Epidemiology and classification for canine and feline mammary gland tumors: a histopathological survey of 437 mammary gland tumor biopsies performed in a secondary care hospital in Chiang Mai, Thailand from 2012 to 2019.

Background: Metastatic disease resulting from mammary gland tumors (MGTs) is a known cause of death among dogs and cats. Keys to successful prevention and management strategies involve the accurate recording of diagnostic data. Methods: This retrospective study reviewed the epidemiology and classification of canine mammary gland tumors (CMTs) and feline mammary gland tumors (FMTs), as well as the factors including sex, age, and breed related to the occurrence of these tumors. Accordingly, 1,736 tumor biopsy cases were reported from 2012 to 2019 at Chiang Mai University Small Animal Hospital, Thailand, with 1,639 canine tumor biopsy cases and 97 feline tumor biopsy cases. Results: The proportion of CMTs was reported at 24.5% (401/1,639) for all canine tumor biopsy cases. Benign and malignant tumors were reported at 14.5% (58/401) and 85.5% (343/401) for all CMT cases, respectively. The mean age of dogs affected by benign CMTs was 9.0 ± 3.0 years, which was significantly lower than for malignant CMTs at 9.9 ± 2.8 years (P = 0.0239). According to histopathological classification, benign mixed tumors and simple carcinoma types were highest among benign and malignant CMT cases, respectively. Moreover, female dogs were at significantly higher risk of developing mammary gland tumors (OR = 45.8, 95% CI [3.9-86.0], P < 0.0001) than male dogs, as well as older dogs (>8 years) (OR = 1.7, 95% CI [1.2-2.2], P = 0.0001) compared to young ones (≤8 years). The proportion of FMTs was 37.1% (36/97) for all feline tumor biopsy cases. Benign and malignant tumors for all FMTs were reported at 16.7% (6/36) and 83.3% (30/36), respectively. According to histopathological classifications, adenoma and simple carcinoma were present in the highest proportion among benign and malignant FMTs, respectively. Female cats were at a significantly higher risk of developing mammary gland tumors than male cats (OR = 25.7, 95% CI [3.9-272.8], P < 0.0001). Conclusions and clinical importance: There was a high proportion of MGT cases compared with other tumor cases reported in a secondary care hospital in Chiang Mai, Thailand from 2012 to 2019, and malignant tumor biopsies have been more frequently observed than benign tumor biopsies in both CMT and FMT cases. The resulting data originating from this study can be an aid for veterinary oncologists in better educating clients and planning treatment and prevention strategies and it can be used as a basis for further experimental studies in the oncology section.

Incidence of hemoparasitic infections in cattle from central and northern Thailand.

Background: Hemoparasites, such as Babesia spp., Theileria spp. and Anaplasma spp., can negatively affect the health of farm animals resulting in significant losses in production. These losses inherently affect the economics of the livestock industry. Since increases in the severity of vector-borne diseases in the southeast Asian region have been reported, investigations of parasitic epidemiology in Thailand will be necessary to improve the existing parasite control strategies for blood parasitic infections. This study aims to investigate incidences of bovine hemoparasites throughout central and northern Thailand by focusing on areas of high-density cattle populations. Methods: Blood parasitic infections among cattle were screened and identified by microscopic examination. Anemia status was then determined by evaluation of the packed cell volume (PCV) of each animal. Furthermore, blood parasites were detected and identified by genus and species-specific primers through the polymerase chain reaction method. Amplicons were subjected to DNA sequencing; thereafter, phylogenetic trees were constructed to determine the genetic diversity and relationships of the parasite in each area. Results: A total of 1,066 blood samples were found to be positive for blood parasitic infections as follows: 13 (1.22%), 389 (36.50%), and 364 (34.15%) for Babesia bovis, Theileria orientalis, and Anaplasma marginale, respectively. Furthermore, multiple hemoparasitic infections in the cattle were detected. The hematocrit results revealed 161 hemoparasitic infected samples from 965 blood samples, all of which exhibiting indications of anemia with no significant differences. Sequence analysis of the identified isolates in this study revealed that B. bovis rap-1, four separate clades of T. orientalis msps, and A. marginale msp4 exhibited considerable sequence similarity to homologous sequences from isolates obtained from other countries. Sequence similarity ranged between 98.57-100%, 83.96-100%, and 97.60-100% for B. bovis rap-1, T. orientalis msps, and A. marginale msp4, respectively. Conclusion: In this study, the analyzed incidence data of cattle hemoparasitic infection in Thailand has provided valuable and basic information for the adaptation of blood-borne parasitic infections control strategies. Moreover, the data obtained from this study would be useful for future effective parasitic disease prevention and surveillance among cattle.

Immunization of Cattle With Recombinant Structural Ectodomains I and II of Babesia bovis Apical Membrane Antigen 1 [BbAMA-1(I/II)] Induces Strong Th1 Immune Response.

Both strong innate and adaptive immune responses are an important component of protection against intraerythrocytic protozoan parasites. Resistance to bovine babesiosis is associated with interferon (IFN)-γ mediated responses. CD4+ T cells and macrophages have been identified as major effector cells mediating the clearance of pathogens. Previously, the apical membrane antigen 1 (AMA-1) was found to significantly induce the immune response inhibiting B. bovis merozoite growth and invasion. However, a detailed characterization of both humoral and cellular immune responses against the structure of B. bovis AMA-1 (BbAMA-1) has not yet been established. Herein, the present study aimed to express the recombinant BbAMA-1 domain I+II protein [rBbAMA-1(I/II)], which is the most predominant immune response region, and to characterize its immune response. As a result, cattle vaccinated with BbAMA-1(I/II) significantly developed high titters of total immunoglobulin (Ig) G antibodies and a high ratio of IgG2/IgG1 when compared to control groups. Interestingly, the BbAMA-1(I/II)-based formulations produced in our study could elicit CD4+ T cells and CD8+ T cells producing IFN-γ and tumor necrosis factor (TNF)-α. Collectively, the results indicate that immunization of cattle with BbAMA-1(I/II) could induce strong Th1 cell responses. In support of this, we observed the up-regulation of Th1 cytokine mRNA transcripts, including IFN-γ, TNF-α, Interleukin (IL)-2 and IL-12, in contrast to down regulation of IL-4, IL-6 and IL-10, which would be indicative of a Th2 cytokine response. Moreover, the up-regulation of inducible nitric oxide synthase (iNOS) was observed. In conclusion, this is the first report on the in-depth immunological characterization of the response to BbAMA-1. According to our results, BbAMA-1 is recognized as a potential candidate vaccine against B. bovis infection. As evidenced by the Th1 cell response, it could potentially provide protective immunity. However, further challenge-exposure with virulent B. bovis strain in immunized cattle would be needed to determine its protective efficacy.

A Preliminary Study of the Cross-Reactivity of Canine MAGE-A with Hominid Monoclonal Antibody 6C1 in Canine Mammary Gland Tumors: An Attractive Target for Cancer Diagnostic, Prognostic and Immunotherapeutic Development in Dogs.

Melanoma-associated antigen-A (MAGE-A), a family of cancer/testis antigens, has been recognized as a potential target molecule for cancer immunotherapy. However, there has been very little information available with regard to this antigen in dogs. This study aimed to investigate the expression of MAGE-A in canine mammary gland tumors (CMTs) using immunohistochemistry and immunoblotting with human monoclonal MAGE-A antibody 6C1. The present study has provided evidence of cross-reactivity of the canine MAGE-A expression with the human MAGE-A antibody in CMTs. The MAGE-A antigens were expressed in moderate- and high-grade malignant CMTs (22.22%, 2/9), but no expression was observed in benign CMTs. The immunohistochemical staining of canine MAGE antigen in CMT cells showed nuclear and nuclear-cytoplasmic expression patterns that may be involved with the mitotic cell division of tumor cells. Molecular weights of the canine MAGE-A antigen presented in this study were approximately 42-62 kDa, which were close to those of other previous studies involving humans and dogs. The findings on this protein in CMTs could supply valuable oncological knowledge for the development of novel diagnostic, prognostic and immunotherapeutic tumor markers in veterinary medicine.

Identification of potential canine mammary tumour cell biomarkers using proteomic approach: Differences in protein profiles among tumour and normal mammary epithelial cells by two-dimensional electrophoresis-based mass spectrometry.

Canine mammary tumours (CMTs) are regarded as invasive with a high rate of recurrent and metastasis in intact female dogs. Tumour diagnosis, therefore, is an important step in predicting and monitoring tumour progression. This study was designed to identify protein expression on CMTs by employing a proteomic approach. The primary cell culture from benign mixed tumour, simple carcinoma, complex carcinoma and normal mammary gland were established, and two-dimensional electrophoresis (2DE) was subsequently performed. The different spots on each sample type were collected for identification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicated that cytokeratin 5 (CK5) and transketolase (TKT) were identified in benign mixed tumour cells and complex carcinoma cells. In contrast, cytokeratin 18 (CK18) and pyruvate kinase PKM were identified in simple carcinoma cells. Moreover, alpha-2-HS-glycoprotein tumour antigen was identified specifically in complex carcinoma cells. In addition, ATP-dependent 6-phosphofructokinase platelet type and elongation factor 2 proteins were observed in benign cells. In conclusion, all expressed proteins in this study have been recognized for acting as their expression that differs from healthy mammary epithelial cells. Expectantly, this study identified the expressed proteins that might be useful in further diagnostic biomarker studies on CMTs.

Comparison of two indirect ELISA coating antigens for the detection of dairy cow antibodies against Pasteurella multocida.

The ELISA is recognized as an efficient diagnostic tool for antibody detection, but there is no standard ELISA assay for detection of antibodies against hemorrhagic septicemia (HS) in cattle. The present study reports on an indirect ELISA assay for antibody detection of HS in dairy cows, and evaluates the sensitivity (Se) and specificity (Sp) of the method using a Bayesian approach. An indirect ELISA was developed with two types of heat extract antigens, Pasteurella multocida strains P-1256 and M-1404, as coating antigens. A checkerboard titration was employed using dairy cow sera immunized with P. multocida bacterin and colostrum-deprived calf sera. The concentrations of heat extract antigen (160µg/mL), sample serum (1:100) and goat anti-bovine immunoglobulin G labeled with horseradish peroxidase (1:2000) were optimal for the assay. The cut-off values were 0.147 and 0.128 for P-1256 and M-1404 coating antigens, and there were no differences in the results of tests with positive and negative sera (p<0.05). The characteristics of three diagnostic tests were evaluated using a one-population Bayesian model, assuming conditional dependence between two types of coating antigen-based ELISAs and indirect hemagglutination assay (IHA). A total of 415 sera samples from dairy cows without HS vaccination and no history of disease were tested. The Se and Sp of the P-1256 and M-1404 ELISAs were higher than those of the IHA. The Se and Sp of the P-1256 ELISA were 90.3% and 90.1%, while the Se and Sp of the M-1404 ELISA were 92.1% and 71.9%. The median values of Se and Sp from the IHA were 36.0% and 58.2%.