Detection and molecular characterisation of an alfalfa phytoplasma in Argentina that represents a new subgroup in the 16S rDNA Ash Yellows group(‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma fraxini’)

A phytoplasma infecting alfalfa crops was detected and characterised in Argentina, the Argentinean Alfalfa witches′-broom (ArAWB) phytoplasma. Typical witches′-broom symptoms were observed in diseased alfalfa plants from fields of the ‘Cuyo’ region in the Andean midwest. Pleomorphic bodies were observed by electron microscopy in sieve tubes of the diseased plants. The results obtained from the sequence homology, similarity coefficients derived from RFLP of the 16S rDNA and phylogenetic analysis led us to include this phytoplasma in the 16Sr VII (Ash Yellows) group. However, the ArAWB phytoplasma showed several differences when compared to other members of group 16Sr VII. The RFLP analysis of partial 16S rRNA gene of two ArAWB isolates, digested with 16 restriction enzymes, showed differences between the ArAWB and the reference strain (AshY1^T) in six enzyme patterns. Restriction patterns unique for the group and an exclusive HinfI restriction site were found in the ArAWB phytoplasma rDNA. Moreover, the similarity coefficients (0.92–0.86) were lower than those obtained among other group members. The significant differences detected suggested that this phytoplasma belonged to a subgroup different from those described so far. We propose therefore, that the ArAWB phytoplasma should be included in a new VII-C subgroup, closely related to the EriWB phytoplasma (VII-B) described in Brazil.     

Characterization of a novel activation antigen on porcine lymphocytes recognized by monoclonal antibody 5A6/8

In this report, we describe the characterization of a novel activation antigen on porcine lymphocytes recognized by mAb 5A6/8. This antigen was detected on B and T cells 24 h after treatment with various stimuli. It was also found on alveolar macrophages, and at low levels on untreated monocytes. MAb 5A6/8 precipitated two bands of 45 and 50 kDa under non-reducing conditions, and of 22 and 28 kDa under reducing conditions. The cellular distribution, expression kinetics and/or molecular size of the 5A6/8 antigen differ from those of other known lymphocyte activation antigens. MAb 5A6/8 was able to inhibit lymphocyte proliferative responses driven by different stimuli, suggesting a role for this molecule in the events that lead to lymphocyte activation.     

The occurrence of Cryptosporidium parvum and C. muris in wild rodents and insectivores in Spain

Five rodent and two insectivore species were investigated for Cryptosporidium at seven sites in north-eastern Spain. Of the 442 animals studied, 82 Apodemus sylvaticus, 1 A. flavicollis, 5 Mus spretus, 1 Rattus rattus, 8 Clethrionomys glareolus and 13 Crocidura russula were infected with only C. parvum. Eleven A. sylvaticus and 2 C. glareolus were infected with only C. muris and 16 A. sylvaticus, 1 M. spretus and 2 C. glareolus showed mixed infections. Both cryptosporidial species were found in most study areas. No causal relationship was found between intrinsic host factors (age and sex) and the parasitic prevalence in the most captured host species (A. sylvaticus and C. russula). Extrinsic factors such as collection site of host, seasonality and covering vegetation exerted different influence on the prevalence of Cryptosporidium. Small mammals could become one of the most important sources of cryptosporidial oocysts in those areas where neither farm animals nor significant human activity are present. This is the first study to report the infection of M. spretus and C. russula by C. parvum and the first finding of C. muris in M. spretus.     

Genetics of Six Components of Autofertility in Vicia faba

The inheritance of six components of autofertility was studied in faba beans in two consecutive years (1989 and 1990) by analysing parents, Fl and segregating generations of 14 crosses between preselected pure lines. Genetic analysis was performed by a joint scaling test proposed by Cavalli (1952). Additive gene effects were more important than dominance effects in flowers/node, pods/flower and ovules/ovary. In contrast, characters directly related with the seed such as pods/node, seeds/pod and, to a lesser extent, seeds/ovule showed high positive values of dominance. The three latter traits in general exhibited strong heterosis, probably due to genetic interactions. In order to produce selection gain, any breeding programme has to consider that dominance and heterosis vary according to the character involved. The present study reveals different degrees of dominance expression depending on cross specificity as well as on environmental conditions. The advantages of studying segregating generations instead of diallel crosses and the need for a simultaneous study of many crosses are emphasized in this paper.     

Experimental equine herpesvirus-1 infection in llamas (Lama glama).

Three llamas (Lama glama) were experimentally infected intranasally with an isolate of equine herpesvirus-1 (EHV-1) from the brain of an alpaca that had experienced severe neurologic signs. Two of the 3 llamas developed severe neurologic disorders following inoculation; 1 died, and 1 was euthanized in a moribund state. The third llama showed only mild neurologic signs. The euthanized llama had preexisting antibodies to EHV-1, and the remaining 2 llamas were seronegative (virus neutralization titer less than 6) at the time of inoculation. One of the seronegative llamas died acutely without production of detectable antibodies, and the other developed antibodies typical of a primary immune response. The EHV-1 virus was recovered only from a sample of the thalamus of the llama that died acutely. Histopathologic lesions were observed in the brain and retina of the dead and euthanized animals. This study verifies the pathogenic potential of EHV-1 for llamas.     

Selection and testing of epiphytic yeasts to control anthacnose in post-harvest of papaya fruit

Anthracnose, caused by Colletotrichum gloeosporioides, is a major post-harvest disease in papaya fruit. The major objectives of the present work were to isolate, select and test the in vitro and in vivo ability of epiphytic microorganisms, isolated from papaya fruit and leaf surfaces, in controlling anthracnose onset after harvest. A total of 75 bacteria, 67 yeasts and 22 mycelial fungi were isolated. Thirty yeast isolates were able to inhibit the mycelial growth of C. gloeosporioide in vitro and seven of those were used in in vivo assays, resulting in the identification of two very effective isolates. Isolate CEN63, identified molecularly as Cryptococcus magnus, was the most effective in controlling the disease and therefore was studied in more detail. The results of the assays with C. magnus provided evidence that when fruit were treated with the antagonists at concentrations of 10⁷ to 10⁸ cells/ml, as early as 24 h, preferentially 48 h, before inoculation with the pathogen, the development of disease was significantly reduced. C. magnus is a potential antagonist for the development of a commercial product. Additional studies on the modes of action of this yeast isolate, as on its ability to interact with fungicides are being conducted to generate solid basis for the development of an environmentally friendly control agent.     

Anti-histone antibodies in dogs with leishmaniasis and glomerulonephritis

The association between serum anti-histone antibodies and glomerulonephritis was studied in 43 dogs with leishmaniasis (Leishmania infantum). Dogs with increased serum creatinine levels and urine protein-creatinine ratio >1 were considered to have glomerulonephritis. Moderately elevated anti-histone antibodies were found in 38.89% (7/18) of infected dogs without glomerulonephritis, whereas 88% of dogs with glomerulonephritis (22/25) showed moderate or strongly elevated anti-histone antibodies. Prevalence of positive anti-histone antibodies reactions and mean serum concentration was significantly higher (P < 0.001; P < 0.0001) in infected dogs with glomerulonephritis. Correlation between anti-histone antibodies and urine protein-creatinine ratio was significant when groups were analysed together (P < 0.046). Positive predictive value for glomerulonephritis of positive anti-histone antibodies was 88%. In conclusion, high anti-histone antibodies are significantly associated with glomerulonephritis. Although other factors must be involved, dogs with moderate or strong positive anti-histone antibodies reactions may have a higher probability to develop glomerular lesions in canine leishmaniasis.     

Molecular epidemiology of Mycobacterium bovis: usefulness in international trade

Tuberculosis (TB) represents a barrier for free trade of livestock between Mexico and the United States of America (US). In spite of efforts from Mexico to export TB-free animals, some of those found with TB lesions in slaughterhouses in the US are traced back to that country. Therefore, the purpose of this study was to determine, through molecular epidemiology, the most probable source of infection for cattle found with TB lesions in the US. Ninety M. bovis isolates, 50 from Mexico obtained from cattle in 8 different states, and 40 from the US from cattle, deer, elk and feral pigs from 7 different states were included in the study. All samples were analyzed in both laboratories, Mexico and the US, following the same protocol for molecular analysis by spoligotyping. Twenty-seven clusters, ranging from 1 to 18 genetically similar strains were found. Some clustering by country was observed, strains from cattle and deer in Michigan in the US fell into the same cluster, suggesting transmission between species. These results, combined with epidemiological information suggest that despite of the possibility that some animals with lesions in the US come from Mexico as false negatives, the US has its own source of infection, must probably in dairy cattle and wildlife. Genetic diversity of isolates from Mexico was larger than that in the US, which could be a consequence of the endemic status of the disease and the indiscriminate movement of animals between regions.     

Sequential medium with GH and IGF‐1 improved in vitro development of bovine preantral follicles enclosed in ovarian tissue

The growth hormone (GH) and growth insulin‐like factor‐1 (IGF‐1) act directly upon the regulation and growth in the different phases of preantral follicles. Thus, it is necessary to define their sequentiality until the in vitro preovulatory development. Therefore, the study aimed to assess the effects of a sequential medium containing GH and/or IGF‐1 in the long‐duration in vitro culture of preantral ovarian follicles. Ovarian fragments were cultivated: first half (days 1–7), second half (days 7–14) or during 14 culture days. Treatments were identified as: αMEM+; GH → IGF‐1; IGF‐1 → GH and GH + IGF‐1. The culture was designed in 24‐well plates, in an incubator at 37°C and 5% CO₂. The parameters of normality, viability, follicles (primordial/in developing) and follicle diameter were evaluated. In addition, the ultrastructure was confirmed with electron transmission microscopy. The results showed that the culture treated with GH → IGF‐1 kept the follicular normality and the viability until the 14th day of culture and increased both in the follicular development until 7th day and in the follicular diameter until 14th day, when compared to the control. The treatments IGF‐1 → GH and GH + IGF‐1 were not effective in the developing and follicular diameter after 7 days of culture, and also reduced the percentage of viability. It is concluded that the bovine preantral follicles cultured in the sequential medium treated with GH → IGF‐1 improved the follicular development until the first half of the culture and kept these parameters with normality, viability and ultrastructure until the second half of the in vitro culture.     

Phenotypic and genotypic characterization of antimicrobial resistance in faecal enterococci from wild boars (Sus scrofa)

The objective was to study the prevalence of antimicrobial resistance and the mechanisms implicated in faecal enterococci of wild boars in Portugal. One hundred and thirty-four enterococci (67 E. faecium, 54 E. hirae, 2 E. faecalis, 2 E. durans and 9 Enterococcus spp.) were recovered from 67 wild boars (two isolates/sample), and were further analysed. High percentages of resistance were detected for erythromycin, tetracycline, and ciprofloxacin (48.5%, 44.8%, and 17.9%, respectively), and lower values were observed for high-level-kanamycin, -streptomycin, chloramphenicol, and ampicillin resistance (9%, 6.7%, 4.5%, and 3.7%, respectively). No isolates showed vancomycin or high-level-gentamicin resistance. The erm(B), tet(M), aph(3')-IIIa, and ant(6)-I genes were demonstrated in all erythromycin-, tetracycline-, kanamycin-, and streptomycin-resistant isolates, respectively. Specific genes of Tn916/Tn1545 and Tn5397 transposons were detected in 78% and 47% of our tet(M)-positive enterococci, respectively. The tet(S) and tet(K) genes were detected in one isolate of E. faecium and E. hirae, respectively. Three E. faecium isolates showed quinupristin-dalfopristin resistance and the vat(E) gene was found in all of them showing the erm(B)-vat(E) linkage. Four E. faecium isolates showed ampicillin-resistance and all of them presented seven amino acid substitutions in PBP5 protein (461Q-->K, 470H-->Q, 485M-->A, 496N-->K, 499A-->T, 525E-->D, and 629E-->V), in relation with the reference one; a serine insertion at 466' position was found in three of the isolates. Faecal enterococci from wild boars harbour a variety of antimicrobial resistance mechanisms and could be a reservoir of antimicrobial resistance genes and resistant bacteria that could eventually be transmitted to other animals or even to humans.