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.

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.

Breeding parameters on a mink farm infected with Aleutian mink disease virus following the use of methisoprinol.

Aleutian mink disease virus is one of the greatest threats to modern mink farming. The disease reduces fecundity and causes high mortality among kits. The aim of this study was to evaluate the effectiveness of methisoprinol in counteracting the effects of Aleutian disease, both by inhibiting replication of the virus and by mitigating the harmful effects of the disease on the fecundity and weight of infected animals. The study included 300 individuals with confirmed infection, divided according to antibody titres into three experimental groups, which received a 20% methisoprinol solution, and three control groups, which did not receive the immunostimulant. In the mink from the experimental groups, the number of copies of the genetic material of the virus in the spleens and lymph nodes was one order of magnitude lower than in the case of the control groups. Mink receiving the supplement also showed higher fecundity (on average 5.83 in the experimental groups and 4.83 in the control groups), and the weight of their offspring before slaughter was over 200 g higher. Given the lack of effective methods for immunoprophylaxis and treatment, methisoprinol supplementation can be an effective means of counteracting the effects of AMDV on persistently infected farms.

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.

A comparative molecular characterization of AMDV strains isolated from cases of clinical and subclinical infection.

The Aleutian mink disease virus (AMDV) is one of the most serious threats to modern mink breeding. The disease can have various courses, from progressive to subclinical infections. The objective of the study was to provide a comparative molecular characterization of isolates of AMDV from farms with a clinical and subclinical course of the disease. The qPCR analysis showed a difference of two orders of magnitude between the number of copies of the viral DNA on the farm with the clinical course of the disease (105) and the farm with the subclinical course (103). The sequencing results confirm a high level of homogeneity within each farm and variation between them. The phylogenetic analysis indicates that the variants belonging to different farms are closely related and occupy different branches of the same clade. The in silico analysis of the effect of differences in the sequence encoding the VP2 protein between the farms revealed no effect of the polymorphism on its functionality. The close phylogenetic relationship between the isolates from the two farms, the synonymous nature of most of the polymorphisms and the potentially minor effect on the functionality of the protein indicate that the differences in the clinical picture may be due not only to polymorphisms in the nucleotide and amino acid sequences, but also to the stage of infection on the farm and the degree of stabilization of the pathogen-host relationship.

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.

Factors affecting prevalence and abundance of A.perfoliata infections in horses from south-eastern Poland.

Equine Anoplocephalosis constitute a significant problem in horses worldwide. The aim of this study was to analyse intrinsic (host age and sex) and extrinsic (management type, pasture type and moisture) factors that influence the prevalence and FEC of A. perfoliata infections. Faecal samples were collected from 994 horses managed in studs or individually between 2012 and 2014. The Sedimentation-flotation method was applied for coproscopic analysis, and faecal egg counts were calculated. The overall prevalence was 25.1% (21.4-29.0) with the highest prevalence (36.1% [28.1-44.8]) found in horses 10-20 years old. The individuals kept in studs showed three times higher A. perfoliata prevalence compared to the ones managed individually. The prevalence significantly differed between pasture types, with individuals kept in studs (37.6% [34.3-40.9]) showing four times higher prevalence than horses kept individually (9.2% [4.8-16.5]). More horses kept on watery (42.0% [36.6-47.6]) and semi-watery (35.9% [31.3-40.7]) pastures were infected than those on dry (6.6% [4.6-9.2]) pastures. The overall A. perfoliata FEC in all examined individual was 2.67 and differed within sex, with mares showing 4.3 - times higher FEC of infection than stallions. Horses bred in studs (3.65±0.289) showed higher FEC than these bred individually (1.28±0.198). There was the effect of pasture type on A. perfoliata FEC, with horses kept on joint pastures (4.06±0.29) showing higher FEC than individuals kept individually (0.88±0.23). Pasture moisture significantly affected A. perfoliata FEC with the highest FECs in horses from watery pastures. Horses bred on dry pastures showed 16 times lower FEC than horses bred on watery pastures. Host age also significantly affected A. perfoliata FEC, with the oldest individuals showing the highest mean FEC. The presented analysis of intrinsic and extrinsic factors may help to overcome A. perfoliata infections in horses in different breeding systems. Understanding the role of management and pasture type risk factors that influence this parasitosis may benefit both breeders and veterinary surgeons.

Comparative molecular analysis of strains of the Aleutian Disease Virus isolated from farmed and wild mink.

INTRODUCTION AND OBJECTIVE: Aleutian Disease is a significant biological factor causing substantial losses in mink farming. The virus inducing the disease also infects wild populations which may constitute an asymptomatic reservoir. To compare genetic variants of the AMD virus occurring in wild and farmed mink populations, an analysis was performed on a fragment of the VP2 protein sequence of the virus infecting both populations, taken from different living environments. MATERIAL AND METHODS: Genetic material was isolated from 11 farmed animals in which anti-AMDV antibodies had been detected and from 20 wild animals. The DNA obtained was amplified using primers specific for the fragment encoding the VP2 protein. The product obtained was sequenced and bioinformatic analysis was performed. RESULTS: Viral material was detected in 11 farmed and 7 free-living animals. Similarity of sequences averaged 99% within groups and 94% between groups. The sequencing results made it possible to identify characteristic changes for each group. In the isolates from the wild animals, the following changes were observed in the epitope region with respect to the reference sequence: C3704T, G3710A, T3722C, T3746C and A3749G. In the isolates from the farmed animals a G3779A transition was noted. Phylogenetic analysis showed that the variants infecting the two groups occupy separate branches of the phylogenetic tree. CONCLUSIONS: The variants of the virus infecting the two groups may have a common origin, but at present they constitute two separate groups, with characteristic differences making it possible to recognize their genotype.

DON-induced Changes in Bone Homeostasis in Mink Dams.

INTRODUCTION: The aim of the study was to investigate the mechanical and geometric properties as well as bone tissue and mineral density of long bones in mink dams exposed to deoxynivalenol (DON) since one day after mating, throughout gestation (ca. 46 d) and lactation to pelt harvesting. MATERIAL AND METHODS: Thirty clinically healthy multiparous minks (Neovison vison) of the standard dark brown type were used. After the mating, the minks were randomly assigned into two equal groups: non-treated control group and DON group fed wheat contaminated naturally with DON at a concentration of 1.1 mg·kg-1 of feed. RESULTS: The final body weight and weight and length of the femur did not differ between the groups. However, DON contamination decreased mechanical endurance of the femur. Furthermore, DON reduced the mean relative wall thickness and vertical wall thickness of the femur, while vertical cortical index, midshaft volume, and cross-sectional moment of inertia increased. Finally, DON contamination did not alter bone tissue density, bone mineral density, or bone mineral content, but decreased the values of all investigated structural and material properties. CONCLUSION: DON at applied concentration probably intensified the process of endosteal resorption, which was the main reason for bone wall thinning and the weakening of the whole bone.

Inflammatory cytokine and acute phase protein concentrations in the peripheral blood and uterine washings of cows with subclinical endometritis in the late postpartum period.

The aim of the study was to evaluate the concentrations of proinflammatory cytokines: tumor necrosis factor (TNF-α) and interleukin-6 (IL-6), anti-inflammatory cytokine interleukin-10 (IL-10), and acute phase proteins (APPs)--haptoglobin (Hp) and serum amyloid A (SAA) in serum and uterine washings of cows with subclinical endometritis, and compare them to healthy animals. The study was performed on 24 cows on day 60 after delivery. The cows were divided into two groups based on the results of cytological tests: 12 cows with subclinical endometritis and 12 healthy cows. Experimental material consisted of blood serum and uterine washings. The levels of the following cytokines in the study material were determined with ELISA: TNF-α, IL-6, IL-10 and APPs - Hp and SAA. The results show that the levels of TNF-α (p < 0.01), IL-6, IL-10 as well as SAA and Hp were significantly higher in the serum of cows with subclinical endometritis compared to the controls (p < 0.001). Uterine washings had significantly higher levels of IL-6, IL-10, and Hp in the experimental cows compared to the controls (p < 0.001). The demonstrated differences in the concentration of cytokines and APP between cows with subclinical endometritis and healthy cows, in both the serum and uterine washings, may suggest the usefulness of these parameters in the diagnosis of subclinical endometritis in cows in the late postpartum period.

Seasonal changes of diagnostic potential in the detection of Anoplocephala perfoliata equine infections in the climate of Central Europe.

For this study, 724 gastrointestinal tracts of slaughter horses were investigated to determine the prevalence, intensity of Anoplocephala perfoliata and tapeworm development stages over the second, third and fourth quarter of 2012 and the first quarter of 2013. For each positive horse, faecal samples were collected from the rectum or small colon for coproscopic examinations. The samples were analysed using dedicated modified sedimentation-flotation methods. In total, 52 horses were infected with A. perfoliata in the course of the study, with an overall prevalence of 7.2 %. The prevalence changed over the study period; however, not markedly. The overall mean of A. perfoliata abundance was 12.3 (3.23) and did not differ significantly between the quarters. Mean invasion intensity did not differ significantly between the quarters. The quantity of mature tapeworms did not differ significantly over the study period; however, there was a significant difference in the number of immature tapeworms. The highest number of mature tapeworms was found in the first quarter of 2013. The number of detected tapeworm eggs rose significantly over the study period. The total number of tapeworms did not have a significant influence on the presence/absence of detected eggs. However, there was a noticeable difference between the number of mature tapeworms and presence/absence of eggs in faeces. This clearly indicates that the efficacy of the modified sedimentation-flotation method is influenced by seasonality, and therefore the most effective and reliable time for detection of A. perfoliata in equines is the first quarter of the year.

Serum insulin-like growth factor-1 and C-reactive protein concentrations before and after ovariohysterectomy in bitches with pyometra.

Pyometra is one of the most common diseases of the reproductive system in bitches. The inflammatory processes occurring in the uterus result in changes in concentrations of numerous serum biomarkers, which are used for monitoring the postoperative period. The aim of the present report was to study the evolution of serum levels of insulin-like growth factor-1 (IGF-1) and C-reactive protein (CRP) in bitches suffered from pyometra and after ovariohysterectomy in comparison with the control group of healthy dogs submitted to the surgery. Our results indicate that the serum level of IGF-1 is decreased (101.6 ng/mL [73.8-177.8 ng/mL]), whereas CRP is increased (114.6 µg/mL [95.3-131.6 µg/mL]) during severe inflammation. When inflammation caused by pyometra and surgery is resolved, the level of IGF-1 is increased (186.0 ng/mL [94.6-344.3 ng/mL], P < 0.05) and CRP decreased (23.3 µg/mL [9.9-77.0 µg/mL], P < 0.01), suggesting that these markers could be useful for assessment of the postoperative period in bitches.

Comparison of the sensitivity of coprological methods in detecting Anoplocephala perfoliata invasions.

The autopsy of 487 slaughter horses revealed the presence of Anoplocephala perfoliata in 36 animals. The invasions varied in the intensity (3 to 2,069 tapeworms) and in the level of tapeworms' proglottid maturity. Twenty nine horses were found to contain tapeworms with gravid proglottid. Fecal samples collected from the rectum were tested using following techniques: flotation with solution-saturated NaCl, decantation, McMaster's, and modified sedimentation-flotation methods (50 g feces samples, flotation solution-saturated NaCl and sucrose, specific gravity 1.25 g/ml). The number of A. perfoliata positive fecal samples was significantly higher using the sedimentation-flotation methods 21 (58.33%) than flotation 6 (16.66%), decantation 3 (8.33%), and McMaster's 1 (2.77%) techniques. The sensitivities of the coprological methods during the patent period were 20.69, 10.34, 3.45, and 72.41% for the flotation, decantation, McMaster's, and sedimentation-flotation method, respectively. Sedimentation-flotation techniques proved to be more sensitive than other one. The lowest intensity of invasion possible to detect using this method was nine tapeworms with gravid proglottid.

Maternal acrylamide treatment reduces ovarian follicle number in newborn guinea pig offspring.

Acrylamide is an industrial chemical which has toxic effects on reproduction. In this study, we investigated whether acrylamide administered prenatally can induce follicular atresia in the newborn guinea pig ovary. Another aim was to describe the localization of vimentin filaments and determine their participation in atresia. After prenatal acrylamide treatment, the pool of primordial and primary follicles was significantly reduced. The number of caspase 3 and TUNEL positive oocytes increased compared to the control group. There were no differences in Lamp1 (autophagy marker) staining. A vimentin immunosignal was present in the granulosa cells of primordial, primary and secondary follicles. Interestingly, in contrast to the control group, the oocytes from all follicles in the ACR-treated females were negative for vimentin. These data suggest that prenatal exposure to acrylamide reduced the number of ovarian follicles by inducing follicular atresia mediated by oocyte apoptosis. Acrylamide-induced apoptosis may be associated with destruction of vimentin filaments.

Concentrations of C-reactive protein, serum amyloid A, and haptoglobin in uterine arterial and peripheral blood in bitches with pyometra.

Pyometra is a life-threatening reproductive disorder that affects the uterus of female dogs. This study was designed to identify the possible indicators of uterine inflammation by comparing C-reactive protein (CRP), serum amyloid A (SAA), and haptoglobin (Hp) concentrations in uterine arterial and peripheral venous blood in bitches with open- and closed-cervix pyometra. CRP, SAA, and Hp concentrations were higher in bitches with closed-cervix pyometra irrespective of the site of blood collection. Higher acute-phase protein concentrations were observed in peripheral compared with uterine arterial blood in bitches with closed-cervix pyometra, whereas the levels were comparable in dogs with open-cervix pyometra. Our results indicate that mean acute-phase protein concentrations differ according to pyometra type/severity and blood source and suggest the possible use of peripheral blood levels of CRP, SAA, and Hp to monitor inflammation during the course of pyometra.

Spectroscopic studies of amphotericin B-Cu²+ complexes.

The aim of this research is to investigate amphotericin B (AmB)-Cu(2+) complexes in aqueous solution at different pH values. Electronic absorption, circular dichroism (CD), Raman and FTIR spectroscopies were used in this study. We found that different concentrations of AmB and Cu(2+) ions in solution leads to formation of complexes with stoichiometry of 2:1 and 1:1. The formation of AmB-Cu(2+) complexes at physiological pH values is accompanied by changes of the molecular organization of AmB especially disaggregation. These observed effects might be significant from a medical point of view.

Changes in CRP, SAA and haptoglobin produced in response to ovariohysterectomy in healthy bitches and those with pyometra.

The aim of the study was to assess changes in serum C-reactive protein (CRP), serum amyloid A component (SAA) and haptoglobin (Hp) concentrations in healthy bitches and in those with pyometra undergoing ovariohysterectomy, and to establish the usefulness of such determinations for monitoring the postoperative period. Our results indicate that CRP and SAA determinations serve to evaluate the severity of the inflammatory process in pyometra since the concentrations of these acute phase proteins were increased immediately after surgery and diminished thereafter. The CRP and SAA response was rapidly produced while Hp concentrations increased in a more gradual manner. Thus, postoperative concentrations of CRP and SAA provide valuable information on the subsidence of the inflammatory response during the uneventful postoperative period. Our findings also suggest that acute phase proteins might be useful diagnostic markers of early postoperative complications.

[Formation of the selected biochemical markers in the patients with atherosclerotic arterial obliteration and the coexistent infection with Chlamydia psittaci]. / Ksztaltowanie sie wybranych wskazników biochemicznych, u chorych z miazdzycowa niedroznoscia tetnic i wspólistniejacym zakazeniem Chlamydia psittaci.

In the arterial wall which is atheromatously changed both basic EB and reticular RB bodies of Chlamydia bacteria may be present. The role of these micro-organisms in the pathogenesis of arteriosclerosis is not thoroughly recognized yet. It is unknown whether Chlamydia initiate or lead to the inflammation of the already existent disease process. That is why it is expedient to carry out the research determining the correlation between the presence of Chlamydia bacteria in the operated arteries and the formation of the selected biochemical indicators. During the operation the imprint specimen was taken from the arterial wall, which was subsequently painted using stamp's method. EB bodies were marked in the immunofluorescence test with the usage of specific antibodies. The concentration of C-reactive protein was marked with the help of the rocket immunoelectrophoresis. The examination of blood lipids and fibrinogens was conducted with the usage of a colorimetric method with the help of ready-made kits. On the basis of the obtained data two groups of patients were distinguished. The first group comprised 16 patients (26%); here elementary EB bodies were detected both in the imprint specimens and the blood serum. In all the patients assigned to the first group the presence of anti-Chlamydia antibodies in the blood serum was observed. The second group consisted of the remaining 47 patients (74%); here no currently lasting Chlamydia infection was stated. On the basis of the conducted research and statistic calculations it is justified to point the correlation between the concentration of C-reactive protein and HDL cholesterol in the group of patients with the detected Chlamydia bacteria in comparison to the group of patients with no infection. However no statistically significant relationship between the concentration of fibrinogen, total cholesterol, LDL cholesterol and triglycerides was observed in the relation to the analyzed groups.

The influence of nonspecific immunostimulation of pregnant sows on the immunological value of colostrum.

The purpose of this experiment was to estimate the influence of nonspecific immunostimulation of pregnant sows on the immunological value of colostrum. The studies were done in the fall-winter season on 20 pregnant sows (Polish Landrace) divided into four groups, five animals each. The sows were clinically healthy and unvaccinated. Group I received isoprinosine, group II (TFX), group III (HMB), and group IV served a control. The immunostimulants were applicated 4-6 weeks before the expected date of delivery. The following parameters were determined in colostrum taken from sows after completed delivery: specific gravidity; total proteins, lysozyme activity, IgG level. It was found that the nonspecific immunostimulation of pregnant sows increased in colostrum the level of IgG, total protein content and lysozyme activity.