Virulence In Hamsters Research Articles

Cold-adapted SARS-CoV-2 variants with different temperature sensitivity exhibit an attenuated phenotype and confer protective immunity.

As novel SARS-CoV-2 Variants of Concern emerge, the efficacy of existing vaccines against COVID-19 is declining. A possible solution to this problem lies in the development of a live attenuated vaccine potentially able of providing cross-protective activity against a wide range of SARS-CoV-2 antigenic variants. Cold-adapted (ca) SARS-CoV-2 variants, Dubrovka-ca-B4 (D-B4) and Dubrovka-ca-D2 (D-D2), were obtained after long-term passaging of the Dubrovka (D) strain in Vero cells at reduced temperatures. Virulence, immunogenicity, and protective activity of SARS-CoV-2 variants were evaluated in experiments on intranasal infection of Syrian golden hamsters (Mesocricetus auratus). In animal model infecting with ca variants, the absence of body weight loss, the significantly lower viral titer and viral RNA concentration in animal tissues, the less pronounced inflammatory lesions in animal lungs as compared with the D strain indicated the reduced virulence of the virus variant. Single intranasal immunization with D-B4 and D-D2 variants induced the production of neutralizing antibodies in hamsters and protected them from infection with the D strain and the development of severe pneumonia. It was shown that for ca SARS-CoV-2 variants, the temperature-sensitive (ts) phenotype was not obligate for virulence reduction. Indeed, the D-B4 variant, which did not possess the ts phenotype but had lost the ability to infect human lung cells Calu-3, exhibited reduced virulence in hamsters. Consequently, the potential phenotypic markers of attenuation of ca SARS-CoV-2 variants are the ca phenotype, the ts phenotype, and the change in species specificity of the virus. This study demonstrates the great potential of SARS-CoV-2 cold adaptation as a strategy to develop a live attenuated COVID-19 vaccine.

M16-Type Metallopeptidases Are Involved in Virulence for Invasiveness and Diffusion of Leptospira interrogans and Transmission of Leptospirosis.

Leptospirosis is a global zoonotic infectious disease caused by Leptospira interrogans. The pathogen rapidly invades into hosts and diffuses from bloodstream into internal organs and excretes from urine to cause transmission of leptospirosis. However, the mechanism of leptospiral invasiveness remains poorly understood. Proteolytic activity of M16-type metallopeptidases (Lep-MP1/2/3) of L. interrogans was determined by spectrophotometry. Expression and secretion of Lep-MP1/2/3 during infection of cells were detected by quantitative reverse-transcription polymerase chain reaction, Western blot assay, and confocal microscopy. Deletion and complementation mutants of the genes encoding Lep-MP1/2/3 were generated to determine the roles of Lep-MP1/2/3 in invasiveness using transwell assay and virulence in hamsters. Leptospira interrogans but not saprophytic Leptospira biflexa strains were detectable for Lep-MP-1/2/3-encoding genes. rLep-MP1/2/3 hydrolyzed extracellular matrix proteins, but rLep-MP1/3 displayed stronger proteolysis than rLep-MP2, with 123.179/340.136 μmol/L Km and 0.154/0.159 s-1 Kcat values. Expression, secretion and translocation of Lep-MP1/2/3 during infection of cells were increased. ΔMP1/3 but not ΔMP2 mutant presented attenuated transmigration through cell monolayers, decreased leptospiral loading in the blood, lungs, liver, kidneys, and urine, and 10/13-fold decreased 50% lethal dose and milder histopathologic injury in hamsters. Lep-MP1 and 3 are involved in virulence of L. interrogans in invasion into hosts and diffusion in vivo, and transmission of leptospirosis.

M16-Type Metallopeptidases are Important Virulence Factors for Invasiveness and Diffusion of <i>Leptospira interrogans</i> and Transmission of Leptospirosis

Background: Leptospirosis is a global zoonotic infectious disease. Leptospira interrogans, the major pathogen of leptospirosis, can rapidly invade into hosts and diffuse from the bloodstream into many internal organs, and excrete leptospires from the urine to form sources of infection in the environment. However, the molecular basis of the powerful invasiveness of L. interrogans remains poorly understood. Methods: M16-type metallopeptidase-encoding genes in different serogroups and serovars of L. interrogans were detected by PCR and sequencing. Proteolytic activity of native/recombinant M16-type metallopeptidases (n/rLep-MP1/2/3) from L. interrogans strain Lai was determined by casein/azocasein and extracellular matrix (ECM) protein hydrolytic tests. Expression and secretion of Lep-MP1/2/3 during infection of vascular endothelial, enteric and renal epithelial cells (FHC, HUVEC and Hek293) were detected by qRT-PCR, Western Blot assay and confocal microscopy. Lep-MP1/2/3-encoding gene-deleted and complemented mutants (ΔMP1/2/3 and CΔMP1/2/3) were generated to determine the roles of Lep-MP1/2/3 in invasiveness by transwell test and virulence in hamsters. Findings: All the tested L. interrogans but not saprophytic L. biflexa strains possess the sequence-conserved M16-type MP-encoding genes. rLep-MP1/3 rather displayed stronger hydrolysis of casein and azocasein than rLep-MP2, with Km of 123.179/340.136 µmol•L-1 and Kcat of 0.154/0.159 s-1 but all the three rLep-MPs hydrolyzed ECM proteins. The expression, secretion or membrane translocation of Lep-MP1/2/3 during infection of different cells were notably increased. The ΔMP1/3 mutant, but not ΔMP2 mutant, presented significantly attenuated transmigration through different cell monolayers, decreased leptospiral loading in the blood, lungs, liver, kidneys and urine, and 10/13-fold-decreased LD50 and milder histopathologic injury in hamsters. Interpretation: M16-type MP1/3 are virulence factors of L. interrogans in invasion into hosts, leptospiral diffusion in vivo, and transmission of leptospirosis. Funding Statement: This work was supported by grants from the National Natural Science Foundation of China (81802021, 81671974, 81760366 and 81971951), a grant from the Zhejiang Medical and Health Science and Technology Project of China (2019KY013) and a grant from the Research Initiation Fund of Zhejiang People’s Hospital in China (ZRY2018B004). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The animal experimental protocol was approved by the Ethics Committee for Animal Experiment of Zhejiang University.

Molecular determinants of Yellow Fever Virus pathogenicity in Syrian Golden Hamsters: one mutation away from virulence

Yellow fever virus (Flavivirus genus) is an arthropod-borne pathogen, which can infect humans, causing a severe viscerotropic disease with a high mortality rate. Adapted viral strains allow the reproduction of yellow fever disease in hamsters with features similar to the human disease. Here, we used the Infectious Subgenomic Amplicons reverse genetics method to produce an equivalent to the hamster-virulent strain, Yellow Fever Ap7, by introducing a set of four synonymous and six nonsynonymous mutations into a single subgenomic amplicon, derived from the sequence of the Asibi strain. The resulting strain, Yellow Fever Ap7M, induced a disease similar to that described for Ap7 in terms of symptoms, weight evolution, viral loads in the liver and lethality. Using the same methodology, we produced mutant strains derived from either Ap7M or Asibi viruses and investigated the role of each of Ap7M nonsynonymous mutations in its in vivo phenotype. This allowed identifying key components of the virulence mechanism in hamsters. In Ap7M virus, the reversion of either E/Q27H or E/D155A mutations led to an important reduction of both virulence and in vivo replicative fitness. In addition, the introduction of the single D155A Ap7M mutation within the E protein of the Asibi virus was sufficient to drastically modify its phenotype in hamsters toward both a greater replication efficiency and virulence. Finally, inspection of the Asibi strain E protein structure combined to in vivo testing revealed the importance of an exposed α-helix in domain I, containing residues 154 and 155, for Ap7M virulence in hamsters.

Clostridium difficile secreted Pro-Pro endopeptidase PPEP-1 (ZMP1/CD2830) modulates adhesion through cleavage of the collagen binding protein CD2831

The Clostridium difficile cd2830 gene product is a secreted metalloprotease, named Pro-Pro endopeptidase (PPEP-1). PPEP-1 cleaves C. difficile cell surface proteins (e.g. CD2831). Here, we confirmed that PPEP-1 has a unique preference for prolines surrounding the scissile bond. Moreover, we show that it exhibits a high preference for an asparagine at the P2 position and hydrophobic residues at the P3 position. Using a PPEP-1 knockout C. difficile strain, we demonstrate that the removal of the collagen binding protein CD2831 is fully attributable to PPEP-1 activity. The PPEP-1 knockout strain demonstrated higher affinity for collagen type I with attenuated virulence in hamsters.

High virulence in hamsters of four dominant Leptospira serovars isolated from rats in the Philippines

Leptospirosis is caused by pathogenic species of Leptospira. The aim of this study was to determine and characterize the pathogenicity of four dominant Leptospira isolates prevailing among rats in the Philippines. The isolates were Leptospira interrogans serovar Manilae strain K64, L. interrogans serovar Losbanos strain K37, L. interrogans serovar Ratnapura strain K5 and Leptospira borgpetersenii serovar Javanica strain K6. Pathogenicities were studied using hamsters, which reproduce severe human leptospirosis. The minimum lethal doses were 10(0) ( = 1) leptospires for K64, K37 and K5, and 10(1) leptospires for K6. Weight loss amongst the Leptospira-infected hamsters was observed from 1 day before death (K64-, K37- and K5-infected hamsters) to as much as 1 week before death for K6-infected hamsters. Similar and varied gross and microscopic lesions were observed amongst infected hamsters, even for strains belonging to the same species (i.e. L. interrogans). The most significant and common histopathological findings were congestion of the glomerulus, disarrangement of hepatic cords and erythrophagocytosis. Other findings were foamy splenic macrophages for K6, severe petechial pulmonary haemorrhage for K64, and hematuria and severe pulmonary congestion for K37. Immunostaining and culture revealed the presence of leptospires in different organs of the infected hamsters. Based on these results, Leptospira isolates from rats in the Philippines were shown to be highly virulent, causing pulmonary haemorrhage, severe hepato-renal damage and death in hamsters even at lower doses. The present findings on experimental leptospirosis support clinical data showing that patients with severe manifestations of leptospirosis, such as pulmonary haemorrhage, are increasing in the Philippines. These findings may serve as a basis to strengthen the early diagnosis and treatment of human leptospirosis.

Identification of Collagenase as a Critical Virulence Factor for Invasiveness and Transmission of Pathogenic Leptospira Species

Leptospirosis is a global zoonotic disease. Transmission of Leptospira from animals to humans occurs through contact with water contaminated with leptospire-containing urine of infected animals. However, the molecular basis for the invasiveness of Leptospira and transmission of leptospirosis remains unknown. Activity of Leptospira interrogans strain Lai colA gene product (ColA) to hydrolyze different collagenic substrates was determined by spectrophotometry. Expression and secretion of ColA during infection were detected by reverse-transcription quantitative polymerase chain reaction and Western blot assay. The colA gene-deleted (ΔcolA) and colA gene-complemented (CΔcolA) mutants were generated to determine the roles of ColA in transcytosis in vitro and virulence in hamsters. Recombinant or native ColA hydrolyzed all the tested substrates in which type III collagen was the favorite substrate with 2.16 mg/mL Km and 35.6 h(-)(1) Kcat values. Coincubation of the spirochete with HUVEC or HEK293 cells directly caused the significant elevation of ColA expression and secretion. Compared with wild-type strain, ΔcolA mutant displayed much-attenuated transcytosis through HEK293 and HUVEC monolayers, and less leptospires in blood, lung, liver, kidney and urine and 25-fold-decreased 50% lethal dose and milder histopathological injury in hamsters. The product of colA gene is a collagenase as a crucial virulence factor in the invasiveness and transmission of L. interrogans.

Burkholderia mallei and Burkholderia pseudomallei Cluster 1 Type VI Secretion System Gene Expression Is Negatively Regulated by Iron and Zinc

Burkholderia mallei is a facultative intracellular pathogen that causes glanders in humans and animals. Previous studies have demonstrated that the cluster 1 type VI secretion system (T6SS-1) expressed by this organism is essential for virulence in hamsters and is positively regulated by the VirAG two-component system. Recently, we have shown that T6SS-1 gene expression is up-regulated following internalization of this pathogen into phagocytic cells and that this system promotes multinucleated giant cell formation in infected tissue culture monolayers. In the present study, we further investigated the complex regulation of this important virulence factor. To assess T6SS-1 expression, B. mallei strains were cultured in various media conditions and Hcp1 production was analyzed by Western immunoblotting. Transcript levels of several VirAG-regulated genes (bimA, tssA, hcp1 and tssM) were also determined using quantitative real time PCR. Consistent with previous observations, T6SS-1 was not expressed during growth of B. mallei in rich media. Curiously, growth of the organism in minimal media (M9G) or minimal media plus casamino acids (M9CG) facilitated robust expression of T6SS-1 genes whereas growth in minimal media plus tryptone (M9TG) did not. Investigation of this phenomenon confirmed a regulatory role for VirAG in this process. Additionally, T6SS-1 gene expression was significantly down-regulated by the addition of iron and zinc to M9CG. Other genes under the control of VirAG did not appear to be as tightly regulated by these divalent metals. Similar results were observed for B. pseudomallei, but not for B. thailandensis. Collectively, our findings indicate that in addition to being positively regulated by VirAG, B. mallei and B. pseudomallei T6SS-1 gene expression is negatively regulated by iron and zinc.

InvA Protein Is a Nudix Hydrolase Required for Infection by Pathogenic Leptospira in Cell Lines and Animals

Leptospirosis caused by pathogenic species of the genus Leptospira is a re-emerging zoonotic disease, which affects a wide variety of host species and is transmitted by contaminated water. The genomes of several pathogenic Leptospira species contain a gene named invA, which contains a Nudix domain. However, the function of this gene has never been characterized. Here, we demonstrated that the invA gene was highly conserved in protein sequence and present in all tested pathogenic Leptospira species. The recombinant InvA protein of pathogenic L. interrogans strain Lai hydrolyzed several specific dinucleoside oligophosphate substrates, reflecting the enzymatic activity of Nudix in Leptospira species. Pathogenic leptospires did not express this protein in media but temporarily expressed it at early stages (within 60 min) of infection of macrophages and nephric epithelial cells. Comparing with the wild type, the invA-deficient mutant displayed much lower infectivity and a significantly reduced survival rate in macrophages and nephric epithelial cells. Moreover, the invA-deficient leptospires presented an attenuated virulence in hamsters, caused mild histopathological damage, and were transmitted in lower numbers in the urine, compared with the wild-type strain. The invA revertant, made by complementing the invA-deficient mutant with the invA gene, reacquired virulence similar to the wild type in vitro and in vivo. The LD(50) in hamsters was 1000-fold higher for the invA-deficient mutant than for the invA revertant and wild type. These results demonstrate that the InvA protein is a Nudix hydrolase, and the invA gene is essential for virulence in pathogenic Leptospira species.

Mutagenic Analysis of the Clostridium difficile Flagellar Proteins, FliC and FliD, and Their Contribution to Virulence in Hamsters

Although toxins A and B are known to be important contributors to the acute phase of Clostridium difficile infection, the role of colonization and adherence to host tissues in the overall pathogenesis of these organisms remains unclear. Consequently, we used the recently introduced intron-based ClosTron gene interruption system to eliminate the expression of two reported C. difficile colonization factors, the major flagellar structural subunit (FliC) and the flagellar cap protein (FliD), to gain greater insight into how flagella and motility contribute to C. difficile's pathogenic strategy. The results demonstrate that interrupting either the fliC or the fliD gene results in a complete loss of flagella, as well as motility, in C. difficile. However, both the fliC and fliD mutant strains adhered better than the wild-type 630Δerm strain to human intestine-derived Caco-2 cells, suggesting that flagella and motility do not contribute to, or may even interfere with, C. difficile adherence to epithelial cell surfaces in vitro. Moreover, we found that the mutant strains were more virulent in hamsters, indicating either that flagella are unnecessary for virulence or that repression of motility may be a pathogenic strategy employed by C. difficile in hamsters.

Comparative Genomics and an Insect Model Rapidly Identify Novel Virulence Genes of Burkholderia mallei

Burkholderia pseudomallei and its host-adapted deletion clone Burkholderia mallei cause the potentially fatal human diseases melioidosis and glanders, respectively. The antibiotic resistance profile and ability to infect via aerosol of these organisms and the absence of protective vaccines have led to their classification as major biothreats and select agents. Although documented infections by these bacteria date back over 100 years, relatively little is known about their virulence and pathogenicity mechanisms. We used in silico genomic subtraction to generate their virulome, a set of 650 putative virulence-related genes shared by B. pseudomallei and B. mallei but not present in five closely related nonpathogenic Burkholderia species. Although most of these genes are clustered in putative operons, the number of targets for mutant construction and verification of reduced virulence in animal models is formidable. Therefore, Galleria mellonella (wax moth) larvae were evaluated as a surrogate host; we found that B. pseudomallei and B. mallei, but not other phylogenetically related bacteria, were highly pathogenic for this insect. More importantly, four previously characterized B. mallei mutants with reduced virulence in hamsters or mice had similarly reduced virulence in G. mellonella larvae. Site-specific inactivation of selected genes in the computationally derived virulome identified three new potential virulence genes, each of which was required for rapid and efficient killing of larvae. Thus, this approach may provide a means to quickly identify high-probability virulence genes in B. pseudomallei, B. mallei, and other pathogens.

Type VI secretion is a major virulence determinant in Burkholderia mallei

Burkholderia mallei is a host-adapted pathogen and a category B biothreat agent. Although the B. mallei VirAG two-component regulatory system is required for virulence in hamsters, the virulence genes it regulates are unknown. Here we show with expression profiling that overexpression of virAG resulted in transcriptional activation of approximately 60 genes, including some involved in capsule production, actin-based intracellular motility, and type VI secretion (T6S). The 15 genes encoding the major sugar component of the homopolymeric capsule were up-expressed > 2.5-fold, but capsule was still produced in the absence of virAG. Actin tail formation required virAG as well as bimB, bimC and bimE, three previously uncharacterized genes that were activated four- to 15-fold when VirAG was overproduced. Surprisingly, actin polymerization was found to be dispensable for virulence in hamsters. In contrast, genes encoding a T6S system were up-expressed as much as 30-fold and mutations in this T6S gene cluster resulted in strains that were avirulent in hamsters. SDS-PAGE and mass spectrometry demonstrated that BMAA0742 was secreted by the T6S system when virAG was overexpressed. Purified His-tagged BMAA0742 was recognized by glanders antiserum from a horse, a human and mice, indicating that this Hcp-family protein is produced in vivo during infection.

Infection of hamsters with epidemiologically important strains of Clostridium difficile.

Five different toxigenic strains of Clostridium difficile of known human epidemiologic importance were tested for virulence in hamsters. Three strains-types B1, J9, and K14-have caused hospital outbreaks. Type Y2 is associated with a high rate of asymptomatic colonization in patients. The fifth strain, type CF2, is a toxin A-negative, toxin B-positive strain implicated in multiple human cases of C. difficile-associated diarrhea. Groups of 10 hamsters per strain were given 1 dose of clindamycin, followed 5 days later with gastric inoculation of 100 cfu of C. difficile. Hamsters given types B1, J9, K14, or Y2 showed 90%-100% colonization (albeit at a slower rate with type Y2) and 100% mortality of colonized animals. Hamsters challenged with type CF2 showed 60% (P= .01) colonization and 30% mortality (P= .0003). The hamster model demonstrated pathogenicity differences between a toxin variant strain and standard toxigenic strains but no significant differences among the standard strains.

Aminoglycoside and Macrolide Resistance in Burkholderia pseudomallei

We were very interested in the recent paper by Moore et al. (3) in which the authors used transposon mutants to demonstrate efflux-mediated resistance to aminoglycosides and macrolides in Burkholderia (formerly Pseudomonas) pseudomallei. Melioidosis is very difficult to treat because of intrinsic resistance to many antimicrobial agents, particularly the aminoglycosides. Aminoglycoside–β-lactam combinations are used empirically to treat suspected community-acquired sepsis in many areas of the world, but they are ineffective in melioidosis. We have been conducting clinical and laboratory studies of melioidosis in Thailand since 1986. During this time we have collected over 3,700 clinical isolates of B. pseudomallei from more than 1,800 patients with melioidosis admitted to Sappasitprasong Hospital, Ubon Ratchathani, northeast Thailand. We have performed susceptibility testing using the disk diffusion method on most of these isolates, according to the guidelines of the National Committee for Clinical Laboratory Standards (4), for ceftazidime, co-amoxiclav, chloramphenicol, doxycycline, ciprofloxacin, gentamicin, polymyxin B, and, more recently, imipenem. MIC determinations, using an agar incorporation method (5), have been performed for representative isolates. During this time we have isolated three strains of B. pseudomallei, from two patients, which were susceptible to gentamicin by disk testing. The isolates were from a splenic aspirate of one patient (708a) (2) and from blood and knee aspirate cultures of the second (2188a and b). All other isolates were resistant to gentamicin by disk testing (disk content, 10 μg). B. pseudomallei is usually highly resistant to aminoglycosides, with a gentamicin MIC at which 50% of the isolates are inhibited of 128 mg/liter (1), and also to the macrolide antibiotics. The MIC results for 100 clinical isolates of B. pseudomallei, collected during 1998, for the macrolides erythromycin, clarithromycin, and azithromycin are listed in Table ​Table1.1. In contrast, the three gentamicin-susceptible isolates were also susceptible to these macrolide antibiotics (Table ​(Table1).1). TABLE 1 MICsa for isolates of B. pseudomallei For all three isolates the MICs of ceftazidime, imipenem, chloramphenicol, ciprofloxacin, and doxycycline were within the expected ranges for each agent. All three were resistant to polymyxin B. Thus, there are very rare wild strains of B. pseudomallei which exhibit antibiotic susceptibility patterns similar to those of the transposon mutants created by Moore et al. (3), i.e., with linked loss of resistance to aminoglycosides and macrolides. By definition the three clinical isolates described here are virulent. This is consistent with the evidence for retention of virulence in hamsters by the mutant strains. Whether these findings have therapeutic implications remains to be seen. This study was part of the Wellcome-Mahidol University Oxford Tropical Medicine Research Programme, funded by the Wellcome Trust of Great Britain.

Physiological Studies on the Culture Form of Four Strains of Leishmania braziliensis. I. Nitrogen Content, Substrate Utilization, and Effect of Metabolic Inhibitors on Respiration and Its Relation to Infectivity

Comparative physiological studies on four strains of Leishmania braziliensis, isolated in pure culture from cutaneous cases of human leishmaniasis, are presented. Shorter lag phases in the growth curves were obtained for strains 0-CR and 6-CR as compared with 3-CR and 2-CR. Nitrogen content of 0-CR was higher than that of the other strains and respiratory activity for 0-CR and 6-CR was also higher. Respiratory quotients and glucose consumption per cell were higher for 0-CR, which showed greater ability to utilize some carbohydrates, nitrogen compounds, and Krebs cycle intermediates. All strains were sensitive, in different degrees, to several metabolic inhibitors while iodoacetate exerted the most marked action. The physiological findings are correlated and discussed in relation to infectivity of the strains. The oldest strain (0-CR), kept in culture for several years, presented the strongest virulence for hamsters. Strains 3-CR and 2-CR, with the lowest metabolic activities, were unable to infect hamsters or showed an extremely low virulence for them. Strain 6-CR, the newest isolate, showed characteristics in between the above strains. Physiological and biochemical observations on species of Leishmania can be found in the literature of the past 40 years. The older studies concerned themselves with the production of acids from media containing sugars (Colas-Belcour and Lwoff, 1925; Kligler, 1926; Noguchi, 1926) and with the production of ammonia by old cultures (Salle and Schmidt, 1928). Other authors have made observations on the relationship between motility of the flagellates and presence of different carbohydrates (Dubois, 1936) and on the respiration of the culture or parasitic forms of the organisms under different circumstances (Adler and Ashbel, 1940; Chang, 1948; Fulton and Joyner, 1949; von Brand and Agosin, 1955; Medina et al., 1955; Chatterjee and Ghosh, 1959). Recently, von Brand (1966) has critically reviewed some of the research on the physiology and biochemistry of Leishmania. In this paper, comparative observations on the respiratory metabolism of four Costa Rican strains of L. braziliensis and its relation to infectivity are presented. A previous note on the comparison of two of the strains has been published (Zeledon and Monge, 1966). Received for publication 24 May 1967. * This work was supported by USPHS Research Grant AI-03304-05, NIAID. MATERIALS AND METHODS The Costa Rican strains 0-CR, 2-CR, 3-CR, and 6-CR, were used in the experiments. The 0-CR strain was isolated by Dr. Alfonso Trejos at the San Juan de Dios Hospital in 1957 and has been kept in culture since then. The other three strains were isolated in pure culture by the authors, from cutaneous lesions of patients in the same hospital. The 2-CR strain was isolated in November 1960, 3-CR in July 1961, and 6-CR in April 1965. The cultures have been kept since isolation in Tobie and Rees' (1948) modification of Senekjie's medium by transferring them every 3 or 4 weeks. In order to determine growth curves the organisms were cultured in Roux bottles in the modified Senekjie medium with 10% rabbit blood taken from at least three animals and incubated at 26.5 C. In all cases, one bottle in the exponential phase of growth served as inoculum and the numbers of organisms inoculated into each batch of flasks were kept within the same limits for all four strains. The organisms of two bottles were counted with a hemocytometer and data from three to seven experiments were combined. For manometric studies, flagellates were harvested at the exponential phase of growth (days 4 to 6 for strains 0-CR and 6-CR; days 8 to 10 for strain 3-CR; days 6 to 8 for strain 2-CR) and was treated as previously described (Zeledon, 1960a, b, c, d). All substrates, in a final concentration of 0.01 M, were added at the beginning of the experiment; the final pH was 7.2. In the case of the Krebs intermediates the final pH was 5.0 since preliminary studies showed strains 0-CR, 6-CR, and 3-CR used these intermediates better at this pH. The experiments were carried out at 30 C for a period of 2 hr with interval readings at 15 to 30 min. The nitrogen content per War-