The pathogenicity of Malaysian isolates of Orientia tsutsugamushi was investigated by a mouse virulence assay. The isolates could be differentiated as low (4 isolates), moderately (3 isolates) and highly virulent (2 isolates) based on the different responses in infected mice. No direct correlation between severity of human scrub typhus infections and virulence of the O. tsutsugamushi in mice was observed. Mice infected with virulent strains of O. tsutsugamushi showed splenomegaly, ascitis accumulation and enlargement of kidneys and livers whereas avirulent O. tsutsugamushi strains were asymptomatic and exhibited ruffled fur for a short period after infection. There was low antibody response in mice infected with isolates of low pathogenicity as compared with those of highly virulent isolates. Upon dissection of the infected mice, enlargement of mouse organs such as spleen, kidney and liver was noted. Presence of rickettsemia in mice was confirmed by the growth of O. tsutsugamushi in the L929 cells when inoculated with blood from infected mice. O. tsutsugamushi was also cultured from the peritoneal exudates of the infected mice. However, DNA of O. tsutsugamushi was only detected in the peritoneal exudates (by PCR) and blood (by cell culture) and not from other tissue samples.
The populations of scrub typhus vector chiggers were compared in two developing oil palm areas, one 5 years old and the other 7 years old at the inception of the study. Both areas were located within the same oil palm scheme in central Peninsular Malaysia. Leptotrombidium (L.) deliense, a principal vector of scrub typhus in Malaysia, was found in reduced numbers in the older oil palm habitat. This reduction is attributed to changes in the microhabitat, specifically the elimination of grasses between the oil palm trees due to canopy shading and to cultural practices.
Part VIII.
1. Cross-immunity experiments in the guinea-pig, rabbit and monkey were carried out with the viruses of the tsutsugamushi disease (including rural typhus) and the urban typhus of Malaya; they showed that immunogenically the two viruses are distinct.
2. The characteristics of setiology, epidemiology, serology and experimental infections are compared, and the conclusion drawn that the two diseases belong to entirely separate groups of rickettsial disease.
Part IX.
1. Cross-immunity experiments in the guinea-pig and rabbit were carried out with the viruses of Rocky Mountain spotted fever, tsutsugamushi (including rural typhus) and urban typhus. They showed that, immunologically, tsutsugamushi
and spotted fever are entirely distinct; whereas urban typhus and spotted fever, though more distinct than alike immunologically, do possess a minor degree of reciprocal cross-immunity.
2. Spotted-fever vaccine was found to have no protective value against the viruses of tsutsugamushi and urban typhus.
1. Cross-immunity tests between strains of rural typhus and tsutsugamushi in the guinea-pig, rabbit and monkey were made. Complete cross-immunity between the strains was demonstrated.
2. The problem of the absence of a primary ulcer in rural typhus and its presence in tsutsugamushi is discussed. Experimental findings are recorded; from consideration of these and certain clinical and epidemiological observations, the conclusion is drawn that one and the same virus may cause gradations of dermal lesion that vary greatly in extent and duration.
3. Correlation of the results of cross-immunity tests and experimental infections with clinical, aetiological, epidemiological and serological findings indicates that the two diseases are identical. Rural typhus is not a disease sui generis, and the term should be discarded, the older designation, "tsutsugamushi disease ", being retained.
1. Transmission of the virus of urban typhus under experimental conditions from rat to rat by the rat flea (X. cheopis) by feeding has been effected. Collateral attempts to transmit the virus of rural typhus by precisely the same procedure failed.
2. Transmission of the virus of urban typhus was also achieved by the inoculation of faeces or crushed tissue of infected fleas into the scarified skin of guinea-pigs.
3. Multiplication of the virus of urban typhus occurs within the rat flea.
4. Infection with the virus of urban typhus is not hereditary in the rat flea.
5. Attempts to transmit the virus of urban or rural typhus by two species of ticks failed. In the case of rural typhus a lessened mortality in the experimental guinea-pigs following test inoculation with passage virus makes it, however, difficult to exclude ticks entirely as a minor factor in the epidemiology of rural typhus.
1. The results of the Weil-Felix reactions of sera from rabbits and monkeys inoculated with human virus or virus of laboratory strains of rural typhus, urban typhus or the tsutsugamushi disease are reported and discussed; as also are corresponding results in rabbits inoculated with the virus of two strains of tropical typhus recovered from wild rats.
2. Instances are given of a change in type of antibody response; in particular, an experience is described in which the agglutinogenic properties of a virus of a tropical typhus strain underwent a temporary change, while the immunogenic properties remained unchanged.
1. A strain of the urban form of tropical typhus has been established in guinea-pigs, and maintained in them for more than one hundred generations. The history and characteristics of the strain are given. The clinical criteria of infection are febrile and scrotal reactions.
2. Methods of demonstration of Rickettsia in material from infected guinea-pigs and rabbits are described. In morphology, distribution and staining characteristics these Rickettsia do not appear to differ from R. prowazeki.
3. The infection of rabbits by intra-ocular inoculation of virus has met with only partial success ; the strains rapidly lose virulence, and do not survive beyond the third generation. The results are closely similar to those reported by Nagayo et al., in corresponding infections of rabbits with the virus of typhus exanthematicus, and to those obtained by the authors in corresponding infections with a strain of Rocky Mountain spotted fever.
4. Infection of white rats has been readily secured, and has been of the "inapparente" form.
5. Two monkeys, inoculated intradermally with infected material, showed a mild general reaction only; no lesion developed at the site of inoculation.
6. The results of the Weil-Felix reactions of sera from rabbits and monkeys convalescent from the infection are summarized. Agglutination is of the OX19 type of Proteus X strains, never of the OXK type.
7. The experimental data obtained indicate that the guinea-pig is the laboratory animal of choice for the study of the urban form of tropical typhus.
1. A strain of the rural form of tropical typhus has been established and maintained in guinea-pigs, and is now in its 97th generation. The history and characteristics of this strain are given. The clinical criterion of infection is a well-marked febrile reaction. Scrotal swelling does not occur. Ascites invariably follows intra-peritoneal inoculation of passage virus.
2. In more than one hundred other such attempts made in this laboratory it has proved impossible to maintain a strain beyond a few generations. Similarly all attempts to maintain the virus of the tsutsugamushi disease in guinea-pigs have failed. The guinea-pig must, therefore, be regarded as very insusceptible to the viruses of the rural typhus and tsutsugamushi group of fevers of Malaya.
3. Infection of rabbits by the intra-ocular inoculation of the virus of rural typhus and of the tsutsugamushi disease has been readily secured. The method, based on that of Nagayo et al., and the criteria of infection, are described in detail.
4. The white rat is readily infected with the virus of rural typhus, the infection being of the "inapparente" form. That the virus could be maintained with unabated virulence for 21 generations indicates, by the criterion of Nicolle, that rural typhus is a murine strain.
5. Monkeys have been successfully infected by the intradermal route with the virus of rural typhus and of the tsutsugamushi disease. At the sites of inoculation, in the case of both viruses, necrotic ulcers have developed that appear to be identical with one another, and with the initial lesion of the tsutsugamushi disease in man; in all other features the experimental infections in the monkey appear to be identical. Rabbits have been similarly infected.
6. The results of the Weil-Felix reactions of sera from rabbits and monkeys convalescent from experimental infection with rural typhus and the tsutsugamushi disease are summarized.
7. Methods of demonstration of Rickettsia from infected guinea-pigs and rabbits are described. In morphology, distribution and staining characteristics the Rickettsia demonstrable in material from animals infected with rural typhus and with the tsutsugamushi disease are identical, and do not appear to differ from the Rickettsia orientalis of Nagayo.
8. The experimental data secured indicate that, provided that intra-ocular inoculation is practised, the rabbit is the laboratory animal of choice in the case of the rural typhus and tsutsugamushi group of fevers (it being assumed that
expense and scarcity make extensive use of monkeys impracticable). Further, these data stress the remarkable similarity of the behaviour of these two viruses in experimental laboratory animals-a similarity that, as will be set forth in a later paper, is fully supported by cross-immunity experiments.
1. Cross-protection tests between a strain of Sumatran mllite-fever and strains of the tsutsugamushi disease were done in the rabbit and monkev with the object of elucidating the inter-relation of these two diseases.
2. It is concluded that Sumatran mite-fever is not a disease sui qeneris, but is identical with the tsutsugamushi disease.