The Italian Network of Laboratories for Veterinary Oncology (NILOV) 2.0: Improving Knowledge on Canine Tumours.

Advances in tumour research are crucial, and comparative oncology can improve the knowledge in several ways. Dogs are not only models of specific naturally occurring tumours but can also be sentinels of environmental exposures to carcinogens, as they share the same environment with their owners. The purpose of this work was to describe the data collected by The Italian Network of Laboratories for Veterinary Oncology in the first 9 years of activity (2013-2021) and to evaluate their potential epidemiological significance. Frequencies of tumour topographies and main morphologies in dogs were described, analysed and compared, calculating age-adjusted proportional morbidity ratios and considering several risk factors (breed, sex, period and region of residence). These observations allowed us to highlight differences not only in morphology and topography of some tumours but also to formulate hypotheses on the potential role of some risk factors, e.g., neutering/spaying or geographical location. In our opinion, the results of this case series confirm the importance of initiating and consolidating animal cancer registration initiatives that would facilitate the possibility of conducting multicentric collaborative studies to deepen the knowledge of the epidemiology of tumours in dogs from a comparative perspective.

A highly endemic area of Echinococcus multilocularis identified through a comparative re-assessment of prevalence in the red fox (Vulpes vulpes), Alto Adige (Italy: 2019-2020).

Surveillance of Echinococcus multilocularis at the edge of its range is hindered by fragmented distributional patterns and low prevalence in definitive hosts. Thus, tests with adequate levels of sensitivity are especially important for discriminating between infected and non-infected areas. In this study we reassessed the prevalence of E. multilocularis at the southern border of its distribution in Province of Bolzano (Alto Adige, northeastern Alps, Italy), to improve surveillance in wildlife and provide more accurate estimates of exposure risk. We compared the diagnostic test currently implemented for surveillance based on coproscopy and multiplex PCR (CMPCR) to a real-time quantitative PCR (qPCR) in 235 fox faeces collected in 2019 and 2020. The performances of the two tests were estimated using a scraping technique (SFCT) applied to the small intestines of a subsample (n = 123) of the same foxes as the reference standard. True prevalence was calculated and the sample size required by each faecal test for the detection of the parasite was then estimated. True prevalence of E. multilocularis in foxes (14.3%) was markedly higher than reported in the last decade, which was never more than 5% from 2012 to 2018 in the same area. In addition, qPCR showed a much higher sensitivity (83%) compared to CMPCR (21%) and agreement with the reference standard was far higher for qPCR (0.816) than CMPCR (0.298) meaning that for the latter protocol, a smaller sample size would be required to detect the disease. Alto Adige should be considered a highly endemic area. Routine surveillance on definitive hosts at the edges of the E. multilocularis distribution should be applied to smaller geographic areas, and rapid, sensitive diagnostic tools using directly host faeces, such as qPCR, should be adopted.

Effects of leukoreduction on storage lesions in whole blood and blood components of dogs.

BACKGROUND: Leukoreduction is a routine procedure in human transfusion medicine but is uncommon in veterinary. OBJECTIVES: To evaluate the effect of leukoreduction on the quality of canine whole blood (WB) and blood products during storage. ANIMALS: Ten canine blood donors. METHODS: This is a case series study. An amount of 450 mL of blood was collected from each dog. Five WB and 5 packed red blood cells (pRBC) bags were divided into 2 units each: leukoreduced (LR) and non-leukoreduced (nLR). RBC count, erythrocytes' mean osmotic fragility (MOF), 2,3-diphosphoglycerate (2,3-DPG), adenosine triphosphate (ATP), percentage of hemolysis, potassium (K), lactate, glucose, and cytokines were measured weekly from day of donation (T0) to day 35 (T35); pH, coagulation times, and clotting factors were evaluated at T0 and T35 from WB and in fresh frozen plasma after 1 year of storage. RESULTS: Leukoreduction showed positive effects on lactate (T35: LR WB 14.42 mmol/L SD 2.71, nLR WB 22.42 mmol/L SD 1.86, LR pRBC 20.88 mmol/L SD 2.65, nLR pRBC 36.81 mmol/L SD 2.34; P < .0001), pH (T35: LR WB 6.88 SD 0.16, nLR WB 6.69 SD 0.20, P = .02; LR pRBC 6.57 SD 0.23, nLR pRBC 6.22 SD 0.11; P < .001), and K (LR pRBC 4.08 mmol/L SD 0.88, nLR pRBC 5.48 mmol/L SD 0.90; P < .001). Increasing values of IL8 were observed in nLR units during storage (T0: 4167 ± 11 888 pg/mL; T35: 6367 ± 11 612 pg/mL). CONCLUSION AND CLINICAL IMPORTANCE: LR blood units are recommended to critically ill dogs with marked inflammatory conditions.

CD30 Cross-Reactivity and Expression in Feline Normal Tissues and Lymphomas.

CD30 is a transmembrane glycoprotein of the tumor necrosis factor receptor superfamily included in the diagnostic algorithm of human cutaneous, anaplastic large cell and Hodgkin lymphomas and represents an optimal therapeutic target for CD30+ tumors. Similar diagnostic and therapeutic approaches are largely missing for feline lymphomas. Cross-reactivity of the antihuman CD30 receptor clone Ber-H2 was investigated in feline lymphomas. Comparative analysis of feline and human CD30 identified 61% identity of the amino acid sequence, with 100% identity of the main sequence of the epitope targeted by the antibody (RKQCEPDYYL). CD30 expression in normal feline tissues was restricted to rare lymphoid cells in perifollicular and interfollicular lymph node areas and in the thymic medulla. In feline lymphoma, CD30 was expressed in 4 of 33 (13%) T-cell lymphomas, 3 of 22 (14%) B-cell lymphomas, and 5 of 7 (71%) mixed-cell lymphomas, showing diffuse (1/5) or multifocal (4/5) positivity restricted to neoplastic multinucleated lymphoid cells and binucleated cells consistent with Reed-Sternberg-like cells. Based on the human classification system, cell morphology, expression of multiple markers (mixed cell components), and CD30 positivity, these cases were considered most consistent with classical Hodgkin-like lymphoma (HLL). The other 2 mixed-cell lymphomas were CD30 negative and thus most consistent with either T-cell-rich large B-cell lymphoma (TCRLBCL) or nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). These findings provide multiple data supporting the cross-reactivity of the Ber-H2 anti-CD30 clone in feline tissues and give evidence of the usefulness of CD30 in the diagnostic evaluation of feline lymphoma.

Evaluation of the performance of three serological tests for diagnosis of Leishmania infantum infection in dogs using latent class analysis / Avaliação da realização de três testes sorológicos para diagnóstico de infecção por Leishmania infantum em cães utilizando análise de classe latente

Abstract Canine leishmaniasis (CanL) is a disease caused by Leishmania infantum. Serological methods are the most common diagnostic techniques used for the diagnosis of the CanL. The objective of our study was to estimate the sensitivity and specificity of one in-house ELISA kit (ELISA UNIZAR) and three commercially available serological tests (MEGACOR Diagnostik GmbH) including an immunochromatographic rapid test (FASTest LEISH®), an immunofluorescent antibody test (MegaFLUO LEISH®) and an enzyme-linked immunosorbent assay (MegaELISA LEISH®), using latent class models in a Bayesian analysis. Two hundred fifteen serum samples were included. The highest sensitivity was achieved for FASTest LEISH® (99.38%), ELISA UNIZAR (99.37%), MegaFLUO LEISH® (99.36%) followed by MegaELISA LEISH® (98.49%). The best specificity was obtained by FASTest LEISH® (98.43%), followed by ELISA UNIZAR (97.50%), whilst MegaFLUO LEISH® and MegaELISA LEISH® obtained the lower specificity (91.94% and 91.93%, respectively). The results of present study indicate that the immunochromatographic rapid test evaluated FASTest LEISH® show similar levels of sensitivity and specificity to the quantitative commercial tests. Among quantitative serological tests, sensitivity and specificity were similar considering ELISA or IFAT techniques.

Resumo A leishmaniose canina (Lcan) é uma causada pela Leishmania infantum. Os métodos sorológicos são as técnicas diagnósticas mais utilizadas para o diagnóstico da leishmaniose canina. O objetivo do nosso estudo foi estimar a sensibilidade e a especificidade de um kit ELISA interno (ELISA UNIZAR) e de três testes sorológicos disponíveis comercialmente, feitos pelo mesmo fabricante (MEGACOR Diagnostik GmbH), incluindo um teste rápido imunocromatográfico (FASTest LEISH®), um teste de anticorpos imunofluorescentes (Megafluo LEISH®) e um ensaio de imunoabsorção enzimática (Megaelisa LEISH®), utilizando-se modelos de classe latentes numa análise bayesiana. Foram incluídas duzentas e quinze amostras de soro. A maior sensibilidade foi alcançada para Fastest LEISH® (99,38%), ELISA UNIZAR (99,37%), Megafluo LEISH® (99,36%) seguida por Megaelisa LEISH® (98,49%). A melhor especificidade foi obtida por FASTest LEISH® (98,43%), seguida por ELISA UNIZAR (97,50%), enquanto Megafluo LEISH® e Megaelisa LEISH® obtiveram a menor especificidade (91,94% e 91,93%, respectivamente). Os resultados do presente estudo indicam que o teste rápido imunocromatográfico, avaliado por FASTest LEISH® mostra níveis similares de sensibilidade e especificidade aos testes comerciais quantitativos incluídos. Entre os testes sorológicos quantitativos, a sensibilidade e a especificidade foram semelhantes, considerando-se as técnicas de ELISA ou IFI.

Detection and Prognostic Relevance of Circulating and Disseminated Tumour Cell in Dogs with Metastatic Mammary Carcinoma: A Pilot Study.

In human breast cancer, both circulating tumour cells (CTCs) in peripheral blood and disseminated tumour cells (DTCs) in the bone marrow are predictive of short survival and may be used as liquid biopsy to guide therapy. Herein we investigate, for the first time, the feasibility to quantify CTCs and DTCs in canine metastatic mammary carcinoma (MMC) with the automated CellSearch platform, which identifies tumour cells by immune-magnetic enrichment and fluorescent labelling. Using this approach before start of treatment, we could detect at least 1 CTC per 7.5 mL of peripheral blood in 12 out of 27 evaluable samples (44.4%) and at least 1 DTC per 1 mL of bone marrow in 11 out of 14 evaluable samples (78.6%). Conversely, we did not find any CTCs in the healthy, negative control dogs (n = 5) that we analysed in parallel. Interestingly, the levels of CTCs/DTCs and the prevalence of positive dogs closely resemble results obtained by CellSearch assay in metastatic breast cancer patients at diagnosis. Moreover, in the canine cohort, the presence of CTCs was significantly associated with poor outcome. These observations identify the first actionable marker in veterinarian oncology to guide treatment of canine MMC. Furthermore, our findings have important implications for human research, since it reinforce the value of canine MMC as model useful to speed up pharmacological studies with primary endpoint of overall survival, given the reduced life-span of the canine species.

In vitro assessment of quality of citrate-phosphate-dextrose-adenine-1 preserved feline blood collected by a commercial closed system.

BACKGROUND: Optimal procedure for storage of feline blood is needed. Open-collection systems have been employed in feline medicine, thus limiting the possibility for storage. OBJECTIVES: To evaluate indicators of quality of feline blood stored for 35 days at +4°C in a closed-collection system specifically designed for cats. ANIMALS: Eight healthy adult European domestic shorthair cats with a weight of 5-6.8 kg. METHODS: This is a case series study. A bacteriological test, CBC, blood smear, pH, osmotic fragility, 2,3-diphosphoglycerate (2,3-DPG), and adenosine triphosphate (ATP) measurement were performed weekly on whole blood (WB) units from day 1 to day 35 after donation. The hemolysis index, lactate and potassium concentrations, prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen were measured on plasma aliquots. RESULTS: One out of eight blood units (BUs) had bacterial growth (Serratia marcescens) at day 35. No significant differences were found regarding CBC, morphology, pH, and osmotic fragility. Despite high inter-individual variability and low starting levels, significant decreases in the mean concentrations of 2,3-DPG (T0 1.99 mmol/g Hb, SD 0.52, T35 1.25 mmol/g Hb, SD 1.43; P = .003) and ATP (T0 1.45 mmol/g Hb, SD 0.71, T35 0.62 mmol/g Hb, SD 0.51; P < .001) were detected during the study, as opposed to an increase in hemolysis (T0 0.11 mmol/L, SD 0.07, T35 0.84 mmol/L, SD 0.19; P < .001), lactate (T0 3.30 mmol/L, SD 0.86, T35 13.36 mmol/L, SD 2.90; P < .001), and potassium (T0 3.10 mmol/L, SD 0.21, T35 4.12 mmol/L, SD 0.35; P < .001) concentrations. CONCLUSIONS AND CLINICAL IMPORTANCE: The commercial BU kit is appropriate for blood collection and conservation of WB in cats. The maintenance of WB quality indicators during storage is essential for future improvements of feline transfusion medicine.

Semiquantitative immunohistochemical detection of progesterone receptors in male accessory sex glands as a screening assay for anabolic steroid use in bulls.

We investigated the immunohistochemical expression of progesterone receptors (PRs) in the prostate and bulbourethral glands of thirty-two 10-14-mo-old Charolais bulls following treatment with a low dosage of estrogens. Animals were divided into 2 groups: 16 animals (group T) were treated for 71 d with a therapeutic dose of trenbolone acetate and estradiol by subcutaneous implant, 16 animals (group C) received no treatment. Urine samples were collected both at the beginning of the trial and 9 times during the study. A semiquantitative analysis of immunohistochemistry (IHC) was performed by counting the number of positive cells in 10 randomly selected high-power fields (hpf). Both groups showed no significant histologic lesions. IHC examination showed positive cells in the epithelium of both glands, with different patterns of distribution between groups. In group C, IHC-positive cells per hpf varied from 0 to 40 in the prostate and from 0 to 32 in the bulbourethral gland. In group T, positive cells varied from 0 to 85 per hpf in the prostate and from 0 to 75 in the bulbourethral gland. The treated group showed significantly higher median numbers of positively stained cells in both organs than the controls ( p < 0.001). Chemical analysis of the urine samples confirmed that the experimental treatment mimics continuous, low-dose administration of anabolic steroids. IHC quantification showed good sensitivity with a high predictive power to correctly classify treated animals and could be used as a preliminary screening test in bulls.

Incidence of mammary tumors in the canine population living in the Veneto region (Northeastern Italy): Risk factors and similarities to human breast cancer.

Although mammary gland tumors (MT) are the most-common type of tumor in intact female dogs, there is little information about their incidence in dog population. Data on MT in female dogs was retrieved from the Animal Tumor registry of dogs and cats of Venice and Vicenza provinces during 2005-2013 and was analyzed to visualize crude incidence rates by breed and across age categories. Overall, 2744 mammary tumors were reported accounting for 54% of all tumors in female dogs. The annual incidence rate (IR) was 250 cases per 100,000 dogs. The most frequent malignant tumors were complex carcinomas, consisting of both epithelial and myoepithelial tissues (IR=71.89), and simple carcinomas (IR=62.59). The MT incidence rate increased through the study period; particularly in the last 4 years, and malignant neoplasms occurred more frequently (70%) than the benign counterparts (30%). Seventy-four percent of tumors were diagnosed in intact females, and the mean age at diagnosis was significantly higher for spayed dogs than for intact ones. MT were less frequent in dogs younger than 6 years and increased up to approximately 60% for ages between 8 and 13 years. The purebred dogs had a higher probability to have a malignant neoplasm than mixed-breed dogs, particularly in dogs younger than 7 years, and the Samoyed, Dobermann, Schnauzer and Yorkshire Terrier breeds were more inclined to develop malignant MT. The incidence of MT in dogs is increasing, and IRs are comparable to that in women. The epidemiological similarities between dogs and women support the validity of canine MT as a model for human breast cancer.

Estimate of the size and demographic structure of the owned dog and cat population living in Veneto region (north-eastern Italy).

The knowledge of the size and demographic structure of animal populations is a necessary prerequisite for any population-based epidemiological study, especially to ascertain and interpret prevalence data, to implement surveillance plans in controlling zoonotic diseases and, moreover, to provide accurate estimates of tumours incidence data obtained by population-based registries. The main purpose of this study was to provide an accurate estimate of the size and structure of the canine population in Veneto region (north-eastern Italy), using the Lincoln-Petersen version of the capture-recapture methodology. The Regional Canine Demographic Registry (BAC) and a sample survey of households of Veneto Region were the capture and recapture sources, respectively. The secondary purpose was to estimate the size and structure of the feline population in the same region, using the same survey applied for dog population. A sample of 2465 randomly selected households was drawn and submitted to a questionnaire using the CATI technique, in order to obtain information about the ownership of dogs and cats. If the dog was declared to be identified, owner's information was used to recapture the dog in the BAC. The study was conducted in Veneto Region during 2011, when the dog population recorded in the BAC was 605,537. Overall, 616 households declared to possess at least one dog (25%), with a total of 805 dogs and an average per household of 1.3. The capture-recapture analysis showed that 574 dogs (71.3%, 95% CI: 68.04-74.40%) had been recaptured in both sources, providing a dog population estimate of 849,229 (95% CI: 814,747-889,394), 40% higher than that registered in the BAC. Concerning cats, 455 of 2465 (18%, 95% CI: 17-20%) households declared to possess at least one cat at the time of the telephone interview, with a total of 816 cats. The mean number of cats per household was equal to 1.8, providing an estimate of the cat population in Veneto region equal to 663,433 (95% CI: 626,585-737,159). The estimate of the size and structure of owned canine and feline populations in Veneto region provide useful data to perform epidemiological studies and monitoring plans in this area.

c-KIT messenger RNA and protein expression and mutations in canine cutaneous mast cell tumors: correlations with post-surgical prognosis.

Cutaneous mast cell tumors (MCTs) are among the most common neoplasms in dogs and show a highly variable biologic behavior. Histological grading, cell proliferation markers, and KIT immunohistochemistry are typically used to predict post-surgical prognosis. In the present study, c-KIT messenger RNA (mRNA) expression was measured in canine MCTs and its relationship with tumor grade, immunohistochemical staining pattern, post-surgical prognosis, and mutations was investigated. A significant increase of c-KIT mRNA was observed in MCTs versus healthy skin and surgical margins. Mutations were observed in 8.3% of cases. The KIT staining pattern was investigated for both grading systems. In particular, staining pattern III was associated with grade II (G2) and G3 MCTs, while staining patterns I and II were associated with G1 and G2 MCTs. Considering the 2-tier histological grading, the high grade was mainly associated with pattern III (71%) while the low grade was associated with patterns II (70%) and I (28%). A weak association between the KIT staining pattern and outcome was also observed. The results obtained suggest that c-KIT mRNA is overexpressed in canine MCT, although the fold variations were not associated with the protein localization or complementary DNA mutations. These observations suggested that the 3 events were independent. The histological grading and the KIT staining pattern have prognostic value as previously published. Staining pattern I could be especially helpful in predicting a good prognosis of G2 MCTs. Sequence mutations were not necessarily suggestive of a worse prognosis, but might be useful in choosing a chemotherapy protocol.

Evaluation of a rapid and inexpensive liquid culture system for the detection of Mycobacterium avium subsp. paratuberculosis in bovine faeces.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis, a chronic granulomatous enteric disease of ruminants. MAP detection by faecal culture provides the definitive diagnosis of the infection. Automated liquid systems for MAP culture are more sensitive and rapid than culture on solid media, but they are expensive and require specialised equipment. In this study, a non-automated culture method using a modified Middlebrook 7H9 liquid medium (7H9+) was compared with Herrold's solid medium (HEYM) and direct real-time PCR on dairy cattle faeces. MAP growth in 7H9+ was monitored weekly by real-time PCR until the 12th week post-inoculation. The analytical sensitivity of the three methods was evaluated using faecal samples from a healthy cow spiked with ten-fold dilutions of MAP organisms (10(4)-10(-1)) and naturally MAP-infected faeces serially diluted 1 to 10 in negative faecal samples. The limits of detection of the solid culture and direct real-time PCR were 10(2) and 10(3)MAP/g, respectively. In comparison, the 7H9+ culture revealed as few as 1MAP/g. A marked reduction in time to detection of the pathogen, compared with HEYM culture, was obtained. In addition, the three methods were applied to environmental faecal samples collected from a high- and a low-prevalence herd. The culture in 7H9+ showed to be the most sensitive test in the low-prevalence herd and provided faster results than HEYM. In the high-prevalence herd the three methods showed the same sensitivity and the real-time PCR had the shortest turnaround time. In conclusion, the use of 7H9+ for MAP-detection from cattle faeces maximizes diagnostic sensitivity and reduces turnaround time and, therefore, could replace culture in solid medium. Hence, we propose a two-step protocol for the assessment of MAP faecal excretion based on: 1) direct real-time PCR on all samples; and 2) inoculation of negative samples into 7H9+ and analysis after 3 and, if necessary, 6weeks by real-time PCR.