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PubMed2001

Cytokeratin subtypes in biliary atresia: immunohistochemical study.

The etiology of biliary atresia (BA) remains unknown, but ductal-plate malformation and insufficient ductal-plate remodeling have been suggested to play important roles, so it is beneficial to examine the maturation and differentiation of bile ducts in BA. Different epithelial types are characterized by the expression of specific cytokeratin (CK) subtypes. CK can therefore serve as a 'lineage marker' of epithelial cells. CK subtypes have not been previously examined in BA. In this study, we examined the maturation of bile-duct cells in BA (n = 45) using immunohistochemistry of CK subtypes, with mouse monoclonal antibodies to CAM5.2, and CK subtypes 7, 8, 13, 14, 17, 19 and 20. We then compared these findings with pediatric non-BA (n = 11) and fetal (n = 21) liver. We semiquantitatively evaluated the findings using a H score method. In the fetal liver, immunoreactivity for CAM5.2, CK-7, CK-8 and CK-19 was detected in bile-duct cells, and CAM5.2 and CK-8 immunoreactivity was also detected in hepatocytes. The distribution of these CK subtypes was the same in fetal, pediatric non-BA and BA liver. However, CK-7 immunoreactivity was markedly weaker in bile ducts of fetal (H scores: ductal plate 0 +/- 0; remodeling 9.5 +/- 40.3; remodeled 37.3 +/- 60.8) and BA (H score: 200.9 +/- 55.3) liver compared to non-BA liver (H score: 251.1 +/- 33.5). In addition, CK-20 was detected in the bile ducts of the fetal and BA liver, but not in non-BA liver. These findings suggest that the expression patterns of CK subtypes in bile-duct cells in BA are similar to that in developing bile-duct cells, which is indicative of bile-duct cell immaturity.

Bile Ducts, Extrahepatic↗
PubMed2002

Molecular phenotype of rat striatal neurons expressing the dopamine D5 receptor subtype.

Dopamine is one of the principal neurotransmitters in the basal ganglia, where it plays a critical role in motor control and cognitive function through its interactions with the specific dopamine receptors D1 to D5. Although the activities mediated by most dopamine receptor subtypes have already been determined, the role of the D5 receptor subtype in the basal ganglia has still not been established. Furthermore, it is often difficult to distinguish between dopamine D5 and D1 receptors as they are stimulated by the same ligands, and they have a similar molecular structure and pharmacology. In an effort to understand the differences between these two receptor subtypes, we have studied the distribution of neurons containing D5 receptors in the striatum, and their molecular phenotype. As a result, we show that the D5 receptor subtype is present in two different populations of striatal neurons, projection neurons and interneurons. Overall, the abundance of this receptor subtype in the striatum is low, particularly in striatal projection neurons of both the direct and indirect projection pathways. In contrast, the expression of D5 receptors in striatal interneurons (cholinergic, somatostatin- or parvalbumin-positive neurons) is high, while low to moderate expression was observed in calretinin-positive neurons. Our results demonstrate the presence of D5 receptors in all the striatal cell populations so far described, although at different intensities in each. The fact that a large number of striatal neurons express the D5 receptor subtype suggests that this receptor fulfils an important function in the process of integrating information in the striatum.

Animals↗
PubMed1999

Hepatitis B virus: genotypes and subtypes in Brazilian hemodialysis patients.

There are no data concerning the genotypic analysis of hepatitis B virus (HBV) infection in Brazilian dialysis centers. Serum samples from all hemodialysis patients (n = 282) in Goiânia City, Central Brazil, were tested for hepatitis B surface antigen (HBsAg). An overall prevalence of 12.0% was found, ranging from 0 to 33.3% depending on dialysis centers. Positive samples (n = 34) were submitted to serological subtyping by monoclonal ELISA and HBV DNA detection by polymerase chain reaction (PCR). Among 30 PCR-positive samples, 26 were genotyped by use of the line probe assay technology (INNO-LiPA HBV, Innogenetics, Gent, Belgium). HBV genotypes A (50. 0%) and D (46.2%) were the most frequently found whereas genotype F (3.8%) was rare in this population. Serological subtypes adw2 (44. 1%) and ayw3 (41.2%) were dominant. By contrast, adw4 and ayw2 were found at a low frequency (2.9%). A correlation was observed in the distribution of genotypes and subtypes by dialysis center. Genotype D and subtype ayw3 were predominant in 2 hemodialysis centers whereas genotype A and subtype adw2 were predominant in the others. The findings of high HBsAg prevalence rates restricted to certain dialysis centers and the data obtained through genotyping and serological subtyping suggest HBV nosocomial transmission in these hemodialysis centers.

Adolescent↗
PubMed2001

Distinguishing subtypes of temporal lobe epilepsy with background hippocampal activity.

PURPOSE: Two subtypes of temporal lobe epilepsy (TLE) can be defined through clinical observations and analysis of hippocampal tissue resected during surgical procedures for intractable TLE: (a) mesial temporal sclerosis (MTS), which is characterized by extensive changes to the hippocampus and good surgical outcome; and (b) paradoxical temporal lobe epilepsy (PTLE), which is characterized by minimal cell loss and comparatively poorer surgical outcome. Patients in both subtypes have seizures that appear to begin in the medial temporal lobe, but documented differences in substrate and outcome between these subtypes has defined a need to distinguish MTS and PTLE patients before surgery. This report describes a retrospective study to investigate the feasibility of doing so during intracranial monitoring. METHODS: Background EEG epochs, 5 min in duration, were recorded from the anterior hippocampus in 14 (10 MTS and four PTLE) patients with consistent localization of seizure onset to medial temporal structures. The power spectral density (PSD) of the EEG epochs was calculated by a Fourier spectral estimator, and the total signal power and power of the delta, theta, alpha, beta, and gamma frequency bands were submitted to group-to-group comparison. RESULTS: Spectral peaks were observed in the delta band in all PSD estimates and in the theta band in nine of 14 (seven MTS, two PTLE) estimates. The MTS and PTLE subtypes could be distinguished by the total signal power and delta band power. These power measurements were greater in the PTLE subtype. CONCLUSIONS: Both delta and theta spectral components are present in hippocampal background EEGs recorded from patients with TLE. The results indicate that group differences exist in spectral measures of background hippocampal signals recorded from MTS and PTLE subtypes. This suggests both that substrate differences in cellular composition and connectivity are reflected in hippocampal background EEGs and that spectral measurements of these signals may hold promise for tests to identify the group membership of individual patients.

Adult↗
PubMed2003

Influence of etofibrate on LDL-subtype distribution in patients with diabetic dyslipoproteinemia.

Low density lipoprotein (LDL-) particles can be subfractionated in large-buoyant (lb), intermediate-dense (id) and small-dense (sd) LDL-subtypes. Fibrates improve the LDL-subtype profile by reducing proatherogenic sd-LDL which are prominent in diabetic dyslipoproteinemia. We evaluated the effect of etofibrate on the LDL-subtype distribution in patients with type 2 diabetes mellitus (n = 13, 55 +/- 18 years, BMI 27.9 +/- 5.5 kg/m2, HbA1c 10.1 +/- 3.9 %) and diabetic dyslipoproteinemia (triglycerides 343 +/- 253 mg/dl, HDL-cholesterol 36 +/- 7 mg/dl, LDL-cholesterol 110 +/- 37 mg/dl). Plasma lipids (enzymatic methods) and LDL-subtypes (7 LDL-subfractions, density gradient ultracentrifugation) were measured before and during etofibrate therapy (500 mg/d, 7 - 16 weeks). Etofibrate significantly (p < 0.05, Wilcoxon-test) reduced triglycerides (- 31 +/- 60 %) and increased HDL-cholesterol (+ 24 +/- 22 %), whereas total cholesterol and LDL-cholesterol did not change. Cholesterol concentration decreased in sd-LDL by 12 % (p < 0.05), while it increased in id- and lb-LDL (+ 26 %,+ 39 %, respectively). Thus, the LDL-subtype profile showed a relative increase of the fraction of lb- (+ 13 +/- 32 %, n.s.) and id-LDL (+ 23 +/- 33 %, p < 0.05) and a relative decrease of the fraction of sd-LDL (- 19 +/- 18 %, p < 0.05). We conclude that etofibrate not only decreases triglycerides and increases HDL-cholesterol but also improves the LDL-subtype profile and thus may reduce the cardiovascular risk in patients with an abundance of sd-LDL such as diabetic patients.

Adult↗
PubMed2001

Use of molecular subtyping in surveillance for Salmonella enterica serotype typhimurium.

BACKGROUND: Because Salmonella enterica serotype typhimurium is the most common serotype isolated from persons with salmonellosis in the United States, it is difficult to detect unusual clusters or outbreaks. To determine whether molecular subtyping could be useful in public health surveillance for S. enterica serotype typhimurium, the Minnesota Department of Health initiated the routine use of pulsed-field gel electrophoresis (PFGE) of isolates. METHODS: Beginning in 1994, all S. enterica serotype typhimurium isolates submitted by clinical laboratories to the Department of Health were subtyped by PFGE. A standard questionnaire was used to interview patients about possible sources of infection. RESULTS: From 1994 through 1998, 998 cases of infection with S. enterica serotype typhimurium were reported to the Minnesota Department of Health (4.4 cases per 100,000 person-years). PFGE was performed on 958 of the isolates (96 percent), and 174 different patterns were identified. Sixteen outbreaks with a common source were identified, accounting for 154 cases. PFGE subtyping made it possible to confirm 10 outbreaks that involved small numbers of cases in institutional settings. Of six larger, community-based outbreaks, four would probably not have been recognized without PFGE subtyping. These four outbreaks accounted for 96 of the 154 culture-confirmed outbreak cases (62 percent). Fifty-six of 209 isolates tested for antimicrobial susceptibility (27 percent) were resistant to at least five antimicrobial agents. The multidrug-resistant isolates identified had unique PFGE patterns. CONCLUSIONS: Routine molecular subtyping of S. enterica serotype typhimurium by PFGE can improve the detection of outbreaks and aid in the identification of multidrug-resistant strains. Combining routine molecular subtyping with a method of rapid communication among public health authorities can improve surveillance for S. enterica serotype typhimurium infections.

DNA, Bacterial↗
PubMed2005

Different GABAA receptor subtypes mediate the anxiolytic, abuse-related, and motor effects of benzodiazepine-like drugs in primates.

Benzodiazepines exert their effects by binding to multiple subtypes of the GABAA receptor, the predominant subtypes in the brain being those that contain alpha1-, alpha2-, alpha3-, and alpha5-subunits. To understand the potentially different roles of these subtypes in the therapeutic and side effects of benzodiazepines, we evaluated GABAA receptor subtype-preferring compounds in nonhuman primate models predictive of anxiolytic, sedative, motor, subjective, and reinforcing effects of benzodiazepine-type drugs. These compounds included zolpidem, which shows preferential binding to GABAA receptors containing alpha1-subunits (alpha1GABAA receptors); L-838,417, which shows functional selectivity for alpha2GABAA, alpha3GABAA, and alpha5GABAA receptors; and nonselective conventional benzodiazepines. The results provide evidence in nonhuman primates that alpha1GABAA receptors do not play a key role in the anxiolytic and muscle-relaxant properties of benzodiazepine-type drugs; instead, these effects involve alpha2GABAA, alpha3GABAA, and/or alpha5GABAA subtypes. Our results also suggest that the alpha1GABAA receptor subtype might be critically involved in the subjective, sedative, and motor effects of benzodiazepine-type drugs. In contrast, stimulation of alpha1GABAA receptors is sufficient, but not necessary, for mediation of the abuse potential of these drugs.

Animals↗
PubMed1993

Structural basis for receptor subtype-specific regulation revealed by a chimeric beta 3/beta 2-adrenergic receptor.

The physiological significance of multiple G-protein-coupled receptor subtypes, such as the beta-adrenergic receptors (beta ARs), remains obscure, since in many cases several subtypes activate the same effector and utilize the same physiological agonists. We inspected the deduced amino acid sequences of the beta AR subtypes for variations in the determinants for agonist regulation as a potential basis for subtype differentiation. Whereas the beta 2AR has a C terminus containing 11 serine and threonine residues representing potential sites for beta AR kinase phosphorylation, which mediates rapid agonist-promoted desensitization, only 3 serines are present in the comparable region of the beta 3AR, and they are in a nonfavorable context. The beta 3AR also lacks sequence homology in regions which are important for agonist-mediated sequestration and down-regulation of the beta 2AR, although such determinants are less well defined. We therefore tested the idea that the agonist-induced regulatory properties of the two receptors might differ by expressing both subtypes in CHW cells and exposing them to the agonist isoproterenol. The beta 3AR did not display short-term agonist-promoted functional desensitization or sequestration, or long-term down-regulation. To assign a structural basis for these subtype-specific differences in agonist regulation, we constructed a chimeric beta 3/beta 2AR which comprised the beta 3AR up to proline-365 of the cytoplasmic tail and the C terminus of the beta 2AR. When cells expressing this chimeric beta 3/beta 2AR were exposed to isoproterenol, functional desensitization was observed. Whole-cell phosphorylation studies showed that the beta 2AR displayed agonist-dependent phosphorylation, but no such phosphorylation could be demonstrated with the beta 3AR, even when beta AR kinase was overexpressed. In contrast, the chimeric beta 3/beta 2AR did display agonist-dependent phosphorylation, consistent with its functional desensitization. In addition to conferring functional desensitization and phosphorylation to the beta 3AR, the C-terminal tail of the beta 2AR also conferred agonist-promoted sequestration and long-term receptor down-regulation.

Adenylyl Cyclases↗
PubMed1994

Classification of hepatitis C viruses based on phylogenetic analysis of the envelope 1 and nonstructural 5B regions and identification of five additional subtypes.

Genotyping of hepatitis C virus-positive sera by means of a line probe assay indicated that < 3% of European samples, but up to 30% of Gabonese sera, could not be classified as either 1a, 1b, 2a, 2b, 3a, 3b, 4c, 5a, or 6a. Such samples were analyzed in the 5' untranslated region and in the nonstructural 5 (NS5) region. Classification based on phylogenetic analysis of the commonly used 222-bp-long NS5B region was possible for most but not all of the selected sera. Therefore, the core/envelope 1 region (579 bp) and a larger NS5B (340 bp) region were also analyzed. Only the phylogenetic analysis of the 340-bp NS5B region of these newly identified and published isolates provided unambiguous classification into types and subtypes. Furthermore, unequivocal evidence for four subtypes in type 2 and eight subtypes in type 4 was provided. A specific recognition sequence in the 5' untranslated region was observed for every newly identified subtype. Based on 1830 pair-wise comparisons in NS5B, isolates belonging to the same subtype showed evolutionary distances of < 0.127 and isolates of the same type exhibited evolutionary distances of < 0.328. These phylogenetic border distances can be conveniently used for classification of hepatitis C virus isolates into types and subtypes.

Amino Acid Sequence↗
PubMed1995

Identification of critical determinants of alpha 1-adrenergic receptor subtype selective agonist binding.

alpha 1-Adrenergic receptor (AR) subtypes mediate many effects of the sympathetic nervous system. The three cloned subtypes (alpha 1a-AR, alpha 1b-AR, alpha 1d-AR), although structurally similar, bind a series of ligands with different relative potencies. This is particularly true for the alpha 1a-AR, which recognizes a number of agonists and antagonists with 5-50-fold higher affinity than the alpha 1b- or alpha 1d- subtypes. Since ligands bind to receptor-residues that are located in the transmembrane spanning domains, we hypothesize that subtype differences in ligand recognition are due to differences in the binding properties of nonconserved transmembrane residues. Using site-directed mutagenesis, selected putative ligand-binding residues in the alpha 1b-AR were converted, either individually or in combination, to the corresponding residues in the alpha 1a-AR. Mutation of two such residues (of approximately 172 amino acids in the transmembrane domains) converted the agonist binding profile entirely to that of the alpha 1a-AR. Over 80% of this conversion was due to an Ala204-->Val substitution; the remainder was due to the additional substitution of Leu314-->Met. To confirm that Ala204 and Leu314 are indeed critical for agonist subtype-selectivity, the equivalent residues in the alpha 1a-AR (Val185 and Met293) were reversed of that of the alpha 1b-AR. Correspondingly, the agonist-binding profile of this double alpha 1a-AR mutant reverted to that of the alpha 1b-AR. From these data, in conjunction with macromolecular modeling of the ligand-binding pocket, a model has been developed, which indicates that the determinants of these two residues for agonist subtype-selectivity are due not only to interactions between their side chains and specific ligand moieties but also to a critical interaction between these two amino acids.

Adrenergic alpha-Agonists↗
PubMed1996

Chimeras of alpha1-adrenergic receptor subtypes identify critical residues that modulate active state isomerization.

We have identified previously two amino acids, one in each of the fifth and sixth transmembrane segments of both the alpha1a-adrenergic receptor and the alpha1b-adrenergic receptor (AR), that account almost entirely for the selectivity of agonist binding by these receptor subtypes (Hwa, J., Graham, R. M., and Perez, D. M. (1995) J. Biol. Chem. 270, 23189-23195). Thus reversal of these two residues, from those found in the native receptor of one subtype to those in the other subtype, produces complementary changes in subtype selectivity of agonist binding. Here we show that mutating only one of these residues in either the alpha1b-AR or the alpha1a-AR to the corresponding residue in the other subtype (Ala204 --> Val for the alpha1b; Met292 --> Leu for the alpha1a-AR) results in chimeras that are constitutively active for signaling by both the phospholipase C and phospholipase A2 pathways. This is evident by an increased affinity for agonists, increased basal phospholipase C and phospholipase A2 activation, and increased agonist potency. Although mutation of the other residue involved in agonist binding selectivity, to the corresponding residue in the other subtype (Leu314 --> Met for the alpha1b-AR; Val185 --> Ala for the alpha1a-AR) does not alter receptor binding or signaling, per se, when combined with the corresponding constitutively activating mutations, the resulting chimeras, Ala204 --> Val/Leu314 --> Met ( alpha1b-AR) and Val185 --> Ala/Met292 --> Leu ( alpha1a-AR), display wild type ligand binding and signaling. A simple interpretation of these results is that the alpha1a- and alpha1b-ARs possess residues that critically modulate isomerization from the basal state, R, to the active state R*, and that the native receptor structures have evolved to select residues that repress active state isomerization. It is likely that the residues identified here modulate important interhelical interactions between the fifth and sixth transmembrane segments that inhibit or promote receptor signaling.

Animals↗
PubMed2000

Requirement of ryanodine receptor subtypes 1 and 2 for Ca(2+)-induced Ca(2+) release in vascular myocytes.

While the roles of subtypes 1 and 2 of the ryanodine receptors (RYRs) have been studied in cellular systems expressing specifically one or the other of these subtypes (i.e. skeletal and cardiac muscle), the function of these receptors has not been evaluated in smooth muscles. We have previously reported RYR-mediated elementary (Ca(2+) sparks) and global Ca(2+) responses in rat portal vein myocytes. Here, we investigated the respective roles of all three RYR subtypes expressed in these cells as revealed by reverse transcriptase-polymerase chain reaction. Antisense oligonucleotides targeting each one of the three RYR subtypes were shown to specifically inhibit the expression of the corresponding mRNA and protein without affecting the other RYR subtypes. Confocal Ca(2+) measurements revealed that depolarization-induced Ca(2+) sparks and global Ca(2+) responses were blocked when either RYR1 or RYR2 expression was suppressed. Caffeine-induced Ca(2+) responses were partly inhibited by the same antisense oligonucleotides. Neither the corresponding scrambled oligonucleotides nor the antisense oligonucleotides targeting RYR3 affected depolarization- or caffeine-induced Ca(2+) responses. Our results show that, in vascular myocytes, the two RYR1 and RYR2 subtypes are required for Ca(2+) release during Ca(2+) sparks and global Ca(2+) responses, evoked by activation of voltage-gated Ca(2+) channels.

Animals↗
PubMed2000

Unique protein determinants of the subtype-selective ligand responses of the estrogen receptors (ERalpha and ERbeta ) at AP-1 sites.

The two subtypes of human estrogen receptor, alpha (hERalpha) and beta (hERbeta), regulate transcription at an AP-1 response element differently in response to estradiol and the anti-estrogens tamoxifen and raloxifene. To better understand the protein determinants of these differences, chimeric and deletional mutants of the N-terminal domain and the F region of ERalpha and ERbeta were made and tested in transient transfection assays at the classical estrogen response element (ERE) site as well as at an AP-1 site. Although the same regions on each receptor subtype appeared to be primarily responsible for estradiol activation at an ERE and in HeLa cells, major differences between ERalpha and ERbeta mutants were seen in the estrogen and anti-estrogen responses at an AP-1 site. This differential ligand response maps to the N-terminal domain and the F region. These results suggest that different estrogenic and anti-estrogenic ligands use different mechanisms of activation and inhibition at the AP-1 site. In contrast to previous studies, this work also shows that many of subtype-specific responses are not transferred to the other subtype by swapping the N-terminal domain of the receptor. This implies that there are other unique surfaces presented by each subtype outside of the N-terminal domain, and these surfaces can play a role in subtype-selective signaling. Together, these data suggest a complex interface between ligand, response element, and receptor that underlies ligand activation in estrogen signaling pathways.

Base Sequence↗
PubMed2002

alpha 1-Adrenergic receptor subtypes differentially control the cell cycle of transfected CHO cells through a cAMP-dependent mechanism involving p27Kip1.

Three distinct subtypes of alpha(1)-adrenergic receptors (alpha(1)A-, alpha(1)B-, and alpha(1)D-AR) play a prominent role in cell growth. However, little is known about subtype-specific effects on cell proliferation. The activation of alpha(1)A- or alpha(1)B-AR inhibits serum-promoted cell proliferation, whereas alpha(1)D-AR activation does not show such an inhibitory effect. Notably, cell-cycle progression was blocked at G(1)/S transition after activation of alpha(1)A/alpha(1)B-AR but not of alpha(1)D-AR. In agreement with the differential cell proliferation effect, cAMP production was increased after activation of alpha(1)A/alpha(1)B-AR but not alpha(1)D-AR, whereas all alpha(1)-AR subtypes are associated with inositol 1,4,5-trisphosphate production and mitogen-activated protein kinase activation in a similar fashion. Furthermore, the serum-induced reduction in the levels of the cyclin-dependent kinase inhibitor, p27(Kip1), was blocked after activation of alpha(1)A/alpha(1)B-AR but not alpha(1)D-AR. These results show that alpha(1)-AR subtypes differentially activate the cAMP/p27(Kip1) pathway and thereby have differential inhibitory effects on cell proliferation. Subtype-dependent effects should be taken into consideration when assessing the physiological response of native cells where alpha(1)-AR subtypes are generally co-expressed.

Animals↗
PubMed2003

Subtypes of emotional and behavioural sequelae in patients with traumatic brain injury.

This study examined patterns of emotional and behavioural sequelae in 300 individuals who sustained a traumatic brain injury (TBI). Participants were obtained through the Adult Acquired Brain Injury Program at Chedoke Hospital in Hamilton, Ontario, based on the following inclusionary criteria: (1) single incident of TBI; (2) no history of additional neurological diseases; (3) time postinjury < or =8.5 years; (4) WAIS-R FSIQ >85 and/or estimated reading skills above grade 5 level; and (5) valid Minnesota Multiphasic Personality Inventory (MMPI) profiles (i.e., F<90, L<66, and K<66). MMPI profiles of these individuals, in randomly split samples of 150 per group, were subjected to a three-step cluster analytic approach. A six-cluster solution was adequately replicated across samples and across clustering techniques. The identified subtypes included profiles indicative of: (1) no concerns or normal functioning; (2) mild somatic and pain concerns; (3) mild internalizing difficulties; (4) marked disinhibition and externalizing behavioural difficulties; (5) marked internalizing difficulties; and (6) marked somatic, internalizing, and externalizing behavioural disturbances. Members of the Externalized subtype were significantly younger in age than those in the other five subtypes, and more likely to be single than those in the Internalized subtype. Individuals in the Internalized subtype tended to be married, have longer times postaccident, and lower WAIS-R Verbal Intelligence Quotients than those comprising the Normal subtype.

Adolescent↗
PubMed2006

Use of a polymerase chain reaction to subtype Mycobacterium avium subspecies paratuberculosis, an increasingly important pathogen from farmed deer in New Zealand.

AIMS: To review the number of microbiologically-confirmed cases of Johne's disease in farmed deer since 2000, and determine the prevalence of the bovine and ovine subtypes of Mycobacterium avium subsp paratuberculosis (M. paratuberculosis), using a highly specific polymerase chain reaction (PCR) test on samples from infected herds. METHODS: The number of cases of M. paratuberculosis in farmed deer identified by culture or IS900 PCR was documented. A highly specific PCR test was applied to subtype M. paratuberculosis from BACTEC 12B cultures selected on the basis of one culture per deer herd, to give a wide coverage of herds in New Zealand. RESULTS: From January 2001 to October 2005, M. paratuberculosis was isolated from 1,141 farmed deer, and has now been identified by microbiological testing in over 600 deer herds in New Zealand. The bovine subtype of M. paratuberculosis was shown by a highly specific PCR test to be present in 91/95 herds examined; the ovine subtype was found in the remaining four herds. CONCLUSIONS: Since 2000, there has been a substantial increase in both the number of microbiologically-confirmed cases of Johne's disease in farmed deer and the number of infected herds. Johne's disease is now widespread and common in deer herds throughout New Zealand. Whilst the bovine subtype of M. paratuberculosis predominates in deer herds in New Zealand in which Johne's disease has been confirmed, the occasional finding of the ovine subtype highlights the need to consider both sheep and cattle as potential sources of infection for farmed deer.

Animals↗
PubMed2004

Characterization of recent H5 subtype avian influenza viruses from US poultry.

In the US, the isolation of H5 subtype avian influenza (AI) viruses has been uncommon in commercial chickens and turkeys, although sporadic isolations have been made from the live bird markets or its supply chain since 1986. In 2002, two different outbreaks of H5 AI occurred in commercial chicken or turkey operations. The first occurred in Texas and was identified as a H5N3 subtype AI virus. The second outbreak was caused by a H5N2 virus isolated from a turkey farm in California. In this study we analyzed recent H5 subtype AI viruses from different avian species and different sources in the US. Most recent H5 subtype isolates shared a high sequence identity and phylogenetically assorted into a separate clade from the Pennsylvania/83 lineage isolates. However, no established lineage was found within this clade and the recent H5 subtype isolates seemed to be the result of separate introductions from the wild bird reservoir. The Texas H5N3 isolate shared the lowest homology with the other recent isolates in the haemagglutinin gene and had a unique haemagglutinin cleavage site sequence of REKR/G (other recent isolates have the typical avirulent motif, RETR/G). Furthermore, this isolate had a 28 amino acid deletion in the stalk region of the neuraminidase protein, a common characteristic of chicken adapted influenza viruses, and may indicate that this virus had actually been circulating in poultry for an extended period of time before it was isolated. In agreement with genetic evidence, the Texas H5N3 isolate replicated better than other H5 isolates in experimentally infected chickens. The outbreak in Texas with a more chicken-adapted H5N3 virus underscores the importance of ongoing surveillance and control efforts regarding the H5 subtype AI virus in the US.

Amino Acid Sequence↗
PubMed2005

Subtyping of new Brazilian avian metapneumovirus isolates from chickens and turkeys by reverse transcriptase-nested-polymerase chain reaction.

The aim of this study was to improve a reverse transcriptase (RT)-nested-polymerase chain reaction (PCR) able to differentiate avian pneumovirus (APV) subtypes A and B, and to characterize new Brazilian isolates. Representative APV strains and clinical field samples from chickens and turkey flocks were amplified in the chicken embryo-related cell line. Viral RNA was extracted from harvested cells, and submitted to cDNA synthesis. The primers utilized for RT-PCR were compatible with the G gene of both the A and B subtypes of APV, while the nested primers were subtype specific. This approach showed that three new APVs from chickens and one from turkeys were subtype A, confirmed by sequencing. This is the first report of APV isolation from turkeys in Brazil. Four other APVs were detected and classified as subtype A by RT-nested-PCR. These optimized techniques could be useful for differentiation of APV subtypes A and B, proving to be a valuable molecular epidemiological tool.

Animals↗