A comparative assessment of culture and serology in the diagnosis of brucellosis in dairy cattle.

To investigate the usefulness of culture for the confirmation of brucellosis in cattle, a comparison of culture and serology was undertaken on 248 animals in four dairy herds where the disease was active. Paired supramammary (SM), retropharyngeal (RP), and internal iliac (IL) lymph nodes were cultured, and five serological tests were deployed: the microserum agglutination test (MSAT), complement fixation test (CFT), the indirect (iELISA) and competitive ELISA, and the fluorescence polarisation assay (FPA). Brucella abortus was isolated from 86.8% of animals on combined culture of all three lymph nodes. Individually, the highest isolation rate was from the RP (90.5% of culture positives). Of culture positive animals, 13.7% and 6.2% were positive from the RP and SM alone, respectively. Approximately half of the positive cultures yielded <10 colonies/culture plate. Although 80.9% of animals were positive in at least one serological test, only 45.2% were positive in all five. For culture-positive animals, the MSAT was the most sensitive test (71.8%). Of the culture-negative animals 67.7% were positive in at least one test, while 12.9% were positive in all five. Titres were higher in animals culture-positive from the SM, and there was a direct correlation between higher titres and higher colony counts in SM cultures. Only 8.9% of animals were both culture-negative and seropositive (in at least one test), while 16.5% were culture-positive and seronegative in all five tests. The results highlight and validate the sensitivity of bacteriological culture in confirming a diagnosis of bovine brucellosis. While the MSAT and FPA were the most sensitive serological tests, a significant percentage of infected animals were undetectable using these standard serological assays.

Application of multiple locus variable number of tandem repeat analysis (MLVA), phage typing and antimicrobial susceptibility testing to subtype Salmonella enterica serovar Typhimurium isolated from pig farms, pork slaughterhouses and meat producing plants in Ireland.

Salmonella enterica subsp. enterica serovar Typhimurium is a common zoonotic pathogen encountered in Irish pigs and the pork industry and its characterisation using highly discriminatory typing methods is necessary for epidemiological studies, outbreak investigation and control. Multiple locus variable number of tandem repeat analysis (MLVA), phage typing and antimicrobial susceptibility testing were applied to characterise 301 S. typhimurium isolates of porcine origin isolated from farms, slaughterhouses and pork meat producing plants in Ireland over a four-year period. 154 MLVA patterns were obtained compared to 19 phage types and 38 AMR patterns, and MLVA was particularly useful for discriminating isolates of the same phage type, e.g. DT104 and DT104b, or isolates that were Untypable or in the category of "react with phage but does not conform to a recognised phage type" (RDNC) by the phage typing method. Cluster analysis of MLVA profiles using a minimum spanning tree (MST) demonstrated two major clusters (I and II), which showed to have a clear association with phage types, cluster I associated to phage types DT104, U302 and DT120 and cluster II associated to DT193 and U288. The results of this present study showed that MLVA is highly discriminatory and permitted the identification of identical profiles among isolates obtained at different points of the pork food chain. The same MLVA profile was observed in some cases among isolates with different phage types. While this can be explained by the fact that some phage types are closely related, it also indicates that combining phage typing and MLVA enhances strain typing of S. typhimurium.

Relationships between clinical signs, pathological changes and tissue distribution of Mycobacterium avium subspecies paratuberculosis in 21 cows from herds affected by Johne's disease.

Twenty-one cows from eight herds affected by Johne's disease were assigned to four groups: seven were not thriving and had persistent diarrhoea, six were not thriving and had intermittent diarrhoea, four were not thriving but did not have diarrhoea, and four were clinically normal. Postmortem, macroscopic lesions consistent with Johne's disease were identified in 17 of the cows and Mycobacterium avium subspecies paratuberculosis (MAP) was isolated from all of them. However, except for the fact that diarrhoea was correlated with the presence of lesions in the large intestine there was little correlation between the presence or absence of clinical signs and the lesions associated with Johne's disease. The tissue distribution of MAP was also poorly correlated with either the clinical signs or the lesions. The organism was widely distributed in 17 of the 21 cows, including three of the clinically normal animals, and was present in the mammary tissues of seven cows including two of the clinically normal animals. Three distinct histopathological patterns were observed in the affected intestines: infiltration of the lamina propria with giant cells, tuberculoid lesions, and lepromatous lesions; the lepromatous lesions were associated with extensive pathological changes.

Genotyping of Mycobacterium bovis isolates from badgers in four areas of the Republic of Ireland by restriction fragment length polymorphism analysis.

An analysis of the molecular epidemiology of Mycobacterium bovis in badgers was made in four selected areas of the Republic of Ireland in which an intensive badger removal programme was being carried out over a period of five years. Tissue samples from 2310 badgers were cultured. Restriction fragment length polymorphism (RFLP) analysis with IS6110, polymorphic GC-rich sequence (PGRS) and direct repeat sequence (DR) probes was applied to the isolates from 398 badgers, and 52 different rflp types were identified. Most of the isolates belonged to seven predominant types, and the other 45 types were represented by few isolates. An analysis suggests that some of these 45 types may have been introduced by the inward migration of badgers and others may have been the result of genetic changes to one of the prevalent types. The badgers were divided into groups on the basis of the sett at which they were captured, and RFLP typing was applied to isolates from two or more badgers from 85 groups. Multiple RFLP types were identified among isolates from 50 of these groups, suggesting that badgers probably moved frequently between group territories.

Detection of salinity by the lobster, Homarus americanus.

Changes in the heart rates of lobsters (Homarus americanus) were used as an indicator that the animals were capable of sensing a reduction in the salinity of the ambient seawater. The typical response to a gradual (1 to 2 ppt/min) reduction in salinity consisted of a rapid increase in heart rate at a mean threshold of 26.6 +/- 0.7 ppt, followed by a reduction in heart rate when the salinity reached 22.1 +/- 0.5 ppt. Animals with lesioned cardioregulatory nerves did not exhibit a cardiac response to changes in salinity. A cardiac response was elicited from lobsters exposed to isotonic chloride-free salines but not to isotonic sodium-, magnesium- or calcium-free salines. There was little change in the blood osmolarity of lobsters when bradycardia occurred, suggesting that the receptors involved are external. Furthermore, lobsters without antennae, antennules, or legs showed typical cardiac responses to low salinity, indicating the receptors are not located in these areas. Lobsters exposed to reductions in the salinity of the ambient seawater while both branchial chambers were perfused with full-strength seawater did not display a cardiac response until the external salinity reached 21.6 +/- 1.8 ppt. In contrast, when their branchial chambers were exposed to reductions in salinity while the external salinity was maintained at normal levels, changes in heart rate were rapidly elicited in response to very small reductions in salinity (down to 29.5 +/- 0.9 ppt in the branchial chamber and 31.5 +/- 0.3 ppt externally). We conclude that the primary receptors responsible for detecting reductions in salinity in H. americanus are located within or near the branchial chambers and are primarily sensitive to chloride ions.

Virulence-associated protein characterisation of Rhodococcus equi isolated from bovine lymph nodes.

Rhodococcus equi has a low pathogenicity in cattle, but it occasionally causes lymph node granulomas, which are detected at abattoir post mortem inspection, and must be distinguished from tuberculous granulomas. Lymph node lesions were detected in 6719 cattle, from a total of 3,263,622 cattle examined post mortem in abattoirs, in the Republic of Ireland, during 1997 and 1998. Histological examination was performed on all lesions, principally for the purpose of identifying animals with tuberculosis. A total of 1122 of the lesions were cultured on blood agar and on Stonebrinks and Lowenstein-Jensen medium containing pyruvate, because the histological findings were difficult to interpret or were suggestive of R. equi infection. R. equi was isolated from 264 lesions. Almost all of the R. equi granulomas were confined to a single lymph node, and were present predominantly in the retropharyngeal, bronchial and mediastinal lymph nodes. R. equi granulomas were present in a significantly higher proportion of the lesions detected in steers and heifers compared to cows. The prevalence in the total population of 3.3 million cattle examined post mortem was 0.008%. The 15-17kDa antigens, associated with virulence in this organism, and the 20kDa antigen, associated with intermediate virulence, were not detected in isolates from 146 cattle, analysed by immunoblot assays. A PCR assay to detect the plasmid gene encoding the 15-17kDa antigens was also negative for isolates from these 146 animals. Plasmids were not detected in 30 isolates which were examined.

Characterization of the Mycobacterium bovis restriction fragment length polymorphism DNA probe pUCD and performance comparison with standard methods.

In this study, the newly described Mycobacterium bovis restriction fragment length polymorphism (RFLP) typing probe pUCD was characterized by sequence analysis and the previously observed polymorphic banding pattern was reproduced with a combination of three oligonucleotide probes in a single, mixed hybridization. In addition, the ability of pUCD to distinguish between 299 M. bovis isolates from the Republic of Ireland was assessed in relation to established methods and a statistical function for objective comparison of RFLP probes was derived. It was found that typing with pUCD alone produced greater discrimination between M. bovis isolates than typing with the commonly used mycobacterial DNA probes IS6110, PGRS, and DR and also by the spoligotyping technique. pUCD and DR in combination produced the highest level of discrimination while maintaining a high level of concordance with known epidemiological data relating to the samples. The reduction of pUCD to the level of oligonucleotides should in future allow pUCD and DR to be included together in a mixed hybridization, thus producing a high level of M. bovis strain type discrimination from a single round of RFLP analysis.

Afferent innervation of outer and inner hair cells is normal in neonatally de-efferented cats.

It has been hypothesized that normal pruning of exuberant branching of afferent neurons in the developing cochlea is caused by the arrival of the olivocochlear efferent neurons and the resulting competition for synaptic sites on hair cells. This hypothesis was supported by a report that afferent innervation density on mature outer hair cells (OHCs) is elevated in animals deefferented at birth, before the olivocochlear system reaches the outer hair cell area (Pujol and Carlier [1982] Dev. Brain Res. 3:151-154). In the current study, this claim was evaluated quantitatively at the electron microscopic level in four cats that were de-efferented at birth and allowed to survive for 6-11 months. A semiserial section analysis of 156 OHCs from de-efferented and normal ears showed that, although de-efferentation essentially was complete in all four cases, the number and distribution of afferent terminals on OHCs was indistinguishable from normal, and the morphology of afferent synapses was normal in both the inner hair cell area and the OHC area. Thus, the postnatal presence of an efferent system is not required for the normal development of cochlear afferent innervation, and the synaptic competition hypothesis is not supported.

Identification of a novel DNA probe for strain typing Mycobacterium bovis by restriction fragment length polymorphism analysis.

Bovine tuberculosis caused by Mycobacterium bovis remains a significant disease of farmed cattle in many countries despite ongoing tuberculosis eradication programs. Molecular typing methods such as restriction fragment length polymorphism (RFLP) analysis and spoligotyping have been used to identify related herd breakdowns in an attempt to identify more precisely the route of infection into cattle herds and to trace the transmission of bovine tuberculosis. A recent geographical survey of Irish M. bovis isolates demonstrated that a significant proportion of isolates ( approximately 20%) exhibit a common strain type, limiting the value of current strain typing methods as an epidemiological tool. We have identified and cloned a region of the M. bovis genome, pUCD, which generates a clear, highly polymorphic banding pattern when used as an RFLP probe on AluI restriction-digested M. bovis genomic DNA and which effectively subdivides this common strain type. When used to type 60 Irish M. bovis isolates, pUCD exhibited greater discriminatory power than the commonly used mycobacterial RFLP probes IS6110, PGRS, and DR and detected an equivalent number of strain types to a combination of these three probes. pUCD also detected significantly more strain types than the spoligotyping technique, while maintaining a high level of concordance between epidemiologically related and unrelated herd breakdowns. The polymorphic element within pUCD remains to be fully characterized, however the potential for this probe to greatly decrease the workload necessary to genotype M. bovis by RFLP analysis is compelling.

Restriction fragment length polymorphism analysis of Mycobacterium avium isolates from animal and human sources.

Restriction fragment length polymorphism (RFLP) analysis using probes derived from the insertion sequences IS901, IS1245 and IS1311, was carried out on Mycobacterium avium isolates obtained from 18 human patients, 44 deer, 14 pigs and five cattle in the Republic of Ireland. Forty-two of the cervine isolates and two of the bovine isolates contained IS901, while this insertion sequence was absent from all of the human and porcine isolates. RFLP analysis with IS901 probe differentiated the 44 field isolates which contained this element into three types. All of the IS901-positive isolates had a characteristic three-band IS1245 hybridisation pattern and a characteristic single-band IS1311 hybridisation pattern. The IS901-negative isolates exhibited highly polymorphic IS1245 and IS1311 hybridisation patterns which differentiated the human and porcine isolates into a wide diversity of strain types.

Study of restriction fragment length polymorphism analysis and spoligotyping for epidemiological investigation of Mycobacterium bovis infection.

Restriction fragment length polymorphism (RFLP) analysis with probes derived from the insertion element IS6110, the direct repeat sequence, and the polymorphic GC-rich sequence (PGRS) and a PCR-based typing method called spacer oligonucleotide typing (spoligotyping) were used to strain type Mycobacterium bovis isolates from the Republic of Ireland. Results were assessed for 452 isolates which were obtained from 233 cattle, 173 badgers, 33 deer, 7 pigs, 5 sheep, and 1 goat. Eighty-five strains were identified by RFLP analysis, and 20 strains were identified by spoligotyping. Twenty percent of the isolates were the most prevalent RFLP type, while 52% of the isolates were the most prevalent spoligotype. Both the prevalent RFLP type and the prevalent spoligotype were identified in isolates from all animal species tested and had a wide geographic distribution. Isolates of some RFLP types and some spoligotypes were clustered in regions consisting of groups of adjoining counties. The PGRS probe gave better differentiation of strains than the IS6110 or DR probes. The majority of isolates from all species carried a single IS6110 copy. In four RFLP types IS6110 polymorphism was associated with deletion of fragments equivalent in size to one or two direct variable repeat sequences. The same range and geographic distribution of strains were found for the majority of isolates from cattle, badgers, and deer. This suggests that transmission of infection between these species is a factor in the epidemiology of M. bovis infection in Ireland.

Correlates of hypnotizability in children: absorption, vividness of imagery, fantasy play, and social desirability.

This study examined correlates of hypnotizability in children that had previously been reported for adults. Forty-two children (ages 7-14) completed the Fantasy Questionnaire (FQ), the Children's Social Desirability Questionnaire (CSDQ), the Zelter and LeBaron (1984) revision of the Stanford Hypnotic Clinical Scale for Children (SHCS:C-R), and the Children's Fantasy Inventory: Absorption and Vividness Scales (CFI: A & V). The nine-item SHCS:C-R yields separate scores for Observed Behavior (OB), and Realness (R), as well as a Total Score (TS). Results indicated significant correlations between SHCS:C-R scores and those for CFI: A & V, and the FQ (r = .42-.53) but not for the CSDQ. On the SHCS:C-R, correlations between R (involuntariness) scores and the above measures were not appreciably different from those found for OB scores. Observations suggested that attitudes towards hypnosis may influence children's hypnotic responsiveness.

Adult infectious diseases: management in a general hospital.

Conflicting views exist as to where patients with infectious diseases (IDs) are best managed. In an effort to define the role of an IDs Unit in this country, we undertook a four-year survey of IDs cases referred to our isolation facility which is part of the Gastroenterology department. Ninety-three per cent of 250 cases referred had either diarrhoea (48%) or jaundice (45%). Two thirds of the total had an infective, and one third a non-infective basis for their illness. Since diarrhoea and jaundice dominated the clinical presentations of these cases, having the IDs facility within the Gastroenterology department made for speedy diagnosis and management of both infectious and non-infectious cases. Where referral and/or transfer to other units was necessary, it still proved helpful to locate the IDs facility within a regional multidisciplinary hospital, particularly in view of the wide range of medical and surgical conditions referred as putative IDs.

Activated sludge biodegradation of 12 commercial phthalate esters.

The activated sludge biodegradability of 12 commercial phthalate esters was evaluated in two test systems: (i) a semicontinuous activated sludge test and (ii) an acclimated 19-day die-away procedure. Both procedures demonstrated that phthalate esters are rapidly biodegraded under activated sludge conditions when loss of the parent phthalate ester (primary degradation) is measured.

Shake flask biodegradation of 14 commercial phthalate esters.

An acclimated shake flask CO(2) evolution test was used to study the biodegradability of 14 commercial phthalate esters that are commonly used as plasticizers. Both CO(2) evolution (ultimate biodegradation) and loss of parent phthalate esters (primary biodegradation) were measured. With only a few exceptions, primary biodegradation was 90% or higher, and ultimate biodegradation was in excess of 55% of theoretical results in 28 days. The results showed that all of the commercial phthalate esters were susceptible to biodegradation by mixed populations of microorganisms from natural sources. The results also provide considerable insight into the utility and reproducibility of a standard biodegradation test that is being recommended for widespread screening of chemicals.