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Zoites showed to be sensitive to temperature. Under refrigeration down to four degrees Celsius they tumed into cysts. The cysts were resistant even under -50 degrees Celsius for months and sur- vived in the pH range 5.0-9.0. They were not affected by strepto- mycin and penicillin in tissue cultures. Zoites showed to be sensitive to temperature. Under refrigeration —_lating respiratory syncytial virus, and from the surfaces in cubicles down to four degrees Celsius they tured into cysts. The cysts in which infants with acute bronchiolitis were being nursed. were resistant even under -50 degrees Celsius for months and sur- A more extensive research has been carried out because of the vived in the pH range 5.0-9.0. They were not affected by strepto- possibility that these amoebas may be associated with disease. mycin and penicillin in tissue cultures. The first amoebas were tsolated at Booth Hall Children's Hospital in the cubicle occupied by a ten-week-old infant with an illness AMOEBAS RECOVERED FROM MONKEY KIDNEYS diagnosed as acute bronchiolitis. First, only syncytial respiratory KILLED ANIMALS virus was isolated and the child was discharged, but later an A series of articles started appearing in Science (1958) and _ unidentified cytopathic effect was noticed in the tissue cultures Journal of Pathology (1959), written by Culbertson and collabora- inoculated from three air samples, and the causative agent was tors, which confirmed that amoebas caused brain disease and _ provisionally identified as the “Ryan virus”. Later isolations were death within days in monkeys and mice. They first found and made from a floor swab and from a sample of air aspirated recognised amoebas accidentally in the lesions of animals that through the bedclothes in a cubicle occupied by another infant died from the inoculation of a tissue-culture fluid of trypsinised with respiratory syncytial virus infection. Amoebas were isolated monkey kidney cells. It was thought this was caused by yet anoth- from one of the samples taken in the laboratory and were identi- er unknown monkey virus, but it was later shown to be _ fied as Hartmanella castellanii. Acanthamoeba. It had been Pereira et al. (1966) who originally isolated "Ryan | Following intracerebral and intraspinal inoculation, "extensive virus" in a post-mortem bronchial swab of a seven-months-old choriomeningitis and destructive encephalomyelitis occurred" and baby boy, Ryan. Six days before admission, baby Ryan developed killed monkeys in four to seven days and mice in three to four a sore throat and ulcers in the mouth which later spread to the days. Intravenous injections of the amoebas resulted in perivascu- face. He was unwell, could not suck and developed loose stools. lar granulomatous lesions. Intranasal inoculation in mice resulted © The day before admission he developed a cough and started to in fatal infection in about four days. These mice exhibited ulcera- | vomit. He was drowsy and dyspnoeic, made jerky movements and tion of the nasal mucous membrane with died soon after admission. The chest direct invasion of the adjacent base of the — X-ray revealed patchy consolidation skull and involvement of the frontal lobes of Amoebas were found in and dilatation of the right side of the the brain. There were amoebas in the lungs, swimming pools in heart. Necropsy showed some and they caused severe pneumonic reaction. * : emphysema, petechiae and small Pulmonary veins were invaded and there Czechoslovakia where 16 areas of congestion and alveolar were numerous thrombi containing many young victims died of acute haemorrhage in the lungs, a fatty amoebas. Haemorrhage was a common fea- liver, prominent mesenteric nodes, ie 4° ture. Sections of the kidney showed amoe- purulent meningoencephalitis, and mucopus in the ears. bas present in the glomerular capillaries and thermal pools In northern Escherichia coli bacteria were cul- (Culbertson et al., Science, 1958), which fur- New Zealand tured from the cars. Death was diag- ther demonstrated that Acanthamoebae were : : . nosed as due to a respiratory infection indeed pathogenic for monkeys and mice. associated with encephalitis and Amoebas showed the ability to migrate hepatitis. Vaccination status was not through the tissues. The size of the inoculum did not matter given, although considering the age of the baby he would have much: both small and large inoculums produced amoebic inva- _ received up to three doses of the DPT and polio vaccines. sions. Small inoculums tended to cause death by pulmonary inva- Ryan 2 and Ryan 3 ‘viruses’ were cultured from a man of 29 sion, while large inoculums were followed by brain invasions. years and a woman aged 30. Intragastric inoculations were unsuccessful most probably because Ryan 4 was isolated from a ten-year-old girl who was ill with amoebic cysts were shown to be dissolved by bile. fever, cough, mild generalised lymphadenopathy and Culbertson et al. (1958 and 1959) concluded that without ques- splenomegaly, and some vomiting for four or five days before tion the Acanthamoeba is able to enter the body by its own power admission. Her mother suffered from a similar illness. and may be able to do so in nature as well. However, the question Vaccination status was not given. of possible contamination and introduction of Acanthamoebae via Ryan 5 was isolated from a two-year-old girl suffering from the polio vaccines was not mentioned. fever and convulsions and some congestion of the throat. Ryan 6 was cultured from a four-year-old child with fever and ISOLATION OF AMOEBAS IN SOIL & AIR, AND cough. She was diagnosed with bronchitis and has subsequently THROATS & NOSES OF PEOPLE recovered. Her two-year-old sister suffered similar symptoms at Furtber research concentrated on isolation of amocbas from the same time. Syncytial respiratory virus was also isolated from water and soil, and throats and noses of healthy people. these children, The attention of amoeba researchers would appear to have been Armstrong and Pereira (1967) identified the Ryan virus as an diverted away from the monkey kidney cultures and the polio vac- amoeba: Hartmanella castellanii. They had no doubt that these cine. amoebas came from the human respiratory tract. Indeed, amoebas do occur widely in nature, ¢.g., in lakes, pools According to Casemore (1969), tissue cultures in laboratories and mud puddles. Amoebas collected with mud from these lakes may be contaminated by amoebas living in the air. grew out in vitro on the fourth day. When inoculated into the Amoebas, including those pathogenic to animals and humans, nose, two mice became ill and were sacrificed before death. were found in swimming pools (Cerva et al., 1969) in Amoebas caused brain abscesses in these mice. Czechoslovakia where 16 young victims died of acute purulent Amoebas were tsolated from the air (Kingston and Warhurst, meningoencephalitis, and thermal pools in northem New Zealand 1969) in the UK during an investigation into the possibility of iso- | (Mandal et al., 1970; Cursons and Brown, 1972; and others). Four ISOLATION OF AMOEBAS IN SOIL & AIR, AND THROATS & NOSES OF PEOPLE Furtber research concentrated on isolation of amoebas from water and soil, and throats and noses of healthy people. The attention of amoeba researchers would appear to have been diverted away from the monkey kidney cultures and the polio vae- cine. Indeed, amoebas do occur widely in nature, ¢.g., in lakes, pools and mud puddles. Amoebas collected with mud from these lakes grew out in vitro on the fourth day. When inoculated into the nose, two mice became ill and were sacrificed before death. Amoebas caused brain abscesses in these mice. Amoebas were tsolated from the air (Kingston and Warhurst, 1969) in the UK during an investigation into the possibility of iso- 44 « NEXUS APRIL-MAY 1996