Malignant Tumors In Dogs Research Articles

Retrospective analysis of data on the prevalence of malignant neoplasms in dogs and cats

The article presents the results of a study on the primary incidence of malignant tumors in dogs and cats in Moscow over the period from January 1, 2023, to July 1, 2024. The study was conducted on the basis of the Department of Diseases of Small Domestic, Laboratory and Exotic Animals and the Research Laboratory of Oncology, Ophthalmology and Animal Biochemistry of the Russian Biotechnological University “ROSBIOTECH”. The study included 208 cats and 325 dogs with primary, spontaneous malignant tumors of various origins. To confirm the diagnosis, a comprehensive approach was used, including clinical examination, hematological tests, and special diagnostic techniques such as ultrasonography, radiography, computed tomography (CT), and magnetic resonance imaging (MRI). Verification of the tumor process was based on cytological and histological methods.The purpose of this work is to analyze the structure of the incidence of small domestic animals with malignant tumors. As a result, we identified the most common types of tumors in cats and dogs. In cats, breast carcinoma was the most common type (44.23%), followed by lymphoma (18.27%), sarcomas (13.46%) and carcinomas (13.94%), as well as squamous cell carcinoma (6.25%). In dogs, breast carcinoma also accounted for the majority of cases (37.23%), along with sarcomas (23.38%), mastocytomas (16.31%), and carcinomas (14.15%).

LINE-1 Methylation Status in Canine Splenic Hemangiosarcoma Tissue and Cell-Free DNA.

Splenic hemangiosarcoma is one of the most common malignant tumors in dogs, and early diagnosis is of great importance for achieving a good prognosis. DNA methylation plays an important role in cancer development. Long interspersed nuclear element 1 (LINE-1) is the most abundant repetitive element in the genome. LINE-1 hypomethylation has been shown to be related to carcinogenesis in humans, and it has been used as a novel cancer biomarker. This study aimed to evaluate the methylation status of LINE-1 in tumor tissue and circulating cell-free DNA and assess its clinical significance in canine splenic hemangiosarcoma. Genomic DNA was isolated from splenic masses of 13 dogs with hemangiosarcoma, 11 with other malignant tumors, and 15 with benign lesions. LINE-1 methylation was quantified using methylation-sensitive and -insensitive restriction enzyme digestion followed by real-time polymerase chain reaction. Additionally, blood samples were collected from eight patients to isolate cell-free DNA to determine LINE-1 methylation status changes during the treatment course. LINE-1 methylation in tumor samples was significantly lower in patients with hemangiosarcoma than in those with other malignant tumors and benign lesions. Non-significant but similar results were observed for the cell-free DNA samples. Our results demonstrate that LINE-1 methylation status is a potential biomarker for splenic hemangiosarcoma.

Canine Somatic Mutations from Whole-Exome Sequencing of B-Cell Lymphomas in Six Canine Breeds-A Preliminary Study.

Canine lymphoma (CL) is one of the most common malignant tumors in dogs. The cause of CL remains unclear. Genetic mutations that have been suggested as possible causes of CL are not fully understood. Whole-exome sequencing (WES) is a time- and cost-effective method for detecting genetic variants targeting only the protein-coding regions (exons) that are part of the entire genome region. A total of eight patients with B-cell lymphomas were recruited, and WES analysis was performed on whole blood and lymph node aspirate samples from each patient. A total of 17 somatic variants (GOLIM4, ITM2B, STN1, UNC79, PLEKHG4, BRF1, ENSCAFG00845007156, SEMA6B, DSC1, TNFAIP1, MYLK3, WAPL, ADORA2B, LOXHD1, GP6, AZIN1, and NCSTN) with moderate to high impact were identified by WES analysis. Through a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of 17 genes with somatic mutations, a total of 16 pathways were identified. Overall, the somatic mutations identified in this study suggest novel candidate mutations for CL, and further studies are needed to confirm the role of these mutations.

Морфобиохимический профиль крови при терапии ингибиторами ЦОГ-2 и циклофосфамидом

Abstract. In modern veterinary medicine, in the last decade, significant achievements have been noted in the treatment of malignant tumors in dogs, but nevertheless the number of oncologically ill animals is quite widespread, and tends to increase further. The use of only traditional methods for the treatment of oncological pathologies (radiation and chemotherapy, surgical) does not have a positive effect, and accordingly does not solve the problem as a whole. The scientific novelty lies in the study of the effect of selective COX-2 inhibitors in combination with cyclophosphamide on the morphobiochemical parameters of the blood of dogs with malignant neoplasms of the breast. The purpose of our work was to study the dynamics of morphobiochemical blood parameters during multimodal conservative therapy of breast cancer recurrence in dogs with selective COX-2 inhibitors (firocoxib, cimicoxib) and cyclophosphamide. Research methods. Clinical observations and studies were carried out in the veterinary clinic of the Center for Animal Beauty and Health “Zoostyle” in Volgograd. The object of the study was 6 female dogs of various breeds aged 10-14 years, with a morphologically confirmed diagnosis of recurrent breast adenocarcinoma. At the same time, an automatic hematological analyzer “Mindray BC-2800 Vet” and a semi-automatic biochemical analyzer “BioChem SA” were used. Stained blood smears were examined under a microscope “MIKMED-5”. Results. The results of the studies show that morphobiochemical blood parameters varied in both groups by the 90th day of therapy. The results obtained convincingly show that the use of a combination of selective COX-2 inhibitors and cyclophosphamide, in clinically significant doses, induced an increase in individual biochemical parameters of blood serum, without symptomatic manifestations. In the firocoxib group, creatinine and urea levels exceeded the reference values by 39.5 and 67.7 %, respectively. Thus, the combination of drugs in the first group demonstrates an increased risk of use in animals with renal insufficiency.

Validation of p53 Immunohistochemistry (PAb240 Clone) in Canine Tumors with Next-Generation Sequencing (NGS) Analysis

In human medicine, p53 immunohistochemistry (IHC) is a common method that is used for the identification of tumors with TP53 mutations. In veterinary medicine, several studies have performed IHC for p53 in canine tumors, but it is not known how well it actually predicts the mutation. The aim of this study was to estimate the accuracy of the IHC method for p53 (clone PAb240) using a lab-developed NGS panel to analyze TP53 mutations in a subset of malignant tumors in dogs. A total of 176 tumors were analyzed with IHC and then 41 were subjected to NGS analysis; among them, 15 were IHC positive and 26 were negative, and 16 out of 41 (39%) were found to be inadequate for NGS analysis. Excluding the non-evaluable cases at NGS, of the remaining eight IHC-positive cases, six were mutants and two were wild-type. Among the 17 IHC-negative cases, 13 were wild type, and 4 were mutants. The sensitivity was 60%, specificity was 86.7%, and the accuracy was 76%. These results suggest that when using IHC for p53 with this specific antibody to predict mutation, up to 25% wrong predictions can be expected.

Impact of near-infrared fluorescence imaging with indocyanine green on the surgical treatment of pulmonary masses in dogs.

To investigate the intraoperative identification and complete resection of pulmonary masses, and to evaluate lymph node metastasis of pulmonary malignant tumors in dogs using indocyanine green (ICG) fluorescence imaging. Forty dogs with pulmonary masses were included, all of which underwent surgical treatment. ICG fluorescence imaging was performed on pulmonary masses before lobectomy and the resection margins after lobectomy. In addition, ICG fluorescence of the excised masses and lymph nodes was evaluated in the shaded box. The fluorescence findings were compared with the histopathological diagnosis. Of 44 nodules resected from 40 dogs, 32 nodules were histopathologically diagnosed as lung adenocarcinoma, five were histiocytic sarcoma, three were undifferentiated sarcoma, two were malignant epithelial tumor metastases, one was carcinosarcoma, and one was a non-neoplastic lesion. Fluorescence was observed in all nodules. In addition to the main lesion, other fluorescent nodules were found in four dogs. Regarding the diagnostic accuracy of complete resection based on ICG fluorescence, the sensitivity was 67.7% and the specificity was 60.0%. The sensitivity and specificity of ICG fluorescence for the diagnosis of lymph node metastasis were 100 and 75.0%, respectively. ICG fluorescence imaging might be a useful intraoperative diagnostic method to identify the location of tumors and lymph node metastasis, but not to evaluate complete tumor resection, in dogs with pulmonary malignant tumors.

Hypercalcemia in bitches with malignant mammary neoplasms

Malignant hypercalcemia is a paraneoplastic syndrome. It has been detected in several types of malignant tumours in dogs, such as lymphomas, and also in mammary neoplasms, without the exact determination of the mechanisms involved in its occurrence as well as its incidence. In this study, 100 bitches with mammary malignant neoplasia, diagnosed by histopathological analysis, were submitted to clinical evaluation for disease staging, haematological evaluation, serum biochemistry, including renal function tests, hepatic and total calcium serum levels and ionized fraction. These parameters were analyzed at the time of the initial attendance and 30 days after the treatment. The most frequent histological pattern was carcinoma in mixed tumors (26%). In 52% of the diseased dogs, hypercalcemia was observed by means of ionized calcium dosing, which was not verified by means of total calcium dosing, where only 4% presented hypercalcemia. No correlation was found between hypercalcemia and the histological pattern of the neoplasms. However, the correlation was verified between hypercalcemia and more advanced stages of the disease, mainly from stage III, suggesting that the detection of hypercalcemia may be correlated with worse prognosis, showing the importance of the research concerning the presence of paraneoplastic syndromes in bitches with mammary neoplasms.

Canine and Feline Oral Cavity Tumours and Tumour-like Lesions: a Retrospective Study of 486 Cases (2015–2017)

Oral cavity tumours and tumour-like lesions are common in dogs and cats, and their diagnosis and classification requires histopathological examination. The aim of this study was to analyse retrospectively oral cavity lesions in dogs and cats in order to evaluate the distribution of inflammatory, hyperplastic and neoplastic lesions manifested as tumours. A total of 486 oral cavity tumours and tumour-like lesions (340 canine; 146 feline), diagnosed routinely from 2015 to 2017, were included. The lesions were classified as inflammatory, hyperplastic or neoplastic (benign and malignant). Histopathological diagnosis was based on haematoxylin and eosin staining and, when necessary, May-Grünwald-Giemsa (for mast cell tumours) or Masson's Fontana (for melanomas) stains or immunohistochemistry (for CD3, CD79α and S100 markers). For dogs, 29.11% (99/340) of the lesions were benign tumours, 24.12% (82/340) were hyperplastic lesions and 14.7% (50/340) were inflammatory lesions. For cats, 4.79% (7/146) were benign tumours, 15.07% (22/146) were hyperplastic lesions and 57.53% (84/146) were inflammatory lesions. Furthermore, 23.24% (79/340) of canine cases were diagnosed with gingival hyperplasia and 19.12% (65/340) were diagnosed with peripheral odontogenic fibroma, while 43.84% (64/146) of feline cases were diagnosed with chronic lymphoplasmacytic stomatitis. Malignant tumours in dogs and cats constituted 32.06% (109/340) and 21.91% (32/146) of the lesions, respectively, with high-grade melanoma in dogs and squamous cell carcinoma in cats being the most common.

Inter- and intra-rater reliability and agreement in determining subcutaneous tumour margins in dogs.

The objective of this prospective study was to evaluate agreement and reliability of calliper-based measurements of locally invasive subcutaneous malignant tumours in dogs. Four raters measured the longest diameter of 12 subcutaneous tumours (7 soft tissue sarcomas and 5 mast cell tumours) from 11 client-owned dogs during 3 randomized, blinded measurement trials, both pre- and post-sedation. Inter- and intra-rater reliability was evaluated using intra-class correlation coefficient (ICC) and agreement was evaluated using Bland-Altman plots. Inter- and intra-rater reliability was good (ICC range of 0.8694-0.89520) and excellent (ICC range of 0.9720-0.9966), respectively. For agreement calculations, an a priori clinically relevant limit of agreement of 10 mm was set. Inter- and intra-rater agreement was unacceptable with inter-rater limits of agreement ranging from 15.9 to 55.6 mm and intra-rater limit of agreement ranging from 11.9 to 28.1 mm. Review of the measurement trial photographs revealed that calliper orientation changes were frequent, occurring in 9/12 (75%) and 8/12 (67%) pre- and post-sedation cases. No significant correlation was found between inter-rater measurement standard deviations and calliper orientation changes or dog body condition score. These findings suggest veterinarians may have poor agreement in determining the gross edge of tumours, which is expected to introduce bias and inconsistency in tumour staging, assessing response to therapy, and surgical margin planning. Due to the potential consequences for veterinary cancer patients, future studies are needed to validate the present findings.

Development of effective tumor immunotherapy using a novel dendritic cell-targeting Toll-like receptor ligand.

Although dendritic cell (DC)-based immunotherapy shows little toxicity, improvements should be necessary to obtain satisfactory clinical outcome. Using interferon-gamma injection along with DCs, we previously obtained significant clinical responses against small or early stage malignant tumors in dogs. However, improvement was necessary to be effective to largely developed or metastatic tumors. To obtain effective methods applicable to those tumors, we herein used a DC-targeting Toll-like receptor ligand, h11c, and examined the therapeutic effects in murine subcutaneous and visceral tumor models and also in the clinical treatment of canine cancers. In murine experiments, most and significant inhibition of tumor growth and extended survival was observed in the group treated with the combination of h11c-activated DCs in combination with interferon-gamma and a cyclooxygenase2 inhibitor. Both monocytic and granulocytic myeloid-derived suppressor cells were significantly reduced by the combined treatment. Following the successful results in mice, the combined treatment was examined against canine cancers, which spontaneously generated like as those in human. The combined treatment elicited significant clinical responses against a nonepithelial malignant tumor and a malignant fibrous histiocytoma. The treatment was also successful against a bone-metastasis of squamous cell carcinoma. In the successful cases, the marked increase of tumor-responding T cells and decrease of myeloid-derived suppressor cells and regulatory T cells was observed in their peripheral blood. Although the combined treatment permitted the growth of lung cancer of renal carcinoma-metastasis, the marked elevated and long-term maintaining of the tumor-responding T cells was observed in the patient dog. Overall, the combined treatment gave rise to emphatic amelioration in DC-based cancer therapy.

Development of an autologous canine cancer vaccine system for resectable malignant tumors in dogs

While conventional therapies exist for canine cancer, immunotherapies need to be further explored and applied to the canine setting. We have developed an autologous cancer vaccine (K9-ACV), which is available for all dogs with resectable disease. K9-ACV was evaluated for safety and immunogenicity for a variety of cancer types in a cohort of companion dogs under veterinary care. The autologous vaccine was prepared by enzymatic digestion of solid tumor biopsies. The resultant single cell suspensions were then UV-irradiated resulting in immunogenic cell death of the tumor cells. Following sterility and endotoxin testing, the tumor cells were admixed with CpG ODN adjuvant and shipped to the participating veterinary clinics. The treating veterinarians then vaccinated each patient with three intradermal injections (10 million cells per dose) at 30-day intervals (one prime and two boost injections). In a cohort of 20 dogs completing the study, 17 dogs (85%) developed an augmented IgG response to autologous tumor antigens as demonstrated using western blot analysis of pre- and post-peripheral blood samples. We also report several dogs have lived beyond expected survival time based on previously published data. In summary, K9-ACV is an additional option to be considered for the treatment of dogs with resectable cancer.

Ki67/KIT double immunohistochemical staining in cutaneous mast cell tumors from Boxer dogs

Cutaneous mast cell tumors (MCTs) are among the most frequent malignant tumors in dogs and Boxer breed dogs have a higher incidence of this disease. Ki67 staining and KIT staining are widely used to predict natural behavior in canine MCT but no previous study has evaluated double staining of these proteins as a prognostic factor. Based on biological behavior predictors in canine MCT, the purpose of this study was to determine the Ki67 proliferative index in KIT positive cells using double stain immunohistochemistry technique. Sixty-nine MCTs from Boxer dogs were selected and a tissue microarray was constructed for the double stained immunohistochemistry. Double positivity (Ki67+/KIT+) was observed in 20/69 (29%) MCT, with a mean of 9.06 double positive cells per tissue core (range 0.48%–43.97%) and Ki67−/KIT+ animals had a longer survival time than Ki67+/KIT+ animals (p=0.03).

Mitochondrial D-loop mutations can be detected in sporadic malignant tumours in dogs

Abstract The aim of this study was to identify mutations in the D-loop region of mtDNA in head, neck, and limb tumours in dogs, and determination of their relationship with the process of neoplastic transformation. Blood and tumour tissue samples from 19 dogs with diagnosed sporadic malignant tumours were analysed. DNA extraction, amplification, and sequencing of the mtDNA D-loop, and bioinformatic analyses were performed. Five mutations and 19 polymorphisms were observed in 68.42% of all tumours. Polymorphic variants were noted in 42.86% of the head and neck tumours and in 58.33% of the limb tumours. Mutations were observed in 21.05% of dogs. The mutations were found in 28.57% of the head and neck tumours and in 16.66% of the limb tumours. The mutations were identified in 50% of the studied epithelial cancers. In the mesenchymal tumours, no mutations in the D-loop region were observed. Mitochondrial haplotype A17 was found in over 40% cases of limb tumours. No association between the age, breed, sex, type of tumour, and detected polymorphic variants were observed. Different mutational changes in the D-loop sequences of mtDNA identified in the blood and tumour tissues may indicate a relationship between the type of tumour and individual changes in the D-loop nucleotide sequences of mtDNA.

Vascular endothelial growth factor in prognosis of splenic malignant tumours in dogs

Abstract The aim of the study was to determine the levels of the vascular endothelial growth factor (VEGF) in the serum of dogs suffering from splenic malignant tumours, prior to splenectomy, as well as three and six months after the surgery. Tumours and blood samples were collected from 10 dogs of various breeds, aged between 7 and 13 years, and from 10 control animals. Tumour sections were fixed in 10% buffered formalin for 24 h. The type of tumour was determined according to the WHO classification. Blood samples were centrifuged and the obtained sera were subjected to immunoenzymatic assays to determine the VEGF levels. The median of VEGF levels in the serum of dogs suffering from splenic malignant tumours was 37.85 pg/mL (15.40-107.18 pg/mL). The highest values were observed in dogs with confirmed metastases (107.18 pg/mL and 65.43 pg/mL). The VEGF values in control group were between 0.1 pg/mL and 13.04 pg/mL. A comparative analysis of the VEGF levels against the animals' survival time indicated that VEGF overexpression may serve as a prognostic factor in cases of malignant tumours of the spleen.

Use of histologic margin evaluation to predict recurrence of cutaneous malignant tumors in dogs and cats after surgical excision

To assess the usefulness of histologic evaluation of surgical margins to predict local recurrence of cutaneous malignant tumors in dogs and cats treated by means of surgical excision. Prospective case series. 40 dogs and 20 cats. 60 surgically excised tumors (20 soft tissue sarcomas [STSs], 20 mast cell tumors [MCTs], and 20 carcinomas) were examined histologically. Margins were classified as clean, close, or infiltrated; histologic grade was assessed in STSs and MCTs. Recurrence rates and recurrence-free intervals (RFIs) during a 24-month follow-up period were recorded, and method accuracy was calculated. Surgical margins were clean in 29 of 60 (48%) tumors, close in 11 (18%), and infiltrated in 20 (33%). Tumors recurred in 27 of 60 (45%) animals, with a mean ± SD RFI of 229 ± 173 days. Recurrence rates for animals that had tumors with infiltrated (16/20) or close (8/11) margins were significantly higher than recurrence rate for animals that had tumors with clean margins (3/29). Margin classification was a significant predictor of RFI. Accuracy of the method to predict recurrence was 94% for carcinomas, 87% for STSs, and 76% for MCTs. Histologic assessment of margin status was useful for predicting local recurrence of cutaneous malignant tumors in dogs and cats treated by means of excision alone. Method accuracy varied among tumor types and grades. Recurrence times suggested postsurgical follow-up should continue for ≥ 2 years. Results were similar for animals with infiltrated and close tumor margins, and careful postsurgical management is recommended for both.

Adjuvant chemotherapy with low-dose cisplatin following surgery for malignant tumors in dogs: 16 cases

Adjuvant chemotherapy with low-dose cisplatin following surgery for malignant tumors in dogs: 16 cases

Ultrasonographic Evaluation of Mammary Tumors in Bitches

Summary The aim of this study was to characterize the ultrasonographic patterns of benign and malignant mammary tumors and to evaluate the efficacy of ultrasonographic imaging to distinguish benign and malignant tumors in dogs. Shape, margins, width-to-craniocaudal (CC) dimension ratio, echotexture, echogenicity, presence of calcification, presence of pseudocapsule, size (the deepest and longest axis) and acoustic transmission of tumors were evaluated. The ultrasonography features and the tissue diagnosis of benign tumor were oval or round (80% of masses with this feature were benign), and circumscribed margins (95% were benign). The features most predictive of a malignant tumor diagnosis were spiculated or microlobulated margins (76% of masses with this feature were malignant), and irregular shape (78% were malignant). No differences between the benign and malignant groups for width-to-CC dimension ratio >1.4 were observed. The benign tumors were seen in different distribution of beign hypoechoic (30%), isoechoic (35%) or varied in echogenicity. It was concluded that, the ultrasonography could be an extremely useful tool for the preevaluation of canine mammary gland tumors, especially in relation to the evaluation of tumor size, shape, border irregularity, echotexture, internal echogenicity and acoustic transmission.

CHARACTERIZATION OF CANINE SUPERFICIAL TUMORS USING GRAY‐SCALE B MODE, COLOR FLOW MAPPING, AND SPECTRAL DOPPLER ULTRASONOGRAPHY—A MULTIVARIATE STUDY

Superficial tumors are not routinely evaluated by two- or three-dimensional diagnostic imaging methods as part of the staging of canine cancer patients, although superficial tumors are readily imaged by ultrasound. The objectives of this study were to characterize the ultrasonographic patterns of superficial tumors and to evaluate whether ultrasound can help discriminate between benign and malignant tumors in dogs. Superficial tumors (n=132) in 86 dogs were evaluated by B mode, color flow mapping, and spectral Doppler ultrasonography. Size, echogenicity, tumor border definition, invasiveness, acoustic transmission, presence and distribution of vascular flow to and within the tumor, as well as perfusion indices were measured. The tumors were classified as lipomas, benign tumors, atypical mammary tumors, and malignant tumors. Multivariate statistics using discriminant analysis was used to determine which parameters may be used to predict the status of the tumor. Tumor echogenicity, border shape, acoustic shadowing, total number of vessels to the tumor and the total flow amount are the parameters that in combination resulted in the lowest classification error (24%), meaning that on average three out of four tumors were correctly classified using these parameters. All the lipomas and atypical mammary tumors were classified correctly by ultrasonography. The results of this study show that ultrasonography has an important role in the evaluation of canine superficial tumors, particularly in the evaluation of tissue homogeneity and tumor vascularity.

Efficacy of mitoxantrone against various neoplasms in dogs

Summary One hundred twenty-six dogs with histologically confirmed, measurable malignant tumors were evaluated in a prospective study to determine the response to the antineoplastic drug mitoxantrone. Ninety-five dogs had been refractory to one or more treatment modalities (surgery, n = 57; chemotherapy other than mitoxantrone, n = 37; radiation, n = 4; whole body hyperthermia, n = 1). The extent of neoplastic disease was determined immediately before each dose of mitoxantrone was administered (1 to 10 doses, 2.5 to 5 mg/m2 of body surface area, iv) 21 days apart. Each dog was treated with mitoxantrone until the dog developed progressive disease or until the dog's quality of life diminished to an unacceptable level as determined by the owner or attending veterinarian. A partial or complete remission (>50% volume reduction) was obtained in 23% (29/126) of all dogs treated. Tumors in which there was a partial or complete remission included lymphoma (11/32), squamous cell carcinoma (4/9), fibrosarcoma (2/9), thyroid carcinoma (1/10), transitional cell carcinoma (1/6), mammary adenocarcinoma (1/6), hepatocellular carcinoma (1/4), renal adenocarcinoma (1/1), rectal carcinoma (1/1), chondrosarcoma (1/2), oral malignant melanoma (1/12), cutaneous malignant melanoma (1/1), myxosarcoma (1/1), mesothelioma (1/1), and hemangiopericytoma (1/1). Our results indicated that mitoxantrone induces measurable regression in various malignant tumors in dogs.

Treatment of spontaneous malignant tumours in dogs with PTMC

ABSTRACTThe chemical analysis of the caffeine salt of a phosphomolybdic‐tungstic acid compound (PTMC) is described. Twenty‐seven dogs with advanced malignant tumours have been treated with PTMC by intramuscular or intravenous injection. Five dogs ‐ one case of metastatic osteosarcoma, two cases of metastatic mammary carcinoma, one case of metastatic squamous cell carcinoma and one case of primary fibrosarcoma showed objective tumour regression for periods varying from four weeks to 20 months. Only two of the 27 dogs treated showed toxic effects.