Panama Strain Research Articles

Cysteine Dioxygenase Enzyme Activity and Gene Expression in the Dimorphic Pathogenic Fungus Histoplasma capsulatum Is in both the Mold and Yeast Morphotypes and Exhibits Substantial Strain Variation.

In the dimorphism (mold/yeast) Histoplasma capsulatum (Hc) literature are reports that yeast (the so-called pathogenic form) uniquely expresses a cysteine dioxygenase (CDO, approx. 10,500 dal) activity which the mold morphotype (the so-called saprophytic soil form) does not express (C.F., Kumar et al., Biochem 22, 762, 1983). This yeast-specific CDO activity is postulated to play a critical role in the mold-to-yeast shift. A number of years ago, our lab isolated the gene encoding the Hc cysteine dioxygenase (CDO1, Genbank accession AY804144) and noted significant expression in the mold morphotype of several Histoplasma strains and also determined that the predicted protein would be over double the 10,500 dal reported by Kumar et al. Our report demonstrates (in the class 1 Downs strain, the class 2 G271B strain and two Panamanian strains, 184AS and 186AS) that the CDO1 gene is expressed in both the mold and yeast morphotypes and both morphotypes show significant CDO activity. Furthermore, we show via a FLAG-tag analysis that the expressed protein is approximately 24.7 ± 2.4 kd, in agreement with the putative protein sequence (determined from cDNA sequence) which yields 23.8 kd and is consistent with most other eukaryotic CDO enzymes. Additionally, we demonstrate that intracellular cysteine levels are actually significantly higher in the mold form of the two Panamanian strains, 184AS and 186AS, equal in both mold and yeast in the class 1 Downs strain and significantly higher in yeast of the more pathogenic class 2 G217B strain.

Molecular Epidemiology of Dengue in Panama: 25 Years of Circulation.

Dengue virus (DENV) is the most prevalent arbovirus in terms of human public health importance globally. In addition to DENV epidemiological surveillance, genomic surveillance may help investigators understand the epidemiological dynamics, geographic distribution, and temporal patterns of DENV circulation. Herein, we aimed to reconstruct the molecular epidemiology and phylogeny of DENV in Panama to connect the epidemiological history of DENV dispersal and circulation in Latin America. We retrospectively analyzed the epidemiological data obtained during 25 years of DENV surveillance in Panama. DENV was reintroduced in Panama in 1993 after a 35 year absence of autochthonous transmission. The increase in the number of total dengue cases has been accompanied by an increase in severe and fatal cases, with the highest case fatality rate recorded in 2011. All four serotypes were detected in Panama, which is characterized by serotype replacement and/or co-circulation of multiple serotypes. Phylogenetic analysis of datasets collected from envelope (E) gene sequences obtained from viruses isolated from human sera demonstrated that circulating viruses were highly diverse and clustered in distinct clades, with co-circulation of clades from the same genotype. Our analyses also suggest that Panamanian strains were related to viruses from different regions of the Americas, suggesting a continuous exchange of viruses within the Americas.

Salmonella Panama: Genetic Diversity of the Isolates Collected from Human and Non-human Sources.

Salmonella enterica serovar Panama belongs to the D1 serogroup and is frequently associated with nontyphoidal salmonellosis in humans. This study aimed to characterize isolates collected from Northeast Brazil by phenotypic and molecular methods. Forty four S. Panama strains were examined for antimicrobial susceptibility, virulence genes, and pulsed field gel electrophoresis (PFGE) types. All strains were susceptible to antibiotics (except for streptomycin), presented classical virulence factors, and could be clustered into four groups and 18 pulsotypes. This work calls for continuous surveillance for the emergence of antibiotic resistance and new clones in a geographical area.

Genetic variability of human respiratory syncytial virus group B in Panama reveals a novel genotype BA14.

In Panama, human respiratory syncytial virus (HRSV) is responsible of 20-40% of acute respiratory infections in children under 5 years old. Currently, little is known about the genetic variability of HRSV in Central America and the Caribbean. Recently, we reported the genetic variability of HRSV-A, however; no studies on HRSV-B in Panama have been described yet. In this study, 24 sequences of Panamanian HRSV-B, from children (<5 years) with acute respiratory infections (ARI), collected from July 2008 to November 2012 were analyzed. All sequences share the characteristic 60-nt duplication of the BA strains. Six Panamanian strains grouped with the BA10 genotype and 12 samples clustered together in a separate monophyletic clade with an aLRT support value of 0.92 and an intra-group p-distance less than 0.07. This fulfills the criteria to consider a new genotype in HRSV, which we named BA14 genotype. Another six strains remain unclassified, but closely related to BA9, BA11, or the new BA14 genotypes, according to their genetic p-distance. Different amino acid substitutions in the Panamanian HRSV-B strains were observed, some previously described and others found only on Panamanian strains. This study contributes to the knowledge of the genetic variability and evolution of HRSV in Central America.

Virulence variation among strains of the emerging infectious fungus Batrachochytrium dendrobatidis (Bd) in multiple amphibian host species.

Emerging infectious diseases have been documented in numerous plant and animal populations. The infectious disease amphibian chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is associated with global amphibian population declines. While much Bd-amphibian research has centered on response variation in hosts, a paucity of information exists on how variation in the pathogen, such as strain differences, affects infection dynamics. To examine how different Bd strains may differentially impact multiple hosts, we conducted laboratory experiments to measure 2 infection outcomes, viz. host survival and pathogen load, in 3 amphibian host species (Pacific treefrog, western toad, and Cascades frog) after exposure to 3 different Bd strains (an additional fourth Bd strain was tested in toads only). Our results confirm that the infection response differs among host species. Western toads experienced significant mortality, but Pacific treefrogs and Cascades frogs did not. Interestingly, our experiment also captured strain-dependent virulence variation but only in 1 host species, the western toad. Increased mortality was observed in 2 of the 4 Bd strains tested in this host species. Toads were also the only host species found to have variable pathogen load dependent on strain type; individuals exposed to the Panama strain harbored significantly higher loads compared to all other strains. These findings underscore the dynamic nature of Bd infection, showing that virulence can vary contingent on host and strain type. We highlight the importance of both host- and pathogen-dependent factors in determining overall infection virulence and show the need for in vivo testing to fully assess pathogenicity.

Characteristics of Salmonella spp. Isolated from Wild Birds Confiscated in Illegal Trade Markets, Rio de Janeiro, Brazil.

The prevalence of Salmonella spp. was investigated in 109 wild birds poached in the illegal wildlife trade in Rio de Janeiro; most of them are passerines from Thraupidae family and three from Psittacidae. One strain of Salmonella ser. Typhimurium and two strains of Salmonella ser. Panama were isolated from passerine species and all of them showed resistance to multiple antimicrobial drugs, like ampicillin, ceftriaxone, ceftiofur, tetracycline, gentamicin, nalidixic acid, ciprofloxacin, and enrofloxacin. PFGE showed 100% similarity among the Salmonella ser. Typhimurium strain isolated from a Temminck's seedeater (Sporophila falcirostris) and the strains isolated from a human outbreak, in southern Brazil. The two Salmonella ser. Panama strains isolated from two chestnut-capped blackbirds (Chrysomus ruficapillus) present in the same catch showed the same clonal origin and have never been associated with epizooties and human outbreaks. Potential for dissemination of resistant Salmonella through situations offered by captive management and the isolation of the same strain from wild birds and human sources may become a problem for the conservation of natural populations and to public health.

Draft Genome Sequence of Brazilian Leptospira noguchii Serogroup Panama Strain U73, Isolated from Cattle.

Leptospira noguchii is a current zoonotic pathogen in Brazil. Here, we report the draft genome sequence of the Brazilian L. noguchii serogroup Panama strain U73, isolated from asymptomatic cattle urine.

Comparison of phylogenetically distinct Histoplasma strains reveals evolutionarily divergent virulence strategies.

ABSTRACTInfection with the dimorphic fungus Histoplasma capsulatum results from the inhalation of contaminated soil. Disease outcome is variable and depends on the immune status of the host, number of organisms inhaled, and the H. capsulatum strain. H. capsulatum is divided into seven distinct clades based on phylogenetic analyses, and strains from two separate clades have been identified in North America (denoted as NAm strains). We characterized an H. capsulatum isolate (WU24) from the NAm 1 lineage in relation to two other well-characterized Histoplasma isolates, the Panamanian strain G186A and the NAm 2 strain G217B. We determined that WU24 is a chemotype II strain and requires cell wall α-(1,3)-glucan for successful in vitro infection of macrophages. In a mouse model of histoplasmosis, WU24 exhibited a disease profile that was very similar to that of strain G186A at a high sublethal dose; however, at this dose G217B had markedly different kinetics. Surprisingly, infection with a lower dose mitigated many of the differences during the course of infection. The observed differences in fungal burden, disease kinetics, symptomology, and cytokine responses all indicate that there is a sophisticated relationship between host and fungus that drives the development and progression of histoplasmosis.

Quantitative molecular networking to profile marine cyanobacterial metabolomes

Untargeted liquid chromatography-MS (LC-MS) is used to rapidly profile crude natural product (NP) extracts; however, the quantity of data produced can become difficult to manage. Molecular networking based on MS/MS data visualizes these complex data sets to aid their initial interpretation. Here, we developed an additional visualization step for the molecular networking workflow to provide relative and absolute quantitation of a specific compound in an extract. The new visualization also facilitates combination of several metabolomes into one network, and so was applied to an MS/MS data set from 20 crude extracts of cultured marine cyanobacteria. The resultant network illustrates the high chemical diversity present among marine cyanobacteria. It is also a powerful tool for locating producers of specific metabolites. In order to dereplicate and identify culture-based sources of known compounds, we added MS/MS data from 60 pure NPs and NP analogs to the 20-strain network. This dereplicated six metabolites directly and offered structural information on up to 30 more. Most notably, our visualization technique allowed us to identify and quantitatively compare several producers of the bioactive and biosynthetically intriguing lipopeptide malyngamide C. Our most prolific producer, a Panamanian strain of Okeania hirsuta (PAB10FEB10-01), was found to produce at least 0.024 mg of malyngamide C per mg biomass (2.4%, w/dw) and is now undergoing genome sequencing to access the corresponding biosynthetic machinery.

Characterization of the First Batrachochytrium dendrobatidis Isolate from the Colombian Andes, an Amphibian Biodiversity Hotspot

The pathogenic chytrid fungus, Batrachochytrium dendrobatidis (Bd), constitutes a significant threat to more than 790 amphibian species occurring in Colombia. To date there is no molecular or morphological description of strains infecting Colombian populations. Here we report the genetic and morphological characterization of the first Colombian isolate of Bd (strain EV001). Our goals were threefold: (1) to characterize the morphology of EV001 using light and scanning electron microscopy, (2) to genotype this strain by direct sequencing of 17 polymorphic nuclear markers developed previously, and (3) to compare our findings with published reports on strains from other areas of the globe. We found that EV001 is morphologically consistent with previously described strains. Multi-locus genotyping suggested that EV001 is grouped genetically with Panamanian strains and is most similar to strain JEL203 isolated from a captive individual. This finding fills an important gap in our knowledge of Neotropical strains of Bd and provides a baseline for further evolutionary and functional analyses.

Histoplasma mechanisms of pathogenesis--one portfolio doesn't fit all.

Histoplasma capsulatum is the leading cause of endemic mycosis in the world. Analyses of clinical isolates from different endemic regions show important diversity within the species. Recent molecular studies of two isolates, the Chemotype I NAm2 strain G217B and the Chemotype II Panamanian strain G186A, reveal significant genetic, structural, and molecular differences between these representative Histoplasma strains. Some of these variations have functional consequences, representing distinct molecular mechanisms that facilitate Histoplasma pathogenesis. The realization of Histoplasma strain diversity highlights the importance of characterizing Histoplasma virulence factors in the context of specific clinical strain isolates.

Molecular cloning and characterization of clyA genes in various serotypes of Salmonella enterica

Cytolysin A (ClyA) is a pore-forming hemolytic protein encoded by the clyA gene. It has been identified in Salmonella enterica serovars Typhi and Paratyphi A. To identify and characterize the clyA genes in various Salmonella enterica strains, 21 different serotypes of strains isolated from clinical specimens were presently examined. Full-length clyA genes were found in S. enterica serovar Brandenburg, Indiana, Panama, and Schwarzengrund strains by polymerase chain reaction amplification. The ClyA proteins from these four strains showed >97% amino acid identity to that of S. enterica serovar Typhi. Although all four serovars expressed detectable levels of ClyA as determined by Western blot analysis, they did not show a strong hemolytic effect on blood agar, indicating that ClyA may not be efficiently expressed or secreted. Escherichia coli transformed with clyA genes from the four serovars enhanced production of ClyA proteins and hemolytic activities to a level similar to S. enterica serovar Typhi ClyA. The present results suggest that ClyA may play a role in the pathogenesis of S. enterica serovar Brandenburg, Indiana, Panama and Schwarzengrund.

Severe respiratory disease and its relationship with respiratory viruses in Colombia

There are important gaps in our understanding of the epidemiology of respiratory virus infections in tropical countries. In September 2003, the Colombian epidemiological surveillance system was notified of several deaths from an acute respiratory disease (ARD). In order to identify the agents associated with ARD cases, a clinical and laboratory-based surveillance system was implemented throughout the country. Between September 19 and December 31, 2003, 64 suspected cases of ARD were reported; of these reported cases, 21 (33%) died. Among 25 patients who underwent virus studies, influenza A (H3N2) (n=7) was the most frequently identified agent. Other viruses included parainfluenza (4), influenza B (1), and respiratory syncytial virus (3). The peak occurrence of cases and deaths coincided with the replacement of the influenza A (H3N2) Panama strain, which had been circulating in Colombia since 1999, by three new influenza A (H3N2) strains (Korea, Fujian, and Wyoming). This outbreak led to the strengthening of surveillance for respiratory viruses and to new national recommendations for influenza vaccination in Colombia.

Antimalarial Linear Lipopeptides from a Panamanian Strain of the Marine Cyanobacterium Lyngbya majuscula

As part of the Panama International Cooperative Biodiversity Groups (ICBG) project, two new (2, 4) and two known (1, 3) linear alkynoic lipopeptides have been isolated from a Panamanian strain of the marine cyanobacterium Lyngbya majuscula. Carmabin A (1), dragomabin (2), and dragonamide A (3) showed good antimalarial activity (IC50 4.3, 6.0, and 7.7 microM, respectively), whereas the nonaromatic analogue, dragonamide B (4), was inactive. The planar structures of all four compounds were determined by NMR spectroscopy in combination with mass spectrometry, and their stereoconfigurations were established by chiral HPLC and by comparison of their optical rotations and NMR data with literature values.

Cross-reactivity of influenza A (H3N2) hemagglutination-inhibition antibodies induced by an inactivated influenza vaccine

The antigenic drift of influenza A (H3N2) virus in 2003–2004 necessitated a change in the vaccine from the A/Panama to the A/Wyoming strain for the 2004–2005 season. Using hemagglutination inhibition, we therefore tested antibodies in sera of 39 individuals (mean age 64.6 years) at the end of the 2003–2004 season for cross-reactivity to vaccine strains and H3N2 antigens subject to antigenic drift. Antibodies against both A (H3N2) Panama and Wyoming developed in 5/13 (38.5%) unvaccinated individuals, whereas, 22/26 (84.6%) vaccinees developed antibodies to Panama and 21/26 (80.8%) to Wyoming. None of these individuals suffered an influenza episode that season. The results suggest that the elderly might develop protective levels of cross-reactive A (H3N2) Wyoming HI antibodies following vaccination with the Panama strain. Such strains, like the ones included in the 2003–2004 influenza vaccine, might be expected to provide a broad-spectrum antibody response that could be effective even in the face of single season antigenic drift.

Psychosocial Factors and the Response to Influenza Vaccination in Older Adults

We examined the influence of psychological state (depression, negative affect, perceived stress) and social support on pre- and post-vaccination response to influenza vaccine. Venous blood was drawn from 37 nursing home residents before and following injection of the trivalent influenza vaccine (comprised of the New Caledonia (NC), Hong Kong (HK), and Panama (Pan) strains of flu). The Geriatric Depression Scale, Perceived Stress Scale, Positive and Negative Affect Schedule, and Multidimensional Scale of Perceived Social Support were completed following the initial blood draw. Social support and perceived stress were correlated with pre-vaccine antibody responses to two of the three vaccine components (HK and NC). Social support was negatively correlated with both pre- and post-vaccine titers to Pan. Depression, positive affect, and negative affect were not related to vaccine response. Perceived stress and social support influence the rate of decline of antibody titers to previous exposures to some strains of influenza occurring either naturally or via deliberate vaccination.

Potential of the Panama strain of Plasmodium vivax for the testing of malarial vaccines in Aotus nancymai monkeys.

Aotus monkeys were infected with a strain of Plasmodium vivax from Panama to determine its potential for the testing of malarial vaccines. After sporozoite inoculation, 3 splenectomized Aotus nancymai that had been infected previously with Plasmodium falciparum and P. vivax had prepatent periods of 13, 15, and 15 days with maximum parasite counts of 12,726/microl, 5,310/microl, and 9,180/microl. Three other A. nancymai previously infected with P. falciparum only had prepatent periods of 17, 15, and 15 days with maximum parasite counts of 44,460/microl, 31,500/microl, and 42,660/microl. One monkey with no previous history of infection had a prepatent period of 14 days after sporozoite inoculation, and a maximum parasite count of 100,000/microl; detectable parasitemia persisted for almost 500 days with 13 recognizable peaks in the parasite count. Anopheles dirus, Anopheles freeborni, Anopheles stephensi, and Anopheles quadrimaculatus mosquitoes were readily infected with the Panama strain.

Modification of the food and agriculture organization larval packet test to measure amitraz-susceptibility against ixodidae.

Modifications of exposure time, substrate, and formulation were made to the Food and Agriculture Organization Larval Packet Test (LPT) to determine a combination suitable for measuring the susceptibility of Boophilus microplus (Canestrini) to amitraz. Exposure time influenced the slope of the dose-response when paper was used as a substrate for amitraz. However, time did not influence the dose-response slope when nylon fabric was used as an amitraz substrate. Formulated amitraz produced results with less deviation from the log-probit model than technical amitraz. The combination of formulated amitraz and nylon fabric as a substrate for amitraz produced results that best fit the log-probit model. The modified FAO procedure (formulated amitraz/nylon substrate combination) was used to assay a Brazilian strain of B. microplus and a Panamanian strain of Rhipicephalus sanguineus (Latreille). Resistance ratios (95% CI) of 26.3 (25.7-26.9) and 7.3 (5.5-9.9) were calculated for the B. microplus and R. sanguineus strains, respectively. A discriminating dose of 0.03% amitraz was determined for B. microplus. This technique will help to locate amitraz resistant tick populations, provide data for improved control practices, and aid in the discovery of resistance mechanisms through synergist studies and verification of molecular techniques.

Characterization of acaricide resistance in Rhipicephalus sanguineus (latreille) (Acari: Ixodidae) collected from the Corozal Army Veterinary Quarantine Center, Panama.

Rhipicephalus sanguineus (Latreille) were collected from the Corozal Army Veterinary Quarantine Center in Panama and characterized for resistance to five classes of acaricides. These ticks were highly resistant to permethrin, DDT, and coumaphos; moderately resistant to amitraz; and not resistant to fipronil when compared with susceptible strains. Resistance to both permethrin and DDT may result from a mutation of the sodium channel. However, synergist studies indicate that enzyme activity is involved. The LC50 estimate for permethrin was lowered further in the Panamanian strain then in susceptible strains with the addition of triphenylphosphate (TPP), but not with the addition ofpiperonyl butoxide (PBO). This suggests that esterases and not oxidases are responsible for at least some pyrethroid resistance. Elevated esterase activity and its inhibition by TPP were confirmed by native gel electrophoresis. The LC50 estimate obtained for coumaphos in the Panamanian strain was not lowered further than what was observed for susceptible strains by the addition of TPP or PBO. This indicates that enzyme activity might not be involved in coumaphos resistance. Resistance to amitraz was measured through a modification of the Food and Agriculture Organization Larval Packet Test. All tick strains were found to be susceptible to fipronil.

Plasmids from bacterial endosymbionts of hump-killer paramecia

Six isolates of Caedibacter taeniospiralis, collected from four continents, were screened for plasmid DNA. Plasmid DNA species containing between 41.5 and 49.5 kilobase pairs (kb) were observed in all strains. Physical maps of plasmids were constructed by determining relative positions of the restriction endonuclease ( BamHI, SalI, XhoI, SacI, PstI, AvaI, and EcoRI) recognition sequences in each plasmid. The physical map of the smallest plasmid (41.5 kb), pKAP30, is reflected in each of the plasmids isolated from the other strains of C. taeniospiralis. Plasmid DNA from three of the isolates (strains 51 and 116 both from Indiana and strain 169 from Japan) each contain 43 kb, where 41.5 kb appear to be identical to pKAP30 (obtained from the Australian strain, A30). The extra 1.5 kb present in pKAP51, pKAP116, and pKAP169 is included as a single polynucleotide sequence. The 1.5-kb inclusion is located at apparently identical positions in pKAP116 and pKAP169 and at a totally different position in pKAP51. The two remaining plasmids, pKAP47 (from California strain 47) and pKAP298 (from Panama strain 298), both contain 49 kb to include a continuous 41.5-kb sequence that is apparently identical to pKAP30. The results indicate that the polynucleotide sequences of these plasmids are highly conserved and that the observed variations among them may be accounted for by transposable elements.