C hair of Medical Biology, M icrobiology, V irology, and I mmunology

1. Chair of Medical Biology, Microbiology, Virology, and Immunology Yersinia. Francisella. Brucella. Bacilli. Prof. S. Klymnyuk

2. Yersinia pestis belongs to Genus Yersinia which includes in family Enterobacteriaceae. The causative agent of plague, Yersinia pestis, was discovered by the French microbiologist A. Yersin in Hong Kong in 1894. Ukrainian scientists D. Samoilovich, D. Zabolotny and others contributed greatly to the study of the mechanisms of its transmission. The French microbiologists G. Girard and T. Robic obtained a live vaccine from the attenuated EV strain.

3. Bubonic plague, caused by Y. pestis, is an ancient disease that has killed millions of people over the centuries. For example, it is believed to have killed more than 100 million persons in an epidemic in the sixth century. Another epidemic in the 14th century killed one fourth of the European population, and the London plague in 1665 killed more than 70,000 persons. In 1893, an epidemic began in Hong Kong and spread to India where more than 10 million individuals died over a 20-year period.

4. Morphology. The plague bacillus, as seen in tissue smears, is ovoid-shaped. It is non-motile, forms no spores, and on solid media cultures is elongated in form. In preparations from tissues and cultures Y. pestis is found to have a delicate capsule. The organism stains with ordinary aniline dyes and gives a bipolar appearance, its ends staining more intensively.

5. Y. pestis

6. Cultivation. The optimum temperature for cultivation is 25-30° C. On agar slants the culture forms a viscid translucent mucilaginous mass. On agar plates it forms colonies with turbid white centres and scalloped borders resembling lace or crumpled lace handkerchiefs. In meat broth the cultures form a pellicle on the surface with thread-like growth resembling stalactites and a flocculent precipitate. Sodium sulphite, fresh haemolytic blood, sarcinic extract, and live sarcina (“feeders”) are used as growth stimulators. They are of special value when the seeded material contains a small number of organisms.

8. Differentiation of Yersinia Species

9. Some diagnostic signs differentiatingthe bacteria of plague from those of pseudotuberculosis in rodents

10. Toxin production. Y. pestis is very virulent for humans. The important virulence factors of Y. pestis seem to be directed toward two goals for the organisms: (1) invasion and proliferation within host cells, and (2) resistance to killing by the host. The incredibly high fatality rate of bubonic plague is probably primarily because of septic shock resulting from the bacteremia occurring in the disease.

11. Virulence factors that seem to be important in human disease.

12. Antigenic Makeup of Y. pestis

13. Antigenic Makeup of Y. pestis

14. Antigenic Makeup of Y. pestis

15. Pathogenicity for animals. Rodents, among them black rats, grey rats, mice, susliks, midday gerbils, tumarisks, and marmots (tarbagans) are susceptible to plague. More than 300 rodent species may spontaneously contract the disease. In addition, 19 rodent species are susceptible to laboratory infection with plague. Camels died in the Astrakhan steppes m 1911, and humans who ate camel meat contracted plague. Pigs, sheep, goats, donkeys, mules, dogs, cats, monkeys, and certain carnivores are susceptible to the disease in natural environments. However, little epidemiological importance is attached to them.

19. The disease is transmitted by the bites of fleas (e.g., Xenopsylla cheopis, the rat flea) which have previously sucked blood from an infected animal. The ingested bacilli proliferate in the intestinal tract of the flea and eventually block the lumen of the proventriculus. The hungry flea, upon biting another rodent, regurgitates into the wound a mixture of plague bacilli and aspirated blood.

20. Bubonic plague

21. Immunity. After recovery from the disease a stable immunity of long duration is acquired. Realizing this, in ancient times people living in countries invaded by plague made use of convalescents for nursing plague patients and burying corpses. Postinfection and postvaccinal immunities are predominantly due to the phagocytic activity of the cells of the lymphoid-macrophage system. An important role is played by the protective capsular antigen which serves as the basis in the preparation of chemical antiplague vaccines.

22. Laboratory diagnosis. Examination is carried out in special laboratories and in antiplague protective clothing. A strict work regimen must be observed. Depending on the clinical form of the disease and the location of the causative agent, test specimens are collected from bubo content (in bubonic plague), ulcer secretions (in cutaneous plague), mucus from the pharynx and sputum (in pneumonic plague), and blood (in septicaemic plague). Test matter is also recovered from necropsy material (organs, blood, lungs, contents of lymph nodes), rodent cadavers, fleas, foodstuff's, water, air, etc.

23. 1. Microscopy of smears, fixed in Nikiforov's mixture and stained by the Gram method or with methylene blue by Loeffler's method. 2. Inoculation of the test material into nutrient media, isolation of a pure culture and its identification. 3. Biological tests of the isolated pure culture and of material from which isolation of the organism is difficult are conducted on guinea pigs

24. Treatment.At present streptomycin is used for treatment of plague, the drug being very effective and curing even pneumonic plague in a high per cent of cases. Good results have been obtained from a combination of streptomycin with chloromycetin or tetracycline with antiplague serum. Antiplague gamma-globulin and a specific bacteriophage are also used for treatment of plague patients. Penicillin, chlortetracycline, and sulphonamides are recommended in cases with complications.

25. Prophylaxis. General prophylaxis comprises the following measures: (1) early diagnosis of plague, particularly the first cases; (2) immediate isolation and hospitalization of patients and enforcement of quarantine; (3) observation (i. e. isolation of individuals or groups of people suspected of having been in contact with infected material, daily inspection from house to house, temperature measurement twice a day, and observation during the possible incubation period); (4) thorough disinfection and extermination of rats in disease foci; (5) individual protection of medical personnel and prophylactic treatment with streptomycin and vaccination;

26. (6) prophylactic measures and systematic observation carried out by plague control laboratories, stations, and institutes in endemic areas; (7) observance of international plague control conventions; (8) security measures from plague invasion at frontiers. Specific prophylaxis is accomplished with live EV vaccine.

27. Causative Agent of Tularaemia The tularaemia bacteria are short coccal-shaped or rod-like cocci measuring 0.2-0.7 mcm. In old cultures the organisms retain the coccal form. They are non-motile, polychromatophilic, and Gram-negative. In the animal body they are sometimes surrounded by a fine capsule.

28. Cultivation. The tularaemia organism is an aerobe which does not grow on ordinary media, but grows well at 37° C on media rich in vitamins, e. g. yolk medium which consists of 60 per cent of yolk and 40 per cent of a 0.85 per cent sodium chloride solution with pH 6.7-7A. The organisms are cultured in a thermostat for 2-14 days.

29. Fermentative properties. Tularaemia bacteria break down proteins with the elimination of hydrogen sulphide, and do not produce indole. They ferment glucose, levulose, mannose, and maltose, with acid formation. Dextrin, saccharose, and glycerin fermentation is not a stable property. Biochemical properties are unstable and liable to comparatively rapid changes. Toxin production. The existence of a soluble toxin in tularaemia bacteria has not been demonstrated. The organism's virulence is associated with its K-antigen. The tularaemia bacterium grows poorly in liquid media, and for this reason it is difficult to isolate any toxin.

30. Pathogenicity for animals. The organism is pathogenic for water rats, field voles, grey rats, common field mice and house mice, hares, susliks, chipmunks, hamsters, muskrats, gerbils, moles, shrews, and other animals. Among the domestic animals camels, sheep, cats, dogs, and pigs are susceptible to the disease, and among laboratory animals, guinea pigs and white mice.

32. Laboratory diagnosis. 1. Allergy develops on the third-fifth day of the disease. For this reason, intracutaneous and cutaneous tests with tularine are made for early diagnosis. In tularaemia patients the test gives a positive reaction 6-12 hours after inoculation of tularine.

33. 2. In the second week of the disease agglutinins begin to accumulate in the blood. They are detected by carrying out the agglutination reaction by the blood-drop and volume methods. In some cases this test may give a positive reaction with material containing brucella organisms, since they possess antigens common to tularaemia bacteria. 3. The tularaemia culture is isolated by the biological method as it is impossible to recover the pathogen directly from a tularaemia patient. For this purpose white mice or guinea pigs are infected by material obtained from people suffering from the disease (bubo punctate, scrapings from ulcers, conjunctiva! discharge, throat films, sputum, and blood).

34. 4. Laboratory diagnosis of rodent tularaemia is made by microscopy of smears from organs, precipitin ring reaction (thermoprecipitation), and biological tests. Water, foodstuffs, and blood-sucking arthropods are examined by biological tests.

35. Prophylaxis comprises the following measures: (1) systematic observation, absolute and relative registration of rodent invasion, and extermination of rats; (2) prevention of mass reproduction of the rodents; (3) protective measures in agricultural enterprises against contamination by tularaemia-infected rodents; (4) protection of foodstuffs and water from rodents; (5) control of ticks, horseflies, stable-flies, mosquitoes, and protection from these insects; (6) specific prophylaxis with a live vaccine. The vaccine is prepared in a dry form. A single application is made by rubbing it into the skin and it produces immunity for a period of 3-6 years.

36. Brucellae Brucella abortus Brucella melitensis Brucella cuis Brucellae are small, coccal, ovoid-shaped micro-organisms 0.5-0.7 mcm in size. Elongated forms are b.6-1.5 mem in length and 0.4 mcm in breadth. Under the electron microscope Brucella organisms of cattle, sheep and goats appear as coccal and coccobacilary forms, while those of pigs are rod-shaped. They are Gram-negative, non-motile, and do not form spores or capsules (in some strains capsules are sometimes present). DNA contains 56 to 58 per cent of G+C.

39. Cultivation. The organisms are aerobic. When cultivated from material recovered from patients, they grow slowly, over a period of 8-15 days.

40. Brucella organisms may be cultivated on ordinary media, but they grow best on liver-extract agar and liver-extract broth. On liver-extract agar the organisms form round, smooth colonies with a white or pearly hue. In liver-extract broth they produce a turbidity, and subsequently a mucilaginous precipitate settles at the bottom of the tubes Brucella organisms grow well on unfertilized eggs and on the yolk sac of a 10-12-day-old chick embryo. The brucellae of bovine origin (Brucella abortus) only grow in an atmosphere of 10 per cent carbon dioxide, which serves as a growth factor.

41. Colonies of Brucella

42. Fermentative properties. Brucellae do not liquefy gelatin and do not produce indole. Some strains produce hydrogen sulphide, break down urea and asparagin, reduce nitrates to nitrites, and hydrolize proteins, peptones and amino acids, with release of ammonia and hydrogen sulphide. No carbohydrates are fermented, although a small number of strains ferment glucose and arabinose. Toxin production. Brucellae do not produce soluble toxins. An endotoxin is produced as a result of disintegration of the bacterial cells. This endotoxin possesses characteristic properties and may be used in allergic skin tests

43. Antigenic structure. The organism contains four antigens: A, M, O, Vi, G and R. The M-antigen is predominant among brucellae of sheep and goats, and the A-antigen, in the other species. Substances of polysaccharide character, with no type specificity, have been extracted from brucellae of cattle, sheep and goats.

44. Pathogenity for animals. Goats, sheep, cattle, pigs, horses, camels, deer, dogs, cats, and rodents (rats, mice, susliks, hamsters, rabbits, field-voles, water rats, and other animals) are all susceptible to infection by brucellae. The high concentration of brucellae in the placenta of cattle is explained by the presence in this tissue of the growth stimulator erythrol.

45. In human beings brucellosis is characterized by undulant fever with atypical and polymorphous symptoms. The disease may assume an acute septic or a chronic metastatic course. The structural and motor systems, haemopoietic, hepatolienal, nervous and genital systems are often involved. Pregnant women may have miscarriages. Often brucellosis recurs, continuing for months and years. The death rate is 1-3 per cent. The diagnosis of mild, asymptomatic forms presents difficulties and is based on laboratory tests.

46. Laboratory diagnosis. The patient's blood and urine (for isolation of the pathogen), serum (for detection of agglutinins), milk and dairy products (for detection of brucellae or agglutinins in milk) are examined. The microbe is isolated in special laboratories. 1. Culture isolation. Since brucellosis is often accompanied by bacteraemia, blood is examined during the first days of the disease (preferably when the patient has a high temperature). The cultures are grown for 3-4 weeks or more. Five to ten per cent of carbon dioxide is introduced into one of the flasks (for growth of the 23 bovine species of the bacteria). Inoculations on agar slants are made every 4-5 days for isolation and identification 'of the pure culture.

47. Brucella susceptibility to stains

48. An antiphage serum is introduced into the cultures for neutralization of the phage which inhibits the growth of brucellae. The best results are obtained when the blood is inoculated into the yolk of an unfertilized egg or the yolk sac of a chick embryo. Growth is examined every 2-3 days. If the blood culture produces a negative result bone marrow obtained by sternum puncture is inoculated onto solid and liquid media for isolation of myelocultures.

49. Susceptibility to phages

50. The urine is also examined. It is obtained with a catheter, centrifuged, and 0.1 ml of the precipitate is seeded onto agar plates containing 1 :200000 gentian violet. In some cases faeces, cow's and human milk, and amniotic fluid of sick humans and animals are examined for the presence of Brucella organisms.