Interleukin-6 as a Milk Marker of Clinical and Subclinical Intramammary Infections (IMI) in Cows Caused by Streptococcus spp.

The aim of the study was to evaluate the concentrations of Interleukin-6 (IL-6) in milk and serum of healthy cows (HE) and cows with mastitis caused by Streptococcus spp. The blood and milk samples were obtained from Holstein-Friesian cows (Lublin region, Poland). A total of 43 milk and serum samples from 28 cows with mastitis and 15 healthy cows were selected for study. IL-6 levels in milk from HE cows ranged from 6.09-80.24 pg/mL (median 26.6 pg/mL) and were significantly lower than in milk from both cows with clinical and subclinical mastitis (487.09 pg/mL vs. 26.6 pg/mL in CM, p < 0.001; and 165.31 pg/mL vs. 26.6 pg/mL in SCM, p < 0.001). The IL-6 concentration in the serum of HE was not significantly different from the serum IL-6 of the entire group of mastitis cows, regardless of whether the inflammation proceeded in a clinical or subclinical form (44.37 pg/mL vs. 78.09 pg/mL; 128.29 pg/mL vs. 78.09 pg/mL, respectively). The present study indicates that cows with mastitis caused by Streptococcus spp. develop a local immune response in the mammary gland in response to the pathogen. Monitoring of IL-6 levels in milk can allow early detection of mastitis, which is especially important in cases of subclinical inflammation.

Comparative analysis of total protein, casein, lactose, and fat content in milk of cows suffering from subclinical and clinical mastitis caused by Streptococcus spp.

Introduction: The aim of the study was to analyse the total protein (TP), casein (CAS), lactose (LAC), and fat content of milk from cows with subclinical (SCM) and clinical mastitis (CM) caused by Streptococcus spp. Material and Methods: A total of 60 milk samples from diseased cows and 30 milk samples from healthy cows were included in the study. Milk samples were taken from Holstein-Friesian cows from four dairy farms in Lublin Province. The bacteriological examination of the milk was performed and the somatic cells count in 1 mL of milk was determined using a SomaCount FC automatic cell counter. Determination of TP, CAS, LAC, FAT and FA levels in milk was carried out using a DairySpec FT automated Fourier transform infrared spectrometer. Results: Total protein in milk from HE was significantly higher than in milk from cows with mastitis (4.04% vs 3.57% in milk from SCM cows and 3.7% in milk from CM cows, P = 0.001). The CAS level was 2.73% in milk from CM cows and 2.92% in milk from SCM cows vs 3.30% in milk from HE cows, P = 0.001. The changes in CAS and TP in milk resulted in a significant difference in the CAS/TP ratio (81.7% in milk from HE cows vs 73.8% in milk from CM cows). A decrease in levels was also recorded for LAC (4.8% in milk from HE cows vs 4.51% in milk from SCM cows and 4.01% in milk from CM cows, P = 0.001). The fat level was significantly higher in milk from healthy cows than in milk from cows with mastitis (4.0% vs 2.3% in milk from SCM cows and 1.64% in milk from CM cows, P = 0.001). Conclusion: It should be emphasised that the decrease in the levels of TP, LAC and FAT was significant not only in milk from CM cows but also in milk from SCM cows. This is very unfavourable, because the reduction in the main milk components results in poor quality dairy products and impairs line processes.

Topoisomerase IIα immunoexpression in feline mammary carcinomas: A correlation with Ki67 immunoexpression and the mitotic count.

The aim of the study was to compare the immunohistochemical expression of topoisomerase IIα protein (Topo IIα) with Ki67 expression and mitotic count in feline mammary carcinomas (FMCs). Topo IIα is considered as a proliferation indicator as well as a molecular target of anthracycline chemotherapy. The studied material included 70 FMCs from female cats treated with mastectomy. Primary mouse monoclonal antibodies directed against Topo IIα and Ki67 were used in immunohistochemical reactions. The number of mitotic figures was counted at 400× magnification in a field of 2.37 mm2. Immunohistochemical reaction for Topo IIα occurred in cell nuclei. The Topo IIα index ranged from 6.12% to 54.60% and was positively correlated with the values of the Ki67 index (r = 0.7193) and the mitotic count (r = 0. 2858). This indicates the potential possibility of use of the immunohistochemical expression of Topo IIα to assess the rate of proliferation in FMCs. The wide range of expression of Topo IIα in individual tumorus found in the conducted studies allows us to hypothesize that its assessment could be used as a predictive marker in chemotherapy of FMCs with the use of anthracyclines. However, this requires confirmation in clinical trials.

Divergences between serum C-reactive protein and ferritin concentrations in canine pyometra.

The main aim of this report was to investigate and compare the response of serum C-reactive protein (CRP) and ferritin, two positive acute phase proteins (APPs) which usually show an increase in inflammatory processes, in dogs with pyometra. For this purpose, two different studies were made. In the first one , both proteins were measured together in an APPs profile in 25 dogs with pyometra, 25 dogs with pancreatitis (as an example of a positive inflammatory control group), and in 25 healthy dogs. In the second study, to advance the knowledge of the changes and evolution of serum ferritin and CRP in dogs with pyometra after treatment, the concentrations of both APPs were analyzed in 30 dogs with pyometra at diagnosis and after ovariohysterectomy and in 10 clinically healthy female dogs before and after elective spaying. In both studies, bitches with pyometra showed significant increases in serum CRP, indicating an inflammatory condition, but not in serum ferritin despite being a moderate positive APP. This divergence between the dynamics of these APPs could be a useful tool for the suspicion of cases of canine pyometra.

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.

Activity of the glycosidases ß-galactosidase, α-l-fucosidase, ß-N-acetyl-hexosaminidase, and sialidase in uterine tissues from female dogs in diestrus with and without pyometra.

This study aimed to compare the activity of selected glycosidases (ß-galactosidase, α-l-fucosidase, ß-N-acetyl-hexosaminidase, and sialidase) in homogenates of uterine tissues obtained from female dogs with and without pyometra. In addition, it examined the availability of substrates for these glycosidases in the homogenates. The study was carried out on female dogs undergoing ovariohysterectomy for pyometra (n = 10) and clinically healthy dogs (n = 10) undergoing elective spaying. The activity of ß-galactosidase, α-l-fucosidase, and ß-N-acetyl-hexosaminidase was analyzed using a spectrofluorometer and that of sialidase using a colorimetric method. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis with Alcian Blue (AB) and Periodic Acid-Schiff (PAS) staining was performed to determine the presence of substrates for these glycosidases in the homogenates of uterine tissues. The results revealed that the activity of all the examined glycosidases was significantly higher (P < 0.05) in the uterine tissues isolated from dogs with pyometra in comparison to healthy dogs. The electrophoretic patterns of the selected samples showed several proteins, which contained different sugar moieties stained by AB and PAS and the profiles differed significantly between the pyometra group and the healthy group. Densitometric analysis of AB staining showed patterns between 233 and 148, 86 and 55, and 43 and 20 kDa, which differed markedly in sugar content between the examined groups of animals. Similarly, PAS staining analysis revealed patterns of different molecular weights, between 233 and 117 and between 55 and 32 kDa, which also differed in sugar content. These findings suggest that canine pyometra is accompanied by the increase in the activity of selected glycosidases in the uterus. This could potentially modify the glycan structures of uterine glycoproteins and in result their biological functions. Further studies are needed to elucidate the potential role of the increased activity of glycosidases in the pathogenesis of this disease.

Content of tryptophan and kynurenines in serum and milk of dairy cows with mastitis caused by Streptococcus spp.

The aim of the study was to investigate serum and milk concentrations of tryptophan (TRP), kynurenine (KYN) and kynurenic acid (KYNA), and activity of indoleamine 2,3-dioxygenase (IDO) in cows suffering from mastitis caused by Streptococcus spp. The blood and milk samples were collected from Holstein-Friesian cows farmed in the Lublin region of Poland. It was found that TRP was lower in cows with mastitis both in serum and milk. KYN was lower in serum but not in milk. KYNA was not significantly altered in diseased cows both in serum and milk. The activity of IDO calculated as KYN to TRP ratio was unchanged in serum but was markedly elevated in milk of cows with mastitis. Our findings may have important implications for diagnosis of mastitis in cows because an increase of activity of IDO and reduction of TRP in milk might be a valuable early marker predicting the occurrence of the disease.

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.

Measurement of ITIH4 and Hp levels in bitches with pyometra using newly developed ELISA methods.

Measurement of acute phase proteins (APPs) as biomarkers in canine medicine is in increasing demand. In the present study, the development and validation of two ELISA methods for the quantification of canine inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) and haptoglobin (Hp) are shown. The adequate imprecision and accuracy and wide analytical range make the developed methods appropriate to quantify ITIH4 and Hp in serum samples. The inter- and intra-assay CVs were lower than 10 %, and the assays maintained linearity under dilution and showed analytical equivalence with the method of radial immunodiffusion. The measurement of CRP, Hp and ITIH4 in sera from bitches affected by pyometra allowed us to determine that ITIH4 behaves as a moderate APP in dogs. The group of bitches affected by pyometra showed very high levels of CRP (147 ± 91 mg/L), corresponding to a strong inflammatory process, which resulted in a moderate increase in the concentrations of Hp (7 times) and ITIH4 (3 times) compared to the control group.

Changes in interferon-gamma and neopterin in female dogs undergoing ovariohysterectomy as elective spay or as treatment of pyometra.

The objective of this study was to determine serum concentrations of interferon-gamma (INF-γ) and neopterin (Np) in dogs with pyometra admitted for surgical treatment and to compare these concentrations to healthy dogs admitted for elective spay. The effects of the surgical procedure were also evaluated by measuring these markers in both groups of dogs before and after ovariohysterectomy. Our study indicates that pre-surgery concentrations of INF-γ (57.4 ± 26.0 pg/mL) and Np (5.6 ± 0.8 nmol/L) in healthy dogs were significantly lower compared to dogs with pyometra (124.3 ± 87.6 pg/mL for INF-γ; 7.0 ± 1.5 nmol/L for Np) (P < 0.05 in both cases). Furthermore, Np was lower in dogs with pyometra 3 days after surgery compared to healthy controls (P < 0.001). During the post-operative period, INF-γ showed no statistically significant changes in any of the groups, while Np showed lower serum concentration on day 3 than on day 0 in the pyometra group (P < 0.001). No statistically significant correlation was detected between serum concentrations of INF-γ and Np. These results indicate that pyometra causes alterations in serum concentrations of INF-γ and Np in female dogs compared to physiological levels before surgery and during the postoperative period.

L'objectif de la présente étude était de déterminer les concentrations sériques d'interféron gamma (INF-γ) et de néoptérine (Np) chez des chiens avec pyomètre admis pour traitement chirurgical et de comparer ces concentrations à celles de chiens en santé admis pour stérilisation élective. Les effets de la procédure chirurgicale furent également évalués en mesurant ces marqueurs dans les deux groupes de chiens avant et après ovariohystérectomie. Notre étude indique que les concentrations pré-chirurgie d'IFN-γ (57,4 ± 26,0 pg/mL) et de NP (5,6 ± 0,8 nmol/L) chez les chiens en santé étaient significativement inférieures comparativement aux chiens avec pyomètre (124,3 ± 87,6 pg/mL pour INF-γ; 7,0 ± 1,5 nmol/L pour Np) (P < 0,05 dans les deux cas). De plus, Np était plus bas chez les chiens avec pyomètre trois jours après la chirurgie comparativement aux témoins en santé (P < 0,001). Durant la période post-chirurgicale, INF-γ ne montra aucun changement statistiquement significatif dans aucun des trois groupes, alors que Np a présenté des concentrations sériques plus faibles au jour 3 qu'au jour 0 dans le groupe avec pyomètre (P < 0,001). Aucune corrélation statistiquement significative ne fut détectée entre les concentrations sériques d'INF-γ et de Np. Ces résultats indiquent que le pyomètre cause une altération des concentrations sériques d'INF-γ et de Np chez les chiennes comparativement aux niveaux physiologiques avant la chirurgie et durant la période post-opératoire.(Traduit par Docteur Serge Messier).

Serum amyloid A as an marker of cowÖ¨ s mastitis caused by Streptococcus sp.

The aim of the study was to evaluate the concentrations of amyloid A in serum (SAA) and in milk (MAA) of cows with mastitis caused by Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis and healthy cows. The blood and milk samples were obtained from Holstein-Friesian cows with clinical signs of mastitis from two tie-stall housing systems herds in the Lublin region in Poland. A total of 80 milk and serum samples from 30 cows with mastitis and 10 healthy cows were selected for study. In the quarter milk samples from cows with mastitis Streptococcus strains were isolated: Strep. agalactiae (7 cows), Strep. dysgalactiae (9 cows) and Strep. uberis (14 cows). The present study indicates that amyloid A concentration was significantly higher in milk of cows with mastitis compared to control cows (1134.25 ng/mL and 324.50 ng/mL, P < 0.001). The highest concentration of amyloid A was found in milk of cows with mastitis caused by Strep. agalactiae and Strep. uberis whereas lowest in the milk of cows with mastitis caused by Strep. dysgalactiae (3882.50 ng/mL, 2587.75 ng/mL and 812.00 ng/mL, respectively). No statistically significant difference in amyloid A concentration in serum was revealed between all unhealthy cows and control group (2140.00 ng/mL and 2510.00 ng/mL, P > 0.05). There was also no statistically significant difference between the level of amyloid A in serum and in milk of cows with mastitis caused by Strep. agalactiae and Strep. uberis. Whereas, in the case of Strep. dysgalactiae, like in the group of healthy cows, the level of amyloid A was significantly higher in serum compared to this in milk (2100 ng/mL and 812.00 ng/mL, P < 0.001; 2510.00 ng/mL and 324.50 ng/mL, P < 0.001; respectively).

Decorin concentrations in canine normal and neoplastic mammary gland tissues.

In the present study, the concentration of decorin in canine normal and neoplastic mammary gland tissues was examined to understand the potential role of decorin in development and progression of canine mammary tumours. The homogenates of 48 mammary gland tumours (10 benign and 38 malignant) and 10 samples of normal canine mammary gland tissue were used in the study. The presence and quantification of decorin was examined in the homogenates using Western blot and specific canine ELISA. Western blotting confirmed the presence of decorin both in the normal mammary gland tissues and in the mammary gland tumours. The concentration of decorin was significantly higher (p < .05) in the benign tumours and non-metastatic malignant tumours than in the normal mammary gland. The concentration of decorin was significantly lower (p < .05) in the malignant tumours with metastasis to regional lymph nodes compared with benign tumours and non-metastatic malignant tumours. No significant differences were found in the level of decorin between the benign and the non-metastatic malignant tumours. Both the histological type of malignant tumours and the histological grade did not significantly affect the concentration of decorin. These findings suggest that neoplastic transformation in the canine mammary gland leads to increase in the decorin protein synthesis. The reducing decorin concentration in canine malignant mammary tumours appears to facilitate the metastatic spread of these tumours.

Serum and salivary adiponectin dynamics in septic and non-septic systemic inflammation in a canine model.

Adiponectin is a 30 kDa protein hormone that has anti-atherogenic properties, being an insulin-sensitizing and anti-inflammatory molecule. Salivary adiponectin concentrations correlate positively with serum, thus, saliva was indicated as appropriate biofluid for its measurement in different clinical situations. However, inflammation was indicated as main confounding factor when evaluating the usefulness and the reliability of determination of salivary adiponectin. The aim of the present report was to evaluate the dynamics of salivary and serum adiponectin in systemic non-septic and septic inflammation using a dogs as a model. Forty bitches were enrolled. Seventeen dogs were healthy (group I, non-septic) and 23 bitches were diagnosed with pyometra (group II, septic). Ovariohysterectomy was performed for all animals. Saliva and blood samples were collected before (D0) and 3 (D3) and 10 (D10) days after ovariohysterectomy. At D0, Group I showed higher serum and salivary adiponectin than group II, although statistical significance was only detected in salivary adiponectin between the two groups at D0 (P = 0.001). In serum, adiponectin was higher on D0 than on D3 and tended to reach pre-surgery values on D10 in both groups. Salivary adiponectin showed similar behaviour to serum in Group I, while in group II salivary adiponectin concentrations were lowest on D0 and tended to increase on D3 and D10. The data obtained in present study describe for the first time the comparative behavior of salivary adiponectin in non-septic and septic inflammation. Salivary, and not serum, adiponectin seemed to mimic better the inflammatory and general health status.

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.

The effect of pyometra on glycosylation of proteins in the uterine tissues from female dogs.

The main aim of this study was to investigate the effect of pyometra on glycosylation of proteins in the uterine tissues from female dogs, using western blotting with selected lectins (Sambucus nigra agglutinin - SNA and Maackia amurensis agglutinin - MAL II). In addition protein pattern of examined tissues was also evaluated. The study was performed on 10 female dogs undergoing ovariohysterectomy because of pyometra and 10 clinically healthy female dogs, undergoing elective spaying (ovariohysterectomy). Uterine tissue samples of 1 cm2 were taken from the middle region of each uterine horn in both group of animals immediately after ovariohysterectomy. Tissue samples were homogenized and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting with SNA and MAL II. SDS-PAGE analysis showed differences between pyometra samples and controls in the amount of obtained protein fractions and the protein content in the individual fractions. Five protein (with a molecular weight of 193.78 kDa, 103.18 kDa, 77.67 kDa, 70.39 kDa, and 53.00 kDa) were found only in the pyometra samples. The remaining fractions differed in intensity of staining, which indicated differ abundance of a given protein. The results of western blotting with SNA and MAL II demonstrated that the pattern obtained from densitometric analysis differs between adequate healthy and pyometra samples with regard to the amount of protein fraction obtained as well as the intensity of staining of particular fraction. The pyometra tissues contained seven SNA-binding proteins (with a molecular weight 189.94 kDa, 165.51 kDa, 100.94 kDa, 59.42 KDa, 41.32 kDa, 35.16 kDa, and 32.6 kDa) that were not in the healthy tissues. Of the nine remaining fractions, six showed significantly higher (P < 0.05) intensity of staining in the healthy uterine tissues. In turn, the MAL II-binding protein with a molecular weight 75.85 kDa, 51.12 kDa, and 49.98 kDa were found only in the pyometra samples. Of the 28 remaining fractions, ten demonstrated significantly higher (P < 0.05), and five fractions had significantly lower (P < 0.05) intensity of staining in the pyometra tissues. The results obtained indicate that proteins in uterine tissues from female dogs with pyometra are differently glycosylated compared to normal uterine tissues. These findings provide the basis for further studies of the possible role of glycosylation in the pathogenesis of canine pyometra.

Immunohistochemical expression of p63 protein and calponin in canine mammary tumours.

The aim of this study was to compare the expression of p63 protein and calponin in terms of their affinity and specificity for myoepithelial cells in canine mammary tumours. The studied material included 10 benign and 32 malignant mammary tumours from female dogs treated with mastectomy. Primary mouse monoclonal antibodies directed against p63 protein clone 4A4 and calponin clone CALP were used in single- and doublestain system of immunohistochemical reaction. The investigations have shown that majority of myoepithelial cells in benign tumours and carcinomas in situ exhibited strong positive labelling for both markers. In other malignant tumours strong immunoreactivity was observed in resting myoepithelial cells (MECs) and hypertrophic myoepithelial cells (HMECs), while the immunoreactivity in spindle-stellate myoepithelial cells (SMECs) and rounded myoepithelial cells (RMECs) was moderate. The granular-diffuse nuclear expression of p63 protein was observed only in myoepithelial cells. In terms of calponin, diffuse cytoplasmic expression was noted not only in myoepithelial cell but also in some stromal fibroblasts and vascular smooth muscle cells. The epithelial cells did not exhibit specific expression of the investigated markers. The obtained results indicate that p63 is a sensitive and more specific marker of myoepithelial cells in canine mammary tumours compared with calponin. These findings suggest that the immunohistochemical analysis peformed with the use of p63 can be a valuable complement of routine histological examinations of canine mammary tumours facilitating identification of tumours with myoepithelial component.

Acute-phase inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) levels in serum and milk of cows with subclinical mastitis caused by Streptococcus species and coagulase-negative Staphylococcus species.

The aim of the study was to investigate the concentrations of acute-phase inter-α-trypsin inhibitor heavy chain 4 (ITIH4) in serum and milk of cows with subclinical mastitis caused by Streptococcus spp. (STR) and coagulase-negative Staphylococcus spp. (CNS) and healthy cows. The blood and milk samples were obtained from 60 mid-lactation, multiparous Holstein-Friesian cows from 7 herds in the Lublin region of Poland. In the milk samples from 40 cows with subclinical mastitis, Streptococcus spp. and CNS were isolated. The ITIH4 was significantly higher in serum of cows with subclinical mastitis caused both by STR and CNS compared with healthy cows. One hundred percent of animals infected with Streptococcus spp. and 89% of animals infected with Staphylococcus spp. showed ITIH4 concentration in sera higher than 0.5 mg/mL. The concentration of ITIH4 in milk also was significantly higher in cows with subclinical mastitis caused by Streptococcus spp. and Staphylococcus spp. compared with the control group. Seventy percent of cows infected by STR and CNS showed ITIH4 concentration in milk higher than 2.5 µg/mL. Milk ITIH4 concentration higher than 5 µg/mL was found in 55% of animals infected with Streptococcus spp. and in 40% of animals infected with Staphylococcus spp. No statistically significant differences were observed in ITIH4 concentrations both in serum and in milk between the studied unhealthy animal groups. These results suggest that ITIH4 may be used in the future as a novel diagnostic marker in serum and in milk of subclinical mastitis in cows.

Tryptophan, kynurenine, kynurenic acid concentrations and indoleamine 2,3-dioxygenase activity in serum and milk of dairy cows with subclinical mastitis caused by coagulase-negative staphylococci.

The aim of the study was to investigate serum and milk concentrations of tryptophan (TRP), kynurenine (KYN), kynurenic acid (KYNA), and indoleamine 2,3-dioxygenase (IDO) activity in cows suffering from subclinical mastitis caused by coagulase-negative staphylococci (MSCNS). TRP and kynurenines were determined by high-performance liquid chromatography (HPLC), and IDO activity was calculated as the KYN/TRP ratio. The blood and milk samples were collected from 40 midlactation Holstein-Fresian cows from two herds in the Lublin region in Poland. In the milk samples from 20 cows with subclinical mastitis, coagulase-negative staphylococci were isolated and in the milk obtained from healthy cows growth of microorganisms was not detected. TRP, KYN and KYNA concentrations were significantly lower in milk of cows with MSCNS compared to control animals (4.47 vs. 7.24 µM, 0.14 vs. 0.21 µM, 1.58 vs. 2.18 nM, respectively). There was no statistically significant difference in TRP and KYNA concentrations in serum between the studied animal groups (32.97 vs. 39.29 µM, 31.3 vs. 26.5 nM, respectively). In turn, the level of KYN was lower in the serum (0.81 vs. 1.13 µM) of cows with mastitis compared to healthy ones. No statistically significant differences in IDO activity, both in serum and in milk (25.24 and 27.55, 28.56 and 27.17, respectively) was revealed between the studied groups. These findings may have potential implications for diagnosis of mastitis in cows because reduction of these parameters in milk might be a marker predicting the occurrence of the disease.

Milk and serum IL-4, IL-6, IL-10, and amyloid A concentrations in cows with subclinical mastitis caused by coagulase-negative staphylococci.

The aim of the study was to evaluate the concentrations of cytokines IL-4, IL-6, and IL-10 and acute phase protein amyloid A in milk and in serum from cows with subclinical mastitis caused by coagulase-negative staphylococci and from healthy cows. The blood and milk samples were obtained from 35 midlactation, multiparous (between parities 2 and 4) Holstein-Friesian cows. In the milk samples from 20 cows with subclinical mastitis, the following species of Staphylococcus were detected: Staphylococcus xylosus (8 samples), Staphylococcus chromogenes (6 samples), Staphylococcus haemolyticus (2 samples), Staphylococcus simulans (2 samples), and Staphylococcus sciuri (2 samples). The results of the present study indicate that the level of IL-6 in cows suffering from subclinical mastitis tended to be high in both serum and milk (432.09 and 254.32 pg/mL) compared with the level in healthy cows (164.47 and 13.02 pg/mL, respectively). Amyloid A value also was significantly higher in milk of unhealthy cows compared with cows without subclinical mastitis (790.2 and 360.5 ng/mL). No significant differences were found in levels of amyloid A in serum of both tested groups of cows (2,680.0 and 2,720.0 ng/mL). In contrast, concentration of IL-4 was significantly lower both in serum and in milk of cows with staphylococcal mastitis (86.1 and 123.17 pg/mL) compared with control animals (413.5 and 670.2 pg/mL). The level of IL-10 also was significantly higher in milk of healthy cows than in infected cows (39.78 and 22.5 pg/mL); however, differences in serum levels of this cytokine between tested groups were significantly less important (220.6 and 175.1 pg/mL).

Preliminary study on plasma proteins in pregnant and non-pregnant female dogs.

In this study, we used a combined approach based on 2-dimensional electrophoresis (2-DE), difference in gel electrophoresis (DIGE), and mass spectrometry (MS) to identify the plasma protein composition in pregnant female dogs and compared it with non-pregnant female dogs. We used the plasma samples obtained from four female dogs during I, II, and III thirds of pregnancy, three days after parturition, as well as from four non-pregnant female dogs in diestrus phase. Analysis of 2-DE gel image exhibited of 249 protein spots. The intensity of staining of 35 spots differed significantly (P < 0.05) between the non-pregnant and pregnant female dogs. We used matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI TOF MS) to identify 47 spots corresponding to 52 different proteins. Five identified protein spots, including zinc finger BED domain-containing protein 5, hemoglobin subunit beta-2, integrator complex subunit 7, apolipoprotein A-I, and glutamyl aminopeptidase were differentially presented in the plasma of pregnant and non-pregnant female dogs. To the best of our knowledge, this is the first report on the plasma protein profile of pregnant and non-pregnant female dogs. In this study, we identified proteins that have not been previously identified in dogs. Our findings showed that numerous protein spots were differentially presented in the plasma of female dogs during normal pregnancy. Although we identified only a limited number of differentially presented proteins, our study demonstrated that the plasma protein profile changed during pregnancy in female dogs, which suggests its importance in maintaining pregnancy. Further studies are necessary to define complete plasma protein profile of pregnant female dogs and to identify all proteins that are differentially presented in the pregnant animals compared with the non-pregnant ones. In addition, studies are warranted to explain the role of those proteins in maintaining the pregnancy and their usefulness in detection of early pregnancy. Furthermore, our results indicated that DIGE technique is useful in the comparison of samples originated from different states and time points in dogs.