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) Two strains of tropical typhus have been isolated from wild rats trapped in endemic areas.
(2) Both were isolated and maintained in guinea-pigs.
(3) One could be isolated in rabbits by the intra-ocular inoculation of virus, and maintained thus indefinitely. The features of this infection appeared to be identical with corresponding infections obtained with the viruses of rural typhus and the tsutsugamushi disease of human origin. Repeated attempts to isolate the other rat strain in rabbits in the same manner failed.
(4) Rickettsia were found with ease and in abundance in infected material from guinea-pigs infected with either strain, and from rabbits infected with the one-strain.
(5) The sera of rabbits infected with the two rat strains gave positive Weil-Felix reactions of significant titre.
(6) Cross-immunity tests in rabbits between one rat strain and six strains of huinan origin of rural typhus and tsutsugamushi showed a cross-immunity to exist, complete in five strains and partial in the sixth strain.
(7) A concomitant infection with sodoku was present in the guinea-pigs of botlh strains ; although this may have modified the clinical signs, the infection by a typhus virus could be determined by four decisive criteria.
(8) The conclusion is drawn that the murine origin of the virus of the rural typhus-tsutsugamuslhi group of diseases is now firmly established.
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. 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.
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. 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.
The mechanical behavior of the heart muscle tissues is the central problem in finite element simulation of the heart contraction, excitation propagation and development of an artificial heart. Nonlinear elastic and viscoelastic passive material properties of the left ventricular papillary muscle of a guinea pig heart were determined based on in-vitro precise uniaxial and relaxation tests. The nonlinear elastic behavior was modeled by a hypoelastic model and different hyperelastic strain energy functions such as Ogden and Mooney-Rivlin. Nonlinear least square fitting and constrained optimization were conducted under MATLAB and MSC.MARC in order to obtain the model material parameters. The experimental tensile data was used to get the nonlinear elastic mechanical behavior of the heart muscle. However, stress relaxation data was used to determine the relaxation behavior as well as viscosity of the tissues. Viscohyperelastic behavior was constructed by a multiplicative decomposition of a standard Ogden strain energy function, W, for instantaneous deformation and a relaxation function, R(t), in a Prony series form. The study reveals that hypoelastic and hyperelastic (Ogden) models fit the tissue mechanical behaviors well and can be safely used for heart mechanics simulation. Since the characteristic relaxation time (900 s) of heart muscle tissues is very large compared with the actual time of heart beating cycle (800 ms), the effect of viscosity can be reasonably ignored. The amount and type of experimental data has a strong effect on the Ogden parameters. The in vitro passive mechanical properties are good initial values to start running the biosimulation codes for heart mechanics. However, an optimization algorithm is developed, based on clinical intact heart measurements, to estimate and re-correct the material parameters in order to get the in vivo mechanical properties, needed for very accurate bio-simulation and for the development of new materials for the artificial heart.
The aqueous and ethanol extracts of Stichopus chloronotus Brandt were investigated for their effectiveness against guinea pig dermatophytosis caused by Microsporum canis and Trichophyton mentagrophytes using the hair root invasion test. The ethanol extract at 10 mg/ml showed 82.8 % efficacy against T. mentagrophytes while the aqueous extract at similar concentration showed 84.8% efficacy against M. canis infection, as compared to econazole which showed 100% efficacy against both infections. No adverse effect on the skin was observed in the treated animals. In conclusion, aqueous and ethanol extracts of S. chloronotus showed high antimycotic activity against experimentally induced dermatophytosis in guinea pigs.
The safety of bacterial cells of Clostridium bifermentans serovar malaysia, which is highly toxic to mosquito larvae, was tested on mice, guinea pigs, rabbits, and goldfish. Inoculations of at least 1 x 10(8) cells per animal by routes recommended by World Health Organization (subcutaneous, percutaneous, inhalation, force-feeding, intraperitoneal, intravenous) and tests of subacute toxicity, anaphylactic shock, persistence in heart blood, and virulence by successive passages, were performed on mice, guinea pigs, or both. Growth was monitored for 1 mo before necropsy. Ocular irritation and skin scarification were tested with rabbits. C. bifermentans serovar malaysia did not induce any mortality or abnormal reactions in mammals at a dose 1,000 times higher than the level established by W.H.O. for the demonstration of safety. Bacterial cells are not toxic to goldfish at a dose 1,000 times higher than the LD50 for the target-mosquito larvae. We conclude that C. bifermentans serovar malaysia bacterial cells are safe for laboratory mammals and goldfish.
Considerable progress has been made in the field of leptospiral vaccines development since its first use as a killed vaccine in guinea pigs. Despite the fact that the immunity conferred is restricted to serovars with closely related lipopolysaccharide antigen, certain vaccines have remained useful, especially in endemic regions, for the protection of high-risk individuals. Other conventional vaccines such as the live-attenuated vaccine and lipopolysaccharide (LPS) vaccine have not gained popularity due to the reactive response that follows their administration and the lack of understanding of the pathogenesis of leptospirosis. With the recent breakthrough and availability of complete genome sequences of Leptospira, development of novel vaccine including recombinant protein vaccine using reverse vaccinology approaches has yielded encouraging results. However, factors hindering the development of effective leptospiral vaccines include variation in serovar distribution from region to region, establishment of renal carrier status following vaccination and determination of the dose and endpoint titres acceptable as definitive indicators of protective immunity. In this review, advancements and progress made in LPS-based vaccines, killed- and live-attenuated vaccines, recombinant peptide vaccines and DNA vaccines against leptospirosis are highlighted.
The aim of this study was to optimize the conditions for the passive adsorption of polyclonal antibody onto plain surface polystyrene latex particles and its performance in a slide latex agglutination test for rotavirus antigen detection. Cleaning of latex particles by washing through repetitive centrifuging, decanting and resuspending in distilled water was adequate in removing surfactants from the particles' surfaces to enable coating. A study of antibody concentration, incubation temperature and buffer pH revealed that optimum coating was achieved with a 3-fold excess of antibody to the calculated total particle surface capacity for the antibody in a glycine-saline buffer of pH 9.2 at 40 degrees C for 4 hours. The ionic strength and pH of the latex suspending buffer and the sample buffer were critical factors determining the sensitivity of the test and the appearance of non-specific agglutination. Ultrasonication, addition of glycerol and Tween 20, either individually or in combination, were able to suppress non-specific agglutination in some batches of latex reagents. Polyethylene glycol 6000 enhanced the quality of agglutination as well as reduced the time of its appearance, especially in reagents that produced poor agglutination.
1. 30 mg kg-1 chlorpromazine (CPZ) depleted more than half of the tissue histamine from lungs, stomach, ileum and skin of the normal guinea-pigs. However, the drug increased the tissue histamine content in scorbutic animals. 2. In contrast, reserpine depleted histamine from the four tested tissues in both normal and scorbutic animals, except those in the lungs of the control animals. 3. Ascorbic acid only depleted histamine from the stomach and ileum. 4. A 24 hr period was the time limit for CPZ to deplete the histamine in all the four tested tissues. 5. Histamine partially or completely recovered in the tissues after the next 24 hr.
The immunological characteristics of 26 strains of Japanese encephalitis virus (JEV) isolated in Japan and Malaya between 1935 and 1966 have been investigated mainly by the antibody-absorption variant of the haemagglutination-inhibition test, and to a certain extent also by conventional haemagglutination-inhibition and complement-fixation tests. The antibody-absorption technique shows promise as a routine method for the immunotyping of JEV.At present, two immunotypes can be distinguished. One comprises 2 strains, Nakayama-NIH and I-58, and is designated as the I-58 immunotype. The other immunotype, JaGAr 01, comprises 17 strains which share the characteristics of the JaGAr 01 strain, including one subline of the Nakayama strain, Nakayama-Yakken. The Nakayama-RFVL strain was found to have the characteristics of both immunotypes. The I-58 immunotype differs more markedly from related arboviruses, such as the Murray Valley encephalitis virus and the West Nile Eg101 strain, than does the JaGAr 01 immunotype.Evidence is presented which suggests that a given JEV strain can change immunotype on repeated passage through mice.
Virulence of Shigella is attributed to the genes presence in chromosome or in the megaplasmid. The apy gene which is located in the megaplasmid of Shigella species encodes for apyrase enzyme, a pathogenesis-associated enzyme causing mitochondrial damage and host cell death. In this study we constructed an apy mutant of Shigella flexneri by insertional activation using a kanamycin resistant gene cassette. The wild type apy gene of S. flexneri 2a was PCR amplified, cloned and mutated with insertion of kanamycin resistant gene cassette (aphA). The mutated construct (apy: aphA) was subcloned into a conjugative suicidal vector (pWM91) at the unique Sma1 and Sac1 sites. The mutation of the wild apy gene in the construct was confirmed by DNA sequencing. The mutated construct was introduced into wild type S. flexneri 2a by conjugation with Escherichia coli. After undergoing homologous recombination, the wild apy gene was deleted from the construct using the sucrose selection method. Non-functional activity of the apyrase enzyme in the constructed strain by colorimetric test indicated the successful mutation of the apyrase enzyme. This strain with mutated apy gene was evaluated for its protective efficacy using the guinea pig keratoconjunctivitis model. The strain was Sereny negative and it elicited a significant protection following challenge with wild S. flexneri strain. This apy mutant strain will form a base for the development of a vaccine target for shigellosis.
A 22-year-old Malay female presented with left eye floaters for 2 weeks, associated with temporal visual field defect and metamorphopsia for 3 days. She has a guinea pig and a hedgehog at home, but denied being bitten or scratched by them. Her visual acuity at presentation was 6/12 on the left eye and 6/6 on the right eye. Her left eye relative afferent pupillary defect was barely positive with mild anterior chamber reaction. Fundus examination of the left eye showed mild vitritis, swollen optic disc with macular star, crops of active choroidal lesions at superonasal retina with a linear arrangement in the form of migratory track nasally. However, there were no nematodes seen on fundus examination. Investigations showed normal full blood count with no eosinophilia and positive serology test for Bartonella henselae. She was diagnosed to have dual infection - diffuse unilateral subacute neuroretinitis (DUSN), based on the presence of crops of choroidal lesions with migratory track, and cat scratch disease (CSD) based on a positive serological test. She was treated with oral albendazole 400 mg 12 hourly for 6 weeks for DUSN and oral doxycycline 100 mg 12 hourly for 4 weeks for CSD. Focal laser had been applied to the area of migratory track in the left eye. Her left eye vision improved to 6/6 at 1 month after treatment, with resolution of neuroretinitis.