Different biological behaviour of Waldenström macroglobulinemia in two dogs

Waldenström Macroglobulinemia is a low‐grade immunosecretory disorder associated with lymphoid tumours, which is rarely reported in veterinary medicine. In this study, we describe two clinical cases of this rare syndrome in dogs, each characterized by a different onset and clinical course. In one case, a hyperacute onset and aggressive behaviour of the neoplasm was observed. Absolute serum viscosity (SV) was retrospectively evaluated in order to explain clinical findings. Rotational viscosimetry showed good precision in measuring SV. Both dogs had SV values higher than a control groups of healthy dogs although only one subject developed hyperviscosity symptoms and complications. At high paraprotein concentrations, a slight reduction of the M‐component was associated with a marked decrease in SV. Thus, this work suggests that SV assessment is a relevant tool for managing monoclonal gammopathies, whose usefulness should be further confirmed in larger cohorts of dogs.     

Validation of a human immunoturbidimetric assay to measure canine albumin in urine and cerebrospinal fluid.

The aim of this study was to validate an automated immunoturbidimetric assay used to quantify human albumin in urine and to accurately measure canine albumin concentrations in both urine and cerebrospinal fluid. The partial homology existing between human and canine albumin limited the accuracy of the human assays in measuring canine albumin without method modifications. Thus, the assay was modified by calibrating the analyzer with calibrators made in the laboratory containing known concentrations of canine albumin. To prepare the set of calibrators, the albumin concentration of pooled sera of healthy dogs was assessed in 5 replicates using the BromocresolGreen assay. Pooled samples were aliquoted and serially diluted to obtain the expected concentrations of albumin (0.5, 1, 5, 13, and 30 mg/dl) for establishing the canine calibration curve. Thereafter, the performance was assessed by analyzing canine urine and CSF The modified assay accurately quantified canine albumin in both specimens, as indicated by the following. Intra- and interassay variability was 0.92% and 2.74%, respectively; recovery was 99.66% and 99.07% in urine and 105.02% in CSF No interference was detected when hemolysate and glucose were added to urine. The test was linear within the verified range (0-225 mg/dl). These results demonstrate that the modified human albumin immunoturbidimetric assay can be a useful tool in the veterinary diagnostic laboratory. It is accurate and tends itself to automatization on chemistry analyzers.     

Anti‐microbial Susceptibility of Streptococcus spp. Isolated from Bovine Mastitis in Argentina

The in vitro susceptibility to penicillin G, erythromycin and clindamycin was determined by the disc diffusion test and by E‐test for a total of 47 streptococcal strains (three Streptococcus uberis, 36 Streptococcus agalactiae, eight Streptococcus dysgalactiae spp. dysgalactiae) isolated from bovine intramammary infections in Argentina. Moreover, resistance phenotypes of erythromycin‐resistant streptococcal isolates was characterized. MIC₉₀ of penicillin G, erythromycin and clindamycin for S. agalactiae were 0.75, 8.0 and 12.0 μg/ml respectively. Resistance to erythromycin and clindamycin was detected in 13 (27.6%) and 12 (25.5%) isolates respectively. No isolate was resistant to penicillin G. Resistance against macrolides, lincosamides and streptogramin B (MLS_B) represented by the constitutive MLS_B phenotype was present in 11 (23.4%) erythromycin‐resistant isolates and two isolates (4.3%) expressed the M phenotype. The inducible MLS_B phenotype was not identified. Results suggest that beta‐lactams are the first‐line antibiotics when treating streptococcal udder infections; however, the continuous monitoring of the antibiotic resistance is essential, as the emergence of resistant strains has become a growing concern on the therapy of bovine mastitis.     

The use of COLD‐PCR,DHPLC and GeneScanning for the highly sensitive detection of c‐KIT somatic mutations in canine mast cell tumours

The conventional polymerase chain reaction (PCR)/sequencing methods may be poorly suited for the detection of somatic mutations in canine mast cell tumour (MCT) samples owing to limited sensitivity. This study was aimed at establishing novel and more sensitive methods, assessing their limit of detection and comparing their sensitivity with conventional methods.Two different ‘driver’ somatic mutations of c‐KIT, together with the wild‐type counterparts, were cloned in plasmids to prepare standard samples with known concentrations of mutated alleles in a background of wild‐type alleles; the plasmids standards were assayed using either conventional or novel, highly sensitive technique. Conventional PCR/sequencing showed a sensitivity of 50–20%. Conversely, all the novel methods obtained higher sensitivities allowed reaching as low as 2.5–1.2% of the mutated DNA.The study demonstrates that early conventional methods could likely have underestimated the prevalence of KIT mutations of MCTs, therefore affecting the assessment of their relevance in prognosis and tyrosine kinase inhibitor (TKI) treatment effectiveness.     

Procalcitonin gene expression after LPS stimulation in the porcine animal model

Procalcitonin (PCT), recognised as a marker of sepsis, was investigated in a porcine model of endotoxic shock. The results showed that continuous IV infusion (1-4 h) of LPS (40 μg/kg) in pigs was able to induce a generalised increase of PCT expression in lung, heart, kidney and liver. The increase in PCT was significant only in kidney and was accompanied by an increase in IL-6 gene expression. In vitro results demonstrated that peripheral blood mononuclear cells (PBMCs), as well as endothelial cells, were potentially capable of contributing to in vivo extrathyroidal PCT production. These findings support previous data from pigs concerning the occurrence of widespread activation of PCT extrathyroidal gene expression during endotoxic shock in pigs. Nevertheless, the levels of PCT detected were very low, suggesting the need for additional studies to validate the pig as a reliable animal model for investigating the role of PCT in sepsis.     

Mitochondrial DNA variation of the dog hookworm Ancylostoma caninum in Brazilian populations

The mitochondrial cytochrome oxidase I gene was partially sequenced for 164 Ancylostoma caninum individuals, originating from five different localities in Brazil, with the aim of describing the genetic diversity and genetic structure of Brazilian hookworm populations. Allelic and nucleotide diversity were moderate (overall h=0.88 and pi=0.016) and were similar among cities. There was moderate genetic differentiation among the populations sampled (approximately Phi(ST)=0.12) and a weak but nonsignificant correlation between geographical and genetic distance. This genetic structure was similar to that observed among populations of the human hookworm, Necator americanus, but distinct from that typically found in trichostrongylid nematode parasites of livestock. Thus, a pattern of different genetic structures among different groups of nematodes is emerging. We also observed a few individuals that had a highly divergent mtDNA sequence (almost 7% sequence divergence from the other sequences). These results in combination with data from other studies suggest that A. caninum populations worldwide consist of a mix of previously differentiated populations, or perhaps even cryptic species. This study contributes to the knowledge of genetic structure and diversity of hookworms, which in turn will be useful in developing methods for their control.     

Treatment of oral squamous cell carcinoma in a horse by surgical debulking followed by metronomic chemotherapy

A 19‐year‐old Quarter Horse gelding presented with a 6‐week history of hypersalivation, halitosis and dysmasesis. Oral examination revealed retention of food and saliva and the presence of a raised, nodular, 6 × 7 cm, ulcerated mass on the dorsal surface of the tongue base. The mass was confirmed histologically as squamous cell carcinoma. Complete resection of the mass was not possible and surgical laser debulking was followed 15 days later by chemotherapy with a combination of meloxicam and cyclophosphamide in metronomic regimen. After one week, there was a significant improvement in clinical signs and food consumption returned to normal. Therapy was well tolerated with no alteration in haematological or urinalysis parameters. After 5 months of excellent life quality, the horse showed progressive difficulties in mastication and swallowing. Endoscopic examination showed extension of the tumour to all the aboral aspect of the tongue and, with the owner's consent, the patient was subjected to euthanasia. This is believed to be the first report on the combined use of meloxicam and cyclophosphamide in a metronomic fashion for management of an oral squamous cell carcinoma in a horse. Since metronomic therapy is less expensive than conventional chemotherapy, easily administrable and well tolerated, it should be considered as a possible treatment option for nonresectable equine malignant tumours.     

Differential diagnosis of dog hookworms based on PCR-RFLP from the ITS region of their rDNA

Species of Ancylostoma infecting dogs and sometimes humans are sympatric in many parts of the world. The establishment of a specific molecular diagnostic tool is important, not only to refine information for epidemiological studies, but also to evaluate the efficacy of vaccine programmes and assist in the development of specific drug treatments. The ITS region from 20 specimens of A. braziliense, collected from three separate geographical areas of Brazil, and from 10 specimens of A. caninum, collected from the same area in Brazil were sequenced and analyzed. Alignment of sequences showed that this gene is highly conserved. The intraspecific polymorphism for both species was less then 1%, whereas the interspecific polymorphism was 6.2, 7.3 and 9.4% between A. ceylanicum and A. braziliense; A. caninum and A. ceylanicum and A. ceylanicum and A. braziliense, respectively. Among the three species it was 12.3%. This revealed the ITS region as highly conserved and consequently a good molecular marker for diagnostic studies. In this work, four restriction enzymes were used in a PCR-RFLP using the ITS region of rDNA, to establish a differential diagnosis which discriminates between three Ancylostoma species, A. braziliense, A. caninum and A. ceylanicum. The best pattern was given by the HinfI enzyme, which produced different fragment sizes for each of the three species. Furthermore, the diagnostic tool differentiates DNA extracted directly from faeces of Ancylostoma-infected dogs.     

GeneScanning analysis of Ig/TCR gene rearrangements to detect clonality in canine lymphomas

The diagnosis of canine lymphoma is achieved using morphological and immunological methods. In a certain percentage of cases, difficulties in making a definitive diagnosis of lymphoproliferative disorders may occur despite extensive immunophenotyping. Therefore, additional diagnostics, such as molecular assessment of Ig/TCR gene rearrangements clonality, may confirm the final diagnosis. Polyacrylamide gel electrophoresis and heteroduplex analysis have already been proven to be suitable for detecting clonality but are cumbersome and labor-intensive. In the present study, GeneScanning analysis of PCR products originating from different primer sets targeting different regions of Ig and TCR was validated in improving sensitivity as well as in reducing the turnaround time of gene rearrangement assays. GeneScanning exploits 5' fluorescently labelled primers for the automated and fast analysis of PCR products either as singleplex or multiplex runs. Initially, the assay was set up using DNA purified from normal tissues (n=6), hyperplastic/reactive tissues (n=10) and a small set of immunophenotyped lymphoma samples (n=12). The optimized methods were then used in a large set of 96 canine lymphoma samples. Normal and hyperplastic/reactive lymphoid tissues showed typically polyclonal or, occasionally, oligoclonal PCR products. Lymphoma samples showed monoclonal peaks arranged as a single or, occasionally, a double narrow base peak sometimes embedded in a polyclonal background. In all immunophenotyped cases, an Ig or TCR clonal finding corresponded to B- and T-cell lymphomas, respectively. Overall, 94/96 (97.9%) samples showed clonal Ig/TCR clonal rearrangements among which clonal Ig was found in 61/96 (63.5%) of samples and clonal TCR in 33/35 Ig negative samples (34.4% of all cases). In one out of ten randomly chosen cases, both Ig and TCR clonal gene rearrangements were found. Among the factors affecting assay accuracy, DNA quality has been shown to be critical and the amplification of DNA controls of different size are recommended to evaluate DNA integrity. Frozen material such as that which remained inside the hub of the needle used for diagnostic procedures is optimal for the analysis herein described. In conclusion, GeneScanning represents a versatile tool for routinely assessing Ig/TCR clonal rearrangements and supporting the diagnostic protocol of canine lymphomas.