Schizodeme analysis of Trypanosoma cruzi Colombian strain clones isolated from the acute phase of murine infection

Colombian strain of Trypanosoma cruzi, biodeme Type III (T. cruzi I), has been cloned by micromanipulation at two phases of the acute infection: early (10 days ) and advanced (30 days). Twelve clones were obtained therefrom. Characterization by their biological and biochemical behavior showed an identity among the several clones and their parental strain, albeit with different degrees of virulence. Molecular characterization of the kinetoplast DNA (kDNA) after amplification by polymerase chain reaction revealed identical profiles of the bands from the kDNA minicircle by the analysis of restriction fragment length polymorphism for the isolated clones, their parental strain, and to the clones isolated at two different phases of the infection. Results suggest the predominance of a "principal clone", in the composition of the Colombian strain, responsible for the biological and biochemical behavior. However, no relationship was detected between the molecular profile of kDNA and the degree of virulence presented by the several clones.     

Role of the fungal Ras-protein kinase A pathway in governing epithelial cell interactions during oropharyngeal candidiasis

Tpk1p, Tpk2p and Efg1p are members of the Ras-protein kinase A pathway that governs the yeast-to-hyphal transition in Candida albicans. We used tpk1Delta/tpk1Delta, tpk2Delta/tpk2Delta and efg1Delta/efg1Delta mutants to investigate the role of these proteins in regulating the interactions of C. albicans with oral epithelial cell lines in vitro and virulence in murine models of oropharyngeal candidiasis (OPC) and haematogenously disseminated candidiasis (HDC). The tpk1Delta/tpk1Delta strain adhered to, invaded and damaged oral epithelial cells in vitro similarly to the wild-type strain. In contrast, both the tpk2Delta/tpk2Delta and efg1Delta/efg1Delta strains had reduced capacity to invade and damage oral epithelial cells, and the efg1Delta/efg1Delta strain also exhibited decreased adherence to these cells. Consistent with these in vitro findings, the tpk2Delta/tpk2Delta and efg1Delta/efg1Delta strains also had significantly attenuated virulence during OPC. Therefore, Tpk2p and Efg1p both govern factors that enable C. albicans to invade and damage oral epithelial cells in vitro and cause OPC. These results also suggest that hyphal formation mediated by the Ras-protein kinase A pathway is a key virulence mechanism during OPC. Interestingly, the efg1Delta/efg1Delta strain, but not the tpk2Delta/tpk2Delta had reduced virulence during HDC. Thus, Tpk2p may be more important for governing virulence during OPC than HDC.     

Metabolic characterization of the genus Brucella. V. Relationship of strain oxidation rate of i-erythritol to strain virulence for guinea pigs

Meyer, Margaret E. (University of California, Davis). Metabolic characterization of the genus Brucella. V. Relationship of strain oxidation rate of i-erythritol to strain virulence for guinea pigs. J. Bacteriol. 92:584-588. 1966.-Strain rate of oxidation of i-erythritol and strain virulence were studied to determine whether or not the two characteristics were related within the species Brucella abortus, B. suis, and B. melitensis. The oxidation rate of i-erythritol was determined manometrically, and strain virulence was assessed by injecting groups of guinea pigs and then recording counts of organisms recovered on culture from spleens 21 and 42 days after inoculation. The range in oxidative rates characteristic of virulent strains in each species was established, and strains displaying oxidative rates representative of the full array of values within the rate ranges were virulence-tested. In addition, a mutant that was capable of oxidizing i-erythritol, obtained from a strain that did not oxidize this substrate, was assessed simultaneously to detect any alterations in virulence of the mutant. The data presented herein warrant the conclusion that strain rate of oxidation of i-erythritol is unrelated to the virulence of the strain for guinea pigs in the species B. abortus, B. suis, and B. melitensis.     

Immunological response to re-infections with clones of the Colombian strain of Trypanosoma cruzi with different degrees of virulence: influence on pathological features during chronic infection in mice

Re-infections with Trypanosoma cruzi are an aggravating factor for Chagas disease morbidity. The Colombian strain of T. cruzi represents multiclonal populations formed by clonally propagating organisms with different tropisms and degrees of virulence. In the present study, the influence of successive inoculations with clones of the Colombian strain, exhibiting different degrees of virulence, on chronic myocarditis and the humoral and cellular immune responses (Col-C1 high virulence, Col-C8 medium virulence and Col-C5 low virulence) were demonstrated. Mice from three groups with a single infection were evaluated during the acute (14th-30th day) and chronic phases for 175 days. An immunofluorescence assay, ELISA and delayed type hypersensitivity (DTH) cutaneous test were also performed. Mice with a triple infection were studied on the 115th-175th days following first inoculation. The levels of IgM and IgG2a were higher in the animals with a triple infection. DTH showed a higher intensity in the inflammatory infiltrate based on the morphometric analysis during a 48 h period of the triple infection and at 24 h with a single infection. The histopathology of the heart demonstrated significant exacerbation of cardiac inflammatory lesions confirmed by the morphometric test. The humoral responses indicate a reaction to the triple infection, even with clones of the same strain.     

Characterization of a spontaneous cyst-forming strain of Toxoplasma gondii isolated from Tokachi subprefecture in Japan

Apicomplexan parasite Toxoplasma gondii has three distinct clonal lineages: high, medium and low virulent strains, type I, II and III, respectively. T. gondii avoids the immune response by transforming from fast multiplying tachyzoite to slow multiplying bradyzoite, and establishing a chronic infection. In the present study, we isolated a new strain of T. gondii from cat feces in the Tokachi subprefecture, Hokkaido, Japan and named it as TgCatJpObi1 (Obi1) strain. Genotyping analysis of 12 loci revealed atypical characters close to type II, genotype 4 according to ToxoDB classification. Phenotypically, Obi1 strain shows slow growth rate and the ability of spontaneous cyst formation in both human foreskin fibroblast (HFFs) and mouse peritoneal macrophages in vitro without bradyzoite induction. Parasite virulence was assessed by means of mouse survival upon infection with either Obi1 or ME49 strains. Obi1 strain displayed no mortalities in comparison to type II clonal lineage, ME49 at LD₅₀ to LD₁₀₀ range (1 × 10³–10⁶ tachyzoites). Although virulence of Obi1 strain is significantly lower than that of ME49, nucleotide sequences analyses revealed that genes of virulence factors such as Gra15, Rop5, 16, 17, and 18 in Obi1 strain were 100% identical to those in the type II strain. Thus, characterization of a newly isolated strain, Obi1, is crucial to clarify the development of toxoplasmosis in both humans and animals.     

Mic1-3KO tachyzoite a live attenuated vaccine candidate against toxoplasmosis derived from a type I strain shows features of type II strain

Vaccination with live attenuated parasites has been shown to induce high level of protection against Toxoplasma gondii. In this study we compared the Mic1-3KO tachyzoite (a live attenuated strain) with the parental wild type (WT) tachyzoite in terms of virulence in mice in vivo, dissemination in mouse tissues and persistence in mouse brain. Survival of mice infected with the Mic1-3KO parasites correlated with reduced parasite burden in mouse tissues compared to the parental strain. Like the WT parasite, Mic1-3KO is able to form tissue cysts in vivo which are not, in our experimental conditions, infectious when given by oral route. Infection with the attenuated tachyzoite induced lower levels of cytokine and chemokine than with the parental strain. These data demonstrate that the deleted strain derived from a type I strain behaves like type II strain in outbred mice in terms of virulence, dissemination in mouse tissue and persistence in brain.     

Studies on the Indochina I/CDC strain of Plasmodium falciparum in Colombian and Bolivian Aotus monkeys and different anophelines

The Indochina I/CDC strain of Plasmodium falciparum was isolated from a physician returning to the United States after working in the refugee camps along the Thailand-Kampuchean border. The strain was established in splenectomized Aotus monkeys from Colombia after being grown in vitro for 50 days. During the first three passages in Colombian monkeys, the parasites were not infective to Bolivian Aotus monkeys. After six intervening passages in Saimiri sciureus monkeys, the parasites produced high parasitemias in both Colombian and Bolivian Aotus, but gametocytes were no longer produced. Mosquito infections were obtained only during the first three passages in the Colombian monkeys. The most susceptible mosquito was Anopheles freeborni, followed by An. dirus, An. stephensi, An. maculatus, An. culicifacies, and, rarely, An. gambiae. Sporozoites were found in the salivary glands of the An. freeborni, An. dirus, An. stephensi, and An. maculatus.     

Listeria monocytogenes: comparative interpretation of mouse virulence assay

Being an opportunistic bacterial pathogen, Listeria monocytogenes demonstrates significant strain variations in virulence and pathogenicity. The availability of laboratory procedures to ascertain the pathogenic potential of L. monocytogenes bacteria would greatly enhance the control and prevention of listerial infections. As a method that measures all virulent determinants, mouse virulence assay has been frequently used for assessing L. monocytogenes virulence. The pathogenic potential of a given L. monocytogenes strain as determined by mouse virulence assay is often calculated from mouse mortality data in combination with colony forming units (CFUs) derived from plate counts, and expressed by medium lethal dose (LD(50)). In this report, we describe an alternative method [i.e., relative virulence (%)] that does not involve CFU estimation, and is comparable to LD(50) for interpretation of mouse virulence assay for L. monocytogenes. The relative virulence (%) is obtained by dividing the number of dead mice with the total number of mice tested for a particular strain using a known virulent strain (e.g., L. monocytogenes EGD) as reference. Besides providing a more direct interpretation in comparison with LD(50) values for mouse virulence assay, this method requires fewer dosage groups per L. monocytogenes strain, and eliminates CFU estimation that is step subject to variations between runs and also between laboratories.     

A microassay for mammalian histidine decarboxylase.

This report describes a microassay procedure for mammalian histidine decarboxylase (HDC) based on the measurement of [¹⁴C]O₂ formed from l-[1-¹⁴C]histidine. This assay is particularly useful for quick measurement of HDC activity both in microgram quantities of cell or tissue extract and in tissues that contain significant levels of endogenous histamine.Using this assay, we have shown that the pH optimum, K_m and thermolability of HDC are similar for extracts prepared both from normal rat peritoneal mast cells and from the Furth mouse mastocytoma. HDC activity could be detected in homogenates prepared from 10⁵ rat mast cells, and it was expressed on a per cell basis. Mast cell HDC activity varied with the strain of rat from which the cells were obtained and with the season when they were assayed.     

Genome location and identification of functions defective in the Bartha vaccine strain of pseudorabies virus.

We have shown previously (Lomniczi et al., J. Virol. 52:198-205, 1984) that the Bartha vaccine strain of pseudorabies virus has a deletion in the short unique (Us) region of its genome--a deletion that is related to the absence of virus virulence. This strain is, however, also defective in other genes involved in virulence. We show here that virulence can be restored by marker rescue of the Bartha strain to which an intact Us has been restored (but not to the parental Bartha strain) by sequences derived from approximate map units 0.460 and 0.505 of the wild-type virus genome. No difference in the ability to grow in cell culture was observed between parental Bartha, Bartha 43/25a (Bartha to which an intact Us has been restored), or the doubly rescued Bartha strains. However, only the doubly rescued Bartha strain was virulent for both chickens and pigs and replicated to high titers when inoculated directly into the brains of chickens. The sequences that could restore virulence to the Bartha 43/25a strain encode four genes, all of which are involved in processes leading to the assembly of nucleocapsids. Since these sequences rescue virulence, it appears that a function that plays a role in nucleocapsid assembly is defective in the Bartha strain and that this defect contributes to the lack of virulence of this virus.     

cDNA-derived amino acid sequence of L-histidine decarboxylase from mouse mastocytoma P-815 cells

The primary structure of L-histidine decarboxylase (HDC: L-histidine carboxy-lyase, EC 4.1.1.22) from mouse mastocytoma P-815 cells has been determined by parallel analysis of the amino acid sequence of the protein and the nucleotide sequence of the corresponding cDNA. HDC contains 662 amino acid residues with a molecular mass of 74017, which is larger by about 21,000 Da than that of the previously purified HDC subunit (53 kDa), suggesting that HDC might be posttranslationally processed. The HDC cDNA hybridized to a 2.7 kilobase mRNA of mastocytoma cells. Homology was found between the sequences of mouse mastocytoma HDC and fetal rat liver HDC.     

In vivo characterization of a Toxoplasma gondii strain TgCatJpTy1/k-3 isolated from a stray cat in Japan

The Toxoplasma gondii strain TgCatJpTy1/k-3 (K-3), isolated from a stray cat in Tokyo, Japan, is categorized as a type II genotype. Since the K-3 strain is empirically known to form relatively larger cysts and exhibit weak pathogenesis in a mouse, it could serve as a useful model organism to study chronic T. gondii infection in the host. However, a detailed biological characterization of this strain had not been performed. In this study, we thoroughly assessed the K-3 strain in vivo using a mouse model. Tests indicated that pathogenicity of the K-3 strain was lower than that of the PLK strain, a clonal laboratory strain with a moderately pathogenic type II genotype. Further, cyst sizes of the K-3 strain were significantly larger than those of the PLK strain. Interestingly, K-3 cyst sizes in T. gondii-resistant ICR mice were larger than those in T. gondii-susceptible C57BL/6N mice.Our study suggests that the K-3 strain is suitable to study T. gondii cystogenesis and chronic infection, which are currently difficult to analyze using cell-adopted T. gondii strains.     

Isoenzymatic pattern of Trypanosoma cruzi Y strain after specific chemotherapy

The isoenzyme pattern of the Trypanosoma cruzi Y strain recovered from mice inoculated with 15 x 10(4) blood trypomastigotes and previously treated with either Bay 2502 (Nifurtimox) or Ro 7-1051 (Benzonidazol) was analyzed in the following situations: a) strain resistant to Bay 2502 and again treated with the same drug; b) strain resistant to Bay 2502 and treated with Ro 7-1051; c) strain resistant to Ro 7-1051 and treated with that same drug. Although marked drug resistance was noted in all cases, the isoenzyme pattern of the Y strain for GPI, PGM, ALAT and ASAT remained throughout the same. The pattern was similar to that of the Peruvian strain, which also belongs to the same strain Type of the Y strain, but differed from those of the 21 SF (Type II) and Colombian (Type III) strains. Thus, the appearance of drug resistance in T. cruzi strain was not associated with a change in its isoenzymatic pattern.     

Comparative genome analysis of the high pathogenicity Salmonella Typhimurium strain UK-1

Salmonella enterica serovar Typhimurium, a gram-negative facultative rod-shaped bacterium causing salmonellosis and foodborne disease, is one of the most common isolated Salmonella serovars in both developed and developing nations. Several S. Typhimurium genomes have been completed and many more genome-sequencing projects are underway. Comparative genome analysis of the multiple strains leads to a better understanding of the evolution of S. Typhimurium and its pathogenesis. S. Typhimurium strain UK-1 (belongs to phage type 1) is highly virulent when orally administered to mice and chickens and efficiently colonizes lymphoid tissues of these species. These characteristics make this strain a good choice for use in vaccine development. In fact, UK-1 has been used as the parent strain for a number of nonrecombinant and recombinant vaccine strains, including several commercial vaccines for poultry. In this study, we conducted a thorough comparative genome analysis of the UK-1 strain with other S. Typhimurium strains and examined the phenotypic impact of several genomic differences. Whole genomic comparison highlights an extremely close relationship between the UK-1 strain and other S. Typhimurium strains; however, many interesting genetic and genomic variations specific to UK-1 were explored. In particular, the deletion of a UK-1-specific gene that is highly similar to the gene encoding the T3SS effector protein NleC exhibited a significant decrease in oral virulence in BALB/c mice. The complete genetic complements in UK-1, especially those elements that contribute to virulence or aid in determining the diversity within bacterial species, provide key information in evaluating the functional characterization of important genetic determinants and for development of vaccines.     

Neuroinvasiveness of the MR766 strain of Zika virus in IFNAR-/- mice maps to prM residues conserved amongst African genotype viruses

Zika virus (ZIKV) strains are classified into the African and Asian genotypes. The higher virulence of the African MR766 strain, which has been used extensively in ZIKV research, in adult IFNα/β receptor knockout (IFNAR^-/-) mice is widely viewed as an artifact associated with mouse adaptation due to at least 146 passages in wild-type suckling mouse brains. To gain insights into the molecular determinants of MR766’s virulence, a series of genes from MR766 were swapped with those from the Asian genotype PRVABC59 isolate, which is less virulent in IFNAR^-/- mice. MR766 causes 100% lethal infection in IFNAR^-/- mice, but when the prM gene of MR766 was replaced with that of PRVABC59, the chimera MR/PR(prM) showed 0% lethal infection. The reduced virulence was associated with reduced neuroinvasiveness, with MR766 brain titers ≈3 logs higher than those of MR/PR(prM) after subcutaneous infection, but was not significantly different in brain titers of MR766 and MR/PR(prM) after intracranial inoculation. MR/PR(prM) also showed reduced transcytosis when compared with MR766 in vitro. The high neuroinvasiveness of MR766 in IFNAR^-/- mice could be linked to the 10 amino acids that differ between the prM proteins of MR766 and PRVABC59, with 5 of these changes affecting positive charge and hydrophobicity on the exposed surface of the prM protein. These 10 amino acids are highly conserved amongst African ZIKV isolates, irrespective of suckling mouse passage, arguing that the high virulence of MR766 in adult IFNAR^-/- mice is not the result of mouse adaptation.     

Herpesvirus saimiri strain variability

Herpesvirus saimiri was isolated from 22 squirrel monkeys by cocultivation of peripheral lymphocytes with permissive owl monkey kidney monolayer cells. Comparison of virion DNA fragments produced from restriction endonuclease digestion was used as a sensitive measure of strain variability. Although all isolates contained similarities and common features, 19 of the 22 were readily distinguished. Three of the isolates, however, were indistinguishable and possibly were related epidemiologically. Distinct subtypes of H. saimiri were not evident by these criteria; Peruvian, Colombian, Guyanan, and Bolivian squirrel monkeys yielded isolates without characteristic features peculiar to the geographic region. Three of three colony-born squirrel monkeys that were tested yielded a strain of virus distinct from that obtained from the mother. In separate experiments, two of three animals chosen at random yielded a strain of virus different from that originally obtained 16 and 22 months previously; only one of the three animals examined yielded the same strain of virus 22 months after the original isolation. The degree of restriction endonuclease fragment variability among H. saimiri strains appeared to be greater than previously observed for other herpesviruses.     

Plasmodium berghei: Isolation and maintenance of an irradiation attenuated strain in the nude mouse

An attenuated strain of malaria causing limited parasitemia in mice was derived from a highly virulent strain of Plasmodium berghei (NK65) which produced 100% lethality in mice. A pool of mouse blood infected with the original highly virulent P. berghei was exposed to 40 Krad irradiation and parasites were inoculated into nude mice as well as into thymus competent normal littermates. Thymus competent mice showed no parasitemia, while one out of the five nude mice inoculated with the irradiated parasites developed a slow and progressive parasitemia. These parasites induced a self-limiting parasitemia in thymus competent mice, even when a large inoculum was administered. Maintenance of the low virulence strain required passage through nude mice. After 50 passages at two weekly intervals, reversion to virulence did not occur. A single vaccination with the attenuated strain induced immunity in mice against a challenge inoculation with the original virulent strain. Specific IgG persisted at high titer for more than 9 weeks in mice receiving a single inoculation of the attenuated strain.     

Expression and regulation of histidine decarboxylase mRNA expression in the uterus during pregnancy in the mouse

It has been hypothesized that hormonally regulated histamine production plays a role in preparation of the uterus for implantation. Histidine decarboxylase (HDC) is the rate-limiting enzyme for histamine production. The current study was designed to determine intrauterine expression of HDC mRNA expression during pregnancy in the mouse. High levels of HDC mRNA expression were observed in the preimplantation mouse uterus with peak expression occurring on day 4. High levels of HDC mRNA expression were also detected in the post-implantation uterus. In an effort to determine whether HDC mRNA is regulated by pro-inflammatory cytokines, the HDC mRNA pattern was compared to intrauterine expression of mRNA's for interleukin-1alpha (IL-1alpha), IL-1beta, macrophage chemotactic protein-1 (MCP-1) and RANTES (regulated on activation, normal T expressed and secreted) during the peri-implantation period. IL-1beta, MCP-1 and RANTES mRNA levels were increased in the uterus on days 1-2 and on days 4-5. Increased expression of IL-1alpha mRNA was observed on days 1-2 and days 5-7. There was no clear relationship between HDC mRNA expression and cytokine/chemokine mRNA expression. Progesterone-stimulated intrauterine expression of HDC mRNA. Intrauterine cytokine/chemokine mRNA was also hormonally regulated. This data allowed the possibility that one or more of these pro-inflammatory cytokines could be involved in regulating intrauterine HDC mRNA production. Recombinant IL-1alpha, IL-1beta, MCP-1 and RANTES all failed to induce HDC mRNA expression in the preimplantation uterus in a mouse pseudopregnancy model. At the same time, IL-1beta induced the expression of mRNA for each of the four cytokines/chemokines. Despite the fact that these were also produced in the uterus during pregnancy and were hormonally regulated, none of these cytokines induced intrauterine HDC mRNA expression. The data suggest that progesterone is involved in the regulation of HDC mRNA expression in the preimplantation uterus, but IL-1alpha/beta, MCP-1 and RANTES, which have been reported to regulate histamine synthesis during inflammatory processes, do not appear to play a role.     

Complete genome sequencing and comparative analysis of the linezolid-resistant Enterococcus faecalis strain DENG1

Genome level analysis of bacterial strains provides information on genetic composition and resistance mechanisms to clinically relevant antibiotics. To date, whole genome characterization of linezolid-resistant Enterococcus faecalis isolated in the clinic is lacking. In this study, we report the entire genome sequence, genomic characteristics and virulence factors of a pathogenic E. faecalis strain, DENG1. Our results showed considerable differences in genomic characteristics and virulence factors compared with other E. faecalis strains (V583 and OG1RF). The genome of this LZD-resistant E. faecalis strain can be used as a reference to study the mechanism of LZD resistance and the phylogenetic relationship of E. faecalis strains worldwide.