Characterization of β-catenin expression in canine osteosarcoma

Osteosarcoma (OSA) is the most frequently occurring malignant primary bone tumour in dogs and children and arises from cells of the osteoblast lineage. Inappropriate Wnt signalling activity has been implicated in human OSA. Altered expression of β-catenin, an integral member of the Wnt signalling pathway, has been associated with numerous human cancers, including OSA. In this study, 30 of the 37 primary canine OSA tissues and 2 of the 3 metastatic OSAs were positive for β-catenin expression as determined by immunohistochemistry, whereas 2 normal bones stained negative for β-catenin. No mutations were identified in exon 3 of β-catenin in the three OSA cases in which DNA sequencing was performed. Finally, there was no relationship between β-catenin expression and overall survival time or disease-free interval. Our results indicate β-catenin is frequently expressed within the cytoplasm of neoplastic cells in canine OSA but contains no detectable mutations in exon 3, similar to human OSA.     

The advantages of using a combination of LDL and glutamine in comparison with TRIS egg yolk and Equex® STAMP extenders in the cryopreservation of canine semen

Twenty semen samples taken from 5 dogs were frozen in liquid nitrogen at ?196 °C in four different extenders: one control extender based on 20% egg yolk, 6% LDL alone (low density lipoproteins: the active cryoprotective principle in chicken egg yolk), 6% LDL combined with 20 mmol glutamine, and Equex® (a reference extender that we wish to compare with the LDL-glutamine combination). After thawing, spermatozoal motility was evaluated using a HAMILTON THORNE CERROS 12 image analyzer; the percentage of motile spermatozoa was 27.7% in the egg yolk extender (p < 0.05), 49.9% with 6% LDL alone (p > 0.05), 54.7% in the 6% LDL + 20 mmol glutamine extender, and 47.9% with Equex® (p > 0.05). The motility parameters (VAP, VCL, VSL and ALH) were also superior in the 6% LDL + 20 mmol glutamine extender in comparison with the other extenders.Finally, the spermatozoa were generally better protected during freezing with the 6% LDL + 20 mmol glutamine association than with the egg yolk, 6% LDL, or Equex extenders in terms of the flagellar plasma membrane (HOS test), DNA (Acridine orange test), and acrosome integrity (Spermac® test: no significant difference). The Equex® extender obtained the best results for the acrosome, followed by 6% LDL + 20 mmol glutamine (FITC-PSA test: p < 0.05 between each extender).     

Rearrangement patterns of the canine TCRγ locus in a distinct group of T cell lymphomas

The T cell antigen receptor chains are assembled through a rearrangement process that combines variable (V), diversity (D) and joining (J) region genes. Recently, the entire canine T cell receptor γ (TRG) locus was described. It is arranged in 8 cassettes with up to 3 V genes, 2 J genes and 1 C gene each. However, no data is available beyond the level of sequence analysis. The objective of this study was to identify rearranged genes of the canine TRG locus through experimental analysis and to assess gene usage and patterns of rearrangement in a series of canine T cell lymphomas. Rearranged genes were identified through computational analysis of recombination signal sequences (RSSs), a gene's potential to generate a polyclonal smear, and through sequencing of clonal rearrangements in a series of T cell lymphomas. Out of a total of 32 Vγ and Jγ genes, 21 genes were found to rearrange, 8 genes were considered not rearranged and 3 genes were suspected to rearrange but their status could not be determined definitely. Rearrangements of the canine TRG locus were assessed in a group of canine T cell lymphomas as well as 3 neoplastic T cell lines. An average of 4.6 rearrangements per lymphoma was found suggesting that canine T cells routinely rearrange multiple cassettes per allele. The most commonly rearranged Vγ genes belonged to subgroups Vγ2, Vγ3, and Vγ7. Genes in cassettes 2 and 3 preferentially rearranged within their respective cassettes, while Vγ genes in cassette 7 rearranged to a Jγ gene in cassette 8. There was a strong preference for Vγ2 genes to rearrange to a 3' Jγ gene and for Vγ3 and Vγ7 genes to rearrange to a 5' Jγ gene. This rearrangement pattern coincided with the conservation of the spacer sequence between V and J gene subgroups rather than the topologic location of genes. These data show that highly divergent spacer sequences allow for equally efficient recombination and suggest that spacer sequences can mediate compatibility between V and J genes.     

New molecular targets in canine hemangiosarcoma—Comparative review and future of the precision medicine

Human angiosarcoma and canine hemangiosarcoma reveal similarities not only in their aggressive clinical behaviour, but especially in molecular landscape and genetic alterations involved in tumorigenesis and metastasis formation. Currently, no satisfying treatment that allows for achieving long overall survival or even prolonged time to progression does not exist. Due to the progress that has been made in targeted therapies and precision medicine the basis for a new treatment design is to uncover mutations and their functions as possible targets to provide tailored drugs for individual cases. Whole exome or genome sequencing studies and immunohistochemistry brought in the last few years important discoveries and identified the most common mutations with probably crucial role in this tumour development. Also, despite a lack of mutation in some of the culprit genes, the cancerogenesis cause may be buried in main cellular pathways connected with proteins encoded by those genes and involving, for example, pathological angiogenesis. The aim of this review is to highlight the most promising molecular targets for precision oncology treatment from the veterinary perspective aided by the principles of comparative science. Some of the drugs are only undergoing laboratory in vitro studies and others entered the clinic in the management of other cancer types in humans, but those used in dogs with promising responses have been mentioned as priorities.     

Incidence of canine rabies in N'Djaména, Chad

This work describes for the first time the incidence risk of passively reported canine rabies, and quantifies reported human exposure in N'Djaména (the capital of Chad). To diagnose rabies, we used a direct immunofluorescent-antibody test (IFAT). From January 2001 to March 2002, we were brought 34 rabies cases in dogs and three cases in cats. Canine cases were geographically clustered. The annual incidence risk of canine rabies was 1.4 (95% CI: 1.2, 1.7) per 1000 unvaccinated dogs. Most of the rabid dogs were owned-although free-roaming and not vaccinated against rabies. Most showed increased aggressiveness and attacked people without being provoked. Eighty-one persons were exposed to rabid dogs and four persons to rabid cats (mostly children<15 years old). Most of the exposed persons were neighbours or family members of the animal owner. Most exposures were transdermal bites, but nearly half of all exposed persons did not apply any first wound care or only applied a traditional treatment. In N'Djaména, humans are often exposed to canine rabies but do not use the full-course post-exposure treatment and wound care is insufficient. Most rabid dogs would be accessible to parenteral vaccination. Pilot vaccination campaigns are needed to determine the success of dog mass vaccination in N'Djaména as a way to prevent animal and human rabies.     

The influence of trilostane on steroid hormone metabolism in canine adrenal glands and corpora lutea—an in vitro study

Trilostane is widely used to treat hyperadrenocorticism in dogs. Trilostane competitively inhibits the enzyme 3-beta hydroxysteroid dehydrogenase (3β-HSD), which converts pregnenolone (P5) to progesterone (P4) and dehydroepiandrosterone (DHEA) to androstendione (A4). Although trilostane is frequently used in dogs, the molecular mechanism underlying its effect on canine steroid hormone biosynthesis is still an enigma. Multiple enzymes of 3β–HSD have been found in humans, rats and mice and their presence might explain the contradictory results of studies on the effectiveness of trilostane. We therefore investigated the influence of trilostane on steroid hormone metabolism in dogs by means of an in vitro model. Canine adrenal glands from freshly euthanized dogs and corpora lutea (CL) were incubated with increasing doses of trilostane. Tritiated P5 or DHEA were used as substrates. The resulting radioactive metabolites were extracted, separated by thin layer chromatography and visualized by autoradiography. A wide variety of radioactive metabolites were formed in the adrenal glands and in the CL, indicating high metabolic activity in both tissues. In the adrenal cortex, trilostane influences the P5 metabolism in a dose- and time-dependent manner, while DHEA metabolism and metabolism of both hormones in the CL were unaffected. The results indicate for the first time that there might be more than one enzyme of 3β-HSD present in dogs and that trilostane selectively inhibits P5 conversion to P4 only in the adrenal gland.     

Mycoplasma haemocanis – the canine hemoplasma and its feline counterpart in the genomic era

Mycoplasma haemocanis is a hemotrophic mycoplasma (hemoplasma), blood pathogen that may cause acute disease in immunosuppressed or splenectomized dogs. The genome of the strain Illinois, isolated from blood of a naturally infected dog, has been entirely sequenced and annotated to gain a better understanding of the biology of M. haemocanis. Its single circular chromosome has 919 992 bp and a low G + C content (35%), representing a typical mycoplasmal genome. A gene-by-gene comparison against its feline counterpart, M. haemofelis, reveals a very similar composition and architecture with most of the genes having conserved synteny extending over their entire chromosomes and differing only by a small set of unique protein coding sequences. As in M. haemofelis, M. haemocanis metabolic pathways are reduced and apparently rely heavily on the nutrients afforded by its host environment. The presence of a major percentage of its genome dedicated to paralogous genes (63.7%) suggests that this bacterium might use antigenic variation as a mechanism to evade the host’s immune system as also observed in M. haemofelis genome. Phylogenomic comparisons based on average nucleotide identity (ANI) and tetranucleotide signature suggest that these two pathogens are different species of mycoplasmas, with M. haemocanis infecting dogs and M. haemofelis infecting cats.     

Owner complaints of canine behavior in Iran–A preliminary survey

The current survey assessed 17 common behavioral complaints reported by 234 dog owners attending the Small Animal Hospital, University of Tehran using a questionnaire. According to the results, nearly 95% of dog owners reported at least 1 undesirable behavior in their dogs. Aggression toward other dogs and people, jumping up, and excessive barking were the most common behavioral complaints. Owners of large-breed dogs were more likely to complain about their dogs’ aggressive behaviors toward people/dogs and excessive barking compared with owners of small-breed dogs. Dogs without access to a yard were more likely to show phobia-associated behaviors, fearfulness, and separation-related behaviors, whereas those having access to a yard were more likely to show aggressive behaviors toward people/dogs and excessive barking. Interaction of dogs with people and type of reaction used by owners in response to the behavior were also related with some other behavioral characteristics of the animals.     

Sero-epidemiological study of canine Leishmania spp. infection in the municipality of Alijó (Alto Douro, Portugal)

Visceral leishmaniosis caused by Leishmania infantum is a zoonotic disease in the Mediterranean basin. We report an epidemiological survey carried out in dogs from the municipality of Alijó in the endemic region of Alto Douro (north Portugal). Performance of the direct agglutination test (DAT) was assessed in 205 matching samples of blood collected on filter paper and serum. A high degree of agreement (97.6%; k = 0.83) was found between the results obtained from both types of samples. DAT was then used to test more blood on filter paper (B-FP) samples from other dogs of the same municipality. The detected sero-prevalence was 18.7% (288/1540), with values ranging from 0.0 to 81.1% in each of the 19 parishes of Alijó. Three distinct geographical zones of mean sero-prevalence could be defined: northwestern (2.5%), intermediate (11.4%) and southern (49.9%). No statistically significant difference was observed between male (19.1%) and female (17.8%) sero-prevalences (P = 0.560). Dogs of 9-11 years of age showed the highest sero-prevalence (28.4%), but all the other age-intervals (0-2, 3-5, 6-8 and 12-17 years) presented values (15.0-22.3%) not significantly different from the mean of the whole study population. Risk factors for canine Leishmania infection were age and geographical zone. Only 5.9% of the sero-positive animals had clinical signs of canine leishmaniosis and the overall prevalence of disease was 1.1%. This study validates the use of B-FP samples and confirms DAT as a simple and sensitive serological test to evaluate the level of canine Leishmania infection in areas of high sero-prevalence.     

The role of ion transport in the formation of sub‐embryonic fluid by the embryo of the Japanese quail

1. Using a modification of the method of New (1955) explanted blastoderms of the Japanese quail were cultured for up to 72 h, and the role of ions in the formation of sub‐embryonic fluid (SEF) investigated.

2. Culture media deficient in either sodium or chloride ions reduced the volume of SEF secreted by up to a quarter. Ionic composition of the fluid was little altered, and sodium was transported against a concentration gradient.

3. Amiloride, an inhibitor of Na⁺/H⁺ exchange, reduced SEF production by half, whereas ionic composition and osmolality of the fluid was unchanged.

4. Likewise, acetazolamide, an inhibitor of carbonic anhydrase, reduced SEF production by the blastoderm, and left the ionic composition and osmolality of the fluid unaltered.

5. Neither furosemide or 4,4´‐diisothiocyanatostilbene‐2,2´‐disulfonic acid at the concentrations used changed the volume of SEF formed or its ionic composition.

6. It is concluded that the secretion of SEF is dependent upon the active transport of sodium across the blastoderm; an amiloride‐sensitive Na⁺/H⁺ exchanger and carbon dioxide hydration catalysed by carbonic anhydrase are also involved.

7. Furthermore, it is proposed that fluid transport across the blastoderm is the result of local osmotic gradients, not from a sub‐embryonic fluid hyperosmotic to albumen, as has been suggested previously.     

An immunogold labelling method for the enumeration of canine T‐lymphocytes

Summary

An immunogold procedure using the monoclonal antibody F3–20–7 to canine Thy‐l has been used to label T‐lymphocytes in peripheral blood samples taken from healthy Beagles. By this method, approximately 64% of peripheral blood lymphocytes were identified as T‐lymphocytes.     

Analysis of blood clotting factor activities in canine Legg-Calvé-Perthes' disease

Legg-Calve-Perthes' (LCP) disease is a noninflammatory aseptic necrosis of the femoral head and neck in small-breed dogs. The etiology of the disease is not known, but ischemia resulting from vascular compression or occlusion has been proposed. A latent ischemic phase during development of the femoral epiphysis seems to be responsible for the onset of the typical clinical features of LCP disease. Ischemia might result from insufficient oxygen supply either caused by a reduced number of afferent arterial vessels or an occlusion of the efferent venous vessels by thrombosis. In humans, LCP disease has been linked to hypercoagulability and hypofibrinolysis caused by deficiencies of protein C, protein S, or resistance to activated protein C. To determine whether canine LCP disease is caused by similar deficiencies, we determined protein C, protein S, activated protein C, factor II, factor V, factor VIII:C, and AT III activities in plasma samples of 18 dogs with clinically and histopathologically verified LCP disease. All dogs had normal plasma activities of these factors, indicating that in these dogs LCP disease was not caused by deficiencies of the analyzed blood clotting factors.     

Delayed embolization of an Amplatz® canine duct occluder in a dog

A 5-year old, 5.8 kg, castrated male Pomeranian was diagnosed with a type IIa patent ductus arteriosus (PDA) with a minimal ductal diameter of 3.5 mm and ampulla width of 7.1 mm based on angiographic assessment. A 6 mm Amplatz^® Canine Duct Occluder (ACDO) was deployed within the PDA. Once deployed, the device assumed it's native shape and back-and-forth maneuvering was performed with the delivery cable to assess device stability. Device position and complete occlusion were confirmed with both angiography and transesophageal echocardiography prior to and after release of the device. The device location was confirmed within the ductus arteriosus by echocardiography prior to discharge. The dog was discharged with instructions for strict activity restriction. Two days after discharge, the dog was left unsupervised in the backyard and shortly afterwards was found coughing with severe respiratory distress. The dog was evaluated at an emergency hospital and thoracic radiographs documented embolization of the ACDO to the main pulmonary artery along with a severe alveolar pattern throughout the right lung fields. Shortly after obtaining thoracic radiographs, the dog experienced cardiopulmonary arrest with unsuccessful resuscitation. This case describes a possible complication of transcatheter PDA occlusion with an ACDO, which has not been previously reported. An incident report, or catalog of adverse events with these devices, may prove useful in identifying additional fatal complications that others may have encountered, but are not reported in the literature. The report of this complication emphasizes the importance of strict activity restriction after device placement in dogs.