A novel automated test battery reveals enduring behavioural alterations and cognitive impairments in survivors of murine pneumococcal meningitis.

Pneumococcal meningitis, caused by Streptococcus pneumoniae infection, is a major form of lethal bacterial meningitis. Survivors are predisposed to developing lifelong disabling sequelae, including cognitive impairment, psychological problems and motor deficits. In our experimental model, ventricular inoculation of 10(5) colony-forming units of S. pneumoniae type 3 caused 90% of mice to develop life-threatening meningitis within 48 h. Antibiotic treatment with ceftriaxone 20 h post infection reduced the incidence of severe meningitis to <10%. At the time of treatment, upregulation of pro-inflammatory cytokines was detected, including interleukin-1ß, interleukin-6 and tumour necrosis factor. We evaluated the long-term behavioural and cognitive sequelae in control mice and those surviving meningitis using an automated system (the IntelliCage) in which mice perform a range of behavioural and spatial tasks to obtain water rewards from conditioning units in their home cage. Surviving mice showed a number of altered behaviours relative to controls, including (i) hypoexploration when first exposed to the IntelliCage, (ii) altered activity patterns (fewer visits to conditioning stations during the light phase and more in the dark phase), (iii) avoidance of light (a constant or flashing LED stimulus), (iv) impaired spatial learning (a complex patrolling task), and (v) impaired discrimination reversal learning. Overall these results suggest photophobia and weakened learning ability in post-meningitic mice, particularly on tasks engaging hippocampal and prefrontal neural substrates. This study also demonstrates a standardised and comprehensive battery of tests that can be readily used to investigate neurological sequelae in undisturbed mice residing in a complex home cage environment.

Brain endothelial cells increase the proliferation of Plasmodium falciparum through production of soluble factors.

We here describe the novel finding that brain endothelial cells in vitro can stimulate the growth of Plasmodium falciparum through the production of low molecular weight growth factors. By using a conditioned medium approach, we show that the brain endothelial cells continued to release these factors over time. If this mirrors the in vivo situation, these growth factors potentially would provide an advantage, in terms of enhanced growth, for sequestered parasitised red blood cells in the brain microvasculature. We observed this phenomenon with brain endothelial cells from several sources as well as a second P. falciparum strain. The characteristics of the growth factors included: <3 kDa molecular weight, heat stable, and in part chloroform soluble. Future efforts should be directed at identifying these growth factors, since blocking their production or actions might be of benefit for reducing parasite load and, hence, malaria pathology.

Temperature dependency of Clostridium botulinum C and D toxin production from anaerobically enriched bovine gastrointestinal samples.

AIMS: To determine whether Clostridium botulinum neurotoxin (BoNT) production in anaerobic culture was affected by temperature and could influence the sandwich ELISA (sELISA) detection of group III toxins in pre-enriched gastrointestinal (GI) contents from clinically suspect cattle botulism cases. METHODS AND RESULTS: Bovine post-mortem GI samples taken from 124 and 96 animals with suspect and nonsuspect botulism, respectively, were pre-enriched anaerobically at 30 and 37°C prior to testing by sELISA. After enrichment at 37°C, BoNT was demonstrated in all clinically suspect bovine botulism cases that had been identified by the mouse bioassay, and enrichment by both temperatures enabled BoNT detection in a number of mouse bioassay-negative suspect cases. CONCLUSIONS: Culture temperature does influence the production of group III BoNT, and incubation at both 30 and 37°C is required for optimum detection. SIGNIFICANCE AND IMPACT OF THE STUDY: The in vitro assay defined in this study has the potential of improving the confirmation rate of clinically suspect cattle botulism cases whilst reducing the use of the costly and ethically sensitive mouse bioassay, the current diagnostic gold standard for BoNT testing.

Biochemical characteristics and inhibitor selectivity of mouse indoleamine 2,3-dioxygenase-2.

The first step in the kynurenine pathway of tryptophan catabolism is the cleavage of the 2,3-double bond of the indole ring of tryptophan. In mammals, this reaction is performed independently by indoleamine 2,3-dioxygenase-1 (IDO1), tryptophan 2,3-dioxygenase (TDO) and the recently discovered indoleamine 2,3-dioxygenase-2 (IDO2). Here we describe characteristics of a purified recombinant mouse IDO2 enzyme, including its pH stability, thermal stability and structural features. An improved assay system for future studies of recombinant/isolated IDO2 has been developed using cytochrome b (5) as an electron donor. This, the first description of the interaction between IDO2 and cytochrome b (5), provides further evidence of the presence of a physiological electron carrier necessary for activity of enzymes in the "IDO family". Using this assay, the kinetic activity and substrate range of IDO2 were shown to be different to those of IDO1. 1-Methyl-D-tryptophan, a current lead IDO inhibitor used in clinical trials, was a poor inhibitor of both IDO1 and IDO2 activity. This suggests that its immunosuppressive effect may be independent of pharmacological inhibition of IDO enzymes, in the mouse at least. The different biochemical characteristics of the mouse IDO proteins suggest that they have evolved to have distinct biological roles.

The ETO protein disrupted in t(8;21)-associated acute myeloid leukemia is a corepressor for the promyelocytic leukemia zinc finger protein.

The ETO protein was originally identified by its fusion to the AML-1 transcription factor in translocation (8;21) associated with the M2 form of acute myeloid leukemia (AML). The resulting AML-1-ETO fusion is an aberrant transcriptional regulator due to the ability of ETO, which does not bind DNA itself, to recruit the transcriptional corepressors N-CoR, SMRT, and Sin3A and histone deacetylases. The promyelocytic leukemia zinc finger (PLZF) protein is a sequence-specific DNA-binding transcriptional factor fused to retinoic acid receptor alpha in acute promyelocytic leukemia associated with the (11;17)(q23;q21) translocation. PLZF also mediates transcriptional repression through the actions of corepressors and histone deacetylases. We found that ETO is one of the corepressors recruited by PLZF. The PLZF and ETO proteins associate in vivo and in vitro, and ETO can potentiate transcriptional repression by PLZF. The N-terminal portion of ETO forms complexes with PLZF, while the C-terminal region, which was shown to bind to N-CoR and SMRT, is required for the ability of ETO to augment transcriptional repression by PLZF. The second repression domain (RD2) of PLZF, not the POZ/BTB domain, is necessary to bind to ETO. Corepression by ETO was completely abrogated by histone deacetylase inhibitors. This identifies ETO as a cofactor for a sequence-specific transcription factor and indicates that, like other corepressors, it functions through the action of histone deactylase.

The promyelocytic leukemia zinc finger (PLZF) protein binds DNA in a high molecular weight complex associated with cdc2 kinase.

A binding site selection from a CpG island library for the promyelocytic leukemia zinc finger protein (PLZF) identified two high affinity PLZF binding sites. These sequences also bound RARalpha/PLZF, a fusion protein formed in chromosomal translocation t(11;17)(q23;q21) associated with acute promyelocytic leukemia. PLZF bound DNA as a slowly migrating complex with an estimated mol. wt of 600 kDa whose formation was dependent on the POZ/dimerization domain of PLZF. The PLZF-DNA complex was unable to form in the presence of cdc2 antibodies. A PLZF-cdc2 interaction was further demonstrated by co-immunoprecipitation and a biotin-streptavidin pull-down assay. PLZF is a phosphoprotein and immunoprecipi-tates with a cdc2-like kinase activity. The PLZF-DNA complex was abolished with the addition of a phosphatase. These studies suggest that the activity of PLZF, a regulator of the cell cycle, may be modulated by cell cycle proteins. RARalpha/PLZF did not complex with cdc2, this potentially contributing to its aberrant transcriptional properties and potential role in leukemo-genesis.

Leukemia translocation protein PLZF inhibits cell growth and expression of cyclin A.

The PLZF gene was identified by its fusion with the RARalpha locus in a therapy resistant form of acute promyelocytic leukemia (APL) associated with the t(11;17)(q23;q21) translocation. Here we describe PLZF as a negative regulator of cell cycle progression ultimately leading to growth suppression. PLZF can bind and repress the cyclin A2 promoter while expression of cyclin A2 reverts the growth suppressed phenotype of myeloid cells expressing PLZF. In contrast RARalpha-PLZF, a fusion protein generated in t(11;17)(q23;q21)-APL activates cyclin A2 transcription and allows expression of cyclin A in anchorage-deprived NIH3T3 cells. Therefore, cyclin A2 is a candidate target gene for PLZF and inhibition of cyclin A expression may contribute to the growth suppressive properties of PLZF. Deregulation of cyclin A2 by RARalpha-PLZF may represent an oncogenic mechanism of this chimeric protein and contribute to the aggressive clinical phenotype of t(11;17)(q23;q21)-associated APL.

Characterisation of avian pathogenic Escherichia coli (APEC) associated with colisepticaemia compared to faecal isolates from healthy birds.

A total of 114 avian pathogenic Escherichia coli (APEC) isolates were collected from cases of colisepticaemia occurring in broilers (77) and layers (37) within Ireland. In addition 45 strains isolated from faeces of healthy birds were included for comparison. All isolates were serogrouped, and examined for known virulence factors, mostly by PCR. The O78 serogroup represented 55 and 27% of broiler and layer colisepticaemic isolates respectively. All isolates were positive for curli fimbriae (crl, csg) and negative for afimbrial adhesin (afa). S-fimbrial (sfa) sequences were present in 8.8% of septicaemic isolates and 8.9% of healthy bird isolates. The majority of E. coli from cases of colisepticaemia (97.4%) and healthy bird (95.6%) isolates were positive for aerobactin (aer), and temperature sensitive haemagglutinin (tsh) was similarly detected in high numbers in 93.9 and 93.3%, respectively. In comparison to E. coli isolates from the faeces of healthy birds, a significantly higher percentage of isolates from septicaemic cases possessed Type 1 fimbriae (fimC) and increased serum survival (iss) gene sequences. Forty-seven (41.2%) isolates from septicaemic birds possessed P-fimbriae (pap) gene sequences, compared with only 15.6% from E. coli isolated from healthy birds. Haemolysin (hlyE) sequences were detected in 46.7% of isolates from healthy birds in comparison with 6.1% of septicaemic isolates. Sequences encoding colicin V (cvaC) were detected in 99.1% of septicaemic isolates and 82.2% of isolates from healthy birds. The K1 capsule was only present in two septicaemic isolates, both taken from layers. Motility was detected in 36.8% of E. coli isolated from cases of septicaemia, compared with 93.3% of isolates from healthy birds. These results demonstrate the presence of 11 virulence genes in E. coli isolated from cases of colisepticaemia within Ireland, and indicate the prevalence of iss and fimC.

Sandwich ELISA detection of Clostridium perfringens cells and alpha-toxin from field cases of necrotic enteritis of poultry.

Sandwich ELISAs (sELISAs) for the detection of Clostridium perfringens cells and alpha-toxin were developed and used to screen intestinal samples from normal broiler chickens and from clinical cases of necrotic enteritis. The assays clearly distinguished between the two sets of samples. The sELISA absorbance values from samples obtained from the majority of healthy birds were low and those from the majority of necrotic enteritis cases were high. Together, the assays provide a suitable test for the rapid screening for the diagnosis of necrotic enteritis in poultry.

New transtracheal bronchoalveolar lavage technique for the diagnosis of respiratory disease in sheep.

A new transtracheal bronchoalveolar lavage technique for the diagnosis of respiratory disease in sheep under field conditions was tested in 76 sheep. The sheep were divided into three groups, normal sheep, sheep with clinical signs of respiratory disease and housed sheep, on the basis of their respiratory disease history and husbandry conditions. The detection of Mannheimia haemolytica and Mycoplasma ovipneumoniae or parainfluenza virus type 3 and bovine respiratory syncytial virus antigen in the lavage samples was closely correlated with clinical disease. The sheep with clinical respiratory disease had a higher mean percentage of neutrophils in the lavage fluid than the sheep in the other two groups.

Immunohistochemical labelling of cyclooxygenase-2 in lung lesions of calves infected with Mycoplasma bovis.

The pathogenesis and persistence of Mycoplasma bovis (Mb) infection of the respiratory tract is incompletely understood. Cyclooxygenase (COX)-2 is overexpressed during inflammatory responses by different cell types in the lung. This study evaluated COX-2 expression immunohistochemically in the inflammatory lesions of calves with naturally occurring and experimentally induced Mb pneumonia. Experimentally infected lungs showed catarrhal bronchointerstitial pneumonia and varying degrees of peribronchiolar mononuclear cell cuffing. Lesions in calves with spontaneously arising disease included exudative bronchopneumonia and extensive foci of coagulative necrosis surrounded by inflammatory cells. Mb antigen was located in epithelial and inflammatory cells in the airway lumina and surrounding areas of necrosis. COX-2 protein was detected in the lung of all infected calves and was localized to goblet cells, bronchial, bronchiolar and alveolar epithelial cells and macrophages. COX-2 protein was overexpressed during Mb infection and was always associated with areas of pneumonia and with the presence of Mb antigen.

The detection of verocytotoxins in bacterial cultures from human diarrhoeal samples with monoclonal antibody-based ELISAs.

Bacterial cultures from 1801 human diarrhoeal faecal specimens were examined for verocytotoxins I and II by monoclonal antibody-based sandwich ELISAs. Of the 68 ELISA-positive cultures selected from initial screening, 32 were positive by ELISA on repeat or from freshly grown cultures. ELISA-positive pure cultures were obtained from 13 of these, of which seven were confirmed as verocytotoxin positive by cytotoxicity assay. These seven strains were typed as O26 (5) and O146 (2). The six false positive results were from isolates of Enterobacter sp. (1), Citrobacter Freundii (1) and Escherichia coli (4), one each of types O1, O18ac and O98 and an untypable strain. Despite the occurrence of false positive reactions, sandwich ELISA was a useful method for the rapid screening of samples, and detected verocytotoxin-positive E. coli strains, other than O157, from patients with clinical conditions in which they could be implicated.

Experimental mastitis caused by Mycoplasma bovigenitalium and M. canadense in the ewe.

Twelve strains of M. bovigenitalium and two of three strains of M. canadense caused an infection resulting in a pathogenic effect when experimentally inoculated into the ovine mammary gland. Differences in the pathogenesis were quantified by the duration of continuous mycoplasma excretion and the duration of high milk cell levels, but variation in the susceptability of the experimental animals prevented the establishment of firm conclusions on the relative virulence of the strains. Seven M. bovigenitalium and two M. canadense strains were eventually eliminated naturally from the infected glands, but four M. bovigenitalium strain infections ultimately became sub-clinical with intermittent mycoplasma excretion and low milk cell levels.

The experimental production of mastitis in ewes as a determinant of the virulence of ovine ureaplasma strains.

Seven out of eight ovine ureaplasma strains inoculated into the mammary gland of suckling ewes produced a mastitis. The pattern of infection was single phase in 5 ewes, persisting for 12-41 days, and biphasic in 3 ewes, persisting in 2 of them until weaning at 60 days and 3 months post-infection. Sucking lambs did not become infected in the eye or nasal areas, and did not transfer infection to the control contralateral glands.

The potential of the ovine udder as a model to determine the pathogenicity of bovine ureaplasmas.

Various methods of inducing mastitis in the ovine mammary gland with two bovine ureaplasma strains were investigated. The most successful method was by inoculation of fresh broth cultures on two successive days, 24 h apart. Eight more bovine strains were inoculated by this means and three successfully infected the glands.

The isolation of sucrose-fermenting Salmonella mbandaka.

During media trials to evaluate the use of Brilliant Green Agar for the primary recognition of Salmonella, strains presenting fermentation reactions were observed. All fermenting strains (84 out of 145) belonged to the serotype Salmonella mbandaka (58%), and the activity was expressed on three batches of Brilliant Green Agar and one of Xylose-Lysine Desoxycholate Agar. It was established using individual lactose and sucrose broth that the reaction in these media was due to sucrose fermentation. The most frequently isolated Salmonella in this laboratory during 1990 was S. mbandaka (61%) i.e. 65 of the 106 isolates during this period. Primary differentiation of Salmonella from other members of the family Enterobacteriaceae on media incorporating sucrose would have resulted in 36% of Salmonella isolates not being recognised. BGA and XLD agar therefore would not be suitable for primary isolation of Salmonella from clinical material with such a high percentage of the major isolate, S. mbandaka, having the ability to ferment sucrose.

CNF producing Escherichia coli isolated from cattle in Northern Ireland.

Tissue culture assays were used to investigate the incidence of cytotoxic necrotising factors (CNFs) 1 and 2 in Escherichia coli strains from cattle. E. coli cultures were obtained from faeces collected from 223 cases of diarrhoea and from 113 healthy animals. In addition, strains cultured from 62 cases of mastitis, 66 cases of septicaemia and 68 cases of abortion were also investigated. E. coli producing CNF 1 or 2 were identified in all sample groups except for the abortion cases. Comparable levels of CNF1 strains were present in E. coli from the faces of diarrhoeic (4%) and healthy faeces (4.4%) whereas lower levels of CNF2 were identified in the faeces from diarrhoeic animals (19.3%) in comparison with healthy animals (30.9%). One CNF1 producing strain was identified among the E. coli isolated from mastitis samples, while 3% and 10.6% of septicaemic strains were positive for CNF1 and 2, respectively. Serogrouping of CNF isolates did not reveal the association of any particular serogroups with the different conditions.

Improved detection of Mycobacterium avium subsp. paratuberculosis In milk by immunomagnetic PCR.

The potential use of a novel immunomagnetic PCR (IMS-PCR) technique as a rapid method to screen milk samples for the presence of Mycobacterium avium subsp. paratuberculosis (M. ptb) was assessed. Immunomagnetic separation (IMS) for M. ptb, developed at Queen's University, Belfast, was applied to milk samples prior to IS900 PCR in order to selectively concentrate any M. ptb cells present and, at the same time, separate the cells from constituents of milk likely to inhibit subsequent PCR. This increased the sensitivity of IS900 PCR. IMS-PCR sensitivity could be further increased by initial centrifugation (2500 g for 20 min) of larger volumes of milk (10 and 50 ml), and resuspension of the sediment into a 1 ml volume appropriate for IMS treatment. Following IMS, template DNA for IS900 PCR was obtained by heating the bead-cell suspension in a thermal cycler at 100 degrees C for 15 min. It was estimated that the IMS-PCR assay could detect approximately 10(3)CFU of M. ptb per 50 ml of milk (equivalent to 20 CFU/ml), whereas the minimum detection limit of direct IS900 PCR was estimated at 10(5)CFU of M. ptb per 50 ml (equivalent to 2000 CFU/ml). A blind trial was carried out in which a total of 40 spiked (10(6)CFU M. ptb) and unspiked, raw and laboratory-pasteurised milk samples were independently tested by IMS-PCR and conventional IS900 PCR. IMS-PCR correctly identified 97. 5% of milk samples (sensitivity 100%, specificity 95%), including spiked milk samples before and after laboratory-pasteurisation. One false positive result was obtained which may have resulted from carryover between samples during the IMS procedure. Conventional IS900 PCR correctly identified only 72.5% of the same 40 milk samples (sensitivity 23%, specificity 100%). IMS-PCR was also shown to be capable of detecting natural M. ptb infection in raw sheep's milk, and raw and commercially pasteurised cows' milk.

Detection of Mycoplasma mycoides subspecies mycoides by monoclonal antibody-based sandwich ELISA.

Monoclonal antibodies (MAbs) were produced from a mouse immunised with Mycoplasma mycoides subsp. mycoides small colony (MmmSC) antigen and their use to detect and differentiate strains within the Mycoplasma mycoides cluster investigated in an antigen capture ELISA format. The MAbs produced could not distinguish between MmmSC and M. mycoides subsp. mycoides large colony (MmmLC) strains. However, the sandwich ELISAs developed were able to specifically distinguish these two biotypes from the other four members of the M. mycoides cluster, and from all other mycoplasma or bacteria species examined. The most sensitive application of the test was a combination of enrichment and capture by overnight or 48-h incubations of samples inoculated into mycoplasma broth in antibody-coated microtiter wells.

The use of pulsed field gel electrophoresis to investigate the epidemiology of Staphylococcus aureus infection in commercial broiler flocks.

Pulsed field gel electrophoresis (PFGE) analysis of SmaI restriction patterns was used to type 109 isolates of Staphylococcus aureus collected from broiler farms and hatcheries in Northern Ireland. Forty-seven isolates from clinical conditions in broilers and 62 strains from hatcheries, were examined. The PFGE patterns demonstrated a similarity between 85% of strains from clinical sources and 71% of the hatchery isolates. The association of disease with the predominant strain type and presence of these same strains in the hatchery, indicates that the hatchery is a potential source of the infection for clinical broiler disease.