Brucella intracellular life: from invasion to intracellular replication

Brucella organisms are pathogens that ultimate goal is to propagate in their preferred niche, the cell. Upon cell contact the bacteria is internalized via receptor molecules by activating small GTPases of the Rho subfamily and by a moderate recruitment of actin filaments. Once inside cells, Brucella localizes in early phagosomes, where it avoids fusion with late endosomes and lysosomes. These early events require the control of Rab small GTPases, and cytokines such as the G-CSF. Then, the bacterium redirects its trafficking to autophagosomes and finally reaches the endoplasmic reticulum, where it extensively replicates. Some of the bacterial molecular determinants involved in the internalization and early events after ingestion are controlled by the BvrS/BvrR two component regulatory system, whereas the intracellular trafficking beyond this early compartments are controlled by the VirB type IV secretion system. Once inside the endoplasmic reticulum, Brucella extensively replicates without restricting basic cellular functions or inducing obvious damage to cells. The integrity of Brucella LPS on the bacterial surface is one of the required factors for Brucella intracellular survival, and therefore for virulence.     

Complementary DNA sequencing: expressed sequence tags and human genome project

Automated partial DNA sequencing was conducted on more than 600 randomly selected human brain complementary DNA (cDNA) clones to generate expressed sequence tags (ESTs). ESTs have applications in the discovery of new human genes, mapping of the human genome, and identification of coding regions in genomic sequences. Of the sequences generated, 337 represent new genes, including 48 with significant similarity to genes from other organisms, such as a yeast RNA polymerase II subunit; Drosophila kinesin, Notch, and Enhancer of split; and a murine tyrosine kinase receptor. Forty-six ESTs were mapped to chromosomes after amplification by the polymerase chain reaction. This fast approach to cDNA characterization will facilitate the tagging of most human genes in a few years at a fraction of the cost of complete genomic sequencing, provide new genetic markers, and serve as a resource in diverse biological research fields.     

Litopenaeus vannamei (Boone) post-larval survival related to age, temperature, pH and ammonium concentration

Transport of post‐larvae shrimp used in aquaculture is an important element of successful cultivation because of the potential for stress during stocking procedures. To find optimum transport conditions, several bioassays were performed in the laboratory to evaluate survival of whiteleg shrimp Litopenaeus vannamei 5–30‐day‐old postlarvae under conditions similar to those encountered during transport from the hatchery to nursery and shrimp ponds. Postlarvae were exposed for 4 h to different temperatures and pH levels ammonia concentrations. Survival was significantly reduced after a 4 h exposure to pH 9 and was inversely related to temperature with or without 7 mg L^?1 of ammonia. The 15‐ and 20‐day‐old postlarvae had higher survival rates than other ages. The lowest survival occurred in alkali conditions (pH 9), with 7 mg L^?1ammonia at 30 and 32°C. To assure optimal survival of postlarvae during transfer from the hatchery to the nursery and shrimp ponds, we recommend temperatures below 28°C, pH no higher than 8, no ammonia and post‐larval age at least 15 days.     

Undernutrition in the parental and first generation provokes an organ‐specific response to oxidative stress on neonates of second filial generation of Wistar rats

Undernutrition induces an increase of the oxidative stress that can predispose offspring to various diseases in adulthood through epigenetic reprogramming. The aim of this study was to evaluate the effects of intergenerational undernutrition on protein oxidation and antioxidant defence response on liver, heart and brain of the second‐generation neonates (F₂) of undernourished rats. For this purpose, both parents in parental (F₀) and first generation (F₁) were fed with a low‐nutrient diet. Body mass and length decreased (p < 0.05) in F₀, F₁ and F₂ being the F₁ males who exhibited a greater mass loss. A decrease in plasma albumin concentration was observed in F₂ neonates (p < 0.05) and also a mass loss of liver, heart and brain (p < 0.05), although proportionally to body length reduction. Undernutrition increased levels of protein oxidation in liver and heart (p < 0.05) but not in brain (p > 0.05) while catalase activity increased only in brain (p < 0.05). In summary, intergenerational undernutrition modifies the antioxidant status through an organ‐specific response, on F₂ neonate rats, where the brain increased catalase activity to prevent a severe oxidative damage and support the vital functions of this key organ to maintain vital functions.     

Comparison of the Antibody Response in Adult Cattle Against Different Epitopes of Brucella abortus Lipopolysaccharide

The comparison of serological responses in a sample of adult, vaccinated and field‐infected bovines with Brucella abortus is reported. Indirect enzyme immunoassay (EIA) titration curves and Western blotting tests for smooth‐type lipopolysaccharide (S‐LPS), rough‐type LPS (R‐LPS) and lipid A were performed. In the initial screening of sera, an overall prevalence of 20.5 % was found, which corresponds to a country with a high incidence of brucellosis. End‐point EIA titres against LPS antigens from vaccinated and field‐infected cows were not significantly different. However, the absorbance values in the titration curves were significantly higher for S‐LPS as compared with the other antigens. A high correlation coefficient (r=0.933) was obtained when the titres to R‐LPS versus lipid A were compared. Western blotting reactions of vaccinated and field‐infected animals were indistinguishable. S‐LPS, R‐LPS and lipid A epitopes were recognized in a heterogeneous manner. In general, the number of bovines that reacted against LPS was higher in the field‐infected group, with a stronger binding to S‐LPS. Based on our observations, the vaccinated and field‐infected bovines are capable of producing similar antibody responses to the Brucella main outer surface antigen, LPS. It should be emphasized that the humoral response of cattle to Brucella LPS contains significant amounts of antibodies to other antigenic moieties of this important surface molecule, which may contribute to the immunity to brucellosis.     

Performance of Prunus rootstocks for apricot in Mediterranean conditions

The influence of five rootstocks (Evrica, Krymsk^® 86, Torinel, PAC 00-08 and PADAC 01-47) on flowering, fruit set, trunk cross-sectional area, yield efficiency and fruit quality parameters of apricot cultivars (Prunus armeniaca L.) grown in a Mediterranean agro-climatic environment was evaluated. The five rootstocks were grafted with ‘E-101’ and ‘E-404’ apricot cvs., and established in an experimental orchard in the Region of Murcia (South-eastern of Spain) in 2004. Rootstock had no significant influence on the number of flowers but affected fruit set. Evrica, PAC 00-08 and PADAC 01-47, induced a higher percentage of fruit set on the apricot cultivar ‘E-101’. The greatest TCSA was exhibited with Torinel, Evrica and PADAC 01-47. The yield efficiency was significantly greater on PADAC 01-47, because of its higher yield and cumulative production compared with the other rootstocks. Thus, differences in precocity among rootstocks became evident, PADAC 01-47 being the most efficient rootstock for the first bearing years. The fruit quality traits were also significantly affected by rootstocks. In the case of ‘E-101’, the highest fruit weight was induced by Evrica, Krymsk^® 86 and Torinel. In relation to fruit size, the smallest equatorial, suture and polar diameters were produced by fruit on PADAC 01-47 for both cultivars. The highest firmness was induced by PAC 00-08 for ‘E-101’, while in ‘E-404’ the highest firmness was induced by PADAC 01-47. The colour of fruit was also affected by the rootstock. The brightest coloured skin (high L* values) was found on Evrica, PADAC 01-47, Torinel and Krymsk^® 86. The fruit weight was positively correlated with pulp yield and negatively correlated with TCSA. According to these results, higher fruit quality was found on PADAC 01-47 and Evrica.     

Comparative efficacy of two single-dose bacterins in the control of Mycoplasma hyopneumoniae in swine raised under commercial conditions in Brazil

A field study was conducted in Brazil to evaluate the efficacy of single vaccination of pigs with two bacterins to prevent Mycoplasma hyopneumoniae lung lesions. The first (T1) treatment group (174 pigs) was injected with 2 mL of saline solution; group T2 (177 pigs) with 2 mL of bacterin A, and group T3 (174 pigs) with 2 mL of bacterin B. On days-on-test (DOT) 0, 35, 66, 97 and 125, blood samples and tonsil swabs were collected from selected pigs for antibody determination (indirect ELISA) and PCR assay for the presence of M. hyopneumoniae. Pigs were slaughtered on DOT 126-129 and lung lesions were scored blindly. Bacterin A vaccinated pigs had significantly (P < or = 0.05) lower lung lesion scores (0.2%) than bacterin B (0.4%) or saline-treated pigs (1.2%); there was also a significantly lower (P < or = 0.05) number of pigs with lung lesions (27.1%), than bacterin B (38.2%) or saline-treated (55.4%) pigs. The two vaccines had similar (P>0.05) results in terms of mean weight gain, average daily weight gain, feed efficiency, frequency of PCR positives, and there was similar antibody conversion (ELISA). It was concluded that although the productivity parameters and antibody conversions were similar, bacterin A was more effective in preventing and reducing the severity of lung lesions than bacterin B.     

Microsatellites in immune-relevant regions and their associations with Maedi-Visna and ovine pulmonary adenocarcinoma viral diseases

Maedi-Visna (MV) and ovine pulmonary adenocarcinoma (OPA) are two retroviral diseases occurring worldwide that affect adult sheep. Differences in incidence, which may be related to sheep-rearing and housing choices, as well as to genetics, and disease progression have been reported for both diseases. In this work four microsatellites located in immune-relevant regions, the major histocompatibility complex (MHC) region, interferon-γ and interleukin-12p35, were genotyped to determine their association with disease progression. The analysed sample included Latxa sheep with and without OPA and MV-characteristic lesions in their lungs. The microsatellites in the MHC were the most diverse, while the ones located in the cytokines were the less polymorphic. In the case of IFN-γ the results suggested the presence of null alleles. Significant results were detected for several microsatellite alleles in the association analysis carried out by logistic regression. All statistical analyses included a flock effect adjustment to avoid false positives due to genetic structuration. MHC Class I microsatellite alleles OMHC1*205 and OMHC1*193 were associated with disease progression for Maedi and OPA, respectively. Moreover, MHC Class II microsatellite allele DRB2*275 was associated with presence of lesions in Maedi. Furthermore, the MHC microsatellites were combined for a bioinformatic haplotype inference with the PHASE software. In total, 73 haplotypes were detected, 18 of them in more than 6 animals. After standard and weighted logistic regression analysis, two of them were significantly associated with susceptibility: OMHC1*205-DRB2*271 for Maedi and OMHC1*193-DRB2*271 for OPA, both with the Class I microsatellite alleles associated in the marker by marker study. Although more extensive analyses are needed to disentangle the relationship between host genetics and disease, as far as we know this is the first study demonstrating a significant association between sheep MHC Class I microsatellite alleles and susceptibility to Maedi-Visna and OPA viral diseases.     

Liver pathology and immune response in experimental Fasciola hepatica infections of goats

The relationship between the immune response and the pathogenesis of the disease was studied in different primary and secondary experimental Fasciola hepatica infections of goats. The establishment of the infection, measured as percentage of recovered flukes at the necropsy, was similar in primarily and secondarily infected animals (between 19.7% and 24.3%), but the hepatic damage was much more severe in secondarily infected goats, as revealed by the levels of serum hepatic enzymes GGT and LDH. Primary infection evolves to chronic fasciolosis that did not induce the development of resistance, since goats were highly susceptible to secondary infection, showing severe acute and chronic hepatic lesions that led to the death of some animals in each group. The immune response to the infection was proved by the production of specific IgG antibodies to ESP of F. hepatica and the involvement of CD3+ T lymphocytes and lambda IgG+ plasma cells in the hepatic infiltrate. Secondary infection did not induce any difference in either IgG response or in the cellular composition of the infiltrate of hepatic lesions, although this was much more extended. However, neither antibodies nor cell-mediated response were protective: there was no correlation between IgG levels and fluke burden and there was no evidence of cell-mediated killing of the parasite. This suggests the existence of some immune evasion mechanisms in goat infection with F. hepatica. The parasite may depress the local inflammatory and immune response, as suggested by the scarcity of CD3+ T cells in the infiltrate surrounding acute migratory tunnels. Moreover, in secondary infected goats can be suspected an immunological damage of the liver, since a very severe infiltrate of immune cells replaced wide areas of hepatic parenchyma and an immune-mediated damage of hepatocytes could occur.