The use of the rapid osmotic fragility test as an additional test to diagnose canine immune-mediated haemolytic anaemia

Background

Diagnosing canine immune-mediated haemolytic anaemia (IMHA) is often challenging because all currently available tests have their limitations. Dogs with IMHA often have an increased erythrocyte osmotic fragility (OF), a characteristic that is sometimes used in the diagnosis of IMHA. Since the classic osmotic fragility test (COFT) is time-consuming and requires specialized equipment, an easy and less labour-intensive rapid osmotic fragility test (ROFT) has been used in some countries, but its diagnostic value has not yet been investigated.This study aimed to evaluate erythrocyte osmotic fragility in dogs with and without IMHA, to compare results of the classic (COFT) and rapid (ROFT) test and to assess the value of the ROFT as diagnostic test for canine IMHA.Nineteen dogs with IMHA (group 1a), 21 anaemic dogs without IMHA (group 1b), 8 dogs with microcytosis (group 2), 13 hyperlipemic dogs (group 3), 10 dogs with lymphoma (group 4), 8 dogs with an infection (group 5) and 13 healthy dogs (group 6) were included.In all dogs, blood smear examination, in-saline auto-agglutination test, Coombs’ test, COFT and ROFT were performed. In the COFT, OF5, OF50 and OF90 were defined as the NaCl concentrations at which respectively 5, 50 and 90% of erythrocytes were haemolysed.

Results

Compared with healthy dogs, OF5 and OF50 were significantly higher in group 1a (P < 0.001) and OF5 was significantly higher in group 3 (P = 0.0266). The ROFT was positive in 17 dogs with IMHA, 10 hyperlipemic dogs, one anaemic dog without IMHA and one healthy dog.

Conclusions

Osmotic fragility was increased in the majority of dogs with IMHA and in dogs with hyperlipidemia, but not in dogs with microcytosis, lymphoma or an infection. Although more detailed information was obtained about the osmotic fragility by using the COFT, the COFT and ROFT gave similar results. The ROFT does not require specialized equipment, is rapid and easy to perform and can be used easily in daily practice. Although, the ROFT cannot replace other diagnostic tests, it may be a valuable additional tool to diagnose canine IMHA.     

Effect of apple particle state on the release of volatile compounds in a new artificial mouth device

Varying the crushing parameters in a model mouth apparatus gave different crushed apple samples, which were compared to apples crushed in the human mouth by six people. An image analysis method was developed to measure the similarity between apple particles after crushing in the artificial mouth and in the human mouth. Thus, experimental conditions were determined that produced fruit in a state closest to that obtained after mastication in a human mouth. The influence of these different conditions on the quantity of released volatile compounds was then studied.     

Development of a real-time PCR method for the differential detection and quantification of four solanaceae in GMO analysis: potato (Solanum tuberosum), tomato (Solanum lycopersicum), eggplant (Solanum melongena), and pepper (Capsicum annuum)

The labeling of products containing genetically modified organisms (GMO) is linked to their quantification since a threshold for the presence of fortuitous GMOs in food has been established. This threshold is calculated from a combination of two absolute quantification values: one for the specific GMO target and the second for an endogenous reference gene specific to the taxon. Thus, the development of reliable methods to quantify GMOs using endogenous reference genes in complex matrixes such as food and feed is needed. Plant identification can be difficult in the case of closely related taxa, which moreover are subject to introgression events. Based on the homology of beta-fructosidase sequences obtained from public databases, two couples of consensus primers were designed for the detection, quantification, and differentiation of four Solanaceae: potato (Solanum tuberosum), tomato (Solanum lycopersicum), pepper (Capsicum annuum), and eggplant (Solanum melongena). Sequence variability was studied first using lines and cultivars (intraspecies sequence variability), then using taxa involved in gene introgressions, and finally, using taxonomically close taxa (interspecies sequence variability). This study allowed us to design four highly specific TaqMan-MGB probes. A duplex real time PCR assay was developed for simultaneous quantification of tomato and potato. For eggplant and pepper, only simplex real time PCR tests were developed. The results demonstrated the high specificity and sensitivity of the assays. We therefore conclude that beta-fructosidase can be used as an endogenous reference gene for GMO analysis.     

Citrantifidiene and citrantifidiol: bioactive metabolites produced by Trichoderma citrinoviride with potential antifeedant activity toward aphids

Two novel metabolites with potential antifeedant activity were isolated from cultures of the fungus Trichoderma citrinoviride strain ITEM 4484 grown in solid-state fermentation on sterile rice kernels. The producing strain was identified at species level by sequence analysis of the internal transcribed spacer regions ITS-1 and ITS-2 of the nuclear rDNA and a fragment of the translation elongation factor gene TEF-1alpha. Fractionation by column chromatography and TLC of the culture organic extract, followed by feeding preference tests on the aphid pest Schizaphis graminum (Rondani), allowed the purification of 5.8 and 8.9 mg/kg of culture of two bioactive metabolites, which were named citrantifidiene and citrantifidiol ( 1 and 2). Citrantifidiene and citrantifidiol, whose structures were determined by spectroscopic methods (NMR and MS) are a symmetrical disubstituted hexa-1,3-dienyl ester of acetic acid and a tetrasubstituted cyclohexane-1,3-diol, respectively. The pure metabolites influenced the feeding preference of S. graminum restraining individuals from feeding on wheat leaves dipped in 5% aqueous methanol solution containing 0.57 mg/mL of citrantifidiene or 0.91 mg/mL of citrantifidiol.     

Postoperative ultrasonographic appearance of uncomplicated enterotomy or enterectomy sites in dogs

The ultrasonographic findings in 20 dogs with 25 healing enterotomy and enterectomy sites resulting from the removal of foreign material or correction of intussusceptions are presented. In this prospective study, dogs had preoperative abdominal ultrasound examinations followed by sequential sonographic examinations on the first, third, sixth, and 10th days postenterotomy or enterectomy with an additional sonographic examination after 20 days postoperatively. Documented sonographic features included length and maximal intestinal wall thickness of the enterotomy or enterectomy sites, echogenicity of omental/mesenteric fat, amount of free gas and abdominal effusion, and gastrointestinal motility. Sonographically, 57% of enterotomies and 100% of enterectomies were visualized. Pneumoperitoneum, hyperechoic omental/mesenteric fat, and abdominal effusion did not appreciably hamper evaluation of the intestinal surgical site. Absent wall layering at the surgical site was noted in 96% of dogs at day 1 postoperatively. At the final sonographic examination, wall layering remained altered to absent in 100% of dogs and normal thickness was noted in only 20% of dogs. The median maximal wall thickness was 7 and 8 mm for enterotomies and enterectomies respectively, which occurred between days 1 and 3 and days 3 and 6 postoperatively. Effusion and increased echogenicity of omental/mesenteric fat localized to the surgical site were noted in 42% and 60% of dogs respectively at day 1 postoperatively, with resolution noted between days 3 and 10 postoperatively in 92% and 80% of dogs. Generalized abdominal effusion and pneumoperitoneum were seen in 100% of dogs immediately postoperatively and resolved in 80% by day 10.     

The role of Mycoplasma bovis in bovine respiratory disease outbreaks in veal calf feedlots

To assess the prevalence and relative importance of Mycoplasma bovis among the pathological agents implicated in bovine respiratory disease (BRD), we surveyed 135 veal calves from nine feedlots in eastern France during naturally occurring outbreaks of respiratory disease. Occurrence of respiratory pathogens, M. bovis, bovine viral diarrhoea (BVD) virus, bovine respiratory syncytial (BRS) virus and parainfluenza-3 (PI3) virus was investigated by seroconversion and isolation of bacteria and viruses from broncho-alveolar fluids. M. bovis and pathogenic respiratory bacteria were isolated in eight of the nine feedlots, and from 106 and 32, respectively, of the 135 tested animals. Seroconversion to PI3 virus occurred in four lots. BVD and BRS viruses were detected in eight and one lot, respectively. M. bovis was the most frequently isolated aetiologic agent in these BRD outbreaks, spreading early and widely throughout the affected units (60-100% rate of isolation and seroconversion). These results stress the importance of M. bovis in the BRD complex.     

Assessment of molecular detection of Fusarium circinatum in insects and passive spore traps in Pinus radiata plantations

Fusarium circinatum is the causal agent of pitch canker, a destructive disease that threatens natural and planted pine forests around the world. Although pitch canker has caused problems in Spain and Portugal, concerning Europe as a whole, the fungus is not established across the pine distribution area. Its dispersion by wind and/or insect vectors could nevertheless play a role in the colonization of currently uninfected stands. It is therefore crucial to develop monitoring tools for its detection. To this end, we assessed the molecular detection of the pathogen in environmental samples of bark beetles and passive spore traps, collected in two infected Pinus radiata plantations in Basque country, Spain. The spread pattern of F. circinatum was assessed by an experimental design that included insect and spore traps installed at the centre, at the edge and outside the plots. Our results showed that F. circinatum was detected in both types of samples, at almost all collection dates. In both type of samples, positive detections were mainly found at the centre of the plots, a lower proportion at the edge, and very few outside. This suggests that long‐distance dispersion of Fusarium circinatum does not rely on wind spore dispersal neither on insect flight. Our study also shows that molecular methods are a powerful tool to monitor the pathogen in environmental samples.