Knowledge gaps that hamper prevention and control of Mycobacterium avium subspecies paratuberculosis infection.

In the last decades, many regional and country-wide control programmes for Johne's disease (JD) were developed due to associated economic losses, or because of a possible association with Crohn's disease. These control programmes were often not successful, partly because management protocols were not followed, including the introduction of infected replacement cattle, because tests to identify infected animals were unreliable, and uptake by farmers was not high enough because of a perceived low return on investment. In the absence of a cure or effective commercial vaccines, control of JD is currently primarily based on herd management strategies to avoid infection of cattle and restrict within-farm and farm-to-farm transmission. Although JD control programmes have been implemented in most developed countries, lessons learned from JD prevention and control programmes are underreported. Also, JD control programmes are typically evaluated in a limited number of herds and the duration of the study is less than 5 year, making it difficult to adequately assess the efficacy of control programmes. In this manuscript, we identify the most important gaps in knowledge hampering JD prevention and control programmes, including vaccination and diagnostics. Secondly, we discuss directions that research should take to address those knowledge gaps.

Successful control of Johne's disease in nine dairy herds: results of a six-year field trial.

The objective was to evaluate if a standardized Johne's disease control program significantly reduced the prevalence of cattle infected with Mycobacterium avium ssp. paratuberculosis in dairy herds with a moderate to high initial infection prevalence of >or=10% ELISA-positive adult cattle. Nine Wisconsin dairy herds of diverse sizes and management styles completed the 6-yr study. The control program involved changes to heifer rearing practices in combination with a routine testing program. For heifers, the program specifically required 1) segregated maternity pens for ELISA-positive and ELISA-negative cattle; 2) removal of calves from the maternity pen in <2h; 3) use of colostrum only from individual ELISA-negative cows (no colostrum pooling); 4) hygienic collection of colostrum; 5) feeding of pasteurized milk as milk replacer or on-farm pasteurized milk until weaning; and 6) minimizing contact with manure from the adult cattle until weaning. The testing program was designed to detect the most infectious cattle by using a commercial ELISA once on every adult during each lactation. Producers were required to cull cows with strong-positive ELISA results before the next calving and to label cows with low- to medium-level ELISA results and manage them to limit infection transmission. Outcomes were measured by comparing the apparent prevalence based on ELISA or fecal culture in the whole herd and in first-lactation cohorts at 2 time points: before implementation of the control program and at the end of the trial. The combined results from the 9 herds showed a significant reduction in ELISA-positive cows, from 11.6% at the start of the trial to 5.6% at conclusion of the trial. The apparent prevalence decline among first-lactation cows was greater and was evident by ELISA (10.4 vs. 3.0%) and by fecal culture (17.0 vs. 9.5%). Although variations among farms were observed, the collective results demonstrated that bovine paratuberculosis can be controlled in dairy herds through effective heifer husbandry practices in combination with diagnostic testing to identify, for culling or management, cows most likely infectious.

Mycobacterium avium subsp. paratuberculosis: pathogen, pathogenesis and diagnosis.

Johne's disease, or paratuberculosis, is a chronic intestinal infection caused by Mycobacterium avium subsp. paratuberculosis. The usually fatal disease is characterised by cachexia, and in some species diarrhoea, after a long pre-clinical phase. Treatment is ineffective and economically impracticable. The infection primarily affects domestic and free-ranging ruminants, but has also been reported in primates, rabbits, stoats and foxes. Since paratuberculosis is often subclinical, under-reporting is suspected, even though the disease is notifiable in numerous countries. Herd prevalence of bovine paratuberculosis in Europe ranges from 7% to 55%. In the United States of America, herd prevalence is strongly associated with herd size; 40% of herds of more than 300 head were found to be infected. In Australia, reported dairy herd infection rates range between 9% and 22%. Paratuberculosis in domestic livestock entails significant economic losses due to several factors (e.g. reduced production, premature culling and increased veterinary costs). Free-ranging and captive wildlife are also at risk from paratuberculosis.

Mycobacterium avium subspecies paratuberculosis: a review of current knowledge.

Although Mycobacterium avium subsp. paratuberculosis infection has had its greatest effect on domestic agricultural animal species, it can also have a significant impact on wildlife species. More cases of infection are being reported, and because of its ability to elude immunologic control and to persist in the environment, M. paratuberculosis may spread within and among captive and free-ranging wildlife populations in the absence of organized control programs. Studies to improve our ability to detect the organism in biologic samples such as milk, blood, and manure through immunomagnetic separation, automated culture methods, and improved polymerase chain reaction procedures are underway in several countries. Studies of the organism's genetic components, virulence factors, and antigens support the development of new diagnostic tools and vaccines.

Epizootic of paratuberculosis in farmed elk.

After multiple cases of chronic diarrhea and weight loss in a farmed elk herd, 3 yearlings and 1 adult elk with similar clinical signs were euthanatized and necropsied. Gross and histologic evidence of paratuberculosis were found in the yearlings. Evidence of serum antibody to Mycobacterium paratuberculosis was detected in the 2 elk with the most disseminated infection. Acid-fast organisms were isolated from multiple organs in all 4 elk and identified as M paratuberculosis by DNA probe. Thirty-five percent of 1 calf crop (n = 31) died or were euthanatized because of paratuberculosis before they were 2 years old. The organism was believed to have been spread by standing water that was used by calves as a wallow and a source of drinking water. The water was believed to have been contaminated by an infected adult female elk introduced to the herd just before calving season.

Central nervous system effects of meclizine and dimenhydrinate: evidence of acute tolerance to antihistamines.

Relative daytime drowsiness and performance impairment produced by meclizine and dimenhydrinate was assessed in 24 healthy male volunteers. Subjects received either dimenhydrinate, 100 mg, at 8:00 AM, 12:00 PM, and 4:00 PM; meclizine, 50 mg, at 8:00 AM, with placebo at 12:00 PM and 4:00 PM; or placebo at all three times in this randomized, double-blind, three-way crossover study. Impairment of mental performance was assessed by choice reaction time testing and digit symbol substitution scores. Drowsiness was self-assessed on the Stanford Sleepiness Scale and on a visual analog scale. Both antihistamines produced changes in digit symbol substitution, recognition time, and subjective assessments of sleepiness different from placebo. Expressed as change from baseline, the greatest reductions in digit symbol substitution scores after dimenhydrinate occurred 3 hours after the first dose (6.6 +/- 7) and were not different from the greatest measured change after meclizine (5.8 +/- 8), which occurred 9 hours after the dose was administered. Similar results were obtained with the other psychometric test scores. Self-rated sleepiness after dimenhydrinate was greatest 1 hour after the first dose, and was significantly greater than the largest degree of sleepiness after meclizine, which occurred at 7 hours after the dose. The effects of the first dose of dimenhydrinate on psychometric test scores were compared with the magnitude of the effects produced by subsequent doses. The magnitude of effect of the first dose of dimenhydrinate was significantly greater than the magnitude of effect produced by subsequent doses. The data suggest the possibility that acute tolerance to central nervous system impairment develops with multiple doses of dimenhydrinate.

Identification of a chromosomal locus for expression of lipopolysaccharide epitopes in Haemophilus influenzae.

Lipopolysaccharide (LPS) is a major virulence determinant of Haemophilus influenzae. The organism is able to display an extensive repertoire of different LPS structures through the loss and acquisition of multiple oligosaccharide epitopes in various combinations. This marked heterogeneity of LPS molecules has complicated the analysis of the structure of LPS and its role in pathogenesis. A genomic library was screened for the ability to transform H. influenzae to express novel LPS epitopes defined by reactivity with oligosaccharide specific monoclonal antibodies. A chromosomal locus, lic-1, involved in expression of at least three different epitopes (recognized by monoclonal antibodies 4C4, 12D9, and 6A2), was identified on a 5.6-kilobase restriction endonuclease fragment. Transformation of H. influenzae with subclones from within lic-1 was used to generate a series of isogenic and phenotypic variants. All transformants displayed phase variation for their newly acquired epitopes. Altered binding specificities of LPS with monoclonal antibodies correlated with changes in sugar compositional analysis. The expression of two epitopes was eliminated by introduction of site-specific mutations in lic-1, confirming the role of lic-1 in oligosaccharide biosynthesis.

A common virulence region on plasmids from eleven serotypes of Salmonella.

Cured derivatives of Salmonella dublin and S. typhimurium showed reduced virulence following oral infection of mice (10(4)-10(5)-fold for S. dublin, 10(2)-fold for S. typhimurium). Large plasmids from S. dublin and S. typhimurium independently restored virulence to the cured S. dublin but truncated S. dublin plasmids with deletions in a previously identified virulence region did not. This common virulence region identified in plasmids from S. dublin and S. typhimurium was shown to be carried on plasmids from 11 other serotypes of Salmonella but was absent from 10 plasmid-containing serotypes. TnA and Tn10 were transduced from the virulence region of two TnA-insertion mutants of S. dublin and one Tn10-insertion mutant of S. typhimurium that showed diminished virulence to recipient wild-type strains of S. dublin, S. enteritidis and S. typhimurium. Each transductant showed a decrease in mouse virulence within the range 10(3)-10(5). It is therefore proposed that similar virulence determinants are expressed in different serotypes. It was also shown that integration that occurred during curing was Tn10 dependent.

The role of plasmid genes in the pathogenicity of Salmonella dublin.

The virulence (expressed as LD50 values) for mice of two mutant strains of Salmonella dublin, both containing TnA insertions in the resident plasmid, was reduced by 10(4)-10(5) when infection was by the oral or intravenous or intraperitoneal route. When the plasmid was lost from one of the mutants no further decrease in virulence was observed. Results also suggested that plasmid genes are not involved in the ability of S. dublin to cross the gut wall.

Evidence for related virulence sequences in plasmids of Salmonella dublin and Salmonella typhimurium.

Transposon-insertion mutants were prepared from virulent field isolates of Salmonella dublin and Salmonella typhimurium. Detailed restriction-enzyme mapping of the single sites of TnA insertion in two mutants (M51 and M173) of S. dublin that showed diminished virulence in a mouse assay indicated that these sites were about 5 kbp apart on the approximately 70 kbp plasmid harboured by the isolate. A Tn10-insertion mutant (M242) of S. typhimurium that showed diminished virulence was also identified. A single copy of Tn10 was inserted into the approximately 90 kbp plasmid harboured by this isolate. Hybridization studies indicated that homology existed between the region encompassing the sites of TnA insertion in M51 and M173 and that encompassing the site of Tn10 insertion in M242. Restriction mapping indicated that the two regions were very similar and could even be identical and, if so, the Tn10 insertion in M242 could be mapped to a point 1.5 kbp from the TnA insertion in M51 and 6.5 kbp from that in M173. It appeared that the maximal extent of the putative similarity/identity was between 13 and 23 kbp. It is proposed that this stretch of high homology could represent a virulence sequence that has been conserved during the evolutionary divergence of the two Salmonella serotypes.

Can scrapie titres be calculated accurately from incubation periods?

Since endpoint titrations of scrapie material are costly and time-consuming, several workers have estimated titre from the correlation between the incubation period of the disease and the infectivity titre. However, we show here that the relationship between incubation period and titre cannot be assumed to be constant for all scrapie preparations. Our results indicate that sodium deoxycholate treatment of scrapie preparations does not reduce the titre, but can lengthen the incubation period by about 10 days. This is equivalent to a discrepancy of 1 log LD50 unit if the estimation of titre was based on the incubation period.

Involvement of protein in scrapie agent infectivity.

The nature of the causative agent of scrapie is not known. Previous work has demonstrated that nuclease digestion does not inactivate scrapie infectivity, but there are conflicting reports about the effects of proteases. It is shown here that the broad range protease, proteinase K, reduces scrapie infectivity under all conditions tested. Control experiments demonstrated that the loss of infectivity is not artefactual and results from protein breakdown. Proteolytic digestion in the presence of detergent greatly increases proteolysis, but does not lead to a further loss of infectivity. This suggests that the protein involved may be a surface component, but whether the component is an integral or secondary part of the agent is not known.

Infectivity of liposomally encapsulated nucleic acids isolated from EMC virus and scrapie-infected mouse brain.

The ability of liposomes to enhance nucleic acid infectivity in vivo was studied. Encephalomyocarditis (EMC) virus RNA was extracted and encapsulated within liposomes. The infectivity of EMC RNA was increased by liposomal entrapment after intraperitoneal injection of mice, but decreased after intracerebral injection. In contrast, when nucleic acids from scrapie-infected brains were entrapped in liposomes and injected into mice by one of four routes, no cases of scrapie were observed. This is the first report of the enhancement of nucleic acid infectivity by liposomes in vivo.

"Normal-pressure" hydrocephalus. Relationship of clinical and radiographic findings to improvement following shunt surgery.

Analysis of 25 patients who fulfilled clinical and radiographic criteria for the diagnosis of "normal pressure" hydrocephalus (NPH) demonstrated (1) a significant relationship between presence of motor signs with good outcome and absence of motor signs with poor outcome following ventricular shunting, (2) symptoms and signs of parkinsonism in 40% of patients in whom the diagnosis of NPH was made, and (3) no reliable relationship between radiographic measurements or cisternogram appearance and outcome following shunting. The clinical picture is the most important factor in selection of NPH patients for surgery.