Rickettsioses studies

Rickettsioses studies 1. Natural foci of rickettsioses in the Armenian Soviet Socialist Republic I. V. TARASEVIt,1 L. F. PLOTNIKOVA,1 N. F. FETISOVA,1 V. A. MAKAROVA,l V. A. JABLONSKAJA,1 J. tEHA&K,' M. ZUPANCItOVA,2 E. KOVA)IOVA,2 J. OJRVOLGYI,2 R. BREZINA,2 A. V. ZAKARJAN,8 & M. E. KOCINJAN' Ixodid ticks collected in the Armenian SSR during 1971 and 1974 were positive for rickettsiae of the spotted fever group, as confirmed by haemocyte tests and by isolation experiments. Serum specimens collected from human beings and from domestic and wild animals in the same areas contained antibodies against such rickettsiae and against Coxiella burnetii. These results indicate the existence of mixed natural foci of rickettsioses of the spottedfever group and of Q fever in the Armenian SSR. Investigation of the natural foci of endemic rickettsioses in the Armenian SSR started in 1950. Several human cases of Q fever (8) and of spotted- fever-like infection (6) were found and rickettsial strains similar to those of the spotted fever group were isolated from Dermacentor marginatus ticks collected on cattle, from Rhipicephalus sanguineus ticks collected on sheep and dogs, and from the brains of hamsters (7, 9, 10). In view of the new observations recorded recently, ideas have changed markedly as regards the geo- graphical distribution of rickettsioses of the spotted fever group in parts of Europe and Asia. Natural foci of tick-borne typhus were found in the Tula region (11) and in Turkmenistan (12). Insufficiently characterized. rickettsiae belonging to the spotted fever group were isolated in Turkmenistan (12), in Czechoslovakia (1), and in Pakistan (17). There is a need for proper identification and classification of the rickettsial strains isolated, in particular in eastern Europe, where the areas of occurrence of tick-borne typhus, boutonneuse fever, and rickettsial- pox may overlap. However, the methods previously 1Scientist, Gamaleja Institute of Epidemiology and Microbiology, Moscow, USSR. Requests for reprints should be addressed to the first author. ' Scientist, WHO Collaborating Centre for Rickettsial Reference and Research, Institute of Virology, Slovak Academy of Sciences, Bratislava, Czechoslovakia. ' Scientist, Institute of Epidemiology, Microbiology, and Medical Parasitology, Erevan, Armenian SSR, USSR. used for interspecies differentiation of rickettsiae of the spotted fever group were inadequate to deter- mine the species of rickettsial strains isolated in the Tula region, in Turkmenistan, and in Czecho- slovakia. To contribute to knowledge of the ecology of spotted fever group rickettsiosis and to the charac- terization of its causative agent, Soviet and Czecho- slovak rickettsiologists carried out two field studies in the Armenian SSR in 1971 and 1974. The results of field and laboratory investigations of natural foci of endemic rickettsioses in different regions of that territory are presented in this paper. COLLECTION OF MATERIALS A total of 894 ticks was collected in 7 districts of the Armenian SSR at the end of September 1971 and in 3 districts at the beginning of October 1974. A detailed description of the localities in question is given in Table 1. The ticks were collected either by trailing a white woollen blanket over pastures (in 1971) or from sheep (in 1971 and 1974). T-he ticks were kept in closed test-tubes at 4°C until required for examination. Small mammals were captured in special traps (4). Blood samples for serological examination were taken from the orbital sinus. Human sera were obtained from hospitals or from hygiene and epidemiology stations in the Leninakan and Erevan districts. Cattle and sheep sera were 3415 - 25 BULL. WORLD HEALTH ORGAN., Vol. 53, 1976 I. V. TARASEVI6 ET AL. Table 1. Areas of the Armenian SSR where ticks and small rodents were collected District Locality Habitat Landscape characteristics Altitude (m) Ahurjan Leninakan mountainous steppe cultivated steppes 1500 Artik Snaberd mountainous steppe wild steppes 1900 Karavansaraj mountainous steppe wild steppes 1900 Amasija Surabad mountainous steppe meadow steppes 2200 Aparan Tudiur alpine alpine meadows 2500 Ararat timankend a mountainous steppe meadow steppes 2000 Agtarak Kot semidesert semicultivated desert 700- 800 Sevan Tzovagjuh mountainous forest wild steppes 1900 Etmiadzin Samagar desert semicultivated desert 700- 800 Kafan Arcvanika mountainous forest semicultivated desert and rare forests 1500 Sisijan Borisovka a mountainous steppe meadow steppes 2500 a Collections took place in 1974. In all other localities, collections took place in 1971. supplied by abattoirs in Leninakan and Erevan, to which animals are brought for slaughter from 9 dis- tricts (Kalinin, Gukasjan, Ahurjan, Ararat, Masin, Abovjan, Ecmiadzin, Amasija, and Sisijan). All sera were stored at -20°C. LABORATORY STUDIES Detection of rickettsiae in ticks, and isolation of rickettsial strains The ticks were sorted into groups of twenty according to species, stage, and sex. Each group of ticks was allowed to feed on guinea-pigs or rabbits, which were observed for symptoms of rickettsioses for 3 weeks. The excreta of partly fed ticks were tested further for the presence of rickettsiae by inoculation into guinea-pigs. All unfed and partly fed ticks were examined for infestation with rickettsiae by the haemocyte test (14) with the Gimenez staining method and by parallel immunofluorescence testing. After the ticks had been thoroughly washed in 70°/. ethanol, repeatedly rinsed in sterile distilled water, and ground, suspensions in phosphate- buffered saline (PBS), pH 7.2, without antibiotics, were prepared from each of the unfed ticks that had been found to be negative in the haemocyte test, for intraperitoneal injection into guinea-pigs. Partly fed ticks that had been found to contain rickettsiae in the haemocyte test were washed in the same way and a suspension from each tick was prepared in 1.5 ml of PBS containing 100 IU of penicillin and 100 ,ug of streptomycin. Then 0.25 ml of each suspension was injected into the yolk sac of a 6-7-day-old chick embryo. In almost every case the embryo died 4-6 days after infection and rickettsiae were present in yolk sac smears. Blind passages were not per- formed. Serological examination All human and animal sera were examined by complement fixation or by the microagglutination test. The former was performed with corpuscular phase-II C. burnetii antigen for the detection of Q fever antibodies and with spotted fever group- specific soluble antigen for the detection of anti- bodies to rickettsiae of that group. The micro- agglutination test was performed as described else- where (5) with stained corpuscular phase-II C. burnetii converted antigen or stained corpuscular antigen of spotted fever group rickettsiae. Antigens were pre- pared in the Gamaleja Institute of Epidemiology and Microbiology, Moscow, in 1971, and in the Institute of Virology, Slovak Academy of Sciences, Bratislava, in 1974. Human sera and the sera of small rodents were also subjected to the microagglutination test with stained Rickettsia canada antigen. 26 RICKETTSIOSES STUDIES. 1 RESULTS As shown in Table 2, rickettsiae or rickettsia-like organisms were found by Gim6nez staining in 163 (18.2%) of the 894 ticks collected. However, fewer ticks were found positive by parallel immuno- fluorescence testing: 23 (2.6%) containing C. burnetii and 105 (11.7%) containing spotted fever group rickettsiae. Thirty-seven strains of the spotted fever group were isolated from D. marginatus and 2 strains from D. reticulatus. Confirmation that ticks collected in 1971 were infected with rickettsiae of the spotted fever group was obtained when guinea-pigs that had been sub- jected to tick bites or inoculated with tick excreta developed symptoms of rickettsial disease and anti- body response (Table 3). Further investigation in guinea-pigs of ticks that had been found to be nega- tive in the haemocyte test revealed rickettsiae of the spotted fever group in 4 out of 12 groups consisting of 10-30 ticks each. In these cases the ticks were of the D. marginatus species, collected in iimankend (district of Ararat). The results of the serological examinations are given in Table 4. The most important finding was the presence of antibodies to spotted fever group rickettsiae in human sera. A higher prevalence of such rickettsiae and of C. burnetii, as confirmed by a greater proportion of positive human and animal sera, was found in 1974. The antibody titres in all sera tested ranged between 4 and 16, except for two sheep sera with titres of 32 to C. burnetii. Human Table 2. Infection with rickettsiae of ticks collected from sheep in the Armenian SSR No. of ticks containing rickettsiae as detected by: No. of strains No. of ticks immunofluorescence of spotted District Locality Tick species investigated Gim6nez technique fever group _______staining__ isolatedstaining C. brnet..,li spottedfever group females males females males females males females males females males Artik Karavansaraj D. marginatus 26 7 4 2 1 - 2 2 - 1 gnaberd D. marginatus 55 48 5 7 - 3 4 3 6 2 H. sulcata 1 - - - Aparan Tudiur D. marginatus 3 7 2 - 1 - 1 - - Attarak Ko§ D. marginatus 3 5 - 3 - 2 - 2 Amasija Surabad D.marginatus 18 9 3 2 1 2 1 1 - - Ahurjan Leninakan D. marginatus 28 18 8 5 - - 7 4 4 Ararat Cimankend D. marginatus 102 40 14 7 2 1 13 6 10 H. sulcata 36 2 2 - - - 1 - - ERmiadzin Samagar D. marginatus 78 15 19 3 1 1 15 3 7 1 H. sulcata 11 1 - - - - - - - Kafan Arcvanik a D. marginatus 13 7 - 1 - - - 1 1 H. sulcata 12 7 - - - - - - Sevan Tzovagjuh D. reticulatus 208 73 49 19 8 - 24 8 2 Sevan a D. reticulatus 18 4 - - - - - - Sisijan Borisovka a D. marginatus 28 10 4 4 - - 4 3 4 1 H. sulcata 1 - - - - Total 641 253 110 53 14 9 72 33 33 6 a Ticks collected in 1974. In other localities, the collections took place in 1971. 27 I. V. TARASEVIe ET AL. Table 3. Evidence of rickettsiosis and antibody response in guinea-pigs bitten by ticks or inoculated with their excreta Symptoms of rickettsiosis and Symptoms of rickettsiosis and antibody response after tick bite antibody response after inoculation of tick excreta Locality of antibodies a to: antibodies a to: (district) fever scrotal spotted fever scrotal spotted reaction C. burnetil fever reaction C. burnetii fever group group Tzovagjuh D. reticulatus - - - 128 - + - 64-128(Sevan) Leninakan D. marginatus - + - 128 - + - > 128(Ahurjan) Snaberd D. marginatus + - - > 128 + + - > 128(Artik) Surabad D. marginatus + - - n.d. b _ + 16 8-64(Amasija) Samagar D. marginatus - - - 64 + + - 64(Edmiadzin) a Reciprocals of initial serum dilution. b Not done because of the early death of the animal. Table 4. Rickettsial antibodies in human and animal sera collected in the Armenian SSR in 1971 and 1974 No. of sera No. and proportion (%) No. of sera No. and proportion (%) Serum examined of positive sera with antigen of: examined of positive sera with antigen of: in 1971 C. burnetii spotted fever group in 1974 C. burnetii spotted fevergroup human 688 2 (0.3) 10 (1.5) 746 42a (5.6) 79 b (10.6) cattle 97 1 (1.0) 0(0) 185 46a (24.9) 26b (14.1) sheep 295 0 (0) 0 (0) 172 25a (14.5) 15 b (8.7) M. arvalis 165 0 (0) 0 (0) 15 1 a (6.7) 5 a (33.3) Mus musculus 31 1 a (3.2) 5a (16.1) a With microagglutination test. b With complement fixation test. sera and the sera of small rodents, tested in parallel, were negative in the microagglutination test with stained R. canada antigen. DISCUSSION It is evident, from the data on the presence of spotted fever group rickettsiae in ticks and small mammals in regions of the Armenian SSR varying in altitude from 700 m to 2500 m, that these rickettsiae possess a marked ability to adapt to various habitats. This applies also to the rickettsiae of the spotted fever group isolated in Czechoslovakia (1) and in Pakistan (16, 17), the conditions for the circulation of rickettsiae in Pakistan being similar to those in Armenia. The typical feature of all the natural foci in Europe and Asia where rickettsiae of the spotted fever group have been isolated is the heavy infection of ticks of the Dermacentor genus with these rickett- siae. Ticks of this genus are among the most common parasites of small wild mammals and of domestic animals, and thus may be the most important reservoirs and vectors of spotted fever group rickett- siae in the Armenian SSR. This is confirmed also by 28 RICKETTSIOSES STUDIES. 1 the high incidence of antibodies to such rickettsiae in Microtus arvalis trapped at an altitude of 1500 m in one mountainous desert region of Armenia. Similar data on the infection of ticks and wild rodents with rickettsiae of the spotted fever group were obtained in Czechoslovakia (15). All the rickett- sial strains from Armenia were isolated from ticks of the Dermacentor genus, namely, D. marginatus. None was isolated from Haemaphysalis sulcata. However, because our material was collected only in autumn, it is not excluded that other tick species and genera occurring in other seasons may play an important part in the ecology of spotted fever group rickettsiae. Serological examination of healthy human beings, domestic animals, and small wild mammals showed differences in the prevalence of spotted fever group rickettsiae in Armenia between 1971 and 1974. These differences can be explained to some extent by the different antigens and serological tests used. The antigen used in 1971 was prepared from the R. siberica strain Necvetajev, which had been passaged more than one hundred times in chick embryos; that used in 1974 was prepared from a freshly isolated strain of spotted fever group rickett- siae (15). Though both antigens were soluble, their differing passage history under laboratory con- ditions may have led to differing serological reactivity. Furthermore, in 1971, the human sera were collected mostly among town dwellers who had had no contact with natural foci, and the animal sera were taken in Ararat valley from domestic animals that had been kept free of ticks by means of regular checks. It must be remembered also that variations occur in the natural foci, e.g., in the degree of infection of ticks with rickettsiae and in the incidence of rickettsial antibodies in wild animals- as found by long-term observation of natural foci of Rocky Mountain spotted fever (2, 3, 13). There remains the serious and unsolved problem of the proper identification and species characteriza- tion of the rickettsial strains isolated. Preliminary experiments have shown that those of the spotted fever group possess a broad spectrum of virulence for laboratory animals (M. Zupancicova & V. A. Makarova, unpublished observations, 1973). Further investigation of the biological properties of these rickettsial isolates, especially their antigenicity and immunogenicity, is necessary. Serological classification of isolates from Pakistan and Thailand (16) revealed that Rickettsia siberica, R. conorii, and a new, so far unclassified, rickettsia of the spotted fever group occur on the Indian subcon- tinent. Similarly, R. siberica and a rickettsial agent different from R. siberica, named R. deserti pavlov- skij (12), were isolated in Turkmenistan, which is close to Armenia. On the basis of our results and of the previous ecological and clinical findings, it may be assumed that various rickettsiae of the spotted fever group may occur in the Armenian SSR. RIESUMJ2 tTUDES SUR LES RICKETTSIOSES. 1. FOYERS NATURELS DE RICKETTSIOSES EN R#PUBLIQUE SOCIALISTE SOVIETIQUE ARMENIENNE On a recherche, par l'epreuve des hemocytes et par des tests d'isolement, la pr6sence de rickettsies dans des tiques ixodes, recueillies dans la Republique socialiste sovietique arm6nienne en 1971 et 1974. L'6preuve des hemocytes, effectuee par la methode de coloration de Gimenez, a r6vele la presence de rickettsies du groupe de la fievre pourpree ou d'organismes apparentes dans 163 tiques sur 894 (18,2 %). Sur les 163 tiques positives, 2,6% l'etaient pour Coxiella burnetii, et 11,7% pour les rickettsies du groupe de la fievre pourpree, ainsi que l'ont montre les methodes d'immunofluorescence. Au total, 39 souches de rickettsies du groupe de la fievre pourpree ont ete isolees, soit 37 souches a partir de Dermacentor marginatus et deux it partir de D. reticulatus. Des tiques de ces deux especes pouvaient transmettre les rickettsies a des cobayes, par morsure, et ont elimine des rickettsies dans leurs dejections. Des serums humains, collectes dans les memes regions en 1971, contenaient des anticorps contre les rickettsies du groupe de la fievre pourpree dans 1,5 % des cas, et des anticorps contre C. burnetii dans 0,3% des cas; en 1974, les taux etaient respectivement de 10,6% et 5,6%. 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