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Plasmid transformation of Streptomyces venezuelae: modified procedures used to introduce the gene(s) for p-aminobenzoate synthase.

Sucrose was unsuitable as an osmotic stabilizer in buffer solutions and media used for transformation of Streptomyces venezuelae ISP5230. Its replacement with NaCl, together with other modifications in the procedure, allowed efficient formation and regeneration of protoplasts but did not support transformation of S. venezuelae ISP5230 by vectors pIJ41 and pIJ941. With pIJ702, transformants with a low plasmid-copy-number and altered growth characteristics were obtained. Both pIJ702 and pIJ941, but not pIJ41, transformed S. venezuelae 13s; when pIJ941 was used, the plasmid in 18 of 20 transformants contained a deletion in the region reported to code for replication and transfer. The modified plasmid transformed S. venezuelae ISP5230 efficiently and was used to introduce a fragment of DNA from the pab locus of the wild-type into a Cml-1 mutant of ISP5230 blocked in chloramphenicol formation. Transformants that overproduced p-aminobenzoic acid were obtained but they remained blocked in chloramphenicol production; thus, the cloned pab fragment did not contain genes able to complement the cml-1 mutation. The results also suggest that the Cml-1 phenotype is not due to a defective reaction common to the biosynthesis of p-aminobenzoic acid and chloramphenicol.

4-Aminobenzoic Acid

Studies of arboviruses in Southwestern Venezuela: I. Isolations of Venezuelan and Eastern Equine Encephalitis viruses from sentinel hamsters in the Catatumbo region.

The purpose of this report is to describe isolations of Venezuelan (VEE) and Eastern (EEE) Equine Encephalitis virus made in the lowland moist tropical forest of the Catatumbo region on the southwestern part of the State of Zulia, Venezuela. We have isolated four strains of EEEV from sentinel hamsters exposed at Caño Mocho and Madre Vieja sites in 1973 and 1974, and three strains of EEEV in Hacienda (Hda.) Las Nubes in 1975. Both viruses were recovered during silent interepidemic periods and we believe these viruses are maintained in this region in sylvatic conditions. The recovered virus strains were detected within 24 to 48 hours, both in SMB and Vero Cell monolayer cultures and the sentinel hamsters yielded virus infectivities up to 10(4) PFU ml. Our VEEV isolate (IVIC PAn 23645-47), recovered during the silent interepizodemic period had an elution profile on the hydroxylapatite column corresponding to that of a I-D (VEEV-3880) or a I-E (VEEV-63A216) 'enzootic' subtype. However, considering other in vitro criteria (KHI; HA pH 5.8-6.0; small plaque size in Vero monolayers with suitable overlay media), this later and other previous isolates had some very distinct properties of the 'epizootic' strains. Thus, the evidence suggests that in Venezuela the VEEV cycle in nature is maintained either by the so called 'enzootic' and/or 'epizootic' virus types, or the virus population of the isolates have particular in vitro properties which do not correlate to the virulence markers in vivo. We consider this important question must be further clarified, and in addition, the isolation of three strains of EEEV are reported; this is the first report of the presence of this virus in Venezuela. Although the EEEV isolates may be of the South American type, they must be considered as potentially dangerous in the case of outbreaks.

Animals

[Onychomyosis caused by Trichopyton soudanense. 1st isolation in Venezuela].

Trichophyton soudanense is a dermatophyte wich is of highest incidence in Africa. Cases of tinea produced by this fungus have been described in England, Switzerland, Germany, Australia, the United States and Brazil. This investigation reports the first isolation of this fungus in Venezuela, from a lesion of the nail in a female patient who has always resided in Venezuela. The identification of this fungus was based on macroscopic and microscopic morphological characteristics; biological properties, scare or no growth when the only nitrogen source in the culture medium is provided by potassium or ammonium nitrate, and the production of dark pigment when inoculated in Lowenstein-Jensen medium. Experimental innoculation in animals produced an endothrix parasitism of the hair. Reference is made ot the incidence of the most frequently occuring dermatophytes in Venezuela.

Adult

[Selenium content in the blood cattle from Venezuela. I. Central and Portugueste zones].

Whole blood concentrations of selenium were determined in 776 dairy cows and calves from 28 farms located in three States (Aragua, Carabobo and Guárico) of the central zone of Venezuela and in Portuguesa State. Selenium levels largely varied according to both the geographic distribution and animals breed. Cattle from Guárico State showed the lowest average concentration (0.21 ppm) and those from Portuguesa the highest one (0.67 ppm). A group of animals from Turén (a District of Portuguesa State) showed the highest average concentration (1.64 ppm, range 1.02 +/- to 3.24 ppm). Concentrations of this magnitude in animals under grazing conditions could be related to high levels of selenium either in the soil or in the pasture. It is concluded that such animals are localized inaseleniferous zone (Turén). It must be noted that Jaffé et al. (op. cit.) found high levels of selenium in sesame seeds (Sesamum indicum) from this area and in blood and urine samples of children from Villa Bruzual, a little town of Turén. The results of this wok significantly differ from those obtained in other countries (of Europe and North America), where animals are managed under conditions very different from those of the tropical areas like Venezuela. The role this factor is discussed. This is the first report on blood levels in large animals of economical importance in Venezuela.

Analysis of Variance

Conjugational fertility and location of chloramphenicol biosynthesis genes on the chromosomal linkage map of Streptomyces venezuelae.

In Streptomyces venezuelae fertility, defined as chromosomal gene recombination, was enhanced over 1000-fold when one parent in a biparental conjugational cross lacked the physically-undetected plasmid SVP1, as compared with crosses in which both parents carried SVP1. The existence of SVP1 and at least two other fertility plasmids, SVP2 and SVP3, was detected in S. venezuelae by 'lethal zygosis' elicited by a plasmid-plus mycelium in contact with a plasmid-minus mycelium. Conjugational crosses were used to construct a linkage map of S. venezuelae which was highly consistent with the map of analogous loci in S. coelicolor A3(2). A cluster of genes governing chloramphenicol biosynthesis was located near arg, cys and pdxB genes at a position roughly equivalent to the 1-2 o'clock region of the S. coelicolor A3(2) map.

Chloramphenicol

Chloramphenicol resistance in Streptomyces: cloning and characterization of a chloramphenicol hydrolase gene from Streptomyces venezuelae.

A 6.5 kb DNA fragment containing a chloramphenicol-resistance gene of Streptomyces venezuelae ISP5230 was cloned in Streptomyces lividans M252 using the high-copy-number plasmid vector pIJ702. The gene was located within a 2.4 kb KpnI-SstI fragment of the cloned DNA and encoded an enzyme (chloramphenicol hydrolase) that catalysed removal of the dichloroacetyl moiety from the antibiotic. The deacylated product, p-nitrophenylserinol, was metabolized to p-nitrobenzyl alcohol and other compounds by enzymes present in S. lividans M252. Examination of the genomic DNA from several sources using the cloned 6.5 kb SstI fragment from S. venezuelae ISP5230 as a probe showed a hybridizing region in the DNA from S. venezuelae 13s but none in the DNA from another chloramphenicol producer, Streptomyces phaeochromogenes NRRLB 3559. The resistance phenotype was not expressed when the 6.5 kb SstI fragment or a subfragment was subcloned behind the lac-promoter of plasmid pTZ18R in Escherichia coli.

Amidohydrolases

Sporulation of Streptomyces venezuelae in submerged cultures.

Shaken cultures of Streptomyces venezuelae ISP5230 in minimal medium with galactose and ammonium sulphate as carbon and nitrogen sources, respectively, showed extensive sporulation after 72 h incubation at 37 degrees C. The spores formed in these cultures resembled aerial spores in their characteristics. The ability of the spores to withstand lysozyme treatment was used to monitor the progress of sporulation in cultures and to determine the physiological requirements for sporulation. In media containing ammonium sulphate as the nitrogen source, galactose was the best of six carbon sources tested. With galactose S. venezuelae ISP5230 sporulated when supplied with any of several nitrogen sources; however, an excess of nitrogen source was inhibitory. In cultures containing galactose and ammonium sulphate, sporulation was suppressed by a peptone supplement. The onset of sporulation was accompanied by a drop in intracellular GTP content. When decoyinine, an inhibitor of GMP synthase, was added to a medium containing starch and ammonium sulphate, a slight increase in sporulation was seen after 2 d. The suppression of sporulation by peptone in liquid or agar cultures was not reversed by addition of decoyinine. A hypersporulating mutant of S. venezuelae ISP5230 was altered in its ability to assimilate sugars. In cultures containing glucose the mutant sporulated more profusely than did the wild-type and did not acidify the medium to the same extent. However, the suppressive effect of glucose on sporulation was not merely a secondary result of acid accumulation.

Adenosine

Purification of Streptomyces venezuelae phage.

Kolstad, R. A. (University of Minnesota, Minneapolis), and S. G. Bradley. Purification of Streptomyces venezuelae phage. J. Bacteriol. 87:1157-1161. 1964.-Streptomyces venezuelae phage MSP8 was concentrated and purified by a combination of methods including dialysis against polyethylene glycol, partitioning between the two phases of aqueous polymer systems, gel filtration, chromatography on ECTEOLA-cellulose, and differential centrifugation. S. venezuelae phage MSP8 is 57% deoxyribonucleic acid and 43% protein. Its head is 55 by 70 mmu, and its tail is 10 by 150 mmu. Its dry weight is 250 mg per 10(15) plaqueforming units, and its density is 1.4. Phage MSP8 contains 15 mug of phosphorus and 40 mug of nitrogen per 10(12) particles. The ratio of light absorbancy at 260 to 280 mmu is 1.5. A mixture of two actinophages, MNP3 and MVP7, was separated by use of ECTEOLA-cellulose. In one fraction, 99% of the phage was MNP3; in another fraction, 99% of the phage was MVP7.

Bacteriophages

Characteristics of a donor population in western Venezuela.

To determine the characteristics of blood donors in western Venezuela, we collected data from 1983 to 1985 on 31,320 volunteer donors at the Blood Bank of the State of Zulia in Maracaibo. Fifty-nine percent of the donors were blood group O, 30 percent were group A, 9 percent were group B, and 2 percent were group AB. Most of the donors (93%) were Rh positive. One percent of donors had positive reactions to hepatitis B surface antigen, 3.15 percent for syphilis, 1.43 percent for antibodies to Trypanosoma cruzi, and 0.32 percent to human immunodeficiency virus antibodies. About one-half of the donors were between 18 and 30 years old, and only 10 percent were women. To determine if iron deficiency anemia was a cause for the small size of the female donor pool, we measured serum ferritin in 50 first-time female donors. Ten of these (20%) had serum ferritin values below normal, and the distribution of serum ferritin levels of all 50 was very similar to that reported for frequent donors in Europe and the United States, with a clustering of ferritin values between 10 and 70 ng per ml. The data indicate that blood donors in western Venezuela are markedly different from those in the United States and that iron supplementation may be indicated for female Venezuelan donors.

Adolescent

Zoonotic cutaneous leishmaniasis due to Leishmania (Viannia) braziliensis associated with domestic animals in Venezuela and Brazil.

After outbreaks of cutaneous leishmaniasis in Solano State, Venezuela, 5% of the population had parasitized ulcers while after similar outbreaks in Mesquita, Rio de Janeiro State, Brazil, 9% had the disease. In these foci children, including some under six years of age, were affected. There was no significant difference in the occurrence of the disease according to sex or type of employment. In Solano, 3% of dogs and 28% of donkeys had parasitized lesions, while in Mesquita these indices were 19.8% and 30.8% respectively. The parasite from man, dogs and equines was identified as Leishmania (Viannia) braziliensis, by zymodeme and serodeme characterization. In these foci there is evidence suggesting that leishmaniasis is a zoonosis, possibly with equines and dogs as reservoirs, although both a wild enzootic cycle and the role of man as a source of infection can not be ruled out. Transmission is assumed to occur peridomestically by sandfly vectors such as Lutzomyia panamensis in Venezuela and Lutzomyia intermedia in Brazil. Information about the origin of these foci suggests that infected equines may be an important factor in the dissemination of the parasite in a peridomestic situation where these sandflies are abundant.

Adolescent

Field evaluation of an exoantigen-containing Babesia vaccine in Venezuela.

Bovine babesiosis is endemic in Venezuela, causing significant losses in highly susceptible imported cattle. Current immunoprophylactic methods include the less desirable use of live parasites. Inactivated vaccines derived from exoantigen-containing supernatant fluids of in vitro Babesia bovis and B. bigemina cultures have been developed and constitute a major improvement in vaccine safety, stability and ease of handling. Vaccination trials conducted under field conditions provide the final evaluation of a culture-derived B. bovis-B. bigemina vaccine. During a 5-year period, approximately 8,000 cattle were vaccinated and 16 clinical trials carried out in 7 states of Venezuela. Clinical, serologic and parasitologic data were collected monthly from 10% of the animals over a 2-year period. Data were also collected from a similar number of nonvaccinated control cattle. Analysis of results from these trials demonstrated a reduction in the incidence of clinical disease among vaccinated animals and complete protection against mortality caused by babesiosis. Vaccine efficacy was measured calculating the incidence rates of disease and mortality among vaccinated and nonvaccinated cattle. Use of this inactivated vaccine offers the best combination of safety, potency and efficacy for the effective immunoprophylactic control of bovine babesiosis.

Animals

A field study of paragonimiasis in Venezuela.

In a natural focus of paragonimiasis in the northeastern region of Venezuela, 130 people were clinically, parasitologically and immunologically evaluated. A specific intradermal test was positive in 13%. Besides an index case, three other active cases of paragonimiasis were identified. Adult Paragonimus worms and metacercariae were recovered from Didelphis marsupialis and Eudaniela garmani, respectively. Previous reports of Paragonimus infection in Venezuela are discussed.

Adolescent

Cryptosporidiosis among patients with acquired immunodeficiency syndrome in Zulia State, Venezuela.

We studied the prevalence of Cryptosporidium in 29 patients with acquired immunodeficiency syndrome (AIDS) from Zulia State, Venezuela. They ranged in age from five months to 46 years. Two were children and 27 were adults, of which six were women. Of the 21 men, 66.6% reported homosexual behavior. Three stool samples from each patient were examined, and modified Ziehl-Neelsen carbolfuchsin staining of formalinether stool concentrates was used to identify Cryptosporidium oocysts. To detect the presence of other intestinal parasites, direct wet mounts and iron-hematoxylin-stained smears were examined. Cryptosporidium was found in 12 (41.3%) of the patients and was identified as a single parasitic infection in seven of the 12 patients (58.3%). Other pathogenic parasites encountered were Giardia lamblia (3 of 12, 25%), Entamoeba histolytica (1 of 12, 8.3%), Ascaris lumbricoides, Trichuris trichiura, and Strongyloides stercoralis (each 1 of 12, 8.3%). Blastocystis hominis, an organism with an uncertain taxonomic position and pathogenicity, was observed in three of 12 patients (25%). An inflammatory exudate was observed in 10 of 12 patients infected with Cryptosporidium. Most of the patients with this infection presented with chronic watery diarrhea and weight loss. Our results suggest that Cryptosporidium is very common in AIDS patients with diarrhea in Venezuela. However, the role of this parasite as an enteropathogen in these patients is uncertain.

AIDS-Related Opportunistic Infections

Brucella suis infections in collared peccaries in Venezuela.

A bacteriologic and serologic study was conducted on two ranches in the states of Apure and Guarico, Venezuela for brucellosis in collared peccaries (Tayassu tajacu). One hundred thirty-nine peccaries were necropsied and tissues were cultured. Forty-three isolations of Brucella suis biovar 1, were made from lymph nodes and spleens of 25 males and 18 females. Antibody to Brucella sp. was detected in sera from 122 animals by the rapid plate agglutination, standard tube agglutination, 2-mer-captoethanol, rivanol, complement fixation and card tests. Young animals had infection and reactor rates nearly as high as the older animals indicating most were infected at a relative early age. Results suggest that this species may transmit brucellosis when living with domestic animals. This is the first report of B. suis biovar 1 from collared peccaries in Venezuela.

Age Factors

IgM antibodies to native phenolic glycolipid-I in contacts of leprosy patients in Venezuela: epidemiological observations and a prospective study of the risk of leprosy.

In a randomized, double-blind vaccine trial in Venezuela, about 29,000 contacts of leprosy patients have been vaccinated with either a mixture of heat-killed Mycobacterium leprae and BCG or BCG alone, and are being re-surveyed annually to detect new cases of leprosy. All contacts had a serum sample collected at the time of entry into the trial, and 13,020 of these sera have been analyzed for antibodies to phenolic glycolipid-I (PGL-I). Antibody levels have been related to various characteristics of the contacts and to their risk of developing leprosy in the following 4 years. A strong association was found between PGL-I antibody level and the risk of developing leprosy, in spite of possible modification of the incidence rate induced by vaccination. Antibody levels were higher in females than in males, and declined progressively with age. Household contacts had higher levels than did non-household contacts, and levels were higher in individuals from the state in Venezuela which has the highest incidence of the disease. No substantial differences were found in antibody levels between contacts of multibacillary and paucibacillary patients, which may in part reflect the influence of treatment, and there was no clear association with the presence of BCG or lepromin scars or with skin-test responses to PPD and leprosy soluble antigen. The assay of antibodies to PGL-I seems unlikely to provide a sensitive or specific test for infection with M. leprae, and measuring PGL-I antibody levels as a screening procedure to identify those at high risk of developing leprosy is unlikely to be particularly useful in most leprosy control programs. Such assays may be useful for the epidemiological monitoring of changes in the intensity of infection with M. leprae in a community and for the study of carefully defined groups of contacts during some phases of control programs.

Adolescent

A genetic study of the malaria vector Anopheles nuneztovari from western Venezuela.

A photomap of larval polytene chromosomes of Anopheles nuneztovari (population B) from western Venezuela is presented. Samples of An. nuneztovari B taken from 4 study sites in the states of Barinas and Táchira comprise a single panmictic population, which is chromosomally identical to the cytotype identified by Kitzmiller et al. (1973) from western Venezuela and Colombia. The frequency of polymorphism 2La has increased significantly in the 16 years that have elapsed since the 1973 study. In addition, inversion 2La does not conform to Hardy-Weinberg equilibrium. These 2 changes are most likely due to environmental alterations which have resulted in the heterozygous condition becoming more locally adaptive.

Animals

Soft contact lens damage: a one-year study in Caracas, Venezuela.

We reviewed 424 requests for soft contact lens cleaning that had been sent to a service laboratory in Caracas, Venezuela in the course of a year. We believe that this sample represents a cross-section of problem lenses in a tropical country (Venezuela). Among the cases for which data were available, there were more extended wear lenses than daily wear lenses sent for cleaning. A majority of the lens wearers were females and/or over age 20. The wearers were experienced handling their lenses. A relatively high percentage of them (52 of 184; 28%) used homemade solutions (unpreserved saline, distilled water, tap water, and/or baby shampoo) for the care and cleaning of their lenses. Although the reasons for referral were similar to those reported from temperate zones, we found a much higher incidence of combined deposits (protein-lipid-inorganic) than reported in studies carried out in other countries. Fungal invasion seemed more prevalent in our area than in temperate zones. From our results, the need for better education of wearers is obvious. In addition, special measures for the control of fungal growth may be required locally. The high incidence of this type of growth may be partly related to tropical conditions that favor the dispersal of fungal spores.

Adolescent