Immunogenicity and protective efficacy of a Streptococcus suis vaccine composed of six conserved immunogens.

A vaccine protecting against different Streptococcus suis serotypes is highly needed in porcine practice to improve animal welfare and reduce the use of antibiotics. We hypothesized that immunogens prominently recognized by convalescence sera but significantly less so by sera of susceptible piglets are putative protective antigens. Accordingly, we investigated immunogenicity and protective efficacy of a multicomponent vaccine including six main conserved immunogens, namely SSU0934, SSU1869, SSU0757, SSU1950, SSU1664 and SSU0187. Flow cytometry confirmed surface expression of all six immunogens in S. suis serotypes 2, 9 and 14. Although prime-booster vaccination after weaning resulted in significantly higher specific IgG levels against all six immunogens compared to the placebo-treated group, no significant differences between bacterial survival in blood from either vaccinated or control animals were recorded for serotype 2, 9 and 14 strains. Furthermore, vaccinated piglets were not protected against morbidity elicited through intranasal challenge with S. suis serotype 14. As ~50% of animals in both groups did not develop disease, we investigated putative other correlates of protection. Induction of reactive oxygen species (ROS) in blood granulocytes was not associated with vaccination but correlated with protection as all piglets with >5% ROS survived the challenge. Based on these findings we discuss that the main immunogens of S. suis might actually not be a priori good candidates for protective antigens. On the contrary, expression of immunogens that evoke antibodies that do not mediate killing of this pathogen might constitute an evolutionary advantage conserved in many different S. suis strains.

Comparative analysis of adeno-associated virus serotypes for gene transfer in organotypic heart slices.

BACKGROUND: Vectors derived from adeno-associated viruses (AAVs) are widely used for gene transfer both in vitro and in vivo and have gained increasing interest as shuttle systems to deliver therapeutic genes to the heart. However, there is little information on their tissue penetration and cytotoxicity, as well as the optimal AAV serotype for transferring genes to diseased hearts. Therefore, we aimed to establish an organotypic heart slice culture system for mouse left ventricular (LV) myocardium and use this platform to analyze gene transfer efficiency, cell tropism, and toxicity of different AAV serotypes. METHODS: LV tissue slices, 300 µm thick, were prepared from 15- to 17-day-old transgenic alpha-myosin heavy-chain-mCherry mice using a vibrating microtome. Tissue slice viability in air-liquid culture was evaluated by calcein-acetoxymethyl ester staining, mCherry fluorescence intensity, and the tetrazolium assay. Four recombinant AAV serotypes (1, 2, 6, 8) expressing green fluorescent protein (GFP) under the CAG promoter were added to the slice surface. Gene transfer efficiency was quantified as the number of GFP-positive cells per slice. AAV cell tropism was examined by comparing the number of GFP-positive cardiomyocytes (CMs) and fibroblasts within heart slices. RESULTS: Slices retained viability in in vitro culture for at least 5 days. After adding AAV particles, AAV6-infected slices showed the highest number of GFP-expressing cells, almost exclusively CMs. Slice incubation with AAV1, 2, and 8 resulted in fewer GFP-positive cells, with AAV2 having the lowest gene transfer efficiency. None of the AAV serotypes tested caused significant cytotoxicity when compared to non-infected control slices. CONCLUSIONS: We have established a readily available mouse organotypic heart slice culture model and provided evidence that AAV6 may be a promising gene therapy vector for heart failure and other cardiac diseases.

Turning fibroblasts into cardiomyocytes: technological review of cardiac transdifferentiation strategies.

To date, no viable therapeutic options exist for the effective and sustained reversal of cardiac failure, other than heart transplantation and mechanical circulatory assist devices. Therefore, divergent strategies aiming at the de novo formation of contractile tissue, as a prerequisite for the restoration of cardiac pump function, are currently being pursued. Clinical trials involving the transplantation of somatic progenitor cells failed. The search for alternative cell-based strategies to combat the consequences of ischemic injury has sparked widespread interest in the genetic and pharmacologic reprogramming of fibroblasts into cardiomyocytes, harnessing the abundant in vivo pool of cardiac fibroblasts. Here, we provide a comprehensive overview of in vitro and in vivo cardiac reprogramming studies identified in an extensive literature search. We systematically review and evaluate feasibility, efficiency, and reproducibility of the different technologies currently being explored. Finally, we discuss potential safety issues deduced from preclinical studies and identify obstacles that must be overcome before clinical translation.-Klose, K., Gossen, M., Stamm, C. Turning fibroblasts into cardiomyocytes: technological review of cardiac transdifferentiation strategies.

Comparative analysis of humoral immune responses and pathologies of BALB/c and C57BL/6 wildtype mice experimentally infected with a highly virulent Rodentibacter pneumotropicus (Pasteurella pneumotropica) strain.

BACKGROUND: Mice are a natural host for Rodentibacter (R.) pneumotropicus. Despite specific monitoring, it is still one of the most important infectious agents in laboratory animals. The objective of this study was to determine the virulence of a prevalent pathotype of R. pneumotropicus and characterize the host response in a new animal model. RESULTS: Intranasal infection of C57BL/6 and BALB/c mice with a R. pneumotropicus strain (JF4Ni) bearing the genes of the three known repeats in toxin (RTX) toxins resulted in an unprecedented high mortality and morbidity above 50 and 80%, respectively. Morbidity was associated with severe weight loss as well as conjunctivitis and dyspnea. A main pathology was a catarrhal purulent to necrotic bronchopneumonia. Specific immune globuline (Ig) A was detected in tracheonasal lavages of most surviving mice which were still colonized by R. pneumotropicus. Furthermore, all surviving animals showed a distinct production of IgG antibodies. To differentiate T-helper cell (Th) 1 and Th2 immune responses we used subclasses of IgGs as indicators. Mean ratios of IgG2b to IgG1 were below 0.8 in sera drawn from both mice strains prior infection and from BALB/c mice post infection. In contrast, C57BL/6 mice had a mean IgG2b/IgG1 ratio of 1.6 post infection indicating a Th1 immune response in C57BL/6 versus a Th2 response in BALB/c mice associated with a tenfold higher bacterial load in the lung. In accordance with a Th1 response high antigen-specific IgG2c titers were detected in the majority of surviving C57BL/6 mice. CONCLUSIONS: R. pneumotropicus JF4Ni is a highly virulent strain causing severe pneumonia and septicemia after intranasal infection of C57BL/6 and BALB/c mice. Persisting infections in the two mice strains are associated with Th1 and Th2 immune responses, respectively, and differences in the bacterial burden of the lung. The described model is ideally suited for future vaccination studies using the natural host.

Regenerative Medicine/Cardiac Cell Therapy: Pluripotent Stem Cells.

For more than 20 years, tremendous efforts have been made to develop cell-based therapies for treatment of heart failure. However, the results of clinical trials using somatic, nonpluripotent stem or progenitor cells have been largely disappointing in both cardiology and cardiac surgery scenarios. Surgical groups were among the pioneers of experimental and clinical myocyte transplantation ("cellular cardiomyoplasty"), but little translational progress was made prior to the development of cellular reprogramming for creation of induced pluripotent stem cells (iPSC). Ever since, protocols have been developed which allow for the derivation of large numbers of autologous cardiomyocytes (CMs) from patient-specific iPSC, moving translational research closer toward clinical pilot trials. However, compared with somatic cell therapy, the technology required for safe and efficacious pluripotent stem cell (PSC)-based therapies is extremely complex and requires tremendous resources and close interactions between basic scientists and clinicians. This review summarizes PSC sources, strategies to derive CMs, current cardiac tissue engineering approaches, concerns regarding immunogenicity and cellular maturity, and highlights the contributions made by surgical groups.

The cytoprotective capacity of processed human cardiac extracellular matrix.

Freshly isolated human cardiac extracellular matrix sheets (cECM) have been shown to support stem cell proliferation and tissue-specific lineage commitment. We now developed a protocol for standardized production of durable, bio-functional hcECM microparticles and corresponding hydrogel, and tested its cytoprotective effects on contractile cells subjected to ischemia-like conditions. Human ventricular myocardium was decellularized by a 3-step protocol, including Tris/EDTA, SDS and serum incubation (cECM). Following snap-freezing and lyophilization, microparticles were created and characterized by laser diffraction, dynamic image analysis (DIA), and mass spectrometry. Moreover, cECM hydrogel was produced by pepsin digestion. Baseline cell-support characteristics were determined using murine HL-1 cardiomyocytes, and the cytoprotective effects of ECM products were tested under hypoxia and glucose/serum deprivation. In cECM, glycoproteins (thrombospondin 1, fibronectin, collagens and nidogen-1) and proteoglycans (dermatopontin, lumican and mimecan) were preserved, but residual intracellular and blood-borne proteins were also detected. The median particle feret diameter was 66 µm (15-157 µm) by laser diffraction, and 57 µm (20-182 µm) by DIA with crystal violet staining. HL-1 cells displayed enhanced metabolic activity (39 ± 12 %, P < 0.05) and proliferation (16 ± 3 %, P < 0.05) when grown on cECM microparticles in normoxia. During simulated ischemia, cECM microparticles exerted distinct cytoprotective effects (MTS conversion, 240 ± 32 %; BrdU uptake, 45 ± 14 %; LDH release, -72 ± 7 %; P < 0.01, each). When cECM microparticles were solubilized to form a hydrogel, the cytoprotective effect was initially abolished. However, modifying the preparation process (pepsin digestion at pH 2 and 25 °C, 1 mg/ml final cECM concentration) restored the cytoprotective cECM activity. Extracellular matrix from human myocardium can be processed to yield standardized durable microparticles that exert specific cytoprotective effects on cardiomyocyte-like cells. The use of processed cECM may help to optimize future clinical-grade myocardial tissue engineering approaches.

Cord blood mesenchymal stromal cell-conditioned medium protects endothelial cells via STAT3 signaling.

BACKGROUND/AIMS: Cell-based therapies may be useful for treating ischemic diseases, but the underlying mechanisms are incompletely understood. We investigated the impact of cord blood mesenchymal stromal cell (CBMSC)- or fibroblast (FB)-secreted factors on starved endothelial cells and determined the relevant intracellular signaling pathways. METHODS: HUVECs were subjected to glucose/serum deprivation (GSD) in hypoxia or normoxia, in presence of CBMSC- or FB-conditioned medium (CM). Viability and proliferation were determined via WST-8 conversion and BrdU incorporation. Apoptosis was quantified by annexin V/ethidium homodimer-III staining, nuclear fragmentation and cell morphology. mRNA expression and protein phosphorylation were determined by real-time qPCR and western blot. Experiments were repeated in presence of small-molecule inhibitors. RESULTS: The negative impact of GSD was most pronounced at 21% O2. Here, medium of CBMSCs and FBs increased viability and proliferation and reduced apoptosis of HUVECs. This was associated with increased STAT3 and ERK1/2 phosphorylation and BCL-2 expression. Under STAT3 inhibition, the beneficial effect of CBMSC-CM on viability and BCL-2 expression was abolished. CONCLUSION: Factors released by CBMSCs protect endothelial cells from the deleterious impact of GSD by activation of the STAT3 survival pathway. However, this phenomenon is not CBMSC-specific and can be reproduced using juvenile fibroblasts.

Impact of heart failure on the behavior of human neonatal stem cells in vitro.

BACKGROUND: Clinical cardiac cell therapy using autologous somatic stem cells is restricted by age and disease-associated impairment of stem cell function. Juvenile cells possibly represent a more potent alternative, but the impact of patient-related variables on such cell products is unknown. We therefore evaluated the behavior of neonatal cord blood mesenchymal stem cells (CB-MSC) in the presence of serum from patients with advanced heart failure (HF). METHODS: Human serum was obtained from patients with severe HF (n = 21) and from healthy volunteers (n = 12). To confirm the systemic quality of HF in the sera, TNF-α and IL-6 were quantified. CB-MSC from healthy neonates were cultivated for up to 14 days in medium supplemented with 10% protein-normalized human HF or control serum or fetal calf serum (FCS). RESULTS: All HF sera contained increased cytokine concentrations (IL-6, TNF-α). When exposed to HF serum, CB-MSC maintained basic MSC properties as confirmed by immunophenotyping and differentiation assays, but clonogenic cells were reduced in number and gave rise to substantially smaller colonies in the CFU-F assay. Cell cycle analysis pointed towards G1 arrest. CB-MSC metabolic activity and proliferation were significantly impaired for up to 3 days as measured by MTS turnover, BrdU incorporation and DAPI + nuclei counting. On day 5, however, CB-MSC growth kinetics approached control serum levels, though protein expression of cell cycle inhibitors (p21, p27), and apoptosis marker Caspase 3 remained elevated. Signal transduction included the stress and cytokine-induced JNK and ERK1/2 MAP kinase pathways. CONCLUSIONS: Heart failure temporarily inhibits clonality and proliferation of "healthy" juvenile MSC in vitro. Further studies should address the in vivo and clinical relevance of this finding.

Relationship between epicardial adipose tissue attenuation and coronary artery disease in type 2 diabetes mellitus patients.

BACKGROUND AND AIMS: High epicardial adipose tissue (EAT) attenuation is a key characteristic of adipose tissue dysfunction and associated with coronary artery disease (CAD). As little is known about the modulation of EAT attenuation by metabolic disorders, we investigated the association between EAT attenuation and CAD risk factors, CAD presence and CAD severity in type 2 diabetes mellitus (T2DM) patients. METHODS: We included 276 inpatients with T2DM and 305 control patients with normal glucose metabolism (NGM), who underwent cardiac computed tomography angiography (CCTA) and coronary artery calcium (CAC) scoring. EAT attenuation and volume were evaluated by contrast-enhanced CCTA image analysis. Furthermore, segment stenosis scores (SSSs) of the left main coronary artery (LMCA), left anterior descending artery (LAD), left circumflex artery (LCX), right coronary artery (RCA), diagonal/intermediate branch (D/I) and obtuse marginal branch (OM) were calculated to assess CAD severity. RESULTS: T2DM patients showed higher significant CAC scores, coronary plaque prevalence, total SSSs and LMCA-SSSs, LAD-SSSs, LCX-SSSs, RCA-SSSs and D/I-SSSs compared with NGM controls. In contrast to NGM controls, EAT volume was significantly increased in T2DM patients, whereas EAT attenuation was similar. In T2DM patients, EAT attenuation was associated with discrete CAD risk factors, the presence of coronary and triple-vessel plaques, as well as LAD-SSSs, LCX-SSSs, RCA-SSSs and total SSSs. In addition, EAT attenuation was only associated with the total SSS of calcified plaques, but not with noncalcified plaques. CONCLUSION: In T2DM patients, high EAT attenuation is associated with the presence and severity of CAD in general and with coronary stenosis caused by calcified plaques in particular.

Cardiomyocyte precursors generated by direct reprogramming and molecular beacon selection attenuate ventricular remodeling after experimental myocardial infarction.

BACKGROUND: Direct cardiac reprogramming is currently being investigated for the generation of cells with a true cardiomyocyte (CM) phenotype. Based on the original approach of cardiac transcription factor-induced reprogramming of fibroblasts into CM-like cells, various modifications of that strategy have been developed. However, they uniformly suffer from poor reprogramming efficacy and a lack of translational tools for target cell expansion and purification. Therefore, our group has developed a unique approach to generate proliferative cells with a pre-CM phenotype that can be expanded in vitro to yield substantial cell doses. METHODS: Cardiac fibroblasts were reprogrammed toward CM fate using lentiviral transduction of cardiac transcriptions factors (GATA4, MEF2C, TBX5, and MYOCD). The resulting cellular phenotype was analyzed by RNA sequencing and immunocytology. Live target cells were purified based on intracellular CM marker expression using molecular beacon technology and fluorescence-activated cell sorting. CM commitment was assessed using 5-azacytidine-based differentiation assays and the therapeutic effect was evaluated in a mouse model of acute myocardial infarction using echocardiography and histology. The cellular secretome was analyzed using mass spectrometry. RESULTS: We found that proliferative CM precursor-like cells were part of the phenotype spectrum arising during direct reprogramming of fibroblasts toward CMs. These induced CM precursors (iCMPs) expressed CPC- and CM-specific proteins and were selectable via hairpin-shaped oligonucleotide hybridization probes targeting Myh6/7-mRNA-expressing cells. After purification, iCMPs were capable of extensive expansion, with preserved phenotype when under ascorbic acid supplementation, and gave rise to CM-like cells with organized sarcomeres in differentiation assays. When transplanted into infarcted mouse hearts, iCMPs prevented CM loss, attenuated fibrotic scarring, and preserved ventricular function, which can in part be attributed to their substantial secretion of factors with documented beneficial effect on cardiac repair. CONCLUSIONS: Fibroblast reprogramming combined with molecular beacon-based cell selection yields an iCMP-like cell population with cardioprotective potential. Further studies are needed to elucidate mechanism-of-action and translational potential.

[Treatment, clinical course and outcome of tibial neuropathy in German Holstein cows]. / Behandlung, Verlauf und Therapieerfolg der Neuropathie des Nervus tibialis bei Deutsch-Holstein Kühen.

OBJECTIVE: As a follow-up to a previous study on the incidence, history and clinical findings of tibial neuropathy (TN), the present work aimed at describing the treatment and prognosis of this disease. MATERIALS AND METHODS: Of 88 German Holstein dairy cows with unilateral (UTN, n = 71) or bilateral (BTN, n = 17) TN, 68 (56 UTN, 12 BTN) with a complete data set were analyzed. They were retrospectively assigned to one of four groups: no treatment - spontaneous healing within 48 h (Spontaneous, 5 UTN), no bandage (0Cast, 8 UTN, 3 BTN) or treatment with anti-inflammatory drugs and support bandage (StV, 3 UTN) or fiberglass cast (Cast, 40 UTN, 9 BTN). Treated cows were re-examined five times (14, 21, 28, 42 and 56 days after the first presentation). The plasma activity of creatine kinase was measured at the last re-examination in 29 cows similar to measurement at day 0. RESULTS: The observed overall success rate of treatment of cows with UTN was considerably higher compared with untreated cows (Cast 98 % and StV 100 % vs. 0Cast 62 %). By comparison, the observed difference between treated and untreated cows with BTN was not so clear (78 % vs. 67 %). Recovering cows exhibited a calculated longer median survival time than cows that did not recover (545 d vs. 100 d). Plasma creatine kinase activities were increased initially and returned within the reference interval (434 U/l and 152 U/L) following treatment. CONCLUSIONS: Cows with ETN have an excellent prognosis provided that treatment with anti-inflammatory drugs and stabilizing bandage is administered. In cows with BTN, the prognosis depended on the type and degree of the primary injury. Loss of skin sensitivity indicated a poor prognosis. From an economic standpoint, treatment of TN is indicated provided that the prognosis is good. In cows that had healed clinically, the average survival time extended into the following lactation. CLINICAL RELEVANCE: This study highlights the advantages of a support bandage for the treatment of cows with TN. Compared with other peripheral neuropathies, muscle damage appears to be of particular importance in TN.

Identification of a large repetitive RTX immunogen in a highly virulent Rodentibacter heylii strain.

Rodentibacter (R.) heylii is frequently detected in laboratory rodents. Repeats in toxin (RTX) toxins are considered important virulence factors of this major murine pathogen. We evaluated the virulence of a R.heylii strain negative for all known RTX toxin genes and Muribacter (M.) muris, a commensal in mice, in experimental infections of C57BL/6 and BALB/c mice. Experimental intranasal infection with 108 CFU of the pnxI-, pnxII- and pnxIII- R. heylii strain resulted in 75% and 100% mortality in C57BL/6 and BALB/c mice, respectively. In early losses, multiple internal organs were infected and purulent bronchopneumonia was the main pathology. Intranasal application of M. muris did not result in mortality or severe weight loss. Immunoproteomics led to the identification of a surface-associated and specific immunogen, which was designated as R. heylii immunogen A (RhiA) and which was exclusively recognised by sera obtained from mice infected with this R. heylii pathotype. RhiA is a 262.6 kDa large protein containing long imperfect tandem repeats and C-terminal RTX consensus sequences. Immunohistochemical analysis confirmed that this R.heylii pathotype expresses RhiA in the lower respiratory tract. In summary, this study describes a specific immunogen in a virulent R. heylii, strain which is an excellent antigen for pathotype-specific serological screenings and which might carry out RTX-related functions.

A Comparative Transcriptome Analysis of Human and Porcine Choroid Plexus Cells in Response to Streptococcus suis Serotype 2 Infection Points to a Role of Hypoxia.

Streptococcus suis (S. suis) is an important opportunistic pathogen, which can cause septicemia and meningitis in pigs and humans. Previous in vivo observations in S. suis-infected pigs revealed lesions at the choroid plexus (CP). In vitro experiments with primary porcine CP epithelial cells (PCPEC) and human CP epithelial papilloma (HIBCPP) cells demonstrated that S. suis can invade and traverse the CP epithelium, and that the CP contributes to the inflammatory response via cytokine expression. Here, next generation sequencing (RNA-seq) was used to compare global transcriptome profiles of PCPEC and HIBCPP cells challenged with S. suis serotype (ST) 2 infected in vitro, and of pigs infected in vivo. Identified differentially expressed genes (DEGs) were, amongst others, involved in inflammatory responses and hypoxia. The RNA-seq data were validated via quantitative PCR of selected DEGs. Employing Gene Set Enrichment Analysis (GSEA), 18, 28, and 21 enriched hallmark gene sets (GSs) were identified for infected HIBCPP cells, PCPEC, and in the CP of pigs suffering from S. suis ST2 meningitis, respectively, of which eight GSs overlapped between the three different sample sets. The majority of these GSs are involved in cellular signaling and pathways, immune response, and development, including inflammatory response and hypoxia. In contrast, suppressed GSs observed during in vitro and in vivo S. suis ST2 infections included those, which were involved in cellular proliferation and metabolic processes. This study suggests that similar cellular processes occur in infected human and porcine CP epithelial cells, especially in terms of inflammatory response.

[Detection of Actinobacillus equuli ssp. equuli in piglets with purulent polyarthritis and tendovaginitis]. / Nachweis von Actinobacillus equuli ssp. equuli bei Saugferkeln mit eitriger Polyarthritis und Tendovaginitis.

Actinobacillus equuli ssp. equuli is an opportunistic pathogen in horses, mainly known to cause "sleepy foal disease". In comparison to horses, there are only few reports describing diseases in pigs associated with this gram-negative bacterium. This case report describes an outbreak of infection in a combined farrow-to-finish-farm. In September 2018, the following symptoms were noticed in one third of all newborn piglets from gilts and sows: 6-8 hours after birth piglets became weak and developed swollen joints with moderate to severe lameness. The piglets exhibited lethargy, a subset were non-ambulatory. An elevated piglet mortality within the first days within birth was noted. Seven piglets that succumbed to the disease (days 2-3 of life) were submitted for examination, 4 of which underwent pathological examination. The main findings were purulent polyarthritis and tendovaginitis. In addition, purulent inflammation was detected in the brain and kidneys of one animal. In the bacteriological examination A. equuli ssp. equuli was isolated in a total of 18 samples (brain, joints, suppurative structures of limbs), in a subset of cases as pure culture. For identification, cultural and biochemical characteristics were tested and a mass spectrometry analysis (MALDI-TOF MS) was performed. Further laboratory testing included 16 S rRNA-gene sequencing, a PCR in order to examine for special apx toxin genes as well as a PCR differentiating the two subspecies of A. equuli. It was not possible to identify the source of infection and routes of spread within the pig herd. The bacterial isolates were used for the production of an autogenous vaccine.

Borna disease outbreak with high mortality in an alpaca herd in a previously unreported endemic area in Germany.

Borna disease virus 1 (BoDV-1) is the causative agent of Borna disease, an often fatal neurologic condition of domestic mammals, including New World camelids, in endemic areas in Central Europe. Recently, BoDV-1 gained further attention by the confirmation of fatal zoonotic infections in humans. Although Borna disease and BoDV-1 have been described already over the past decades, comprehensive reports of Borna disease outbreaks in domestic animals employing state-of-the-art diagnostic methods are missing. Here, we report a series of BoDV-1 infections in a herd of 27 alpacas (Vicugna pacos) in the federal state of Brandenburg, Germany, which resulted in eleven fatalities (41%) within ten months. Clinical courses ranged from sudden death without previous clinical signs to acute or chronic neurologic disease with death occurring after up to six months. All animals that underwent necropsy exhibited a non-suppurative encephalitis. In addition, six apparently healthy seropositive individuals were identified within the herd, suggesting subclinical BoDV-1 infections. In infected animals, BoDV-1 RNA and antigen were mainly restricted to the central nervous system and the eye, and sporadically detectable in large peripheral nerves and neuronal structures in other tissues. Pest control measures on the farm resulted in the collection of a BoDV-1-positive bicoloured white-toothed shrew (Crocidura leucodon), while all other trapped small mammals were negative. A phylogeographic analysis of BoDV-1 sequences from the alpacas, the shrew and BoDV-1-positive equine cases from the same region in Brandenburg revealed a previously unreported endemic area of BoDV-1 cluster 4 in North-Western Brandenburg. In conclusion, alpacas appear to be highly susceptible to BoDV-1 infection and display a highly variable clinical picture ranging from peracute death to subclinical forms. In addition to horses and sheep, they can serve as sensitive sentinels used for the identification of endemic areas.

Vaccination with the immunoglobulin M-degrading enzyme of Streptococcus suis, Ide Ssuis, leads to protection against a highly virulent serotype 9 strain.

Vaccination of weaning piglets with the recombinant IgM degrading enzyme of Streptococcus suis (S. suis), rIde Ssuis, elicits protection against disease caused by serotype (cps) 2 infection. In Europe, S. suis cps9 is at least as important as cps2 in causing severe herd problems associated with meningitis, septicemia and arthritis. The objective of this study was to determine humoral and cellular immunogenicities of rIde Ssuis suckling piglet vaccination and to investigate protection against a virulent cps9 strain. Vaccination in the 2nd and 4th week of life with rIde Ssuis and an oil-in-water adjuvant induced seroconversion against Ide Ssuis in 13 of 20 vaccinated piglets. In the 5th week, survival of the S. suis cps9 strain was significantly reduced in the blood of prime-booster vaccinated piglets. After a 2nd booster vaccination Ide Ssuis -reactive T helper (Th) cells partially producing TNF-α, IL-17A or IFN-É£ were detectable in rIde Ssuis -vaccinated but not in placebo-treated piglets and frequencies of Ide Ssuis -reactive Th cells correlated with α-Ide Ssuis-IgG levels. An intravenous challenge, conducted with a cps9 strain of sequence type (ST) 94, led to 89% mortality in placebo-treated piglets due to septicemia and meningitis. In contrast, all rIde Ssuis prime-booster-booster vaccinated littermates survived the challenge despite signs of disease such as fever and lameness. In conclusion, the described rIde Ssuis vaccination induces humoral and detectable Ide Ssuis -reactive Th cell responses and leads to protection against a highly virulent cps9 strain.

Effect of the duration of estradiol priming prior to progesterone administration on endometrial gene expression in anestrous mares.

Field data indicate that a longer period of estrus prior to ovulation correlates positively with fertility. To test the hypothesis that the duration of exposure to estrogens prior to progesterone dominance influences endometrial function, we used anestrous mares to simulate varying durations of estrus (3 groups of 5 mares): long (LE), short (SE), and no estrus (NE), as determined by the duration of estradiol priming prior to progesterone treatment: 7, 2 and 0 days for the LE, SE and NE, respectively. Endometrial biopsies were recovered 4 days after progesterone administration in all groups for real time quantitative reverse transcription PCR (RT-qPCR) and immunohistochemical analyses. A total of 17 genes believed to contribute to a "receptive endometrium" for embryo development and viability were evaluated by RT-qPCR. Of the genes evaluated, the expression of FGF-2 (fibroblast growth factor-2) decreased with increased length of preceding estrus, whereas P19 (uterocalin) expression was higher in the LE than in the SE or NE groups. In conclusion, a lower abundance of FGF-2 and higher abundance of uterocalin, a lipocalin protein known to play an important role in providing lipids to the embryo, could contribute to a more receptive endometrium in mares following a long estrus.

Mesenchymal Stromal Cells Cultured in Serum from Heart Failure Patients Are More Resistant to Simulated Chronic and Acute Stress.

Despite regulatory issues surrounding the use of animal-derived cell culture supplements, most clinical cardiac cell therapy trials using mesenchymal stromal cells (MSCs) still rely on fetal bovine serum (FBS) for cell expansion before transplantation. We sought to investigate the effect of human serum from heart failure patients (HFS) on cord blood MSCs (CB-MSCs) during short-term culture under regular conditions and during simulated acute and chronic stress. Cell survival, proliferation, metabolic activity, and apoptosis were quantified, and gene expression profiles of selected apoptosis and cell cycle regulators were determined. Compared to FBS, HFS and serum from healthy donors (CS) showed similar effects by substantially increasing cell survival during chronic and acute stress and by increasing cell yields 5 days after acute stress. Shortly after the termination of acute stress, both HFS and CS resulted in a marked decrease in apoptotic cells. Transcriptome analysis suggested a decrease in TNF-mediated induction of caspases and decreased activation of mitochondrial apoptosis. Our data confirm that human serum from both healthy donors and heart failure patients results in increased cell yields and increased resistance to cellular stress signals. Therefore, we consider autologous serum a valid alternative to FBS in cell-based therapies addressing severe heart disease.

Comparative analysis of clinics, pathologies and immune responses in BALB/c and C57BL/6 mice infected with Streptobacillus moniliformis.

Streptobacillus (S.) moniliformis is a rat-associated zoonotic pathogen that occasionally causes disease in other species. We investigated the working hypothesis that intranasal infection might lead to different immune responses in C57BL/6 and BALB/c mice associated with distinct pathologies. This study confirmed with 75% mortality the known high susceptibility of C57BL/6 mice to Streptobacillus moniliformis infection in comparison to BALB/c mice which did not develop signs of disease. Main pathologies in C57BL/6 mice were purulent to necrotizing lymphadenitis and pneumonia. Significant seroconversion was recorded in surviving mice of both strains. Differentiation of IgG-subclasses revealed mean ratios of IgG2b to IgG1 below 0.5 in sera of all mice prior to infection and of BALB/c mice post infection. In contrast, C57BL/6 mice had a mean IgG2b/IgG1 ratio of 2.5 post infection indicating a Th1 immune response in C57BL/6 versus a Th2 response in BALB/c mice. Evaluation of different sentinel systems revealed that cultural and serological investigations of these animals might not be sufficient to detect infection. In summary, an intranasal S. moniliformis infection model in C57BL/6 mice leading to purulent to necrotizing inflammations in the lung, the lymph nodes and other organs associated with a Th1 immune response is described.

A Novel Squirrel Respirovirus with Putative Zoonotic Potential.

In a globalized world, the threat of emerging pathogens plays an increasing role, especially if their zoonotic potential is unknown. In this study, a novel respirovirus, family Paramyxoviridae, was isolated from a Sri Lankan Giant squirrel (Ratufa macroura), which originated in Sri Lanka and deceased with severe pneumonia in a German zoo. The full-genome characterization of this novel virus, tentatively named Giant squirrel respirovirus (GSqRV), revealed similarities to murine (71%), as well as human respiroviruses (68%) with unique features, for example, a different genome length and a putative additional accessory protein. Congruently, phylogenetic analyses showed a solitary position of GSqRV between known murine and human respiroviruses, implicating a putative zoonotic potential. A tailored real-time reverse transcription-polymerase chain reaction (RT-qPCR) for specific detection of GSqRV confirmed a very high viral load in the lung, and, to a lesser extent, in the brain of the deceased animal. A pilot study on indigenous and exotic squirrels did not reveal additional cases in Germany. Therefore, further research is essential to assess the geographic distribution, host range, and zoonotic potential of this novel viral pathogen.