Protective effect of Azadirachta indica extract against Eimeria papillata-induced coccidiosis

Coccidiosis in poultry is caused by protozoan parasites of the genus Eimeria, which is responsible for worldwide economic losses. The methanolic extract of Azadirachta indica (neem) leaves was used in vivo for its pharmacological, antioxidant, and anticoccidial properties. Four groups of mice were investigated. The first group was inoculated only with sterile saline and served as the control group. The second group was treated by oral gavage with neem extract (500 mg/kg) daily for 4 days. The third and fourth groups were infected with 10³ sporulated oocysts of Eimeria papillata. The fourth group was also treated once daily with neem extract for 4 days. Paraffin sections from the jejunum as well as jejunal homogenate were prepared for the histopathological and biochemical investigations, respectively. The data showed that mice infected with E. papillata revealed an output of 6.5?×?10⁵?±?29,753 oocysts per gram feces on day?4 postinoculation. This output is significantly decreased to 2.7?×?10⁵?±?37,341 oocysts in neem-treated mice. Infection with E. papillata induced marked histopathological alterations in the jejunum in the form of inflammation, vacuolation of the epithelium, and destruction of some villi. Also, the neem extract greatly diminished body weight loss of infected mice. Moreover, the number of goblet cells stained with Alcian blue within the infected villi was significantly lowered (P?≤?0.05). In addition, E. papillata enhanced lipid peroxidation and nitric oxide production in both serum and jejunum with concomitant reduction in glutathione. Neem induced marked improvements in all of the studied parameters as well as the histopathological features of the jejunum. Our study revealed that neem as a natural product has protective effects against E. papillata-induced coccidiosis.     

Anti-coccidial, anthelmintic and antioxidant activities of pomegranate (Punica granatum) peel extract

Coccidiosis and helminthosis in poultry are responsible for worldwide economic losses. The methanolic extract of Punica granatum (pomegranate) peel was used in vivo for its pharmacological, antioxidant and anti-coccidial properties and in vitro for its anthelmintic activity. For the in vivo study, four groups of mice were investigated. The first group was inoculated only with sterile saline and served as the control group. The second group was treated by oral gavage with pomegranate extract (300 mg/kg) daily for 5 days. The third and fourth groups were infected with 10³ sporulated oocysts of Eimeria papillata. The fourth group was also treated once daily with pomegranate peel extract for 5 days. For the in vitro study, the anthelmintic effect of pomegranate peel extract was observed on live adult Allolobophora caliginosa. Paraffin sections from jejunum as well as jejunal homogenate were prepared for the histopathological and biochemical investigations, respectively. The data showed that mice infected with E. papillata revealed an output of approximately 2.9?×?10⁵ oocysts per gram faeces on day 5 p.i. This output is significantly decreased to 50 % in pomegranate-treated mice. Infection with E. papillata induced marked histopathological alterations in jejunum in the form of inflammation, vacuolation of the epithelium and destruction of some villi. In addition, pomegranate extract caused a great diminish in body weight loss of infected mice. Moreover, the number of goblet cells stained with Alcian blue within the infected villi was significantly increased by about 26 % after pomegranate treatment. In addition, Pomegranate significantly lowered the increased number of apoptotic cells due to E. papillata infection by about 36 %. The results showed that E. papillata enhanced hydrogen peroxide, lipid peroxidation and nitric oxide production with concomitant reduction in glutathione. Pomegranate induced marked improvements in all of the studied parameters as well as the histopathological features of jejunum. In addition, pomegranate was able to exert a significant anthelmintic effect on live adult A. caliginosa worms in terms of the paralysis and death of the worms at different concentrations (100, 200 and 300 mg/ml). The study revealed that pomegranate as a natural product has protective effects against E. papillata-induced coccidiosis as well as it possesses an anthelmintic activity.     

Taxonomy and phylogeny of some Eimeria (Apicomplexa: Eimeriidae) species of rodents as determined by polymerase chain reaction/restriction-fragment-length polymorphism analysis of 18S rDNA

The 18S rDNA genes of 10 Eimeria species from rodents (E. albigulae, E. arizonensis, E. falciformis, E. langebarteli, E. nieschulzi, E. onychomysis, E. papillata, E. reedi, E. separata, E. sevilletensis) were polymerase-chain-reaction (PCR)-amplified, digested with 12 restriction endonucleases, and electophoresed in agarose gels. The resulting fragment patterns (riboprints) distinguished all species except E. sevilletensis from E. falciformis, and E. arizonensis from E. albigulae; the sporulated oocysts of the latter two species and of E. onychomysis are often indistinguishable morphologically. When the restriction fragment data were analyzed using distance and parsimony phylogenetic methods a clade was found consistently, which contained E. arizonensis, E. albigulae, E. onychomysis, E. reedi, and E. papillata. This finding and other results of the phylogenetic analyses agreed and supplemented previous phylogenetic work on the Eimeria of rodents. Riboprinting appears to provide useful data for taxonomic and phylogenetic studies on the genus Eimeria and may be especially practical when samples do not contain enough oocysts for other molecular-based methods. Received: 24 February 1999 / Accepted:20 May 1999     

Dietary selenium affects intestinal development of Eimeria papillata in mice

Here, we investigated the effect of the trace element selenium (Se) on course and outcome of Eimeria-paplllata-induced coccidiosis in mice. Male mice were fed on Se-adequate (0.15 ppm), Se-deficient, and Se-high diets (1.0 ppm) for 6 weeks. Mice were orally infected with 1,000 oocysts. The prepatent period lasts for 3 days, but the course of infections varied. At Se-adequate diet, the maximum fecal output of oocysts amounted to 68,300 ooccysts/g feces on day 5 p.i.. However, fecal shedding of oocysts was accelerated in mice on Se-deficient diet and occurred already on day 4 p.i.. By contrast, maximal shedding is impaired in mice on high-Se diet, which takes place on day 5 p.i., but with a decreased output of only 7,300 oocysts/g feces. Light microscopy reveals that all developmental stages are affected: meronts, micro- and macrogamonts, and developing oocysts are increased in comparison with mice fed on selenium-adequate diet. At high Se, the number of parasitic stages in the jejunum is substantially higher than at Se-deficient diet. Se does not affect the number of jejunal Alcian blue-stained goblet cells. Se deficiency increased the number of apoptotic cells in the jejunum. Substantially increased histological injury scores reveal more injuries in jejunum tissue infected by E. papillata. Our data indicate that high dietary Se exerts potential anticoccidial activity. This may be taken advantage of in control measures towards Eimeriosis as a feed additive, potentially alleviating the need for concomitantly utilized anti-coccidial drugs in the feed.     

Hepatic miRNA expression reprogrammed by <Emphasis Type="Italic">Plasmodium chabaudi</Emphasis> malaria

Evidence is accumulating that miRNAs are critically implicated in the outcome of diseases, but little information is available for infectious diseases. This study investigates the hepatic miRNA signature in female C57BL/6 mice infected with self-healing Plasmodium chabaudi malaria. Primary infections result in approximately 50% peak parasitemia on day 8 p.i., approximately 80% survival, and development of protective immunity. The latter is evidenced as 100% survival and 1.5% peak parasitemia upon homolog re-infections of those mice which are still alive on day 56 after primary infection. Such immune mice exhibit increased levels of IgG2a and IgG2b isotypes and still contain P. chabaudi-infected erythrocytes in their livers as revealed by light microscopy and PCR analysis. Primary infections, but not secondary infections, induce an upregulation of hepatic mRNAs encoding IL-1β, TNFα, IFNγ, NF-κB, and iNOS, and a downregulation of mRNAs for CYP7A1 and SULT2A2, respectively. Using miRXplore microarrays containing 634 mouse miRNAs in combination with quantitative RT-PCR, the liver is found to respond to primary infections with an upregulation of the three miRNA species miR-26b, MCMV-miR-M23-1-5p, and miR-1274a, and a downregulation of the 16 miRNA species miR-101b, let-7a, let-7g, miR-193a-3p, miR-192, miR-142-5p, miR-465d, miR-677, miR-98, miR-694, miR-374^*, miR-450b-5p, miR-464, miR-377, miR-20a^*, and miR-466d-3p, respectively. Surprisingly, about the same pattern of miRNA expression is revealed in immune mice, and this pattern is even sustained upon homolog re-infections of immune mice. These data suggest that development of protective immunity against malarial blood stages of P. chabaudi is associated with a reprogramming of the expression of distinct miRNA species in the female mouse liver.     

<Emphasis Type="Italic">Eimeria biarmicus</Emphasis> sp.n. (Apicomplexa: Eimeriidae) infecting falcons from the genus <Emphasis Type="Italic">Falco</Emphasis> in Saudi Arabia

The oocysts of Eimeria biarmicus sp. n. were described from the feces of the lanner falcon, Falco biarmicus, collected from the falcon market in Riyadh City, Saudi Arabia. The prevalence of infection was 5% (2/40). The majority of the oocysts examined had completed sporulation within 84 h at 24 ± 2°C. Sporulated oocysts are ovoid in shape, measuring 22.4 × 17.9 (20.5–24.7 × 15.8–18.5) μm; shape index (L/W) is 1.25 (1.14–1.36) μm. The oocyst wall is smooth and bi-layered. Micropyle and oocyst residuum are absent. A polar granule is present, consisting of 2–4 globules. Sporocysts are ovoid, 10.1 × 6.1 (9.4–11.2 × 5.4–6.8) μm; with a smooth single-layered wall and a minute Stieda body, but there is no substieda body. The sporocyst residuum consists of numerous small granules. Sporozoites are comma shaped, each contains two refractile bodies. E. biarmicus sp. n. is the second eimerian species described from F. biarmicus.     

Detection of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> in water by an immunomagnetic separation method targeting the sporocysts

An immunomagnetic separation (IMS) method was developed to detect Toxoplasma gondii in fresh waters by using the monoclonal antibody 4B6 targeting the sporocyst wall of T. gondii, Hammondia hammondi, Hammondia heydorni, and Neospora caninum. Water concentrates obtained by filtering 10- to 20-l samples samples were spiked with Toxoplasma oocysts, sonicated to release the sporocysts, and analyzed by IMS-4B6. Mean sporocyst recoveries were 74.5 ± 5.3% in drinking water, 30.6 ± 2.4 and 37.1 ± 3.2% in surface waters, and 81.6 ± 2.1% in IMS buffer. Then, this IMS method was integrated in a multistep procedure (i.e., filtration, IMS, immunofluorescence and autofluorescence) to detect Toxoplasma in unspiked and spiked water samples (10–30 l) of various qualities. Sporocyst recoveries ranged from 14.4 to 44.7% in drinking water samples spiked with 1–10 oocysts/l, and from 17.8 to 32.5% in surface water samples spiked with 10 oocysts/l. Sporocysts were not detected in 25 unspiked water samples. A sporocyst-like structure was seen in one of these unspiked samples, but its coccidian nature could not be proved by three polymerase chain reaction (PCR) methods targeting sequences of coccidian small and large subunit rRNA genes and Toxoplasma repetitive elements. In conclusion, IMS-4B6 is relevant for the detection of Toxoplasma in water generating small concentrates (<1 ml). Due to 4B6 cross-reactions, a PCR would be useful to further characterize coccidian sporocysts found microscopically.     

Light microscopic study on Eimeria species infecting Japanese quails reared in Saudi Arabian farms

Japanese quails Coturnix coturnix japonica reared in economic farms were individually investigated for coccidian infections. The results indicated the absence of infections in birds younger than 1 month. An Eimeria infection rate of up to 80% was detected in birds 7–9 weeks old with a general infection rate of 29%. The infection rate decreased to 21.42% in birds older than 10 weeks. Morphometric characteristics of freshly shed, unsporulated oocysts were taken. These oocysts appeared pale yellow in color, were oval to subspherical in shape being limited by a bilayered oocyst wall of 1.2 μm. The unsporulated oocysts measured 17.73 ± 12.92 × 12.79 ± 1.69 μm (mean of 100) and possessed a polar granule, a micropyle and an oocyst residuum. The sporulation took 72 h and resulted in the formation of four elongated sporocysts containing two sporozoites, in addition to a stieda body and a sporocyst residuum. The life cycle of this Eimeria species was followed in experimentally infected quails. Three asexual generations (at 60, 78, and 96 h p.i.) were detected in the epithelium of the small intestine before the sexual cycle started at 84 h p.i. The prepatent period was 5 days, while the patent period covered 6–7 days. Besides this well-defined species, another Eimeria species occurred, the oocysts of which were excreted in low numbers and were characterized by the absence of a micropyle and an oocyst residuum. These oocysts measured 15.73 ± 2.22 × 14.18 ± 1.89 μm (mean of 100) and sporulated already within 60 h.     

Parasitological and clinical parameters of experimental <Emphasis Type="Italic">Eimeria zuernii</Emphasis> infection in calves and influence on weight gain and haemogram

Infection trials were performed to characterize experimental Eimeria zuernii coccidiosis parasitologically and clinically and to investigate the effects on weight gain and haemotologic parameters in affected calves. Three groups of calves were formed: Group 1 (n = 14) served as uninfected control group, group 2 (n = 11) was infected with 150,000 sporulated E. zuernii oocysts per calf, and group 3 (n = 16) was infected with 250,000 sporulated E. zuernii oocysts per calf. All infected animals shed oocysts and showed diarrhoea; a positive correlation could be shown between quantified oocyst excretion and faecal consistency. Measurements throughout the prepatent and the patent period revealed a marked influence of E. zuernii infection on weight gain, leukocyte concentration, haemoglobin, haematocrit, and mean cellular volume. Aberrations in these parameters were most pronounced in the highly infected group. The results of this study confirm that acute sublethal E. zuernii coccidiosis causes distinct loss of fluid and blood via intestine. This dominates also the haematological picture of the disease, which is mainly characterized by haemoconcentration. Leukocyte concentration was depressed during the early patent period, whereas it increased markedly from day 24 after infection on.     

Influence of experimental <Emphasis Type="Italic">Eimeria zuernii</Emphasis> infection in calves on electrolyte concentrations,acid–base balance and blood gases

Coccidiosis, often caused by Eimeria zuernii, is an important disease in calf rearing and is clinically mainly associated with diarrhoea (PR Fitzgerald in Adv Vet Sci Comp Med, 24:121–143, 1980). Calves were experimentally infected with E. zuernii oocysts to investigate the effects of artificial E. zuernii coccidiosis on electrolyte concentrations, acid–base balance and blood gases. Therefore, animals were assigned to three groups: group 1 (n = 14) served as uninfected control group, group 2 (n = 11) was infected with 150,000 sporulated E. zuernii oocysts per calf, and group 3 (n = 16) was infected with 250,000 sporulated E. zuernii oocysts per calf. Aberrances which were attributed to coccidiosis were observed in the following parameters: sodium and chloride concentrations, pH (only high-dose infected group 3), base excess, standard bicarbonate, total carbon dioxide and partial pressure of carbon dioxide. Alterations were most pronounced in the high-dose infected group 3. Anion gap and oxygen saturation did not show significant differences between the groups. Due to diarrhoea and malabsorption in coccidiosis-affected calves, there is a distinct loss not only of fluid and blood but also of electrolytes and alkaline buffer substances which provokes the development of an acidosis. This is counteracted by metabolism and respiration but cannot be compensated in severely affected and moribund calves.     

Cryptosporidium parvum oocysts in zebra mussels (Dreissena polymorpha): evidence from the St. Lawrence River

Molluscan shellfish can recover and concentrate environmentally derived waterborne pathogens and can be used for the sanitary assessment of water quality. Oocysts of Cryptosporidium parvum (genotype 1) were identified in zebra mussels (Dreissena polymorpha) from the St. Lawrence River, Quebec. Approximately 67 oocysts/ml of hemolymph and 129 oocysts/g of soft tissue were recovered. The adjusted concentration of oocysts per gram of tissue was 2.2 × 10², and approximately 4.4 × 10² oocysts were recovered from a single mussel. Zebra mussels can serve as biological indicators of waterborne contamination with Cryptosporidium. Received: 22 May 2000 / Accepted: 17 July 2000     

Frequent concomitant inactivation of <Emphasis Type="Italic">miR-34a</Emphasis> and <Emphasis Type="Italic">miR-34b/c</Emphasis> by CpG methylation in colorectal,pancreatic, mammary,ovarian, urothelial,and renal cell carcinomas and soft tissue sarcomas

The microRNA encoding genes miR-34a and miR-34b/c represent direct p53 target genes and possess tumor suppressive properties as they mediate apoptosis, cell cycle arrest, and senescence. We previously reported that the miR-34a gene is subject to epigenetic inactivation by CpG methylation of its promoter region in primary prostate cancer and melanomas, and in 110 different cancer cell lines of diverse origin. Here we analyzed the methylation status of miR-34a and miR-34b/c in additional primary tumors of divergent sites. We found methylation of miR-34a or miR-34b/c in formalin-fixed, paraffin-embedded (FFPE) tumor samples from 178 patients with the following frequencies: colorectal cancer (74% miR-34a, 99% miR-34b/c; n = 114), pancreatic cancer (64%, 100%; n = 11), mammary cancer (60%, 90%; n = 10), ovarian cancer (62%, 69%; n = 13), urothelial cancer (71%, 57%; n = 7), and renal cell cancer (58%, 100%; n = 12). Furthermore, soft tissue sarcomas showed methylation of miR-34 gene promoters in FFPE samples (64%, 45%; n = 11), in explanted, cultured cells (53%, 40%; n = 40), and in frozen tissue samples (75%, 75%, n = 8). In the colorectal cancer samples a statistically significant correlation of miR-34a methylation and the absence of p53 mutation was detected. With the exception of sarcoma cell lines, the inactivation of miR-34a and miR-34b/c was concomitant in most cases. These results show that miR-34 inactivation is a common event in tumor formation, and suggest that CpG methylation of miR-34a and miR-34-b/c may have diagnostic value. The mutual exclusiveness of miR-34a methylation and p53 mutation indicates that miR-34a inactivation may substitute for loss of p53 function in cancer.     

Occupational exposure to diesel engine exhaust and serum levels of microRNAs in a cross-sectional molecular epidemiology study in China

Diesel engine exhaust (DEE) is an established lung carcinogen, but the biological mechanisms of diesel-induced lung carcinogenesis are not well understood. MicroRNAs (miRNAs) are small noncoding RNAs that play a potentially important role in regulating gene expression related to lung cancer. We conducted a cross-sectional molecular epidemiology study to evaluate whether serum levels of miRNAs are altered in healthy workers occupationally exposed to DEE compared to unexposed controls. We conducted a two-stage study, first measuring 405 miRNAs in a pilot study of six DEE-exposed workers exposed and six controls. In the second stage, 44 selected miRNAs were measured using the Fireplex circulating miRNA assay that profiles miRNAs directly from biofluids of 45 workers exposed to a range of DEE (Elemental Carbon (EC), median, range: 47.7, 6.1–79.7 μg/m³) and 46 controls. The relationship between exposure to DEE and EC with miRNA levels was analyzed using linear regression adjusted for potential confounders. Serum levels of four miRNAs were significantly lower (miR-191-5p, miR-93-5p, miR-423-3p, miR-122-5p) and one miRNA was significantly higher (miR-92a-3p) in DEE exposed workers compared to controls. Of these miRNAs, miR-191-5p (p_trend = .001, FDR = 0.04) and miR-93-5p (p_trend = .009, FDR = 0.18) showed evidence of an inverse exposure–response with increasing EC levels. Our findings suggest that occupational exposure to DEE may affect circulating miRNAs implicated in biological processes related to carcinogenesis, including immune function.     

Neuromuscular adaptation during prolonged strength training, detraining and re-strength-training in middle-aged and elderly people

Effects of a 24-week strength training performed twice weekly (24 ST) (combined with explosive exercises) followed by either a 3-week detraining (3 DT) and a 21-week re-strength-training (21 RST) (experiment A) or by a 24-week detraining (24 DT) (experiment B) on neural activation of the agonist and antagonist leg extensors, muscle cross-sectional area (CSA) of the quadriceps femoris, maximal isometric and one repetition maximum (1-RM) strength and jumping (J) and walking (W) performances were examined. A group of middle-aged (M, 37–44 years, n=12) and elderly (E, 62–77, n=10) and another group of M (35–45, n=7) and E (63–78, n=7) served as subjects. In experiment A, the 1-RM increased substantially during 24 ST in M (27%, P < 0.001) and E (29%, P < 0.001) and in experiment B in M (29%, P < 0.001) and E (23%, P < 0.01). During 21 RST the 1-RM was increased by 5% at week 48 (P < 0.01) in M and 3% at week 41 in E (n.s., but P < 0.05 at week 34). In experiment A the integrated electromyogram (IEMG) of the vastus muscles in the 1-RM increased during 24 ST in both M (P < 0.05) and E (P < 0.001) and during 21 RST in M for the right (P < 0.05) and in E for both legs (P < 0.05). The biceps femoris co-activation during the 1-RM leg extension decreased during the first 8-week training in M (from 29 ± 5% to 25 ± 3%, n.s.) and especially in E (from 41 ± 11% to 32 ± 9%, P < 0.05). The CSA increased by 7% in M (P < 0.05) and by 7% in E (P < 0.001), and by 7% (n.s.) in M and by 3% in E (n.s.) during 24 ST periods. Increases of 18% (P < 0.001) and 12% (P < 0.05) in M and 22% (P < 0.001) and 26% (P < 0.05) in E occurred in J. W speed increased (P < 0.05) in both age groups. The only decrease during 3 DT was in maximal isometric force in M by 6% (P < 0.05) and by 4% (n.s.) in E. During 24 DT the CSA decreased in both age groups (P < 0.01), the 1-RM decreased by 6% (P < 0.05) in M and by 4% (P < 0.05) in E and isometric force by 12% (P < 0.001) in M and by 9% (P < 0.05) in E, respectively, while J and W remained unaltered. The strength gains were accompanied by increased maximal voluntary neural activation of the agonists in both age groups with reduced antagonist co-activation in the elderly during the initial training phases. Neural adaptation seemed to play a greater role than muscle hypertrophy. Short-term detraining led to only minor changes, while prolonged detraining resulted in muscle atrophy and decreased voluntary strength, but explosive jumping and walking actions in both age groups appeared to remain elevated for quite a long time by compensatory types of physical activities when performed on a regular basis. Accepted: 2 May 2000     

Infestation status of gnathiid isopod juveniles parasitic on Dusky grouper (<Emphasis Type="Italic">Epinephelus marginatus</Emphasis>) from the northeast Mediterranean Sea

This is the first detailed documented record of Gnathiid isopod praniza larvae infestating dusky grouper, (Epinephelus marginatus Lowe 1834) in the northeast Mediterranean Sea (36°36′N–36°07′E, 35°52′N–36°25′E). Fish were sampled monthly from Iskenderun Bay during a 3-year period from 2000 to 2003 [N = 468, W ± SD (range) = 503.69 ± 342.35 g (177–2,832 g), TL ± SD (range) = 32.39 ± 9.22 cm (16.1–67.0 cm), W _total = 0.213L _total ^2.19, r _total ² = 0.85]. Juveniles of the Gnathia sp. were only extracted from the epithelium of the buccal cavity. The monthly and seasonal patterns in infestation rates (mean prevalence, P = 27.35% and mean intensity, MI ± SD = 21.35 ± 16.19), and the relationship between length–weight and infested/non-infested fish were calculated. This study suggests that gnathiid parasite has no effect on the growth and general health condition of infested fish, although high intensities were observed in fish.     

Elevated expression of the miR-17-92 polycistron and miR-21 in hepadnavirus-associated hepatocellular carcinoma contributes to the malignant phenotype

Alterations in microRNA (miRNA) expression in both human and animal models have been linked to many forms of cancer. Such miRNAs, which act directly as repressors of gene expression, have been found to frequently reside in fragile sites and genomic regions associated with cancer. This study describes a miRNA signature for human primary hepatitis B virus-positive human hepatocellular carcinoma. Moreover, two known oncomiRs—miRNAs with known roles in cancer—the miR-17–92 polycistron and miR-21, exhibited increased expression in 100% of primary human and woodchuck hepatocellular carcinomas surveyed. To determine the importance of these miRNAs in tumorigenesis, an in vitro antisense oligonucleotide knockdown model was evaluated for its ability to reverse the malignant phenotype. Both in human and woodchuck HCC cell lines, separate treatments with antisense oligonucleotides specific for either the miR-17–92 polycistron (all six members) or miR-21 caused a 50% reduction in both hepatocyte proliferation and anchorage-independent growth. The combination of assays presented here supports a role for these miRNAs in the maintenance of the malignant transformation of hepatocytes.     

Release of cardiac troponin I from viable cardiomyocytes is mediated by integrin stimulation

Elevated cardiac troponin-I (cTnI) levels have been demonstrated in serum of patients without acute coronary syndromes, potentially via a stretch-related process. We hypothesize that this cTnI release from viable cardiomyocytes is mediated by stimulation of stretch-responsive integrins. Cultured cardiomyocytes were treated with (1) Gly–Arg–Gly–Asp–Ser (GRGDS, n = 22) to stimulate integrins, (2) Ser–Asp–Gly–Arg–Gly (SDGRG, n = 8) that does not stimulate integrins, or (3) phosphate-buffered saline (control, n = 38). Cells and media were analyzed for intact cTnI, cTnI degradation products, and matrix metalloproteinase (MMP)-2. Cell viability was examined by assay of lactate dehydrogenase (LDH) activity and by nuclear staining with propidium iodide. GRGDS-induced integrin stimulation caused increased release of intact cTnI (9.6 ± 3.0%) as compared to SDGRG-treated cardiomyocytes (4.5 ± 0.8%, p < 0.001) and control (3.0 ± 3.4%, p < 0.001). LDH release from GRGDS-treated cardiomyocytes (15.9 ± 3.8%) equalled that from controls (15.2 ± 2.3%, p = n.s.), indicating that the GRGDS-induced release of cTnI is not due to cell necrosis. This result was confirmed by nuclear staining with propidium iodide. Integrin stimulation increased the intracellular and extracellular MMP2 activity as compared to controls (both p < 0.05). However, despite the ability of active MMP2 to degrade cTnI in vitro, integrin stimulation in cardiomyocytes was not associated with cTnI degradation. The present study demonstrates that intact cTnI can be released from viable cardiomyocytes by stimulation of stretch-responsive integrins.     

Fine structure of sporogonic stages of Goussia clupearum (Apicomplexa: Eimeriidae) in the liver of infected fish (Belone belone L.), using light and electron microscopy

A coccidian species, Goussia clupearum (L.) is reported to parasitize the liver of a new host, Belone belone (Teleostei: Belonidae), caught on the Atlantic coast at the north of Portugal. The parasitophorous vacuole containing oocysts was attached to the host's liver cells. Spherical oocysts (∼ 21.2 μm diameter), each containing four ellipsoidal elongated sporocysts (10.5 × 6.3 μm), were enclosed in the parasitophorous vacuole. Each sporocyst contained two sporozoites. The micropyle was absent, but a polar granule (without Stieda body) was present. Each sporozoite possessed four refractile bodies. During sporoblastogenesis and sporogenesis, one or two dense polar bodies were found within the oocysts. They were composed of a dense homogeneous core, surrounded by a ring of dense granular material. On occasion, we observed some sporocysts in direct contact with host cells. This paper describes the morphology and ultrastructural details of the oocysts, sporocysts and sporozoites of G. clupearum. This species seems to represent the only coccidium described in fish from this Atlantic coast. Received: 5 August 2000 / Accepted: 12 October 2000     

Gerbils (Meriones unguiculatus) are highly susceptible to oral infection with Neospora caninum oocysts

Laboratory-reared gerbils (Meriones unguiculatus) were found to be highly susceptible to oral infection with Neospora caninum (NC-Liv strain) oocysts. Gerbils fed ∼1000 oocysts became sick or died at 6–13 days post feeding of oocysts (PFO). N. caninum was isolated in cell culture and from γ-interferon-knockout mice inoculated with homogenates of mesenteric lymph nodes of gerbils examined as early as 1 day PFO. Numerous N. caninum tachyzoites were found in ulcerative lesions in the intestines of gerbils examined at 7–9 days PFO. In a gerbil fed 10 oocysts, N. caninum tachyzoites were found in lesions in the brain. Gerbils fed 10 oocysts developed antibodies to N. caninum by 18 days PFO as determined by the Neospora agglutination test (titers ≥1:500). All gerbils remained negative for antibodies to Toxoplasma gondii as determined by the Toxoplasma agglutination test. Received: 10 September 1999 / Accepted: 10 September 1999