Identification and validation of housekeeping genes as internal control for gene expression in an intravenous LPS inflammation model in chickens

Real-time PCR has become a powerful tool for the detection of inflammatory parameters, including cytokines. Reference or housekeeping genes are used for the normalization of real-time RT-PCR results. In order to obtain reliable results, the stability of these housekeeping genes needs to be determined. In this study the stability of five genes, including beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phophoribosyl-transferase (HPRT), ubiquitin (UB) and glucose-6-phosphate dehydrogenase (G6PDH), was determined in a lipopolysaccharide inflammation model in chickens. beta-Actin appeared to be the most stable single gene in our model. Because the use of a single gene for normalization can lead to relatively large errors, the use of the geometric mean of multiple reference genes or normalization factor is preferred. The most stable combination for gene expression analysis in this lipopolysaccharide inflammation model in chickens is G6PDH and UB, since their correlation coefficients were 0.953 and 0.969, respectively (BestKeeper) and an M value of 0.34 and a low V(2/3) value of 0.155 (geNorm) were obtained. The use of HPRT and GAPDH should be avoided. The stable housekeeping genes, G6PDH and UB together, can be used to normalize the expression of pro-inflammatory cytokines in a lipopolysaccharide inflammation model in chickens.     

Thromboelastographic tracings in retired racing greyhounds and in non-greyhound dogs

BACKGROUND: Bleeding disorders in patients with normal coagulation test results are frequently reported in Greyhounds. The purpose of this study was to compare Greyhounds to non-Greyhounds by thromboelastography (TEG). HYPOTHESIS: TEG parameters in Greyhounds are different from those in non-Greyhounds. ANIMALS: Forty-three healthy dogs (28 Greyhounds and 15 non-Greyhounds) based on the results of physical examination, CBC, activated partial thromboplastin time, prothrombin time, fibrinogen, and platelet count. MATERIALS AND METHODS: Recalcified citrated native TEGs were performed in both groups; data were compared using Student's, Mann-Whitney, and Pearson's statistical tests. RESULTS: In Greyhounds, mean +/- SD were as follows: R-time 4.3 +/- 1.7 minutes, K-time 3.8 +/- 1.4 minutes, angle (alpha) 50.0 +/- 8.0 degrees , maximum amplitude (MA) 47.6 +/- 5.6 mm, clot strength (G) 4,647 +/- 1,097 dyn/cm2, and percent lysis at 60 minutes (LY60) 2.8 +/- 5.0%. In the non-Greyhounds they were R-time 3.7 +/- 1.6 minutes, K-time 2.5 +/- 0.9 minutes, angle 59.8 +/- 7.0 degrees , MA 53.1 +/- 5.6 mm, G 5,811 +/- 1,256 dyn/cm2, and LY60 3.1 +/- 2.5%. All parameters were significantly different between the groups, except for R-time and LY60. CONCLUSION: In Greyhounds, clotting kinetics are slower and clot strength are weaker than in non-Greyhounds, supporting the increased tendency to bleed observed after minor trauma or surgical procedures in the breed. The findings may also be attributed to blood viscosity or to the concentration of citrate in the sample (ie, Greyhounds have higher hematocrit and less plasma per unit volume).     

Laryngeal rhinosporidiosis in a Belgian warmblood horse

In Belgium and even in northern Europe Rhinosporidium seeberi has not been reported in autochtonous people or animals. In this paper, the authors report the first observation of laryngeal masses, caused by Rhinosporidium seeberi, in a Belgian Warmblood horse. Moreover, laryngeal rhinosporidiosis is extremely rare since this localisation is only described in four human cases.     

First outbreak of Trypanosoma evansi in camels in metropolitan France

The first outbreak of trypanosomosis caused by Trypanosoma evansi in camels in France was reported on a farm in the Aveyron Department. Five camels were imported from the Canary Islands to the farm in early July 2006, and trypanosomes were observed on a stained blood smear from one of them, which died in October. On further investigations, trypanosomes were observed in the blood of five camels, three of them indigenous to the farm and two that had been imported. On the basis of microscopical examination (morphological criteria and measurements) and serological results based on the card agglutination T evansi test and PCR typing, the parasites were identified as T evansi. After treatment with melarsomine, the infected camels rapidly became negative by parasitological tests and were negative two to four months later by serological tests. The parasite was probably transmitted by tabanids and Stomoxys calcitrans, which were abundant in July to September 2006. No parasites were observed in other animals on the farm or on neighbouring farms, but some of the sheep on these farms were positive by PCR or serology.     

Leukemia inhibitory factor protein and receptors are expressed in the bovine adrenal cortex and increase cortisol and decrease adrenal androgen release

The release of adrenal steroids during acute stress is primarily regulated by adrenocorticotropic hormone (ACTH). In contrast, during chronic inflammatory stress additional factors are involved in regulating adrenal function. Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that increases ACTH release from the pituitary. In addition, LIF and LIF receptors (LIFR) are expressed in the human adrenal cortex and the human adrenocortical tumor cell line H295R. Furthermore, LIF increases basal and ACTH-stimulated cortisol release from H295R cells. However, the expression of LIF and LIFR in non-human adrenal glands and the effects of LIF on the release of cortisol from adrenal cells of non-human species have not been determined. Furthermore, the effects of LIF on adrenal androgen release from all species are unknown. In this study, immunohistochemistry, Western blots, RT-PCR, and nucleotide sequencing was utilized to demonstrate that LIF and its receptor are expressed throughout the bovine adrenal cortex. Although LIF did not modify basal cortisol release from dispersed cells isolated from the bovine adrenal zona fasciculate, this cytokine increased ACTH-stimulated release of cortisol from these cells in a manner dependent on the LIF concentration and exposure interval. In contrast, LIF in a concentration-dependent and time-dependent manner decreased basal and ACTH-stimulated adrenal androgen release from dispersed cells isolated from the bovine adrenal zona reticularis. Because LIF release increases during inflammatory stress and this cytokine stimulates adrenal cortisol release and inhibits adrenal androgen release, this cytokine may play an important role in regulating the release of adrenal steroids during inflammatory stress.     

Molecular evaluation of five cardiac genes in Doberman Pinschers with dilated cardiomyopathy

OBJECTIVE: To sequence the exonic and splice site regions of 5 cardiac genes associated with the human form of familial dilated cardiomyopathy (DCM) in Doberman Pinschers with DCM and to identify a causative mutation. ANIMALS: 5 unrelated Doberman Pinschers with DCM and 2 unaffected Labrador Retrievers (control dogs). PROCEDURES: Exonic and splice site regions of the 5 genes encoding the cardiac proteins troponin C, lamin A/C, cysteine- and glycine-rich protein 3, cardiac troponin T, and the beta-myosin heavy chain were sequenced. Sequences were compared for nucleotide changes between affected dogs and the published canine sequences and 2 control dogs. Base pair changes were considered to be causative for DCM if they were present in an affected dog but not in the control dogs or published sequences and if they involved a conserved amino acid and changed that amino acid to a different polarity, acid-base status, or structure. RESULTS: A causative mutation for DCM in Doberman Pinschers was not identified, although single nucleotide polymorphisms were detected in some dogs in the cysteine- and glycine-rich protein 3, beta-myosin heavy chain, and troponin T genes. CONCLUSIONS AND CLINICAL RELEVANCE: Mutations in 5 of the cardiac genes associated with the development of DCM in humans did not appear to be causative for DCM in Doberman Pinschers. Continued evaluation of additional candidate genes or a focused approach with an association analysis is warranted to elucidate the molecular cause of this important cardiac disease in Doberman Pinschers.     

Western blot detection of PrP Sc in archived paraffin-embedded brainstem from scrapie-affected sheep

Scrapie is a naturally occurring fatal neurodegenerative disease of adult sheep and goats, one of a group of mammalian diseases known as transmissible spongiform encephalopathies (TSE) or prion diseases. Immunoassays that identify disease-associated prion protein (PrP Sc) are integral to the diagnosis of scrapie and other prion diseases. Results obtained by either immunohistochemistry (IHC) or Western blot (WB) assay are generally adequate for the definitive diagnosis. Approved or accepted methods for WB diagnosis of TSEs requires the use of fresh or frozen nonfixed tissue samples, whereas formalin-fixed, paraffin-embedded tissue is required for the localization of PrP Sc by IHC. Because disparate processing methods are used for these accepted diagnostic techniques, separate tissue samples are collected from the same animal. Occasions arise in which there is either insufficient quantity of tissue available to complete analysis by both techniques or initial tissue processing is incompatible with one of the assays. Also, results between the assays may differ because of the vagaries of sampling, especially in case material that contains moderate-to-low levels of PrP Sc. The present article describes a method to conduct a WB assay from the same paraffin-embedded brainstem sample used for the IHC diagnosis of experimentally induced sheep scrapie.