Surface plasmon resonance immunosensor for label-free detection of BIRC5 biomarker in spontaneously occurring canine mammary tumours.

We report detection of Baculoviral inhibitor of apoptosis repeat containing-5 (BIRC5) protein biomarker in dog serum by label-free surface plasmon resonance (SPR) immunosensor. Initially, overexpression of BIRC5 in canine mammary tumour (CMT) tissues was confirmed by real-time PCR. Recombinant BIRC5 was produced and protein specific antibodies developed in guinea pig specifically reacted with native protein in immunohistochemistry and immunocytochemistry. SPR immunosensor was developed by fabricating anti-BIRC5 antibodies on gold sensor disc. The equilibrium dissociation constant, (KD = kd/ka) was 12.1 × 10-12 M; which indicates that antibodies are of high affinity with sensitivity in picomolar range. The SPR assay could detect as low as 6.25 pg/ml of BIRC5 protein in a calibration experiment (r2 = 0.9964). On testing real clinical samples, 95% specificity and 73.33% sensitivity were recorded. The average amount of serum BIRC5 in dogs with CMT was 110.02 ± 9.77 pg/ml; whereas, in non-cancerous disease conditions, 44.79 ± 4.28 pg/ml and in healthy dog sera 30.28 ± 2.99 pg/ml protein was detected. The SPR immunosensor for detection of BIRC5 in dog sera is reported for the first time and this may find prognostic and diagnostic applications in management of CMT. In future, 'on-site' sensors can be developed using this technique for near-patient testing.

Multiplexed Autoantibody Signature for Serological Detection of Canine Mammary Tumours.

Spontaneously occurring canine mammary tumours (CMTs) are the most common neoplasms of female unspayed dogs and are of potential importance as models for human breast cancer as well. Mortality rates are thrice higher in dogs as compared to humans with breast cancer, which can partly be attributed to lack of diagnostic techniques for their early detection. Human breast cancer studies reveal role of autoantibodies in early cancer diagnosis and also the usefulness of autoantibody panels in increasing the sensitivity, as well as, specificity of diagnostic assays. Therefore, in this study, we took advantage of high-throughput Luminex technique for developing a multiplex assay to detect autoantibody signatures against 5 canine mammary tumour-associated autoantigens (TAAs). These TAAs were expressed separately as fusion proteins with halo tag at the N-terminus, which allows easy and specific covalent coupling with magnetic microspheres. The multiplex assay, comprising a panel of candidate autoantigens (TPI, PGAM1, MNSOD, CMYC & MUC1) was used for screening circulating autoantibodies in 125 dog sera samples, including 75 mammary tumour sera and 50 healthy dog sera. The area under curve (AUC) of the combined panel of biomarkers is 0.931 (p < 0.0001), which validates the discriminative potential of the panel in differentiating tumour patients from healthy controls. The assay could be conducted in 3hrs using only 1ul of serum sample and could detect clinical cases of canine mammary tumour with sensitivity and specificity of 78.6% and 90%, respectively. In this study, we report for the first time a multiplexed assay for detection of autoantibodies in canine tumours, utilizing luminex technology and halo-tag coupling strategy. Further to the best of our knowledge, autoantibodies to CMYC and MUC1 have been reported for the first time in canines in this study.

Trypanosoma evansi infection in a German shepherd dog - Apparent successful treatment using serial low dose of diminazene aceturate.

The present study reports a new case of Trypanosoma evansi infection in a dog possibly due to oral transmission from a wild antelope, Nilgai. A four year old male German shepherd dog presented to the Referral Veterinary Polyclinic with history of inappetance, persistent fever, ocular discharge, standing for prolong periods and head pressing. Physical examination revealed corneal opacity, absence of menace reflex and partial blindness. The blood smear examination revealed the presence of Trypanosoma species. T. evansi infection was confirmed by RoTat 1.2 T. evansi specific Polymerase Chain Reaction (PCR). Haemato-biochemical examination showed anaemia, leukopenia, neutrophilia, a mild increase in concentration of alanine aminotransferase, blood urea nitrogen, and creatinine, and a decrease in concentration of blood glucose level. The dog was treated with diminazene aceturate at 3.5mg/kg body weight by deep intramuscular for 5days along with supportive therapy. Marked recovery in clinical signs as well as restoration of normal organ function and oxidant/antioxidant equilibrium was observed after two weeks of treatment. The dog was followed up to 6month and was negative for T. evansi by microscopy and PCR. The present treatment of a consecutive five dose regime of diminazene aceturate along with supportive therapy may help clinicians to overcome the hurdle of relapsing parasitaemia due to T. evansi and successful recovery in clinical cases.