Coxiella burnetii: Serological reactions and bacterial shedding in primiparous dairy cows in an endemically infected herd-impact on milk yield and fertility.

Coxiella burnetii (C. burnetii) is the causative agent of Q fever both in humans and animals. The objectives of this study were to investigate seropositivity and bacterial shedding in heifers and primiparous cows in an endemically infected herd and to assess the effects on post-partum diseases, fertility and milk production. At the age of 9 months, 96 Holstein heifers were included. Sampling was performed reproduction-orientated: at the beginning of the study, at detection of first pregnancy, 3 weeks before expected calving date (blood serum), at parturition and after 21, 42, 100 and 150 days in milk (DIM) (blood serum, vaginal swabs and milk). Serum samples were investigated by a commercial ELISA for the presence of specific antibodies and vaginal swabs and milk samples by PCR to detect C. burnetii DNA. Individual animal data (calving ease, stillbirth, retained foetal membranes, puerperal metritis, endometritis after 42 DIM, presence of corpus luteum after 42 DIM, interval calving-first service, interval calving-conception, number of inseminations until 150 DIM, proportion of pregnant cows until 100 and 150 DIM, proportion of pregnant cows after first service and data of the dairy herd improvement test) were documented. All heifers were seronegative at the age of 9 months and 3 weeks before the expected calving date. Subsequently, the proportion of seropositive animals and the antibody score increased significantly towards 42 and 100 DIM, respectively. Vaginal C. burnetii shedding was highest at parturition (30.9%), while the most positive milk samples were detected after 100 DIM (15.3%). Coxiella burnetii seropositivity and shedding had no impact on parameters of reproduction. However, milk fat yield was declined in puerperal vaginal shedders and cows which seroconverted during their first 42 DIM, respectively.

Effects of Saussurea lappa roots extract in ethanol on leukocyte phagocytic activity, lymphocyte proliferation and interferon-gamma (IFN-gamma).

Effects of Saussurea lappa root extracts prepared in ethanol according to the homeopathic principles were assessed on leukocyte phagocytic activity, lymphocyte transformation and mitogen-induced interferon-gamma (IFN-gamma) in the cultures of peripheral blood mononuclear cells of goats (PBMC) in vitro. Leukocyte phagocytic activity was measured by flow cytometry, lymphocyte proliferation by MTT and IFN-gamma level in cell culture supernatants was determined by ELISA. The results obtained demonstrated that all test dilutions (D4, D6, D8) of Saussurea lappa in ethanol have exerted a stimulating effect on leukocyte phagocytic activity in dose-dependent manner. A 10 microl dose of Saussurea lappa of each dilution markedly enhanced phagocytic activity, while other doses tested made only a feeble stimulating effect. The increases with 10 microl dose were found significantly (P<0.01) different between each dilution, maximal stimulation was observed by D8 dilution. Different doses (10 microl, 2 microl, 1 microl, 0.5 microl) of all test dilutions (D4, D6, D8) of Saussurea lappa in sterile 0.9% NaCl solution inhibited lymphocyte proliferation. Maximal inhibitory effect was observed with the 2 microl dose. Similarly, Saussurea lappa suppressed the secretion of IFN-gamma by mitogen-activated (PHA; 2.5 microg/ml) of peripheral mononuclear cells in dose-dependent manner. In conclusion these findings suggest that enhanced leukocyte phagocytic activity may be helpful to clear the soluble immune complexes produced during a sustained immune response against self antigens which causes chronic inflammatory injury of tissue. On the other hand, inhibition of lymphocyte proliferation and IFN-gamma by Saussurea lappa may contribute to suppress immune-mediated inflammatory reactions possibly through a cell-mediated cytokine pathway. Thus it is concievable that ethanolic extracts of Saussurea lappa roots in homeopathetic dilutions may be considered as a potential candidate for therapeutic support in autoimmune and chronic inflammatory disorders.

Stimulation of bovine sperm mitochondrial activity by homeopathic dilutions of monensin.

Mitochondrial activity is an important viability parameter of spermatozoa and is linked to sperm motility. Monensin is commonly used as an inhibitor for sperm mitochondrial activity in the laboratory. This study was conducted to evaluate the influence of some homeopathic dilutions of monensin on sperm mitochondrial activity. Fresh ejaculates from 6 mature bulls were used in the study. Samples of the semen were tested using a flow cytometer for mitochondrial activity and sperm viability using Rhodamine 123 and SYBR-14, respectively. The 9x dilution of monensin resulted in very highly significant (P<0.001) stimulation of mitochondrial activity. Monensin 5x, 7x, 8x and 13x caused highly significant (P<0.01) stimulation of the sperm mitochondrial activity. Other homeopathic dilutions of monensin (6x, 10x, 11x, 12x and 14x) also had a significant (P<0.05) stimulatory effect. The use of monensin did not have any negative effect on sperm viability. We conclude that some homeopathic dilutions of monensin increase mitochondrial activity of bovine spermatozoa without negative effect on sperm viability, the 9x dilution was the most effective. Further in vivo studies are required to estimate the effect of homeopathic dilutions of monensin on semen quality.

Application of MTT reduction assay to evaluate equine sperm viability.

The assay of MTT reduction depends on the ability of metabolically active cells to reduce the tetrazolium salt (3[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide) to formazan. This study was conducted to examine and validate of a simple and less costly MTT test in determining equine sperm viability and compare the efficiency of this test with a flow cytometer. Fresh ejaculates from 11 stallions (warm blood) were included in this study. Semen was diluted to 100 million cells/ml in a Hepes 0.1% BSA. The rates of MTT reduction were measured in microtiter plates after incubation for 1 and 4h at 37 degrees C using spectrophotometer (MS2 Reader) at wavelength 550 nm. Simultaneously split samples of the same semen were tested, using a flow cytometer for mitochondrial activity, sperm viability, and acrosomal integrity using Rhodamine 123, SYBR-14 and LysoTracker Green DNA-26, respectively. The results revealed a strong correlation (P < 0.001) between the results of MTT test at 1 and 4 h of incubation time and the result of mitochondrial activity (r = 0.978, 0.977), sperm viability (r = 0.954, 0.977) and acrosomal integrity (r = 0.867, 0.886). In conclusion, the MTT test was found to be a reliable method in evaluating semen viability and can be used successfully, especially in routine analysis, where practical aspects such as time, costs and practicability are important.

Experimental infection of mice with Borna disease virus (BDV): replication and distribution of the virus after intracerebral infection.

To develop an animal model resembling natural asymptomatic Borna disease virus (BDV) infections, BDV He/80 rat brain homogenate was passaged four times in adult SJL/J mice. Within 12 months of observation, mice did not develop overt signs of disease. Nucleotide sequencing of the rat isolate and the mouse isolates at the fourth passage revealed no difference in the deduced amino acids. Viral RNA was found in brain, heart, kidney, lung, liver, and urinary bladder. Infectious virus was isolated from brain, but also from heart and lung tissue. Immunohistochemically, BDV was demonstrated in nerves in the abdominal cavity, ganglion coeliacum, and adrenal glands, but not in organ parenchyma. Occasionally, viral RNA was detected in mononuclear blood cells.

Experimental and natural borna disease virus infections: presence of viral RNA in cells of the peripheral blood.

Cells of the peripheral blood of experimentally and naturally borna disease virus (BDV)-infected animals and of human psychiatric patients and healthy individuals were analyzed for the presence of viral RNA using a BDV-p40-specific nested reverse transcription-polymerase chain reaction (RT-PCR). The assay proved to be highly sensitive as 10 RNA molecules were reproducibly amplified. BDV RNA was detected in blood cells of experimentally infected immunocompetent mice and rats. Mice were persistently infected without showing clinical signs of borna disease (BD), whereas the rats suffered from acute BD. Among 19 horses examined, five were positive for viral RNA in the blood. In a flock of sheep with a history of BD, 1 out of 25 clinically healthy animals was positive. BDV RNA was also detected in cells of the peripheral blood of 10 out of 27 selected humans with psychiatric disorders, and in 2 out of 13 healthy individuals. Remarkably, BDV-specific RNA was present in some cases in the absence of BDV-specific antibodies. Sequence analysis of PCR products confirmed the specificity of the amplification system. The presence of BDV RNA in the blood of naturally and experimentally BDV-infected individuals may point to an incidental but relevant role of blood for the spread of BDV in the infected organism, as well as for the transmission of BDV to other individuals.

Neuron-glia interactions in the rat retina infected by Borna disease virus.

Neuron-glia interactions in the Borna disease virus (BDV)-infected rat retina were investigated with emphasis on the ultrastructural characterization of degenerative alterations in the ganglion cell and photoreceptor layer. Immuno- and cytochemical techniques were applied to label microglia, macrophages and Müller (macroglial) cells. Four weeks after intracerebral infection of adult rats, the total thickness of the retina was considerably diminished, primarily due to the loss of photoreceptor segments and ganglion cells. A gradual reduction of both plexiform layers was also observed. There was a remarkable increase in the number of microglial cells, predominantly in the ganglion cell and the inner plexiform layers. Ultrastructural analysis confirmed that microglia, but also macrophages, were involved in phagocytosis accompanying severe neuronal degeneration in the ganglion cell and the photoreceptor layer. In contrast, Müller cells showed moderate morphological and cytochemical alterations, indicating that Müller cells play only a minor role in early stages of BDV-induced retinitis. Monitoring neuron-glia interactions in BDV-induced retinopathy, combined with the application of different protocols of immunosuppression effecting the BDV virus and/or the microglia, might help to establish specific strategies to suppress BDV-induced neuronal degeneration.

[Lysozyme activity in the milk of sucking mares during lactation]. / Zur Lysozymaktivität in der Milch von Fohlenstuten im Verlaufe der Laktation.

It was the aim of this project to investigate the changes of the lysozyme activity in the milk of mares during the lactation period. Further on the influence of race, date of conception and foaling, age and number of lactations on the lysozyme activities in milk was analysed. Milk samples were collected from 44 mares (trotters, warmblood, quarter horses) from eight farms between the 1st and 90th day p. p. The activity of the lysozyme was measured by a turbidometric method. Summarizing the following results are obtained: Lysozyme activities in mare milk of the 1st and 3rd day p. p. were higher than in mature milk. On average the highest lysozyme activity (Xa = 113.600 +/- 25.171 U/ml) was measured on the 3rd day p. p. Until the 9th day p. p. the activity decreased about 25%, afterwards there was only a slight decrease. The lowest activity (Xa = 57.509 +/- 14.606 U/ml) was measured at the 83rd day p. p. The influence of race and conception time proved to be statistically significant resp. highly significant.

[Activities of the enzymes LDH, gamma-GT, GOT, GPT and lactoperoxidase in the milk of breeding mares during the course of lactation]. / Die Aktivitäten der Enzyme LDH, gamma-GT, GOT, GPT und Laktoperoxidase in der Milch von Zuchtstuten im Verlaufe der Laktation.

Milk samples were collected from 44 mares (trotters, warm blood horses, quarter horses) during lactation between the 1st and 90th day p.p. at 20 defined days. The activity of the enzymes LDH, gamma-GT, GOT, GPT and lactoperoxidase was investigated. The aim of this study was to find out the changes of these parameters during lactation and whether an influence of race, conception, date of foaling, age and number of lactations existed on the enzyme activities in mare's milk. The following results were obtained: In mare's milk the LDH-activity was highest (xg = 629 x 1.5 +/- 1 U/l) on the 1st day p.p. and showed a marked decrease on the 3rd day p.p. followed by a slight decrease until the 20th day p.p. It remained then at a constant level of about 80 U/l. The lowest activity (xg = 65.2 x 1.51 +/- 1 U/l) was measured on the 76th day p.p. The influence of conception and date of foaling time were statistically significant. The gamma-GT-activity was highest on the 3rd day p.p. (xg = 143.4 x 1.45 +/- 1 U/L) and decreased during lactation. The lowest activity was measured on the 90th day p.p. (xg = 23.2 x 1.51 +/- 1 U/L). The influence of race and conception--time were statistically significant. The GOT-activity of the 1st day p.p. (xg = 38.3 x 1.77 +/- 1 U/L) was higher than in mature milk. After the 3rd day p.p. activities between 15.5 x 1.38 +/- 1 U/l and 11.9 x 1.38 +/- 1 U/L were measured. The GPT-activity was highest on the 1st day p.p. (xg = 37.5 x 1.81 +/- 1 U/L) and decreased on the following days. After the 9th day p.p. activities between 9.1 x 1.37 +/- 1 U/l and 8.5 x 1.32 +/- 1 U/l were found. The influence of age, race, race (time, date of foaling (time and conception (time were significant. No Lactoperoxidase activity could be found in mare's milk. The origin and importance of the mare's milk enzymes are discussed.

[Effect of energy level of rations and time of blood sampling on the relative activity lf LDH isoenzymes in the serum of lambs]. / Zur Wirkung des Energiegehaltes in Rationen sowie des Zeitpunktes der Blutentnahme auf die relative Aktivität der LDH-Isoenzyme im Blutserum von Lämmern.

The effect of the energy level of the feed ration and the time of blood sampling on the Lactate Dehydrogenase (LDH)-Isoenzyme pattern in the blood serum of "Thessaloniki" crossbred type lambs was examined. For this purpose 24 lambs, 45 days old, were randomly allocated in three groups (8 animals each group). The lambs were (after weaning) fed by rations differing in energy content. (Gruppe A: 12.0 MJ, M.E./kg, Gruppe B: 10.8 MJ, M.E./kg, Gruppe C: 9.9 MJ, M.E./kg). For the estimation of the main effects and interactions a least square analysis was applied. The effect of the energy level upon the relative distribution of LDH-Isoenzymes in the serum of lambs was significant only for the enzymes LDH1 and LDH5, between the groups A and C. As to the effect of time of blood sampling there were significant differences in all isoenzymes. The interaction "ration X" "time of blood sampling" was only significant for LDH1-isoenzymes.

[Course of blood levels of calcium, inorganic phosphate, alkaline phosphatase, parathyroid hormone and calcidiol (25-OH-D3) in one and two year old thoroughbred horses]. / Untersuchungen zum Verlauf der Blutspiegel von Calcium, anorganischem Phosphat, alkalischer Phosphatase, Parathormon und Calcidiol (25-OH-D3) bei ein- bis zweijährigen Vollblutpferden.

The present study was aimed to determine the contents of calcium, inorganic phosphate, parathormon, 25-OH-D3 and the activity of alkaline phosphatase in the plasma of one- and two-years-old thoroughbred horses. Data were obtained monthly from 44 one-year-old thoroughbred of 4 different studs from May during grazing-season and from October during stable-, resp. training-season up to april of the following year. Calcium, inorganic phosphate and the activity of alkaline phosphatase were measured with a photometric method and the concentration of PTH and 25-OH-D3 were determined with a radioimmunoassay. The following results were obtained: Calcium: The concentration of calcium in the plasma of one-year-old thoroughbred horses was 3.03 +/- 0.23 mmol/l during grazing-season and 3.14 +/- 0.14 mmol/l during the following stable-, resp. training-season. Inorganic phosphate: The concentration of inorganic phosphate was significantly affected by the age. The average was 1.7 +/- 0.19 mmol/l during grazing-season and 1.3 +/- 0.19 mmol/l during the following stable- and training-season. Activity of alkaline phosphatase: The activity of alkaline phosphatase was also significantly affected by the age. The average of the activity was 403 +/- 86 U/l during grazing-season and 308 +/- 65 U/l during the following winter period. Parathormon: There were big differences between the averages of parathormon during grazing-season (1.27 +/- 0.45 ng/ml) and the following winter-season (0.9 +/- 39 ng/ml). Besides from that there were big individual differences. 25-OH-D3: The concentration of 25-OH-D3 during grazing-season (10.38 +/- 3.08 ng/ml) was lower than during the winter period (13.03 +/- 2.86 ng/ml). The significance of the obtained results is discussed in relation to the corresponding literature.

[Diagnosis of early pregnancy by testing the progesterone level]. / Diagnose der Fruehtraechtigkeit mit Hilfe der Progesteronebestimmung.

PIP: Measuring serum or plasma levels of progesterone can be used to determine if conception has occurred in horses. If the mare's progesterone level is below 2 ng/ml 18 days after mating has occurred, conception has not taken place. This method can be used as an addunct to genital examination, and it can be used to determine if hormonal irregularities are present in mares who have not been able to conceive.^ieng