[Growth in infants with cow's milk allergy]. / Cómo crecen los lactantes diagnosticados de alergia a proteínas de leche de vaca?

INTRODUCTION: Approximately 2-3 % of infants develop cow's milk allergy (CMA). Treatment consists of eliminating milk from the diet. Some studies have shown growth failure in children with CMA and a milk-free diet. OBJECTIVES: To evaluate growth status at 1 and 2 years of age in infants diagnosed with CMA. MATERIAL AND METHODS: An observational, longitudinal, retrospective study of all infants diagnosed with CMA from 2000-2001 was performed. The following data were analyzed: chronology and type of feeding, the presence of allergy to other foods, atopic dermatitis or other symptoms of allergy, duration of CMA, and anthropometric data (weight and height) at diagnosis, and at 1 and 2 years of age. Anthropometric data were expressed as Z-scores. RESULTS: A total of 141 infants (71 boys and 70 girls) were studied. Atopic dermatitis was found in 67 infants (47.5%) and wheezing in 36 (25.5%). Allergy to foods other than milk was found in 27%. Only 21.3% of the infants grew out of CMA at the age of 2 years, of which 37% did so in the first year of life. Z-scores for weight were -0.5 at birth, -0.25 at the first follow-up visit, -0.25 at 1 year, and -0.19 at 2 years. Z-scores for height were 10.26 at the first follow-up visit, 10.64 at 1 year, and 10.35 at 2 years. A significant difference in Z scores for weight was found in infants with allergies to other foods, atopic dermatitis or wheezing compared with those with CMA only. CONCLUSIONS: Infants with CMA receiving a substitute formula (hydrolyzed or soy formulae) showed normal weight and height at 2 years, although the percentile for height tended to be better than that for weight. The presence of other food allergies, atopic dermatitis or wheezing seems to affect the nutritional status of infants with CMA.

Identification of the minimal sequence required for vascular-specific activity of Tomato mottle Taino virus Replication-associated protein promoter in transgenic plants.

A 597 nt fragment from Tomato mottle Taino virus (ToMoTV) DNA-A, with 459 nt located upstream of the Replication-associated protein translation start codon, was tested for promoter activity in solanaceous plants. The promoter activity of this fragment (pRep(459::Rep)) was demonstrated when it was introduced upstream the uidA reporter gene into tobacco, potato and tomato plants by genetic transformation. It became active in 7-day-old transgenic tobacco seedlings as revealed by a vascular-specific pattern of gene expression which was maintained during the continued growth of the plant. Transformed potato and tomato plants also showed a vascular-specific pattern of expression. In comparative assays, pRep(459::Rep) showed an expression activity 10-40-fold less than the 35S promoter from Cauliflower mosaic virus. To delimit the minimal cis-acting elements necessary for vascular specificity of this promoter, a set of PCR deletion mutants of pRep(459::Rep) (pRep(459), pRep(324), pRep(203), pRep(145), pRep(132) and pRep(115)), were generated and used to transform tobacco plants. Transgenic tobacco plants belonging to all the pRep versions were blue stained in the vascular system except those from the pRep(115) version. The results described in this report demonstrate that the minimal sequences necessary for the pRep promoter activity are confined in a segment of 132 nts (located between the nts 2454 and 2585 of the ToMoTV DNA A) and that this promoter harbors those elements sufficient for vascular-specific expression.

Taino Tomato Mottle Virus, a New Bipartite Geminivirus from Cuba.

Geminiviruses have become the most important virus group affecting tomatoes (Lycopersicon lycopersicum (L.) Karsten) in Cuba since they have been detected in all tomato-producing areas, causing serious losses. Recently, a whitefly-transmitted, monopartite geminivirus was detected in Cuba and identified as tomato yellow leaf curl virus-Israel (TYLCV-Is) (1). Samples collected from the main tomato-producing areas during the period 1995 to1996 were further analyzed by polymerase chain reaction (PCR) with degenerate primers (PAL1v1978 and PAR1c496) (2). Whereas in samples from most areas only TYLCV was detected, in some samples from the Havana area, two DNA fragments (approximately 1.4 and 1.1 kb) were amplified by PCR. The larger fragment was identified as part of the TYLCV-Is genome, confirming the previous report (1). The 1.1-kb fragment was cloned and its nucleotide sequence suggested that a new bipartite geminivirus was also present in those tomato samples. To clone the entire genome, tomato plants were inoculated by biolistics with DNA extract from field samples. After symptom expression, a viral DNA-enriched preparation from the inoculated tomatoes was independently digested with several restriction enzymes and the products were ligated into pZero plasmid (Invitrogen, San Diego, CA). Several clones in the 2.6-kb size range were characterized by restriction mapping and hybridization against component A and B heterologous probes. Two clones were selected as containing putative A and B components and their infectivity was tested by biolistic inoculation of tomato and pepper plants. The inoculated tomatoes developed a mild mottle in the younger leaves, whereas no symptoms were visible on the inoculated pepper plants. However, the presence of viral DNA was confirmed in both tomatoes and peppers by Southern blot hybridization analysis with A- and B-specific probes. Partial sequences of both components were obtained and their analysis showed that both components shared a 170-bases common region with a 95% identity. In addition, the nucleotide sequences of two open reading frames, one in each component (AC1 and BC1), were determined and compared with geminivirus sequences deposited in Gen-Bank. A dendogram generated with the CLUSTAL program and obtained with the AC1 and BC1 amino acid sequences, placed the new geminivirus in a cluster with tomato mottle virus (ToMoV; accession nos. L14460, L14461), Abutilon mosaic virus (AbMV; X15983, X15984), potato yellow mosaic virus (PYMV; D00940, D00941), and bean dwarf mosaic virus (BDMV; M88179, M88180). The percentages of identity obtained with the amino acid sequences were as follows. For AC1: ToMoV, 87%; PYMV, 79.5%; BDMV, 78.7%; and AbMV, 78%. For BC1 protein: BDMV, 92.8%; ToMoV, 89.1%; PYMV, 88.1%; and AbMV, 67.5%. In addition, the sequences were compared with partial nucleotide sequences (AC1, coat protein [CP], and common region) of a bipartite geminivirus affecting tomatoes in Jamaica (accession nos. U83855, U83854, and U83850). Interestingly, the common regions showed a higher percentage of identity (88%) than the CP and AC1 partial nucleotide sequences (86 and 74%, respectively). These data suggest that the virus reported here is a new geminivirus and the first bipartite geminivirus reported in Cuba. Thus, the name of Taino tomato mottle virus is proposed. (Taino refers to the name of the inhabitants of Cuba at the time of Columbus's arrival in the Caribbean). References: (1) P. L. Ramos et al. Plant Dis. 80:1208, 1996. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Cuban Isolate of Bean golden yellow mosaic virus is a Member of the Mesoamerican BGYMV Group.

In Cuba, the emergence of bean golden mosaic was associated with high populations of Bemisia tabaci in common bean (Phaseolus vulgaris L.) plantings in the 1970s (1). During the last two decades, the disease has caused significant economic losses, forcing some growers to abandon bean production. In Holguín, one of the main bean producing provinces of the country, about 2,000 ha of beans were abandoned in 1991 due to the high incidence of this whitefly-transmitted virus. At that time, yield losses associated with this disease reached 90 to 100% in farmer's fields. In spite of various control measures, the disease affected 33, 28, and 6.5% of the total area planted in Cuba to common bean in 1990, 1992, and 1996, respectively. For this investigation, common bean leaves showing systemic yellowing symptoms were collected in fields located in the provinces of Havana, Matanzas, and Holguín during 1998-1999. Sap and total DNA leaf extracts were used to inoculate healthy bean plants by manual and biolistic procedures, respectively. Characteristic yellowing symptoms were more efficiently reproduced using a particle gun device than by manual inoculation (18/20 plants and 5/20 plants, respectively, for a Holguín virus isolate). DNA extracts were further analyzed by polymerase chain reaction using two degenerate primer sets: PAL1v1978-PAR1c715 and PAL1c1960-PAR1v722 (2). Fragments of approximately 1.4 and 1.2 kb were amplified and cloned. Restriction fragment length polymorphism analysis of the cloned 1.4-kb fragments was performed with BglII, HincII, SalI, EcoRI, PstI, and XbaI, indicating that selected isolates from the three Cuban provinces shared identical restriction patterns. The nucleotide sequence obtained from two clones of a virus isolate from Holguín, was compared to sequences available for other begomoviruses using BLAST. The Cuban isolate shared up to 94% nt sequence identity with various strains of Bean golden yellow mosaic virus (BGYMV) in the first 250 nt of the rep gene. For the common region (CR), scores were 93% for BGYMV-GA (Guatemala), 92% for BGYMV-MX (southern Mexico) and BGYMV-PR (Puerto Rico), and 91% for BGYMV-DR (Dominican Republic). The iterative sequence ATGGAG was identified in the CR of the Cuban BGYMV isolate, as reported for other BGYMV isolates. Finally, the Cuban begomovirus, hereafter referred to as BGYMV-CU, shared nt and aa sequence identities of 94 and 100%, respectively, with the coat protein gene of BGYMV-MX. We conclude that the begomovirus isolated from mosaic-affected common bean plants in the province of Holguín is a member of the Mesoamerican BGYMV group (3). References: (1) N. Blanco and C. Bencomo. Cienc. Agric. 2:39, 1978. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993. (3) Morales and Anderson, Arch. Virol. 146:415, 2001.

Molecular Characterization of Tobacco leaf rugose virus, a New Begomovirus Infecting Tobacco in Cuba.

Tobacco (Nicotiana tabacum L.) is the main raw material for the cigar industry and one of the most important crops in Cuba comprising 49,654 ha. During the past 20 years, foliar rugosity and stunting symptoms have been observed in several tobacco producing areas. These symptoms were correlated with the presence of typical geminivirus nuclear inclusions and the transmission of the causal agent by whiteflies (Bemisia tabaci Genn) (1). To identify the suspect geminivirus, diseased leaf samples were collected in Havana province in 2000 and 2001. Sap extracts or leaf pieces were used to inoculate healthy tomato and tobacco plants by mechanical and graft inoculation procedures. Characteristic symptoms were reproduced in tobacco plants only by grafting (8 to 10 plants). DNA extracts from symptomatic plants were analyzed by Southern blot and polymerase chain reaction. The presence of a bipartite begomovirus was supported by the observation of hybridization signals (1.6 kb to 3 kb) at low stringency to probes derived from DNA-A and DNA-B of Taino tomato mottle virus. Furthermore, typical begomovirus amplicons of approximately 1.4 kb and 1.2 kb were amplified using the primer sets PAL1v1978-PAR1c715 and PAL1c1960-PAR1v722 (2), respectively. Amplicons were cloned, and their nucleotide sequences (nt) obtained from two clones each. Sequence for component A was assembled, and some fragments were compared with those for other begomoviruses using CLUSTAL W. For the CP gene (756 nt) (GenBank Accession No. AJ488768), the comparison revealed the highest percentages of nt identity with Sida golden mosaic virus from Florida (SiGMV-F, GenBank Accession No. AF049336) (86%), Tomato mottle virus (GenBank Accession No. L14460) (83.5%), and the yellow vein strain of Sida golden mosaic virus from Honduras (GenBank Accession No. Y11099) (83.3%). In addition, the percentages of nt identity obtained using the core region (a 540-nt fragment located between positions 147 and 687) of the CP gene from the tobacco virus were calculated. The best scores were as follows: SiGMV-F, 87.8%; Jatropha mosaic virus (JMV) from Puerto Rico (GenBank Accession No. AF058025), 86.9%; and Tomato rugose mosaic virus (GenBank Accession No. AF291705), 86.3%. Finally, comparisons of the common region (CR, 144 nt) revealed the highest values with JMV from Jamaica (JMV-JM) DNA-A and DNA-B (GenBank Accession Nos. AF324410 and AF324411; 89% and 91.1%, respectively). Interestingly, the CR analysis revealed the presence of the Ori-associated iterative motif GGGGT, which is the same in the CR of JMV-JM. Although the data suggest that the tobacco begomovirus is related to the JMV-JM isolate, it is a new species, and the name of Tobacco leaf rugose virus (TbLRV) is proposed. References: (1) S. Quintero and J. Santiesteban, Agrotec. Cuba 11(1), 1979. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Macroptilium yellow mosaic virus, a New Begomovirus Infecting Macroptilium lathyroides in Cuba.

Macroptilium lathyroides (L) is a weed that is widely distributed in Cuba. Frequently, leaves show bright yellow mosaic symptoms, which suggest the incidence of a viral disease. Since begomovirus occurrence in Macroptilium lathyroides has been previously reported in other islands of the Caribbean (1,3), symptomatic plants from three distant places in Cuba (Havana, Villa Clara, and Camaguey), were collected and tested for the presence of begomoviruses. Plant DNA extracts were analyzed by Southern blot hybridization and polymerase chain reaction with two sets of degenerate primers (2). The presence of a bipartite begomovirus was evident through strong hybridization signals obtained with the DNA-A and DNA-B of Taino tomato mottle virus as probes at low stringency. Furthermore, 1.4-kb and 1.2-kb PCR amplified fragments were obtained with DNA-A degenerate primers, PAL1v1978-PAR1c715 and PAL1c1960-PAR1v722, respectively. Both PCR fragments from the samples from the three locations were cloned, and restriction fragment length polymorphism analysis of the 1.4-kb fragments were performed using PstI, EcoRI, HincII, XbaI and BglII. Restriction fragment patterns were the same for the three clones. The DNA-A sequence (GenBank Accession No. AJ344452) of the isolate from Villa Clara was compared with sequences available for other geminiviruses using CLUSTAL program. For the coat protein (CP) gene, the comparisons had the highest percentage of identity with various strains of Bean golden yellow mosaic virus (BGYMV, GenBank Accession Nos. AF173555, M91604, and L01635) (85 to 87% and 93 to 94%, nucleotide and amino acid sequences, respectively). For Rep gene (1,044 nt), the best percentages of identities were with BGYMV (81 to 82% and 80 to 82% nucleotide and amino acid sequences, respectively), Tomato leaf crumple virus (GenBank Accession No. AF101476) (78 and 81%, nucleotide and amino acid sequences, respectively), and Sida golden mosaic virus from Florida (GenBank Accession No. AF049336) (78 and 79%, nucleotide and amino acid sequences, respectively). Finally, the comparative analysis of the intergenic region (i.e. the common region plus the CP gene promoter) had the highest identity with BGYMV (56 to 55%) and Tomato severe rugose virus (GenBank Accession No. AY029750) (49%). Interestingly, this virus has in this region the three G-box elements that are characteristic of BGYMV but it differs in the Rep protein-binding iterative motif that is GGTGA instead of GGAGA, for BGYMV. These data indicate that this virus is a new begomovirus and the name of Macroptilium yellow mosaic virus (MaYMV) is proposed. References: (1) A. M. Idris et al. Plant Dis. 83:1071, 1999. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993. (3) M. E. Roye et al. Plant Dis. 81:1251, 1997.

[Current status of the degree of malnutrition in hospitalized patients of the Community of La Rioja]. / Estado actual del grado de desnutrición en los pacientes hospitalizados de la Comunidad de La Rioja.

UNLABELLED: Recent studies have reflected the prevalence of malnutrition within the hospital sphere. The high incidence of malnutrition in hospitals and the negative effect this has on the evolution of patients, since complications are significantly greater in undernourished patients, make it important to find out the nutritional status of patients when they are admitted to hospital and to follow their progress. In the La Rioja region, there have been no prior studies establishing the nutritional status of patients in hospital, so this study was undertaken. It is important to know whether malnutrition is present, the degree of any malnutrition found, and, where necessary, to make the medical and nursing staff aware of the importance of proper nutritional care during a hospital stay. MATERIALS AND METHODS: In a sample of 240 patients selected at random, a nutritional assessment was made on admission. If the patients were still hospitalized one week later, the nutritional assessment was repeated. RESULTS: After the initial assessment, only 12.5% of the 240 patients showed normal nutritional status; mild malnutrition was seen in 55% of cases while moderate and severe cases represented 28.3% and 0.8%, with the most prevalent types of malnutrition being kwasiorkor and mixed (31.6% and 29.6%, respectively). There was no statistical association between the sex of the patient and the degree of malnutrition (p > 0.05). On analysis of the cases by age group, those patients aged less than 65 years mainly presented a marasmus-like and mixed malnutrition, both to a slight degree, whereas the most prevalent malnutrition in patients over the age of 65 was the kwashiorkor type. Among patients with slight and moderate degrees of malnutrition, most of them were diagnosed as suffering from digestive-system related pathologies. After one week, 37.5% of them continued in hospital and approximately half of those presenting normal nutritional status on admission were found to have a status of slight malnutrition.

Lysis efficiency of standard DNA extraction methods for Halococcus spp. in an organic rich environment.

The extraction of nucleic acids from a given environment marks a crucial and essential starting point in any molecular investigation. Members of Halococcus spp. are known for their rigid cell walls, and are thus difficult to lyse and could potentially be overlooked in an environment. Furthermore, the lack of a suitable lysis method hinders subsequent molecular analysis. The effects of six different DNA extraction methods were tested on Halococcus hamelinensis, Halococcus saccharolyticus and Halobacterium salinarum NRC-1 as well as on an organic rich, highly carbonated sediment from stromatolites spiked with Halococcus hamelinensis. The methods tested were based on physical disruption (boiling and freeze/thawing), chemical lysis (Triton X-100, potassium ethyl xanthogenate (XS) buffer and CTAB) and on enzymatic lysis (lysozyme). Results showed that boiling and freeze/thawing had little effect on the lysis of both Halococcus strains. Methods based on chemical lysis (Triton X-100, XS-buffer, and CTAB) showed the best results, however, Triton X-100 treatment failed to produce visible DNA fragments. Using a combination of bead beating, chemical lysis with lysozyme, and thermal shock, lysis of cells was achieved however DNA was badly sheared. Lysis of cells and DNA extraction of samples from spiked sediment proved to be difficult, with the XS-buffer method indicating the best results. This study provides an evaluation of six commonly used methods of cell lysis and DNA extraction of Halococcus spp., and the suitability of the resulting DNA for molecular analysis.

Tomato mottle Taino virus pseudorecombines with PYMV but not with ToMoV: implications for the delimitation of cis- and trans-acting replication specificity determinants.

Over the last decade, the tomato production in Cuba has been affected by new whitefly-associated diseases. In addition to the well-documented presence of Tomato yellow leaf curl virus (TYLCV) along the island, the occurrence of bipartite begomoviruses has also been reported. One of them, tentatively named Tomato mottle Taino virus (ToMoTV), has now been cloned and characterized at the molecular level. Its genomic organization is similar to other bipartite geminiviruses. Phylogenetic analyses placed ToMoTV in a subcluster with other geminiviruses isolated in the Caribbean Basin: Tomato mottle virus (ToMoV), Bean dwarf mosaic virus, Abutilon mosaic virus, Sida golden mosaic virus and Potato yellow mosaic virus (PYMV). Biolistic inoculation of tobacco and tomato plants with cloned viral DNA showed that ToMoTV pseudorecombines with PYMV-GP as predicted by the identity of their iterative elements, whereas it does not show the same ability with ToMoV, even when their replication-associated proteins (Rep and REn) show the highest percentage of similarity. A comparative analysis of Rep proteins from begomoviruses that are able to produce viable reassortants suggests that some key elements for virus replication specificity are located in the first ten amino acids of this protein.