Clinical and pathological factors associated with Ehrlichia canis in companion dogs.

INTRODUCTION: Canine monocytic ehrlichiosis (CME) is a disease caused by the Gram-negative bacteria Ehrlichia canis, a bacterium that affects domestic dogs but can also infect humans. The diagnosis implies a challenge due to its diversity in clinical manifestations. METHODOLOGY: The frequency of E. canis infection, risk factors, and clinical-pathological parameters associated with seropositivity were calculated with the PROC FREQ TABLES and PROC LOGISTIC procedures of the SAS statistical software. RESULTS: The study showed a seroprevalence of 26.62% (156/586). Association between seropositivity and risk factors was found. The age and the presence of ticks including clinical signs such as anorexia, seizures, cough, petechiae, epistaxis, and hematochezia, as well as multiple blood and biochemical alterations were analyzed. The logistic regression analysis showed a high predictive power (c = 0.98) for CME for thrombocytopenia, leukopenia, and anemia. CONCLUSIONS: The high prevalence of E. canis in endemic areas makes its diagnosis difficult. Thus, clinical signs must be considered, along with blood and biochemical alterations, as a possible predictor of the disease.

Antibiotic resistance in tick-borne bacteria: A One Health approach perspective.

The emergence and re-emergence of tick-borne bacteria (TBB) as a public health problem raises the uncertainty of antibiotic resistance in these pathogens, which could be dispersed to other pathogens. The impact of global warming has led to the emergence of pathogenic TBB in areas where they were not previously present and is another risk that must be taken into account under the One Health guides. This review aimed to analyze the existing information regarding antibiotic-resistant TBB and antibiotic-resistance genes (ARG) present in the tick microbiome, considering the potential to be transmitted to pathogenic microorganisms. Several Ehrlichia species have been reported to exhibit natural resistance to fluoroquinolones and typhus group Rickettsiae are naturally susceptible to erythromycin. TBB have a lower risk of acquiring ARG due to their natural habitat, but there is still a probability of acquiring them; furthermore, studies of these pathogens are limited. Pathogenic and commensal bacteria coexist within the tick microbiome along with ARGs for antibiotic deactivation, cellular protection, and efflux pumps; these ARGs confer resistance to antibiotics such as aminoglycosides, beta-lactamase, diaminopyrimidines, fluoroquinolones, glycopeptides, sulfonamides, and tetracyclines. Although with low probability, TBB can be a reservoir of ARGs.

The Effect of Acute Physical Exercise on Natural Killer Cells Populations and Cytokine Levels in Healthy Women.

Physical exercise generates a systemic response in the immune system. It has been observed that cell populations respond to exercise stimuli, especially Natural Killer cells, whose number increase within minutes of starting physical exertion. This study aimed to evaluate the acute effect of moderate- and high-intensity exercise on immunological markers in healthy women. As specific objectives, the percentages of CD3-CD56+ Natural Killer total cells, CD56brightCD16dim effector subpopulation, CD56dimCD16bright cytotoxic subpopulation, NKG2A inhibition receptor, NKG2D activation receptor, and NKT cells were analyzed. In addition, the levels of the cytokines IL-1ß, IL-6, IL-8, IL-10, IL-12p70, and TNF and the chemokines CCL5/RANTES, CXCL9/MIG, CCL2/MCP-1, and CXCL10/IP-10 were also analyzed. Natural Killer total cells showed an increase in their percentage in both exercise protocols (p = 0.001 for the moderate-intensity group and p = 0.023 for the high-intensity group); however, only in the high-intensity exercise session was there an increase in the CD56dimCD16bright cytotoxic subpopulation (p = 0.014), as well as a decrease in CD56brightCD16dim effector subpopulation (p = 0.001) and their NKG2A inhibition receptor (p = 0.043). An increase in IL-6 was observed after the high-intensity exercise session (p = 0.025). Conclusions. Physical exercise influences immunological markers and shows an acute response to moderate- or high-intensity exercise.

Identification and Venom Characterization of Two Scorpions from the State of Chihuahua Mexico: Chihuahuanus coahuliae and Chihuahuanus crassimannus.

Chihuahua is the largest state in Mexico. The ecosystem of this region is composed of large area of bushes, forests, and grasslands, which allows for a specific diversity of fauna; among them are interesting species of non-lethal scorpions. Most of the Chihuahuan scorpions have been previously morphologically and molecularly described; however, this manuscript could be the first to describe the composition of those venoms. This work aimed at the collection of two scorpion species from the region of Jiménez (Southwest of the State of Chihuahua), which belong to the species Chihuahuanus cohauilae and Chihuahuanus crassimanus; the two species were taxonomically and molecularly identified using a 16S DNA marker. Reverse-phase high-performance liquid chromatography (RP-HPLC) of C. coahuilae and C. crassimanus venoms allowed the identification of three fractions lethal to mice. Additionally, three fractions of each scorpion displayed an effect on house crickets. In the end, three new fractions from the venom of C. coahuilae were positive for antimicrobial activity, although none from C. crassimanus venom displayed growth inhibition. Despite being a preliminary study, the venom biochemical analysis of these two uncharacterized scorpion species opens the opportunity to find new molecules with potential applications in the biomedical and biotechnological fields.

Levofloxacin induces differential effects in the transcriptome between the gut, peripheral and axial joints in the Spondyloarthritis DBA/1 mice: Improvement of intestinal dysbiosis and the overall inflammatory process.

To analyze the effect of levofloxacin-induced intestinal microbiota modifications on intestinal, joint, and systemic inflammation in the DBA/1 mice with spontaneous arthritis. The study included two groups of mice, one of which received levofloxacin. The composition and structure of the microbiota were determined in the mice's stool using 16S rRNA sequencing; the differential taxa and metabolic pathway between mice treated with levofloxacin and control mice were also defied. The effect of levofloxacin was evaluated in the intestines, hind paws, and spines of mice through DNA microarray transcriptome and histopathological analyses; systemic inflammation was measured by flow cytometry. Levofloxacin decreased the pro-inflammatory bacteria, including Prevotellaceae, Odoribacter, and Blautia, and increased the anti-inflammatory Muribaculaceae in mice's stool. Histological analysis confirmed the intestinal inflammation in control mice, while in levofloxacin-treated mice, inflammation was reduced; in the hind paws and spines, levofloxacin also decreased the inflammation. Microarray showed the downregulation of genes and signaling pathways relevant in spondyloarthritis, including several cytokines and chemokines. Levofloxacin-treated mice showed differential transcriptomic profiles between peripheral and axial joints and intestines. Levofloxacin decreased the expression of TNF-α, IL-23a, and JAK3 in the three tissues, but IL-17 behaved differently in the intestine and the joints. Serum TNF-α was also reduced in levofloxacin-treated mice. Our results suggest that the microbiota modification aimed at reducing pro-inflammatory and increasing anti-inflammatory bacteria could potentially be a coadjuvant in treating inflammatory arthropathies.

Immune Monitoring of Paediatric Patients Infected with Rickettsia rickettsii, Ehrlichia canis and Coinfected.

In 2021, 273 Rocky Mountain spotted fever cases were reported nationwide in Mexico. In Chihuahua City, fourteen samples were obtained from children suspected of rickettsial infection. The analysis of samples (January to December 2021) showed prevalence rates of 28.5%, 43%, and 28.5% for Rickettsia rickettsii, Ehrlichia canis, and both pathogens in coinfection, respectively. The analysis of clinical haematological and biochemistry analytes showed alterations; 100% of the children had elevated liver enzymes and coagulation times, 64% showed leukocytosis due to neutrophilia, 55% had thrombocytopenia, lymphopenia, and hypoalbuminemia, and 45% showed normocytic normochromic anaemia. Statistically significant differences were observed in the expression of the chemokines IL-8, RANTES, CXCL9/MIG, and CXCL10/IP-10 across the coinfected and control groups, and the difference in IP-10 expression was significant for patients infected by R. rickettsii compared to the control group. Additionally, significant differences were observed for expression levels of IL-1ß, IL-6, IL-17, IFNγ, and TNFα among the R. rickettsii-positive group compared to the control group. On the other hand, the coinfected group exhibited modified levels of IL-6, IL-8, and IL-10 compared with the control group. Finally, significant differences were observed for CD8+ T lymphocyte subpopulations between individuals positive for R. rickettsii and those positive for E. canis.

Treatment with silica-gold nanostructures decreases inflammation-related gene expression in collagen-induced arthritis.

Gold salts have been used to treat rheumatoid arthritis (RA) since the 1940s, and, with advances in nanotechnology, the use of nanogold provides multiple options for anti-inflammatory therapies. This paper presents the synthesis and characterization of silica-gold nanostructures (SGNs) and their therapeutic effect in collagen-induced arthritis (CIA) in DBA/1 mice. At the end of the treatment, the synovial membranes, kidneys, livers, and spleens were dissected and analyzed by inductively coupled plasma mass spectroscopy (ICP) showing less than 0.0001 and 0.1% of the administered doses of Au and Si, respectively. Remains of the SGNs were visually identified in the synovial membrane by scanning electron microscopy (SEM), and the bone density of the hind paws was observed by computerized tomography (CT) indicating a reduction of porosity in the CIA-experimental group. The DNA microarray analysis carried out with RNA obtained from the hind paws showed 2628 differentially expressed genes (DEGs) by SGNs. The bioinformatic analysis showed that DEGs were significantly associated with several inflammatory signalling pathways including chemokines, cytokine-cytokine receptor interaction, PI3K-Akt, TNF, IL-17, NFκß, MAPK, and RA. SGNs downregulated relevant inflammatory genes in the arthritic joints, including Tnf, Ifng, Il6, and Cxcl5; immunohistochemistry (IHC) confirmed the reduction of TNFα, IL-6, NFκß, and VEGF in the joints due to the effect of SGNs. TNFα and IL-6 were also reduced in the serum of DBA/1 mice treated with SGNs.

Antimicrobial and Immunomodulatory Effects of Selected Chemokine and Antimicrobial Peptide on Cytokine Profile during Salmonella Typhimurium Infection in Mouse.

The antimicrobial and immunomodulatory capacities of the peptide Css54 and the chemokine MCP-1 were tested. The first, a peptide isolated from the venom of the scorpion Centruroides suffusus suffusus was synthesized chemically. In contrast, the second is a monocyte chemoattractant expressed as a recombinant protein in our lab. It was observed in vitro that Css54 inhibited the growth of Salmonella enterica serovar Typhimurium (6.2 µg/mL). At high concentrations, it was toxic to macrophages (25 µg/mL), activated macrophage phagocytosis (1.5 µg/mL), and bound Salmonella LPS (3 µg/mL). On the other hand, the recombinant MCP-1 neither inhibited the growth of Salmonella Typhimurium nor was it toxic to macrophages (up to 25 µg/mL), nor activated macrophage phagocytosis or bound Salmonella LPS (up to 3 µg/mL). Although it was observed in vivo in mice Balb/C that both Css54 and MCP-1 did not resolve the intraperitoneal infection by S. Typhimurium, Css54 decreased the expression of IL-6 and increased IL-10, IL-12p70, and TNF-α levels; meanwhile, MCP-1 decreased the expression of IFN-γ and increased IL-12p70 and TNF-α. It was also observed that the combination of both molecules Css54 and MCP-1 increased the expression of IL-10 and TNF-α.

Immunomodulatory Responses of Two Synthetic Peptides against Salmonella Typhimurium Infection.

In vitro assays of phagocytic activity showed that the peptide Pin2[G] stimulates phagocytosis in BMDM cells from 0.15 to 1.25 µg/mL, and in RAW 264.7 cells at 0.31 µg/mL. In the same way, the peptide FA1 induced phagocytosis in BMDM cells from 1.17 to 4.69 µg/mL and in RAW 264.7 cells at 150 µg/mL. Cytokine profiles of uninfected RAW 264.7 showed that Pin2[G] increased liberation TNF (from 1.25 to 10 µg/mL) and MCP-1 (10 µg/mL), and FA1 also increased the release of TNF (from 18.75 to 75 µg/mL) but did not increase the liberation of MCP-1. In RAW 264.7 macrophages infected with Salmonella enterica serovar Typhimurium, the expression of TNF increases with Pin2[G] (1.25-10 µg/mL) or FA1 (18.75-75 µg/mL). In these cells, FA1 also increases the expression of IL-12p70, IL-10 and IFN-γ when applied at concentrations of 37.5, 75 and 150 µg/mL, respectively. On the other hand, stimulation with 1.25 and 10 µg/mL of Pin2[G] promotes the expression of MCP-1 and IL-12p70, respectively. Finally, peptides treatment did not resolve murine gastric infection, but improves their physical condition. Cytokine profiles showed that FA1 reduces IFN-γ and MCP-1 but increases IL-10, while Pin2[G] reduces IFN-γ but increases the liberation of IL-6 and IL-12p70. This data suggests a promising activity of FA1 and Pin2[G] as immunomodulators of gastric infections in S. Typhimurium.

Ashwin Gene Expression Profiles in Oocytes, Preimplantation Embryos, and Fetal and Adult Bovine Tissues.

The ashwin gene, originally identified in Xenopus laevis, was found to be expressed first in the neural plate and later in the embryonic brain, eyes, and spinal cord. Functional studies of ashwin suggest that it participates in cell survival and anteroposterior patterning. Furthermore, ashwin is expressed zygotically in this species, which suggests that it participates in embryonic development. Nevertheless, the expression of this gene has not been studied in mammals. Thus, the aim of this study was to analyze the ashwin expression pattern in bovine fetal and adult tissues, as well as in three independent samples of immature and mature oocytes, and in two- to four-, and eight-cell embryos, morula, and blastocysts. Spatiotemporal expression was analyzed using real-time polymerase chain reaction (PCR); ashwin mRNA was detected in all tissues analyzed, immature and mature oocytes, and two- to eight-cell embryos. It was down-regulated in morula and blastocysts, suggesting that this expression profile is similar to that of maternal genes. Immunohistochemical localization of the ashwin protein in fetal and adult ovaries and testes reveals that this protein is consistently present during all stages of follicular development and during bovine spermatogenesis. These observations lead us to propose ashwin as an important gene involved in mammalian reproduction.

Exercise Exacerbates the Transcriptional Profile of Hypoxia, Oxidative Stress and Inflammation in Rats with Adjuvant-Induced Arthritis.

Physical exercise (PE) is recommended for Rheumatoid Arthritis (RA), but the molecular and biological mechanisms that impact the inflammatory process and joint destruction in RA remain unknown. The objective of this study was to evaluate the effect of PE on the histological and transcriptional changes in the joints of adjuvant-induced arthritis (AIA) rat model. AIA rats were subjected to PE on a treadmill for eight weeks. The joints were subjected to histological and microarray analysis. The differentially expressed genes (DEGs) by PE in the arthritic rats were obtained from the microarray. The bioinformatic analysis allowed the association of these genes in biological processes and signaling pathways. PE induced the differential expression of 719 genes. The DEGs were significantly associated with pathogenic mechanisms in RA, including HIF-1, VEGF, PI3-Akt, and Jak-STAT signaling pathways, as well as response to oxidative stress and inflammatory response. At a histological level, PE exacerbated joint inflammatory infiltrate and tissue destruction. The PE exacerbated the stressed joint environment aggravating the inflammatory process, the hypoxia, and the oxidative stress, conditions described as detrimental in the RA joints. Research on the effect of PE on the pathogenesis process of RA is still necessary for animal models and human.

Selected scorpion toxin exposures induce cytokine release in human peripheral blood mononuclear cells.

A cytokine screening on human peripheral blood mononuclear cells (PBMCs) stimulated with selected scorpion toxins (ScTx's) was performed in order to evaluate their effect on human immune cells. The ScTx's chosen for this report were three typical buthid scorpion venom peptides, one with lethal effects on mammals Centruroides suffussus suffusus toxin II (CssII), another, with lethal effects on insects and crustaceans Centruroides noxius toxin 5 (Cn5), and one more without lethal effects Tityus discrepans toxin (Discrepin). A Luminex multiplex analysis was performed in order to determine the amounts chemokines and cytokines IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12-p40, IL-13, interferon alpha (IFN-α), interferon gamma (IFN-γ), tumor necrosis factor alpha TNF-α, and interferon-inducible protein-10 (IP-10) secreted from human PBMCs exposed to these toxins. Although, the ScTx Cn5 is not lethal for mammals, it was able to induce the secretion of cytokines IL-1ß, IL-6, and TNF-α, IL-10 and IP-10 in comparison to the lethal CssII, which was able to induce only IP-10 secretion. Discrepin also was able to induce only IP-10. Interestingly, only low amounts of interferons α and ß were induced in the presence of the ScTx's assayed. In a synergic experiment, the combination of Discrepin and Cn5 displayed considerable reverse effects on induction of IL-1ß, IL-6, IL-10 and TNF-α, but they had a slight synergic effect on IP-10 cytokine production in comparison with the single effect obtained with the Cn5 alone. Thus, the results obtained suggest that the profile of secreted cytokines promoted by ScTx Cn5 is highly related with a cytokine storm event, and also it suggests that the mammalian lethal neurotoxins are not solely responsible of the scorpion envenomation symptomatology.

Targeting antigens to Dec-205 on dendritic cells induces a higher immune response in chickens: Hemagglutinin of avian influenza virus example.

It is widely known that targeting a variety of antigens to the DEC-205 receptor on dendritic cells (DCs) significantly potentiate immunity. This communication reports the development of a new murine monoclonal antibody (mAb) against the chicken DEC-205, using as immunogen the carbohydrate recognition domain-2 (CRD-2) heterologously expressed. This mAb recognizes a protein band of 250kDa by immunoprecipitation analysis and shows strong cross-reactivity with human and pig DEC-205. Furthermore, the hemagglutinin (HA) of avian influenza H5N2 virus was cloned and expressed using insect cell-baculovirus expression system. We chemically conjugated the anti-chicken DEC-205 antibody with the highly purified HA to direct the antigen to the dendritic cells and evaluate the immune response elicited in vivo by this conjugate. A single dose of chemical conjugate was sufficient to elicit a strong immune response in chickens as early as fourteen days after priming. In addition, the conjugate induced an earlier and higher response compared to unconjugated HA. These results suggest that the strategy described here has potential to be used in the future design and development of successful vaccines against different chicken infectious diseases with direct impact in biotechnology and veterinary fields.

Novel monoclonal antibody against alphaX subunit from horse CD11c/CD18 integrin.

The αX I-domain of the horse integrin CD11c was successfully expressed in Escherichia coli, purified, biochemically characterized and used as immunogen to generate murine monoclonal antibodies against horse CD11c, which are not yet commercially available. One monoclonal antibody mAb-1C4 against the αX I-domain, is an IgG2a able to interact with the recombinant I-domain, showing an EC50=2.4ng according to ELISA assays. By western blot with horse PBMCs lysates the mAb-1C4 recognized a protein of 150kDa which corresponds well with the CD11c molecule. Using immunohistochemistry in horse lymph node tissue sections, mAb-1C4 marked cells in situ, some with apparent dendritic morphology. Thus the mAb generated to a recombinant epitope from horse CD11c identified the molecule in intact cells within horse lymphoid tissue. By the labelling intensity, the histological location (paracortical and interfollicular areas) and the apparent morphology of the marked cells, we can say that these are putative horse dendritic cells (DCs). The development of a mAb to horse CD11c provides a new tool to better study the horse DC biology and opens other biotechnological avenues, such as DC targeting-based vaccines.

Lectins: a brief review / Lectinas: Una revisión breve

The ability of plant agglutinins to distinguish between erythrocytes of different blood types led Boyd and Shapleigh (1954) to propose for them the name lectins, from the Latin "legere", to pick out or choose [1]. This term was later generalized to embrace all sugar-specific agglutinins of non-immune origin, irrespective of source and blood type specificity [2]. It was toward the end of the 19th century that evidence first started to accumulate for the presence in nature of proteins possessing the ability to agglutinate erythrocytes. Such proteins were referred to as hemagglutinins, or phytoagglutinins, because they were originally found in extracts of plants…

Antimicrobial peptides from arachnid venoms and their microbicidal activity in the presence of commercial antibiotics.

Two antimicrobial peptides (AMPs), named La47 and Css54, were isolated from the venom of the spider Lachesana sp. and from the scorpion Centruroides suffusus suffusus, respectively. The primary structures of both La47 and Css54 were determined using N-terminal sequencing and mass spectrometry. La47 is identical to the AMP latarcin 3a obtained previously from the venom of the spider Lachesana tarabaevi, but the primary structure of Css54 is unique having 60% identities to the AMP ponericin-W2 from the venom of the ant Pachycondyla goeldii. Both La47 and Css54 have typical α-helix secondary structures in hydrophobic mimicking environments. The biological activities of both La47 and Css54 were compared with the AMP Pin2 isolated from the venom of the scorpion Pandinus imperator. La47 has lower antimicrobial and hemolytic activities compared with Css54 and Pin2. In addition, La47 and Pin2 were evaluated in the presence of the commercial antibiotics, chloramphenicol, ampicillin, novobiocin, streptomycin and kanamycin. Interestingly, the best antimicrobial combinations were obtained with mixtures of La47 and Pin2 with the antibiotics chloramphenicol, streptomycin and kanamycin, respectively. Furthermore, the novel peptide Css54 was evaluated in the presence of antibiotics used for the treatment of tuberculosis, isoniazid, rifampicin, pyrazinamide and ethambutol. Although the mixtures of Css54 with isoniazid, pyrazinamide or ethambutol inhibit the growth of Staphylococcus aureus, the best effect was found with rifampicin. Overall, these data show a motivating outlook for potential clinical treatments of bacterial infections using AMPs and commercial antibiotics.

Isolation and molecular cloning of beta-neurotoxins from the venom of the scorpion Centruroides suffusus suffusus.

This communication reports the identification and characterization of two new toxins from the venom of the scorpion Centruroides suffusus suffusus, named: CssVIII and CssIX, according to the original nomenclature of toxins previously described for this scorpion. The isolation was obtained by means of two chromatographic steps, and a cDNA library was used to fully identify their precursors. CssVIII and CssIX contain signal peptides of 19 and 17 amino acid residues, and mature peptides of 66 and 65 residues, respectively. Intracranial injections into mice of both purified toxins showed toxicity results similar to those found for toxins CssII and CssIV. Additionally, they compete with the parent toxin CssIV, in binding and displacement experiments, conducted with brain synaptosomes showing nanomolar affinities. These results strongly support the conclusion that they are new ß-neurotoxins and certainly would be of the interest of researchers in the field of venomics for studying sodium channels.

Neuroprotective effect of DAHK peptide in an occlusive model of permanent focal ischemia in rats.

This study examined the neuroprotective ability of tetrapeptide L-Asp-Ala-His-Lys (DAHK) in permanent middle cerebral artery occlusion in rats. One DAHK dose (16 mg/kg) or saline solution were i.v. administered 30 min after occlusion and neurological deficit was evaluated at 2, 24, 48, 72 and 96 h using Longa scoring scale. The striatum infarction area was evaluated until 96 h after occlusion in both groups after staining with hematoxylin-eosin. DAHK-treated group showed a significant (P < 0.05) protection of 70% of neurological deficit at 96 h after occlusion, in comparison with the control-group that showed permanent neurological deficit. The DAHK-treated group showed a significant (P < 0.05) reduction of 52% infarction area in the striatum, as compared to control values. Results presented here support the possible therapeutic application of DAHK as a neuroprotective agent in human patients with stroke, as the peptide is part of human serum albumin, already being tested in clinical trials.