Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1x MIC) gave 99.99%o reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.

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SUMMARYA large outbreak of Legionnaires’ disease was associated with Stafford District General Hospital. A total of 68 confirmed cases was treated in hospital and 22 of these patients died. A further 35 patients, 14 of whom were treated at home, were suspected cases of Legionnaires’ disease. All these patients had visited the hospital during April 1985. Epidemiological investigations demonstrated that there had been a high risk of acquiring the disease in the out patient department (OPD), but no risk in other parts of the hospital. The epidemic strain ofLegionella pneumophila, serogroup 1, subgroup Pontiac la was isolated from the cooling water system of one of the air conditioning plants. This plant served several departments of the hospital including the OPD. The water in the cooling tower and a chiller unit which cooled the air entering the OPD were contaminated with legionellae. Bacteriological and engineering investigations showed how the chiller unit could have been contaminated and how an aerosol containing legionellae could have been generated in the U–trap below the chiller unit. These results, together with the epidemiological evidence, suggest that the chiller unit was most likely to have been the major source of the outbreak.Nearly one third of hospital staff had legionella antibodies. These staff were likely to have worked in areas of the hospital ventilated by the contaminated air conditioning plant, but not necessarily the OPD. There was evidence that a small proportion of these staff had a mild legionellosis and that these ‘influenza–like’ illnesses had been spread over a 5–month period. A possible explanation of this finding is that small amounts of aerosol from cooling tower sources could have entered the air–intake and been distributed throughout the areas of the hospital served by this ventilation system. Legionellae, subsequently found to be of the epidemic strain, had been found in the cooling tower pond in November 1984 and thus it is possible that staff were exposed to low doses of contaminated aerosol over several months.Control measures are described, but it was later apparent that the outbreak had ended before these interventions were introduced. The investigations revealed faults in the design of the ventilation system.

and § Public Health Laboratory, PrestonB R U C E L L O S I S presents in three forms-acute brucellosis, chronic brucellosis following an acute attack and chronic brucellosis of insidious onset. Because the rate of isolation of Brucella abortus from blood or tissues is low, laboratory diagnosis of this disease is based mainly on the results of serological tests, the results of which depend on the clinical form and stage of the infection. The use of some of these tests in chronic brucellosis has already been discussed (Kerr, Coghlan, Payne and Robertson, 1966a and b).The variety of technical procedures employed at present leads to results that cannot be equated between laboratories. The purposes of this paper are to present details of these tests so that comparable results may be obtained and to indicate the way in which the results may be interpreted to help to determine the stage of the infection. The paper is presented in two parts; the first deals with a description of the tests and the second with their interpretation. There is also an appendix in which certain technical minutiae omitted from the main text may be referred to. The following tests are dealt with: (A) the standard agglutination test ; (B) the mercaptoethanol test (agglutination in the presence of 2-mercaptoethanol) ; (C) the anti-human globulin (Coombs) test (AHG test) for non-agglutinating antibodies ; (D) the complement fixation (CF) test. TECHNICAL PROCEDURESIn all cases Brucella abortus (BY. abortus) agglutinable suspensions are used for the tests, but in certain circumstances, to be discussed later, Brucella melitensis (Br. melitensis) suspensions are included. The standard agglutination test Mate vialsAntigen. Three different Br. abortus suspensions have been available to the authors for investigation. They are " Weybridge " standardised BY. abortus " agglutination concentrate", produced by the Veterinary Laboratories of the Ministry of Agriculture, Fisheries and Food, Weybridge, England; Br. abortus " concentrated 0 suspension " produced by the Standards Laboratory, Central Public Health Laboratory, London (PHLS); and " Wellcome " Br. abortus " agglutinable concentrated smpension " produced by Burroughs Wellcome and Co. These three suspensions differ in their cell concentrations, and since an inverse linear relation exists between the concentration of the bacterial cells and the

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