The effect of spikelet position on the success frequencies of wheat haploid production using the maize cross system

Barbela is an old Portuguese landrace of wheat that is highly genetically heterogeneous. Different Barbela populations when subjected to aluminium stress show variable levels of tolerance. In order to study the inheritance of this character, doubled haploid (DH) lines were developed. These DH were obtained by intergeneric crosses of 14 different lines of Barbela with maize. During this process the efficiency of the technique was evaluated and suggestions for its improvement were obtained. Several parameters were studied in the crosses: % of crossability, % of embryos per florets pollinated and % of embryos per seed set. The different genotypes of Barbela showed significant variation for the parameters analysed. When the reciprocal crosses were analysed, no differences were found, indicating that cytoplasm differences do not influence the parameters of DH production. However, different spikelet positions (lower, middle and upper) gave highly significant differences in all parameters analysed. Highest success frequencies were obtained for pollinated spikelets in the middle of the spike. This can indicate that concentrating on the middle part of the spike can increase the frequency of DHs obtained using inter generic crosses of wheat with maize. This revised version was published online in August 2006 with corrections to the Cover Date.     

Varietal discrimination and genetic relationships of Vitis vinifera L. cultivars from two major Controlled Appellation (DOC) regions in Portugal

Thirty-nine grapevine cultivars widely grown in Portugal, especially in Vinhos Verdes and Douro regions, and two well known international cultivars as standards, were genotyped at 12 microsatellite loci. The number of alleles per locus ranged from 6 to 12, and the number of allelic combinations per locus from 13 to 26. The total number of unique genotypes in the 12 analysed loci was 120, having most of the cultivars (38 out of 41) at least one unique genotype in any of the loci. The microsatellite profiles were adequate to discriminate 41 cultivars. The level of observed heterozygosity at each locus varied from 70.7% to 95.1%. VVMD28 has been revealed as one of the most informative markers. Several synonymies between Spanish and Portuguese cultivars were confirmed, and some homonymies are discussed. The genetic profiles of all 41 cultivars were searched for possible parent-offspring groups. The data obtained revealed the possible descendence of Touriga Franca from Touriga Nacional and Marufo.     

Identification of coagulase-negative staphylococci from bovine mastitis using RFLP-PCR of the groEL gene

Coagulase-negative staphylococci (CNS) have become the predominant pathogens causing bovine mastitis in many countries. CNS infections are associated with damage to milk secretory tissue of the mammary gland by increased connective tissue stroma, moderate increases of somatic cells count in milk and significant production decreases. These consequences impose serious economic losses for the farmers and the dairy industry. Routine veterinary laboratories do not usually identify CNS at the species level. Thereby, the aims of this study were to identify the most common staphylococcal pathogens involved in bovine mastitis using PCR-restriction fragment length polymorphism (RFLP) analysis of a partial groEL gene sequence and to compare our results with the identification carried out by the conventional method. A total of 54 isolates of Staphylococcus, involved in bovine mastitis, were analyzed by this method. The size and number of the fragments obtained by either AluI or HindIII/PvuII digestions made possible to form clear patterns differentiating, among the isolates, 11 of the most common species of animal staphylococcal pathogens. Most of the isolates clustered together with the reference strain of Staphylococcus chromogenes (28) and the type strain of Staphylococcus epidermidis (8). Besides, some isolates clustered together with the type strain of Staphylococcus aureus (5). All patterns were confirmed by the conventional biochemical method, showing concordant results. Thus, the PCR-RFLP of the groEL gene constitutes a reliable and reproducible molecular method for identification of CNS species responsible for bovine mastitis.     

Association of Trypanosoma vivax in extracellular sites with central nervous system lesions and changes in cerebrospinal fluid in experimentally infected goats

ABSTRACT: Changes in cerebrospinal fluid (CSF) and anatomical and histopathological central nervous system (CNS) lesions were evaluated, and the presence of Trypanosoma vivax in CNS tissues was investigated through PCR. Twelve adult male goats were divided into three groups (G): G1, infected with T. vivax and evaluated during the acute phase; G2, infected goats evaluated during the chronic phase; and G3, consisting of non-infected goats. Each goat from G1 and G2 was infected with 1.25 × 105 trypomastigotes. Cerebrospinal fluid (CSF) analysis and investigation of T. vivax was performed at the 15th day post-infection (dpi) in G1 goats and on the fifth day after the manifestation of nervous system infection signs in G2 goats. All goats were necropsied, and CNS fragments from G1 and G2 goats were evaluated by PCR for the determination of T. vivax. Hyperthermia, anemia and parasitemia were observed from the fifth dpi for G1 and G2, with the highest parasitemia peak between the seventh and 21st dpi. Nervous system infection signs were observed in three G2 goats between the 30th and 35th dpi. CSF analysis revealed the presence of T. vivax for G2. Meningitis and meningoencephalitis were diagnosed in G2. PCR were positive for T. vivax in all the samples tested. In conclusion, T. vivax may reach the nervous tissue resulting in immune response from the host, which is the cause of progressive clinical and pathological manifestations of the CNS in experimentally infected goats.     

Follicular degeneration in the ovaries of goats experimentally infected with Trypanosoma vivax from the Brazilian semi-arid region

Infection by Trypanosoma vivax and other African trypanosomes plays an important role in reproductive disorders in male and female livestock. Outbreaks of T. vivax in the semi-arid region of northeastern Brazil are characterized by wasting disease in cattle, sheep and goats with hematological, cardiac and nervous compromises in addition to reproductive failures. Similar to reports from Africa, we previously observed a reduction in fertility rates and severe testicular degeneration and epididymitis in male sheep infected with T. vivax from this region. Although anestrus is frequently reported in goats and sheep infected with T. vivax, the effects of this infection on the female reproductive organs need clarification. In this study, we addressed this issue through a histopathological evaluation of ovarian follicular morphology and classification in goats experimentally infected with a T. vivax isolate from the Brazilian semi-arid region. The infected animals presented typical clinical signs of trypanosomosis by T. vivax, including anemia, hyperthermia, pallor of the mucous membranes, enlarged lymph nodes, and progressive loss of weight. All the infected goats remained anestrus throughout the experimental period and exhibited important disturbances in the ovaries, evidenced by reduced size and a smooth surface without follicles or corpora lutea, and abnormal follicular development. In addition, through PCR, we detected T. vivax DNA in the ovarian tissues of the infected goats. Our findings contributed to understand the female reproductive failure associated with trypanosomosis caused by T. vivax.     

Pathogenesis of reproductive failure induced by Trypanosoma vivax in experimentally infected pregnant ewes

The present study was aimed at investigating the effect of experimental infection by Trypanosoma vivax in different stages of pregnancy, determining the pathogenesis of reproductive failure, and confirming transplacental transmission. We used 12 pregnant ewes distributed into four experimental groups: G1, was formed by three ewes infected with T. vivax in the first third of pregnancy (30 days); G2 comprised three infected ewes in the final third of pregnancy (100 days); G3 and G4 were composed of three non-infected ewes with the same gestational period, respectively. Each ewe of G1 and G2 was inoculated with 1.25 × 10⁵ tripomastigotes. Clinical examination, determination of parasitemia, serum biochemistry (albumin, total protein, glucose, cholesterol, and urea), packed cell volume (PCV), serum progesterone, and pathological examination were performed. Placenta, amniotic fluid, blood and tissues from the fetuses and stillbirths were submitted to PCR. Two ewes of G1 (Ewe 1 and 3) presented severe infection and died in the 34^th and 35^th days post-infection (dpi), respectively; but both fetuses were recovered during necropsy. In G2, Ewe 5 aborted two fetuses on the 130^th day (30 dpi) of pregnancy; and Ewe 6 aborted one fetus in the 140^th day (40 dpi) of gestation. Ewes 2 and 4 delivered two weak lambs that died five days after birth. Factors possibly involved with the reproductive failure included high parasitemia, fever, low PCV, body score, serum glucose, total protein, cholesterol, and progesterone. Hepatitis, pericarditis, and encephalitis were observed in the aborted fetuses. The presence of T. vivax DNA in the placenta, amniotic fluid, blood, and tissues from the fetuses confirms the transplacental transmission of the parasite. Histological lesion in the fetuses and placenta also suggest the involvement of the parasite in the etiopathogenesis of reproductive failure in ewes.     

Determination of relationships among autochthonous grapevine varieties (Vitis vinifera L.) in the Northwest of the Iberian Peninsula by using microsatellite markers

Fifty six grapevine varieties traditionally grown in the Northwest region of the Iberian Peninsula were analysed for six microsatellite loci, in order to determine the relationships among them as well as the plant material that should be collected and preserved in germplasm banks. Previous morphological and molecular results were taken into account for assessment of the existing synonymies among accessions from different European countries. Percent distribution of the main alleles was calculated. Multivariate analysis was carried out and similarities among the studied material were described and commented.     

Antimicrobial activity of marine sponges against coagulase-negative staphylococci isolated from bovine mastitis

Bovine mastitis remains worldwide a major challenge for the dairy industry despite the widespread implementation of control strategies. The increasing number of coagulase-negative staphylococci (CNS) causing mastitis and of bacteria resistant to conventional antibiotics has become a serious problem in recent years. Marine sponges are a rich source of bioactive compounds, and many species can be useful for the development of new antimicrobial drugs. In the present study, 49 CNS strains were isolated from bovine mastitis cases from 21 different dairy herds kept at farms in Southeast Brazil. Strains were analyzed for antimicrobial susceptibility and mecA gene detection. Fifty-nine percent of the CNS strains were resistant to at least one of the drugs tested and 12.2% were classified as multiresistant. Three strains carried the mecA gene, confering resistance to the beta-lactamic antibiotics. In addition, the CNS strains were submitted to in vitro screening for antimicrobial activities of extracts from marine sponges. Extracts from the sponge species Cinachyrella sp., Haliclona sp. and Petromica citrina showed antibacterial activity against 61% of the CNS strains, including strains resistant to conventional antibiotics. Extracts from P. citrina showed the largest spectrum of inhibitory activity. The aqueous extract inhibited 51% of the CNS strains and presented a bactericidal effect over susceptible and multiresistant-bacteria at a minimal inhibitory concentration of 1.024μg/ml. This study shows the potential of marine sponges as new sources of antibiotics and disinfectants for the control of CNS involved in bovine mastitis.     

Aluminum tolerance variability in rye and wheat Portuguese germplasm

Regional rye populations and wheat cultivars/lines were screened for aluminum tolerance using the hematoxylin staining method. Portuguese regional rye populations showed better tolerance than the Polish cv. Dank. Zlote, used as tolerant tester. In the group of bread wheats, EPM 305/81, a Barbela reselection, was the most tolerant genotype with the same behaviour as the cv. BH 1146, a tolerant tester. In a study with lines selected from a local Barbela landrace, aluminum tolerance variability was detected. Some lines were as tolerant, or higher, as wheat tester. As Portuguese rye populations and the Barbela wheat landrace have grown for centuries on an acid soil region, the data supports the idea that natural biotic or abiotic stresses associated to man selection, lead to the adaptation of genotypes to specific regional conditions and, in this case, to acid soils where aluminum toxicity occurs.