSF) analysis and retroviral testing. Except for testing positive for feline immunodeficiency virus (FIV) antibodies, all other results were within the normal range. The patient was euthanased two days later because of progression of clinical signs, and a necropsy was performed. Histologically, lesions were limited to the cerebellum and consistent with cerebellar abiotrophy. No secondary diseases that could explain the rapid development of clinical signs were found. It was considered unlikely that cerebellar degeneration was related to FIV positivity, as virus invasion of the central nervous system (CNS) is mainly limited to the cerebral cortex. This case report is the first to describe late onset and rapid progression cerebellar abiotrophy in a cat.

Validation of Thromboelastometry in Cattle

Thromboelastometry (TEM) and thromboelastography are useful tools for the evaluation of the entire clotting process from the activation of plasmatic factors until fibrinolysis. TEM has been validated in several domestic species but not in cattle. In this study, we defined normal TEM values in adult cattle and in veal calves; we then compared rates of coagulation in these two groups. Furthermore, the precision of the instruments was evaluated. Finally, we studied the correlation between the hematological and TEM results. In calves, we identified a faster activation of plasmatic factors (shorter clotting times) and lower fibrinogenemia. The precision of TEM on the bovine blood samples was good. Additionally, hematocrit was positively linked to clotting time of the intrinsic and extrinsic pathways, whereas fibrinogenemia was strongly associated with fib-TEM® tracing.

Assessment of coagulation utilizing thromboelastometry in dogs undergoing orthopedic surgery

OBJECTIVE To evaluate blood coagulation using thromboelastometry in dogs following orthopedic surgery. DESIGN Longitudinal observational study. SETTING University veterinary teaching hospital. ANIMALS Thirty-four adult client-owned dogs. MEASUREMENTS AND MAIN RESULTS Dogs undergoing elective or emergency orthopedic surgery had whole blood collected before (T0), at 24 hours (T1), and 1 week (T2) after surgery. Whole blood from each dog was collected by jugular venipuncture using a 20-Ga needle and minimum venous stasis. The blood was placed into tubes containing 3.8% trisodium citrate (1 part citrate: 9 parts blood) and rested at 37°C. Coagulation was evaluated by means of thromboelastometry using the in-TEM, ex-TEM, and fib-TEM assays. Statistically significant increases (P < 0.05) in maximum clot firmness (MCF) from T0 to T1 in the in-TEM and fib-TEM profiles (both P = 0.0001), from T0 to T2 in the in-TEM, ex-TEM, and fib-TEM profiles (P = 0.012, P = 0.037, and P = 0.0001, respectively), and from T1 to T2 in the fib-TEM profile (P = 0.039) were noted. The α angle increased from T0 to T2 in the in-TEM and ex-TEM profiles (P = 0.019 and P = 0.036, respectively). All results were, however, within the institutional reference ranges. CONCLUSIONS In this study, unlike the hypercoagulability observed in human orthopedic patients, a hypercoagulable state as measured by thromboelastometry did not develop in dogs following orthopedic surgery.

Thromboelastometric evaluation of hemostasis in dogs infected with Leishmania infantum

OBJECTIVE Evaluation of hemostasis using thromboelastometry in dogs with leishmaniasis before and after treatment. DESIGN Longitudinal observational study. SETTING University veterinary teaching hospital. ANIMALS Eighty-four adult, client-owned dogs. MEASUREMENTS AND MAIN RESULTS Whole blood samples for the coagulation profile were collected from symptomatic dogs with leishmaniasis (group S), asymptomatic dogs with leishmaniasis after treatment (group T), and a control group of healthy dogs (group H). Hemostasis was evaluated by means of standard coagulation profile (prothrombin time, activated partial thromboplastin time, and fibrinogen) and by thromboelastometry (ROTEM). Prothrombin time and activated partial thromboplastin time were within the upper reference range in all 3 groups. Comparison of the ROTEM variables between the 3 groups showed statistically significant differences between group S versus groups T and H, but remaining within the reference ranges. Statistically significant differences in hematocrit and fibrinogen concentrations were noted between groups (group S vs. H: hematocrit P = 0.001, fibrinogen P = 0.002; Group S vs. T: hematocrit P = 0.001, fibrinogen P = 0.001). These variations have interfered with some parameters of the ROTEM profile. CONCLUSIONS This study showed normal standard coagulation profiles in all 3 groups evaluated. The ROTEM results did not fall outside of the maximum values of the reference ranges.

Polymorphism Analysis of Ch1 and Ch2 Genes in the Siberian Cat

Cats are usually spreaders of allergens that are critical for sensitive people; the Siberian cat is a breed supposed to be low level allergenic, according to some breeders’ statements. The sequence of the two genes, namely Ch1 and Ch2, that code for the allergen Fel d 1, the major allergen responsible for outbreaks of allergy symptoms, is not yet known in the Siberian cat, and finding this was the aim of our investigation. Notably, our work is the first survey of the genetic structure of these genes in Siberian cats. The comparison of the sequences of Siberian cats, non-Siberian cats, and sequences present in the National Center for Biotechnology Information database revealed a considerable number of mutations; some of those detected in the Siberian cat, due to their position in exon regions, could affect the Fel d 1 allergenic properties. Therefore, further investigations are recommended to assess if the identified mutations can be responsible for a reduced-allergen synthesis and can be used as markers for selection of low level allergenic cats.