Down Regulation of Catsper1 Expression by Calmodulin Inhibitor (Calmidazolium): Possible Implications for Fertility.

The CatSper channel localizes exclusively in the flagella of sperm cells. The Catsper1 protein, together with three pore units, is essential for the CatSper Channel formation, which produces flagellum hyperactivation and confers sperm fertility. Catsper1 expression is dependent on Sox transcription factors, which can recognize in vitro at least three Sox binding sites on the promoter. Sox transcription factors have calmodulin-binding domains for nuclear importation. Calmodulin (CaM) is affected by the specific inhibitor calmidazolium (CMZ), which prevents the nuclear transport of Sox factors. In this work, we assess the regulation of the Catsper1 promoter in vivo by Sox factors in the murine testis and evaluate the effects of the inhibitor calmidazolium on the expression of the Casper genes, and the motility and fertility of the sperm. Catsper1 promoter has significant transcriptional activity in vivo; on the contrary, three Sox site mutants in the Catsper1 promoter reduced transcriptional activity in the testis. CaM inhibition affects Sox factor nuclear transport and has notable implications in the expression and production of Catsper1, as well as in the motility and fertility capability of sperm. The molecular mechanism described here might conform to the basis of a male contraceptive strategy acting at the transcriptional level by affecting the production of the CatSper channel, a fundamental piece of male fertility.

Changes in nutrient and calorie intake, adipose mass, triglycerides and TNF-α concentrations after non-caloric sweetener intake: A pilot study.

Establishing the safety of non-caloric sweetener consumption in humans is a difficult task, since many contradictory results have been reported. The objective of this study was to compare the effect of frequent intake of sucrose, sucralose or steviol glycosides, on selected anthropometric, biochemical and immunological parameters in healthy, young adults. 38 individuals with normal body mass index were recruited and randomly divided into three experimental groups. After a washout week (where food with added sweeteners was restricted), each group was supplemented with sucrose (8 × 5 g packets/day), sucralose or steviol glycosides (4 × 1 g packets/day each) for 6 weeks. Selected variables were measured before and after treatment in each group and differences within and among groups were assessed. Our results showed that, compared to baseline, there was a modest but significant increase in weight (p = 0.0293) in the sucralose group, while the steviol glycosides group reduced their fat mass (p = 0.0390). No differences were observed in glycaemia; however, there was a significant increase in serum triglycerides (77.8-110.8 mg/dL) and cholesterol (162.0-172.3 mg/dL) in the sucrose group, whereas the steviol glycosides group presented lower triglycerides (104.7-92.8 mg/dL) and TNF-α concentrations (51.1-47.5 pg/mL). Comparison among groups showed differences in serum triglycerides (p = 0.0226), TNF-α (p = 0.0460) and IL-ß (p = 0.0008). Our results suggest that, even in a short time span, frequent intake of steviol glycosides may have positive effects on metabolic parameters that may be relevant for human health.

High prevalence of human papillomavirus type 66 in low-grade cervical lesions of Mexican women.

Our aim was to analyze the prevalence of high-risk human papillomavirus (HR-HPV) and its association with risk factors related to cervical lesions. We used 362 cervical samples from a transversal study to detect nineteen types from the high-risk HPV clade by highly sensitive PCR. Unexpectedly, we found a very high prevalence of HPV type 66 (32.8%), particularly in low-grade squamous intraepithelial lesions. A significant association of HPV66 with previously sexually transmitted disease was observed (p < 0.05). Our results strongly suggest that HPV66 might be indicative of cervical lesions that will not progress to cancer. HPV genotyping by methods that grouped type 66 with other HR-HPV clade types should be interpreted with caution.

Seroprevalence and geographical distribution of sero-positive blood donors to Trypanosoma cruzi at the central blood bank of the National Medical Center "La Raza".

BACKGROUND: Trypanosoma cruzi is a protozoan parasite that causes Chagas disease endemic to Latin-America. It is estimated that 1.0 to 1.5% of Mexicans are infected with T. cruzi, which constitutes a potential risk of disease transmission via contaminated blood. New cases are being reported worldwide due to the migration of infected people from endemic areas. STUDY DESIGN AND METHODS: Serum samples were collected from donors at the Central Blood Bank of the National Medical Center "La Raza" from July 2008 to December 2015 and analyzed for T. cruzi antibodies using Enzyme-linked Immunosorbent Assays. Blood donors were classified serologically as either negative or positive for Chagas disease based on the Official Mexican Standard NOM-032-SSA2-2014. The geographical distribution of sero-positive donors for Chagas disease was then determined based on the donor's areas of residence. RESULTS: Of the 510, 047 donors, 595 tested positive for Chagas disease. We found a prevalence of 0.12%, was higher in males (0.13%) than females (0.08%) In both genders, there were more sero-positive donors aged 51-65 years as compared to other age groups. Overall there were more positive donors from the State of Mexico, northern area of Mexico City, and southern area of Hidalgo State, with rates of 67.4%, 20.6%, and 5.9%, respectively. CONCLUSIONS: The seroprevalence of Chagas disease in blood donors attending to La Raza BB is low. Chagas disease is more prevalent in the older age groups; most sero-positive donors are from areas considered non-endemic to Chagas disease.

Oral Administration of Efavirenz Dysregulates the Tph2 Gene in Brain Serotonergic Areas and Alters Weight and Mood in Mice.

Most HIV-antiretroviral drugs have adverse effects. Efavirenz (EFV) is an example of a drug with neuropsychiatric effects, such as anxiety, depression, and suicidal thoughts, in people living with HIV (PLWH). The mechanisms by which EFV causes neuropsychiatric alterations in PLWH are complex, multifactorial, and not fully understood, although several studies in animals have reported changes in brain energy metabolism, alterations in monoamine turnover, GABA, and glutamate levels, and changes in 5-HT receptors. In this report, we studied the effects of EFV on the serotonergic system in healthy mice, specifically, whether EFV results in alterations in the levels of the tryptophan hydroxylase 2 (Tph2) gene in the brain. EFV (10 mg/kg) and distilled water (1.5 µL/kg) (control group) were orally administered to the mice for 36 days. At the end of the treatment, Tph2 expression levels in mouse brains were measured, and mood was evaluated by three trials: the forced swim test, elevated plus maze, and open field test. Our results revealed dysregulation of Tph2 expression in the brainstem, amygdala, and hypothalamus in the EFV group, and 5-HT levels increased in the amygdala in the EFV group. In the behavioral tests, mice given EFV exhibited a passive avoidance response in the forced swim test and anxiety-like behavior in the elevated plus maze, and they lost weight. Herein, for the first time, we showed that EFV triggered dysregulation of the Tph2 gene in the three serotonergic areas studied; and 5-HT levels increased in the amygdala using the ELISA method. However, further studies will be necessary to clarify the increase of 5-HT in the amygdala as well as understand the paradoxical decrease in body weight with the simultaneous increase in food consumption. It will also be necessary to measure 5-HT by other techniques different from ELISA, such as HPLC.

Molecular detection of hemotropic Mycoplasma and Bartonella species in lice from sheep and goats of Mexico.

The knowledge of lice associated with small ruminants, especially sheep and goats, is scarce. In Mexico, there are historical reports of six species of chewing and sucking lice associated with Capra hircus and Ovis canadensis. However, the reports did not analyze the ecology of the infestations or the presence of potentially pathogenic bacteria. For this reason, the objectives of this study were i) to identify the species of lice associated with sheep and goats in three states of the Mexican Republic, ii) to characterize the infestations, and iii) to identify the presence of bacterial pathogens. From October 2019 to August 2021, six ranches with sheep and goats were sampled in the states of Hidalgo and Veracruz. Hosts were visually inspected, and lice were retrieved with forceps. The specimens were sexed and identified using morphological taxonomic keys. DNA extraction was performed individually, and a fragment of the cytochrome oxidase subunit 1 gene (COI) was amplified for the molecular identification of the specimens. Subsequently, Anaplasma, Bartonella, Ehrlichia, Mycoplasma, and Rickettsia were molecularly detected. Additionally, the infestations were characterized by calculating the prevalence and mean abundances. We collected 563 specimens of three species, Bovicola caprae, Bovicola ovis, and Linognathus africanus. The highest infestation levels were recorded for B. ovis (66.7%; 4.4) from Veracruz. Additionally, two Bartonella species were detected: Bartonella mellophagi in B. ovis and Bartonella capreoli in L. africanus. In contrast, Mycoplasma ovis was detected exclusively in one pool of B. ovis. This study provides new bacterial-ectoparasite associations and highlights the possible role of these neglected ectoparasites as vectors in the populations of sheep and goats from Mexico.

The Low Variability of Tc24 in Trypanosoma cruzi TcI as an Advantage for Chagas Disease Prophylaxis and Diagnosis in Mexico.

(1) Background: Chagas disease is the main neglected tropical disease in America. It is estimated that around 6 million people are currently infected with the parasite in Latin America, and 25 million live in endemic areas with active transmission. The disease causes an estimated economic loss of USD 24 billion dollars annually, with a loss of 75,200 working years per year of life; it is responsible for around ~12,000 deaths annually. Although Mexico is an endemic country that recorded 10,186 new cases of Chagas disease during the period of 1990-2017, few studies have evaluated the genetic diversity of genes that could be involved in the prophylaxis and/or diagnosis of the parasite. One of the possible candidates proposed as a vaccine target is the 24 kDa trypomastigote excretory-secretory protein, Tc24, whose protection is linked to the stimulation of T. cruzi-specific CD8+ immune responses. (2) Methods: The aim of the present study was to evaluate the fine-scale genetic diversity and structure of Tc24 in T. cruzi isolates from Mexico, and to compare them with other populations reported in the Americas with the aim to reconsider the potential role of Tc24 as a key candidate for the prophylaxis and improvement of the diagnosis of Chagas disease in Mexico. (3) Results: Of the 25 Mexican isolates analysed, 48% (12) were recovered from humans and 24% (6) recovered from Triatoma barberi and Triatoma dimidiata. Phylogenetic inferences revealed a polytomy in the T. cruzi clade with two defined subgroups, one formed by all sequences of the DTU I and the other formed by DTU II-VI; both subgroups had high branch support. Genetic population analysis detected a single (monomorphic) haplotype of TcI throughout the entire distribution across both Mexico and South America. This information was supported by Nei's pairwise distances, where the sequences of TcI showed no genetic differences. (4) Conclusions: Given that both previous studies and the findings of the present work confirmed that TcI is the only genotype detected from human isolates obtained from various states of Mexico, and that there is no significant genetic variability in any of them, it is possible to propose the development of in silico strategies for the production of antigens that optimise the diagnosis of Chagas disease, such as quantitative ELISA methods that use this region of Tc24.

Seroprevalence of Trypanosoma cruzi in Eight Blood Banks in Mexico.

BACKGROUND: The true prevalence of Chagas disease in Mexico is unknown. However, it has been estimated that 1.1-4 million people are infected with Trypanosoma cruzi, which represents a potential risk for transmission of the disease via contaminated blood. AIM OF THE STUDY: To determine the Chagas disease seroprevalence in donors from eight blood banks in the north of Mexico City, and the northeast of the State of Mexico. STUDY DESIGN AND METHODS: Serum samples from blood donors (n = 515,038) were tested to detect the presence of anti-Trypanosoma cruzi antibodies in eight blood banks. The serologic screening test was performed in each of the blood banks. To confirm the seropositive blood donors, only two out of the eight blood banks used a test with a different principle with the aim of identifying anti-Trypanosoma cruzi antibodies. All tests were validated by the Mexican Institute for Epidemiological Diagnosis and Reference. RESULTS: One thousand two hundred and ten blood donors were seropositive for Trypanosoma cruzi, which represents a 0.23% seroprevalence (95% CI 0.22-0.25%). Of the seropositive blood donors, 97.03 % resided in the northeast area of the State of Mexico, Mexico City, and southern part of the State of Hidalgo. CONCLUSIONS: Active transmission of Chagas disease may be occurring in non-endemic regions in the northeast of the State of Mexico.

Increase of 5-HT levels is induced both in mouse brain and HEK-293 cells following their exposure to a non-viral tryptophan hydroxylase construct.

Tryptophan hydroxylase type 2 (Tph2) is the rate-limiting enzyme for serotonin (5-HT) biosynthesis in the brain. Dysfunctional Tph2 alters 5-HT biosynthesis, leading to a deficiency of 5-HT, which could have repercussions on human behavior. In the last decade, several studies have associated polymorphisms of the TPH2 gene with suicidal behavior. Additionally, a 5-HT deficiency has been implicated in various psychiatric pathologies, including alcoholism, impulsive behavior, anxiety, and depression. Therefore, the TPH2 gene could be an ideal target for analyzing the effects of a 5-HT deficiency on brain function. The aim of this study was to use the construct pIRES-hrGFP-1a-Tph2-FLAG to treat CD1-male mice and to transfect HEK-293-cells and then to evaluate whether this treatment increases 5-HT production. 5-HT levels were enhanced 48 h post-transfection, in HEK-293 cells. Three days after the ocular administration of pIRES-hrGFP-1a-Tph2-FLAG to mice, putative 5-HT production was significantly higher than in the control in both hypothalamus and amygdala, but not in the brainstem. Further research will be needed on the possible application of this treatment for psychiatric diseases involving a Tph2 dysfunction or serotonin deficiency.

Prevalence of oncogenic human papillomavirus in patients with cervical lesion. / Prevalencia del virus del papiloma humano oncogénico en pacientes con lesión cervical.

BACKGROUND: High risk human papillomavirus (hrHPV) infection is associated with the development of cervical cancer (CC) in 99.7%. The prevalence of HPV varies according to the geographic region, lesion degree, method of detection, among other variables. OBJECTIVE: To determine the prevalence of hrHPV and identify some risk factors in a group of women with cervical lesions from Mexico City. MATERIAL AND METHODS: Of 421 women, 310 were included. Questionnaires of risk factors were administered, and cervical samples which included the entire spectrum of cervical lesions according to the Bethesda system were obtained. HPV genotyping was made with INNO-LiPA system. Population characteristics were analyzed with descriptive statistics. Risk factors' odds ratio (OR) was calculated with chi squared using SPSS software, version 24.0. RESULTS: 91.6% of the samples were positive for hrHPV. The prevalent types were 16, 66, 52 and 51. By age group there were not statistically significant differences in the risk of HPV infection. Having had three or more sexual partners increased the risk of infection by hrHPV (OR: 2.99; 95% confidence interval [95% CI]: 1.247.24). Sexually transmitted diseases increased the probability of infection by hrHPV different to types 16, and 18 (OR: 2.47; 95% CI, 1.24-7.24 and 1.50-4.06). CONCLUSIONS: The high prevalence of types 66, 52 and 51 is a finding that has not been described previously in our population. We hope that this study will help to improve health services in order to decrease the incidence of cervical ­cancer.

INTRODUCCIÓN: La infección por el virus del papiloma humano (VPH) de alto riesgo oncogénico (VPHar) se asocia al cáncer cervicouterino en el 99.7% de los casos. La prevalencia de VPH varía según la región geográfica, el grado de lesión y el método de detección, entre otras variables. OBJETIVO: Determinar la prevalencia de VPHar e identificar factores de riesgo en mujeres con lesión cervical de la Ciudad de Mexico. MATERIAL Y MÉTODOS: De 421 mujeres, se incluyeron 310. Se aplicaron cuestionarios y se obtuvieron muestras que incluyeron todo el espectro de las lesiones cervicales según el sistema Bethesda. La tipificación del VPH se hizo mediante el sistema INNO-LiPA. Las características de la población se analizaron con estadística descriptiva. Con la prueba de chi cuadrada se calculó la razón de momios (RM) de los factores de riesgo con el programa SPSS, versión 24.0. RESULTADOS: El 91.6% de las muestras fueron positivas para VPHar. Los VPH prevalentes fueron los tipos 16, 66, 52 y 51. Por edad no hubo significación estadística para riesgo de infección por VPHar. Haber tenido tres o más parejas sexuales elevó el riesgo de infección por HPVar (RM: 2.99; intervalo de confianza del 95 [IC 95%]: 1.247.24). Las infecciones de transmisión sexual favorecieron el riesgo de infección por otros VPHar distintos de los tipos 16 y 18 (RM: 2.47; IC 95%: 1.24-7.24 y 1.50-4.06). CONCLUSIÓN: La elevada prevalencia de VPH 66, 52 y 51 es un hallazgo que no ha sido descrito previamente en nuestra población. Esperamos que este estudio contribuya a mejorar los programas de los servicios de salud dirigidos a disminuir la incidencia de cáncer cervicouterino.

Antitumor effect of an adeno-associated virus expressing apolipoprotein A-1 fused to interferon alpha in an interferon alpha-resistant murine tumor model.

Interferon alpha (IFNα) is a cytokine approved for the treatment of several types of cancer. However, the modest effect on overall survival and the high toxicity associated with the treatment has reduced the clinical use of this cytokine. In this study, we have developed a tumor model that reproduces this clinical setting. A high dose of an adeno-associated virus encoding IFNα (AAV-IFNα) was able to eradicate a liver metastases model of colon cancer but induced lethal pancytopenia. On the other hand, a safe dose of AAV-IFNα was not able to eliminate the liver metastases of colon cancer. In this IFNα-resistant tumor model, administration of an adeno-associated vector encoding apolipoprotein A-1 fused to IFNα was able to fully eradicate the tumor in 43% of mice without toxicity. This antitumor effect was limited by suboptimal long-term CD8+ T cell activation and the expansion of T regulatory cells. In contrast, IFNα upregulated suppressor molecules such as PD-1 and interleukin 10 on CD8+ T lymphocytes. In conclusion, we show that apolipoprotein A-1 fused to IFNα is a novel antitumor drug that differs from IFNα in the modulation of suppressor mechanisms of the immune response. These differential properties pave the way for rational combinations with other immunomodulatory drugs.

Role of CD8 Regulatory T Cells versus Tc1 and Tc17 Cells in the Development of Human Graft-versus-Host Disease.

CD8+ T cells that secrete proinflammatory cytokines play a central role in exacerbation of inflammation; however, a new subpopulation of CD8 regulatory T cells has recently been characterized. This study analyzes the prominent role of these different subpopulations in the development of graft-versus-host disease (GVHD). Samples from 8 healthy donors mobilized with Filgrastim® (G-CSF) and 18 patients who underwent allogeneic hematopoietic stem cell transplantation (HSCT) were evaluated by flow cytometry. Mobilization induced an increase in Tc1 (p < 0.01), Th1 (p < 0.001), Tc17 (p < 0.05), and CD8+IL-10+ cells (p < 0.05), showing that G-CSF induces both pro- and anti-inflammatory profiles. Donor-patient correlation revealed a trend (p = 0.06) toward the development of GVHD in patients who receive a high percentage of Tc1 cells. Patients with acute GVHD (aGVHD), either active or controlled, and patients without GVHD were evaluated; patients with active aGVHD had a higher percentage of Tc1 (p < 0.01) and Tc17 (p < 0.05) cells, as opposed to patients without GVHD in whom a higher percentage of CD8 Treg cells (p < 0.01) was found. These findings indicate that the increase in Tc1 and Tc17 cells is associated with GVHD development, while regulatory CD8 T cells might have a protective role in this disease. These tests can be used to monitor and control GVHD.

Leukocyte production of IFN-γ and TNF-α in 8- to 12-y-old children with low serum iron levels.

OBJECTIVE: Iron deficiency is likely the most common nutritional deficiency worldwide; low iron concentrations have been related to alterations in immune system functions; therefore, the aim of this study was to determine the effect of low serum iron (LSI) concentrations on the production of proinflammatory cytokines by peripheral blood leukocytes in 8- to 12-y-old children from a local community. METHODS: We obtained 120 blood samples and determined full blood counts and serum iron concentrations. An LSI and a control group, paired by age and sex were established using serum iron <60 µg/dL as the cutoff point. Ferritin and C-reactive protein concentrations were quantified. Serum interferon (IFN)-γ and tumor necrosis factor (TNF)-α concentrations were measured in these groups by enzyme-linked immunosorbent assay. A second blood sample was taken from children in both groups to isolate peripheral blood mononuclear cells (PBMCs) and measure IFN-γ and TNF-α production by unstimulated and lipopolysaccharide/phorbol myristate acetate/ionomycin-stimulated leukocytes in vitro. RESULTS: Of the participants in the present study, 17.5% (21 children) presented LSI, as well as decreased ferritin concentrations. Differential counts from total blood samples showed a significant increase in leukocyte numbers in the LSI group, along with increased neutrophil frequencies and numbers but decreased lymphocyte frequencies. Decreased serum IFN-γ concentrations and decreased in vitro production of IFN-γ by PBMCs were found in the LSI group. CONCLUSIONS: The results of the present study suggest that low iron levels alter leukocyte subpopulations in circulation and have a detrimental effect on leukocyte production of proinflammatory cytokines after an antigenic challenge.

Antigen-specific activation and proliferation of CD4+ and CD8+ T lymphocytes from brucellosis patients.

Salt-extractable antigen from Brucella melitensis 16M (RCM-BM) was used to evaluate the immune response from acute and chronic patients suffering from Brucella infections (in Mexico); their responses were compared with those of healthy controls. As a readout we used upregulation of CD69 (a well-established early activation marker for lymphocytes), lymphocyte proliferation by 3[H]thymidine or 5-bromo-2-deoxyuridine (BrdU) incorporation measured by liquid scintillation or flow cytometry, respectively, and production of gamma interferon (IFN gamma). We compared the antigen-specific response with the response induced by phytohaemagglutinin (PHA) as a positive control. There was no difference between acute patients and the healthy controls in the percentages of CD3+, CD4+ or CD8+ lymphocytes. However, we found that chronic patients had a significant (P < 0.05) increase in the CD8+ T cells, in line with previous studies. Antigen-specific responses to RCM-BM showed a significant (P < 0.05) upregulation of CD69 in both CD4+ and CD8+ T lymphocytes in acute brucellosis patients and in CD8+ T lymphocytes in chronic patients, indicating that both populations became activated by this antigen preparation. Moreover, lymphocyte proliferation from both acute and chronic patients in response to RCM-BM was highly significant (P < 0.001) when compared with healthy controls. However, there were no apparent differences between acute and chronic patients. Although the incorporation of BrdU showed similar results it provided additional information, since we demonstrated that both CD4+ and CD8+ T lymphocytes from acute and chronic patients proliferated equally well in response to RCM-BM. Similar results were observed with intracellular IFN gamma determination. As a whole, our data suggest an important role for both CD4+ and CD8+ T lymphocytes in Brucella infection in humans. As has been reported in mice, it is feasible that activated CD8+ T cells participate in protection against Brucella in humans through cytotoxicity or/and by the production of factors such as interferon and granulysin. The role of these cells should be carefully analysed to understand better their participation in human infection by Brucella.

Activation and proliferation of T lymphocyte subpopulations in patients with brucellosis.

BACKGROUND: T-cell proliferation is a standard method to evaluate cellular immune responses against intracellular infectious agents. The present study was undertaken to look for expression of an early activation marker (CD69) and proliferation using a nonradioactive method to evaluate cellular immune response against a salt-extractable antigen from Brucella melitensis 16M (RCM-BM) in patients suffering from brucellosis. METHODS: Expression of CD69 on membrane of CD4+ and CD8+ T-cells was determined by flow cytometry. Lymphoproliferation was determined by tritiated thymidine and 5-bromo-2'-deoxyuridine (BrdU) incorporation using liquid scintillation counter or flow cytometry, respectively, to evaluate DNA synthesis. RESULTS: Thirty healthy donors and 24 patients suffering from brucellosis were included in this study. In all cases, incubation with mitogen induced expression of CD69 and proliferation of both CD4+ and CD8+ T-cells. In contrast, only brucellosis patients responded with expression of CD69 and proliferation against RCM-BM antigen from Brucella melitensis (p < 0.001). CONCLUSIONS: Methods used in this study were useful to evaluate immune response against specific antigen or polyclonal stimulation. CD4+ and CD8+ T cells from patients became equally activated and proliferated in response to RCM-BM antigen. Our data suggest that both T-cell subpopulations play an important role in immune response against Brucella.

Comparative proteome analysis of Brucella abortus 2308 and its virB type IV secretion system mutant reveals new T4SS-related candidate proteins.

Brucella abortus is an alpha-2 proteobacteria with a type IV secretion system (T4SS) known as virB, which is necessary to gain virulence by building up a replicative vacuole associated with the endoplasmic reticulum of the host cell. A virB T4SS mutant of the B. abortus 2308 strain and its wild-type strain were grown in acid medium in order to obtain and analyze their proteomes, looking for putative proteins that may serve as T4SS substrates and those that may be subjected to T4SS regulation. A total of 47 overexpressed and 22 underexpressed proteins from the virB T4SS mutant strain were selected and sequenced. Some of the 69 analyzed proteins have not been described before either as over or under-expressed in relation to a virB T4SS mutation, whereas some of them have been already described by other groups as potentially important secretory proteins in other Brucella species. An important number of the proteins identified are outer membrane and periplasmic space protein, which makes them become particularly important new T4SS-related candidate proteins.