Mitochondrial DNA suggests cryptic speciation in Prodiplosis longifila Gagné (Diptera: Cecidomyiidae) associated with geographic distance and host specialization.

Prodiplosis longifila is reported as a pest of a wide range of species cultivated in America, including citrus, solanaceous species and asparagus. This species has different behavioural traits that are primarily centred on the oviposition habit and the feeding of larvae, which can change depending on the host. However, scarce information is available on population studies and the natural history of this insect, and uncertainty exists about the taxonomic identity and the geographic distribution of this species. The main objective was to perform a phylogenetic and genetic study of P. longifila populations and to define whether the North American and South American populations belong to the same species or whether a differentiation process had occurred due to geographic distance. A second objective was to determine whether this species showed genetic differentiation by host specialization in South America. The phylogenetic and population analyses based on DNA barcodes (cytochrome oxidase I gene) and a region of the ribosomal DNA (ITS2) revealed divergent clades attributable to geographic distance and host specificity. The North American and South American P. longifila insects were confirmed to be genetically distinct, and the genetic distances exceeded the values expected for intraspecific variation. In South America, the population analysis of P. longifila from tomato, sweet pepper (Solanaceae), Tahiti lime and key lime (Rutaceae) hosts evidenced high genetic differentiation between populations associated with different hosts and an absence of gene flow between these groups, suggesting the corresponding formation of cryptic species.

Preliminary enquiry into the availability, price and quality of malaria rapid diagnostic tests in the private health sector of six malaria-endemic countries.

OBJECTIVES: This enquiry aimed to provide a snap-shot of availability, price and quality of malaria rapid diagnostic tests (RDTs) in private health facilities at selected sites in six malaria-endemic countries in Africa, South East Asia and South America. METHODS: In each study site, data collectors surveyed private healthcare facilities which were selected based on accessibility from their home institution. Using a questionnaire, information was recorded about the facility itself and the malaria RDT(s) available. Where possible, a small number of RDTs were procured and quality control tested using a standardized procedure. RESULTS: Of the 324 private healthcare facilities visited, 35 outlets (mainly private clinics and hospitals) were found to supply 10 different types of RDTs products. RDT prices across the six countries ranged from US$1.00 to $16.81. Five of the 14 malaria RDTs collected failed quality control testing. CONCLUSIONS: In the private outlets sampled, the availability of RDTs was limited. Some of the RDTs whose quality we tested demonstrated inadequate sensitivity. This presents a number of risks. Given the more widespread distribution of antimalarials currently planned for private sector facilities, parasite-based diagnosis in this sector will be essential to adhere to the WHO guidelines for effective case management of malaria. Considerable regulation and quality control are also necessary to assure the availability of accurate and reliable RDTs, as well as adequate case management and provider adherence to RDT results. Public sector engagement is likely to be essential in this process.

Isolation by distance among California sea lion populations in Mexico: redefining management stocks.

Understanding the spatial structure of a population is critical for effective assessment and management. However, direct observation of spatial dynamics is generally difficult, particularly for marine mammals. California sea lions (Zalophus californianus) are polygynous pinnipeds distributed along the Pacific coast of North America. The species' range has been subdivided into three management stocks based on differences in mitochondrial DNA, but to date no studies have considered nuclear genetic variation, and thus we lack a comprehensive understanding of gene flow patterns among sea lion colonies. In light of recent population declines in the Gulf of California, Mexico, it is important to understand spatial structure to determine if declining sea lion colonies are genetically isolated from others. To define population subdivision and identify sex biases in gene flow, we analysed a 355-bp sequence of the mitochondrial DNA control region and 10 polymorphic microsatellite loci from 355 tissue samples collected from six colonies distributed along Mexican waters. Using a novel approach to estimate sex biases in gene flow, we found that male sea lions disperse on average 6.75 times more frequently than females. Analyses of population subdivision strongly suggest a pattern of isolation by distance among colonies and challenge current stock definitions. Based on these results, we propose an alternative classification that identifies three Mexican management units: Upper Gulf of California, Southern Baja Peninsula, and Upper Pacific Coast of Baja. This revised classification should be considered in future assessment and management of California sea lion populations in Mexican waters.

Harmful alcohol use among acutely ill hospitalized medical patients in Oslo and Moscow: A cross-sectional study.

BACKGROUND: The aim was to estimate the prevalence of harmful alcohol use in relation to socio-demographic characteristics among acutely ill medical patients, and examine identification measures of alcohol use, including the alcohol biomarker phosphatidylethanol 16:0/18:1 (PEth). METHODS: A cross-sectional study, lasting one year at one hospital in Oslo, Norway and one in Moscow, Russia recruiting acute medically ill patients (≥ 18 years), able to give informed consent. Self-reported data on socio-demographics, mental distress (Symptom Check List-5), alcohol use (Alcohol Use Disorder Identification Test-4 (AUDIT-4) and alcohol consumption past 24 h were collected. PEth and alcohol concentration were measured in whole blood. RESULTS: Of 5883 participating patients, 19.2% in Moscow and 21.1% in Oslo were harmful alcohol users, measured by AUDIT-4, while the prevalence of PEth-positive patients was lower: 11.4% in Oslo, 14.3% in Moscow. Men in Moscow were more likely to be harmful users by AUDIT-4 and PEth compared to men in Oslo, except of those being ≥ 71 years. Women in Oslo were more likely to be harmful users compared to those in Moscow by AUDIT-4, but not by PEth for those aged < 61 years. CONCLUSIONS: The prevalence of harmful alcohol use was high at both study sites. The prevalence of harmful alcohol use was lower when assessed by PEth compared to AUDIT-4. Thus, self-reporting was the most sensitive measure in revealing harmful alcohol use among all groups except for women in Moscow. Hence, screening and identification with objective biomarkers and self-reporting might be a method for early intervention.

Evaluation of an in vitro and in vivo model for experimental infection with Leishmania (Viannia) braziliensis and L. (V.) peruviana.

Leishmania (Viannia) braziliensis and L. (V.) peruviana are two parasite species characterized by a very different pathogenicity in humans despite a high genetic similarity. We hypothesized previously that L. (V.) peruviana would descend from L. (V.) braziliensis and would have acquired its 'peruviana' character during the southward colonization and adaptation of the transmission cycle in the Peruvian Andes. In order to have a first appreciation of the differences in virulence between both species, we evaluated an in vitro and in vivo model for experimental infection. A procedure was adapted to enrich culture forms in infective stages and the purified metacyclics were used to infect macrophage cell lines and golden hamsters. The models were tested with 2 representative strains of L. (V.) braziliensis from cutaneous and mucosal origin respectively and 2 representative strains of L. (V.) peruviana from Northern and Southern Peru respectively. Our models were reproducible and sensitive enough to detect phenotypic differences among strains. We showed in vitro as well as in vivo that the L. (V.) braziliensis was more infective than L. (V.) peruviana. Furthermore, we found that in vitro infectivity patterns of the 4 strains analysed, were in agreement with the geographical structuring of parasite populations demonstrated in our previous studies. Further work is needed to confirm our results with more strains of different geographical origin and their specific clinical outcome. However, our data open new perspectives for understanding the process of speciation in Leishmania and its implications in terms of pathogenicity.

Cortisol and pain-related behavior in disbudded goat kids with and without cornual nerve block.

Plasma cortisol and behavior were measured in disbudded goat kids with and without the use of cornual nerve block. A total of 45 kids were used in 5 experimental groups (n=9, males and females). Group LidoD was infiltrated with 1 mL of 2% lidocaine locally at the cornual branches of lacrimal and infratrochlear nerves, 15 min before thermal disbudding. Group Lido was similarly infiltrated and was not disbudded. In group Sim, the disbudding procedure was simulated. A control group (CD) was disbudded without lidocaine infiltration, and group SD was infiltrated with saline before disbudding. The cornual nerve block did not prevent the short-term increase in cortisol levels during and after disbudding. LidoD, CD and SD groups showed higher cortisol concentrations than Lido and Sim (p<0.05) during the first 20 min after the procedure. Frequency of vocalizations during the procedure was significantly different between groups and was higher in SD (29.6 ± 3.1; mean±SE) and CD (28.4 ± 3.1) than in Sim (16.6 ± 3.1; p<0.05). Infiltrating lidocaine did not decrease this response to disbudding (21.1 ± 3.1; p>0.05). Struggles tended to be higher in SD (16.5 ± 2.5), CD (17.8 ± 2.5) and LidoD (15.6 ± 2.5) than Sim (10.6 ± 2.5; p=0.1). The total behavioral response was different between groups (CD, 59.6 ± 6.8; LidoD, 52 ± 6.8; SD, 62.6 ± 6.8; Sim, 36.8 ± 6.8; p=0.05), and disbudded animals showed the strongest reactions (disbudded, 58.1 ± 3.9 vs non-disbudded, 36.8 ± 6.8; p=0.01). It was concluded that cornual nerve block (lacrimal and infratrochlear) using 2% lidocaine did not prevent pain during thermal disbudding of goat kids.

Host response to Treponema pallidum in intradermally-infected rabbits: evidence for persistence of infection at local and distant sites.

Intradermal infection of rabbits with Treponema pallidum imitiates rapid and active cellular response at the site of injection. During the first 2 weeks following infection, there is a marked increase in the numbers of organisms at the site of infection. Systemic dissemination of treponemes occurs during the early stage of infection, presumably before the immune response is fully mobilized. The mononuclear infiltration, which is apparent at the lesion site one week postinfection, becomes more pronounced at 2 weeks. The infiltrating cells are predominantly T lymphocytes and macrophages. By 4 weeks postinfection, most of the organisms have been cleared from the primary site; however, low numbers of treponemes survive locally and in distant tissues. Thus, whereas infection with T. pallidum appears to activate immune mechanisms which are capable of clearing most of the organisms from the primary lesion, some organisms are able to evade these mechanisms and persist in vivo.

Hepatic carcinoma with spleen metastasis in a California sea lion from the Gulf of California.

A primary hepatic carcinoma with a neuroendocrine pattern was detected in an adult female California sea lion (Zalophus californianus) found dead on Granito Island in the Gulf of California (Mexico) in January 1996. At necropsy, several light yellow nodules of different sizes were observed on the entire surface of the liver and spleen. Microscopic examination of these nodules using routine haematoxylin-eosin stain, revealed cubic, polyhedral and pleomorphic cells with three to four bizarre mitotic figures per field (40X). An immunohistochemistry test revealed a positive reaction of indirect immunoperoxide to cytokeratin (CK2). This is the first known case of a primary hepatic carcinoma in free-ranging California sea lions from Mexican waters.

Antibodies against Leptospira interrogans in California sea lion pups from Gulf of California.

One hundred and twenty-five serum samples from California sea lion (Zalophus californianus californianus) pups, and one from an adult female from eight reproductive rookeries located in seven islands in the Gulf of California (Mexico), were collected during the 1994-96 reproductive seasons. These were tested for antibodies to 19 serovars of Leptospira interrogans using a Microscopic Agglutination Test (MAT). Forty-one samples (32%) had antibody levels from 1:20 to 1:320 to one or more serovars. The most frequently detected serotypes were Leptospira interrogans hardjo (n = 13), cynopteri (8), ballum (6), and szwajizak (5). Serovars with the highest prevalence were Leptospira interrogans hardjo and serjoe (1:320), ballum (1:160), and cynopteri, girppotyphosa, and tarassovi (1:80). Based on these results, exposure of sea lions to L. interrogans serovar hardjo seems to be relatively common among colonies located in the islands of the Gulf of California in contrast with those located on the Pacific coast, where the most frequently detected serovar is L. interrogans serovar pomona.

Use of expander cottonseed meal in broiler diets formulated on a digestible amino acid basis.

This experiment was designed to evaluate the use of expander cottonseed meal (CSM) in broiler diets formulated on a digestible amino acid basis and to determine the tissue distribution of gossypol enantiomers and their relationship to gossypol consumption. Gossypol is an antinutritional polyphenolic pigment found in cottonseed. A total of 800 1-d-old broilers (Arbor Acres) was randomly divided into 40 groups of 20 birds each and placed in a broiler house with wood shavings litter. The CSM was included in starter and grower diets at 0, 7, 14, 21, and 28% of the diet. Diets were formulated to have similar levels of digestible methionine and lysine as the 0% CSM control diet. At 21 d of age, five birds per pen were randomly selected for the determination of gossypol enantiomers in plasma, liver, heart, and breast muscle. Tissues were again collected at 42 d of age. Results of this experiment indicated that at 21 d of age cumulative body weight and feed-to-BW ratios of birds fed CSM diets were not significantly different from the control. By 35 d of age, feed-to-BW ratios of experimental birds fed the 28% CSM diet were significantly greater than the control birds, and by 42 d, BW were lower than those of the control group. Tissue concentrations of total, (+) and (-) gossypol increased linearly as the level of CSM increased. At 42 d, liver had the most gossypol with a ratio of 87% (+) gossypol to 13% (-) gossypol. Plasma contained 73% (+) gossypol and 27% (-) gossypol. Heart contained 45% (+) gossypol and 55% (-) gossypol. Breast muscle had the least gossypol. Results of this experiment indicated that expander solvent CSM could be fed to broilers at up to 21% of the total diet if amino acid digestibility was considered.

Tissue distribution of gossypol enantiomers in broilers fed various cottonseed meals.

Plasma and tissue concentrations of total, (+)- and (-)-gossypol were determined in broilers fed cottonseed meals (CSM) from eight oil mills (five expander solvent, two expeller, and one direct solvent). Free gossypol in the meals ranged from 0.033 to 0.180%, and total gossypol ranged from 0.974 to 1.459%. The (+)-enantiomer of gossypol varied from 53.8 to 61.3% of total gossypol. Eight CSM diets containing 28% CSM and a soybean meal control diet were fed to 162 1-d-old male broiler chicks during a 3-wk starter period. Concentrations of free gossypol in the CSM diets ranged from 92 to 504 microg/g, and total gossypol ranged from 2,626 to 4,085 microg/g. All diets were formulated with the same concentrations of digestible lysine and methionine and were fed ad libitum. At 21 d of age, there were no significant differences in body weights, feed conversions, or mortality of birds fed the CSM diets when compared to birds fed the soybean meal diet. Concentrations of (+)- and (-)-gossypol in plasma, liver, kidney, and muscle increased linearly as the level of free gossypol increased in the diets. Liver had the highest concentration of total gossypol (71.4 to 313.6 microg/g DM) followed by kidney (9.2 to 36.3 microg/g DM), plasma (3.0 to 14.6 microg/mL), and muscle (2.1 to 9.8 microg/g DM). The proportion of (-)-gossypol was higher in plasma (26.7%) and kidney (25.6%) than in muscle (19.1%) and liver (16.0%). Performance data from this study indicate CSM can be used successfully in chick starter diets at levels up to 28% when diets are formulated on a digestible amino acid basis.

Evaluation of BACTEC™ MGIT™ 960 and the nitrate reductase assay in the National Laboratory Network of Colombia.

SETTING: The Mycobacteria Group of the National Reference Laboratory, National Laboratory Network, National Institute of Health and the Public Health Laboratories of Antioquia, Atlántico and Valle del Cauca, Colombia. OBJECTIVE: To compare BACTEC™ MGIT™ (Mycobacteria Growth Indicator Tube) 960 and the nitrate reductase assay (NRA) with the proportion method on Löwenstein-Jensen medium and to determine resistance to first-line anti-tuberculosis drugs. METHODS: Evaluation of diagnostic assays using time-adjusted convenience sampling. We analysed 183 isolates of Mycobacterium tuberculosis to compare MGIT 960 and NRA with the proportion method. RESULTS: MGIT 960 had a sensitivity and specificity of 90% for isoniazid (INH), and respectively 100% and 99.4% for rifampicin (RMP). NRA had a sensitivity and a specificity of respectively 86% and 94.7% for INH and 100% and 99% for RMP. CONCLUSION: Given its high sensitivity and specificity, and easy accessibility and interpretation of results, the NRA should be implemented in the National Laboratory Network for the detection of resistance to INH and RMP.

Putative markers of infective life stages in Leishmania (Viannia) braziliensis.

Gene expression is known to vary significantly during the Leishmania life-cycle. Its monitoring might allow identification of molecular changes associated with the infective stages (metacyclics and amastigotes) and contribute to the understanding of the complex host-parasite relationships. So far, very few studies have been done on Leishmania (Viannia) braziliensis, one of the most pathogenic species. Such studies require, first of all, reference molecular markers. In the present work, we applied differential display analysis (DD analysis) in order to identify transcripts that might be (i) candidate markers of metacyclics and intracellular amastigotes of L. (V.) braziliensis or (ii) potential controls, i.e. constitutively expressed. In total, 48 DNA fragments gave reliable sequencing data, 29 of them being potential markers of infective stages and 12 potential controls. Eight sequences could be identified with reported genes. Validation of the results of DD analysis was done for 4 genes (2 differentially expressed and 2 controls) by quantitative real-time PCR. The infective insect stage-specific protein (meta 1) was more expressed in metacyclic-enriched preparations. The oligopeptidase b showed a higher expression in amastigotes. Two genes, glucose-6-phosphate dehydrogenase and a serine/threonine protein kinase, were found to be similarly expressed in the different biological samples.

Experimental neonatal syphilis. I. Evidence of resistance to symptomatic infection in neonatal rabbits following intradermal inoculation with Treponema pallidum (Nichols strain).

Resistance of 5- to 8-day-old neonatal rabbits to dermal lesion development after intradermal inoculation of Treponema pallidum was demonstrated. Clinical evidence of infection following inoculation of 1 X 10(6) Treponema pallidum at each of two sites was either minimal or absent. Atypical, nonprogressive, nonulcerative lesions occurred in 59% of the inoculated neonates and at 45% of inoculated sites. Differences in incubation periods, duration, and maximum diameters of lesions among adult controls versus neonatal rabbits were significant. The age of waning resistance was determined by inoculating groups of neonates ranging from 1 to 7 weeks of age. Five-week-old (31-36 days) neonates demonstrated waning resistance by the appearance of typically ulcerative, progressive lesions, though their parameters (duration, size) were not yet those of adult control lesions. The resistance demonstrated by neonates may be due in part to group housing (nesting) which could create unfavorable temperatures for T. pallidum survival; comparison of lesion development between nesting and individually housed neonates, 31 to 46 days of age, revealed a greater percentage of typical lesions developing among those individually housed (95 versus 52%). However, these differences may reflect the variability of typical lesion development found among animals of this age when resistance begins to wane. In both groups, the duration of typical lesions was significantly shorter than for adult controls. A heat-stable serum factor(s) was demonstrated in 19 of 20 basal sera from neonates 4 to 6 days of age; this presented another possible mechanism of resistance. The neutralizing serum factor(s) was not demonstrable in the sera of does either before mating, during gestation, or shortly after kindling.(ABSTRACT TRUNCATED AT 250 WORDS)