Antimicrobial susceptibility profiles of porcine mycoplasmas isolated from samples collected in southern Europe.

BACKGROUND: Mycoplasma (M.) hyopneumoniae, M. hyorhinis and M. hyosynoviae are significant pathogens for the porcine industry worldwide. The aim of the present study was to determine the antimicrobial susceptibility of six key antimicrobials (tylosin, tilmicosin, tylvalosin, lincomycin, tiamulin and valnemulin) routinely used for treating infections caused by these pathogens. Twenty-seven M. hyopneumoniae, 48 M. hyorhinis and 40 M. hyosynoviae field strains isolated from clinical samples from different Southern European countries between 2013 and 2018 using broth microdilution method were evaluated. RESULTS: Tylvalosin exhibited the highest in vitro activity among the macrolides assayed, with MIC90 values 4 to 5 two-fold dilutions lower than those of tylosin and tilmicosin. The pleuromutilin valnemulin showed one of the highest in vitro activities against the three mycoplasma species. On the contrary, lincomycin exhibited the highest MIC values of the antimicrobials tested. CONCLUSIONS: The data obtained in the present study supports the use of pleuromutilins and macrolides for the control of infections caused by porcine mycoplasmas. The use of lincomycin for the treatment of porcine mycoplasma infections should be carefully evaluated due to the presence of circulating field isolates with decreased susceptibility to this antimicrobial.

Interaction of glucocorticoids with FXR/FGF19/FGF21-mediated ileum-liver crosstalk.

At high doses, glucocorticoids (GC) have been associated with enhanced serum bile acids and liver injury. We have evaluated the effect of GC, in the absence of hepatotoxicity, on FXR/FGF91(Fgf15)/FGF21-mediated ileum-liver crosstalk. Rats and mice (wild type and Fxr-/-, Fgf15-/- and int-Gr-/- strains; the latter with GC receptor (Gr) knockout selective for intestinal epithelial cells), were treated (i.p.) with dexamethasone, prednisolone or budesonide. In both species, high doses of GC caused hepatotoxicity. At a non-hepatotoxic dose, GC induced ileal Fgf15 down-regulation and liver Fgf21 up-regulation, without affecting Fxr expression. Fgf21 mRNA levels correlated with those of several genes involved in glucose and bile acid metabolism. Surprisingly, liver Cyp7a1 was not up-regulated. The expression of factors involved in transcriptional modulation by Fxr and Gr (p300, Drip205, CBP and Smrt) was not affected. Pxr target genes Cyp3a11 and Mrp2 were not up-regulated in liver or intestine. In contrast, the expression of some Pparα target genes in liver (Fgf21, Cyp4a14 and Vanin-1) and intestine (Vanin-1 and Cyp3a11) was altered. In mice with experimental colitis, liver Fgf21 was up-regulated (4.4-fold). HepG2 cells transfection with FGF21 inhibited CYP7A1 promoter (prCYP7A1-Luc2). This was mimicked by pure human FGF21 protein or culture in medium previously conditioned by cells over-expressing FGF21. This response was not abolished by deletion of a putative response element for phosphorylated FGF21 effectors present in prCYP7A1. In conclusion, GC interfere with FXR/FGF19-mediated intestinal control of CYP7A1 expression by the liver and stimulate hepatic secretion of FGF21, which inhibits CYP7A1 promoter through an autocrine mechanism.

Comparative analysis of cytokine transcript profiles within mediastinal lymph node compartments of pigs after infection with porcine reproductive and respiratory syndrome genotype 1 strains differing in pathogenicity.

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a weak immune response enabling it to persist in different organs of infected pigs. This has been attributed to the ability of PRRSV to influence the induction of cytokine responses. In this study, we investigated the cytokine transcriptional profiles in different compartments of the mediastinal lymph node of pigs infected with three genotype 1 PRRSV strains of differing pathogenicity: the low virulence prototype Lelystad virus (LV), and UK field strain 215-06 and the highly virulent subtype 3 SU1-Bel isolate from Belarus. We have used a combination of laser capture micro-dissection (LCM) followed by real time quantitative PCR (RT-qPCR) and immunohistochemical (IHC) detection of immune cell markers (CD3, CD79a and MAC387) and RT-qPCR quantification of PRRSV and cytokine transcripts. Compared to mock infected pigs, we found a significant downregulation of TNF-α and IFN-α in follicular and interfollicular areas of the mediastinal lymph node from 3 days post-infection (dpi) in animals infected with all three strains. This was accompanied by a transient B cell depletion and T cell and macrophage infiltration in the follicles together with T cell depletion in the interfollicular areas. A delayed upregulation of IFN-γ and IL-23p19 was observed mainly in the follicles. The PRRSV load was higher in all areas and time-points studied in the animals infected with the SU1-Bel strain. This paper describes the first application of LCM to study the cytokine transcript profiles and virus distribution in different compartments of the lymph node of pigs.

Flow cytometry follow-up analysis of peripheral blood leukocyte subpopulations in calves experimentally infected with field isolates of Mycoplasma bovis.

Changes in peripheral blood leukocyte subpopulations were investigated in calves challenged intratracheally with three different Mycoplasma bovis isolates in Groups E1, E2, and E3. The controls received a placebo. Blood samples were collected before challenge and then at days 1 to 7, 14, 21 and 28. White blood cells (WBC), polymorphonuclear leukocytes (PMNLs), lymphocytes (LYMs), monocytes, eosinophils and basophils (mid-size cells, MID), as well as CD2(+), CD4(+), CD8(+), WC4(+) lymphocyte subsets with CD4:CD8 ratio were also analysed. A transient increase of WBC and PMNLs in all challenged calves was observed on day 1. Increased LYM counts were observed in E1 throughout the study, whereas in E2 the LYM counts were higher only between days 14 and 28, and consistently lower in E3. The MID count had broadly comparable values for all groups. Stimulation of the CD2(+) response was observed in E2 and E3 in contrast with E1 which had a lower CD2(+) throughout. The CD4(+) response was dominant in E1 and E2, whereas in E3 a parallel CD4(+) and CD8(+) stimulation was observed. The B-cell response (WC4(+)) and an increased CD4:CD8 ratio was most apparent in E1. The main host responses to M. bovis infections are a stimulation of CD4(+) cells and an enhancement of the WC4(+) response.

FATAL CASE OF STREPTOCOCCUS SUIS INFECTION IN A YOUNG WILD BOAR (SUS SCROFA) FROM SOUTHWESTERN SPAIN.

Streptococcus suis is a recognized pathogen that may cause important diseases in pigs and humans. This microorganism has been repeatedly isolated from wild boar (Sus scrofa). However, its health implications for this wild species are still unknown. This article reports a detailed description of a fatal case of septicemia by S. suis affecting a young wild boar. The affected animal, about 15 days old, was found near death and exhibiting neurologic signs at a wild boar estate in southwestern Spain. Postmortem examination showed generalized congestion, brain hemorrhages and lobular pneumonia. Histopathological evaluation demonstrated the presence of meningitis and encephalitis with marked congestion and suppurative bronchopneumonia. Streptococcus suis serotype 2 isolates exhibiting important virulence factors (extracellular factor, muramidase-released protein, and suylisin) were isolated from the affected animal. This study confirms the presence of potentially virulent and zoonotic strains of S. suis in wild boar from Spain.

Clinicopathologic findings and causes of mortality in 100 pet rabbits from the Canary Islands, Spain, 2011-2022.

The European domestic rabbit (Oryctolagus cuniculus domesticus) is commonly kept as a pet, with increasing popularity among pet owners. Despite the increasing body of information on lagomorph medicine and pathology, comprehensive published compilations of causes of mortality in pet rabbits are limited. We analyzed health disorders, pathology findings, and most probable causes of death in 100 pet rabbits submitted to the Anatomopathological Diagnostic Service of the Veterinary School of the University of Las Palmas de Gran Canaria, Canary Islands, Spain, from 2011 to 2022. We reviewed clinical data and gross and histopathologic findings to classify the various disorders into infectious (n = 39), noninfectious (n = 45), and idiopathic conditions (n = 16). Within infectious disease processes, Encephalitozoon cuniculi infection (n = 14) and rabbit hemorrhagic viral disease (n = 14) had the highest prevalence. Regarding the noninfectious conditions, perioperative mortality (n = 10), gastrointestinal syndrome (n = 7), and trauma (n = 6) were the most common clinicopathologic entities observed.

Differential Gene Expression in Porcine Lung Compartments after Experimental Infection with Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (Mhyo) is the causative agent of porcine enzootic pneumonia (EP), as well as one of the main pathogens involved in the porcine respiratory disease complex. The host-pathogen interaction between Mhyo and infected pigs is complex and not completely understood; however, improving the understanding of these intricacies is essential for the development of effective control strategies of EP. In order to improve our knowledge about this interaction, laser-capture microdissection was used to collect bronchi, bronchi-associated lymphoid tissue, and lung parenchyma from animals infected with different strains of Mhyo, and mRNA expression levels of different molecules involved in Mhyo infection (ICAM1, IL-8, IL-10, IL-23, IFN-α, IFN-γ, TGF-ß, and TNF-α) were analyzed by qPCR. In addition, the quantification of Mhyo load in the different lung compartments and the scoring of macroscopic and microscopic lung lesions were also performed. Strain-associated differences in virulence were observed, as well as the presence of significant differences in expression levels of cytokines among lung compartments. IL-8 and IL-10 presented the highest upregulation, with limited differences between strains and lung compartments. IFN-α was strongly downregulated in BALT, implying a relevant role for this cytokine in the immunomodulation associated with Mhyo infections. IL-23 was also upregulated in all lung compartments, suggesting the potential involvement of a Th17-mediated immune response in Mhyo infections. Our findings highlight the relevance of Th1 and Th2 immune response in cases of EP, shedding light on the gene expression levels of key cytokines in the lung of pigs at a microscopic level.

Microbiological Survey and Evaluation of Antimicrobial Susceptibility Patterns of Microorganisms Obtained from Suspect Cases of Canine Otitis Externa in Gran Canaria, Spain.

A retrospective study of microbiological laboratory results from 2020 to 2022, obtained from a veterinary diagnostic laboratory of the island of Gran Canaria, Spain, focused on canine otitis cases, was performed. The objective of this study was to analyze the pathogen distribution, antimicrobial susceptibility, prevalence of multidrug resistant phenotypes and the role of coinfections in otitis cases in order to provide up-to-date evidence that could support effective control strategies for this prevalent pathology. A total of 604 submissions were processed for the diagnosis of canine external otitis. Of the samples analyzed, 472 were positive for bacterial or fungal growth (78.1%; 95% CI: 74.8-81.4%). A total of 558 microbiological diagnoses were obtained, divided in 421 bacterial (75.4%; 95% CI: 71.8-79.0%) and 137 fungal (24.6%; 95% CI: 20.9-28.1%) identifications. Staphylococcus pseudintermedius, Malassezia pachydermatis and Pseudomonas aeruginosa were the most prevalent microorganisms detected in clinical cases of otitis. High level antimicrobial resistance was found for Pseudomonas aeruginosa (30.7%), Proteus mirabilis (29.4%), Staphylococcus pseudintermedius (25.1%) and Escherichia coli (19%). Multidrug-resistant phenotypes were observed in 47% of the bacteria isolated. In addition, a 26.4% prevalence of methicillin-resistant Staphylococcus pseudintermedius was detected. The high prevalence of antimicrobial resistant phenotypes in these bacteria highlights the current necessity for constant up-to-date prevalence and antimicrobial susceptibility data that can support evidence-based strategies to effectively tackle this animal and public health concern.

Vaccination Upregulates Th1 Cytokines in the Lung of Pigs Experimentally Infected with Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (Mhy) is the causative agent of enzootic pneumonia, characterized by high morbidity and low mortality rates in intensive swine production systems. To better understand the mechanisms underlying the protection of an inactivated whole cell vaccine, we investigated the immunohistochemical differences in the cytokine expression in vaccinated and non-vaccinated pigs experimentally infected with Mhy. Four-week-old Mhy-negative pigs (n = 24) were allocated to negative control (n = 8), or one of two Mhy-infected groups: vaccinated (n = 8) and non-vaccinated (n = 8). Infection was carried out by a combination of trans-tracheal and aerosol route. Lung samples were processed for histopathological and immunohistochemical studies, by using antibodies against Mhy, IL1-α, IL1-ß, IL-2, IL-4, IL-5, IL-6, Il-8, IL-10, IL-12p35, IL-13, IL-17A, TNF-α, IFN-γ, and CD-4 lymphocytes. Although all cytokines increased in both infected groups, IL-2, IL-12, and IFN-γ were significantly overexpressed in vaccinated pigs. These findings, in conjunction with the decrease of macroscopic and histological lesions in vaccinated animals, indicate the importance to enhance Th1 response in the immunization strategies to control Mhy infection.

Nurturing a Respectful Connection: Exploring the Relationship between University Educators and Students in a Spanish Veterinary Faculty.

The respect of the teacher for the student is essential for effective teaching from the perspective of the students, even in comparison to the knowledge and communication capacity of the teacher. Consequently, the optimal development of this characteristic fosters a more effective and efficient student-teacher relationship. We initiated this research following a conversation with a group of university students, who expressed their discontent regarding the lack of respect shown towards them by some teachers. Therefore, we conducted a descriptive study using online surveys, focusing on the central axis in the teacher-student relationship. The results highlighted the need for faculty members to analyze and question their attitudes towards their students. This paper presents initial results of the data collected at the Veterinary Faculty of the University of Las Palmas de Gran Canaria.

Mycoplasma bradburyae sp. nov. isolated from the trachea of sea birds.

In the search for mollicutes in wild birds, six Mycoplasma strains were isolated from tracheal swabs taken from four different species of seabirds. Four strains originated from three Yellow-legged gulls (Larus michahellis) and a Cory's shearwater (Calonectris borealis) from Spain, one from a South African Kelp gull (Larus dominicanus), and one from an Italian Black-headed gull (Chroicocephalus ridibundus). These Mycoplasma strains presented 99 % 16S rRNA gene sequence similarity values with Mycoplasma (M.) gallisepticum. Phylogenetic analyses of marker genes (16S rRNA gene and rpoB) confirmed the close relationship of the strains to M. gallisepticum and M. tullyi. The seabirds' strains grew well in modified Hayflick medium, and colonies showed typical fried egg morphology. They produced acid from glucose and mannose but did not hydrolyze arginine or urea. Transmission electron microscopy revealed a cell morphology characteristic of mycoplasmas, presenting spherical to flask-shaped cells with an attachment organelle. Gliding motility was also observed. Furthermore, serological tests, MALDI-ToF mass spectrometry and genomic studies demonstrated that the strains were different to any known Mycoplasma species, for which the name Mycoplasma bradburyae sp. nov. is proposed; the type strain is T158T (DSM 110708 = NCTC 14398).

Machine Learning Approach for Analyzing 3-Year Outcomes of Patients with Brain Arteriovenous Malformation (AVM) after Stereotactic Radiosurgery (SRS).

A cerebral arteriovenous malformation (AVM) is a tangle of abnormal blood vessels that irregularly connects arteries and veins. Stereotactic radiosurgery (SRS) has been shown to be an effective treatment for AVM patients, but the factors associated with AVM obliteration remains a matter of debate. In this study, we aimed to develop a model that can predict whether patients with AVM will be cured 36 months after intervention by means of SRS and identify the most important predictors that explain the probability of being cured. A machine learning (ML) approach was applied using decision tree (DT) and logistic regression (LR) techniques on historical data (sociodemographic, clinical, treatment, angioarchitecture, and radiosurgery procedure) of 202 patients with AVM who underwent SRS at the Instituto de Radiocirugía del Perú (IRP) between 2005 and 2018. The LR model obtained the best results for predicting AVM cure with an accuracy of 0.92, sensitivity of 0.93, specificity of 0.89, and an area under the curve (AUC) of 0.98, which shows that ML models are suitable for predicting the prognosis of medical conditions such as AVM and can be a support tool for medical decision-making. In addition, several factors were identified that could explain whether patients with AVM would be cured at 36 months with the highest likelihood: the location of the AVM, the occupation of the patient, and the presence of hemorrhage.

Genome sequence of Mycoplasma hyorhinis strain GDL-1.

Mycoplasma hyorhinis impacts swine health and production in many countries, either as a primary pathogen or as a component of a polymicrobial infection. Isolates of this species are also common contaminants of tissue culture lines. The genome sequence of the cell culture isolate M. hyorhinis GDL-1 is presented herein.

Characterization of the role of ABCG2 as a bile acid transporter in liver and placenta.

ABCG2 is involved in epithelial transport/barrier functions. Here, we have investigated its ability to transport bile acids in liver and placenta. Cholylglycylamido fluorescein (CGamF) was exported by WIF-B9/R cells, which do not express the bile salt export pump (BSEP). Sensitivity to typical inhibitors suggested that CGamF export was mainly mediated by ABCG2. In Chinese hamster ovary (CHO cells), coexpression of rat Oatp1a1 and human ABCG2 enhanced the uptake and efflux, respectively, of CGamF, cholic acid (CA), glycoCA (GCA), tauroCA, and taurolithocholic acid-3-sulfate. The ability of ABCG2 to export these bile acids was confirmed by microinjecting them together with inulin in Xenopus laevis oocytes expressing this pump. ABCG2-mediated bile acid transport was inhibited by estradiol 17ß-d-glucuronide and fumitremorgin C. Placental barrier for bile acids accounted for <2-fold increase in fetal cholanemia despite >14-fold increased maternal cholanemia induced by obstructive cholestasis in pregnant rats. In rat placenta, the expression of Abcg2, which was much higher than that of Bsep, was not affected by short-term cholestasis. In pregnant rats, fumitremorgin C did not affect uptake/secretion of GCA by the liver but inhibited its fetal-maternal transfer. Compared with wild-type mice, obstructive cholestasis in pregnant Abcg2(-/-) knockout mice induced similar bile acid accumulation in maternal serum but higher accumulation in placenta, fetal serum, and liver. In conclusion, ABCG2 is able to transport bile acids. The importance of this function depends on the relative expression in the same epithelium of other bile acid exporters. Thus, ABCG2 may play a key role in bile acid transport in placenta, as BSEP does in liver.

Resistance to 16-Membered Macrolides, Tiamulin and Lincomycin in a Swine Isolate of Acholeplasma laidlawii.

Acholeplasma (A.) laidlawii is an opportunistic pathogen with the ability to disseminate resistance determinants to antibiotics; however, its resistance to macrolides has been less studied. The aim of the present study was to characterize the mechanisms responsible for the resistance to macrolides, tiamulin and lincomycin found in a strain of A. laidlawii isolated from a pig with pneumonia. MICs of erythromycin, 15- and 16-membered macrolides, tiamulin and lincomycin were determined by microdilution method with and without reserpine, an inhibitor of ABC efflux pumps and regions of the genome were sequenced. Reserpine only decreased lincomycin MIC but it did not change the MICs of macrolides and tiamulin. The analysis of the DNA sequence of 23S rRNA showed nucleotide substitutions at eight different positions, although none of them were at positions previously related to macrolide resistance. Five mutations were found in the L22 protein, one of them at the stop codon. In addition, two mutations were found in the amino acid sequence of L4. The combination of multiple mutations in the ribosomal proteins L22 and L4 together with substitutions in 23S rRNA DNA sequence was associated with the resistance to macrolides, the pleuromutilin and lincomycin in the studied A. laidlawii strain.

Comparison of Methods for the Histological Evaluation of Odontocete Spiral Ganglion Cells.

Cetaceans greatly depend on their hearing system to perform many vital activities. The spiral ganglion is an essential component of the auditory pathway and can even be associated with injuries caused by anthropogenic noise. However, its anatomical location, characterized by surrounding bony structures, makes the anatomical and anatomopathological study of the spiral ganglion a difficult task. In order to obtain high-quality tissue samples, a perfect balance between decalcification and the preservation of neural components must be achieved. In this study, different methodologies for spiral ganglion sample preparation and preservation were evaluated. Hydrochloric acid had the shortest decalcification time but damaged the tissue extensively. Both formic acid and EDTA decalcification solutions had a longer decalcification time but exhibited better preservation of the neurons. However, improved cell morphology and staining were observed on ears pretreated with EDTA solution. Therefore, we suggest that decalcifying methodologies based on EDTA solutions should be used to obtain the highest quality samples for studying cell morphology and antigenicity in cetacean spiral ganglion neurons.

Microorganisms Resistant to Antimicrobials in Wild Canarian Egyptian Vultures (Neophron percnopterus majorensis).

Due to their predatory habits, raptors may serve as indicators of the presence of antimicrobial-resistant bacteria in the environment, but they also represent a public health risk for livestock and humans because they can act as reservoirs, sources and spreaders of these bacteria. Our objective was to determine the presence of antimicrobial-resistant bacteria in cloacal samples of Canarian Egyptian vultures (Neophron percnopterus majorensis), an endemic bird of prey. One hundred and forty-two cloacal swabs were obtained; Escherichia coli was isolated from 80.28% and Salmonella from 6.3% of these samples. Low levels of susceptibility to ampicillin, tetracycline and trimethoprim/sulfamethoxazole were found. About 20% of the isolates were resistant or presented intermediate susceptibility to fluoroquinolones. Surprisingly, we found isolates resistant to imipenem (6.96%). Isolates from chicks were more susceptible to antimicrobial drugs than adult and immature birds. About 50% of E. coli isolates were resistant to ampicillin, tetracycline and trimethoprim/sulfamethoxazole, and about 20% to piperacillin, enrofloxacin and marbofloxacin. High percentages of isolates of Salmonella were found to be resistant to cephalexin (88%) and aminoglycosides (greater than 77%). Our results support the idea that raptors could act as reservoirs of Salmonella and antimicrobial-resistant bacteria, posing a risk not only to wildlife but also to livestock and the human population, thus reinforcing the need to minimize the exposure of wildlife to antimicrobial agent through human and livestock waste.

First description of two moderately halophilic and psychrotolerant Mycoplasma species isolated from cephalopods and proposal of Mycoplasma marinum sp. nov. and Mycoplasma todarodis sp. nov.

Two moderately halophilic and psychrotolerant new Mycoplasma species were isolated from common cephalopods. Three strains were isolated in pure culture from two individual European flying squid (Todarodes sagittatus), and two individual octopuses (Octopus vulgaris). The strains showed optimal growth at 25 °C and a salinity of 3% (w/v) NaCl. Molecular analyses revealed that the isolates belonged to two new, but phylogenetically related species, divergent from all previously described Mollicutes, representing the first marine isolates of the class, and also the first Mycoplasma strains for which NaCl requirement has been demonstrated. A genome search against all available marine metagenomes and 16S rRNA gene databases indicated that these two species represent a novel non-free-living marine lineage of Mollicutes, specifically associated with marine animals. Morphology and physiology were compatible with other members of this group, and genomic and phenotypic analyses demonstrated that these organisms represent two novel species of the genus Mycoplasma, for which the names Mycoplasma marinum sp. nov. and Mycoplasma todarodis sp. nov. are proposed; the type strains are PET (DSM 105487T, CIP 111404T) and 5HT (DSM 105,488T, CIP 111405T), respectively.

A semi-defined medium without serum for small ruminant mycoplasmas.

The composition of the medium used to cultivate Mycoplasma species is very important. Serum is one of the most important additives as it contains lipids (cholesterol) and serum proteins, which are essential for the growth of the organisms. This work reports the development of a semi-defined medium, called MWS (Medium Without Serum) produced without animal serum and bovine serum albumin. MWS seems to be suitable for cultivating several species of caprine mycoplasma, especially M. mycoides subsp. mycoides (LC) and M. mycoides subsp. capri.

Applications of flow cytometry to mycoplasmology.

Flow cytometry has become a valuable tool in different fields of microbiology, such as clinical microbiology, aquatic and environmental microbiology, food microbiology, and biotechnology. It combines direct and rapid assays to determine numbers, biochemical and physiological characteristics of individual cells, revealing the heterogeneity present in a population. This review focuses on the applications of flow cytometry to the field of mycoplasmology. It tries to give a scope of the important breakthroughs which occurred in this field in the last decades, and in the advantages of introducing flow cytometry in research and routine diagnostic procedures of mycoplasmas.