Diabetic retinopathy (DR) is the leading cause of blindness in human and animal patients. Early detection and treatment of the disease are important and can be facilitated by proteomic approaches providing biomarkers.

Proteomics, which is called the identification phase of the proteome identifying all the proteins encoded by the genome, has an important place in solving the difficulties experienced in toxicology. Proteomics refers to the study of interactions with other proteins and macromolecules, structure, location, amount, post-translational modifications (PTM), function in tissues/cells of the proteome. With proteomic studies, new relationships between proteins and toxicopathological effects can be determined and it is revealed the information on the toxic action mechanisms of various substances, from metals to peroxisome proliferators. On the other hand, toxicoproteomics seeks to identify critical proteins and pathways in biological systems that respond to it and adversely affected by chemical and environmental exposures using protein expression technologies. Toxicoproteomics combines 3 discipline areas. These are 1) traditional toxicology and pathology 2) differential protein and gene expression analysis 3) systems biology. Toxicoproteomic studies are an important area that can provide critical tools for identification of biomarkers associated with exposure to toxic substances, assessing their reliability and designing appropriate measures that can minimize adverse effects. In this statement, it is aimed to emphasize the importance of proteomics in toxicology.

OBJECTIVE To measure changes in interleukin-8 (IL-8), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) concentrations in stored canine packed RBCs (PRBCs) over time and assess the effect of leukoreduction on these cytokine concentrations. ANIMALS 12 anesthetized healthy Greyhounds. PROCEDURES 1 unit of whole blood from each dog was processed into PRBCs. Half of each PRBCs unit was passed through a leukoreduction filter to produce a leukoreduced unit, and the remaining blood was kept as a nonleukoreduced unit. All units had a CBC performed on day 0 (day of collection) and were stored at 2° to 6°C. Samples were collected from leukoreduced and nonleukoreduced units on days 0, 10, 20, 30, and 37 and centrifuged; the supernatant was stored at −80°C until analysis. Canine TNF-α and IL-8 concentrations were assessed with a multiplexed genomic and proteomic biomarker analyzer, and canine IL-1β concentration was measured by ELISA. RESULTS Leukocyte counts were decreased by ≥ 99.9% in all leukoreduced units. Median TNF-α and IL-1β concentrations were not significantly different between leukoreduced and nonleukoreduced units and did not change significantly during storage; median IL-8 concentration was significantly higher in nonleukoreduced versus leukoreduced units on all days, and was greater at all time points after ≥ 10 days of storage than on day 0. Median IL-8 concentration in leukoreduced units did not increase during storage. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that leukoreduction was effective for the removal of leukocytes from canine PRBCs and prevented significant increases in IL-8 concentration during storage. Further studies are needed to evaluate whether leukoreduction reduces cytokine-associated complications of transfusion.

OBJECTIVE To use proteomic analysis to determine the protein constituents of synovial fluid samples from the stifle joints of dogs with and without osteoarthritis secondary to cranial cruciate ligament rupture (CCLR). ANIMALS 12 dogs with clinically normal stifle joints (controls) and 16 dogs with osteoarthritis secondary to CCLR. PROCEDURES A synovial fluid sample was obtained from all dogs. Synovial fluid total protein concentration was determined by the Bradford assay. Proteins were separated by use of a 1-D SDS-PAGE to detect protein bands that differed between dogs with and without osteoarthritis. Those protein bands then underwent trypsin digestion and were analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry, the results of which were compared with a curated protein sequence database for protein identification. One of the most frequently identified proteins, apoprotein (apo) A-I, was then quantified in all synovial fluid samples by use of a competitive-inhibition ELISA. Results were compared between dogs with and without osteoarthritis. RESULTS Median synovial fluid total protein and apo A-I concentrations for dogs with osteoarthritis were significantly greater than those for control dogs. The most abundant proteins identified in the synovial fluid were albumin and apo A-I. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that quantification of synovial fluid total protein and apo A-I concentrations might facilitate diagnosis of osteoarthritis secondary to CCLR in dogs. Further research and validation of synovial fluid apo A-I concentration as a biomarker for osteoarthritis in dogs are necessary before it can be recommended for clinical use.

While pancreatitis is now recognized as a common ailment in cats, the diagnosis remains challenging due to discordant results and suboptimal sensitivity of ultrasound and specific feline pancreatic lipase (Spec fPL) assay. Pancreatitis also shares similar clinical features with pancreatic carcinoma, a rare but aggressive disease with a grave prognosis. The objective of this pilot study was to compare the plasma proteomes of normal healthy cats (n = 6), cats with pancreatitis (n = 6), and cats with pancreatic carcinoma (n = 6) in order to identify potential new biomarkers of feline pancreatic disease. After plasma protein separation by 2-dimensional gel electrophoresis, protein spots were detected by Coomassie Brilliant Blue G-250 staining and identified by mass spectrometry. Alpha-1-acid glycoprotein (AGP), apolipoprotein-A1 (Apo-A1), and apolipoprotein-A1 precursor (Pre Apo-A1) appeared to be differentially expressed, which suggests the presence of a systemic acute-phase response and alteration of lipid metabolism in cats with pancreatic disease. Future studies involving greater case numbers are needed in order to assess the utility of these proteins as potential biomarkers. More sensitive proteomic techniques may also be helpful in detecting significant but low-abundance proteins.

Objective: To evaluate proteomic delineation of feline urine by mass spectrometry as a method for identifying biomarkers in cats at risk of developing azotemia. Samples: Urine samples from geriatric cats (> 9 years old) with chronic kidney disease and nonazotemic cats that either remained nonazotemic (n = 10) or developed azotemia (10) within 1 year. Procedures: Optimization studies with pooled urine were performed to facilitate the use of surface enhanced laser desorption-ionization time-of-flight mass spectrometry (SELDI-TOF-MS) for analysis of the urinary proteome of cats. Urine samples from nonazotemic cats at entry to the study were analyzed via SELDI-TOF-MS with weak cation exchange and strong anion exchange arrays. Spectral data were compared to identify biomarkers for development of azotemia. Results: Low protein concentration in feline urine precluded direct application to array surfaces, and a buffer exchange and concentration step was required prior to SELDI-TOF-MS analysis. Three preparation conditions by use of weak cation and strong anion exchange arrays were selected on the basis of optimization studies for detection of biomarkers. Eight potential biomarkers with an m/z of 2,822, 9,886, 10,033, 10,151, 10,234, 11,653, 4,421, and 9,505 were delineated. Conclusions and Clinical Relevance: SELDI-TOF-MS can be used to detect urinary low-molecular weight peptides and proteins that may represent biomarkers for early detection of renal damage. Further study is required to purify and identify potential biomarkers before their use in a clinical setting.

Objective: To evaluate protein expression in bronchoalveolar lavage fluid (BALF) obtained from West Highland White Terriers with idiopathic pulmonary fibrosis (IPF), dogs with chronic bronchitis, and healthy control dogs to identify potential biomarkers for IPF. Samples: BALF samples obtained from 6 West Highland White Terriers with histologically confirmed IPF, 5 dogs with chronic bronchitis, and 4 healthy Beagles. Procedures: Equal amounts of proteins in concentrated BALF samples were separated via 2-D differential gel electrophoresis. Proteins that were differentially expressed relative to results for healthy control dogs were identified with mass spectrometry and further verified via western blotting. Results: Expression of 6 proteins was upregulated and that of 1 protein was downregulated in dogs with IPF or chronic bronchitis, compared with results for healthy dogs. Expression of proteins β-actin, complement C3, α-1-antitrypsin, apolipoprotein A-1, haptoglobin, and transketolase was upregulated, whereas expression of lysozyme C was downregulated. Conclusions and Clinical Relevance: Proteomics can be used to search for biomarkers and to reveal disease-specific mechanisms. The quantitative comparison of proteomes for BALF obtained from dogs with IPF and chronic bronchitis and healthy dogs revealed similar changes for the dogs with IPF and chronic bronchitis, which suggested a common response to disease processes in otherwise different lung diseases. Specific biomarkers for IPF were not identified.

Tick-borne diseases (TBDs) or otherwise called equine piroplasmosis (EP) are the foremost economic limitations to equids production. Thus, reducing the breeding capability and athletic performance of equids globally. Identification of these haemoparasites is crucial in understanding their distribution in the population and it is imperative to discern between species and subspecies that are responsible for the occurrence of the disease conditions. Conventional procedures such as microscopic and serological evaluations do not usually meet these prerequisites. Diagnostic contrivances, such as the complement fixation test (CFT), the indirect fluorescent antibody test (IFAT) and the enzyme linked immunosorbent assay (ELISA) have been efficaciously used for many years. Furthermore, DNA-based investigations for identification, differentiation and classification of different haemoparasites have also been established. Molecular diagnostic procedures, such as DNA hybridization, polymerase chain reaction (PCR), transcriptomics, proteomics, metagenomics and metabolomics, permit the uncovering of parasites in blood, tissues or ticks with optimal sensitivity, specificity and consistency. In addition, these procedures can be exploited to detect definite species and subspecies. The prerequisite of these investigations must include proper premeditation and validation, these investigations provide an effective device for molecular studies, with greater benefits of flexibility to standardization. The application of these procedures for studying TBDs or EP globally will be irreplaceable for a long period from now. Therefore, the aim of this review is to draw up the specifics of the procedures in more convenient form for practitioners and researchers. KEY WORDS: Diagnosis, equids, molecular, transcriptomics, proteomics, metagenomics, metabolomics, haemoparasites, tick-borne diseases [J Adv Vet Anim Res 2016; 3(2.000): 84-91]

Objective: This study aimed to elucidate the proteome profile of the 150 kDa protein isolated from the subordinate follicle of Bali cattle (Bos sondaicus/javanicus). Some researchers have revealed several factors in the follicular subordinate with a 150 kDa protein substance, which play important roles in the bovine ovulation. Materials and methods: In the present study, subordinate follicles (~ 5 mm in diameter) were collected from 10 female Bali cattle from slaughterhouses in Taliwang, Sumbawa of West Nusa Tenggara Province, Indonesia. The follicular liquid was pooled; fractionated using SDSPAGE 10%, the 150 kDa band was sliced and then analyzed using MALDI-TOF/TOF-MS. Results: Mascot search results significantly revealed the presence of four species of proteins/peptides. Of the four peptides, two were predominant i.e. complement C3 and anti-testosterone antibody, which both were 100% identical to complement C3 and anti-testosterone antibody of Bos Taurus cattle. Conclusion: Complement C3 and anti-testosterone antibody are present in the follicle fluid of Bos sondaicus/javanicus cows. These findings are novel in Bali cattle follicles. [J Adv Vet Anim Res 2018; 5(1.000): 19-24]