An ultrasound-guided serratus anterior plane block with continuous local anaesthetic infusion and epidural analgesia for rib fracture pain.

BACKGROUND: We compared analgesia with an ultrasound (US)-guided serratus anterior plane block (SAPB) to thoracic epidural analgesia (EA) with continuous local anaesthetic infusion in patients with unilateral multiple traumatic rib fractures. EA often carries contraindications in patients with multiple rib fractures (MRFs), whereby having alternative effective methods to treat rib fracture pain remains important to patient care. Thus, we hypothesised that both regional anaesthetic techniques would provide effective pain relief. METHODS: In this study, we included 59 patients with unilateral MRFs and a numerical rating scale (NRS) pain score ≥4 at rest or upon movement. Patients were randomised to receive a US-guided SAPB or continuous infusion EA with 2 mg/mL ropivacaine. Patients were given a patient-controlled analgesia (PCA) device with intravenous oxycodone boluses for rescue medication. The primary outcome was a change in the NRS score at rest and upon movement from baseline to Day 2 following the block. We also measured the forced expiratory volume in 1 s of expiration (FEV1) and FEV1% at the same time points when NRS was measured. The total consumption of oxycodone with PCA was measured at 24 and 48 h after the block. RESULTS: We detected a significant reduction (≥2) in NRS for both groups; however, EA associated with a greater reduction in NRS upon movement after block initiation. The mean reduction in NRS upon movement within 1 h was 3 (1.8, p < .01) in the SAPB group versus 4.7 (2.4, p < .01) in the EA group. We found no significant difference between groups in pain scores on Days 1 and 2 following the block. In the EA group, FEV1% increase in the first 12 h from baseline. Finally, PCA oxycodone consumption did not differ between groups. CONCLUSIONS: SAPB with continuous local anaesthetic infusion is an effective alternative to treat rib fracture pain when EA is contraindicated. We found that SABP significantly reduces pain in patients with unilateral MRFs, although EA achieves better analgesia over the first 12 h following the block.

Phenotypic characterization of Eimeria tenella-specific chicken T-cells responding to in vitro parasite antigen re-stimulation.

Introduction. Coccidiosis, caused by protozoan parasites of genus Eimeria, is a disease with large impact on poultry production worldwide. It is well known that Eimeria immunity is dependent on Th1-type responses.Gap Statement. In vitro assessment of Eimeria-specific T-cell activity would therefore be a valuable research tool but has so far proven difficult to establish.Aim. The present study aimed to evaluate in vitro induced blast transformation and CD25 expression in defined chicken T-cell populations as a measure of Eimeria immunity.Methodology. Three E. tenella infection experiments were performed and PBMC and/or spleen cells were collected between 6 and 16 days after infection of chickens. Cells were stimulated in vitro with E. tenella antigens and T-cell activation was assessed by immunofluorescence labelling and flow cytometry.Results. The results consistently showed statistically significant E. tenella specific activation of TCRα/ß+T cells within a 'window' from 8 to 14 days after infection for both spleen cells and PBMC. Responding T-cells were identified as CD4+CD8-, CD4+CD8αα+ and CD4-CD8αß+ where the CD4+CD8αα+ cells generally showed the highest responses. All three of these TCRα/ßT-cell subsets showed significant E. tenella induced blast transformation and/or CD25 expression albeit not always in concert on the same days after infection indicating complex kinetics of T-cell responses. In general, responses were higher for spleen cells compared to PBMC for all responding T-cell populations.Conclusions. This methodology shows promise to study Eimeria-specific T-cells, e.g. to evaluate vaccine responses. Results indicated that a Th1-type response was induced and suggested a role for CD4+CD8αα+ cells in Eimeria immunity.

Systematic Review and Modelling of Age-Dependent Prevalence of Toxoplasma gondii in Livestock, Wildlife and Felids in Europe.

Toxoplasma gondii is a zoonotic parasite of importance to both human and animal health. The parasite has various transmission routes, and the meat of infected animals appears to be a major source of human infections in Europe. We aimed to estimate T. gondii prevalence in a selection of animal host species. A systematic literature review resulting in 226 eligible publications was carried out, and serological data were analyzed using an age-dependent Bayesian hierarchical model to obtain estimates for the regional T. gondii seroprevalence in livestock, wildlife, and felids. Prevalence estimates varied between species, regions, indoor/outdoor rearing, and types of detection methods applied. The lowest estimated seroprevalence was observed for indoor-kept lagomorphs at 4.8% (95% CI: 1.8-7.5%) and the highest for outdoor-kept sheep at 63.3% (95% CI: 53.0-79.3%). Overall, T. gondii seroprevalence estimates were highest within Eastern Europe, whilst being lowest in Northern Europe. Prevalence data based on direct detection methods were scarce and were not modelled but rather directly summarized by species. The outcomes of the meta-analysis can be used to extrapolate data to areas with a lack of data and provide valuable inputs for future source attribution approaches aiming to estimate the relative contribution of different sources of T. gondii human infection.

Dual RNA-seq transcriptome analysis of caecal tissue during primary Eimeria tenella infection in chickens.

BACKGROUND: Coccidiosis is an infectious disease with large negative impact on the poultry industry worldwide. It is an enteric infection caused by unicellular Apicomplexan parasites of the genus Eimeria. The present study aimed to gain more knowledge about interactions between parasites and the host immune system during the early asexual replication phase of E. tenella in chicken caeca. For this purpose, chickens were experimentally infected with E. tenella oocysts, sacrificed on days 1-4 and 10 after infection and mRNA from caecal tissues was extracted and sequenced. RESULTS: Dual RNA-seq analysis revealed time-dependent changes in both host and parasite gene expression during the course of the infection. Chicken immune activation was detected from day 3 and onwards with the highest number of differentially expressed immune genes recorded on day 10. Among early (days 3-4) responses up-regulation of genes for matrix metalloproteinases, several chemokines, interferon (IFN)-γ along with IFN-stimulated genes GBP, IRF1 and RSAD2 were noted. Increased expression of genes with immune suppressive/regulatory effects, e.g. IL10, SOCS1, SOCS3, was also observed among early responses. For E. tenella a general up-regulation of genes involved in protein expression and energy metabolism as well as a general down-regulation genes for DNA and RNA processing were observed during the infection. Specific E. tenella genes with altered expression during the experiment include those for proteins in rhoptry and microneme organelles. CONCLUSIONS: The present study provides novel information on both the transcriptional activity of E. tenella during schizogony in ceacal tissue and of the local host responses to parasite invasion during this phase of infection. Results indicate a role for IFN-γ and IFN-stimulated genes in the innate defence against Eimeria replication.

Dual RNA-Seq transcriptome analysis of chicken macrophage-like cells (HD11) infected in vitro with Eimeria tenella.

The study aimed to monitor parasite and host gene expression during the early stages of Eimeria tenella infection of chicken cells using dual RNA-Seq analysis. For this, we used chicken macrophage-like cell line HD11 cultures infected in vitro with purified E. tenella sporozoites. Cultures were harvested between 2 and 72 h post-infection and mRNA was extracted and sequenced. Dual RNA-Seq analysis showed clear patterns of altered expression for both parasite and host genes during infection. For example, genes in the chicken immune system showed upregulation early (2­4 h), a strong downregulation of genes across the immune system at 24 h and a repetition of early patterns at 72 h, indicating that invasion by a second generation of parasites was occurring. The observed downregulation may be due to immune self-regulation or to immune evasive mechanisms exerted by E. tenella. Results also suggested pathogen recognition receptors involved in E. tenella innate recognition, MRC2, TLR15 and NLRC5 and showed distinct chemokine and cytokine induction patterns. Moreover, the expression of several functional categories of Eimeria genes, such as rhoptry kinase genes and microneme genes, were also examined, showing distinctive differences which were expressed in sporozoites and merozoites.

Seroprevalence of Toxoplasma gondii in domestic pigs, sheep, cattle, wild boars, and moose in the Nordic-Baltic region: A systematic review and meta-analysis.

BACKGROUND: Toxoplasma gondii is an important foodborne zoonotic parasite. Meat of infected animals is presumed to constitute a major source of human infection and may be a driver of geographical variation in the prevalence of anti-T. gondii antibodies in humans, which is substantial in the Nordic-Baltic region in northern Europe. However, data on seroprevalence of T. gondii in different animal species used for human consumption are scattered. METHODS: We conducted a systematic review of seroprevalence studies and meta-analysis to estimate the seroprevalence of T. gondii in five animal species that are raised or hunted for human consumption in the Nordic-Baltic region: domestic pigs (Sus scrofa domesticus), sheep (Ovis aries), cattle (Bos taurus), wild boars (Sus scrofa), and moose (Alces alces). We searched for studies that were conducted between January 1990 and June 2018, and reported in articles, theses, conference abstracts and proceedings, and manuscripts. Subgroup analyses were performed to identify variables influencing the seroprevalence. FINDINGS: From a total of 271 studies identified in the systematic review, 32 were included in the meta-analysis. These comprised of 13 studies on domestic pigs, six on sheep, three on cattle, six on wild boars, and four on moose. The estimated pooled seroprevalence of T. gondii was 6% in domestic pigs (CI95%: 3-10%), 23% in sheep (CI95%: 12-36%), 7% in cattle (CI95%: 1-21%), 33% in wild boars (CI95%: 26-41%), and 16% in moose (CI95%: 10-23%). High heterogeneity was observed in the seroprevalence data within each species. In all host species except wild boars, the pooled seroprevalence estimates were significantly higher in animals >1 year of age than in younger animals. Not all studies provided information on animal age, sensitivity and specificity of the serological method employed, and the cut-off values used for defining an animal seropositive. CONCLUSIONS: A substantial proportion of animals raised or hunted for human consumption in the region had tested positive for T. gondii. This indicates widespread exposure to T. gondii among animals raised or hunted for human consumption in the region. Large variations were observed in the seroprevalence estimates between the studies in the region; however, studies were too few to identify spatial patterns at country-level.

Parasite-specific proliferative responses of chicken spleen cells upon in vitro stimulation with Eimeria tenella antigen.

This study aimed to set up methodology to monitor parasite-specific T-cell activation in vitro using Eimeria tenella-infected chickens. A sonicated E. tenella sporozoite protein preparation was used for the activation of chicken spleen cell cultures. Proliferation assessed by 3H-thymidin incorporation or blast transformation of T-cells assessed by immunofluorescence labelling and flow cytometry were used as read-outs for activation. Results showed that E. tenella-specific proliferation was detected in cultures of spleen cells collected in a 'window' between 8 and 14 days after primary infection. However, due to high variation in proliferative responses between individuals and to high background proliferation, large numbers of observations were needed to obtain significant results. Moreover, the outcome was not improved by increasing the infection dose to chickens or by depletion of T-cell receptor (TCR) γ/δ expressing cells from cultures. An E. tenella-specific blast transformation response was observed for TCRα/ß expressing cells within the same 'window', confirming the identity of the responding cells as classic T-cells. Thus, it is possible to study the kinetics of E. tenella-specific T-cell responses in vitro. However, more in-depth phenotypic identification of the responding T-cells could improve the methodology.

Serglycin proteoglycans limit enteropathy in Trichinella spiralis-infected mice.

BACKGROUND: Serglycin proteoglycans are essential for maturation of secretory granules and for the correct granular storage of cationic proteases in hematopoietic cells, e.g. mast cells. However, little is known about the in vivo functions of serglycin proteoglycans during infection. Here we investigated the potential role of serglycin proteoglycans in host defense after infection with the nematode Trichinella spiralis. RESULTS: Twelve days post infection lack of serglycin proteoglycans caused significantly increased enteropathy. The serglycin-deficient mice showed significantly increased intestinal worm burden, reduced recruitment of mast cells to the intestinal crypts, decreased levels of the mast cell proteases MCPT5 and MCPT6 in intestinal tissue, decreased serum levels of TNF-α, IL-1ß, IL-10 and IL-13, increased levels of IL-4 and total IgE in serum, and increased intestinal levels of the neutrophil markers myeloperoxidase and elastase, as compared to wild type mice. At five weeks post infection, increased larvae burden and inflammation were seen in the muscle tissue of the serglycin-deficient mice. CONCLUSIONS: Our results demonstrate that the serglycin-deficient mice were more susceptible to T. spiralis infection and displayed an unbalanced immune response compared to wild type mice. These findings point to an essential regulatory role of serglycin proteoglycans in immunity.

Pasture is a risk factor for Toxoplasma gondii infection in fattening pigs.

As consumer awareness of animal welfare increases throughout Europe, housing of pigs in more animal-friendly systems is becoming more common. There is concern that these free-range and organic management systems increase the prevalence of zoonotic meat-borne pathogens, such as Toxoplasma gondii. In this study we compared the seroprevalence of T. gondii between commercial fattening pigs raised on conventional and on organic farms in Sweden. Furthermore, potential associations between presence of T. gondii antibodies and type of production, access to pasture, and geographical region were analysed. A significant difference in T. gondii seroprevalence was found between conventional (1%) and organic pigs (8%). The higher odds of seropositivity in organic production was attributed to pasture access specifically (OR=1.8 for a one-month increase in length of pasture exposure). This study shows that the prevalence of T. gondii in Swedish conventional pigs is low. However, as pigs with access to pasture are at higher risk of infection and because the demand for animal-friendly production systems is increasing, there is an obvious need to practically manage the higher T. gondii presence in products from pigs raised in organic systems with outdoor access.

CD107a as a marker of activation in chicken cytotoxic T cells.

The study aimed to evaluate cell surface mobilisation of CD107a as a general activation marker on chicken cytotoxic T cells (CTL). Experiments comprised establishment of an in vitro model for activation-induced CD107a mobilisation and design of a marker panel for the detection of CD107a mobilisation on chicken CTL isolated from different tissues. Moreover, CD107a mobilisation was analysed on CTL isolated from airways of infectious bronchitis virus (IBV)-infected birds direct ex vivo and upon in vitro stimulation. Results showed that phorbol 12-myristate 13-acetate (PMA) in combination with ionomycin was a consistent inducer of CD107a cell surface mobilisation on chicken CTL in a 4h cell culture model. In chickens experimentally infected with IBV, higher frequencies of CTL isolated from respiratory tissues were positive for CD107a on the cell surface compared to those from uninfected control chickens indicating in vivo activation. Moreover, upon in vitro PMA+ ionomycin stimulation, higher proportions of CTL isolated from the airways of IBV-infected chickens showed CD107a mobilisation compared to those from uninfected control chickens. Monitoring of CD107a cell surface mobilisation may thus be a useful tool for studies of chicken CTL cytolytic potential both in vivo and in vitro.

"Meat juice" is not a homogeneous serological matrix.

Meat juice samples are used in serological assays to monitor infectious diseases within the food chain. However, evidence of inferior sensitivity, presumably due to low levels of antibodies in the meat juice compared to serum, has been presented, and it has been suggested that adjusting the dilution factor of meat juice in proportion to its blood content could improve sensitivity. In the present study, the agreement between Toxoplasma gondii-specific immunoglobulin G (IgG) levels in meat juice and serum was evaluated, and whether the level of immunoglobulins in meat juice was dependent on its blood content. Serum and meat juice from diaphragm, heart, tongue, Musculus triceps brachii and M. semitendinosus were collected from 20 pigs experimentally infected with T. gondii. Analysis of total IgG, heme-containing proteins (hematin), and hemoglobin (Hb) revealed significant differences between samples from different muscles, with the highest levels in samples from heart and tongue, and the lowest in samples from leg muscles. Comparison of T. gondii-specific antibody titers in meat juice and serum revealed a strong positive correlation for meat juice from heart (rs=0.87; p<0.001), while it was lower for M. semitendinosus (rs=0.71; p<0.001) and diaphragm (rs=0.54; p=0.02). Meanwhile, the correlation between total IgG and T. gondii titer ratio (meat juice/serum) was highest in diaphragm (rs=0.77; p<0.001) followed by M. semitendinosus (rs=0.64; p=0.005) and heart (rs=0.50; p=0.051). The correlation between Hb and T. gondii titer ratio was only significant for diaphragm (rs=0.65; p=0.008), and for hematin no significant correlation was recorded. In conclusion, the specific IgG titers in meat juice appeared to depend on the total IgG level, but the correlation to blood (Hb or hematin) was poor. Importantly, large significant differences in total IgG levels as well as in specific antibody titers were recorded, depending on the muscle the meat juice had been extracted from.

Serglycin-independent release of active mast cell proteases in response to Toxoplasma gondii infection.

Earlier studies identified serglycin proteoglycan and its heparin chains to be important for storage and activity of mast cell proteases. However, the importance of serglycin for secretion and activity of mast cell proteases in response to parasite infection has been poorly investigated. To address this issue, we studied the effects on mast cell proteases in serglycin-deficient and wild type mice after peritoneal infection with the obligate intracellular parasite Toxoplasma gondii. In line with previous results, we found severely reduced levels of cell-bound mast cell proteases in both noninfected and infected serglycin-deficient mice. However, serglycin-deficient mice secreted mast cell proteases at wild type levels at the site of infection, and enzymatic activities associated with mast cell proteases were equally up-regulated in wild type and serglycin-deficient mice 48 h after infection. In both wild type and serglycin-deficient mice, parasite infection resulted in highly increased extracellular levels of glycosaminoglycans, including hyaluronan and chondroitin sulfate A, suggesting a role of these substances in the general defense mechanism. In contrast, heparan sulfate/heparin was almost undetectable in serglycin-deficient mice, and in wild type mice, it was mainly confined to the cellular fraction and was not increased upon infection. Furthermore, the heparan sulfate/heparin population was less sulfated in serglycin-deficient than in wild type mice indicative for the absence of heparin, which supports that heparin production is dependent on the serglycin core protein. Together, our results suggest that serglycin proteoglycan is dispensable for normal secretion and activity of mast cell proteases in response to peritoneal infection with T. gondii.

Improving anaerobic and aerobic degradation by ultrasonic disintegration of biomass.

Biological cell lysis is known to be the rate-limiting step of anaerobic biosolids degradation. Due to the slow pace by which this reaction occurs, it is necessary to equip treatment plants with large digesters or alternatively incorporate technological aids. High-power ultrasound used to disintegrate bacterial cells has been utilized as a pre-treatment process prior to anaerobic digestion. Through this application, as seen on pilot- and full-scales, it is possible to attain up to 30% more biogas, an increase in VS-destruction of up to 30% and a reduced sludge mass for disposal. Utilizing ultrasound technology in aerobic applications is a new and innovative approach. Improved denitrification through a more readily available internal carbon source, and less excess sludge mass can be traced to the positive effects that sonication of sludge has on the overall biological wastewater treatment process. Reference full-scale installations suggest that the technology is both technically feasible and economically sound.

Immunogenicity and protective effect against murine cerebral neosporosis of recombinant NcSRS2 in different iscom formulations.

Recombinant NcSRS2, a major immunodominant surface antigen of the intracellular protozoan parasite Neospora caninum, was used as a model antigen to compare the immunogenicity of iscoms prepared according to three different methods. Two NcSRS2 fusion proteins were used, one that was biotinylated upon expression in Escherichia coli and linked to Ni2+-loaded iscom matrix (iscom without any protein) via a hexahistidyl (His6)-tagged streptavidin fusion protein, and another that contained both a His6-tag and streptavidin (His6-SA-SRS2') and was coupled to either Ni2+-loaded or biotinylated matrix. While all three iscom preparations induced N. caninum specific antibodies at similar levels, His6-SA-SRS2' coupled to biotinylated matrix generated the strongest cellular responses measured as in vitro proliferation and production of interferon-gamma and interleukin-4 after antigen stimulation of spleen cells. However, the relationship between the levels of these cytokines as well as between IgG1 and IgG2a titres in serum induced by the three iscom preparations were similar, indicating that the balance between Th1 and Th2 responses did not differ. After challenge infection, mice immunised with His6-SA-SRS2' coupled to biotinylated matrix had significantly lower amounts of parasite DNA in their brains compared to the other immunised groups. Possible reasons for the performance of the different iscom formulations are discussed.

Achieving directed immunostimulating complexes incorporation.

In recent years, several studies have been reported with the common aim of generating general expression systems for straightforward production and subsequent coupling of expressed antigens to an adjuvant system. Here, we describe a series of such efforts with a common theme of using gene fusion technology for association of recombinant antigens to immunostimulating complexes (iscoms). In the early stages of vaccine development, uniform antigen preparations are crucial to allow the comparison of immune responses to different antigens, or even subdomains thereof, and we believe that the described systems constitute an important development in this context.

Serological survey of Toxoplasma gondii infection in free-ranging Eurasian lynx (Lynx lynx) from Sweden.

To investigate the prevalence of Toxoplasma gondii infection in free-ranging Eurasian lynx (Lynx lynx) in Sweden, serosanguinous fluids and feces were collected from 207 carcasses of lynx killed or found dead from 1996 to 1998. Sera were tested for antibodies against T. gondii by the direct agglutination test, and 156 (75.4%) of the sera tested positive at antibody titers>or=40. Antibody prevalence was significantly lower in lynx originating from the northern parts of Sweden than in lynx from the more southern regions that are more densely populated by humans. Age-related differences also were found, with a significantly lower prevalence (55%) in juvenile (<1-yr-old) than in subadult and adult animals (82%). There was no significant difference in seroprevalence between males and females. Oocysts typical of T. gondii were not detected in any of the fecal samples.

Seroprevalences of Toxoplasma gondii and Neospora sp. infections in Swedish horses.

Sera from 414 Swedish horses were investigated for the presence of antibodies to Toxoplasma gondii and Neospora sp. by the T. gondii direct agglutination test (DAT), and an Neospora caninum iscom-ELISA. Five sera (1%) had a titre >1:40 in DAT, but when analysed by immunoblotting against T. gondii antigens only two of them were positive, giving a seroprevalence of 0.5%. Since the Neospora iscom ELISA had not been validated for equine sera it was used for an initial screening, and all sera with an optical density exceeding 0.200 absorbance units were selected for further investigation by immunoblot analysis. Of the 39 sera tested by immunoblotting, four reacted with at least two of the immunodominant Neospora antigens recognized by the positive control sera and were judged as positive, resulting in a seroprevalence of 1%. This is the first evidence of Neospora infection in Swedish horses. The study illustrates the necessity of critically evaluating results of serological analyses performed by methods that are not validated for the animal species under investigation.

Immunisation of mice against neosporosis with recombinant NcSRS2 iscoms.

The coccidian parasite Neospora caninum is an intracellular protozoan, causing abortion in cattle in many countries around the world. In this study, the protective potential of the major N. caninum surface antigen NcSRS2, expressed in Escherichia coli and formulated into immunostimulating complexes (iscoms), was investigated in an experimental mouse model. The recombinant protein was specially designed for binding to iscoms via biotin-streptavidin interaction. Two groups of 10 BALB/c mice were immunised twice, on days 0 and 28 with iscoms containing either the recombinant NcSRS2 (NcSRS2 iscoms) or similar iscoms with NcSRS2 substituted by an unrelated recombinant malaria peptide (M5) as a control (M5 iscoms). A third group of 10 age-matched BALB/c mice served as an uninfected control group. Immunisation with recombinant NcSRS2 iscoms resulted in production of substantial antibody titres against N. caninum antigen, while the mice immunised with M5 iscoms produced only very low levels of antibodies reacting with N. caninum antigen. After challenge infection with N. caninum tachyzoites on day 69, mice immunised with NcSRS2 iscoms showed only mild and transient symptoms, whereas the group immunised with M5 iscoms showed clinical symptoms until the end of the experiment at 31 days post inoculation. A competitive PCR assay detecting Nc5-repeats was applied to evaluate the level of parasite DNA in the brain. The amount of Nc5-repeats in the group vaccinated with NcSRS2 iscoms was significantly lower than in the control group given M5 iscoms. In conclusion, it was found that the recombinant NcSRS2 iscoms induced specific antibodies to native NcSRS2 and immunity sufficient to reduce the proliferation of N. caninum in the brains of immunised mice.

General strategies for efficient adjuvant incorporation of recombinant subunit immunogens.

We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic peptides or lipid tags to improve their capacity to be incorporated into an adjuvant formulation, e.g., immunostimulating complexes (iscoms). Recently, we also explored the strong interaction between biotin and streptavidin to achieve iscom association of recombinant immunogens. Plasmodium falciparum,Toxoplasma gondii and Neospora caninum antigens have served as model immunogens in the different studies. Generated fusion proteins have been found to be successfully incorporated into iscoms and high-titer antigen-specific antibody responses have been obtained upon immunization of mice. We believe that the different concepts presented, utilizing either hydrophobic peptide or lipid tags, or the recently explored biotin-streptavidin principle, offer convenient methods to achieve efficient adjuvant incorporation of recombinant immunogens.