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Abstract: Brucella is an intracellular facultative bacterium able to survive and multiply in professional and non-professional phagocytes. However, its adhesion and invasion mechanisms have not been elucidated yet. In this work, we assess the interruption of a BMEI0216 gene of Brucella melitensis, by using HeLa epithelial cells and murine macrophages for invasion and replication assays. The mutation did not affect survival or multiplication within macrophages. Likewise, invasion assays with HeLa cells revealed no differences at 30 and 45min, whereas, at 1 and 2h, the infection ability of the mutant was drastically reduced. These results suggest that the BMEI0216 gene is required for B. melitensis internalization. [Copyright &y& Elsevier]

Brucellosis is an infectious disease caused by bacteria of the genus Brucella which affects several domestic and wild life animals, as well as humans. Until the past decade, strain 19 (S19) of B. abortus was used as the vaccine for brucellosis in cattle in Mexico. Brucella abortus S19 carries a deletion in two genes of the operonery, responsible for the erythritol catabolic pathway. Since 1997, B. abortus strain RB51 was officially approved in Mexico for its use as a vaccine for cattle. This strain has an insertion sequence (IS) named IS711, which interrupts the wboA gen. Based on this knowledge, two PCR assays were standardized to allow the identification of strains S19 and RB51, and distinguish them from other Brucella species and biotypes. These assays were used for characterization of 11 field strains of B. abortus isolated from milk of cattle. A 456-bp DNA fragment was amplified from all strains when PCR, that identifies RB51, was used, discarding the presence of vaccine strain RB51 among the field strains. In contrast, when the assay to identify strain S19 was performed, a 1063-bp DNA fragment was amplified from nine strains, while the other two were identified as vaccine strain S19 by amplification of a 361-bp DNA fragment. The finding of vaccine strains among this isolates is important to both epidemiological and government level, due to their direct and indirect implications on the National Campaign for Control and Eradication of Bovine Brucellosis. [ABSTRACT FROM AUTHOR]

La brucelosis es una enfermedad infectocontagiosa de origen bacteriano que afecta tanto al humano como a diferentes especies animales domésticas y silvestres. En la década pasada, la vacuna que se usaba ampliamente en los bovinos era la cepa vacunal B. abortus S19, que tiene una deleción en dos de los genes del operón ery que participa en el catabolismo del eritritol. En México, desde 1997 se aprobó la cepa B. abortus RB51 como vacuna para el ganado. Esta cepa presenta una secuencia de inserción (IS) conocida como elemento IS711, que interrumpe el gen wboA. La presencia de una deleción en la cepa S19, así como del elemento IS711 que interrumpe el gen wboA en la cepa RB51, puede evidenciarse fácilmente mediante reacción en cadena de la polimerasa (PCR). Con base en este hecho se estandarizaron dos ensayos de PCR que permiten identificar y diferenciar a las cepas S19 y RB51, respectivamente, de todas las demás especies y biotipos de Brucella. Estos ensayos se utilizaron para la caracterización de 11 cepas de campo de Brucella abortus procedentes de leche bovina. A partir de las once muestras se amplifi có un producto de 456 pb con la PCR que identifica a la cepa vacunal RB51, descartando con ello la presencia de dicha cepa en las de campo. Con la PCR que identifica a la cepa S19, nueve de las 11 muestras amplificaron un producto de 1063 pb, mientras que dos lo hicieron para un producto de 361 pb, ello significa que estas dos cepas de campo corresponden a cepas vacunales S19 de B. abortus. El hallazgo de cepas vacunales dentro de dichas cepas es de importancia tanto a nivel epidemiológico como oficialmente, debido a las implicaciones directas e indirectas que tiene sobre la Campaña Nacional de Control y Erradicación de la Brucelosis Bovina. [ABSTRACT FROM AUTHOR]

Escherichia coli strains are part of the normal biota of humans and animals; however, several clinical reports have implicated E. coli as the etiological agent of diarrhea in humans and companion animals. Thus, the aim of the present study was to know if companion dogs in the city of San Luis Potosi are colonized with virulent potentially harmful E. coli strains. Rectal swabs from 30 dogs, 13 with and 17 without diarrhea were analyzed. Phylogenetic and virulence genes analysis was performed to the E. coli isolates. Additionally, the Kirby-Bauer test was used to analyze the sensitivity to 32 different antimicrobials from 14 families. Eighty-five isolates were identified as E. coli and detected in 97% of healthy and diarrheic dog samples. E. coli isolates from healthy dogs carried several virulence genes, in contrast with those from diarrheic animals that presented only eaeA. In healthy dogs, phylogenetic analysis showed that 57% and 43% of E. coli isolates belonged to commensal (A and B1) and virulent (B2 and D) groups respectively. Meanwhile, diarrheic dogs showed that 69% of the isolates were identified as virulent B2 and D phylogroups. Moreover, E. coli resistant to β-lactams, aminoglycosides, tetracycline, quinolones, and folate inhibitors were detected in both groups of dogs. The presence of E. coli with eaeA virulence gene in diarrheic dogs, suggest that these strains are associated with the animal´s condition. Finally, major attention must be drawn to the careful handling of dogs because of their capability to harbor and disseminate virulent E. coli strains. [ABSTRACT FROM AUTHOR]

Background: Severe COVID-19 is a disease characterized by profound dysregulation of the innate immune system. There is a need to identify highly reliable prognostic biomarkers that can be rapidly assessed in body fluids for early identification of patients at higher risk for hospitalization and/or death. This study aimed to assess whether differential gene expression of immune response molecules and cellular enzymes, detected in saliva samples of COVID-19 patients, occurs according to disease severity staging. Methods: In this cross-sectional study, subjects with a COVID-19 diagnosis were classified as having mild, moderate, or severe disease based on clinical features. Transcripts of genes encoding 6 biomarkers, IL-1β, IL-6, IL-10, C-reactive protein, IDO1 and ACE2, were measured by RT‒qPCR in saliva samples of patients and COVID-19-free individuals. Results: The gene expression levels of all 6 biomarkers in saliva were significantly increased in severe disease patients compared to mild/moderate disease patients and healthy controls. A significant strong inverse relationship between oxemia and the level of expression of the 6 biomarkers (Spearman's correlation coefficient between -0.692 and -0.757; p < 0.001) was found. Conclusions: Biomarker gene expression determined in saliva samples still needs to be validated as a potentially valuable predictor of severe clinical outcomes early at the onset of COVID-19 symptoms. [ABSTRACT FROM AUTHOR]

Summary: The genus Pestivirus within Flaviviridae is comprised of four recognized species, namely, bovine viral diarrhoea virus 1 (BVDV‐1), bovine viral diarrhoea virus 2 (BVDV‐2), border disease virus (BDV) and classical swine fever virus (CSFV). BDV, while primarily infecting sheep and goats, has also been reported in cattle and wild animals. Infections of sheep and goats result in economic loss due to abortions and the birth of persistently infected animals that have poor production and reduced life expectancy. In this study, we report the detection of BDV in cattle serum collected as part of pestivirus surveillance programme from six regions of Mexico, where a 67.1% of BVDV seroprevalence was calculated previously. Phylogenetic analyses based on comparison of the 5′UTR region typed the Mexican strains as BDV‐1. Border disease (BD) is listed as an exotic disease in Mexico, and the origin of BDV found in these cattle is unclear. This is the first identification of BDV in Mexican cattle. [ABSTRACT FROM AUTHOR]

Ehrlichia canis has gained importance over the years as a zoonotic bacterium, nevertheless in Mexico is unknown the extent of the problem in animals and public health. The country had a few studies carried out locally using serology and molecular tests as diagnostic methods. Ehrlichiosis is not considered endemic in the central valley of Mexico, because the climatic conditions in the region have not allowed the vector (Rhipicephalus sanguineus) to establish itself adequately, therefore, diagnosis is not used in clinical practice in this area. A nested PCR (nPCR) offers rapid results with high sensitivity and specificity regardless of cost. The use of a recombinant positive control provides the advantage of timely diagnosis, follow-up treatment and allows the clinician to decide. In this work, the nPCR reported by Wen et al. (J Clin Microbiol 35(7):1852–2185, 1997) was used for the diagnosis of E. canis by modifying the reaction conditions to improve the detection of the test. We constructed a recombinant positive control to nPCR as diagnostic technique for E. canis, also we modified the reaction conditions to improve detection of the test which allowed the diagnosis of E. canis in dogs in the Mexican Republic using 53 samples from dogs with positive serological diagnosis of Ehrlichiosis, some of them from the valley of Mexico. Currently, this nPCR is offered to public at the Faculty of Veterinary Medicine and Zootechnics of the National Autonomous University of Mexico at an accessible cost and allows to begin to generate epidemiological information to know distribution of the bacterium. [ABSTRACT FROM AUTHOR]

La pertactina (Prn) es una proteína de la membrana externa de Bordetella bronchiseptica con propiedades de adhesina y de autotransportador del sistema de secreción tipo V, que posee dos regiones variables en la estructura primaria de la proteína y que induce inmunidad protectora en cerdos y ratones. En este trabajo, el gen prn fue clonado, secuenciado y expresado en E. coli a partir de un aislamiento de campo, cepa P98 de Bordetella bronchiseptica de origen canino. La detección de Prn fue realizada con el empleo de los anticuerpos contra Bordetella, mediante Inmunoblot. Con el análisis de la secuencia de aminoácidos se identificó el dominio RGD de adherencia a células epiteliales en los aminoácidos 261 al 263 y las dos regiones de repeticiones variables ubicadas entre el aminoácido Gly 264 a la Pro 278 que corresponde a la región I y de la Pro 568 a la Pro 587 para la región II. [ABSTRACT FROM AUTHOR]

Pertactin (Prn) is an outer membrane protein of Bordetella bronchiseptica, it has adhesive properties and the auto-transporter properties of the type V secretion system. This protein has two variable regions in the primary structure, which induces protective immunity in pigs and mice. In this study, the prn gene of B. bronchiseptica (P98 wild type) of canine origin was cloned, sequenced and expressed in E coli. Prn was detected by immunoblot using antibodies against Bordetella. The RGD domain was identified due to the sequence of aminoacids, which is the epithelial cell adherence domain between 261 and 263 aminoacids and two repeat regions between gly 264 and pro 278 that correspond to the region I and pro 568 to pro 587 for the region II. [ABSTRACT FROM AUTHOR]

In this communication, we report the presence of RNA of bovine viral diarrhea virus (BVDV) as a contaminant of different biological products used in Mexico for veterinary vaccine production. For this purpose, six batches of monovalent vaccines, eight cell line batches used for vaccine production, and 10 fetal bovine serum lots (FBS) commercially available in Mexico from different suppliers were tested by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was detected in 62.5% of the samples analyzed. Phylogenetic analysis revealed the presence of the subgenotypes BVDV-1a, 1b, and BVDV-2a in the tested samples. Collectively, these findings indicate that contamination by BVDV RNA occurs in commercial vaccines and reagents used in research and production of biological products. The ramifications of this contamination are discussed. [ABSTRACT FROM AUTHOR]

Brucellosis is an infectious disease that affects practically all species of mammals, including human, and is a major zoonosis worldwide. Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in phagocytic and nonphagocytic cells such as trophoblast and epithelial cells. Among the six recognized species of the genus Brucella, Brucella melitensis is the main etiological agent involved in goat brucellosis and is also the most pathogenic for human. It causes significant losses in livestock production as a result of abortions, metritis, infertility, and birth of weak animals. Outer membrane proteins (OMPs) are exposed on the bacterial surface and are in contact with cells and effectors of the host immune response, whereby they could be important virulence factors of Brucella species. To evaluate this hypothesis, the gene encoding for the major outer membrane protein Omp31 was amplified, cloned into pUC18 plasmid, and inactivated by inserting a kanamycin cassette, rendering pLVM31 plasmid which was transformed into B. melitensis wild-type strain to obtain LVM31 mutant strain. The Outer membrane (OM) properties of the mutant strain were compared with B. melitensis Bm133 wild-type and B. melitensis Rev1 vaccine strains, in assessing its susceptibility to polymyxin B, sodium deoxycholate, and nonimmune serum. The mutant strain was assessed in vitro with survival assays in murine macrophages J774.A1 and HeLa cells. Our results demonstrate that LVM31 mutant is more susceptible to polymyxin B, sodium deoxycholate, and nonimmune serum than control strains; moreover, Omp31 mutation caused a decrease in the internalization and a significant decrease in the intracellular survival compared with the reference strains in both cell lines. These results allow us to conclude that Omp31 is important for maintaining OM integrity, but also it is necessary for bacterial internalization, establishment and development of an optimal replication niche, and essential for survival and intracellular multiplication. [ABSTRACT FROM AUTHOR]

Salmonella gallinarum is the causative agent of fowl typhoid. Being a Gram-negative bacteria, its outer membrane proteins (OMP) can be regulated by different microenvironments. S. gallinarum was cultured under the following conditions: nutrient broth (NB), NB supplemented with serum from specific pathogen-free birds (NBS) and NB with serum incubated at 56 °C prior to incubation with the bacteria (NBSD); OMP were subsequently extracted. Several changes were observed in the apparent expression of OMP, mainly a decrease in an OMP with a size of 30 kDa, approximately, under the NBS condition. In contrast, the same event was not observed in NB and NBSD when using one- and two-dimensional polyacrylamide gels (SDS-PAGE). Using the OMP with a size of 30 kDa, approximately, as antigen in indirect ELISA, we were able to differentiate serum from healthy and vaccinated birds, as well as birds infected with S. gallinarum and S. enteritidis. The amino-terminal of this protein was sequenced, showing 100 % identity with OmpA of S. typhimurium. Subsequently, we designed primers to amplify the gene by PCR. The partial sequence of the amplified gene showed 100 % identity with OmpA of S. gallinarum. (1) Heat-labile serum components influence the presence of OmpA in the OM of S. gallinarum; (2) by the way of ELISA, OmpA allows to specifically differentiate healthy from diseased birds. [ABSTRACT FROM AUTHOR]

A sensory evaluation of the cooked pork meat fed with ractopamine at doses: 0, 5, 10, and 20 parts per million over the course of 28 days without withdrawal time, a panel of 72 judges evaluated the color, odor, and flavor of the meat from the ham area. There were no significant sensorial differences between ractopamine and control meats. Panelist's age showed an association with sensorial ratings (i.e. higher scores by older panelists, P < 0.05) and illustrates the need of using each panelist as his or her own control. Moreover, meat from fed swine with ractopamine for 28 days without withdrawal was generally well accepted by the panel of judges. [ABSTRACT FROM AUTHOR]

It has been proposed that intracellular pathogens may interfere with expression or function of proteins that mediate vesicular traffic in order to survive inside cells. Brucella melitensis is an intracellular pathogen that evades phagosomelysosome fusion, surviving in the so-called Brucella-containing vacuoles (BCV). Vesicle-associated membrane protein 3 (VAMP3) is a v-SNARE protein that promotes the exocytosis of the proinflammatory cytokine TNF at the phagocytic cup when docking to its cognate t-SNARE proteins syntaxin-4 and SNAP-23 at the plasma membrane. We determined the expression level of VAMP3 in J774.1 murine macrophages stimulated with B. melitensis lipopolysaccharide (LPS) and detected a transitory increase of VAMP3 mRNA expression at 30 min. A similar result was obtained when cells were incubated in the presence of LPS from Salmonella enterica serovar Minnesota (SeM). This increase of VAMP3 mRNA was also observed on infected cells with B. melitensis even after one hour. In contrast, infection with Salmonella enterica serovar Enteritidis (SeE) did not cause such increase, suggesting that membrane components other than LPS modulate VAMP3 expression differently. To determine the effect of VAMP3 inhibition on macrophages infection, the expression of VAMP3 in J774.A1 cells was silenced and then infected with wild-type B. melitensis. Although a slight decrease in the rate of recovery of surviving bacteria was observed between 12 h and 36 h post-infection with B. melitensis, this was not significant indicating that VAMP3 is not involved in Brucella survival. [ABSTRACT FROM AUTHOR]

Nasal swab samples from clinically healthy California sea lions pups (Zalophus californianus) from six different reproductive rookeries in the Gulf of California were collected to determine the type and frequency of the representative aerobic bacterial microflora of their nasal mucosa. A total of 114 samples were examined and 100 bacterial isolates were identified and typified by microbiological and biochemical standard tests. Fifty four isolates corresponded to Gram positive bacteria (54%) and 46 isolates to Gram negative bacteria (46%). Fifteen bacterial genera were identified, including Micrococcus, Arcanobacterium, Corynebacterium, Moraxella, Neisseria, Escherichia, Kurthia, Acinetobacter, Staphylococcus, Brevibacillus, Bacillus, Klebsiella, Stenotrophomonas, Pseudomonas and Aeromonas. The most frequently isolated genera were Moraxella (24%), Micrococcus (18%), and Corynebacterium (15%). These results show the presence in the nasal cavity of sea lions of several microorganisms. Although considered part of the normal microflora, they may also be opportunistic pathogens for their hosts and may act as a potential natural sentinel of environmental changes. [ABSTRACT FROM AUTHOR]

• A new canine distemper virus sequence from wild carnivores was found. • Difference in prevalence between two close related procyonids was detected. • Difference in humoral and cellular response between procyonids were detected. Canine Distemper Virus (CDV) can produce a fatal multisystem disease in carnivores and other mammals and is an important threat for wildlife conservation. However, integrative and comparative studies in wild carnivores are scarce and some areas of the world lack of genetic studies. We explore the dynamic of host-CDV in a procyonid community during an outbreak. This study reports for the first time an index case occurred in a common raccoon (Procyon lotor) and for which a complete CDV diagnosis was performed. The long-term epidemiological analysis in two sympatric populations of common raccoons and white-nosed coatis (Nasua narica) was achieved through seroneutralization, RT-PCR and direct immunofluorescence assays. Additionally, hematologic analyses were performed and phylogenetic reconstruction of CDV was done using molecular data from this study. Overall prevalence for white-nosed coatis was 19.6 % and for common raccoons was 25.3 % by seroneutralization, and 13.3 % and 17.3 % by RT-PCR. Antibodies titer average for white-nosed coatis was 1:512 and 1:156 for common raccoons. Significant difference in prevalence between white-nosed coatis and common raccoons was detected during one season (summer 2013). White-nosed coatis showed differences in erythrocytes and monocytes counts between positives and negative animals. A 100 % similarity was found between CDV of white-nosed coati and CDV of common raccoon and is a new CDV sequence not previously described; this sequence is close to Asian and European lineage. An endemic state of distemper in both species was observed but showed different dynamics over time per host species. Differences in cellular and humoral responses were also detected between procyonids. The evidence found here may have serious implications for CDV understanding in wild carnivores, it reveals clear differences in the response over time to the same CDV strain, in two close related carnivore species. [ABSTRACT FROM AUTHOR]