Increased Brucella abortus asRNA_0067 expression under intraphagocytic stressors is associated with enhanced virB2 transcription.

Intracellular pathogens like Brucella face challenges during the intraphagocytic adaptation phase, where the modulation of gene expression plays an essential role in taking advantage of stressors to persist inside the host cell. This study aims to explore the expression of antisense virB2 RNA strand and related genes under intracellular simulation media. Sense and antisense virB2 RNA strands increased expression when nutrient deprivation and acidification were higher, being starvation more determinative. Meanwhile, bspB, one of the T4SS effector genes, exhibited the highest expression during the exposition to pH 4.5 and nutrient abundance. Based on RNA-seq analysis and RACE data, we constructed a regional map depicting the 5' and 3' ends of virB2 and the cis-encoded asRNA_0067. Without affecting the CDS or a possible autonomous RBS, we generate the deletion mutant ΔasRNA_0067, significantly reducing virB2 mRNA expression and survival rate. These results suggest that the antisense asRNA_0067 expression is promoted under exposure to the intraphagocytic adaptation phase stressors, and its deletion is associated with a lower transcription of the virB2 gene. Our findings illuminate the significance of these RNA strands in modulating the survival strategy of Brucella within the host and emphasize the role of nutrient deprivation in gene expression.

Effect of the Lacticaseibacillus paracasei JLM Strain Against Brucella abortus Strains in Ripened Cheese.

This study evaluated the antagonistic effect of the Lacticaseibacillus paracasei JLM strain isolated from aguamiel, against Brucella abortus RB51, S19, and 2308 strains, during the manufacture of soft-ripened cheese. First, the tolerance of Lc. paracasei JLM was tested with pH values and bile salt concentrations for 3 h to simulate digestive tract conditions. The antagonistic effect against B. abortus strains was evaluated through double-layer diffusion and agar well diffusion assays. In addition, the stability of the cell-free supernatant (CFS) was tested with the agar well diffusion method under different conditions of temperature, pH, and treatment with digestive enzymes. Finally, the antagonistic effect against B. abortus strains was observed during the manufacture of ripened cheese for 31 days at 4°C and 25°C using the Lc. paracasei JLM strain as starter culture. The results showed that the Lc. paracasei JLM strain remains viable after exposure to different pH values (from 3.00 to 7.00) and concentrations of bile salts (from 0.5% to 7%). Moreover, the results demonstrate that the growth of the three B. abortus strains was inhibited in both antagonism tests and that CFS maintained 86% activity after heat treatment at 100°C, 121°C, or enzymatic digestion (proteinase K, trypsin, chymotrypsin), but it was inactivated at pH levels above 6. Finally, Lc. paracasei JLM completely inhibited the growth of B. abortus in ripened cheese at 25°C from day 17 and showed greater inhibition on the B. abortus RB51 strain in the ripened cheese at 4°C, showing statistical differences for the B. abortus S19 and B. abortus 2308 strains. The current research concluded that the Lc. paracasei JLM strain has an antagonistic effect on B. abortus, enhancing the potential of its use in the future as a probiotic.

Survival of Brucella abortus RB51 and S19 Vaccine Strains in Fresh and Ripened Cheeses.

Brucellosis is a zoonotic infection caused by the consumption of contaminated raw milk and dairy products. This study aims to compare survival rates of Brucella abortus RB51 and S19 vaccine strains to that of virulent B. abortus 2308 strain during the manufacture of fresh and ripened cheeses. To do this, we inoculated fresh pasteurized milk with B. abortus RB51, S19, or 2308 at a 6 × 108 colony-forming unit per milliliter concentration during the cheese making process. Cheese was manufactured at room temperature, then, fresh cheeses were conserved at either 4°C or 25°C for 7 days, while ripened cheeses were conserved for 31 days at the same temperatures. We measured B. abortus survival and pH values during different stages of the process. Our results confirm that all three strains can maintain viable cells in both types of cheeses throughout the process. Survival of B. abortus RB51 was 10 times lower than was the survival of the B. abortus S19 and B. abortus 2308 strains in both fresh and ripened cheeses. Our results also suggest that both temperature and pH can condition Brucella survival. In conclusion, B. abortus RB51 and S19 vaccine strains can survive throughout the manufacture and conservation processes of both fresh and ripened cheeses. In turn, this implies a potential health risk if cheeses contaminated with these strains were to be consumed.

Brucella melitensis invA gene (BME_RS01060) transcription is promoted under acidic stress conditions.

The invA gene of Brucella melitensis codes for a NUDIX (nucleoside diphosphate linked to moiety X) hydrolase related to invasiveness. The objective of this work was to evaluate invA transcription under acidic conditions. The invA gene transcription was up regulated at pH 3 and pH 5 observed with semiquantitative real-time PCR in B. melitensis 133 strain. Results indicated that invA gene transcription at pH 3 showed a basal and decreased transcription compared to that of pH 5 incubation. Transcription levels of the dnaK gene were similar to those obtained with invA gene. The survival rates of wild type and invA mutant strains at pH 5 were above 90% in all post-incubation times. In contrast, at pH 3 there was a time-dependent reduction on both strains at 15 min (P < 0.05). These results suggest that invA gene transcription is promoted under acidic conditions in Brucella melitensis.

Comparative proteomic analysis of outer membrane vesicles from Brucella suis, Brucella ovis, Brucella canis and Brucella neotomae.

Gram-negative bacteria release nanovesicles, called outer membrane vesicles (OMVs), from their outer membrane. Proteomics has been used to determine their composition. OMVs contain proteins able to elicit an immune response, so they have been proposed as a model to develop acellular vaccines. In this study, OMVs of Brucella suis, B. ovis, B. canis, and B. neotomae were purified and analyzed by SDS-PAGE, transmission electron microscopy and liquid chromatography coupled to mass spectrometry to determine the pan-proteome of these vesicles. In addition, antigenic proteins were detected by western blot with anti-Brucella sera. The in silico analysis of the pan-proteome revealed many homologous proteins, such as Omp16, Omp25, Omp31, SodC, Omp2a, and BhuA. Proteins contained in the vesicles from different Brucella species were detected by anti-Brucella sera. The occurrence of previously described immunogenic proteins derived from OMVs supports the use of these vesicles as candidates to be evaluated as an acellular brucellosis vaccine.

Survival of Brucella abortus aqpX mutant in fresh and ripened cheeses.

The objective of this work was to evaluate the survival of a Brucella abortus aqpX mutant during the elaboration and conservation of fresh and ripened cheeses at 4 °C and 24 °C. The pH values and water activity were monitored for each type of cheese. The fresh cheese was elaborated with raw milk inoculated with 6×108 colony-forming units (CFU)/mL each of parental and mutant strain. Ripening cheeses were elaborated with both raw and pasteurized milk and inoculated with 12×108 CFU/mL each of parental and mutant strains. In fresh cheese, survival was observed during elaboration and conservation for 7 days at 4 °C in mutant and parental strains. The number of survivors of the mutant strain was 10 times lower compared with the parental strain at pH 5 and a(w) of 0.930. In the cheese elaborated with raw milk and ripened at 24 °C, both strains survived until day 17 at pH 4.0 and a(w) of 0.89. However, when the cheese was elaborated with pasteurized milk, the parental strain survived until day 31 of ripening, and the mutant strain survived 24 days at pH 4 and a(w) of 0.886. The survival of the mutant strain showed a diminution of one logarithm during elaboration and ripening of cheese as compared with the parental strain. When the cheese was elaborated with raw milk and ripened at 4 °C, survival of the parental strain was 24 days, whereas the mutant strain survived only 17 days (pH 5 and a(w) 0.90). Regarding the cheese elaborated with pasteurized milk and maturated at 4 °C, both strains survived 31 days (pH 5 and a(w) 0.90), with the same survival diminution during elaboration and ripening. Our results show that in both types of cheese, the mutated aqpX strain survived 10 times less than the parental strain, which shows that the aqpX gene can be related to the survival of Brucella abortus in this type of cheese.

Milk yield and reproductive performance of brucellosis-vaccinated but seropositive Holstein cows.

The aim of this research was to study if seropositivity for brucellosis in vaccinated cows against this disease hampers reproductive performance and milk production in high-yielding Holstein cows. For this purpose 1,026 healthy cows and 372 cows seropositive for brucellosis were enrolled in this study. Cows positive to card test and subsequently to the rivanol test were further subjected to the radial immunodiffusion (RID) test. It was found that only 11% of the presumably infected cows by brucellosis screening tests were really infected with this disease. The reproductive performance of the group of cows with 11% Brucella-infected animals was not impaired; overall pregnancy rate did not differ between seropositive and healthy cows (30.9 vs. 29.6%). The abortion rates were similar between seropositive cows (5.3%) and seronegative animals (6.9%). Cows in the herd with 11% Brucella-infected animals produced significantly more milk than unaffected cows over a 305-day lactation (10,684 ± 1,720 vs. 10,345 ± 1,736; mean ± SD; P < 0.05). It was concluded that in dairy herds vaccinated against brucellosis with both 19 and RB51 strains, supplemental tests such as RID need to be conducted on all reactors in order to maintain diagnostic accuracy. These results also indicate that 11% animal prevalence of brucellosis did not exert a detrimental effect on 305-day milk yield and reproductive performance in high milk-yielding Holstein cows.

Molecular Detection of Coxiella burnetii in Vaginal Swab Samples from Sheep That Aborted.

Background: Coxiella burnetii, an obligate intracellular bacterium, is the etiological agent of Q fever in humans and one of the causes of abortion in small ruminants. Although coxiellosis is considered an exotic disease, there are a few reports in Mexico. Methods: The objective of this work was to determine the presence of C. burnetii DNA in vaginal samples from sheep that presented abortion and ram semen. A total of 180 vaginal exudate samples and 20 semen samples were obtained from five Central and Southern States of Mexico. Total DNA was extracted from vaginal swabs and C. burnetii was identified by PCR amplification and sequencing of the IS1111 insertion sequence. Results and Conclusion: In total, 110 (110/180) vaginal samples and 12 (12/20) semen samples were positive for C. burnetii. This is the first report of C. burnetii in sheep that aborted and in ram semen in Mexico.

Isolation of a field strain of Brucella abortus from RB51-vaccinated- and brucellosis-seronegative bovine yearlings that calved normally.

A study was carried out in Pichucalco, Chiapas (Mexico) to determine whether recently calved cows or those that aborted shed Brucella. Serological diagnosis of brucellosis was made in all animals (209). Six of the cows that calved normally and two that aborted underwent a bacteriological study of milk and vaginal exudate. Brucella abortus was isolated from vaginal exudate samples in two 3- to 4-year-old seronegative first-birth cows that had calved normally. This was confirmed through bacteriological identification and PCR as a field strain and smooth phenotypes. We conclude that seronegative cows vaccinated with RB51 which calved normally and shed B. abortus in the vaginal exudate after calving could be a serious problem because these cows are overlooked in routine diagnoses and are a source of Brucella infection.

Enhancing Therapeutic Efficacy against Brucella canis Infection in a Murine Model Using Rifampicin-Loaded PLGA Nanoparticles.

The in vivo efficacy of rifampicin encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanoparticles was evaluated for the treatment of BALB/c mice experimentally infected with Brucella canis. The PLGA nanoparticles loaded with rifampicin (RNP) were prepared using the single emulsification-solvent evaporation technique, resulting in nanoparticles with a hydrodynamic diameter of 138 ± 6 nm. The zeta potential and polydispersity index values indicated that the system was relatively stable with a narrow size distribution. The release of rifampicin from the nanoparticles was studied in phosphate buffer at pH 7.4 and 37 °C. The release profile showed an initial burst phase, followed by a slower release stage attributed to nanoparticle degradation and relaxation, which continued for approximately 30 days until complete drug release. A combined model of rifampicin release, accounting for both the initial burst and the degradation-relaxation of the nanoparticles, effectively described the experimental data. The efficacy of RNP was studied in vivo; infected mice were treated with free rifampicin at concentrations of 2 mg per kilogram of mice per day (C1) and 4 mg per kilogram of mice per day (C2), as well as equivalent doses of RNP. Administration of four doses of the nanoparticles significantly reduced the B. canis load in the spleen of infected BALB/c mice. RNP demonstrated superior effectiveness compared to the free drug in the spleen, achieving reductions of 85.4 and 49.4%, respectively, when using C1 and 93.3 and 61.8%, respectively, when using C2. These results highlight the improved efficacy of the antibiotic when delivered through nanoparticles in experimentally infected mice. Therefore, the RNP holds promise as a potential alternative for the treatment of B. canis.

Evaluation of the Fluorescence Polarization Assay for the Diagnosis of Brucellosis in Goat Milk.

The milk ring test is a detection assay for antibodies against Brucella in bovine milk. It has good sensitivity but tends to give false positive results. In this study, we standardized the application of the fluorescence polarization assay (FPA) for the detection of antibodies against B.melitensis in goat milk. We obtained negative serum and milk samples from healthy goat flocks in the northern zone of Nuevo León. Positive milk and negative, weak, and strong controls were obtained by mixing volumes of positive control serum with negative control milk. Milk samples were treated with citric acid, after which an FPA was performed. Results were then compared with the Rose Bengal test and the FPA in serum. Milk treatment allowed the quantification of antibodies in samples. Significant differences were found between the 2%, 4%, and 6% groups, compared with the control group (F3, 67 = 17.45, p < 0.0001) but not between the 2% and 4% groups (p = 0.0718). The cut-off value was 74.1 mP, with a sensitivity (Se) of 95% and a specificity (Sp) of 100%. Se and Sp values in field milk samples were 84% and 74.55%, respectively. Despite the FPA test on milk samples showed lower Se and Sp than the FPA test on serum samples, its cutoff may be adjusted. It may be recommended as a screening test in goat milk and become useful for the control and eradication of the disease.

Isolation of Histophilus somni from the nasal exudates of a clinically healthy adult goat.

METHODS: The nasal exudate from 42 goats of the Mixteca Region in the state of Puebla, Mexico, was evaluated. A strain was isolated after 4 days of incubation. This strain was identified according to its phenotypic characteristics and by means of a species-specific polymerase chain reaction (PCR), as well as by sequencing of the amplified product. RESULTS: The species-specific PCR amplified a 407-bp fragment of 16S RNAr subunit, and the product sequencing revealed 100% homology with Histophilus somni 129PT. The nucleotide sequence was deposited in the GenBank under accession number HM032735. CONCLUSION: This is the first worldwide isolation of H. somni from nasal exudates of a clinically healthy goat.

Edaphoclimatic seasonal trends and variations of the Salmonella spp. infection in Northwestern Mexico.

Currently, Salmonella spp. is the bacterium causing the highest number of food-borne diseases (FADs) in the world. It is primarily associated with contaminated water used to that irrigates crops from intensive livestock farming. However, literature emphasizes that the reservoirs for Salmonella spp. remain in wildlife and there are unconventional sources or secondary reservoirs, such as soil. Human soil-borne diseases have not been modeled in spatial scenarios, and therefore it is necessary to consider soil and other climatic factors to anticipate the emergence of new strains or serotypes with potential threat to public and animal health. The objective of this research was to investigate whether edaphic and climatic factors are associated with the occurrence and prevalence of Salmonella spp. in Northwestern Mexico. We estimated the potential distribution of Salmonella spp. with an interpolation method of unsampled kriging areas for 15 environmental variables, considering that these factors have a seasonal dynamic of change during the year and modifications in longer periods. Subsequently, a database was generated with human salmonellosis cases reported in the epidemiological bulletins of the National System of Epidemiological Surveillance (SIVE). For the Northwest region, there were 30,595 human cases of paratyphoid and other salmonellosis reported have been reported in Baja California state, 71,462 in Chihuahua, and 16,247 in Sonora from 2002 to 2019. The highest prevalence was identified in areas with higher temperatures between 35 and 37 °C, and precipitation greater than 1000 mm. The edaphic variables limited the prevalence and geographical distribution of Salmonella spp., because the region is characterized by presenting a low percentage of organic matter (≤4.3), and most of the territory is classified as aridic and xeric, which implies that the humidity comprises ≤ 180 days a year. Finally, the seasonal time series indicated that in the states of Baja California and Chihuahua the rainy quarter of the year is 18.7% and 17.01% above a typical quarter respectively, while for Sonora the warmest quarter is 23.3%. It is necessary to deepen the relationship between different soil characteristics and climate elements such as temperature and precipitation, which influence the distribution of different soil-transmitted diseases.

Serotyping versus genotyping in infected sheep and goats with small ruminant lentiviruses.

Despite SRLV infection being endemic in Mexico, there is little information regarding which genotypes are present. We compared serotyping and PCR-sequencing results from sheep and goats infected with SRLV. We separated plasma and peripheral blood leukocytes (PBL) from 1940 blood samples from sheep and goats from 12 states across Mexico. To detect SRLV infection, we tested plasma samples using two commercial ELISA kits (VMRD and Eradikit SRLV Screening). Then, we serotyped the infecting virus (A/ B) using Eradikit SRLV Genotyping. PBL DNA was used to detect the proviral genome via PCR. Positive amplicons were sequenced to identify viral genotypes using a phylogenetic analysis. Also, we analysed for residues differences in the sequences of a capsid epitope between genotypes. The serological results indicated a higher detection of seropositive animals using the VMRD ELISA compared to Eradikit, with 21 % and 15.3 % more in sheep and goats respectively. Only 25.7 % of the ELISA serotyping results matched those from PCR-sequencing. PCR-sequencing was able to identify genotype A, B and coinfections in animals classified as indeterminate by the ELISA test. This lack of sensitivity may be related to the lack of epitopes from the matrix and transmembrane peptides used by ELISA screening. Sequences analysis revealed that SRLVs found in sheep cluster with genetic subtypes A2 and B1, while those in goats cluster with subtypes A1 and B1. Serotyping did not prove to be an adequate method for predicting the viral genotype (A and / or B) in infections caused by SRLV.

Outer Membrane Vesicles From Brucella melitensis Modulate Immune Response and Induce Cytoskeleton Rearrangement in Peripheral Blood Mononuclear Cells.

Similar to what has been described in other Gram-negative bacteria, Brucella melitensis releases outer membrane vesicles (OMVs). OMVs from B. melitensis 16M and the rough-mutant B. melitensis VTRM1 were able to induce a protective immune response against virulent B. melitensis in mice models. The presence of some proteins which had previously been reported to induce protection against Brucella were found in the proteome of OMVs from B. melitensis 16M. However, the proteome of OMVs from B. melitensis VTRM1 had not previously been determined. In order to be better understand the role of OMVs in host-cell interactions, the aim of this work was to compare the proteomes of OMVs from B. melitensis 16M and the derived rough-mutant B. melitensis VTRM1, as well as to characterize the immune response induced by vesicles on host cells. Additionally, the effect of SDS and proteinase K on the stability of OMVs was analyzed. OMVs from B. melitensis 16M (smooth strain) and the B. melitensis VTRM1 rough mutant (lacking the O-polysaccharide side chain) were analyzed through liquid chromatography-mass spectrometry (LC-MS/MS). OMVs were treated with proteinase K, sodium deoxycholate, and SDS, and then their protein profile was determined using SDS-PAGE. Furthermore, PBMCs were treated with OMVs in order to measure their effect on cytoskeleton, surface molecules, apoptosis, DNA damage, proliferation, and cytokine-induction. A total of 131 proteins were identified in OMVs from B. melitensis16M, and 43 in OMVs from B. melitensis VTRM1. Proteome comparison showed that 22 orthologous proteins were common in vesicles from both strains, and their core proteome contained Omp31, Omp25, GroL, and Omp16. After a subsequent detergent and enzyme treatment, OMVs from B. melitensis VTRM1 exhibited higher sensitive compared to OMVs from the B. melitensis 16M strain. Neither OMVs induced IL-17, proliferation, apoptosis or DNA damage. Nonetheless, OMVs from the smooth and rough strains induced overproduction of TNFα and IL-6, as well as actin and tubulin rearrangements in the cytoskeleton. Moreover, OMVs from both strains inhibited PD-L1 expression in T-cells. These data revealed significant differences in OMVs derived from the rough and smooth Brucella strains, among which, the presence or absence of complete LPS appeared to be crucial to protect proteins contained within vesicles and to drive the immune response.

Campylobacter fetus is Internalized by Bovine Endometrial Epithelial Cells.

Campylobacter fetus is an important venereal pathogen of cattle that causes infertility and abortions. It is transmitted during mating, and it travels from the vagina to the uterus; therefore, an important cell type that interacts with C. fetus are endometrial epithelial cells. Several virulence factors have been identified in the genome of C. fetus, such as adhesins, secretion systems, and antiphagocytic layers, but their expression is unknown. The ability of C. fetus to invade human epithelial cells has been demonstrated, but the ability of this microorganism to infect bovine endometrial epithelial cells has not been demonstrated. Bovine endometrial epithelial cells were isolated and challenged with C. fetus. The presence of C. fetus inside the endometrial epithelial cells was confirmed by the confocal immunofluorescence. C. fetus was not internalized when actin polymerization was disturbed, suggesting cytoskeleton participation in an internalization mechanism. To evaluate the intracellular survival of C. fetus, a gentamicin protection assay was performed. Although C. fetus was able to invade epithelial cells, the results showed that it did not have the capacity to survive in the intracellular environment. This study reports for the first time, the ability of C. fetus to invade bovine endometrial epithelial cells, and actin participation in this phenomenon.Campylobacter fetus is an important venereal pathogen of cattle that causes infertility and abortions. It is transmitted during mating, and it travels from the vagina to the uterus; therefore, an important cell type that interacts with C. fetus are endometrial epithelial cells. Several virulence factors have been identified in the genome of C. fetus, such as adhesins, secretion systems, and antiphagocytic layers, but their expression is unknown. The ability of C. fetus to invade human epithelial cells has been demonstrated, but the ability of this microorganism to infect bovine endometrial epithelial cells has not been demonstrated. Bovine endometrial epithelial cells were isolated and challenged with C. fetus. The presence of C. fetus inside the endometrial epithelial cells was confirmed by the confocal immunofluorescence. C. fetus was not internalized when actin polymerization was disturbed, suggesting cytoskeleton participation in an internalization mechanism. To evaluate the intracellular survival of C. fetus, a gentamicin protection assay was performed. Although C. fetus was able to invade epithelial cells, the results showed that it did not have the capacity to survive in the intracellular environment. This study reports for the first time, the ability of C. fetus to invade bovine endometrial epithelial cells, and actin participation in this phenomenon.

Polymorphisms in Brucella Carbonic Anhydrase II Mediate CO2 Dependence and Fitness in vivo.

Some Brucella isolates are known to require an increased concentration of CO2 for growth, especially in the case of primary cultures obtained directly from infected animals. Moreover, the different Brucella species and biovars show a characteristic pattern of CO2 requirement, and this trait has been included among the routine typing tests used for species and biovar differentiation. By comparing the differences in gene content among different CO2-dependent and CO2-independent Brucella strains, we have confirmed that carbonic anhydrase (CA) II is the enzyme responsible for this phenotype in all the Brucella strains tested. Brucella species contain two CAs of the ß family, CA I and CA II; genetic polymorphisms exist for both of them in different isolates, but only those putatively affecting the activity of CA II correlate with the CO2 requirement of the corresponding isolate. Analysis of these polymorphisms does not allow the determination of CA I functionality, while the polymorphisms in CA II consist of small deletions that cause a frameshift that changes the C-terminus of the protein, probably affecting its dimerization status, essential for the activity. CO2-independent mutants arise easily in vitro, although with a low frequency ranging from 10-6 to 10-10 depending on the strain. These mutants carry compensatory mutations that produce a full-length CA II. At the same time, no change was observed in the sequence coding for CA I. A competitive index assay designed to evaluate the fitness of a CO2-dependent strain compared to its corresponding CO2-independent strain revealed that while there is no significant difference when the bacteria are grown in culture plates, growth in vivo in a mouse model of infection provides a significant advantage to the CO2-dependent strain. This could explain why some Brucella isolates are CO2 dependent in primary isolation. The polymorphism described here also allows the in silico determination of the CO2 requirement status of any Brucella strain.

First genome sequencing and comparative analyses of Corynebacterium pseudotuberculosis strains from Mexico.

Corynebacterium pseudotuberculosis is a pathogenic bacterium which has been rapidly spreading all over the world, causing economic losses in the agricultural sector and sporadically infecting humans. Six C. pseudotuberculosis strains were isolated from goats, sheep, and horses with distinct abscess locations. For the first time, Mexican genomes of this bacterium were sequenced and studied in silico. All strains were sequenced using Ion Personal Genome Machine sequencer, assembled using Newbler and SPAdes software. The automatic genome annotation was done using the software RAST and in-house scripts for transference, followed by manual curation using Artemis software and BLAST against NCBI and UniProt databases. The six genomes are publicly available in NCBI database. The analysis of nucleotide sequence similarity and the generated phylogenetic tree led to the observation that the Mexican strains are more similar between strains from the same host, but the genetic structure is probably more influenced by transportation of animals between farms than host preference. Also, a putative drug target was predicted and in silico analysis of 46 strains showed two gene clusters capable of differentiating the biovars equi and ovis: Restriction Modification system and CRISPR-Cas cluster.

Protection of Brucella abortus RB51 revaccinated cows, introduced in a herd with active brucellosis, with presence of atypical humoral response.

It is a dogma, that RB51 vaccination does not induce antibodies that interfere with Brucellosis diagnosis, therefore any animal positive to serological test is considered as an infected animal. To determine protection against Brucellosis virulent field strain, 35 pregnant cows from a free-Brucellosis herd, previously vaccinated as calves with 1 x 10(10) CFU of RB51, were revaccinated with RB51 reduced dose, and then introduced into a herd with an active outbreak. Seventeen cows resulted positive in card test after revaccination. All 35 pregnant revaccinated cows had normal parturition; nevertheless, RB51 vaccine strain was isolated from milk and vaginal exudates from two cows after delivery at day 120 post-revaccination. At 150 days post-revaccination, two cows were positives to card and rivanol test and the field virulent strain was isolated. Revaccination with a reduced dose of RB51 in endemic zones did not cause abortion and protected 94% of animals against field infection, but caused an atypical response to conventional serological tests.

Intracellular trafficking study of a RB51 B. abortus vaccinal strain isolated from cow milk.

Brucella is responsible for one of the major worldwide zoonoses. Over the last century, several vaccines have been used against brucellosis. Among these, the rough vaccine Brucella abortus RB51 was introduced with the idea that it would not interfere with the diagnosis of brucellosis. Recently, RB51 has been isolated from milk and vaginal exudates from vaccinated cows, thus raising the possibility of extensive bacterial replication in these animals. We hypothesized that shedding of RB51 might be related to a change in its intracellular cell cycle. Therefore, we have compared the intracellular trafficking in CHO cells of the virulent B. abortus 2308 and two RB51 strains, the vaccinal strain and the one isolated from cow milk. Both RB51 strains were transiently observed in phagosomes characterized by the presence of the early endosomal marker EEA1 and then were found in cathepsin D-enriched lysosomal compartments, in which they eventually underwent degradation at later post-infection times. In contrast, the virulent 2308 strain replicated within the endoplasmic reticulum. These results suggest that a change in intracellular trafficking cannot account for Brucella shedding in adult vaccinated cows.