Laboratory and Clinical Evaluation of a Feia Method for Canine Serum Progesterone Assay.

The evaluation of progesterone (P4) concentration is a valuable tool in assessing physiological reproductive events and reproductive disorders in bitches. A reliable and rapid (preferable, point of care) determination of P4 is advisable in most cases. Aims of this study were to evaluate a fluorescence enzyme immunoassay (FEIA) for canine serum P4 concentration by (i) the agreement with liquid chromatography-tandem mass spectrometry (LC/MS/MS), (ii) the association with vaginal cytology and (iii) the accuracy in the prediction of the parturition date calculated from the estimated day of ovulation. Serum samples were collected from client-owned bitches presented between 2011 and 2014 for the evaluation of their oestrous cycle, pregnancy or reproductive disorders. The agreement between FEIA and LC/MS/MS, evaluated on 19 samples, was statistically significant (R(2) = 95.7%, p < 0.001), although FEIA showed significantly higher values than LC/MS/MS (p < 0.05). In the different phases of oestrous cycle, as determined by vaginal cytology, P4 concentrations (by FEIA) were statistically different (p < 0.05): anoestrus (n = 7) 0.38 ± 0.14 ng/ml, proestrus (n = 14) 1.04 ± 0.67 ng/ml and oestrus (n = 72) 6.8 ± 7.26 ng/ml. Mean pregnancy length from the estimated day of ovulation was 62.9 ± 1.8 days. In 13 of 22 (59.1%), 19 of 22 (86.3%) and 21 of 22 (95.5%) bitches pregnancy lasted 63 ± 1, 63 ± 2 and 63 ± 3 days, respectively. Three pregnancies were outside the 61-65 days range (60, 60 and 67 days). In conclusion, the FEIA method employed can be considered reliable and, in association with vaginal cytology, effective in evaluating the canine oestrous cycle.

The erythrocyte sedimentation rate (ESR) in canine inflammation.

The clinical application of the erythrocyte sedimentation rate (ESR) assay is challenging due to its long processing time. However, in 2020 a new automated instrument for veterinary ESR was released and validated. This study sought: (1) to refine the proposed reference range (reference interval, RI) for canine ESR; (2) to compare the ESR values of healthy and sick dogs; and (3) to correlate ESR with other inflammatory markers such as C-reactive protein (CRP), fibrinogen, albumin:globulin ratio (A/G), and neutrophil-to-lymphocyte ratio (NLR); and also (4) to study ESR behavior across illnesses of varying durations. A prospective cohort study of 396 client-owned dogs (n = 120 healthy and n = 276 sick dogs) was conducted. Animals with a full clinical evaluation, complete hematobiochemical profile and a final diagnosis were included. ESR was performed according to manufacturer's instructions using the same 1 mL K3-EDTA tube used for the complete blood count. The RI was established at 1-8 mm/h in 14 min. Sick dogs had a significantly faster ESR (median 10 mm/h) than healthy dogs (median 1 mm/h; P < 0.0001). ESR was positively correlated with NLR (r = 0.36), CRP (r = 0.18) and fibrinogen (r = 0.56) and negatively correlated with A/G (r = -0.37). Dogs with an acute-on-chronic disease had the highest ESR values (median 17 mm/h) compared with either acute (median 11 mm/h; P < 0.001) or chronic diseases (median 7 mm/h; P = 0.001). ESR was confirmed as a reliable canine inflammatory marker, positively correlating with the main inflammatory markers in dogs and significantly different between sick and healthy dogs. The ESR assay appears useful especially in dogs with an acute clinical presentation, with or without an underlying chronic condition.

Neutrophil-to-lymphocyte ratio, nucleated red blood cells and erythrocyte abnormalities in canine systemic inflammatory response syndrome.

Systemic inflammatory response syndrome (SIRS) is the manifestation of the systemic response to an infectious or non-infectious disease. We evaluated the association between erythrocyte parameters, including nucleated red blood cells (NRBCs) and leukocyte ratios (NLR, neutrophil-to-lymphocyte ratio; BLR, band neutrophil-to-lymphocyte ratio; BLNR, band neutrophil-to-neutrophil-to-lymphocyte ratio). A review of the medical records was conducted searching SIRS dogs among those admitted to our intensive care unit and a SIRS grading was obtained based on how many criteria were fulfilled. The Acute Patient Physiology and Laboratory Evaluation (APPLEfast) score was assessed in each dog. Survival rate was assessed 15 days after admission. Dogs with clinical and/or clinicopathological signs of hemolytic or hemorrhagic disorders were excluded. Dogs with ≥2 criteria of SIRS along with a documented underlying infectious cause were recorded as septic (32/90, 35%). A SIRS grading >2 (p = .001) and an APPLEfast score > 25 (p = .03) were associated with mortality. Twenty-two of SIRS dogs (24%) showed circulating NRBCs. The occurrence of circulating NRBCs was associated with the mortality in SIRS groups (p = .0025). The median NLR was 11.69 and NLR was lower in septic dogs compared to non-septic ones (p = .0272). APPLEfast, SIRS grading and circulating NRBCs may be considered as negative prognostic factors in canine SIRS. NLR could be a useful tool in dogs with SIRS, which was significantly lower in the septic group. Further prospective, large-scale studies investigating BLR and BNLR in canine SIRS are warranted.

Overexpression of HER-2 via immunohistochemistry in canine urinary bladder transitional cell carcinoma - A marker of malignancy and possible therapeutic target.

Transitional cell carcinoma (TCC) is the most commonly diagnosed neoplasm in the urinary bladder. Distant metastases to the regional lymph nodes, lungs, abdominal organs or bones are noted in up to 50% of dogs at time of death. Surgical excision is often not practical as TCC typically involve the trigone of the bladder and/or occurs multifocally throughout the bladder with field cancerization. Therapeutic approaches are very challenging and the requirement to evaluate alternative therapeutic protocols that may prolong survival times in dogs bearing these tumours is compelling. We assessed the immunohistochemical expression of HER-2 in 23 cases of canine TCCs of the urinary bladder and compare it with non-neoplastic urothelium in order to evaluate a rationale for targeted therapies and gene-based vaccines. HER-2 positivity was recorded in 13/23 (56%) neoplastic lesions. The receptor was significantly overexpressed in neoplastic than in non-neoplastic samples (P = .015). According to our preliminary results, it would be of interest to further evaluate the role of HER-2 in canine TCCs as a marker of malignancy and a therapeutic target for cancer vaccine and antibodies. Moreover, the significantly different overexpression of HER-2 in TCCs than in non-neoplastic urothelium further supports to investigate its role in the progression toward malignancy of non-neoplastic lesions.

Faecal microbiota in dogs with multicentric lymphoma.

Malignant lymphoma B-cell type is the most common canine haematopoietic malignancy. Changes in intestinal microbiota have been implicated in few types of cancer in humans. The aim of this prospective and case-control study was to determine differences in faecal microbiota between healthy control dogs and dogs with multicentric lymphoma. Twelve dogs affected by multicentric, B-cell, stage III-IV lymphoma, and 21 healthy dogs were enrolled in the study. For each dog, faecal samples were analysed by Illumina sequencing of 16S rRNA genes and quantitative PCR (qPCR) for selected bacterial groups. Alpha diversity was significant lower in lymphoma dogs. Principal coordinate analysis plots showed different microbial clustering (P = .001) and linear discriminant analysis effect size revealed 28 differentially abundant bacterial groups in lymphoma and control dogs. The qPCR analysis showed significant lower abundance of Faecalibacterium spp. (q < .001), Fusobacterium spp. (q = .032), and Turicibacter spp. (q = .043) in dogs with lymphoma compared with control dogs. On the contrary, Streptococcus spp. was significantly higher in dogs with lymphoma (q = .041). The dysbiosis index was significantly higher (P < .0001) in dogs with lymphoma. In conclusion, both sequencing and qPCR analyses provided a global overview of faecal microbial communities and showed significant differences in the microbial communities of dogs presenting with multicentric lymphoma compared with healthy control dogs.

The treatment of canine mast cell tumours with electrochemotherapy with or without surgical excision.

To describe the results of electrochemotherapy (ECT) in dogs with mast cell tumours (MCTs) either as first line therapy or as an adjuvant to surgery. The treatment combines administration of low dose chemotherapeutic drugs with the application of microsecond electric pulses, which cause the temporary permeabilization and increased porosity of the tumour cell membranes. The design of this study is a retrospective case series. A total of 51 dogs with MCTs were included and classified according to ECT procedure into 4 groups (ECT only, 15 cases, intra-surgery ECT, 11, ECT Adjuvant to surgery, 14, Surgery followed by ECT, 11). The four groups (staged with location, size and grade) were evaluated to assess complete or partial remission, disease free interval, overall survival time and local toxicity. In this case series, Boxers, mixed breed and Labrador Retrievers, male dogs, between 4 and 9 years old were more represented. MCTs were predominantly grade 2 (Patnaik) and T stage 0-1, I-1 (World Health Organization). Treated lesions were most commonly identified on the hindlimb and head where curative surgery would involve cosmetic or functional compromise. The intra-surgery group of dogs showed the best disease free interval with Kaplan-Meyer analysis. Local toxicity induced by ECT ranged mostly from 1 to 4 in a 5-point arbitrary scale with 0 - no toxicity to 5 - highest toxicity. In this study, ECT can be applied successfully as an exclusive therapy in smaller MCTs as an alternative to surgery. ECT can be combined with surgery either intra-operatively or post operatively for larger lesions without significant toxicity.

Conformity and controversies in the diagnosis, staging and follow-up evaluation of canine nodal lymphoma: a systematic review of the last 15 years of published literature.

Diagnostic methods used in the initial and post-treatment evaluation of canine lymphoma are heterogeneous and can vary within countries and institutions. Accurate reporting of clinical stage and response assessment is crucial in determining the treatment efficacy and predicting prognosis. This study comprises a systematic review of all available canine multicentric lymphoma studies published over 15 years. Data concerning diagnosis, clinical stage evaluation and response assessment procedures were extracted and compared. Sixty-three studies met the eligibility criteria. Fifty-five (87.3%) studies were non-randomized prospective or retrospective studies. The survey results also expose variations in diagnostic criteria and treatment response assessment in canine multicentric lymphoma. Variations in staging procedures performed and recorded led to an unquantifiable heterogeneity among patients in and between studies, making it difficult to compare treatment efficacies. Awareness of this inconsistency of procedure and reporting may help in the design of future clinical trials.

Validation of an automated spectrophotometric assay for the determination of cholinesterase activity in canine serum.

The determination of enzymatic activity of cholinesterase is a useful diagnostic method to detect exposure to anticholinesterase compounds in human and in veterinary medicine. We validated a modification of the Ellman method in canine serum and applied it to the diagnosis of dogs poisoned with anticholinesterase substances. The method used butyrylthiocholine as substrate and potassium hexacyanoferrate as chromophore. The reference range calculated on 60 clinically healthy dogs was set between 3405 and 6561 U/L (chi-square test for normal distribution, p > 0.05). The overall mean intra-assay and inter-assay coefficients of variation were 0.53% and 3.83%, respectively. The assay was linear when using two sera with 12,538 U/L and 6604 U/L serum cholinesterase activity (r(2) = 0.997) and 0.999, respectively). The mean recovery values of pooled sera with a mean pseudocholinesterase (PChE) activity of 12,081 U/L and pooled sera with a mean PChE activity of 3415 U/L were 103.5% and 102.8%, respectively. Six dogs with a diagnosis of anticholinesterase compound intoxication showed a decrease in cholinesterase activity of at least 50% of normal activity with a mean +/- SD of 487 +/- 291 U/L ranging from 169 to 847 U/L. This technique conforms to the current standard for precision, linearity and accuracy and is a useful method for the complementary diagnosis of organophosphate or carbamate insecticide intoxication in dogs.

Clinicopathological findings in naturally occurring cases of babesiosis caused by large form Babesia from dogs of northeastern Italy.

There are few extensive studies about clinicopathological findings of spontaneous canine babesiosis caused by a large form of the parasite found in Europe. To further characterize and describe clinicopathological findings in dogs affected with this large form of Babesia in northeastern Italy, we evaluated 23 Italian dogs with canine babesiosis by means of clinical history, physical examination, hematological, biochemical, hemostatic tests, serum electrophoresis and urinalysis. Seventeen dogs (74%) had recently traveled on a hunting trip (within 5-15 days of being presented to the clinic) to Bosnia and Herzegovina (n=7), to Croatia (n=8) and to Hungary (n=2). The duration of clinical signs ranged from 1 to 5 days prior to the arrival at the clinic. The main clinical signs were dehydration (100%), apathy (74%), anorexia or decrease appetite (70%) and fever (68%). The anemia was present in 74% of the dogs and classified as mild (35%), moderate (59%) and severe (6%). In all cases, the anemia was normocytic and normochromic. Only three dogs presented erythrocyte regeneration. Seventy percent of dogs had hemolytic anemia and 30% had non-hemolytic anemia. Sixty-nine percent of dogs showed leucopenia and 74% neutropenia. Leucocitosis, due to mature neutrophilia and lymphocytosis, was present in one dog. Activated lymphocytes were noted in 61% of dogs. In all dogs, thrombocytopenia and an elevated hyperfibrinogenemia were present. Significant prolonged activated partial thromboplastin time (aPTT) was only found in one case. In four dogs, both plasma fibrinogen/fibrin degradation products (FDPs) and D-Dimer were increased. Antithrombin (AT) was slightly decreased in 11 of the 23 dogs. In the majority of cases, mild elevation of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatinekinase (CK), total bilirubin and lactic acid and decrease of total iron and total iron binding capacity (TIBC) were present. In conclusion, the main clinicopathological findings were a mild to severe thrombocytopenia, a mild to moderate hemolytic anemia, neutropenia and hyperfibrinogenemia.

Double filtration plasmapheresis in a dog with multiple myeloma and hyperviscosity syndrome.

A 12 year old, 38 kg, mix-breed, intact male dog presented with a 20 day history of clinical signs consistent with hyperviscosity syndrome secondary to multiple myeloma. The dog received three double filtration plasmapheresis treatments on day 0, 7 and 22 after presentation. A significant (p<0.05) reduction in serum total protein, alpha-2 and gamma globulins was found following each treatment. These reductions were accompanied by a complete resolution, although temporary, of the clinical signs of hyperviscosity syndrome. The present study reported for the first time the use of double filtration plasmapheresis to reduce clinical signs of hyperviscosity syndrome in a dog with multiple myeloma.

Association between canine malignant lymphoma, living in industrial areas, and use of chemicals by dog owners.

A case-control study was carried out to determine whether residential exposure to environmental pollutants increased risk for canine lymphoma in pet dogs. One hundred one cases with cytologically or histologically confirmed lymphoma diagnosed at a veterinary teaching hospital between the middle of 1996 and the middle of 1998 were examined. Controls were obtained by choosing twice the number of dogs without neoplastic disease, with overlapping distributions of province of residence, age, sex, and breed. Information regarding animal management, residence type, professional or hobby use of chemicals by owners, and treatment with herbicides or other pesticides in the area frequently visited by the dogs was obtained with a multiple-choice questionnaire by telephone interview. Two variables were positively and independently associated with the disease, namely residency in industrial areas (odds ratio [OR]; = 8.5; 95% confidence interval [CI], 2.3-30.9) and use of chemicals by owners, specifically paints or solvents (OR = 4.6; 95% CI, 1.7-12.6). A significantly lower value of the mean age of disease onset was found in the group of dogs at risk in comparison with the group of all other dogs (6.1 +/- 0.4 years, n = 36 versus 7.5 +/- 0.4 years, n = 65, respectively; P = .008). Variables describing animal care and pesticide use were either not associated with the disease or were uninformative. We suggest that canine lymphoma may be considered a sentinel of potentially hazardous situations for humans, because of the relatively short latency between exposure and disease onset.

Hemophilia A in a Belgian Shepherd Malinois dog: case report.

This case report presents a Belgian Shepherd Malinois dog affected by hemophilia A recognized at the age of seven months. The clinical follow-up including all the diagnostic procedures leading to the final diagnosis and the course of this disorder are presented. This is a typical proband case demonstrating the appearance of this genetic disease in a breed never involved by this coagulation disorder so far documented that started an intensive and laborious plan to reduce the incidence of hemophilia A and the further appearance of new cases.

Use of CD9 and CD61 for the characterization of AML-M7 by flow cytometry in a dog*.

Acute megakaryoblastic leukaemia (AML-M7) is a rare myeloproliferative disorder in domestic animals. Recently, thanks to the greater availability of immunophenotype techniques, precise diagnosis is more easily made. The morphological evaluation has its limitations, especially in the study of poorly differentiated cells. Few reports have described AML-M7 in dogs using flow cytometry. This clinical case points out the utility of flow cytometry in the characterization of AML-M7 in a 3-year-old German Shepherd dog. Flow cytometry investigation has established megakaryocytic lineage involvement by showing the presence of two megakaryocyte/platelet associated antigens (CD9 and CD61). In human medicine CD9 may be used as a platelet and megakaryocyte marker. There is an evidence of cross-reactivity of human anti-CD9 monoclonal antibody with canine samples. To our knowledge, the use of CD9 has never been described before, for this purpose in the dog.