Mannheimia haemolytica OmpP2-like is an amyloid-like protein, forms filaments, takes part in cell adhesion and is part of biofilms.

Mannheimia haemolytica causes respiratory disease in cattle. Amyloid proteins are a major component of biofilms; they aid in adhesion and confer resistance against several environmental insults. The amyloid protein curli is highly resistant to protease digestion and physical and chemical denaturation and binds Congo red (CR) dye. The purpose of this study was to characterize an approximately 50-kDa CR-binding amyloid-like protein (ALP) expressed by M. haemolytica. This protein resisted boiling and formic acid digestion and was recognized by a polyclonal anti-Escherichia coli curli serum, suggesting its relationship with amyloid proteins. Immunolabeling and transmission electron microscopy showed that antibodies bound long, thin fibers attached to the bacterial surface. Mass spectrometry analysis indicated that these fibers are M. haemolytica OmpP2-like proteins. The purified protein formed filaments in vitro, and antiserum against it reacted positively with biofilms. An in silico analysis of its amino acid sequence indicated it has auto-aggregation properties and eight amyloid peptides. Rabbit polyclonal antibodies generated against this ALP diminished the adhesion of ATCC 31612 and BA1 M. haemolytica strains to A549 human epithelial cells, indicating its participation in cell adhesion. ALP expressed by M. haemolytica may be important in its pathogenicity and ability to form biofilms.

Pet serine protease from enteroaggregative Escherichia coli stimulates the inflammatory response activating human macrophages.

BACKGROUND: Pet is a toxin from the family of Serine Protease Autotransporters of Enterobacteriaceae which was initially identified in Enteroaggregative Escherichia coli strains. This protease exhibits enterotoxin properties, damages the cell cytoskeleton and induces intestinal epithelium alterations, which are associated with a severe inflammatory process. An in-vitro study was conducted to evaluate the effect of Pet on the migration of human peripheral blood monocytes-derived macrophages and its participation in the activation of the early inflammatory response and cytokine expression. RESULTS: In the macrophage migration activation assay, Pet produced a similar effect to that induced by opsonized zymosan (ZAS). Regarding the cytokine expression, an increase of IL-8, TNF-α (pro-inflammatory) and IL-10 (anti-inflammatory) was identified. In addition to the above results, the nuclear translocation of NF-kB pp65 was also identified. These events are probably related to the inflammatory response identified in the histological examination of intestine rat samples inoculated with Pet during a ligated loop assay. CONCLUSION: The results showed that Pet participates as an immunostimulant molecule for macrophages, which activates both their mobility and cytokine expression. These observations suggest that the toxin participates in the inflammatory process that is observed during the host infection by EAEC Pet producing.

Characterisation of quinolone-resistant Escherichia coli of 1997 and 2005 isolates from poultry in Mexico.

Forty-two enrofloxacin-resistant Escherichia coli strains isolated from eggs and first-week mortality associated with yolk sac infection of two vertically integrated poultry companies of Central Mexico in 1997 and 2005 were characterised. E. coli resistance to 19 antibiotics was determined, as well as the minimum inhibitory concentrations (broth dilution) for ciprofloxacin. The presence of gyrA,B, parC,E chromosomal point mutations, qnrA,B,S plasmid genes and the aminoglycoside acetyltransferase aac(6')-Ib-cr were determined by PCR and sequencing. Resistance to ampicillin (95%), piperacillin (95%), gatifloxacin (95%), levofloxacin (95%), ampicillin/sulbactam (90%), cefazolin (85%), trimethoprim/sulfamethoxazole (80%), amoxicillin/clavulanic acid (80%), aztreonam (80%), cefepime (80%), cefotaxime (80%), ceftazidime (80%), ceftriaxone (80%) and cefoxitin (75%) was high in the 2005 strains and 19 (95%) strains were resistant to 7 or more antimicrobials. The strains from 1997 expressed high rates of resistance only to the fluoroquinolones and 4 strains (18%) expressed resistance to 7 or more antimicrobials. All strains had a gyrA mutation (Ser83Leu) and a parC mutation (Ser80Ile or Ser80Arg) and 41 (97.6%) strains had a second gyrA mutation (Asp87Asn, Asp87Tyr or Asp87Gly). Only two (4.7%) strains had a parE mutation (Ser458Ala). A total of 10 strains were positive for the aac(6')-Ib wild-type gene, 6 strains for the aac(6')-Ib-cr variant and 6 strains possessed both the wild type and the variant. No gyrB mutations or qnrA,B,S genes were detected. This is the first report in Latin America of chromosomal and plasmid quinolone resistance genes in E. coli strains recovered from poultry.

Genetic diversity of Chlamydia pecorum detected in sheep flocks from Mexico.

Chlamydia pecorum, an obligate intracellular bacterium, is associated with reproductive and systemic diseases in sheep, goats, pigs, cattle, and koalas. The main conditions include polyarthritis, conjunctivitis, enteritis, pneumonia, encephalomyelitis, orchitis, placentitis, and abortion. Even though there are several studies showing that C. pecorum infections are widely spread in the world, in Mexico there are no reports. During 2016, as part of a sheep restocking program in Mexico, sheep were imported from New Zealand. Briefly after their arrival in the herds in the State of Mexico, these sheep presented abortions during the last third of gestation. A total of 62 sheep vaginal swabs that had presented abortion from different municipalities of the State of Mexico were collected. Bacterial isolation was performed using L929 mouse fibroblasts, and molecular identification was achieved by 23S rRNA (Chlamydiaceae family) and ompA gene (species-specific) real-time polymerase chain reaction (PCR). In addition, the 16S rRNA subunit and ompA gene were amplified and sequenced. Seven of 62 samples were positive for C. pecorum by bacterial isolation, 23S rRNA, and ompA gene real-time PCR. The 16S rRNA subunit and ompA gene amplicons were purified and the nucleotide sequence was determined in both directions. The consensus sequences homology search was performed using BLASTn analysis and showed a 100% of homology with the C. pecorum 16S rRNA subunit and 99% with the C. pecorum ompA gene. The population structure analyses using ompA gene demonstrated 15 genetic populations or clusters of 198 sequences from GenBank and our sequences were in a particular genetic structure corresponding to genotype "O." Herein, we describe the presence of C. pecorum in sheep imported from New Zealand into Mexico. Genetic analysis of the ompA gene showed that the isolates belong to genotype O and are related to strains isolated from sheep, cattle, and koalas.

Characterization of aortic and brachiocephalic filariasis by Filarioidea sp (Nematoda:Spirurida:Filarioidea) in Mexican ramphastids.

The manuscript presented herein documents the findings of filaria nematodes in 5 keel-billed toucans, and one emerald toucanet, originated from 2 private aviaries in Mexico City during two years. The birds displayed ruffled feathers, depression, inability to perch, convulsions, and sudden death. Furthermore, thickened wall of the aortic and brachiocephalic arteries, with connective tissue proliferation and chondroid metaplasia were observed. Molecular characterization matched Filarioidea sp (Nematoda: Spirurida: Filarioidea). To the authors' knowledge, this is the first documented report of filariae Filarioidea sp. causing mortality in ramphastids in Mexico. This manuscript may contribute to expand current knowledge of filariasis and the health risks and livability of wild birds.

An impressive case of chromoblastomycosis due to Fonsecaea pedrosoi in a patient with a long history of fungal infection.

Chromoblastomycosis is defined as a chronic cutaneous and subcutaneous fungal infection caused by melanized or brown-pigmented fungi. A 63-year-old man farmer showed on external and internal part of the right arm, a well-delimited verrucous and hyperkeratotic plaque, with atrophic and cicatricial areas. Direct examination of skin scrapings samples showed the presence of muriform cells, a classic feature of chromoblastomycosis. Fungal isolation was performed in Sabouraud dextrose agar, and dark olivaceous colonies were isolated. Skin biopsy samples were obtained for histopathological and molecular diagnosis. DNA extracted from both, paraffin-embedded skin biopsy samples and fungal colonies, was used for molecular identification by 18S-ITS1-5.8S-ITS2-28S rRNA amplification and sequencing. Fonsecaea pedrosoi was identified from paraffin-embedded skin samples and fungal colonies. A combined therapy with terbinafine and itraconazole, plus cryotherapy was applied with an important improvement. Herein, we report an impressive case of chromoblastomycosis due to Fonsecaea pedrosoi with a successful outcome.

Treponema pallidum ssp. pallidum identification by real-time PCR targetting the polA gene in paraffin-embedded samples positive by immunohistochemistry.

Syphilis is a systemic and sexually transmitted infection caused by Treponema pallidum ssp. pallidum. This spirochete causes different clinical and subclinical stages depending on the duration of infection and immune status of the host. Several tests have been developed for diagnosis, and are classified into direct and indirect methods. The first one includes dark field microscopy, direct fluorescent antibody test in fluids or tissue, and molecular biology techniques. In the indirect method (serologic), the routine tests are used, and are divided in two categories: non-treponemal and treponemal ones. The objective of this work was to identify T. pallidum ssp. pallidum in paraffin-embedded skin biopsies positive by immunohistochemistry, using conventional polymerase chain reaction (PCR) and quantitative real time PCR (qPCR). We included a sample of 17 paraffin-embedded biopsies. DNA was extracted and processed by conventional PCR and real-time PCR with a TaqMan® probe to identify the polA gene. Using PCR, 11 tested positive (64.7%) and 6 (35.3%) were negative. With qPCR and TaqMan® probe, 100% of samples tested positive. The minimum number of spirochetes detected in each sample was 2. With this work, we can conclude that qPCR is a fast and very accurate method for diagnosis of syphilis in tissue specimens.

In search of new structural states of exchangeable apolipoproteins.

Based upon state of the art biophysical experimentation, this article focuses on the different structural arrangements exchangeable apolipoproteins achieve when placed on Langmuir monolayers and subjected to changes in lateral pressure. We have studied the monolayers of apolipoproteins CI, CIII, AI, AII, and E that show as secondary structure a high percentage of amphipathic alpha-helix. This has been achieved employing techniques such as Brewster angle microscopy, synchrotron X-ray diffraction, and surface pressure measurements. In addition, the lateral order of protein arrays has been also studied by atomic force microscopy. These monolayers show that a phase transition from a two-dimensional disorder fluid to an ordered state is detected at relatively high lateral pressure, where unusual one-dimensional solid phases are discovered. While several helices that conform the apolipoprotein are confined to the interface, others are uniformly tilted toward the hydrophobic air or the phospholipid fatty acid chains. Our results suggest that a similar ordering might also occur when these apolipoproteins are attached to a lipoprotein particle such as a high density lipoprotein (HDL) particle. Therefore, changes from a nascent or discoidal HDL to a mature spherical HDL might in parallel involve structural changes as those described in our Langmuir interfaces. Current experimentation is being carried out in order to elucidate if the structural states already found are related to the efficiency of lipid transfer between lipoprotein particles or lipoproteins and the plasma membrane of cells, as well as receptor ligand recognition.

Human antibody response to longus type IV pilus and study of its prevalence among enterotoxigenic Escherichia coli in Bangladesh by using monoclonal antibodies.

Mouse monoclonal antibodies (MAbs) were derived against longus (CS20), a type IV pilus expressed by human enterotoxigenic Escherichia coli (ETEC). One MAb (ICA39) detected longus in 56 (8.5%) of 662 ETEC isolates obtained from a routine surveillance of diarrheal stools from children and adults. Five patients with diarrhea from whom longus-positive ETEC were isolated were also recruited. Of these 61 isolates, 50 were positive for other colonization factors (CFs; 61% for CFA/II and 21% for CFA/I), and 11 were negative for any of the other 8 CFs that were tested. They were either positive for the heat-stable enterotoxin (ST; n=29) or for the heat-labile enterotoxin (LT) and ST (n=32). All longus-positive ETEC were confirmed by polymerase chain reaction to harbor lngA, the longus structural pilin gene. Sera and/or fecal extracts from the patients reacted with the 22-kDa pilin polypeptide in immunoblots and ELISA. These studies show that longus is prevalent among ETEC in Bangladesh and that longus gives rise to IgA antibody responses in patients.