Child-caregiver interaction in two remote Indigenous Australian communities.

This paper reports on a study in two remote multilingual Indigenous Australian communities: Yakanarra in the Kimberley region of Western Australia and Tennant Creek in the Barkly region of the Northern Territory. In both communities, processes of language shift are underway from a traditional language (Walmajarri and Warumungu, respectively) to a local creole variety (Fitzroy Valley Kriol and Wumpurrarni English, respectively). The study focuses on language input from primary caregivers to a group of preschool children, and on the children's productive language. The study further highlights child-caregiver interactions as a site of importance in understanding the broader processes of language shift. We use longitudinal data from two time-points, approximately 2 years apart, to explore changes in adult input over time and developmental patterns in the children's speech. At both time points, the local creole varieties are the preferred codes of communication for the dyads in this study, although there is some use of the traditional language in both communities. Results show that for measures of turn length (MLT), there are notable differences between the two communities for both the focus children and their caregivers. In Tennant Creek, children and caregivers use longer turns at Time 2, while in Yakanarra the picture is more variable. The two communities also show differing trends in terms of conversational load (MLT ratio). For measures of morphosyntactic complexity (MLU), children and caregivers in Tennant Creek use more complex utterances at Time 2, while caregivers in Yakanarra show less complexity in their language at that time point. The study's findings contribute to providing a more detailed picture of the multilingual practices at Yakanarra and Tennant Creek, with implications for understanding broader processes of language shift. They also elucidate how children's language and linguistic input varies diachronically across time. As such, we contribute to understandings of normative language development for non-Western, non middle-class children in multilingual contexts.

Production and proteomic characterisation of purified protein derivative from Mycobacterium avium subsp. paratuberculosis.

BACKGROUND: Effective diagnosis of Johne's disease (JD), particularly at the stage of early subclinical infection, remains one of the greatest challenges for the control of JD worldwide. The IFN-γ test of cell mediated immunity is currently one of the most suitable diagnostics for subclinical infections, however a major limitation of this test is the lack of a standardised purified protein derivative (PPD) antigen (also referred to as Johnin PPD or PPDj). While attempting to replace PPDj with more specific individual antigens is an attractive proposition, bacterial culture derived PPDj remains the most effective antigen preparation for the diagnosis of subclinical JD. It may be possible to increase the reproducibility and specificity of PPDj preparations by further characterising and standardising the PPDj production. RESULTS: Using a standardised protocol, five in-house preparations of PPDj were prepared from cultures of Mycobacterium avium subsp. paratuberculosis (MAP). Compared to PPDs obtained from other institutes/laboratories, these preparations appeared to perform similarly well in the IFN-γ test. Although the broad proteomic composition of all PPDj preparations was remarkably similar, the absolute abundance of individual proteins varied markedly between preparations. All PPDj preparations contained common immunogenic proteins which were also observed in PPD preparations from Mycobacterium avium subsp. avium (PPDa) and Mycobacterium bovis (PPDb). Temporal difference in protein secretion of in vitro cultured MAP was observed between 20 and 34 weeks suggesting that the age of MAP culture used for PPDj preparations may markedly influence PPDj composition. CONCLUSIONS: This study describes a protocol for the production of PPDj and its subsequent proteomic characterisation. The broad proteomic composition of different preparations of PPDj was, for the most part, highly similar. Compositional differences between PPDj preparations were found to be a direct reflection of genetic differences between the MAP strain types used to produce these preparations and the age of MAP cultures they were derived from. A number of conserved immunogenic proteins, such as members of the cutinase-like protein family, were found to be more abundant in PPDj compared to PPDa and should be considered as possible diagnostic antigens for the future.

Mycobacterium avium subspecies paratuberculosis in children with early-onset Crohn's disease.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) is the most enduring infectious candidate that may be associated with inflammatory bowel disease (IBD). It is possible that the inconsistencies in the prevalence studies of MAP in adults reflect clinical differences in adult patients studied, including duration of disease and treatment regimens, and also in lack of specificity of some of the assays used. The aim was to determine the presence of MAP in children with symptoms of Crohn's disease (CD) and ulcerative colitis (UC), using gut biopsy tissue and peripheral blood mononuclear cells (PBMC) collected at initial endoscopic examination prior to clinical treatment. METHODS: Mucosal biopsies and/or PBMC specimens were collected from a total of 142 children, comprising 62 with CD, 26 with UC, and 54 with non-IBD. MAP-specific IS900 polymerase chain reaction (PCR) analysis was performed on all biopsies and PBMC specimens. Conventional MAP culture technique was performed on a subset of 10 CD, 2 UC, and 4 non-IBD patients to isolate MAP. RESULTS: MAP was identified by IS900 PCR significantly more often in mucosal biopsies from CD 39% (22/56) than from non-IBD 15% (6/39) patients (P < 0.05), and in PBMC from CD 16% (8/50) than from non-IBD 0% (0/31) patients (P < 0.05). Viable MAP were cultured from mucosal biopsies from 4/10 CD, 0/2 UC, and 0/4 non-IBD patients, but were not cultured from PBMC specimens. CONCLUSIONS: This unique study on the occurrence of MAP in gut tissue and blood from pediatric IBD patients suggests the possible involvement of MAP in the early stages of development of CD in children.

The recovery of Mycobacterium avium subspecies paratuberculosis from the intestine of infected ruminants for proteomic evaluation.

Johne's disease is a slowly developing intestinal disease, primarily of ruminants, caused by Mycobacterium avium subspecies paratuberculosis. The disease contributes to significant economic losses worldwide in agricultural industry. Analysis of bacterial proteomes isolated directly from infected animals can provide important information about the repertoire of proteins present during infection and disease progression. In this study, M. avium subspecies paratuberculosis has been extracted from Johne's disease-infected cattle and goat intestinal tissue sections in a manner compatible with direct 2-DE proteomic analysis for comparison with in vitro-cultured bacteria. M. avium subspecies paratuberculosis was harvested from the submucosa and mucosa of intestinal sections and enriched from macerated tissue by hypotonic lysis, sonication and centrifugation through a viscosity gradient. Subsequent comparison of the proteomes of the in vivo- and in vitro-derived bacteria identified a number of proteins that were differentially expressed. Among them, a number of hypothetical proteins of unknown function and a hypothetical fatty acyl dehydrogenase (FadE3_2) and 3-hydroxyacyl-CoA dehydrogenase, possibly important for in vivo metabolism, utilising the pathway for the beta-oxidation of fatty acids.

Recovery of Mycobacterium avium subspecies paratuberculosis from the natural host for the extraction and analysis in vivo-derived RNA.

RNA has been extracted and analysed from in vivo-derived Mycobacterium avium subspecies paratuberculosis recovered from the natural host. The bacteria were selectively extracted from the intestinal tissue of two goats exhibiting clinical signs of Johne's disease. Small intestine was rapidly removed, luminal contents washed away and the mucosa and submucosa harvested. Mycobacteria in this material were released from the macrophages by isotonic lysis and differential centrifugation. RNA was extracted and compared with RNA extracted from bacteria grown in vitro. Real-time polymerase chain reaction was used to analyse the katG gene from the bacterial messenger RNA. The katG mRNA encoding the putative catalase/peroxidase showed differential expression in the in vivo and in vitro-derived samples. We hypothesize that the increase in katG expression for in vivo-derived M. paratuberculosis may represent a response to the oxidative stress encountered within the intra-macrophage environment.

Mycobacterial proteome extraction: comparison of disruption methods.

Mycobacterium avium subsp. paratuberculosis has long been recognized as the causative agent of Johne's disease, a chronic inflammatory intestinal disease of sheep, cattle and other ruminants. Mycobacterial cells are extremely hardy, and proteomic analyses require the use of harsh conditions to effect their disruption. We compared the effectiveness of bead beating and sonication as cell lysis methods for the extraction of the proteomes of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis. Broad and narrow range two-dimensional gel electrophoresis was used to compare the numbers of silver stained protein spots that were observed in mycobacterial lysates. Despite differences in the yield of total protein from either species, and at different ages, the two methods appeared to give similar representations of the mycobacterial proteomes analyzed. Bead beating therefore represents a rapid and effective method of extracting the proteomes of mycobacterial species without the risks associated with an open tube sonication procedure.