Cross-sectional study: prevalence of oedema disease Escherichia coli (EDEC) in weaned piglets in Germany at pen and farm levels.

BACKGROUND: Escherichia coli bacteria capable of producing the toxin Stx2e and possessing F18-fimbriae (edema disease E. coli, EDEC) are considered causative agents of porcine oedema disease. This disease, which usually occurs in piglets shortly after weaning, has a high lethality in affected animals and can lead to high economic losses in piglet rearing. The aim of this cross-sectional field study was to determine the prevalence of EDEC in weaned piglets in Germany at pen and farm levels. RESULTS: Ninety-nine farms with unknown history of infections with shigatoxin-producing E. coli (STEC) and oedema disease were sampled. On each farm, up to five pens were selected for sampling (n = 481). The piglets in these pens were at an age 1-3 weeks after weaning. Single faecal samples (n = 2405) and boot swabs (n = 479) were collected from the floor. On 50 farms, cotton ropes were additionally used to collect oral fluid samples (n = 185) and rope wash out samples (n = 231) from the selected pens. All samples were analyzed by bacterial culture combined with a duplex PCR for the presence of the corresponding genes stx2e and fedA (major subunit protein of F18 fimbriae). In addition, whole DNA specimens extracted from boot swabs, oral fluid samples, and rope wash out samples were directly examined by duplex PCR for DNA of stx2e and fedA. A pen was classified as positive if at least one of the samples, regardless of the technique, yielded a positive result in the PCR, and farms were considered positive if at least one pen was classified as positive. Overall, genes stx2e and fedA were found simultaneously in 24.9% (95% CI 22.1-29.1%) of sampled pens and in 37.4% (95% CI 27.9-47.7%) of sampled farms. Regardless of the presence of F18-fimbriae, Escherichia coli encoding for Stx2e (STEC-2e) were found in 35.1% (95% CI 31.0-39.1%) of the pens and 53.5% (95% CI 44.4-63.6%) of the farms sampled. CONCLUSIONS: Escherichia coli strains considered capable to cause oedema disease in swine (EDEC) are highly prevalent in the surveyed pig producing farms in Germany. Due to intermittent shedding of EDEC and a potentially low within-farm prevalence, we recommend a combination of different sampling techniques for EDEC monitoring at pen and farm levels. Further studies are needed to understand which STEC-2e strains really pose the risk of causing severe porcine disease.

Vector-Borne Pathogens in Stray Cats in Eastern Germany (Thuringia).

Bacterial, protozoal, and viral vector-borne pathogens (VBPs) can cause infections in cats. There is little information on feline VBP prevalence in Germany. Stray cats are frequently exposed to vectors but receive no veterinary care. The aim of this study was to determine the prevalence of selected VBPs in stray cats. EDTA blood and serum samples were taken from apparently healthy stray cats during a spay/neuter campaign in the federal state of Thuringia. Overall, 11/50 (22%) and 32/50 (64%) cats tested positive for at least one VBP by direct and indirect detection methods, respectively. PCR testing of EDTA blood detected hemotropic Mycoplasma spp. in 12% of cats, Hepatozoon spp. in 10%, and Anaplasma phagocytophilum in 4%. PCR testing for Rickettsia spp. and piroplasms was negative. IFAT on serum samples showed 46% of cats had detectable antibodies for Bartonella spp., 30% for Rickettsia spp., and 16% for A. phagocytophilum. The cats were additionally tested for feline coronavirus, FIV, and FeLV to identify potential risk factors for pathogen contact and/or infections. No correlation between FIV and FeLV status and VBP positivity was detected. Anaplasma phagocytophilum, Rickettsia spp., and Bartonella spp. have zoonotic potential, and surveillance is recommended in the context of the One Health approach.

Molecular Features and Antimicrobial Susceptibilities of Streptococcus equi ssp. equi Isolates from Strangles Cases in Indonesia.

Strangles, caused by Streptococcus equi ssp. equi (S. equi equi), is a highly infectious and frequent disease of equines worldwide. No data are available regarding the molecular epidemiology of strangles in Indonesia. This study aimed to characterize S. equi equi isolates obtained from suspected strangles cases in Indonesia in 2018. Isolates originated from seven diseased horses on four different farms located in three provinces of Indonesia. Whole genome sequences of these isolates were determined and used for seM typing, multilocus sequence typing (MLST), and core genome MLS typing (cgMLST). Genomes were also screened for known antimicrobial resistance genes and genes encoding for the recombinant antigens used in the commercial Strangvac® subunit vaccine. All seven S. equi equi isolates from Indonesia belonged to ST179 and carried seM allele 166. Isolates differed from each other by only 2 to 14 cgSNPs and built an exclusive sub-cluster within the Bayesian Analysis of Population Structure (BAPS) cluster 2 (BAPS-2) of the S. equi equi cgMLST scheme. All isolates revealed predicted amino acid sequence identity to seven and high similarity to one of the eight antigen fragments contained in Strangvac®. Furthermore, all isolates were susceptible to beta-lactam antibiotics penicillin G, ampicillin, and ceftiofur. Our data suggest that the horses from this study were affected by strains of the same novel sublineage within globally distributed BAPS-2 of S. equi equi. Nevertheless, penicillin G can be used as a first-choice antibiotic against these strains and Strangvac® may also be protective against Indonesian strains.

Occurrence of mcr-1 and mcr-2 colistin resistance genes in porcine Escherichia coli isolates (2010-2020) and genomic characterization of mcr-2-positive E. coli.

Introduction: The global emergence of plasmid-mediated colistin resistance is threatening the efficacy of colistin as one of the last treatment options against multi-drug resistant Gram-negative bacteria. To date, ten mcr-genes (mcr-1 to mcr-10) were reported. While mcr-1 has disseminated globally, the occurrence of mcr-2 was reported scarcely. Methods and results: We determined the occurrence of mcr-1 and mcr-2 genes among Escherichia coli isolates from swine and performed detailed genomic characterization of mcr-2-positive strains. In the years 2010-2017, 7,614 porcine E. coli isolates were obtained from fecal swine samples in Europe and isolates carrying at least one of the virulence associated genes predicting Shiga toxin producing E. coli (STEC), enterotoxigenic E. coli (ETEC) or enteropathogenic E. coli (EPEC) were stored. 793 (10.4%) of these isolates carried the mcr-1 gene. Of 1,477 additional E. coli isolates obtained from sheep blood agar containing 4 mg/L colistin between 2018 and 2020, 36 (2.4%) isolates were mcr-1-positive. In contrast to mcr-1, the mcr-2 gene occurred at a very low frequency (0.13%) among the overall 9,091 isolates. Most mcr-2-positive isolates originated from Belgium (n = 9), one from Spain and two from Germany. They were obtained from six different farms and revealed multilocus sequence types ST10, ST29, ST93, ST100, ST3057 and ST5786. While the originally described mcr-2.1 was predominant, we also detected a new mcr-2 variant in two isolates from Belgium, which was termed mcr-2.8. MCR-2 isolates were mostly classified as ETEC or ETEC-like, while one isolate from Spain represented an atypical enteropathogenic E. coli (aEPEC; eae+). The ST29-aEPEC isolate carried mcr-2 on the chromosome. Another eight isolates carried their mcr-2 gene on IncX4 plasmids that resembled the pKP37-BE MCR-2 plasmid originally described in Belgium in 2015. Three ST100 E. coli isolates from a single farm in Belgium carried the mcr-2.1 gene on a 47-kb self-transmissible IncP type plasmid of a new IncP-1 clade. Discussion: This is the first report of mcr-2 genes in E. coli isolates from Germany. The detection of a new mcr-2 allele and a novel plasmid backbone suggests the presence of so far undetected mcr-2 variants and mobilizable vehicles.

Erratum to "The GadX regulon affects virulence gene expression and adhesion of porcine enteropathogenic Escherichia coli in vitro" [Vet. Anim. Sci. 3C (2017) 10-17].

[This corrects the article DOI: 10.1016/j.vas.2017.04.001.].

Toxoplasma gondii-induced host cellular cell cycle dysregulation is linked to chromosome missegregation and cytokinesis failure in primary endothelial host cells.

Toxoplasma gondii is a zoonotic and intracellular parasite with fast proliferating properties leading to rapid host cell lysis. T. gondii modulates its host cell on numerous functional levels. T. gondii was previously reported to influence host cellular cell cycle and to dampen host cell division. By using primary endothelial host cells, we show for the first time that T. gondii tachyzoite infections led to increased host cell proliferation and to an enhanced number of multi-nucleated host cells. As detected on DNA content level, parasite infections induced a G2/M cell cycle arrest without affecting expression of G2-specific cyclin B1. In line, parasite-driven impairment mainly concerned mitotic phase of host cells by propagating several functional alterations, such as chromosome segregation errors, mitotic spindle alteration and blockage of cytokinesis progression, with the latter most likely being mediated by the downregulation of the Aurora B kinase expression.

Besnoitia besnoiti infection alters both endogenous cholesterol de novo synthesis and exogenous LDL uptake in host endothelial cells.

Besnoitia besnoiti, an apicomplexan parasite of cattle being considered as emergent in Europe, replicates fast in host endothelial cells during acute infection and is in considerable need for energy, lipids and other building blocks for offspring formation. Apicomplexa are generally considered as defective in cholesterol synthesis and have to scavenge cholesterol from their host cells for successful replication. Therefore, we here analysed the influence of B. besnoiti on host cellular endogenous cholesterol synthesis and on sterol uptake from exogenous sources. GC-MS-based profiling of cholesterol-related sterols revealed enhanced cholesterol synthesis rates in B. besnoiti-infected cells. Accordingly, lovastatin and zaragozic acid treatments diminished tachyzoite production.  Moreover, increased lipid droplet contents and enhanced cholesterol esterification was detected and inhibition of the latter significantly blocked parasite proliferation. Furthermore, artificial increase of host cellular lipid droplet disposability boosted parasite proliferation. Interestingly, lectin-like oxidized low density lipoprotein receptor 1 expression was upregulated in infected endothelial hostcells, whilst low density lipoproteins (LDL) receptor was not affected by parasite infection. However, exogenous supplementations with non-modified and acetylated LDL both boosted B. besnoiti proliferation. Overall, current data show that B. besnoiti simultaneously exploits both, endogenous cholesterol biosynthesis and cholesterol uptake from exogenous sources, during asexual replication.

Interaction of Coxiella burnetii Strains of Different Sources and Genotypes with Bovine and Human Monocyte-Derived Macrophages.

Most human Q fever infections originate from small ruminants. By contrast, highly prevalent shedding of Coxiella (C.) burnetii by bovine milk rarely results in human disease. We hypothesized that primary bovine and human monocyte-derived macrophages (MDM) represent a suitable in vitro model for the identification of strain-specific virulence properties at the cellular level. Twelve different C. burnetii strains were selected to represent different host species and multiple loci variable number of tandem repeat analysis (MLVA) genotypes. Infection efficiency and replication of C. burnetii were monitored by cell culture re-titration and qPCR. Expression of immunoregulatory factors after MDM infection was measured by qRT-PCR and flow cytometry. Invasion, replication and MDM response differed between C. burnetii strains but not between MDMs of the two hosts. Strains isolated from ruminants were less well internalized than isolates from humans and rodents. Internalization of MLVA group I strains was lower compared to other genogroups. Replication efficacy of C. burnetii in MDM ranged from low (MLVA group III) to high (MLVA group IV). Infected human and bovine MDM responded with a principal up-regulation of pro-inflammatory cytokines such as IL-1ß, IL-12, and TNF-α. However, MLVA group IV strains induced a pronounced host response whereas infection with group I strains resulted in a milder response. C. burnetii infection marginally affected polarization of MDM. Only one C. burnetii strain of MLVA group IV caused a substantial up-regulation of activation markers (CD40, CD80) on the surface of bovine and human MDM. The study showed that replication of C. burnetii in MDM and the subsequent host cell response is genotype-specific rather than being determined by the host species pointing to a clear distinction in C. burnetii virulence between the genetic groups.

The GadX regulon affects virulence gene expression and adhesion of porcine enteropathogenic Escherichia coli in vitro.

The ability of enteropathogenic Escherichia coli (EPEC) to express virulence factor genes and develop attaching and effacing (AE) lesions is inhibited in acidic environmental conditions. This inhibition is due to the activation of transcription factor GadX, which upregulates expression of glutamic acid decarboxylase (Gad). Gad, in turn, produces γ-aminobutyric acid (GABA), which was recently shown to have a beneficial effect on the jejunal epithelium in vitro due to increased mucin-1 levels. In the present study, we sought to test whether forced GadX activation/overexpression abolishes virulence associated features of EPEC and provokes increased GABA production. EPEC strains were isolated from diarrheic pigs and submitted to activation of GadX by acidification as well as gadX overexpression via an inducible expression vector plasmid. GABA concentrations in the growth medium, ability for adhesion to porcine intestinal epithelial cells (IPEC-J2) and virulence gene expression were determined. Growth in acidified media led to increased GABA levels, upregulated gadA/B expression and downregulated mRNA synthesis of the bacterial adhesin intimin. EPEC strains transformed with the gadX gene produced 2.1-3.4-fold higher GABA levels than empty-vector controls and completely lost their ability to adhere to IPEC-J2 cells and to induce actin accumulation. We conclude that intensified gadX activation can abolish the ability of EPEC to adhere to the intestinal epithelium by reducing the expression of major virulence genes.

Identification of a novel feline large granular lymphoma cell line (S87) as non-MHC-restricted cytotoxic T-cell line and assessment of its genetic instability.

Feline large granular lymphocyte lymphomas are rare but very aggressive tumors with a poor prognosis. In this study, a cell line from an abdominal effusion of a cat with large granular lymphoma was characterized. Immunophenotype staining was positive for CD3 and CD45R, and negative for CD4, CD8, CD56, CD79α, BLA.36 and NK1. A TCR γ gene rearrangement was detectable by PARR. Neither FeLV antigen nor exogenous FeLV provirus could be detected. A chromosomal instability associated with a centrosome hyperamplification could also be determined. The cell line is able to lyse target cells without antigen presentation or interaction with antigen presenting cells. Therefore, these cells were classified as genetically instable non-MHC-restricted cytotoxic T cells with large granular lymphocyte morphology.

Coxiella burnetii Infects Primary Bovine Macrophages and Limits Their Host Cell Response.

Although domestic ruminants have long been recognized as the main source of human Q fever, little is known about the lifestyle that the obligate intracellular Gram-negative bacterium Coxiella burnetii adopts in its animal host. Because macrophages are considered natural target cells of the pathogen, we established primary bovine monocyte-derived macrophages (MDM) as an in vitro infection model to study reservoir host-pathogen interactions at the cellular level. In addition, bovine alveolar macrophages were included to take cell type peculiarities at a host entry site into account. Cell cultures were inoculated with the virulent strain Nine Mile I (NMI; phase I) or the avirulent strain Nine Mile II (NMII; phase II). Macrophages from both sources internalized NMI and NMII. MDM were particularly permissive for NMI internalization, but NMI and NMII replicated with similar kinetics in these cells. MDM responded to inoculation with a general upregulation of Th1-related cytokines such as interleukin-1ß (IL-1ß), IL-12, and tumor necrosis factor alpha (TNF-α) early on (3 h postinfection). However, inflammatory responses rapidly declined when C. burnetii replication started. C. burnetii infection inhibited translation and release of IL-1ß and vastly failed to stimulate increased expression of activation markers, such as CD40, CD80, CD86, and major histocompatibility complex (MHC) molecules. Such capability of limiting proinflammatory responses may help Coxiella to protect itself from clearance by the host immune system. The findings provide the first detailed insight into C. burnetii-macrophage interactions in ruminants and may serve as a basis for assessing the virulence and the host adaptation of C. burnetii strains.

Eimeria bovis infection modulates endothelial host cell cholesterol metabolism for successful replication.

During first merogony Eimeria bovis forms large macromeronts in endothelial host cells containing >120 000 merozoites I. During multiplication, large amounts of cholesterol are indispensable for the enormous offspring membrane production. Cholesterol auxotrophy was proven for other apicomplexan parasites. Consequently they scavenge cholesterol from their host cell apparently in a parasite-specific manner. We here analyzed the influence of E. bovis infection on endothelial host cell cholesterol metabolism and found considerable differences to other coccidian parasites. Overall, free cholesterol significantly accumulated in E. bovis infected host cells. Furthermore, a striking increase of lipid droplet formation was observed within immature macromeronts. Artificial host cell lipid droplet enrichment significantly improved E. bovis merozoite I production confirming the key role of lipid droplet contents for optimal parasite proliferation. The transcription of several genes being involved in both, cholesterol de novo biosynthesis and low density lipoprotein-(LDL) mediated uptake, was significantly up-regulated at a time in infected cells suggesting a simultaneous exploitation of these two cholesterol acquisition pathways. E. bovis scavenges LDL-derived cholesterol apparently through significantly increased levels of surface LDL receptor abundance and LDL binding to infected cells. Consequently, LDL supplementation significantly improved parasite replication. The up-regulation of the oxidized LDL receptor 1 furthermore identified this scavenger receptor as a key molecule in parasite-triggered LDL uptake. Moreover, cellular cholesterol processing was altered in infected cells as indicated by up-regulation of cholesterol-25-hydroxylase and sterol O-acyltransferase. Overall, these results show that E. bovis considerably exploits the host cell cholesterol metabolism to guarantee its massive intracellular growth and replication.

Evaluation of biological safety in vitro and immunogenicity in vivo of recombinant Escherichia coli Shiga toxoids as candidate vaccines in cattle.

Cattle are the most important reservoir for enterohemorrhagic Escherichia coli (EHEC), a subset of shigatoxigenic E. coli (STEC) capable of causing life-threatening infectious diseases in humans. In cattle, Shiga toxins (Stx) suppress the immune system thereby promoting long-term STEC shedding. First infections of animals at calves' age coincide with the lack of Stx-specific antibodies. We hypothesize that vaccination of calves against Shiga toxins prior to STEC infection may help to prevent the establishment of a persistent type of infection. The objectives of this study were to generate recombinant Shiga toxoids (rStx1mut & rStx2mut) by site-directed mutagenesis and to assess their immunomodulatory, antigenic, and immunogenic properties. Cultures of bovine primary immune cells were used as test systems. In ileal intraepithelial lymphocytes both, recombinant wild type Stx1 (rStx1WT) and rStx2WT significantly induced transcription of IL-4 mRNA. rStx1WT and rStx2WT reduced the expression of Stx-receptor CD77 (syn. Globotriaosylceramide, Gb3) on B and T cells from peripheral blood and of CD14 on monocyte-derived macrophages. At the same concentrations, rStx1mut and rStx2mut exhibited neither of these effects. Antibodies in sera of cattle naturally infected with STEC recognized the rStxmut toxoids equally well as the recombinant wild type toxins. Immunization of calves with rStx1mut plus rStx2mut led to induction of antibodies neutralizing Stx1 and Stx2. While keeping their antigenicity and immunogenicity recombinant Shiga toxoids are devoid of the immunosuppressive properties of the corresponding wild type toxins in cattle and candidate vaccines to mitigate long-term STEC shedding by the reservoir host.