Five protein serine,'threonine phosphatases (PP) have been identified by cloning cDNA from mammalian and Drosophila libraries. These novel enzymes. which have not yet been detected by the techniques of protein chemistry and enzymology.are termed PPV. PP2Bw, PPX, PPY and PPZ. The complete amino acid sequences of PPX, PPY and PPZ and an almost complete sequence of PPV are presented. In the catalytic domain PPV and PPX are more similar to PPZA (57-69~ identity) than PPI (45 49% identity), while PPY and PPZ are more similar to PPI (6&68% identity) than PP2A (44% identity). The cDNA for PPZB, encodes a novel CaZ+ /calmodulin-dependent protein phosphatase only 62% identical to PP2B in the catalytic domain. Approaches for determining the cellular functions of these protein phosphatases are discussed.

Five pigmented isolates of Mycobacterium avium subsp. paratuberculosis were examined by pulsed-field gel electrophoresis (PFGE), IS900 restriction fragment length polymorphism (IS900-RFLP), and IS1311 polymorphism analysis using PCR. All of the pigmented isolates exhibited one of three distinct PFGE profiles with SnaBI, designated 9, 10, and 11, and with SpeI, designated 7, 8, and 9, which generated three multiplex profiles designated . IS1311-PCR analysis typed all of the pigmented isolates as sheep (S) strains. The genetic relationship between pigmented and nonpigmented isolates was investigated by using multiplex PFGE data from the analysis of both the 5 pigmented isolates and 88 nonpigmented isolates of M. avium subsp. paratuberculosis from a variety of host species and geographic locations. It was possible to classify the isolates into two distinct types designated type I, comprising the pigmented isolates, and type II, comprising the nonpigmented isolates, which exhibit a very broad host range.Mycobacterium avium subsp. paratuberculosis is the etiological agent of paratuberculosis (Johne's disease), a fatal chronic granulomatous enteritis affecting principally ruminants. The organism is distinguished from other subspecies of M. avium by its mycobactin requirement for in vitro growth (22) and by the presence of the insertion sequence IS900 (9). Molecular typing procedures such as pulsed-field gel electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP) coupled with hybridization to IS900 (IS900-RFLP) have shown that in comparison with other pathogens, there is relatively little genetic variability within this subspecies. However, there is a small subset of isolates distinguished by their prominent yellow or orange pigment that have not been characterized fully.Pigmented M. avium subsp. paratuberculosis has been isolated in cases of lepromatous ovine paratuberculosis (18)(19)(20). In such cases, the affected intestine appears yellow or orange, probably due to the very large number of organisms present in the lesions. The organisms are difficult to culture, and few isolates have been maintained in collections. The pigmentation appears to be an inherent characteristic, as it is present at all stages of growth and is not altered by animal passage. The pigmented strains appear to have a host preference for sheep, although they can produce disease in cattle following experimental infection (21) and there has been one report of a naturally occurring bovine case (24). The purpose of this study was to analyze available pigmented isolates and investigate their genetic relatedness to other, nonpigmented M. avium subsp. paratuberculosis isolates. MATERIALS AND METHODSM. avium subsp. paratuberculosis isolates. Five pigmented and 88 nonpigmented strains of M. avium subsp. paratuberculosis were analyzed in this study. Four of the pigmented strains were isolated at the Moredun Research Institute, Penicuik, Scotland, and Finn Saxegaard (National Veterinary Institute, Oslo, Norway) kindly provided pigmented...

A survey of 22 farms confirmed the presence of paratuberculosis in wild rabbits in Scotland. Regional differences were apparent in the prevalence of the disease in rabbits, with a significantly higher incidence occurring in the Tayside region. Statistical analysis showed a significant relationship between a previous history or current problem of paratuberculosis in cattle and the presence of paratuberculosis in rabbits on the farms. Molecular genetic typing techniques could not discriminate between selected rabbit and cattle isolates from the same or different farms, suggesting that the same strain may infect and cause disease in both species and that interspecies transmission may occur. The possibility of interspecies transmission and the involvement of wildlife in the epidemiology of paratuberculosis have important implications for the control of the disease.

We have used rocket immunoelectrophoresis and immunoblotting to detect myeloperoxidase in synovial fluid from patients with rheumatoid arthritis. This protein was enzymatically inactive but its identity as myeloperoxidase was confirmed by comparing its subunit structure with that of the purified enzyme. When neutrophils were stimulated to secrete myeloperoxidase in vitro, a polypeptide with an apparent molecular mass of 62 kDa was detected extracellularly by immunoblotting. Neutrophils isolated from synovial fluid showed a reduced level of this 62 kDa polypeptide but it was detected extracellularly in synovial fluid by immunoblotting. Thus, we conclude that neutrophils in synovial fluid from patients with rheumatoid arthritis have been activated in vivo to secrete myeloperoxidase and propose that the products of this enzyme system can contribute to the tissue damage associated with this disease.

Summary Here, we describe the development of a method that exploits bacteriophage D29 as a lysis agent for efficient DNA extraction from low numbers of mycobacterial cells. This method (Actiphage®) used in combination with PCR achieved rapid and sensitive (LOD ≤ 10 cell ml−1) detection and identification of viable, pathogenic mycobacteria in blood samples within 6 h. We demonstrate that mycobacteriophage D29 can be used to detect a range of mycobacteria from clinical blood samples including both Mycobacterium tuberculosis complex and Mycobacterium avium subsp. paratuberculosis without the need for culture and confirms our earlier observations that a low‐level bacteraemia is associated with these infections in cattle. In a study of M. bovis‐infected cattle (n = 41), the sensitivity of the Actiphage® method was 95 % (95 % CI; 0.84–0.99) and specificity was 100 % (95% CI; 0.92–1). We further used Actiphage® to demonstrate viable Mycobacterium avium subsp. paratuberculosis is present in the blood of Johne’s infected cattle. This method provides a revolutionary new tool for the study of infections caused by these difficult to grow pathogens.