The mammalian gastrointestinal (GI) tract is inhabited by over a hundred species of symbiotic bacteria. Differences among individuals in the composition of the GI flora may contribute to variation in in vivo experimental analyses and disease susceptibility. To investigate potential interindividual differences in GI flora composition, we developed real-time quantitative PCR-based assays for the detection of the eight members of the Altered Schaedler Flora (ASF) as representative members of different bacterial niches within the mammalian GI tract. Quantitative and reproducible strain-specific variations in the numbers of the ASF members were observed across 23 different barrier-housed inbred mouse strains, suggesting that the ASF assays can be used as sentinels for changes in GI flora composition. A significant cage effect was also detected. Isogenic mice that cohabited at weaning, whether from the same or different litters, showed little variation in ASF profiles. Conversely, litters split among different cages at weaning showed divergence in ASF profiles after three weeks. Individual ASF profiles, once established, were highly stable over time in the absence of environmental perturbation. Furthermore, cohabitation of different inbred strains maintained most of the interstrain variation in the GI flora, supporting a role of host genetics in determining GI flora composition.

An indirect hemagglutination (IHA) test and a complement fixation (CF) test were evaluated from test results on sera from 212 human melioidosis patients of which 119 were culturally proved cases. Significant antibody titers (IHA titers of 1:40 or greater and CF titers of 1:4 or greater) were demonstrated with either test in all except five patients. IHA and CF titers ranged as high as 1:20,480 and 1:1,024, respectively. Antibodies were usually demonstrated by both tests 1 week after onset of disease. Transient seronegative reactions during the course of disease were seen in sera of approximately 19% of the patients with either IHA and CF but rarely with both tests. High titers in either test were obtained by the third week of disease and reached maximum levels in 4 to 5 months. Titers usually were detectable for 9 or more months. Antibodies were detected by IHA and CF tests in 80 to 100% of the sera obtained at various time intervals from 9 months to 2 or more years after disease onset. Antibody persistence occurred in patients who had a short disease course, as well as in patients with prolonged, complicated infections. The IHA test had excellent specificity when evaluated with normal human sera and diverse antimicrobial sera from hyperimmunized rabbits and human patients. The CF antigen appeared to contain common antigens with some but not all types of Pseudomonas aeruginosa. The specificity of the CF antigen could be enhanced without appreciable effect on its sensitivity by use of a titer of 1:8 in lieu of 1:4 as a criterion for a significant reaction. Either test could be used advantageously for the laboratory diagnosis of melioidosis.

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INTRODUCTION of concurrent research projects or study facilities of the Division of Veterinary Surveys for selected zoonoses were Medicine, Walter Reed Army Institute of conducted on small wild mammals, ex-Research (WRAIR). These included cluding rodents, located in an undevelop-leptospirosis, listeriosis, rickettsiosis, viral ed forest and swamp area of Aberdeen infections, toxoplasmosis, and filariasis. Proving Grounds, Maryland. Diseases Findings of this extensive study are selected for study were within the scope summarized in this report.

The deoxyribonucleic acids (DNA) from phenotypically unusual Leptospira strains were compared to each other and to reference strains of existing genetic groups. In the "pathogenic" genetic complex, three groups emerged as genetically distinct. Representative examples of these groups were bataviae strain Van Tienen, javanica strain Veldrat Bataviae, and ranarum strain Iowa City Frog. The serologically different muenchen strain Muenchen 90C, icterohaemorrhagiae strain RGA, and kabura strain Kabura were found to be closely related to bataviae strain Van Tienen. The "saprophytic"genetic complex also contained three inter-related groups. The representative examples were patoc strain Patoc I, codice strain CDC, and Turtle strain A-183. Strain 3055 of serotype illini appeared t o have unique nucleotide sequences and was placed in a third genetic complex of its own. Partial relatedness between DNA could be emphasized by increasing the salt concentrations in the incubation media. This could not be attributed t o the methods of annealing but appeared to be dependent upon the genetic relatedness between the heterologous DNA.The genus Leptospira can be separated into two major divisions, the pathogenic and the so-called "sap rop hy t icy' or "bifle xa " lep t 0-spiras. The latter are usually found in fresh surface waters and are rarely found in animals. In addition to infectivity, various phenotypic properties serve to separate members of the two divisions (1 8). Using deoxyribonucleic acid (DNA) base composition determinations and specific DNA-DNA annealing tests in agar matrices, Haapala et al. (7) demonstrated genetic differences between, as well as within, serotypes in the two divisions. Two distinct genetic groups were demonstrated among selected strains within each of the pathogenic and biflexa serotypes. The serological relationships among lep tospiras did not necessarily denote genetic relatedness; however, strains of the same or very similar serotypes appeared to be in the same genetic group. The two genetic groups of pathogenic lep tospiras could be differentiated from each other and from the two biflexa groups by a number of phenotypic characteristics which also served as a basis for the leptospiral groups described by Johnson and Harris (9). The principal differentiating attributes were lipase production and relative resistance to the bacteriostatic action of 8-azaguanine (AZA) and 2,6-diaminopurine (DAP).The genetic groups of pathogenic strains represented by strains of serotypes bataviae and javanica corresponded to the Johnson and Harris biological groups 1 and 2, respectively. Group 1 strains were sensitive to AZA and DAP and had lipase activity, whereas group 2 strains were sensitive to AZA, resistant t o DAP, and lacked lipase activity. The two genetic groups of biflexa strains were phenotypically indistinguishable and fit into the Johnson and Harris biological group 3 , which was characterized by resistance to both purine analogues and by positive lipase activity.

In 1969, five cases of melioidosis in three separate outbreaks were diagnosed in nonhuman primates in the United States. In the first outbreak, two stump-tailed macaque monkeys (Macaca arctoides) developed signs of the disease approximately 6 months after purchase. A third animal, a chimpanzee (Pan troglodytes), probably acquired its infection from one of these monkeys. Two other unrelated cases involving a pig-tailed monkey (Macaca nemestrina) and a rhesus monkey (Macaca mulatta) were diagnosed. These monkeys had been imported 3 years and 6 months, respectively, prior to the recognized onset of their disease. These cases represent the first known occurrences of spontaneous melioidosis in nonhuman primates in the United States.

A predictable 6- to 7-day course of a fatal Leptospira interrogans serovar bataviae infection in experimentally infected mature 110- to 150-g hamsters was used to evaluate the therapeutic efficacy of conventionally used and newer antibiotics. Active drugs were ampicillin, bacampicillin, cyclacillin, piperacillin, mezlocillin, doxycycline, chlortetracycline, cefotaxime, and moxalactam. Cephalexin, cefadroxil, cefamandole, and cefoperazone showed little or no activity in preliminary studies. In delayed treatment studies, all nine active drugs prevented death of hamsters even when treatment was delayed until 1 to 2.5 days before expected time of death. Leptospires in kidneys of surviving animals could be demonstrated in one or more hamsters treated with doxycycline, chlortetracycline, cyclacillin, and piperacillin, but in none of the animals treated with ampicillin, bacampicillin, mezlocillin, cefotaxime, and moxalactam. The potential usefulness of newer penicillins and cephalosporins, as well as ampicillin, chlortetracycline, and doxycycline, for treatment of severe leptospirosis is reported.

Annealing experiments on membrane filters were carried out with deoxyribonucleic acids (DNA) from selected strains of the nomen-species of Pseudomonas, Actinobacillus, Chromobacteriwn, and Micrococcus, with the use of DNA of Pseudomonas pseudomallei and Actinobacillus mallei as reference materials. Under the usual conditions employed in these experiments, the results were not quantitatively reproducible. Incorporation of dimethylsulfoxide (DMSO) into the incubation medium greatly increased differences in comparative binding. DNA binding in agar matrices was examined in the presence and absence of DMSO at various incubation temperatures. It was found that the greatest specificity, stability, and total binding for DNA containing high amounts of guanine and cytosine occurred in the presence of DMSO. Under the most stringent annealing conditions permitted in agar, DNA species from P. pseudomallei and A. mallei in the presence of DMSO demonstrated interspecific relative bindings of 76 to 86% when compared to the homologous reactions. The thermal elution midpoints (Em) of these duplexed interspecific DNA species were quite close to the homologous Em values. The relative bindings of P. multivorans DNA types to either reference DNA ranged between 6 to 27%, and the Em values were 4 to 7 C less than those for the homologous reactions. were extracted from these organisms and characterized by composition and homologous and heterologous annealing abilities.