Species diversity, novel interactions and absence of well-supported host-guided phylogenetic groupings of Neotropical Alternaria isolates causing foliar lesions in Solanaceae.

AIM: To report the characterization of 120 Alternaria isolates inducing early blight-like foliar lesions in nine species of five Solanaceae genera collected across all macrogeographical Brazilian regions. MATERIAL AND RESULTS: Phylogenetic relationships were assessed via analyses of the Alternaria alternata allergenic protein-coding, glyceraldehyde-3-phosphate dehydrogenase and the calmodulin gene sequences. Most of the tomato isolates were placed into the Alternaria linariae cluster, whereas most of the potato isolates were grouped with Alternaria grandis. Novel host-pathogen interactions were also reported. Seventeen isolates were selected for morphometrical characterization, and a subsample of 13 isolates was employed in pathogenicity assays on tomato, potato, eggplant, scarlet eggplant, Capsicum annuum, Datura stramonium, Physalis angulata and Nicotiana tabacum. Eleven isolates were able to induce foliar lesions in tomatoes but none in C. annuum. Potato was susceptible to a subgroup of isolates but displayed a subset of isolate-specific interactions. Morphological traits were in overall agreement with molecular and host range data. CONCLUSION: Alternaria linariae and A. grandis were confirmed as the major causal agents of tomato and potato early blight, respectively. However other Alternaria species are also involved with early blight in solanaceous hosts in Brazil. SIGNIFICANCE AND IMPACT OF THE STUDY: The diversity and host-specific patterns of the Alternaria isolates from Solanaceae may have practical implications in establishing effective early blight genetic resistance and cultural management strategies especially for tomato and potato crops.

A three-decade survey of Brazilian Fusarium oxysporum f. sp. lycopersici races assessed by pathogenicity tests on differential tomato accessions and by molecular markers.

AIM: Physiological race determination of 143 Fusarium oxysporum f. sp. lycopersici (FOL) isolates collected along 30 years in major tomato-producing regions of Brazil. MATERIALS AND RESULTS: Physiological races were determined via root-dipping inoculation of differential tomato accessions and by the PCR-based marker system of Hirano and Arie (2006). According to pathogenicity/virulence assays, five race 1, 23 race 2 and 115 race 3 isolates were identified. FOL race 1 and 2 isolates prevailed up to early 2000s. Afterwards, the large majority of the isolates was classified as the invasive race 3. Novel reports of race 3 were done in five states, thus expanding its geographical distribution. Using this PCR-based marker system, a precise discrimination was observed for all race 3 isolates. However, all race 1 and 2 isolates displayed only the cosmopolitan race 1-specific amplicon pattern. CONCLUSION: The development and/or validation of novel race-specific marker systems are necessary to allow a precise discrimination of the potentially endemic Brazilian FOL race 2. SIGNIFICANCE AND IMPACT OF THE STUDY: The present characterization of isolates indicates that distinct evolutionary mechanisms are acting to select new FOL races and/or genetic variants across agroecosystems around the globe.

First Report of Tomato chlorosis virus Infecting Tomato Crops in Uruguay.

A survey of viral diseases was carried out during 2012 to 2013 in two major tomato (Solanum lycopersicum L.) producing regions in Uruguay (Salto and Canelones). Lower leaves of fruit-bearing plants were observed displaying yellowing and interveinal chlorosis under greenhouse conditions. The symptoms were similar to those associated with magnesium deficiency. However, the chlorosis displayed a tendency to move up affecting medial and apical leaves and prevailed even after supplementary magnesium applications to the soil, indicating potential infection by either Tomato chlorosis virus (ToCV) or Tomato infectious chlorosis virus (TICV) (3). Four leaf samples were collected from two sites in Canelones and 28 samples were collected from distinct commercial fields in Salto. Whiteflies (Bemisia tabaci biotype Q and Trialeurodes vaporariorum) were present in all sampling sites. Total RNA was extracted from symptomatic and healthy (control) plants and used for cDNA preparation with the HS-11/HS-12 primer pair followed by PCR amplification using the same primer pair. The 587-bp amplicon, corresponding to a highly conserved region of the heat shock protein (HSP-70) homolog gene reported in both TICV and ToCV genomes, was observed only in the symptomatic samples. These PCR products were then subjected to nested PCR using the ToCV specific primer pair (ToC-5/ToC-6) and TICV specific primer pair (TIC-3/TIC-4) (1). The expected 463-bp ToCV-specific amplicon was observed in all symptomatic plants but not in the healthy controls. The 223-bp amplicon corresponding to TICV was not observed in any sample, indicating the sole presence of ToCV. The amplicon of one Uruguayan ToCV isolate from Salto (named as CRS03) was purified and directly sequenced (GenBank KC626018). BLAST analysis revealed 99% identity of CRS03 with one Spanish isolate (AF233435.1) (2). Virus-free B. tabaci biotype Q adults were exposed to symptomatic plants infected with the CRS03 isolate for a 24-h period and then cage-confined with 10 healthy tomato plants (line 'LT17') for a 48-h period. Symptoms were reproduced in all tested plants after a 65-day period and ToCV infection was confirmed via PCR assays and by sequence analysis of the gel-purified amplicons. This is the first formal report of ToCV infecting tomatoes in Uruguay. Incidence of symptomatic plants in tomato crops varied from 30 to 100%, even under low whitefly pressure. Epidemiological information needs to be generated in order to evaluate the impact of ToCV in the fresh-market tomato yield and quality. References: (1) C. I. Dovas et al. Plant Dis. 86:1345, 2002. (2) G. Lozano et al. Arch. Virol. 151:581, 2006. (3) G. C. Wisler et al. Arch. Virol. 151:409, 2006.

Report of Tomato yellow spot virus Infecting Leonurus sibiricus in Paraguay and Within Tomato Fields in Brazil.

Leonurus sibiricus L. (Lamiaceae) is a subtropical weed frequently found with golden mosaic symptoms. Leonurus mosaic virus (LeMV) was the first begomovirus reported on L. sibiricus in Brazil (3). Later, a new bipartite species (Tomato yellow spot virus, ToYSV) was reported affecting tomatoes, beans, and also L. sibiricus (1,2). A survey of begomovirus isolates was conducted within tomato fields also displaying high incidence of plants with begomovirus-induced symptoms. Thirty L. sibiricus and 33 tomato samples were collected (2007 to 2012) in nine districts in Paraná State, Brazil. Two L. sibiricus isolates were also obtained within citrus orchards in Major Otaño, Itapúa, Paraguay. Total DNA was extracted from all 65 isolates and PCR assays were conducted with primers for conserved DNA-A (PAL1v1978/PAR1c496) and DNA-B (PBL1v2040/PCRc1) regions (3). Nucleotide sequence identity of the 1,193-bp DNA-A amplicons of our L. sibiricus isolates ranged from 93.4 to 98.2% with LeMV (GenBank Accession No. U925321) and from 92.4 to 94.8% with ToYSV isolates from tomato (DQ336350.1) and bean (FJ538207). None of the 33 tomato samples was found to be infected by ToYSV, with all having high nucleotide sequence identity (92 to 99%) only with Tomato severe rugose virus (GU358449). Complete DNA-A genome sequence was obtained via a rolling circle amplification-based strategy for one Brazilian L. sibiricus isolate (PR-088) and one isolate from Paraguay (PAR-07). The entire DNA-A genome of PR-088 (JQ429791) had 96.8% nucleotide sequence identity with PAR-07 (KC683374) and ranged from 95.6 to 96.3% with ToYSV isolates from bean, tomato, and L. sibiricus (JX513952). The nucleotide sequence identity of the 487-bp DNA-B amplicon ranged from 87 to 92% among PR-088 (KC 683374); PAR-07 (KC740619) and ToYSV isolates from tomato (DQ336351.1) and L. sibiricus (JX513953.1). Leonurus cuttings infected with the ToYSV (PR-088) were caged together with healthy L. sibiricus and tomato 'Alambra' seedlings. Hybridization assays with ToYSV-specific probes (2) and sequencing of PCR amplicons indicated that Bemisia tabaci biotype B adults were able to transmit ToYSV to both hosts as reported (1). Our results suggest that L. sibiricus is the main ToYSV reservoir under natural conditions and tomato seems to be an occasional alternative host. In fact, ToYSV has not often detected in tomatoes as observed in a number of extensive surveys (4). So far, the complete LeMV genome is not available for comparison (3). However, our analyses with a DNA-A segment indicated that LeMV and ToYSV isolates might represent strains of single virus at the current threshold of 89% nucleotide sequence identity for Begomovirus species discrimination (4). Thus, a reappraisal of the taxonomic status of ToYSV is necessary to clarify its genetic relationship with LeMV. This is the first report of ToYSV on L. sibiricus in Paraguay. References: (1) J. C. Barbosa et al. Plant Dis. 97:289, 2013. (2) R. F. Calegario et al. Pesq. Agrop. Bras. 42:1335, 2007. (3) J. C. Faria and D. P. Maxwell, Phytopathology 89:262, 1999. (4) F. R. Fernandes et al. Virus Genes 36:251, 2008.

Physalis angulata: A New Natural Host of Tomato chlorosis virus in Brazil.

Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV) are the two Solanaceae-infecting Crinivirus species (family Closteroviridae) of worldwide importance. In Brazil, only ToCV has been detected under natural conditions infecting tomato (Solanum lycopersicum), sweet pepper (Capsicum annuum), and potato (S. tuberosum), causing foliar chlorosis (1, 3). However, there are no formal reports of alternative weed hosts of ToCV. During crop surveys in Capão Bonito, São Paulo State, Brazil (May 2011), a high incidence (above 20%) of plants of the weed, cut leaf ground cherry (Physalis angulata L.) growing around and within a tomato (cv. Alambra) field with a high incidence of ToCV, were found displaying interveinal chlorosis on the lower leaves, similar to those induced by magnesium deficiency. The P. angulata plants also had high populations of whiteflies (Bemisia tabaci biotype B). Ten leaf samples were taken from individual symptomatic ground cherry and tomato plants for Crinivirus testing. Total nucleic acids were extracted from 2 g of symptomatic and healthy leaf tissues of both hosts using Whatman CF-11 cellulose (Sigma) as described (4). The purified double stranded RNA samples were used as a template in reverse transcription (RT)-PCR using specific primers targeting the p22 gene region in the genome of ToCV (2). Only the 566-bp ToCV-specific amplicon was detected in all field samples. Sequences of samples from the P. angulata and tomato cDNA amplicons were identical to each other (GenBank Accession No. JX187514) and they showed 99% identity with the ToCV RNA 1 from a tomato isolate from Florida (AY903447). This confirmed the initial hypothesis of Crinivirus infection. Cuttings of symptomatic P. angulata plants were also obtained and kept in a voile cage under greenhouse conditions together with healthy seedlings of P. angulata and the begomovirus-resistant inbred tomato line 'TX-468RG.' Fifty aviruliferous B. tabaci (biotype B) adults were placed in the cage. Similar symptoms were observed 50 days after exposure to whiteflies in both hosts. Transmission to P. angulata and to 'TX-468RG' was also confirmed via sequencing of ToCV-specific amplicon, demonstrating the infectivity of the isolate to both hosts. To our knowledge, this is the first report of P. angulata as a natural host of ToCV in Brazil. This weed is often present in the commercial fields because of its natural tolerance to herbicides currently used in tomato production. The ToCV-infected P. angulata plants might serve as alternative sources of inoculum for the surrounding tomato fields. The environmental persistence of P. angulata combined with its intense whitefly colonization might allow a year-round ToCV exposure for tomato plants under field conditions in this major production area of Brazil where at least 25 million tomato plants are cultivated annually. References: (1) J. C. Barbosa et al. Trop. Plant Pathol. 36: 256, 2011. (2) M. I. Font et al. Plant Dis. 86:696, 2002. (3) D. M. S. Freitas et al. Plant Dis. 96:593, 2012. (4) R. A. Valverde et al. Plant Dis. 74:285, 1990.

Identification of Fusarium oxysporum f. sp. lycopersici Race 3 Infecting Tomatoes in Northeast Brazil.

The three races of Fusarium oxysporum f. sp. lycopersici (FOL) are important tomato pathogens throughout the world, causing severe economic losses (1). In Brazil, races 1 and 2 are widespread, but the current geographic distribution of race 3 is restricted to the mild climate areas of Espírito Santo and Rio de Janeiro States in the southeast region (2,3). Here we report the spread of FOL race 3 to the warm northeast region of Brazil. Plants in commercial fields of the hybrid 'Alambra' (resistant to FOL races 1 and 2) were found displaying chlorosis, vascular browning, and wilt symptoms in Jaguaquara County, Bahia State, Brazil. Disease incidence ranged from 10 to 50%. The virulence profile of six isolates obtained from three distinct tomato-producing fields was investigated by root-dipping inoculation (106 conidia/ml) of 21-day-old seedlings from a set of FOL race differential accessions: 'Ponderosa' (susceptible to all races), 'IPA-5' (FOL race 1 resistance; I-1 locus); 'Alambra' and 'Floradade' (FOL races 1 and 2 resistance; I-2 gene), and Solanum pennellii 'LA 716' (resistant to all three races; I-3 locus). All six isolates were able to induce severe wilt symptoms in 100% of the plants from all lines but S. pennellii 'LA 716'. FOL race 3 identity was confirmed via PCR assays employing a specific set of primers that are able to discriminate all the three FOL races as well as F. oxysporum f. sp. radicis-lycopersici isolates (1). Total DNA was extracted from pure fungal colonies growing in agar medium. The typical FOL race 3 amplicon profiles (i.e. positive for the primers uni, sp13, and sp23 and negative for the primer sprl) were observed only in the six FOL 3 isolates from Bahia as well as in five reference isolates of race 3 (previously obtained from tomato in Espírito Santo and Rio de Janeiro States), thus confirming their race identities. This recent, fast, and wide geographic expansion of the FOL race 3 in Brazil suggests that the pathogen has been introduced into new tomato producing areas via either contaminated seeds or seedlings. Because of the complexity of establishing effective chemical and cultural control strategies, these epidemics caused by FOL race 3 in distinct areas of Brazil might cause the replacement of the currently grown susceptible hybrids by resistant ones. References: (1) Y. Hirano and T. Arie. J. Gen. Plant Pathol. 72:273, 2006; (2) A. Reis et al. Fitopatol. Bras. 30:426, 2005; (3) A. Reis and L. S. Boiteux. Hort. Bras. 25:451, 2007.

A Tomato Fruit Rot Caused by Trichothecium roseum in Brazil.

Fruit rots caused by distinct fungal pathogens are commonly observed on tomatoes (Solanum lycopersicum L.) throughout all major production areas in Brazil. Samples of fruits displaying white mycelial growth associated with a profuse salmon-color sporulation were collected in greenhouse-grown tomatoes in Brasília-DF in February 2011. The isolated fungus displayed pink-to-white colonies containing several conidiophores with conidia. Mycelia displayed hyaline hyphae as much as 4 µm in diameter; conidiophores were simple or branched, 112 to 300 (360) µm long, and 2 to 4 µm wide. Conidia were produced in basipetal chains (frequently clustered), were ellipsoidal to pyriform with oblique and prominent truncate basal scars, two-celled, hyaline, and (14-) 16 to 26 (-28) × (6-) 7 to 10 (-12) µm. These characteristics allocated the specimen to Trichothecium roseum (Pers.). Koch's postulates were fulfilled for one fungal isolate by either spraying 10 intact fruits or by placing a drop of a spore suspension (adjusted to 105 conidia/ml) into three to five wounds created on 10 mature fruits of each of two tomato cultivars (Santa Clara and Dominador) by puncturing each fruit with a sterile needle. Five fruits of each cultivar were treated with sterile water as the mock-inoculated control treatment. Identical symptoms to those of the original fruit were observed only in the T. roseum-inoculated samples 5 to 7 days after using both inoculation procedures. Total DNA was extracted from a pure colony of the fungus growing on potato dextrose agar medium and used as template in PCR assays with the internal transcribed spacer (ITS)-4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS-5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') primer pair (2). A single amplicon of approximately 630 bp was observed and directly sequenced. Sequence analysis of the Brazilian isolate (GenBank No. JN081877) indicated identity levels of 99% with T. roseum isolates reported on Leucadendron xanthoconus in South Africa (GenBank No. EU552162) and isolates from strawberry fruits in South Korea (GenBank No. HM355750). However, phylogenetic analysis was unable to discriminate isolates of T. roseum from Passalora (GenBank No. EF432764) and Fusarium (GenBank No. GU183369) isolates, confirming the low genetic variability of the ITS region in Hypocreales (3). T. roseum has been reported to be infecting greenhouse tomatoes in the United States (4) and causing postharvest disease of tomatoes in Argentina (1). To our knowledge, this is the first report of T. roseum infecting greenhouse tomatoes in Brazil. References: (1) G. Dal Bello. Australas. Plant Dis. Notes 3:103, 2008. (2) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (3) L. Lombard et al. Stud. Mycol. 66:31, 2010. (4) A. W. Welch, Jr. et al. Plant Dis. Rep. 59:255, 1975.

First Report of Sida micrantha mosaic virus in Phaseolus vulgaris in Brazil.

Snap and common beans (Phaseolus vulgaris L.) are severely affected by Bean golden mosaic virus (BGMV) infection, so far the only begomovirus reported on these crops in Brazil (1). Samples of snap and common beans colonized by the whitefly Bemisia tabaci biotype B and displaying golden mosaic, chlorotic spots, and leaf distortion were collected in three production regions in Goiás State (Goianápolis, Luziânia, and Itaberaí) between 2003 and 2007. Total DNA extracted from leaf samples was used as template in PCR assays using universal primers targeting conserved regions of the DNA-A and DNA-B genomes (3). Begomovirus-specific amplicons were observed only with DNA template from symptomatic plants. Two single amplicons were observed for both genomic segments, indicating the presence of bipartite species in all samples. Sequence analysis of four isolates (named as GO-176, GO-260, GO-354, and GO-368) obtained from common bean samples indicated identity levels of approximately 95% with the DNA-A segment of BGMV (GenBank Accession No. FJ665283). However, the complete DNA-A sequence (GenBank Accession No. HM357459.1) of the GO-060 isolate (from a symptomatic snap bean plant collected in Goianápolis) displayed 76% identity with BGMV (GenBank Accession No. FJ665283) and 95% identity with the DNA-A of a Sida micrantha mosaic virus (SimMV) isolate (GenBank Accession No. EU908733.1) reported to be infecting okra (Abelmoschus esculentus L.) and 94.8% with a SimMV isolate reported to be infecting soybean (GenBank Accession No. FJ686693) in Brazil (2). Koch's postulates were fulfilled for the isolate GO-060 by inoculating a set of soybean and bean accessions via a biolistic approach. The ratio of positive PCR amplicons per total of inoculated plants were 15 of 16 for snap bean cv. Trepador, 9 of 10 for snap bean cv. Fartura, 18 of 24 for common bean cv. Olate Pinto, and 19 of 25 for common bean cv. Carioca. The isolate was also able to infect eight of nine soybean 'Doko' plants. Sequence analysis using symptomatic leaf samples (15 days after inoculation) confirmed SimMV as the causal agent. To our knowledge, this is the first report of a SimMV isolate infecting P. vulgaris. This virus is apparently fast expanding its host range from Malvaceae to Solanaceae species and leguminous hosts after the introduction of B. tabaci biotype B (2). More extensive surveys are necessary to access the current epidemiological importance of SimMV in both snap and common beans in Brazil. References: (1) J.C. Faria and D. P. Maxwell. Phytopathology 89:262, 1999. (2) F. R. Fernandes et al. Arch. Virol. 154:1567, 2009. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Epidemics of Meloidogyne brasilensis in Central Brazil on Processing Tomato Hybrids That Have the Root-Knot Nematode Mi Resistance Gene.

The species Meloidogyne brasilensis Charchar & Eisenback 2002 was described as causing root rot, severe wilt, and numerous galls in pea (Pisum sativum L.) in Brasília-Federal District and tomato (Solanum lycopersicum L.) cv. Rossol (known to have the root-knot nematode resistance Mi gene) in Londrina-Paraná State, Brazil. To our knowledge, this current work is the first report of the epidemics on tomato hybrids that have the Mi gene caused by infection of M. brasilensis in central Brazil. Samples were obtained from fields with two commercial hybrids that have the Mi gene ('Calroma' and 'Nemapride') that were cultivated under center-pivot irrigation in Silvânia, Goiás State. These hybrids exhibited slow vegetative development and malformed roots because of the high number of large galls. Symptomatic plants were collected from a tomato crop area of more than 100 ha. Random sampling indicated field sectors with up to 100% of symptomatic plants. Morphological and morphometric evaluations of this Meloidogyne population were carried out with the female perineal pattern, stylet, and excretory pore and also with the male body traits, labial disc, and stylet. The esterase phenotype was unique for this population with four clear bands (J. M. Charchar, unpublished data). Altogether, the morphological and biochemical characteristics of this population were in agreement with that reported for M. brasilensis (1). Koch's postulates were fulfilled using tomato 'Rutgers' (susceptible) and 'Rossol' (with the Mi resistance locus) under greenhouse conditions. The massive use of tomato hybrids with the Mi gene could be a strong selection factor favoring this pathogen under growing conditions in central Brazil. Germplasm screen searching for sources of resistance specific to this nematode species is advisable. Reference: (1) J. M. Charchar and J. D. Eisenback. Nematology 4:629, 2002.

Multiple Resistance to Meloidogyne spp. and to Bipartite and Monopartite Begomovirus spp. in Wild Solanum (Lycopersicon) Accessions.

The Ty-1 locus confers tolerance to monopartite and bipartite Begomovirus spp. (genus Begomovirus, family Geminiviridae) and this phenotype is improved in homozygous tomato lines. However, the gene Mi (Meloidogyne spp. resistance) is in repulsion phase linkage with Ty-1, which hampers the large-scale development of multiresistant inbred lines. Seventy-one Solanum (section Lycopersicon) accessions were whitefly inoculated with the bipartite Begomovirus sp. Tomato rugose mosaic virus (ToRMV) and simultaneously infested with a mixture of Meloidogyne incognita and M. javanica under greenhouse conditions in Brazil. Accessions were then transplanted into a nematode-infested field with natural ToRMV infection. A severity index was used to evaluate ToRMV reaction. Nematode evaluation was done by counting the number of galls per root system. Seventeen accessions with Meloidogyne spp. and ToRMV resistance were selected and evaluated in Spain against three monopartite Begomovirus spp. associated with the tomato yellow leaf curl virus disease, using infectious clones. Systemic infection was monitored by DNA hybridization. Five S. peruvianum accessions (PI-306811, PI-365951, LA-1609, LA-2553, and CNPH-1194) displayed nematode and broad-spectrum resistance to all Begomovirus spp. tested in both continents. From the breeding standpoint, accessions combining resistance to Meloidogyne spp. and to bipartite and monopartite Begomovirus spp. would be useful for the development of elite lines expressing all traits in homozygous condition.

Insulin resistance in offspring of hypertensive subjects.

OBJECTIVE: To assess whether apparently healthy subjects with a family history of systemic hypertension have a higher risk of presenting the insulin resistance syndrome. SUBJECTS: Three hundred and eighty-six subjects aged 20-65 years. SETTING: A middle socio-economic class urban community from Mexico City. METHOD: All subjects and, when necessary, their first-degree relatives, answered a questionnaire and underwent a physical examination with measurement of height, weight and blood pressure. Serum insulin, glucose, cholesterol and triglycerides were measured during fasting and 2 h after an oral load of 75 g glucose. RESULTS: A family history of systemic hypertension was present for 167 (43%) of the subjects, of whom 123 (31%) were obese. Subjects with a family history of hypertension had higher systolic blood pressures than did those without such a history (120 +/- 15 versus 115 +/- 10 mmHg). In the logistic regression model, the body mass index and age showed statistically significant effects on the fasting glucose:insulin ratio and on serum insulin levels after an oral load of glucose. When men and women were analysed separately, only in men were higher systolic and mean blood pressures and lower glucose:insulin ratios observed. In the logistic regression analysis the body mass index was a significant predictor of the glucose:insulin ratio and serum insulin levels after an oral load of glucose, especially in men. CONCLUSION: Apparently healthy male offspring of hypertensive parents have higher blood pressure levels and lower insulin sensitivities than do offspring of normotensive parents. Insulin resistance was related to obesity, but not to a family history of hypertension, as had previously been reported by other research groups.

Placental phagocytic cells infected with herpes simplex type 2 and echovirus type 19: virological and ultrastructural aspects.

Placental macrophage cells were kept in a short-term culture and infected with herpes simplex type 2 virus and echovirus type 19. These were observed under optical and electron microscopy. Immunofluorescence, virus titration and autoradiographic technique were used to determine if the virus was replicating in the system. The results showed that the placental phagocytic cells do not allow virus growth and that the virus particles are destroyed right after virus uptake, within 4 h post-infection. The increase of lipid bodies and other cellular alterations suggested the intensive action of these cells against viruses.

Enterovirus associated placental morphology: a light, virological, electron microscopic and immunohistologic study.

The purpose of this study was to identify the possible effect of enteroviruses on placental tissue. Seventy-eight pregnant women were studied throughout their pregnancy: enteroviral infection was detected by faecal viral isolation and seric neutralization of previously identified virus in cell culture. In 19 cases of confirmed maternal infection, placentae were examined grossly, by optical microscopy, immunohistochemical and electron microscopic methods. Ten term placentae from women included in the study, with no clinical, serological or virological evidence of enteroviral infection, were used as control, and examined by gross and optical microscopy. In 17 specimens (echovirus-coxsackievirus) an haematogenous placentitis was suspected on the basis of gross observation. Microscopic lesions were similar to those found in other viral infections, with specific features. The nature of the inflammatory reaction pointed to the presence of an acute type of haematogenous placentitis, not present in placentae of the control group. The authors (AA) comment on the results and present the hypotheses about the available data: (1) maternal enteroviremia and faecal virus shedding without placental invasion, placentary damage being an unspecific consequence of infection; (2) direct virus-induced injury is not the only possible cause for the lesions: (3) placental enteroviral infection occurred with placental pathology but the virus did not cross the organ as the newborn had no signs of infection.

Placental pathology in congenital rubella.

Two groups of placentae from 18 cases of maternal rubella were examined morphologically and virologically. Placentae in Group I (four cases) had a mean gestational age of 21 +/- 1.9 weeks, whilst those in Group 2 (14 cases) had a mean gestational age of 38 +/- 2.8 weeks. A tendency to hypoplasia was observed. The microscopic lesions were similar to those found in other viral infections but in each group some specific features were noted. Only placentae of Group I showed nodules of villi agglutinated by fibrin. This lesion suggested recent maternal infection. Attention is drawn to the presence of abnormal areas of lobular rarefaction due to dysmaturity of villous stem and terminal villi. This aspect was more diffuse and accentuated in Group 2 placentae. Villitis of reactive, necrotic, proliferative and reparative types was seen only in placentae of Group 2. Devastating villitis was not observed. Inclusions in placental cells suggested rubella infection. The lesions were non-specific and hence stress the need for virological examination of the placenta, immunofluorescence studies and electron microscopy to confirm the diagnosis.

Ultrastructure of a spiral micro-organism from pig gastric mucosa ("Gastrospirillum suis").

The ultrastructural features of a helical-shaped bacterium occurring in the stomach of pigs, within the mucus on the mucosal surface of antral pits, were examined. The bacterial cell had three to eight spiral turns, flattened and truncated ends and was approximately 4.0 microns long and 0.6 microns wide. In some sections, up to six flagella, about 22 nm in diameter, were seen arising from each pole. The cytoplasm contained sparse, irregular granules, numerous ribosomes and large single-layered membrane-bound granules. In the flagella insertion area, there was a highly electron-dense component, the "polar membrane". This micro-organism differed from similar bacteria described in cats, dogs and monkeys, and may cause inflammation in the antral mucosa of pigs similar to Helicobacter pylori infection in man. Furthermore, it was morphologically similar to the spiral micro-organism distinct from H. pylori which has been described recently in human antral mucosa from patients with gastritis and may be of potential significance as a pathogen in man. The name "Gastrospirillum suis" is proposed for this bacterium.

A rapid and sensitive dot-blot hybridization assay for the detection of citrus exocortis viroid in Citrus medica with digoxigenin-labelled RNA probes.

A rapid and sensitive dot-blot hybridization assay using in vitro-transcribed digoxigenin-labelled RNA probes (riboprobes) was developed aiming at detection of citrus exocortis viroid (CEVd) in crude sap of infected Citrus medica plants. The protocol includes a very quick and simple preparation of RNA extracts from samples using a denaturation step with formaldehyde. From our results, the employment of this step is highly recommended because the hybridization signals in formaldehyde-denatured samples were significantly stronger when compared with that of extracts without formaldehyde treatment. The assay was found to be sensitive enough to detect 0.1 ng of purified CEVd RNA and was able to detect viroid in 0.2 mg of symptomatic Citrus medica leaves. The use of riboprobes also allowed hybridization under high temperature conditions, avoiding non-specific background.

Effect of naloxone on serum prolactin levels in adult male rabbits.

In order to clarify the possible physiological role of endogenous opioid peptides (EOP), we studied the effect of low doses of naloxone (specific opiate antagonist) on plasma prolactin levels in male rabbits. Five groups of five male rabbits each was injected daily between 8-9 a.m., with naloxone 12.5, 25, 50, 100 and 200 microg/kg/day. An additional group of ten animals was injected with saline solution and considered the control group. Blood samples were taken at baseline before naloxone administration and later at 90 min and 1, 2, 4, 7, 10 days after its administration. Samples were also taken 4 days after stopping naloxone administration (day 14). Plasma prolactin levels were measured by RIA. A significant constant decrease in PRL levels was seen with the 12.5 microg at 90 minutes, while with the remaining doses a progressive decrease was recorded throughout the study.

Changes in the prolactin serum isoforms secreted by a pituitary adenoma associated with therapy.

Variations in serum molecular forms of prolactin (PRL) from an adolescent woman presenting amenorrhea-galactorrhea are reported. Persistent hyperprolactinemia and hypoestrogenism were demonstrated as well as the presence of a pituitary tumor with suprasellar extension. Bromocriptine was given at progressive doses up to 37 mg daily, decreasing the hyperprolactinemia and galactorrhea. After 2 years of treatment the patient noticed symptoms of gastric intolerance, bromocriptine was discontinued and a rebound of hyperprolactinemia was observed. Lisuride was administered instead resulting in a new decrease in PRL serum levels, disappearance of galactorrhea and beginning of regular menses. Serum gel chromatographic analysis was carried out before and during lisuride treatment. The first chromatographic analysis showed a predominance of high molecular weight (approximately 66 KD) PRL, accounting for more than 90% of the immunoreactive PRL. The second chromatography showed the major peak of immunoreactive PRL displaced to the right (molecular weight of 22 KD), which was eluted near the PRL standard. With these chromatographic patterns it is concluded that the pituitary macroprolactinoma secreted different molecular forms of PRL and treatment with lisuride appeared to exert some effect on the PRL molecular size secreted by the pituitary.

A scanning electron microscopic evaluation of in vitro dentinal tubules penetration by selected anaerobic bacteria.

In vitro root canal dentinal tubule invasion by selected anaerobic bacteria commonly isolated from endodontic infections was evaluated. Dentinal cylinders obtained from bovine incisors were inoculated with bacteria, and microbial penetration into tubules was demonstrated by scanning electron microscopy. The results indicated that all bacterial strains tested were able to penetrate into dentinal tubules, but to different extents.