Detection of Mycobacterium avium subsp. paratuberculosis in Slovakian wildlife.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of infectious enteritis called paratuberculosis that has a high economic impact on the worldwide livestock production. A central important question arises: Can wildlife animals serve as a reservoir for transmission of MAP to domestic ruminants? With this in mind, we devised a study to detect MAP in various Slovakian wildlife species found in the areas where paratuberculosis had been documented in domestic ruminants. The samples of parenchymatous organs (intestines, ileocecal valve and mesenteric lymphatic nodes) from 83 wildlife animals representing 13 species, inclu- ding 7 herbivorous, 5 carnivorous and 1 omnivorous species were collected during a four-year period. The clinical and pathological examinations failed to demonstrate any manifestations of paratuberculosis in any of the wildlife samples. The detection of MAP was done by widely used tests, i.e. cultivation and the PCR analysis. The bacterial cultures revealed the growth of Mycobacterium spp. colonies in 58 (70%) of all of the wild animals, but the PCR testing demonstrated paratuberculosis only in one (7.69%) of the roe deer population.

Survival and differentiation of human embryonic stem cell-derived neural precursors grafted spinally in spinal ischemia-injured rats or in naive immunosuppressed minipigs: a qualitative and quantitative study.

In previous studies, we have demonstrated that spinal grafting of human or rat fetal spinal neural precursors leads to amelioration of spasticity and improvement in ambulatory function in rats with spinal ischemic injury. In the current study, we characterize the survival and maturation of three different human embryonic stem (ES) cell line-derived neural precursors (hNPCs) once grafted into ischemia-injured lumbar spinal cord in rats or in naive immunosuppressed minipigs. Proliferating HUES-2, HUES-7, or HUES-9 colonies were induced to form embryoid bodies. During the nestin-positive stage, the rosettes were removed and CD184(+)/CD271(-)/CD44(-)/CD24(+) population of ES-hNPCs FAC-sorted and expanded. Male Sprague-Dawley rats with spinal ischemic injury or naive immunosuppressed Gottingen-Minnesota minipigs received 10 bilateral injections of ES-NPCs into the L2-L5 gray matter. After cell grafting, animals survived for 2 weeks to 4.5 months, and the presence of grafted cells was confirmed after staining spinal cord sections with a combination of human-specific (hNUMA, HO14, hNSE, hSYN) or nonspecific (DCX, MAP2, CHAT, GFAP, APC) antibodies. In the majority of grafted animals, hNUMA-positive grafted cells were identified. At 2-4 weeks after grafting, double-labeled hNUMA/DCX-immunoreactive neurons were seen with extensive DCX(+) processes. At survival intervals of 4-8 weeks, hNSE(+) neurons and expression of hSYN was identified. Some hSYN-positive terminals formed putative synapses with the host neurons. Quantitative analysis of hNUMA(+) cells at 2 months after grafting showed comparable cell survival for all three cell lines. In the presence of low-level immunosuppression, no grafted cell survival was seen at 4.5 months after grafting. Spinal grafting of proliferating pluripotent HUES-7 cells led to consistent teratoma formation at 2-6 weeks after cell transplantation. These data show that ES-derived, FAC-sorted NPCs can represent an effective source of human NPCs to be used in CNS cell replacement therapies.

Influence of storage conditions and shotshell wounding on the hygienic condition of hunted, uneviscerated pheasant (Phasianus colchicus).

The effects of shot wounds on the hygienic conditions of pheasants (particularly those in the body cavity) were studied. Slaughtered (n = 33) and hunted pheasants (31 specimens with, and 33 specimens without shots in the body cavity) were stored uneviscerated at 0 and 4 degrees C. Specimens were taken at d 0, 3, 7, and 14. Hunted pheasants differed from slaughtered pheasants with respect to muscular hemorrhages and blood and fecal matter in the body cavity but also with regard to the presence of Escherichia coli in breast and thigh muscles. In addition, a higher thigh muscle pH (P < 0.05) was noted in hunted pheasants, with no significant (P > 0.05) increase observed during storage. Concentrations of biogenic amines in muscle tissue remained below the determination limit of 1 mg/kg for 90% of samples analyzed, with the maximum concentration for the remaining 10% of samples reaching 5.7 mg/kg, indicating a low incidence of contaminant bacteria. The observed changes in pH values and levels of biogenic amines failed to correlate with the presence or absence of shot lesions in the body cavity or abdominal region. Total aerobic counts increased significantly during storage, but the absolute numbers were consistently below 10(6) log(10) cfu/g. Although E. coli were <1 log(10) cfu/g in muscles of hunted pheasants on d 3 at 4 degrees C, counts of up to 3.7 log(10) cfu/g on d 7 at 4 degrees C indicated a loss of hygienic quality. Therefore, it is recommended that hunted, uneviscerated pheasants be stored 3 d at 4 degrees C, but not longer than 7 d after the hunt.

Protein A/G dependent ELISA a promising diagnostic tool in Lyme disease seroprevalence in game animals and hunting dogs.

One of the major problems in serodiagnosis in wild animals is unavailability of specific antiglobulin conjugate. Our study focuses on validation of Protein A/G dependent ELISA in game animals like deer and mouflons as well as in hunting dogs. Binding ability of Protein A/G-conjugate to antibodies was the highest in dogs followed by fallow deer and mouflons. Three different whole cell Borrelia antigens were used to evaluate antigen dependent variation. In new Protein A/G-ELISA the highest sensitivities (90.50%, deer; 85.37%, mouflon & 94.29%, dog) were obtained by B. garinii antigen, with no statistically significant variation (chi(2), P>0.05) among all other antigens used. Average seroprevalences observed in deer, mouflons and dogs were 44.90%, 29.41% and 30.43%, respectively. Marked influence of age on seroprevalence was noticed. Protein A/G-ELISA proved to be sensitive and promising diagnostic tool in serodiagnosis of Lyme disease in game ungulates and it can be used effectively for serosurvey in different wild mammals.

Development and in vitro efficacy of novel MMP2 and MMP9 specific doxorubicin albumin conjugates.

Two doxorubicin albumin conjugates (A-DP1 and A-DP2), which differ in their substrate specificity for the matrix metalloproteinases MMP2 and MMP9, were prepared by binding maleimide doxorubicin peptide derivatives to the cysteine-34 position of human serum albumin. The incorporated octapeptide, Gly-Pro-Gln-Arg-Ile-Ala-Gly-Gln, in A-DP2 is not cleaved by activated MMP2 and MMP9 in contrast to Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln incorporated in A-DP1 that is cleaved efficiently by activated MMP2 and MMP9 liberating a doxorubicin tetrapeptide. A-DP1 showed antiproliferative activity in a murine renal cell carcinoma line in the low micromolar range (IC(50) value approximately 0.2 microM).

Specific amino acids moderate the effects on Ni2+ on the testosterone production of mouse leydig cells in vitro.

The purpose of this investigation was to study the effectiveness of two nickel-binding amino acids, histidine (His) and cysteine (Cys), to prevent the inhibitory action of Ni2+ on testosterone (T) production by mouse primary Leydig cell culture. The maximal human chorionic gonadotropin (hCG)-stimulated T response was measured by radioimmunoassay (RIA) in the culture media. Three types of experiments were performed. In a concentration-response study, Ni2+ (62.5 to 1,000 microM) was added to the cells simultaneously with equimolar or twice the equimolar concentrations of His or Cys and the cultures were maintained for 48 h. Nickel-induced reduction in T production was completely prevented by equimolar concentrations of His at Ni2+ concentrations of 125, 250, and 500 microM; equimolar or twice the equimolar concentrations of His were only partially effective at 1,000 microM Ni2+. Protective action of Cys was complete only at the lowest concentration of Ni2+ (125 microM). In a second series, the cells were incubated for various times (0.5 to 48 h) with 1,000 microM Ni2+ in the presence of 2,000 microM His or Cys. Increasing the time of incubation, the protective effect of both amino acids against Ni2+ was reduced. In a third series, attempts were made to reverse the action of 1,000 microM Ni2+ after incubation with cells for various times (0.5 to 24 h), followed by exposure to 2,000 microM His or Cys. Cell cultures were maintained for 48 h. A partial recovery of hCG-stimulated T production could be observed only if the amino acid was added not later than 4 h after the metal. This time was also required to elicit the T depression produced by Ni2+. Administration of either His or Cys at later times had no effect. Our results show that both His and Cys are able to moderate the effects of Ni2+ on Leydig cell T production, depending on the concentration of this metal ion, as well as on amino acid. However, at higher Ni2+ concentrations the complete protection by His or Cys is only temporary. Administration of these amino acids after the Ni2+-produced decrease in T production was not able to reverse the process.

Effect of Ni2+ on the testosterone production of mouse primary Leydig cell culture.

This study evaluated the effects of Ni2 on testosterone (T) production of mouse Leydig cells in vitro following an in vivo or in vitro exposure. CFLP mice were subjected to repeated exposure (4 treatments, subcutaneously, every 3 d) to 10, 20 or 40 mg/kg body weight of NiSO4 or 1.0 ml of 0.9% NaCl solution. Depressed human chorionic gonadotropin (hCG)-stimulated T response was seen over a 48-h culture of testicular interstitial cells obtained from the animals exposed to 20 mg/kg or higher dose of NiSO4, while the basal T production remained unaltered. There were no Ni2+-related changes in the body weights or in the weights of testes, epididymides, adrenals, and kidneys. No histopathological alteration was found in the examined organs of NiSO4-treated groups except the dose-dependent tubular lesions in kidney as a result of a specific rather than a general cytotoxic action. To assess the direct effect of Ni2+ on Leydig-cell T production, testicular interstitial cells were cultured with Ni2+ (62.5 to 1000 microM) for 48 h in the presence or absence of maximally stimulating concentration of hCG. Dose-dependent depression in hCG-stimulated T production was seen at 125 microM or higher dose of Ni2+, while basal T production was unaffected. In order to evaluate the time dependency of this effect the cells were cultured for various times in the presence or absence of 250 and 1000 microM Ni2+. Decreased hCG-stimulated T production was found in the cultures maintained at least for 4 h in the presence of 1000 microM Ni2+, whereas at 250 microM at least 16 h was required to elicit the depression. Cell viability was assessed by a metabolic activity (MTT) assay. The viability of cells was unaltered by 250 microM Ni2+, and only a slight decrease was found even at the end of the 48-h culture period in the presence of 1000 microM Ni2+. Our results show a dose-related depression in stimulated T production of mouse Leydig cells in culture following either in vivo or in vitro Ni2+ treatment at a dose that does not induce any general toxic or significant cytotoxic action. The data of the time-course study indicate that the effect of Ni2+ on Leydig-cell T production is both time and concentration dependent, and not due to cytotoxicity.

Effect of cadmium on morphology and steroidogenesis of cultured human ovarian granulosa cells.

Cadmium (Cd) is able to decrease preovulatory luteinizing hormone (LH) levels in blood and inhibit ovulation in rats. In this study the direct effects of Cd on steroidogenesis in granulosa cells were investigated. The cells obtained from ovarian follicular aspirates of 41 women undergoing in vitro fertilization (IVF) were cultured. Cadmium-induced alterations in the cellular morphology and in the production of progesterone by the cells was determined after exposure to concentrations of 8, 16, 32 and 64 microM CdCl2 for 2, 4, 8, 24 and 48 h. Progesterone secretion by granulosa cells could be stimulated with increasing concentrations of follicle-stimulating hormone (FSH). Combined effects of Cd and FSH were also studied. Cadmium diminished progesterone production in unstimulated and FSH-supported cells depending on its concentration and the exposure time. Follicle-stimulating hormone (100 ng ml[-1]) protected against Cd-induced suppression of progesterone production. Cadmium interfered with cell-cell junctions and the adherence of cells. No protective effect of FSH on Cd-induced alteration in cell morphology could be observed. Retraction of cytoplasmic extensions occurred at a lower dose and within a shorter exposure than a decrease in progesterone production. In conclusion, Cd exerted a direct effect on both granulosa cell morphology and on steroid biosynthesis. The lowest Cd concentration (16 microM) that was able to reduce progesterone production was about 3.5 times higher than levels reported in the ovary of a female smoker. The presented data can help to define environmental, occupational and life-style (smoking) risk factors in gonadal function during the preconception period of the female reproductive lifespan.

Zinc protection against cadmium-induced infertility in female rats. Effect of zinc and cadmium on the progesterone production of cultured granulosa cells.

Adult female rats were treated subcutaneously (s.c.) with zinc chloride (ZnCl2, 10 or 20 mg kg-1 body weight, bw) four times during two ovarian cycles. The third injection was accompanied by cadmium chloride (CdCl2) administration sc (2.5, 5 and 10 mg kg-1 bw). The fourth zinc (Zn) treatment was followed by mating. ZnCl2 (20 mg kg-1) itself impaired fertility by 20%, while CdCl2 dose-dependently blocked the receptivity of female rats. In combination with 2.5 and 5 mg kg-1 CdCl2 the metal salts decreased fertility in an additive fashion, whereas at the highest CdCl2 dose (10 mg kg-1) a marked ameliorating effect of ZnCl2 (10 and 20 mg kg-1) on cadmium (Cd)-caused sterility was observed. In the pregnant animals apart from the higher Cd-induced blood progesterone levels and reduced body weight gain of dams, no significant treatment-related maternal and fetal effects could be observed. ZnCl2 (10 to 80 microM) and CdCl2 (10 to 80 microM) were added to the culture medium of ovarian granulosa cells. CdCl2 suppressed follicle-stimulating-hormone- (FSH-) and cAMP-stimulated progesterone accumulation. No protective effect of Zn against Cd-induced drop in progesterone production could be seen, while Zn by itself induced a significant increase in FSH-supported progesterone synthesis. In conclusion, while Zn protected against Cd-induced sterility in vivo, it failed to counteract the direct effect of Cd on steroid biosynthesis. The data indicate that Zn protection does not take place at the level of ovary. Moreover, Zn and Cd seem to affect FSH-stimulated progesterone production by different mechanisms.

[Follicular maturation, dizygotic twin conceptions and maternal age]. / Maturation folliculaire, conceptions gémellaires dizygotes et âge maternel.

This note aims to given an interpretation of the "paradoxical" increase of dizygotic twin pregnancies (and more generally of multiple pregnancies) according to maternal age, when in parallel the number of growing follicles decreases strongly. From the analysis, through an allometric model, of the relative variations with age of the size of the various ovarian follicular compartments, it reinforces the hypothesis of the existence of an inhibition factor, whose level would depend on the size of the residual stock of follicles and which would act on one or several phases of the follicular development. It brings evidence for the major role that this factor might play in the regulation of the whole reproductive life of women (and probably other mammals).

Effect of cadmium on female fertility, pregnancy and postnatal development in the rat.

Adult female rats having regular ovarian cycles were treated with 2.5, 5 or 10 mg/kg cadmium chloride (CdCl2) during estrus or diestrus and mated 32, 80 or 132 h post-treatment. Sperm positivity was checked next day on the predicted estrus. Maternal effects during pregnancy, fetal outcome on day 10 or at term as well as postnatal development of the F1 generation were recorded. CdCl2 caused sterility in 40 or 87% of animals at doses 5 and 10 mg/kg, respectively. Influence of Cd on fertility depended on the day of the cycle, and on the time elapsed between treatment and mating. The Cd-caused overt toxicity in fertile female rats was expressed by dose-dependent decrease in maternal body weight gain and increased progesterone blood levels. No treatment-related alteration in number and weight of conception day 10 of pregnancy or in weight and size of litters, rate of males and females at term and during the 21-day post-parturition study could be seen. It is concluded that Cd given prior mating may lead to sterility in a dose-dependent fashion. This is suggested to be caused by anovulation resulting from reversible pituitary disfunction. Animals proving fertile in spite of Cd-treatment have developed tolerance against Cd in terms of fetal outcome and postnatal development.

Marker-derived phylogeny of European cattle supports demic expansion of agriculture.

Using frequencies of 86 genes from 23 loci of blood group systems, blood and milk proteins, the genetic relationships among 14 cattle breeds including four native Balkan and four synthetic Balkan-Alp breeds were studied. The dendrogram and nonlinear map construction shows a consensus 'Balkan breed cluster', an 'Alp breed cluster', an unstable position of synthetic breeds and well-separated American breeds. Positive partial correlations between genetic distance and time elapsed since introduction of farming while keeping geographical distances constant, and regular patterns over thousands of kilometers indicate that large-scale cattle population movements together with human migration (in the Neolithic age) from the Near East into Europe across the Balkans are the most likely explanation for the genetic distances observed in our data. More recent breed differentiation and selection do not yet blur this initial pattern of European cattle populations.