Oxidative Stress, Persistent Inflammation and Blood Coagulation Alterations in Serum Proteome of Patients with Neovascular Age-Related Macular Degeneration.

Neovascular age-related macular degeneration (AMD) is a major cause of irreversible blindness in elderly populations in developed countries. AMD's etiopathology is multifactorial, with strong environmental and genetic components, but the exact molecular pathomechanisms underlying the disease are still unknown. In this study, we analyzed blood serum collected from 74 neovascular AMD patients and 58 healthy controls to identify proteins that may serve as potential biomarkers and expand our knowledge about the etiopathogenesis of the disease. The study revealed 17 differentially expressed proteins-11 up-regulated and 6 down-regulated-in neovascular AMD, which are involved in the biological processes previously linked with the disease-oxidative stress and persistent inflammation, impaired cellular transport, lipid metabolism and blood coagulation. In conclusion, the differences in the expressions of the proteins identified in this study may contribute to our understanding of the mechanisms underlying AMD and possibly serve in future as promising biomarkers.

Changes in Tear Proteomic Profile in Ocular Diseases.

The search for proteomic biomarkers in ocular disease is one of the most important research directions in recent years. Reliable biomarkers can be an immense adjuvant for both diagnostic and therapeutic approaches. There is no more readily available ocular tissue for proteomic analysis than tear film, which makes an interesting target for the biomarker search. Tear film is a complex fluid consisting of a superficial lipid layer, which covers the aqueous-mucous layer. Its complexity makes it a perfect candidate for all the "omics" approaches. Glaucoma, cataract, age-related macular degeneration, and other diseases are commonly thought to have a multifactorial background. Currently, no reliable non-invasive tests are available that would help physicians with screening and further patient management. The aim of the study is to present modern methods of measuring biomarkers in tears, with particular emphasis on spectrometric methods, and to discuss their diagnostic and therapeutic usefulness.

Procoagulant and anticoagulant plasma indicators in diabetic dogs showing increased antithrombin III levels in canine diabetes mellitus.

BACKGROUND: Diabetes mellitus (DM) often leads to dangerous thromboembolic complications in humans. DM is also a relatively common endocrinopathy of dogs. There is scarce information regarding procoagulant and anticoagulant plasma indicators in this disease. The aim of the study was to evaluate the levels of the selected plasma haemostatic parameters in dogs suffering from diabetes. The study group consisted of 20 dogs meeting all the inclusion criteria, with fasting glycaemia exceeding 11.1 mmol/l. The control group consisted of 15 healthy dogs presented for routine examination. An evaluation of the prothrombin time (PT); and fibrinogen, D-dimer and antithrombin III (ATIII) levels was performed. RESULTS: Except for ATIII activity, the haemostatic parameter differences were not statistically significant. High values of ATIII activity were observed in 90% of diabetic dogs. On average, the values amounted to 166.6% and were 31.4% higher than those in the control group. The ATIII activity in the diabetic group was significantly higher than that in the control group (p = 0.0004). CONCLUSIONS: Here, we report elevated levels of ATIII in diabetic dogs. This finding may suggest the protective role of ATIII against potential thrombotic events. However, the exact role of ATIII in dog diabetes remains unclear.

Babesia gibsoni Infection in Dogs-A European Perspective.

Canine babesiosis is a disease caused by infection with parasites of the genus Babesia. These protozoa are classified as either large (e.g., Babesia canis) or small (e.g., Babesia gibsoni). So far, only three small Babesia species of clinical importance, able to infect dogs, have been described: B. gibsoni, B. conradae, and B. vulpes. This review presents the current epidemiological situation of Babesia gibsoni infections in dogs in Europe. In most European countries where B. gibsoni has been reported, the percentage of infected dogs is around 1%. The higher prevalence of the B. gibsoni infection among American Pit Bull Terriers suggests breed susceptibility. An analysis of the available data makes it possible to conclude that B. gibsoni infections may appear in the future in other non-endemic regions of Europe, which may pose significant diagnostic and therapeutic challenges for veterinary practitioners.

Urinary Proteome Differences in Canine Diabetes with and without the Presence of Microalbuminuria.

In this study we aimed to analyze the protein composition of the urine collected from the healthy animals and compare it to the two diabetic groups (DM I normoalbuminuric diabetic dogs; DM II diabetic dogs with microalbuminuria). We tried to identify potential urinary proteins which could be up- or downregulated in diabetic patients even before the appearance of microalbuminuria. Methods: After obtaining urine, we performed two-dimensional electrophoresis, followed by Delta2D software analysis, which allowed for selection and identification with MALDI-TOF spectrometry, statistically significant differentially expressed proteins. Our study revealed 286 common protein spots on 2D gels from the diabetic and control group. From these proteins five were positively identified by MALDI-TOF MS. To further evaluate the five differentiating proteins, the Panther program was used to assign them to appropriate biological process. Conclusion: Significant number of identified proteins play a role in intracellular signaling-vesicle formation, bonding, transport through membranes. This may suggest that first signs of kidney diabetic cellular impairment may be seen in the urine composition before any clinical signs occur.

Proteomic Analysis of Tear Film in Canine Diabetic Patients with And Without Retinopathy.

Introduction: 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. Material and Methods: Tear films were collected on Schirmer strips from 32 canine patients (12 diabetic dogs without changes in the retina, 8 diabetic dogs with signs of DR, and 12 control dogs). Two-dimensional electrophoresis was used to separate tear film proteins prior to their identification with matrix-assisted laser desorption/ionisation-tandem time-of-flight mass spectrometry and interrogation of protein function databases to find matches. Results: Five significantly differentially expressed proteins were identified; of those, one was downregulated (2'-5'-oligoadenylate synthase 3) and four were upregulated in the tear film of two diabetic groups (Ras-related protein RAB-13; aldo-keto-reductase family 1 member C3; 28S ribosomal protein S31, mitochondrial; and 60S ribosomal protein L5). The differentially expressed proteins identified in the tear film were involved in signalling pathways associated with impaired protein clearance, persistent inflammation and oxidative stress. Conclusion: The results of our study confirm that the pathological process in the retina in the course of diabetes mellitus causes changes in the tear film proteome.

Prevalence of selected tick-borne pathogens in wild ungulates and ticks in southern Spain.

A survey study was carried out to assess the occurrence of selected tick-borne pathogens (TBP) in wild ungulates in Mediterranean ecosystems in southern Spain. Spleen samples were collected from 1,132 wild ungulates, including 578 red deer, 269 wild boar, 135 mouflon, 121 fallow deer and 29 roe deer, between 2009 and 2015. Eighty-nine ticks collected from TBP-positive animals were also analysed. Samples were tested by PCR and sequenced whenever possible. TBP DNA was detected in 127 of 863 wild ruminants (14.7%; 95% CI: 12.4-17.3) including the following: Anaplasma phagocytophilum (9.2%), Babesia divergens (2.9%), Theileria sp. OT3 (1.7%), Borrelia afzelii (0.7%) and Theileria capreoli (0.2%), but no positive samples were detected in wild boar (0/269). All the strains from mouflon were identified as Theileria sp. OT3, while B. divergens and T. capreoli were mainly found in red deer. Co-infection with A. phagocytophilum and B. divergens, and A. phagocytophilum and Theileria spp. was detected in red deer and mouflon, respectively. The risk factor analysis showed that the prevalences of A. phagocytophilum and piroplasms were species-related. Eighty-nine tick specimens collected from ungulates found to be infected with the selected TBP were identified as Hyalomma lusitanicum (95.5%) and Ixodes ricinus (4.5%). Thirty ticks were positive for Anaplasma/Ehrlichia spp. (33.7%), 25 for Babesia/Theileria (28.1%) and two for B. burgdorferi s.l. (2.3%). Eleven specimens showed co-infections with Anaplasma/Ehrlichia and Babesia/Theileria (10.1%) or Anaplasma/Ehrlichia and B. burgdorferi s.l. (2.3%). The estimated prevalences obtained in the present study suggest the possible contribution of wild ruminants to the maintenance of some selected TBP in Mediterranean ecosystems in southern Spain, while the role of wild boar in the epidemiology of these pathogens seems to be limited in this region.

Dysregulated Tear Film Proteins in Macular Edema Due to the Neovascular Age-Related Macular Degeneration Are Involved in the Regulation of Protein Clearance, Inflammation, and Neovascularization.

Macular edema and its further complications due to the leakage from the choroidal neovascularization in course of the age-related macular degeneration (AMD) is a leading cause of blindness among elderly individuals in developed countries. Changes in tear film proteomic composition have been reported to occur in various ophthalmic and systemic diseases. There is an evidence that the acute form of neovascular AMD may be reflected in the tear film composition. Tear film was collected with Schirmer strips from patients with neovascular AMD and sex- and age-matched control patients. Two-dimensional electrophoresis was performed followed by MALDI-TOF mass spectrometry for identification of differentially expressed proteins. Quantitative analysis of the differential electrophoretic spots was performed with Delta2D software. Altogether, 11 significantly differentially expressed proteins were identified; of those, 8 were downregulated, and 3 were upregulated in the tear film of neovascular AMD patients. The differentially expressed proteins identified in tear film were involved in signaling pathways associated with impaired protein clearance, persistent inflammation, and neovascularization. Tear film protein analysis is a novel way to screen AMD-related biomarkers.

Occurrence of Listeria monocytogenes in Artisanal Cheeses from Poland and Its Identification by MALDI-TOF MS.

Listeria monocytogenes is a foodborne pathogen. A source of infection can be artisanal cheeses. Identification of the Listeria species is important for the protection of public health and the food industry. This study aimed to examine artisanal cheeses for the presence of L. monocytogenes and the effectiveness of the MALDI-TOF MS method in the identification of the L. monocytogenes isolates. A total of 370 samples of artisanal cheeses were examined. L. monocytogenes was found in 23 cheese samples (6.2%). The reliability of L. monocytogenes identification achieved by MALDI-TOF MS was varied, and the vast majority of the isolates (27/32) were identified only to the secure genus, probable species level. This study showed that (i) the occurrence of L. monocytogenes in the artisanal cheeses was at a higher level than that in the other EU countries, (ii) the standard of species identification of L. monocytogenes isolates from artisanal cheeses achieved by MALDI-TOF MS was not satisfactory and (iii) the presence of L. monocytogenes in artisanal cheeses remains a problem with regard to the food safety criterion and a potential public health risk.

Granulocytic anaplasmosis in captive ring-tailed lemur (Lemur catta) in Poland.

BACKGROUND: Anaplasma are obligate intracellular bacteria and aetiological agents of tick-borne diseases of both veterinary and medical interest. The genus Anaplasma comprises six species: Anaplasma marginale, Anaplasma centrale, Anaplasma ovis, Anaplasma phagocytophilum, Anaplasma bovis and Anaplasma platys. They can infect humans, carnivores, ruminants, rodents, insectivores, birds and reptiles. The aim of this study was to present the first clinical case of granulocytic anaplasmosis in a captive ring-tailed lemur in Poland. CASE PRESENTATION: A 4-year-old female lemur presented anorexia, epistaxis and tick infestation. The microscopic examination of a blood smear revealed morulae in neutrophils. Polymerase chain reaction test and sequencing of obtained PCR product confirmed infection by the GU183908 Anaplasma phagocytophilum strain. Therapeutic protocol included doxycycline (2.5 mg/kg p.o., b.i.d.) for 3 weeks and the lemur recovered within 24 h. CONCLUSIONS: This is the first report on granulocytic anaplasmosis in a ring-tailed lemur in Europe, indicating that A. phagocytophilum infection must also be considered in differential diagnosis in this animal species, especially in individuals with thrombocytopenia associated with Ixodes ricinus parasitism.

Molecular surveillance of tick-borne diseases affecting horses in Poland-Own observations.

The purpose of the study was to carry out the molecular surveillance of piroplasmosis, granulocytic anaplasmosis and lyme borreliosis in horses which originated from Poland and exhibited symptoms raising the suspicion of the aforementioned disease units. The presence of Theileria equi genetic material was detected in 37 out of 512 examined horses (7.2%), and Anaplasma phagocytophilum in 9 (1.8%). The DNA of Borrelia burgdorferi was found in 11 out of 204 examined horses (5.4%). The above-cited results indicate that the problem of tick-borne diseases affecting horses in Poland is not as significant as in other parts of Europe, however they have to be considered in differential diagnosis of the diseases with lethargy, fever, anaemia and thrombocytopenia.

Molecular detection of Anaplasma phagocytophilum in roe deer (Capreolus capreolus) in eastern Poland.

Wild ungulates may serve as reservoirs for particular tick-borne pathogens (TBP) of importance for public and animal health. The aim of the study was to investigate, for the first time, the occurrence of Anaplasma phagocytophilum (AP) in a large population of roe deer in eastern Poland. Spleen samples from 424 roe deer (Capreolus capreolus) were collected during the 2018-2019 hunting season. Genetic screening for AP was carried out by PCR amplification of a fragment of the 16S rRNA and groEL genes. Twenty-six of the 424 spleen samples (6.13%) tested positive both for 16S rRNA and groEL. Phylogenetic analysis confirmed the existence of a specific roe deer ecotype of AP in eastern Poland. Despite the low prevalence of AP in roe deer populations in the study area, these animals may act as anaplasmosis reservoirs for ticks. Based on the zoonotic potential of granulocytic anaplasmosis, it seems advisable to introduce continuous monitoring of this infection among domestic, farm and wild animals.

Proteomic Analysis of Tear Film Obtained from Diabetic Dogs.

Canine diabetes mellitus is a significant health burden, followed with numerous systemic complications, including diabetic cataracts and retinopathy, leading to blindness. Diabetes should be considered as a disease damaging all the body organs, including gastrointestinal tract, through a complex combination of vascular and metabolic pathologies, leading to impaired gut function. Tear film can be obtained in a non-invasive way, which makes it a feasible biomarker source. In this study we compared proteomic changes ongoing in tear film of diabetic dogs. The study group consisted of 15 diabetic dogs, and 13 dogs served as a control group. After obtaining tear film with Schirmer strips, we performed 2-dimensional electrophoresis, followed by Delta2D software analysis, which allowed to select statistically significant differentially expressed proteins. After their identification with MALDI-TOF (matrix assisted laser desorption and ionisation time of flight) spectrometry we found one up-regulated protein in tear film of diabetic dogs-SRC kinase signaling inhibitor 1 (SRCIN1). Eight proteins were down-regulated: phosphatidylinositol-4 kinase type 2 alpha (PI4KIIα), Pro-melanin concentrating hormone (Pro-MCH), Flotillin-1, Protein mono-ADP ribosyltransferase, GRIP and coiled coil domain containing protein 2, tetratricopeptide repeat protein 36, serpin, and Prelamin A/C. Identified proteins were analyzed by Panther Gene Ontology software, and their possible connections with diabetic etiopathology were discussed. We believe that this is the first study to target tear film proteome in canine diabetes. We believe that combined with traditional examination, the tear film proteomic analysis can be a new source of biomarkers both for clinical practice, and experimental research.

Utility of Urinary Markers in the Assessment of Renal Dysfunction in Familial Glomerulonephritis in Dobermann Dogs.

INTRODUCTION: Dobermann dogs are reportedly predisposed to familial glomerulonephropathy. Proteinuria is a hallmark of canine familial glomerular diseases. The identification of glomerular abnormalities in breeds so predisposed is of great importance in improving breeding policy. Therefore, markers that allow the detection and localisation of renal damage are needed. The purpose of this study was to investigate the urinary concentrations of immunoglobulin G (uIgG), retinol-binding protein (uRBP), and Tamm-Horsfall protein (uTHP) in a family of Dobermanns with proteinuria and compare these concentrations with the corresponding values in healthy controls. MATERIAL AND METHODS: Ten dogs of the Dobermann breed with proteinuria (five with a urine protein-to-creatinine ratio (UPC) of 0.5-1 and five with a UPC >1) and twelve healthy dogs were enrolled. An ELISA was performed to measure uIgG, uRBP, and uTHP, and these proteins were quantified in relation to urinary creatinine (uCrea). RESULTS: uIgG/uCr and uRBP/uCr were significantly higher in the family of Dobermanns than in the healthy dogs. A significant difference in the uTHP/uCr value was found only in dogs with a UPC of >1. CONCLUSION: IgG seems to facilitate the diagnosis of primary hereditary glomerulopathy in Dobermanns. Moreover, in affected dogs, proteinuria characterisation seems to be a promising alternative option for the detection and localisation of renal lesions.

Comparison of Intraocular Pressure Measurement with Schiotz Tonometer and Tono-Pen Vet Tonometer in Healthy Dogs.

INTRODUCTION: Measurement of intraocular pressure (IOP) in dogs has high diagnostic value because of the possibility of detecting ocular and systemic diseases. Various types of tonometers are available for this measurement in small animal practice. The aim of the study was to compare the IOP values measured with Schiotz and Tono-Pen Vet tonometers in healthy dogs. Clinical diagnostic usefulness of both models was also evaluated. MATERIAL AND METHODS: The examination was performed in 62 eyes in 31 clinically healthy dogs of different races, gender, and ages. RESULTS: The values for intraocular pressure obtained with Schiotz tonometer were in the range of 12 to 24 mmHg, with the mean of 16.3 ± 2.1 mmHg. The intraocular pressure measured with Tono-Pen Vet tonometer was in the range of 11-25 mmHg, with a mean of 18.1 ± 3.8 mmHg. The mean results of measurements taken using the two tonometers differed statistically significantly, the difference being 1.79 mmHg and the higher values being read from the Tono-Pen Vet tonometer. CONCLUSION: Correlation coefficients calculated for the results obtained in the right and left eyes using two tonometers indicated highly correlative relationships between the results. The study shows that both tonometers can be advantageously used in clinical practice to measure intraocular pressure in dogs.

Tear film proteome in age-related macular degeneration.

PURPOSE: Age-related macular degeneration (AMD) is the main reason for blindness in elderly people in the developed countries. Current screening protocols have limitations in detecting the early signs of retinal degeneration. Therefore, it would be desirable to find novel biomarkers for early detection of AMD. Development of novel biomarkers would help in the prevention, diagnostics, and treatment of AMD. Proteomic analysis of tear film has shown promise in this research area. If an optimal set of biomarkers could be obtained from accessible body fluids, it would represent a reliable way to monitor disease progression and response to novel therapies. METHODS: Tear films were collected on Schirmer strips from a total of 22 patients (8 with wet AMD, 6 with dry AMD, and 8 control individuals). 2D electrophoresis was used to separate tear film proteins prior to their identification with matrix-assisted laser desorption/ionization time of flight spectrometer (MALDI-TOF/TOF) and matching with functional databases. RESULTS: A total of 342 proteins were identified. Most of them were previously described in various proteomic studies concerning AMD. Shootin-1, histatin-3, fidgetin-like protein 1, SRC kinase signaling inhibitor, Graves disease carrier protein, actin cytoplasmic 1, prolactin-inducible protein 1, and protein S100-A7A were upregulated in the tear film samples isolated from AMD patients and were not previously linked with this disease in any proteomic analysis. CONCLUSION: The upregulated proteins supplement our current knowledge of AMD pathogenesis, providing evidence that certain specific proteins are expressed into the tear film in AMD. As far we are aware, this is the first study to have undertaken a comprehensive in-depth analysis of the human tear film proteome in AMD patients.

Utility of urinary markers in the assessment of renal dysfunction in canine babesiosis.

OBJECTIVE: Canine babesiosis is a common and clinically significant tick-borne disease caused by haemoprotozoan parasites of the genus Babesia. Acute renal failure is considered to be one of the most prevalent complications of canine babesiosis. This complication leads to a decrease in the glomerular filtration rate and in consequence causes azotemia and uremia. The objective of this study was to assess the localization and extent of renal damage in dogs infected with Babesia canis using an urinary marker for glomerular (urinary immunoglobulin G, uIgG), proximal tubular dysfunction (urinary retinol binding protein, uRBP) and distal tubular dysfunction (urinary Tamm-Horsfal protein, uTHP). Material und methods: In 10 dogs naturally infected with B. canis and 10 healthy control dogs the levels of urinary biomarkers were measured using commercially available ELISA tests. RESULTS: Higher concentrations of uIgG, uRBP and uTHP were found in the urine of all dogs with babesiosis than in those from the control group. This indicates that in the course of the disease, the glomeruli as well as the renal tubules become damaged. CONCLUSION AND CLINICAL RELEVANCE: The study results allow a better understanding of the pathogenesis of canine babesiosis. However, in order to fully determine the extent and the nature of the damage to the kidneys of the infected dogs, it is advisable to conduct additional histopathological examinations of these organs.

Dog Tear Film Proteome In-Depth Analysis.

In this study, mass spectrometry was used to explore the canine tear proteome. Tear samples were obtained from six healthy dogs, and one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (1D SDS-PAGE) was used as a first step to separate intact proteins into 17 bands. Each fraction was then trypsin digested and analysed by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) to characterize the protein components in each fraction. In total, 125 tear proteins were identified, with MCA (Major Canine Allergen), Serum albumin, UPF0557 protein C10orf119 homolog, Collagen alpha-2(I) chain, Tyrosine -protein kinase Fer, Keratine type II cytoskeletal, Beta-crystallin B2, Interleukin-6 and Desmin occurring as the most confident ones with the highest scores. The results showed that the proteomic strategy used in this study was successful in the analysis of the dog tear proteome. To the best of our knowledge, this study is the first to report the comprehensive proteome profile of tears from healthy dogs by 1D SDS PAGE and MALDI-TOF. Data are available via ProteomeXchange with identifier PXD003124.

Application the mass spectrometry MALDI-TOF technique for detection of Babesia canis canis infection in dogs.

The aim of this study was to use rapid mass spectrometry (MS)-based proteomics analyses for diagnosis of Babesia canis canis infections in dogs. The study was conducted on two groups of dogs--healthy dogs and dogs infected with B. canis canis which demonstrated symptoms of babesiosis. The matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS technique revealed the presence of a protein fraction of 51-52 kDa in the blood serum of all the animals infected with the protozoa, which was not found in the serum of healthy dogs. The proteins are suspected to be disease markers, whereas the MALDI-TOF technique itself has high specificity and sensitivity and can be applied in analytical laboratories in the diagnosis of canine babesiosis.