A new avian Cryptosporidium genotype in a 1-month-old caged brown wood owl (Strix leptogrammica) with severe dehydration and diarrhea.

A 1-month-old brown wood owlet (Strix leptogrammica) purchased from a wholesaler and housed as a companion bird by an individual owner in Japan showed severe dehydration and anorexia following a week of vomiting and severe diarrhea. A great number of approximately 5 × 4-µm-sized Cryptosporidium oocysts were found in the feces by microscopy. The owlet was administered subcutaneous fluid and intragastric tube feeding for 2 weeks, resulting in improvement of the condition with a decreased number of oocysts in the feces. At days 51 and 119, no oocysts were found in the feces by microscope and PCR detection. These results suggested that this parasite was a possible agent of severe diarrhea in the affected bird. Molecular analysis of DNA extracted from oocysts based on the 18SrRNA loci identified C. avium; however, analysis of actin and hsp (heat shock protein) genes identified a novel genotype indicating a mixed infection with C. avium and a novel genotype.

Ascaridia nymphii n. sp. (Nematoda: Ascaridida) from the alimentary tract of a severely emaciated dead cockatiel Nymphicus hollandicus.

This report describes Ascaridia nymphii n. sp., a new species isolated from the alimentary tract of cockatiel Nymphicus hollandicus in Japan. More than 63 nematodes were found in the formalin-fixed small intestine, ventriculus, proventriculus and crop of a 48-day-old young cockatiel that died after exhibiting severe emaciation. No nematode eggs were observed in the faecal examination performed while the cockatiel was alive, but Cryptosporidium oocysts were found. The intestinal mucosa was damaged considerably. Male worms had two alate spicules, well-developed precloacal sucker and a tail with ventrolateral caudal alae and mainly 11 pairs of papillae. Nuclear partial (813 bp) 18S ribosomal RNA gene (18S rDNA) sequences obtained from two female samples were mutually identical. They respectively showed 99.1 and 98.6% identities to those from Ascaridia numidae and Ascaridia galli. Phylogenetic analysis using this locus indicated the present nematode as Ascaridia species. The mitochondrial NADH dehydrogenase subunit 2 gene (nad2) sequences obtained for four samples were mutually identical. They respectively showed 98.7, 85.7 and 82.2% identities with those from Ascaridia columbae, Ascaridia sp. and A. galli. Combining the morphological and sequencing data from two loci, the present nematode was identified as A. nymphii n. sp., which is closely related with A. columbae. This report is the first of a study examining the distribution of Ascaridia species in captive parrots in Japan. This study also identified the trachea and cloaca, like Cryptosporidium baileyi, as the possible location of Cryptosporidium avian genotype V in avian hosts.

Molecular characterization of Giardia psittaci by multilocus sequence analysis.

Multilocus sequence analyses targeting small subunit ribosomal DNA (SSU rDNA), elongation factor 1 alpha (ef1α), glutamate dehydrogenase (gdh), and beta giardin (ß-giardin) were performed on Giardia psittaci isolates from three Budgerigars (Melopsittacus undulates) and four Barred parakeets (Bolborhynchus lineola) kept in individual households or imported from overseas. Nucleotide differences and phylogenetic analyses at four loci indicate the distinction of G. psittaci from the other known Giardia species: Giardia muris, Giardia microti, Giardia ardeae, and Giardia duodenalis assemblages. Furthermore, G. psittaci was related more closely to G. duodenalis than to the other known Giardia species, except for G. microti. Conflicting signals regarded as "double peaks" were found at the same nucleotide positions of the ef1α in all isolates. However, the sequences of the other three loci, including gdh and ß-giardin, which are known to be highly variable, from all isolates were also mutually identical at every locus. They showed no double peaks. These results suggest that double peaks found in the ef1α sequences are caused not by mixed infection with genetically different G. psittaci isolates but by allelic sequence heterogeneity (ASH), which is observed in diplomonad lineages including G. duodenalis. No sequence difference was found in any G. psittaci isolates at the gdh and ß-giardin, suggesting that G. psittaci is indeed not more diverse genetically than other Giardia species. This report is the first to provide evidence related to the genetic characteristics of G. psittaci obtained using multilocus sequence analysis.

Cryptosporidium avian genotype III as a possible causative agent of chronic vomiting in peach-faced lovebirds (Agapornis roseicollis).

In the present study, Cryptosporidium oocysts were found, by light microscopy, in 37 fecal samples of peach-faced lovebirds (Agapornis roseicollis). Cryptosporidium avian genotype III was isolated in 13 of the 37 infected birds by sequence analysis of the small subunit ribosomal RNA and the actin genes. All of the birds showed chronic vomiting and weight loss with enlargement of isthmi, narrowed proventricular lumens, and thickened proventricular walls radiographically. Cryptosporidium parasites were found only in the ductal epithelium of the proventricular glands in three of the tissue samples provided for necropsy. To date, there have been no reports concerning the pathogenicity, nor the location, of avian genotype III in avian hosts. Our report confirms, for the first time, the presence of avian genotype III in peach-faced lovebirds in Japan and also reveals the location in the avian host.

Multilocus genotypic analysis of Cryptosporidium isolates from cockatiels, Japan.

Cryptosporidium is a significant pathogen in humans and animals. Cases of infection by Cryptosporidium parvum, Cryptosporidium meleagridis, and Cryptosporidium baileyi with zoonotic potential have also been reported in domestic birds, and recent studies indicate the presence of new host-adapted species or genotypes in birds. It is generally difficult to discriminate accurately among Cryptosporidium species and genotypes by light microscopy because of the morphological similarity of their oocysts. Although C. parvum and Cryptosporidium hominis are the primary Cryptosporidium species associated with infection in humans, recent studies have shown C. meleagridis to be a significant cause of cryptosporidiosis in both immunocompetent and immunocompromised individuals. Moreover, genetic variation among C. meleagridis isolates from humans and birds has been reported. Therefore, accurate identification of Cryptosporidium parasites using molecular methodologies is important to assess genetic diversity and to elucidate the transmission dynamics of Cryptosporidium parasites. In Japan, the cockatiel is a popular companion sold in many pet shops, but to the best of our knowledge only 11 Cryptosporidium isolates from cockatiels have been identified molecularly. In the present study, we identified five isolates from cockatiels by multilocus (18S ribosomal RNA, actin, Cryptosporidium oocyst wall protein, 70-kDa heat shock protein, and 60-kDa glycoprotein precursor) sequence and phylogenetic analyses. Analyses identified three new genotypes in C. meleagridis, avian genotype III, and a new avian genotype V.

Adrenomedullin-induced relaxation of rat brain pericytes is related to the reduced phosphorylation of myosin light chain through the cAMP/PKA signaling pathway.

Brain pericytes are known to embrace the abluminal endothelial surfaces of cerebral microvessels. The rich expression of contractile proteins in these cells suggests pericytal regulation of cerebral blood flow. Here, we investigated the molecular mechanisms by which an endothelium-derived relaxing factor, adrenomedullin, was able to induce the relaxation of rat primary cultured brain pericytes. Adrenomedullin increased the relative proportion of pericytes that were relaxed, as shown by an increased cell surface area. A smaller fragment of adrenomedullin (adrenomedullin(22-52)) blocked the adrenomedullin-induced relaxation. Adrenomedullin increased intracellular cAMP concentrations and decreased the phosphorylation of myosin light chain (MLC). H89 (a PKA inhibitor) inhibited the adrenomedullin-induced increase in the number of relaxed pericytes, and returned the level of phosphorylation of MLC to the control level. The results of the present study suggest that adrenomedullin-induced relaxation of brain pericytes is related to the reduced phosphorylation of MLC through cAMP/PKA.

Amnioreduction in patients with bulging prolapsed membranes out of the cervix and vaginal orifice in cervical cerclage.

OBJECTIVE: To determine whether an amnioreduction via bulging membranes (AVBM) and cerclage could be useful in 17 women with singleton gestations demonstrating hourglass membranes bulging out of the cervix or vaginal orifice. METHODS: We used the following selection criteria for AVBM under ultrasonographic guidance using a peit needle because of undetectable cervical edges: (type 1) the bag of membranes protruded beyond the inlet of the vagina; (type 2) the bag of huge membranes completely occupied the vagina. RESULTS: Eight patients (three cases of type 1 and five of type 2) were successful in AVBM and cerclage at 22.1 +/- 2.2 weeks gestation (range 19-24 weeks), and mean birth weight was 1,048.1 +/- 801.6 g (range 302-2,688 g). Although the diameter of the forewater by transabdominal ultrasonography (cm) was higher than in the nine patients without AVBM (6.7 +/- 1.1 versus 4.1 +/- 0.7 cm, p = 0.002), the prolongation of pregnancy (32.9 +/- 46.2 days; range 2-133 days) was the same as in patients without AVBM (36.9 +/- 39.3 day, p = 1.000). CONCLUSION: It is important that every effort should be made to perform cervical cerclage at or before 26 weeks of gestation, even in women with type 1 or 2.

Regulation of ghrelin secretion during pregnancy and lactation in the rat: possible involvement of hypothalamus.

We investigated the plasma concentration of ghrelin peptide during pregnancy and lactation in rats. Plasma ghrelin levels on days 10 and 15 of pregnancy were significantly lower than those of the non-pregnant rats. Thereafter, the plasma ghrelin levels on day 20 of pregnancy sharply increased to levels comparable with those in non-pregnant rats. Ghrelin peptide concentrations in the stomach did not change significantly during pregnancy. In the hypothalamus, ghrelin mRNA levels were significantly lower on day 15 of pregnancy than in the non-pregnant rats. Also, plasma ghrelin levels were significantly lower in lactating dams than non-lactating controls on days 3 and 8 of lactation. We examined the possible involvement of prolactin and oxytocin in the regulation of plasma ghrelin concentrations during lactation. Although plasma prolactin levels were decreased by the administration of bromocriptine, plasma ghrelin levels did not differ significantly between vehicle- and drug-treated lactating rats. Administration of haloperidol produced a marked increase in plasma prolactin levels as compared with the non-lactating controls. However, plasma ghrelin levels were not significantly different between vehicle- and drug-treated rats. Administration of an oxytocin antagonist into the lateral ventricle significantly inhibited the increase in the plasma oxytocin level induced by acute suckling. However, plasma ghrelin levels did not significantly between the groups. These observations indicated that the decrease in serum ghrelin is caused by a loss of the contribution of hypothalamic ghrelin. Furthermore, the present results suggested that the suckling stimulus itself, but the release of prolactin or oxytocin, is the factor most likely to be responsible for the suppression of ghrelin secretion during lactation.

An automated liquid handling system for polymerase chain reaction sample preparation.

An automated liquid handling system for dispensing liquid samples of sub-microliter volume has been developed. The system has eight nozzles composed of glass capillaries connected to syringe-style pumps. The distance between the nozzles can be changed from 4.5 to 9 mm, which corresponds to the distance between the wells in 96- and 384-well microplates, respectively. The eight nozzles of the system can aspirate and dispense liquid samples in both 96- and 384-well microplates. Sub-microliter volumes of reagents and samples were transferred between 96- and 384-well microplates using the system. This system was successfully used for PCR sample preparation.

Decreased mature adrenomedullin levels in feto-maternal tissues of pregnant women with histologic chorioamnionitis.

Adrenomedullin (AM) gains its bioactivity by amidation at its C-terminal, forming "mature AM." The mature AM and the expression of AM receptor component mRNAs, receptor activity-modifying protein 2 and calcitonin receptor-like receptor, from feto-maternal tissues of normal pregnant women and women with histologic chorioamnionitis were examined to clarify the pathophysiological features of this intrauterine infection. Samples of the placenta and fetal membranes were obtained from 10 normal pregnant women and eight women with histologic chorioamnionitis under informed consent. Mature AM in the fetal membranes was significantly lower in patients with chorioamnionitis than in normal pregnant women. On the other hand, there were no differences in mature AM levels in the placenta between the two groups. The total AM levels as a sum of mature and immature AM were not significantly different between the two groups in either area. The ratio of mature AM/total AM was significantly decreased in the fetal membranes of the patients with chorioamnionitis compared with normal pregnancies, but not in the placenta. Also, levels of mature AM were negatively correlated with C-reactive protein concentrations. The present results thus suggested that mature AM may have some role in chorioamnionitis.

[Adrenomedullin in pregnancy].

In this study, we used the animal model of preeclampsia. The blood pressure in animals receiving L-NAME at 25 mg/day were significantly higher compared to that of rats receiving saline solution only. In addition, L-NAME treated rats showed a high fetal mortality as compared with intact rats. Also, we demonstrated infusion of AM reverse the hypertension and decrease in pup mortality induced by L-NAME during pregnancy. We showed that the AM mRNA levels predominantly exists in a high level in the placenta, uterus and ovary as compared with other tissues. These evidences suggest that AM may have a possible important role during pregnancy. In conclusion, the present study suggest that L-NAME-induced elevated blood pressure and increased fetal mortality can be reversed by low dose of AM. Thus AM may play an important role in the regulation of blood pressure, the blood supply to the utero-placental unit and fetal development.

Alteration of plasma ghrelin levels associated with the blood pressure in pregnancy.

Ghrelin, an endogenous ligand for the growth hormone secretagogues, was originally isolated from rat stomach. It stimulates the release of growth hormone from primary pituitary cell cultures. We investigated the plasma concentration of ghrelin peptide in 16 nonpregnant women, 18 normal pregnant women, 20 patients with pregnancy-induced hypertension, and 10 postpartum women. The plasma concentration of ghrelin in nonpregnant women was 239.5+/-16.9 fmol/mL. The plasma concentration of ghrelin in normal pregnant women at the third trimester was 127.1+/-5.6 fmol/mL. There was negative correlation between plasma ghrelin concentration and systemic blood pressure in normal pregnant women (systolic: r=-0.564, P<0.05; diastolic: r=-0.610, P<0.01). Pregnant women with pregnancy-induced hypertension (177.9+/-14.6 fmol/mL, P<0.05) also had significantly higher levels of ghrelin compared with those of normal pregnant women. In addition, there was a significant correlation between plasma ghrelin levels and systemic blood pressure (systolic: r=-0.482, P<0.05; diastolic: r=-0.466, P<0.05). These results suggest for the first time that ghrelin might have some role in cardiovascular control during normal pregnancy and in pathophysiological conditions in pregnancy, such as pregnancy-induced hypertension.

Real-time detection of PCR products for comparative analysis of expressed genes using module-shuffling TaqMan probes (MTPs).

A method for comparative analysis of gene expression was developed. It is based on competitive PCR amplification and real-time detection of PCR products and it uses module-shuffling sequences as "universal TaqMan probes". Namely, cDNA-tagged module-shuffling sequences, which derived from different sources, were amplified in one reaction tube by the same primer set. Two kinds of fluorescent TaqMan probes with different module-shuffling sequences (MTPs) detect their own targets. The method can detect different amounts of expressed genes derived from different sources; accordingly, it was successfully used for comparative analysis of expressed mouse genes.