In Vitro Antibacterial Activity of Microbial Natural Products against Bacterial Pathogens of Veterinary and Zoonotic Relevance.

Antimicrobial resistance (AMR) is considered one of the greatest threats to both human and animal health. Efforts to address AMR include implementing antimicrobial stewardship programs and introducing alternative treatment options. Nevertheless, effective treatment of infectious diseases caused by bacteria will still require the identification and development of new antimicrobial agents. Eight different natural products were tested for antimicrobial activity against seven pathogenic bacterial species (Brachyspira sp., Chlamydia sp., Clostridioides sp., Mannheimia sp., Mycobacterium sp., Mycoplasma sp., Pasteurella sp.). In a first pre-screening, most compounds (five out of eight) inhibited bacterial growth only at high concentrations, but three natural products (celastramycin A [CA], closthioamide [CT], maduranic acid [MA]) displayed activity at concentrations <2 µg/mL against Pasteurella sp. and two of them (CA and CT) also against Mannheimia sp. Those results were confirmed by testing a larger collection of isolates encompassing 64 Pasteurella and 56 Mannheimia field isolates originating from pigs or cattle, which yielded MIC90 values of 0.5, 0.5, and 2 µg/mL against Pasteurella and 0.5, 4, and >16 µg/mL against Mannheimia for CA, CT, and MA, respectively. CA, CT, and MA exhibited higher MIC50 and MIC90 values against Pasteurella isolates with a known AMR phenotype against commonly used therapeutic antimicrobial agents than against isolates with unknown AMR profiles. This study demonstrates the importance of whole-cell antibacterial screening of natural products to identify promising scaffolds with broad- or narrow-spectrum antimicrobial activity against important Gram-negative veterinary pathogens with zoonotic potential.

Genome-wide association reveals host-specific genomic traits in Escherichia coli.

BACKGROUND: Escherichia coli is an opportunistic pathogen which colonizes various host species. However, to what extent genetic lineages of E. coli are adapted or restricted to specific hosts and the genomic determinants of such adaptation or restriction is poorly understood. RESULTS: We randomly sampled E. coli isolates from four countries (Germany, UK, Spain, and Vietnam), obtained from five host species (human, pig, cattle, chicken, and wild boar) over 16 years, from both healthy and diseased hosts, to construct a collection of 1198 whole-genome sequenced E. coli isolates. We identified associations between specific E. coli lineages and the host from which they were isolated. A genome-wide association study (GWAS) identified several E. coli genes that were associated with human, cattle, or chicken hosts, whereas no genes associated with the pig host could be found. In silico characterization of nine contiguous genes (collectively designated as nan-9) associated with the human host indicated that these genes are involved in the metabolism of sialic acids (Sia). In contrast, the previously described sialic acid regulon known as sialoregulon (i.e. nanRATEK-yhcH, nanXY, and nanCMS) was not associated with any host species. In vitro growth experiments with a Δnan-9 E. coli mutant strain, using the sialic acids 5-N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) as sole carbon source, showed impaired growth behaviour compared to the wild-type. CONCLUSIONS: This study provides an extensive analysis of genetic determinants which may contribute to host specificity in E. coli. Our findings should inform risk analysis and epidemiological monitoring of (antimicrobial resistant) E. coli.

Mycobacterium tuberculosis-Induced Prostaglandin J2 and 15-Deoxy-Prostaglandin J2 Inhibit Inflammatory Signals in Human M1 Macrophages via a Negative Feedback Loop.

Tuberculosis caused by Mycobacterium tuberculosis is a leading cause of death globally and a major health concern. In humans, macrophages are the first line invaded by M. tuberculosis. Upon infection, macrophages upregulate cyclooxygenase-2 (COX-2) expression and consequently elevate the formation of PGs, including PGE2 and PGD2. Although the role of proinflammatory PGE2 in M. tuberculosis infection has been reported, the roles of PGJ2 and 15-deoxy-PGJ2 (collectively named J2-PGs), the metabolites of PGD2 with anti-inflammatory features, remain elusive. In this study, we show that M. tuberculosis (H37Rv strain)-conditioned medium stimulates human monocyte-derived macrophages (MDMs) to elevate COX-2 expression along with robust generation of PGJ2, exceeding PGD2 formation, and to a minor extent also of 15-deoxy-PGJ2. Of interest, in M1-MDM phenotypes, PGJ2 and 15-deoxy-PGJ2 decreased M. tuberculosis (H37Rv strain)-conditioned medium-induced COX-2 expression and related PG formation by a negative feedback loop. Moreover, these J2-PGs downregulated the expression of the proinflammatory cytokines IL-6, IL-1ß, and IFN-γ, but elevated the anti-inflammatory cytokine IL-10 and the M2 markers arginase-1 and CD163. These anti-inflammatory effects of J2-PGs in M1-MDM correlated with impaired activation of TGF-ß-activated kinase 1/NF-κB/MAPK pathways. Finally, we found that J2-PGs regulate COX-2 expression, at least partially, via PGD2 receptor (DP1) and chemoattractant receptor homologue expressed on Th2 cells/DP2 receptors, but independent of the J2-PG receptor peroxisome proliferator-activated receptor-γ. Together, our findings reveal that M. tuberculosis induces COX-2 expression in human M1-MDMs, along with robust formation of J2-PGs that mediates anti-inflammatory effects via a negative feedback loop.

Safety and Immunogenicity of Recombinant Bacille Calmette-Guérin Strain VPM1002 and Its Derivatives in a Goat Model.

A more effective vaccine against tuberculosis than Bacille Calmette-Guérin (BCG) is urgently needed. BCG derived recombinant VPM1002 has been found to be more efficacious and safer than the parental strain in mice models. Newer candidates, such as VPM1002 Δpdx1 (PDX) and VPM1002 ΔnuoG (NUOG), were generated to further improve the safety profile or efficacy of the vaccine. Herein, we assessed the safety and immunogenicity of VPM1002 and its derivatives, PDX and NUOG, in juvenile goats. Vaccination did not affect the goats' health in regards to clinical/hematological features. However, all three tested vaccine candidates and BCG induced granulomas at the site of injection, with some of the nodules developing ulcerations approximately one month post-vaccination. Viable vaccine strains were cultured from the injection site wounds in a few NUOG- and PDX- vaccinated animals. At necropsy (127 days post-vaccination), BCG, VPM1002, and NUOG, but not PDX, still persisted at the injection granulomas. All strains, apart from NUOG, induced granuloma formation only in the lymph nodes draining the injection site. In one animal, the administered BCG strain was recovered from the mediastinal lymph nodes. Interferon gamma (IFN-γ) release assay showed that VPM1002 and NUOG induced a strong antigen-specific response comparable to that elicited by BCG, while the response to PDX was delayed. Flow cytometry analysis of IFN-γ production by CD4+, CD8+, and γδ T cells showed that CD4+ T cells of VPM1002- and NUOG-vaccinated goats produced more IFN-γ compared to BCG-vaccinated and mock-treated animals. In summary, the subcutaneous application of VPM1002 and NUOG induced anti-tuberculous immunity, while exhibiting a comparable safety profile to BCG in goats.

Pharmacokinetics and Efficacy of the Benzothiazinone BTZ-043 against Tuberculous Mycobacteria inside Granulomas in the Guinea Pig Model.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is the world's leading cause of mortality from a single bacterial pathogen. With increasing frequency, emergence of drug-resistant mycobacteria leads to failures of standard TB treatment regimens. Therefore, new anti-TB drugs are urgently required. BTZ-043 belongs to a novel class of nitrobenzothiazinones, which inhibit mycobacterial cell wall formation by covalent binding of an essential cysteine in the catalytic pocket of decaprenylphosphoryl-ß-d-ribose oxidase (DprE1). Thus, the compound blocks the formation of decaprenylphosphoryl-ß-d-arabinose, a precursor for the synthesis of arabinans. An excellent in vitro efficacy against M. tuberculosis has been demonstrated. Guinea pigs are an important small-animal model to study anti-TB drugs, as they are naturally susceptible to M. tuberculosis and develop human-like granulomas after infection. In the current study, dose-finding experiments were conducted to establish the appropriate oral dose of BTZ-043 for the guinea pig. Subsequently, it could be shown that the active compound was present at high concentrations in Mycobacterium bovis BCG-induced granulomas. To evaluate its therapeutic effect, guinea pigs were subcutaneously infected with virulent M. tuberculosis and treated with BTZ-043 for 4 weeks. BTZ-043-treated guinea pigs had reduced and less necrotic granulomas than vehicle-treated controls. In comparison to the vehicle controls a highly significant reduction of the bacterial burden was observed after BTZ-043 treatment at the site of infection and in the draining lymph node and spleen. Together, these findings indicate that BTZ-043 holds great promise as a new antimycobacterial drug.

Bovine blood derived macrophages are unable to control Coxiella burnetii replication under hypoxic conditions.

Background: Coxiella burnetii is a zoonotic pathogen, infecting humans, livestock, pets, birds and ticks. Domestic ruminants such as cattle, sheep, and goats are the main reservoir and major cause of human infection. Infected ruminants are usually asymptomatic, while in humans infection can cause significant disease. Human and bovine macrophages differ in their permissiveness for C. burnetii strains from different host species and of various genotypes and their subsequent host cell response, but the underlying mechanism(s) at the cellular level are unknown. Methods: C. burnetii infected primary human and bovine macrophages under normoxic and hypoxic conditions were analyzed for (i) bacterial replication by CFU counts and immunofluorescence; (ii) immune regulators by westernblot and qRT-PCR; cytokines by ELISA; and metabolites by gas chromatography-mass spectrometry (GC-MS). Results: Here, we confirmed that peripheral blood-derived human macrophages prevent C. burnetii replication under oxygen-limiting conditions. In contrast, oxygen content had no influence on C. burnetii replication in bovine peripheral blood-derived macrophages. In hypoxic infected bovine macrophages, STAT3 is activated, even though HIF1α is stabilized, which otherwise prevents STAT3 activation in human macrophages. In addition, the TNFα mRNA level is higher in hypoxic than normoxic human macrophages, which correlates with increased secretion of TNFα and control of C. burnetii replication. In contrast, oxygen limitation does not impact TNFα mRNA levels in C. burnetii-infected bovine macrophages and secretion of TNFα is blocked. As TNFα is also involved in the control of C. burnetii replication in bovine macrophages, this cytokine is important for cell autonomous control and its absence is partially responsible for the ability of C. burnetii to replicate in hypoxic bovine macrophages. Further unveiling the molecular basis of macrophage-mediated control of C. burnetii replication might be the first step towards the development of host directed intervention measures to mitigate the health burden of this zoonotic agent.

Clostridioides difficile in South American Camelids in Germany: First Insights into Molecular and Genetic Characteristics and Antimicrobial Resistance.

Little is known about zoonotic pathogens and their antimicrobial resistance in South American camelids (SAC) in Germany including Clostridioides (C.) difficile. The aim of this study was to investigate prevalence, molecular characteristics and antimicrobial resistance of C. difficile in SAC. Composite SAC faecal samples were collected in 43 husbandries in Central Germany and cultured for C. difficile. Toxinotyping and ribotyping was done by PCR. Whole genome sequencing was performed with Illumina® Miseq™. The genomes were screened for antimicrobial resistance determinants. Genetic relatedness of the isolates was investigated using core genome multi locus sequence typing (cgMLST) and single nucleotide polymorphism analysis. Antimicrobial susceptibility testing was done using the Etest® method. Eight C. difficile isolates were recovered from seven farms. The isolates belonged to different PCR ribotypes. All isolates were toxinogenic. cgMLST revealed a cluster containing isolates recovered from different farms. Seven isolates showed similar resistance gene patterns. Different phenotypic resistance patterns were found. Agreement between phenotypic and genotypic resistance was identified only in some cases. Consequently, SAC may act as a reservoir for C. difficile. Thus, SAC may pose a risk regarding zoonotic transmission of toxinogenic, potentially human-pathogenic and resistant C. difficile isolates.

Portable Differential Detection of CTX-M ESBL Gene Variants, blaCTX-M-1 and blaCTX-M-15, from Escherichia coli Isolates and Animal Fecal Samples Using Loop-Primer Endonuclease Cleavage Loop-Mediated Isothermal Amplification.

Cefotaximase-Munich (CTX-M) extended-spectrum beta-lactamase (ESBL) enzymes produced by Enterobacteriaceae confer resistance to clinically relevant third-generation cephalosporins. CTX-M group 1 variants, CTX-M-1 and CTX-M-15, are the leading ESBL-producing Enterobacteriaceae associated with animal and human infection, respectively, and are an increasing antimicrobial resistance (AMR) global health concern. The blaCTX-M-1 and blaCTX-M-15 genes encoding these variants have an approximate nucleotide sequence similarity of 98.7%, making effective differential diagnostic monitoring difficult. Loop-primer endonuclease cleavage loop-mediated isothermal amplification (LEC-LAMP) enables rapid real-time multiplex pathogen detection with single-base specificity and portable on-site testing. We have developed an internally controlled multiplex CTX-M-1/15 LEC-LAMP assay for the differential detection of blaCTX-M-1 and blaCTX-M-15. Assay analytical specificity was established using a panel of human, animal, and environmental Escherichia coli isolates positive for blaCTX-M-1 (n = 18), blaCTX-M-15 (n = 35), and other closely related blaCTX-Ms (n = 38) from Ireland, Germany, and Portugal, with analytical sensitivity determined using probit regression analysis. Animal fecal sample testing using the CTX-M-1/15 LEC-LAMP assay in combination with a rapid DNA extraction protocol was carried out on porcine fecal samples previously confirmed to be PCR-positive for E. coli blaCTX-M. Portable instrumentation was used to further analyze each fecal sample and demonstrate the on-site testing capabilities of the LEC-LAMP assay with the rapid DNA extraction protocol. The CTX-M-1/15 LEC-LAMP assay demonstrated complete analytical specificity for the differential detection of both variants with sensitive low-level detection of 8.5 and 9.8 copies per reaction for blaCTX-M-1 and blaCTX-M-15, respectively, and E. coli blaCTX-M-1 was identified in all blaCTX-M positive porcine fecal samples tested. IMPORTANCE CTX-M ESBL-producing E. coli is an increasing AMR public health issue with the transmission between animals and humans via zoonotic pathogens now a major area of interest. Accurate and timely identification of ESBL-expressing E. coli CTX-M variants is essential for disease monitoring, targeted antibiotic treatment and infection control. This study details the first report of portable diagnostics technology for the rapid differential detection of CTX-M AMR markers blaCTX-M-1 and blaCTX-M-15, facilitating improved identification and surveillance of these closely related variants. Further application of this portable internally controlled multiplex CTX-M-1/15 LEC-LAMP assay will provide new information on the transmission and prevalence of these CTX-M ESBL alleles. Furthermore, this transferable diagnostic technology can be applied to other new and emerging relevant AMR markers of interest providing more efficient and specific portable pathogen detection for improved epidemiological surveillance.

Phenotype of Coxiella burnetii Strains of Different Sources and Genotypes in Bovine Mammary Gland Epithelial Cells.

Despite the high prevalence of C. burnetii in dairy herds and continuous shedding via milk by chronically infected cows, bovine milk is not recognized as a relevant source of human Q fever. We hypothesized that the bovine mammary gland epithelial cell line PS represents a suitable in vitro model for the identification of C. burnetii-strain-specific virulence properties that may account for this discrepancy. Fifteen C. burnetii strains were selected to represent different host species and multiple loci variable number of tandem repeat analysis (MLVA) genotypes (I, II, III and IV). The replication efficiencies of all strains were similar, even though strains of the MLVA-genotype II replicated significantly better than genotype I strains, and bovine and ovine isolates replicated better than caprine ones. Bovine milk isolates replicated with similar efficiencies to isolates from other bovine organs. One sheep isolate (Cb30/14, MLVA type I, isolated from fetal membranes) induced a remarkable up-regulation of IL-1ß and TNF-α, whereas prototypic strains and bovine milk isolates tended to suppress pro-inflammatory responses. While infection with strain Nine Mile I rendered the cells partially refractory to re-stimulation with E. coli lipopolysaccharide, Cb30/14 exerted a selective suppressive effect which was restricted to IL-6 and TNF-α and spared IL-1ß. PS cells support the replication of different strains of C. burnetii and respond in a strain-specific manner, but isolates from bovine milk did not display a common pattern, which distinguishes them from strains identified as a public health concern.

Vaccine-Induced Subcutaneous Granulomas in Goats Reflect Differences in Host-Mycobacterium Interactions between BCG- and Recombinant BCG-Derivative Vaccines.

Tuberculous granulomas are highly dynamic structures reflecting the complex host-mycobacterium interactions. The objective of this study was to compare granuloma development at the site of vaccination with BCG and its recombinant derivatives in goats. To characterize the host response, epithelioid cells, multinucleated giant cells (MNGC), T cell subsets, B cells, plasma cells, dendritic cells and mycobacterial antigen were labelled by immunohistochemistry, and lipids and acid-fast bacteria (AFB) were labelled by specific staining. Granulomas with central caseous necrosis developed at the injection site of most goats though lesion size and extent of necrosis differed between vaccine strains. CD4+ T and B cells were more scarce and CD8+ cells were more numerous in granulomas induced by recombinant derivatives compared to their parental BCG strain. Further, the numbers of MNGCs and cells with lipid bodies were markedly lower in groups administered with recombinant BCG strains. Microscopic detection of AFB and mycobacterial antigen was rather frequent in the area of central necrosis, however, the isolation of bacteria in culture was rarely successful. In summary, BCG and its recombinant derivatives induced reproducibly subcutaneous caseous granulomas in goats that can be easily monitored and surgically removed for further studies. The granulomas reflected the genetic modifications of the recombinant BCG-derivatives and are therefore suitable models to compare reactions to different mycobacteria or TB vaccines.

Comparative Genomic Analysis of Antimicrobial-Resistant Escherichia coli from South American Camelids in Central Germany.

South American camelids (SAC) are increasingly kept in Europe in close contact with humans and other livestock species and can potentially contribute to transmission chains of epizootic, zoonotic and antimicrobial-resistant (AMR) agents from and to livestock and humans. Consequently, SAC were included as livestock species in the new European Animal Health Law. However, the knowledge on bacteria exhibiting AMR in SAC is too scarce to draft appropriate monitoring and preventive programs. During a survey of SAC holdings in central Germany, 39 Escherichia coli strains were isolated from composite fecal samples by selecting for cephalosporin or fluoroquinolone resistance and were here subjected to whole-genome sequencing. The data were bioinformatically analyzed for strain phylogeny, detection of pathovars, AMR genes and plasmids. Most (33/39) strains belonged to phylogroups A and B1. Still, the isolates were highly diverse, as evidenced by 28 multi-locus sequence types. More than half of the isolates (23/39) were genotypically classified as multidrug resistant. Genes mediating resistance to trimethoprim/sulfonamides (22/39), aminoglycosides (20/39) and tetracyclines (18/39) were frequent. The most common extended-spectrum-ß-lactamase gene was blaCTX-M-1 (16/39). One strain was classified as enteropathogenic E. coli. The positive results indicate the need to include AMR bacteria in yet-to-be-established animal disease surveillance protocols for SAC.

Mycobacterium tuberculosis-Induced Upregulation of the COX-2/mPGES-1 Pathway in Human Macrophages Is Abrogated by Sulfasalazine.

Macrophages are the primary human host cells of intracellular Mycobacterium tuberculosis (M.tb) infection, where the magnitude of inflammatory reactions is crucial for determining the outcome of infection. Previously, we showed that the anti-inflammatory drug sulfasalazine (SASP) significantly reduced the M.tb bactericidal burden and histopathological inflammation in mice. Here, we asked which genes in human inflammatory macrophages are affected upon infection with M.tb and how would potential changes impact the functional state of macrophages. We used a flow cytometry sorting system which can distinguish the dead and alive states of M.tb harbored in human monocyte-derived macrophages (MDM). We found that the expression of cyclooxygenase-2 and microsomal prostaglandin E2 synthase (mPGES)-1 increased significantly in tagRFP+ MDM which were infected with alive M.tb. After exposure of polarized M1-MDM to M.tb (H37Rv strain)-conditioned medium (MTB-CM) or to the M.tb-derived 19-kD antigen, the production of PGE2 and pro-inflammatory cytokines increased 3- to 4-fold. Upon treatment of M1-MDM with SASP, the MTB-CM-induced expression of COX-2 and the release of COX products and cytokines decreased. Elevation of PGE2 in M1-MDM upon MTB-CM stimulation and modulation by SASP correlated with the activation of the NF-κB pathway. Together, infection of human macrophages by M.tb strongly induces COX-2 and mPGES-1 expression along with massive PGE2 formation which is abrogated by the anti-inflammatory drug SASP.

Video Endoscopy-Guided Intrabronchial Spray Inoculation of Mycobacterium bovis in Goats and Comparative Assessment of Lung Lesions With Various Imaging Methods.

Bovine tuberculosis (bTB) not only poses a zoonotic threat to humans but also has a significant economic impact on livestock production in many areas of the world. Effective vaccines for humans, livestock, and wildlife are highly desirable to control tuberculosis. Suitable large animal models are indispensable for meaningful assessment of vaccine candidates. Here, we describe the refinement of an animal model for bTB in goats. Intrabronchial inoculation procedure via video-guided endoscopy in anesthetized animals, collection of lungs after intratracheal fixation in situ, and imaging of lungs by computed tomography (CT) were established in three goats using barium sulfate as surrogate inoculum. For subsequent infection experiments, four goats were infected with 4.7 × 102 colony-forming units of M. bovis by intrabronchial inoculation using video-guided endoscopy with spray catheters. Defined amounts of inoculum were deposited at five sites per lung. Four age-matched goats were mock-inoculated. None of the goats developed clinical signs until they were euthanized 5 months post infection, but simultaneous skin testing confirmed bTB infection in all goats inoculated with M. bovis. In tissues collected at necropsy, M. bovis was consistently re-isolated from granulomas in lymph nodes, draining the lungs of all the goats infected with M. bovis. Further dissemination was observed in one goat only. Pulmonary lesions were quantified by CT and digital 2D radiography (DR). CT revealed mineralized lesions in all the infected goats ranging from <5 mm to >10 mm in diameter. Small lesions <5 mm predominated. The DR failed to detect small lesions and to determine the exact location of lesions because of overlapping of pulmonary lobes. Relative volume of pulmonary lesions was low in three but high in one goat that also had extensive cavitation. CT lesions could be correlated to gross pathologic findings and histologic granuloma types in representative pulmonary lobes. In conclusion, video-guided intrabronchial inoculation with spray catheters, mimicking the natural way of infection, resulted in pulmonary infection of goats with M. bovis. CT, but not DR, presented as a highly sensitive method to quantify the extent of pulmonary lesions. This goat model of TB may serve as a model for testing TB vaccine efficacy.

Using unique ORFan genes as strain-specific identifiers for Escherichia coli.

BACKGROUND: Bacterial identification at the strain level is a much-needed, but arduous and challenging task. This study aimed to develop a method for identifying and differentiating individual strains among multiple strains of the same bacterial species. The set used for testing the method consisted of 17 Escherichia coli strains picked from a collection of strains isolated in Germany, Spain, the United Kingdom and Vietnam from humans, cattle, swine, wild boars, and chickens. We targeted unique or rare ORFan genes to address the problem of selective and specific strain identification. These ORFan genes, exclusive to each strain, served as templates for developing strain-specific primers. RESULTS: Most of the experimental strains (14 out of 17) possessed unique ORFan genes that were used to develop strain-specific primers. The remaining three strains were identified by combining a PCR for a rare gene with a selection step for isolating the experimental strains. Multiplex PCR allowed the successful identification of the strains both in vitro in spiked faecal material in addition to in vivo after experimental infections of pigs and recovery of bacteria from faecal material. In addition, primers for qPCR were also developed and quantitative readout from faecal samples after experimental infection was also possible. CONCLUSIONS: The method described in this manuscript using strain-specific unique genes to identify single strains in a mixture of strains proved itself efficient and reliable in detecting and following individual strains both in vitro and in vivo, representing a fast and inexpensive alternative to more costly methods.

Evaluation of the discriminatory power of spoligotyping and 19-locus mycobacterial interspersed repetitive unit-variable number of tandem repeat analysis (MIRU-VNTR) of Mycobacterium bovis strains isolated from cattle in Algeria.

Bovine tuberculosis (bTB) caused by Mycobacterium (M.) bovis and M. caprae is a transmissible disease of livestock, notifiable to the World Organization for Animal Health (OIE). BTB particularly affects cattle and small ruminants and can be transmitted to humans thereby posing a significant threat to veterinary and public health worldwide. M. bovis is the principal cause of bTB in Algeria. In order to better understand the route of spreading and elaborate an eradication program, isolation and characterization of mycobacteria from Algerian cattle was performed. Sixty strains belonging to the M. tuberculosis complex were analyzed by spoligotyping, thereof 42 by 19-locus-MIRU-VNTR-typing. Spoligotyping revealed 16 distinguishable patterns (Hunter-Gaston discriminatory index [HGDI] of 0.8294), with types SB0120 (n = 20) and SB0121 (n = 13) being the most frequent patterns, representing 55% of the strains. Analyses based on 19-locus-MIRU-VNTR yielded 32 different profiles, five clusters and one orphan pattern, showing higher discriminatory power (HGDI = 0.9779) than spoligotyping. Seven VNTR-loci [VNTR 577 (alias ETR C), 2163b (QU11b), 2165 (ETR A), 2461 (ETR B), 3007 (MIRU 27), 2163a (QUB11a) and 3232 (QUB 3232)] were the most discriminative loci (HGDI ˃ 0.50). In conclusion, 19-locus-MIRU-VNTR yielded more information than spoligotyping concerning molecular differentiation of strains and better supports the elucidation of transmission routes of M. bovis between Algerian cattle herds.

Shiga Toxin-Producing E. coli in Animals: Detection, Characterization, and Virulence Assessment.

Cattle and other ruminants are primary reservoirs for Shiga toxin-producing Escherichia coli (STEC) strains which have a highly variable, but unpredictable, pathogenic potential for humans. Domestic swine can carry and shed STEC, but only STEC strains producing the Shiga toxin (Stx) 2e variant and causing edema disease in piglets are considered pathogens of veterinary medical interest. In this chapter, we present general diagnostic workflows for sampling livestock animals to assess STEC prevalence, magnitude, and duration of host colonization. This is followed by detailed method protocols for STEC detection and typing at genetic and phenotypic levels to assess the relative virulence exerted by the strains.

The Role of Escherichia coli Shiga Toxins in STEC Colonization of Cattle.

Many cattle are persistently colonized with Shiga toxin-producing Escherichia coli (STEC) and represent a major source of human infections with human-pathogenic STEC strains (syn. enterohemorrhagic E. coli (EHEC)). Intervention strategies most effectively protecting humans best aim at the limitation of bovine STEC shedding. Mechanisms enabling STEC to persist in cattle are only partialy understood. Cattle were long believed to resist the detrimental effects of Shiga toxins (Stxs), potent cytotoxins acting as principal virulence factors in the pathogenesis of human EHEC-associated diseases. However, work by different groups, summarized in this review, has provided substantial evidence that different types of target cells for Stxs exist in cattle. Peripheral and intestinal lymphocytes express the Stx receptor globotriaosylceramide (Gb3syn. CD77) in vitro and in vivo in an activation-dependent fashion with Stx-binding isoforms expressed predominantly at early stages of the activation process. Subpopulations of colonic epithelial cells and macrophage-like cells, residing in the bovine mucosa in proximity to STEC colonies, are also targeted by Stxs. STEC-inoculated calves are depressed in mounting appropriate cellular immune responses which can be overcome by vaccination of the animals against Stxs early in life before encountering STEC. Considering Stx target cells and the resulting effects of Stxs in cattle, which significantly differ from effects implicated in human disease, may open promising opportunities to improve existing yet insufficient measures to limit STEC carriage and shedding by the principal reservoir host.

Metabolic Traits of Bovine Shiga Toxin-Producing Escherichia Coli (STEC) Strains with Different Colonization Properties.

Cattle harbor Shiga toxin-producing Escherichia coli (STEC) in their intestinal tract, thereby providing these microorganisms with an ecological niche, but without this colonization leading to any clinical signs. In a preceding study, genotypic characterization of bovine STEC isolates unveiled that their ability to colonize cattle persistently (STECper) or only sporadically (STECspo) is more closely associated with the overall composition of the accessory rather than the core genome. However, the colonization pattern could not be unequivocally linked to the possession of classical virulence genes. This study aimed at assessing, therefore, if the presence of certain phenotypic traits in the strains determines their colonization pattern and if these can be traced back to distinctive genetic features. STECspo strains produced significantly more biofilm than STECper when incubated at lower temperatures. Key substrates, the metabolism of which showed a significant association with colonization type, were glyoxylic acid and L-rhamnose, which were utilized by STECspo, but not or only by some STECper. Genomic sequences of the respective glc and rha operons contained mutations and frameshifts in uptake and/or regulatory genes, particularly in STECper. These findings suggest that STECspo conserved features leveraging survival in the environment, whereas the acquisition of a persistent colonization phenotype in the cattle reservoir was accompanied by the loss of metabolic properties and genomic mutations in the underlying genetic pathways.

Molecular Biology of Escherichia Coli Shiga Toxins' Effects on Mammalian Cells.

Shiga toxins (Stxs), syn. Vero(cyto)toxins, are potent bacterial exotoxins and the principal virulence factor of enterohemorrhagic Escherichia coli (EHEC), a subset of Shiga toxin-producing E. coli (STEC). EHEC strains, e.g., strains of serovars O157:H7 and O104:H4, may cause individual cases as well as large outbreaks of life-threatening diseases in humans. Stxs primarily exert a ribotoxic activity in the eukaryotic target cells of the mammalian host resulting in rapid protein synthesis inhibition and cell death. Damage of endothelial cells in the kidneys and the central nervous system by Stxs is central in the pathogenesis of hemolytic uremic syndrome (HUS) in humans and edema disease in pigs. Probably even more important, the toxins also are capable of modulating a plethora of essential cellular functions, which eventually disturb intercellular communication. The review aims at providing a comprehensive overview of the current knowledge of the time course and the consecutive steps of Stx/cell interactions at the molecular level. Intervention measures deduced from an in-depth understanding of this molecular interplay may foster our basic understanding of cellular biology and microbial pathogenesis and pave the way to the creation of host-directed active compounds to mitigate the pathological conditions of STEC infections in the mammalian body.

Tuberculosis in a pet ferret (Mustela putorius furo).

A 9-month old pet ferret was presented to a veterinarian with symptoms of weight loss, apathy, and hyporexia. Explorative laparotomy identified a firm mass of approximately 2 × 2 × 2 cm in size in the mesentery of the jejunum. Because of the poor general condition and the unfavorable prognosis, the ferret was euthanized during surgery. The mass was resected in total and submitted to histological examination which revealed a granulomatous and necrotizing lymphadenitis. Acid fast bacteria were detected by Fite-Faraco staining leading to the suspicion of an infection with Mycobacteria sp. PCR confirmed presence of DNA of members of the Mycobacterium tuberculosis complex, subsequently specified as M. bovis. The detected spoligotype SB2548 was described for the first time. Ferrets are presented to veterinarians with increasing frequency because of their growing popularity as pet animals. Since these animals are highly susceptible to mycobacterial infections, mycobacteriosis and especially zoonotic relevant tuberculosis should be considered as differential diagnosis.