Effects of subclinical inflammation on C-reactive protein and haptoglobin levels as well as specific humoral immunity in dogs vaccinated against canine distemper and parvovirus.

BACKGROUND: The aim of the present study was to assess the effects of subclinical inflammation on specific humoral immunity in dogs vaccinated with Nobivac® DHP based on serum levels of CRP and Hp. Dogs from the group I were administered Nobivac® DHP, the vaccine against distemper, infectious hepatitis and parvovirus whereas group II animals received subcutaneous turpentine oil to induce subclinical inflammation, followed by Nobivac® DHP after 24 h. Animals in group III received only turpentine oil in the way and amount identical to that as in group II. RESULTS: Nobivac DHP relatively poorly induced the immune inflammatory response showing good immunogenic properties, which was evidenced by only a double increase in mean CRP and Hp levels associated with antigenic stimulation in group I. In group II, serum neutralization (SN) and haemagglutination inhibition (HI) results were quite closely correlated with serum levels of CPR and Hp. CONCLUSIONS: Our findings suggest that the efficacy of vaccinations in dogs can be significantly affected by subclinical inflammations, which is indicated by a correlation between serum CRP and Hp levels versus antibody titres for canine distemper and parvovirus in both experimental groups of dogs (group I and II). The correlation of mean CRP and Hp values in dogs with subclinical inflammation and after vaccination with the kinetics of increasing antibody titres against distemper and parvovirus in group II dogs reflects the severity of inflammatory response and the extent of specific humoral immunity. Routine determinations of serum CRP and Hp levels as the indices of inflammation severity can be the essential biochemical markers for assessment of dogs' health in the period preceding specific immunoprophylaxis and efficacy of the vaccine.

The diagnostic value of selected immune parameters in peripheral blood of dogs with malignant mammary tumours - a preliminary study.

Introduction: The main adaptive immune cells are T and B lymphocytes and they play key roles in the induction of immune responses against canine mammary tumours. Investigating these cell subpopulations may lead to more precise diagnosis of these malignancies. Material and Methods: The percentages of CD3+, CD4+ and CD8+ T cells and of CD21+ B cells in the peripheral blood of bitches with malignant mammary tumours were compared with those in the blood of healthy animals. The phenotypic features of peripheral blood leukocytes were evaluated by flow cytometry. Results: There was a significant difference in the mean percentages of CD3+ lymphocytes between healthy (66.7%) and metastatic dogs (46.1%), and between tumour-bearing non-metastatic (66.6%) and metastatic dogs. There was also a significant difference in CD4+ T helper cell percentages between healthy dogs (40.4%) and dogs with metastases (23.2%), and between the latter and dogs without them (35.5%). In the case of CD21+ lymphocyte subsets, a significant difference was noted between healthy animals (10.9%) and those with metastases (20.1%), and between the latter and patients without metastases (8.5%). There were also significant differences in CD3+/CD21+ ratios between the group with metastases (3.0), the healthy group (7.8), and the group without metastases (8.5). Similarly, a significant difference was noted in CD4+/CD8+ ratios between animals with metastases (1.4), bitches in the control group (2.2), and dogs without metastases (1.9). Conclusion: Peripheral blood leukocyte phenotypic characteristics are putative novel biomarkers. These findings may be useful in future studies improving mammary tumour diagnostic procedures, especially in metastasis detection.

Polymorphism of glutathione S-transferase P1 of dogs with mammary tumours.

Mammary tumours constitute more than half of neoplasms in female dogs from different countries. Genome sequences are associated with cancer susceptibility but there is little information available about genetic polymorphisms of glutathione S-transferase P1 (GSTP1) in canine cancers. The aim of this study was to find single nucleotide polymorphisms (SNPs) in GSTP1 of dogs (Canis lupus familiaris) with mammary tumours compared to healthy dogs and to determine the association between GSTP1 polymorphisms and the occurrence of these tumours. The study population included 36 client-owned female dogs with mammary tumours and 12 healthy female dogs, with no previous diagnosis of cancer. DNA was extracted from blood and amplified by PCR assay. PCR-products were sequenced by Sanger method and analysed manually. The 33 polymorphisms were found in GSTP1: 1 coding SNP (exon 4), 24 non-coding SNPs (9 in exon 1), 7 deletions and 1 insertion. The 17 polymorphisms have been found in introns 1, 4, 5 and 6. The dogs with mammary tumours have significant difference from healthy in SNPs I4 c.1018 + 123 T > C (OR 13.412, 95%CI 1.574-114.267, P = .001), I5 c.1487 + 27 T > C (OR 10.737, 95%CI 1.260-91.477, P = .004), I5 c.1487 + 842 G > C (OR 4.714, 95% CI 1.086-20.472, P = .046) and I6 c.2481 + 50 A > G (OR 12.000, 95% CI 1.409-102.207, P = .002). SNP E5 c.1487 T > C and I5 c.1487 + 829 delG also differed significantly (P = .03) but not to the confidence interval. The study, for the first time, showed a positive association of SNPs in GSTP1 with mammary tumours of dogs, that can possibly be used to predict the occurrence of this pathology.

Assessment of Selected Immunological Parameters in Dairy Cows with Naturally Occurring Mycotoxicosis before and after the Application of a Mycotoxin Deactivator.

Introduction: Mycotoxins in dairy cows can cause many non-specific symptoms often resulting from immune system overreaction. The study assessed the concentration of selected cytokines and acute phase proteins (APP) in cows with natural mycotoxicosis before and after using a mycotoxin neutraliser. The cytokines were tumour necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and interleukin 10 (IL-10), and the APP were serum amyloid A (SAA) and haptoglobin (Hp). Material and Methods: The research was carried out on an experimental group (Exp) of 10 herdmate Holstein-Friesian cows with mycotoxicosis. The control group (Con) was 10 healthy cows of the same breed from a different herd. Cows in the Exp group were administered the mycotoxin deactivator Mycofix for three months. Blood was drawn from Exp cows once before administering Mycofix and a second time after three months of its use. Blood was also drawn from Con cows at the same times. Serum levels of TNF-α, IL-6, IL-10, SAA and Hp were assessed using ELISA. Results: The concentrations of all cytokines and Hp in Exp cows were higher before treatment (P < 0.001) than those in Con cows. After three months of administering Mycofix, the concentrations of TNF-α and IL-6 were significantly lower than their pre-treatment levels (P < 0.001). The concentrations of IL-6, IL-10, and Hp were still significantly higher than those in the Con group (P < 0.001). In cows with mycotoxicosis, simultaneous stimulation of antagonistic processes was noted: a pro-inflammatory process in the upregulation of TNF-α and IL-6, and an anti-inflammatory one in the upregulation of IL-10. Conclusion: Despite the absorbent's use and the resolution of clinical symptoms in Exp cows, high levels of IL-10 and Hp and IL-6 were maintained. Assessment of the level of cytokines and APP appears to be a useful and precise tool for the evaluation and application of the appropriate dose of the mycotoxin absorbent or the evaluation of its effectiveness.

Phagocytic Activity, Oxygen Metabolism and Serum Amyloid a Concentration in Peripheral Blood of Mink with Subclinical Aleutian Virus Infection.

Aleutian disease (AD) is a chronic disease of mink caused by the Aleutian Mink Disease Virus (AMDV) that results in dysfunction of the immune system. The prevalence of asymptomatic AMDV infections suggests a necessity to explore their effects on the cellular mechanisms of non-specific immunity in farmed mink. The study evaluated the phagocytic activity and oxygen metabolism of peripheral blood granulocytes and monocytes in mink with chronic subclinical AMDV infection. Moreover, the intensity of inflammatory processes was assessed based on the serum amyloid A (SAA) concentration. The analyses involved 24 brown mink females aged 12−24 months. The experimental group (group I) consisted of mink with chronic subclinical AMDV infections, and the control group (group II) included healthy animals. The statistical analysis was performed using the Mann-Whitney U rank test. Phagocytic activity of granulocytes and monocytes was carried out using flow cytometry, and SAA concentration was determined with enzyme-linked immunosorbent assay (ELISA). Compared with the control group, there was a significant decrease in the phagocytic activity and oxygen metabolism of granulocytes and monocytes in the AMDV-infected mink (p < 0.05). Additionally, it was found that the mean SAA value was significantly higher in the group infected with AMDV than in the control group (p < 0.05). The obtained data indicate that monitoring the serum SAA levels in mink with asymptomatic inflammation may help assess the health of mink and detect asymptomatic inflammation caused by AMDV infection.

Comparative evaluation of cytokine and acute-phase protein concentrations in sera of dairy cows with subclinical and clinical ketosis as a different view of the causes of the disease.

BACKGROUND AND AIM: Ketosis is a common disease occurring during the first stage of lactation in highly productive dairy cows. The aim of the present study was the comparative assessment of selected pro-inflammatory cytokines (including tumor necrosis factor-α [TNF-α] and interleukin 6 [IL-6]), anti-inflammatory cytokines (including IL-10), and acute-phase proteins (APPs) (including haptoglobin [Hp] and serum amyloid A [SAA]), in the sera of cows with subclinical ketosis (SCK), in those with clinical ketosis (CK), and in healthy cows. MATERIALS AND METHODS: Thirty dairy cows of Holstein-Friesian breed were investigated. The cows were divided into three groups depending on the serum ß-hydroxybutyric acid (ßHBA) level. The control, SCK, and CK groups included healthy cows, cows with SCK, and cows with CK, respectively. BHBA concentration in blood serum was determined using colorimetric method. The blood serum was used for proper tests. Cytokine (TNF-α, IL-6, and IL-10) and APPs (SAA and Hp) concentrations in the investigated samples were determined by enzyme-linked immunosorbent assay method. RESULTS: The SCK group had significantly higher TNF-α, IL-6; IL-10, and SAA values than had the CK group (p<0.01). The SCK group had a lower Hp concentration than had the CK group (p<0.05). CONCLUSION: This study showed that the inflammation intensity is higher in the initial phase of the disease and decreases during the advancement, probably due to active anti-inflammatory mechanisms (an increase of IL-10 concentration), which protect animal organism from self-destruction. On the basis of our study, it can be assumed that ketosis development in dairy cows was preceded by the systemic inflammation that may influence the progress of this disease.

Comparative Expression Analysis of Innate Immune Markers and Phagocytic Activity in Peripheral Blood of Dogs with Mammary Tumors.

Canine innate immune system role in cancer prevention and progression remains poorly understood. It has been revealed that innate immune cells could play a dual role in cancer immunology promoting or inhibiting tumor development and growth. Current immunotherapies target mainly the adaptive anti-tumor response and that may be a reason why they remain ineffective in a majority of patients. It is important to acquire detailed knowledge about innate immune mechanisms to broaden the diagnostic and therapeutic options and employ innate immune cells in anti-cancer therapies. In the present study, 21 female dogs of different breeds and types of spontaneous mammary tumors were investigated. The study aimed to find simple and cheap markers that can be used for preliminary diagnosis, prior to the surgical resection of the tumor. The differences in innate immune cell quantity and function were investigated between female dogs with malignant mammary tumors of epithelial and mesenchymal origin. Flow cytometry was used to evaluate the percentages of CD5+ lymphocytes including CD5low lymphocytes, CD11b integrin expression on leukocytes, phagocytosis, and oxidative burst. The number of CD11b lymphocytes was increased in tumors with epithelial origin compared to the control group. No significant differences were found between the percentages of phagocytic cells neither for granulocytes nor for monocytes. However, the phagocytes of canine patients with tumors of epithelial origin showed increased phagocytosis compared to the control group. The percentages of granulocytes that produced reactive oxygen species (ROS) in response to E.coli and PMA were not altered in patients with malignant tumors compared to control. A statistically significant difference between the number of ROS produced by the single granulocyte was demonstrated only between the group of bitches with epithelial tumors and the control group in case of E. coli stimulation. The obtained results suggest that some innate immune cells may be involved in anti-tumor immune mechanisms and have the potential to be supportive diagnostic markers in canine mammary tumors.

Structure and Pathologies of Articular Cartilage.

The aim of the review was to describe a complex microstructure and biomechanical properties of the articular cartilage as well as a current review of its pathologies encountered in veterinary practice. The articular cartilage with its unique features: complex microarchitecture, significant mechanical durability and elasticity, lacking blood, lymphatic vessels, and innervation, seems to stand in contradiction to the laws of biology. It can be involved in a vast majority of diseases, from osteoarthrosis as a result of natural aging process to more complex in nature like osteochondromatosis. The primary role of articular cartilage is to provide the surface for movement in any single joint in the body. Therefore, its diseases lead to physical impairment and deterioration of the quality of life. Treatment of articular cartilage poses a formidable challenge in both modern human and animal medicine.

Analysis of the Leukocyte Response in Calves Suffered from Mycoplasma bovis Pneumonia.

Mycoplasma bovis is known to be a cause of chronic pneumonia in cattle. To date, the disease pathomechanism has not been fully elucidated. Leukocytes play a key role in host antimicrobial defense mechanisms. Many in vitro studies of the effect of Mycoplasma bovis (M. bovis) on leukocytes have been performed, but it is difficult to apply these results to in vivo conditions. Additionally, only a few studies on a local immune response in M. bovis pneumonia have been undertaken. In this study, the experimental calf-infection model was used to determine the effect of field M. bovis strains on changes of the peripheral blood leukocyte response, including phagocytic activity and oxygen metabolism by cytometry analyses. An additional aim was to evaluate the lung local immunity of the experimentally infected calves using immunohistochemical staining. The general stimulation of phagocytic and killing activity of peripheral blood leukocytes in response to the M. bovis infection points to upregulation of cellular antimicrobial mechanisms. The local immune response in the infected lungs was characterized by the T- and B-cell stimulation, however, most seen in the increased T lymphocyte response. Post-infection, strong expression of the antigen-presenting cells and phagocytes also confirmed the activation of lung local immunity. In this study-despite the stimulation-both the peripheral and local cellular antimicrobial mechanisms seem to appear ineffective in eliminating M. bovis from the host and preventing the specific lung lesions, indicating an ability of the pathogen to avoid the host immune response in the M. bovis pneumonia.

Selected leukocyte subpopulations in peripheral blood and uterine washings in cows before and after intrauterine administration of cefapirin and methisoprinol.

The aim of the study was to evaluate the selected lymphocyte subpopulations TCD4, TCD8, BCD21, BCD25, CD18, CD11b, and MHC II in blood and uterine flush of cows with endometritis, before and after intrauterine (i.u.) administration of cefapirin and methisoprinol. The research was carried out on 28 cows with clinical endometritis. Animals were divided into four groups, each composed of seven cows, depending on the i.u. preparation used: Group A, cefapirin; Group B, methisoprinol; Group C, cefapirin and methisoprinol simultaneously; and a control group-without medication. The study was performed using flow cytometry method. Summarizing the results of the research, i.u. infusion of cefapirin caused a weakening of the effector phase of the local uterine immune response; however, it enhanced leukocyte chemotaxis and antigen presentation. After i.u. administration of methisoprinol, the stimulation of specific uterine immunity mechanisms was mainly observed. The use of both mentioned preparations showed the strengthening of specific uterine immunological mechanisms presumably caused by methisoprinol, despite the inhibitory effect of the antibiotic. Intrauterine use of immunostimulatory substances can improve the effectiveness of the endometritis treatment in cows by improving specific local mechanisms of uterine immunity. As a consequence, it may enhance the effector function of immune competent cells and finally eliminate inflammation.

Immunogenic Evaluation of Ribosomal P-Protein Antigen P0, P1, and P2 and Pentameric Protein Complex P0-(P1-P2)2 of Plasmodium falciparum in a Mouse Model.

Malaria remains one the most infectious and destructive protozoan diseases worldwide. Plasmodium falciparum, a protozoan parasite with a complex life cycle and high genetic variability responsible for the difficulties in vaccine development, is implicated in most malaria-related deaths. In the course of study, we prepared a set of antigens based on P-proteins from P. falciparum and determined their immunogenicity in an in vivo assay on a mouse model. The pentameric complex P0-(P1-P2)2 was prepared along with individual P1, P2, and P0 antigens. We determined the level of cellular- and humoral-type immunological response followed by development of specific immunological memory. We have shown that the number of Tc cells increased significantly after the first immunization with P2 and after the second immunization with P1 and P0-(P1-P2)2, which highly correlated with the number of Th1 cells. P0 appeared as a poor inducer of cellular response. After the third boost with P1, P2, or P0-(P1-P2)2, the initially high cellular response dropped to the control level accompanied by elevation of the number of activated Treg cells and a high level of suppressive TGF-ß. Subsequently, the humoral response against the examined antigens was activated. Although the titers of specific IgG were increasing during the course of immunization for all antigens used, P2 and P0-(P1-P2)2 were found to be significantly stronger than P1 and P0. A positive correlation between the Th2 cell abundance and the level of IL-10 was observed exclusively after immunization with P0-(P1-P2)2. An in vitro exposure of spleen lymphocytes from the immunized mice especially to the P1, P2, and P0-(P1-P2)2 protein caused 2-3-fold higher cell proliferation than that in the case of lymphocytes from the nonimmunized animals, suggesting development of immune memory. Our results demonstrate for the first time that the native-like P-protein pentameric complex represents much stronger immune potential than individual P-antigens.

Improvement of Candida parapsilosis by genome shuffling for the efficient production of arabitol from l-arabinose.

Arabitol is used in the food industry as a low-calorie sweetener. It is produced by yeasts during the biotransformation process of l-arabinose. Genome shuffling was performed in Candida parapsilosis DSM 70125, an efficient producer of arabitol, to obtain fusants with improved arabitol production ability. Four mutants from the parental library were used for the first round of genome shuffling. The best fusants, GSI-1 and GSI-10A, were subjected to a second round of genome shuffling. Finally, two fusants, GSII-3 and GSII-16, produced concentrations of arabitol that were 50% higher than that of the wild-type strain during selection culture. Under the optimal conditions established for C. parapsilosis, the two fusants produced 11.83 and 11.75 g/L of arabitol and were approximately 15-16% more efficient than the wild-type strain. Flow cytometry analysis showed that the ploidy of the new strains did not change.

Phagocytic Activity and Oxygen Metabolism of Peripheral Blood Granulocytes from Rabbits Experimentally Infected with Trichophyton Mentagrophytes.

INTRODUCTION: Phagocytic activity and oxygen metabolism of peripheral blood granulocytes from rabbits with experimental trichophytosis were assessed by flow cytometry. MATERIAL AND METHODS: Virulent species of T. mentagrophytes var. granulosum (Tm-K) isolated from rabbits with natural trichophytosis was used for experimental infection. The phagocytic activity of granulocytes was measured in whole blood by flow cytometry using the commercial Phagotest kit. Oxidative burst was measured in whole blood by flow cytometry using the commercial Bursttest kit. RESULTS: It was found that rabbits were susceptible to infection with Trichophyton mentagrophytes under experimental conditions. The analysis of the phagocytic activity indices and oxygen metabolism of granulocytes in peripheral blood of infected rabbits showed that changes of the indices were connected with the progression and regression of the disease. A significant decrease in phagocytic activity and oxygen metabolism was observed during development of fungal lesions and it remained similar throughout the progress of the disease. The highest means of the percentage of activated and ingesting phagocytes and a significant increase in the mean fluorescence intensity (representing the number of ingested bacteria) were observed during spontaneous recovery. Therefore, the decrease or increase in the indices of phagocytic activity and oxygen metabolism of granulocytes from rabbits experimentally infected with T. mentagrophytes is somehow related to the progress of infection and suppressive activity of the fungus, whose elimination during recovery caused significant increases in investigated indices of non-specific cellular immunity. CONCLUSION: The results of the present investigation confirm that the mechanism of oxygen-dependent killing is crucial in infections caused by T. mentagrophytes.

Expression of receptor interacting protein 1 and receptor interacting protein 3 oval cells in a rat model of hepatocarcinogenesis.

When apoptosis is suppressed in a neoplastic state, necroptosis may enable an anticancer response. In the present study, the association between apoptosis and necroptosis was assessed in a partial hepatectomy (PH)/diethylnitrosamine (DEN) rat model of hepatocarcinogenesis. Isolated oval cells (OCs) were analysed at 24, 48 and 72 h and at the first and second week of incubation. Phenotypic studies, apoptosis and necroptosis detection and proliferative activity assays were also performed on the OCs. The OCs were isolated from non-neoplastic (PH) and neoplastic (PH/DEN) livers, which expressed receptor interacting protein (RIP) 1 and RIP3. Western blot analysis revealed that the RIP1 and RIP3 expression in the PH/DEN OCs started to increase at 72 h and continually increased to the end of cell culture. Compared with the PH OCs, the cells isolated from PH/DEN rats exhibited significantly less potential for apoptosis (P<0.05). There were a minimal number of apoptotic PH/DEN OCs (2.82±1.1%) at 72 h. In addition, the PH/DEN OCs demonstrated progressive proliferative activity during incubation, which was significantly increased compared with the PH OCs at ≥72 h. The present study revealed that PH/DEN OCs, which trigger hepatic cancer, have a high proliferative activity and suppress apoptosis. It was also observed that, based on the expression of RIP3 and RIP1, necroptosis may be maintained and may serve as an alternative pathway for programmed PH/DEN OC death.

Phagocytic and oxidative burst activity of phagocytic cells in peripheral blood and uterine washings in cows with clinical endometritis before and after intrauterine use of cephapirin and methisoprinol.

The aim of the study was to evaluate phagocytic and killing activity of phagocytic cells in blood and uterine flush of cows with endometritis before and after intrauterine (i.u.) administration of cephapirin and methisoprinol. The research was carried out on 28 cows with clinical endometritis. Animals were divided into four groups, each composed of seven cows, depending on the i.u. treatment used: Group A-cephapirin; Group B-methisoprinol; Group C-cephapirin and methisoprinol at the same time; and a control group-without medication. Using flow cytometry technique, the phagocytic activity of granulocytes and monocytes was identified, as well as the oxidative burst activity of neutrophils in the peripheral blood and uterine washings. Summarizing the results of the research, i.u. infusion of cephapirin caused a reduction in the phagocytic and killing activity of phagocytes. The i.u. use of methisoprinol increased phagocytic and killing activity of phagocytes in the uterus. Administering both listed substances simultaneously showed a decrease in phagocytosis, presumably due to the dominating inhibitor effect of the antibiotic. However, also an increase of mean fluorescence intensity was observed, presumably caused by the methisoprinol. Intrauterine use of immunostimulatory substances, can improve the effectiveness of the treatment of endometritis in cows.