Characterization of mycophage endolysin cell wall binding domains targeting Mycobacterium bovis peptidoglycan.

Mycophage endolysins are highly diverse and modular enzymes composed of domains involved in peptidoglycan binding and degradation. Mostly, they are characterized by a three-module design: an N-terminal peptidase domain, a central catalytic domain and a C-terminal peptidoglycan binding domain. Previously, the affinity of cell wall binding domains (CBDs) to the mycobacterial peptidoglycan layer was shown for some of these endolysins. In this study, an in depth screening was performed on twelve mycophage endolysins. The discovered CBDs were characterized for their binding affinity to Mycobacterium (M.) bovis bacille Calmette-Guérin (BCG), a largely unexplored target and an attenuated strain of M. bovis, responsible for bovine tuberculosis. Using homology-based annotation, only four endolysins showed the presence of a known peptidoglycan binding domain, the previously characterized pfam 01471 domain. However, analysis of the secondary structure aided by AlphaFold predictions revealed the presence of a C-terminal domain in the other endolysins. These were hypothesized as new, uncharacterized CBDs. Fusion proteins composed of these domains linked to GFP were constructed and positively assayed for their affinity to M. bovis BCG in a peptidoglycan binding assay. Moreover, two CBDs were able to fluorescently label M. bovis BCG in milk samples, highlighting the potential to further explore their possibility to function as CBD-based diagnostics.

Impact of phage predation on P. aeruginosa adhered to human airway epithelium: major transcriptomic changes in metabolism and virulence-associated genes.

Phage therapy is a promising adjunct therapeutic approach against bacterial multidrug-resistant infections, including Pseudomonas aeruginosa-derived infections. Nevertheless, the current knowledge about the phage-bacteria interaction within a human environment is limited. In this work, we performed a transcriptome analysis of phage-infected P. aeruginosa adhered to a human epithelium (Nuli-1 ATCC® CRL-4011™). To this end, we performed RNA-sequencing from a complex mixture comprising phage-bacteria-human cells at early, middle, and late infection and compared it to uninfected adhered bacteria. Overall, we demonstrated that phage genome transcription is unaltered by bacterial growth and phage employs a core strategy of predation through upregulation of prophage-associated genes, a shutdown of bacterial surface receptors, and motility inhibition. In addition, specific responses were captured under lung-simulating conditions, with the expression of genes related to spermidine syntheses, sulphate acquisition, biofilm formation (both alginate and polysaccharide syntheses), lipopolysaccharide (LPS) modification, pyochelin expression, and downregulation of virulence regulators. These responses should be carefully studied in detail to better discern phage-induced changes from bacterial responses against phage. Our results establish the relevance of using complex settings that mimics in vivo conditions to study phage-bacteria interplay, being obvious the phage versatility on bacterial cell invasion.

qDNase assay: A quantitative method for real-time assessment of DNase activity on coated surfaces.

DNase coatings show great potential to prevent biofilm formation in various applications of the medical implant, food and marine industry. However, straightforward and quantitative methods to characterize the enzymatic activity of these coatings are currently not available. We here introduce the qDNase assay, a quantitative, real-time method to characterize the activity of DNase coatings. The assay combines (1) the use of an oligonucleotide probe, which fluoresces upon cleavage by coated DNases, and (2) the continuous read-out of the fluorescent signal within a microplate fluorometer format. The combination of these two properties results in a real-time fluorescent signal that is used to directly quantify the activity of DNase coatings. As a proof of concept, bovine DNase I coatings were immobilized on titanium by means of chemical grafting and their activity was estimated at 3.87 × 10-4 U. To our knowledge, the qDNase assay provides the first approach to report the activity of a DNase coating in absolute DNase activity units. This assay will not only serve to compare existing DNase coating methods more accurately, but will also enable the rational design of new DNase coating methods in the future.

Advanced engineering of third-generation lysins and formulation strategies for clinical applications.

One of the possible solutions for the current antibiotic resistance crisis may be found in (often bacteriophage-derived) peptidoglycan hydrolases. The first clinical trials of these natural enzymes, coined here as first-generation lysins, are currently ongoing. Moving beyond natural endolysins with protein engineering established the second generation of lysins. In second-generation lysins, the focus lies on improving antibacterial and biochemical properties such as antimicrobial activity and stability, as well as expanding their activities towards Gram-negative pathogens. However, solutions to particular key challenges regarding clinical applications are only beginning to emerge in the third generation of lysins, in which protein and biochemical engineering efforts focus on improving properties relevant under clinical conditions. In addition, increasingly advanced formulation strategies are developed to increase the bioavailability, antibacterial activity, and half-life, and to reduce pro-inflammatory responses. This review focuses on third-generation and advanced formulation strategies that are developed to treat infections, ranging from topical to systemic applications. Together, these efforts may fully unlock the potential of lysin therapy and will propel it as a true antibiotic alternative or supplement.

Quinolinecarboxamides Inhibit the Replication of the Bovine Viral Diarrhea Virus by Targeting a Hot Spot for the Inhibition of Pestivirus Replication in the RNA-Dependent RNA Polymerase.

The bovine viral diarrhea virus (BVDV), a pestivirus from the family of Flaviviridae is ubiquitous and causes a range of clinical manifestations in livestock, mainly cattle. Two quinolinecarboxamide analogues were identified in a CPE-based screening effort, as selective inhibitors of the in vitro bovine viral diarrhea virus (BVDV) replication, i.e., TO505-6180/CSFCI (average EC50 = 0.07 µM, SD = 0.02 µM, CC50 > 100 µM) and TO502-2403/CSFCII (average EC50 = 0.2 µM, SD = 0.06 µM, CC50 > 100 µM). The initial antiviral activity observed for both hits against BVDV was corroborated by measuring the inhibitory effect on viral RNA synthesis and the production of infectious virus. Modification of the substituents on the quinolinecarboxamide scaffold resulted in analogues that proved about 7-fold more potent (average EC50 = 0.03 with a SD = 0.01 µM) and that were devoid of cellular toxicity, for the concentration range tested (SI = 3333). CSFCII resistant BVDV variants were selected and were found to carry the F224P mutation in the viral RNA-dependent RNA polymerase (RdRp), whereas CSFCI resistant BVDV carried two mutations in the same region of the RdRp, i.e., N264D and F224Y. Likewise, molecular modeling revealed that F224P/Y and N264D are located in a small cavity near the fingertip domain of the pestivirus polymerase. CSFC-resistant BVDV proved to be cross-resistant to earlier reported pestivirus inhibitors (BPIP, AG110, LZ37, and BBP) that are known to target the same region of the RdRp. CSFC analogues did not inhibit the in vitro activity of recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes (RCs). CSFC analogues likely interact with the fingertip of the pestivirus RdRp at the same position as BPIP, AG110, LZ37, and BBP. This indicates that this region is a "hot spot" for the inhibition of pestivirus replication.

Stem cell-derived hepatocytes: A novel model for hepatitis E virus replication.

BACKGROUND & AIMS: Yearly, approximately 20million people become infected with the hepatitis E virus (HEV) resulting in over 3million cases of acute hepatitis. Although HEV-mediated hepatitis is usually self-limiting, severe cases of fulminant hepatitis as well as chronic infections have been reported, resulting annually in an estimated 60,000 deaths. We studied whether pluripotent stem cell (PSC)-derived hepatocytes, mesodermal and/or neuroprogenitor cells support HEV replication. METHODS: Human PSC were differentiated towards hepatocyte-like cells, mesodermal cells and neuroprogenitors and subsequently infected with HEV. Infection and replication of HEV was analyzed by qRT-PCR, RNA in situ hybridization, negative strand RT-PCR, production of infectious virions and transfection with a transient HEV reporter replicon. RESULTS: PSC-derived hepatocytes supported the complete replication cycle of HEV, as demonstrated by the intracellular presence of positive and negative strand HEV RNA and the production of infectious virions. The replication of the virus in these cells was inhibited by the antiviral drugs ribavirin and interferon-α2b. In contrast to PSC-derived hepatocytes, PSC-derived mesodermal cells and neuroprogenitors only supported HEV replication upon transfection with a HEV subgenomic replicon. CONCLUSION: We demonstrate that PSC can be used to study the hepatotropism of HEV infection. The complete replication cycle of HEV can be recapitulated in infected PSC-derived hepatocytes. By contrast other germ layer cells support intracellular replication but are not infectable with HEV. Thus the early steps in the viral cycle are the main determinant governing HEV tissue tropism. PSC-hepatocytes offer a physiological relevant tool to study the biology of HEV infection and replication and may aid in the design of therapeutic strategies.

Functional elucidation of antibacterial phage ORFans targeting Pseudomonas aeruginosa.

Immediately after infection, virulent bacteriophages hijack the molecular machinery of their bacterial host to create an optimal climate for phage propagation. For the vast majority of known phages, it is completely unknown which bacterial functions are inhibited or coopted. Early expressed phage genome regions are rarely identified, and often filled with small genes with no homology in databases (so-called ORFans). In this work, we first analysed the temporal transcription pattern of the N4-like Pseudomonas-infecting phages and selected 26 unknown, early phage ORFans. By expressing their encoded proteins individually in the host bacterium Pseudomonas aeruginosa, we identified and further characterized six antibacterial early phage proteins using time-lapse microscopy, radioactive labelling and pull-down experiments. Yeast two-hybrid analysis gaveclues to their possible role in phage infection. Specifically, we show that the inhibitory proteins may interact with transcriptional regulator PA0120, the replicative DNA helicase DnaB, the riboflavin metabolism key enzyme RibB, the ATPase PA0657and the spermidine acetyltransferase PA4114. The dependency of phage infection on spermidine was shown in a final experiment. In the future, knowledge of how phages shut down their hosts as well ass novel phage-host interaction partners could be very valuable in the identification of novel antibacterial targets.

CRISPR-Cas12/Cas13: Bibliometric analysis and systematic review of its application in infectious disease detection.

BACKGROUND: Infectious diseases impose a significant burden on the global public health and economy, resulting in an estimated 15 million deaths out of 57 million annually worldwide. This study examines the current state of CRISPR-Cas12/Cas13 research, focusing on its applications in infectious disease detection and its evolutionary trajectory. METHODS: A bibliometric analysis and systematic review were conducted by retrieving CRISPR-Cas12/Cas13-related articles published between January 1, 2015 to December 31, 2022, from the Web of Science database. The research protocol was registered with International Platform of Registered Systematic Review and Meta-analysis Protocols (INPLASY202380062). RESULTS: Our search identified 1987 articles, of which, 1856 were included in the bibliometric analysis and 445 were used in qualitative analysis. The study reveals a substantial increase in scientific production on CRISPR-Cas12/Cas13, with an annual growth rate of 104.5%. The United States leads in the number of published articles. The systematic review identified 580 different diagnostic assays targeting 170 pathogens, with SARS-CoV-2 dominating with 158 assays. Recombinase polymerase amplification (RPA)/reverse transcription-RPA (RT-RPA) emerged as the predominant amplification method, while lateral flow assay was the most common readout method. Approximately 72% of the diagnostic assays developed are suitable for point-of-care testing. CONCLUSION: The rapid increase in research on CRISPR-Cas12/Cas13 between 2015 and 2022 suggests promising potential for advancements in infectious disease diagnosis. Given the numerous advantages of CRISPR-Cas technology for disease detection over other methods, and the dedicated efforts of scientists from around the world, it is reasonable to anticipate that CRISPR-Cas technology may emerge as a formidable alternative, offering the possibility of expedited point-of-care testing in the not-too-distant future.

Spatiotemporal analysis and forecasting of lumpy skin disease outbreaks in Ethiopia based on retrospective outbreak reports.

Introduction: Lumpy skin disease is a viral disease that affects cattle belonging to genus Capripoxvirus (Poxviridae) and lead to significant economic losses. Objective: The objective of this study was to evaluate the distribution of lumpy skin disease (LSD) outbreaks and predict future patterns based on retrospective outbreak reports in Ethiopia. Methods: Data were collected through direct communication with regional laboratories and a hierarchical reporting system from the Peasant Associations to Ministry of Agriculture. Time-series data for the LSD outbreaks were analyzed using classical additive time-series decomposition and STL decomposition. Four models (ARIMA, SARIMA, ETS, STLF) were also used to forecast the number of LSD outbreaks that occurred each month for the years (2021-2025) after the models' accuracy test was performed. Additionally, the space-time permutation model (STP) were also used to study retrospective space-time cluster analysis of LSD outbreaks in Ethiopia. Results: This study examined the geographical and temporal distribution of LSD outbreaks in Ethiopia from 2008 to 2020, reporting a total of 3,256 LSD outbreaks, 14,754 LSD-positive cases, 7,758 deaths, and 289 slaughters. It also covered approximately 68% of Ethiopia's districts, with Oromia reporting the highest LSD outbreaks. In the LSD's temporal distribution, the highest peak was reported following the rainy season in September to December and its lowest peak in the dry months of April and May. Out of the four models tested for forecasting, the SARIMA (3, 0, 0) (2, 1, 0) [12] model performed well for the validation data, while the STLF+Random Walk had a robust prediction for the training data. Thus, the SARIMA and STLF+Random Walk models produced a more accurate forecast of LSD outbreaks between 2020 and 2025. From retrospective Space-Time Cluster Analysis of LSD, eight possible clusters were also identified, with five of them located in central part of Ethiopia. Conclusion: The study's time series and ST-cluster analysis of LSD outbreak data provide valuable insights into the spatial and temporal dynamics of the disease in Ethiopia. These insights can aid in the development of effective strategies to control and prevent the spread of the disease and holds great potential for improving efforts to combat LSD in the country.

An in vivo duck hepatitis B virus model recapitulates key aspects of nucleic acid polymer treatment outcomes in chronic hepatitis B patients.

Nucleic acid polymers (NAPs) are an attractive treatment modality for chronic hepatitis B (CHB), with REP2139 and REP2165 having shown efficacy in CHB patients. A subset of patients achieve functional cure, whereas the others exhibit a moderate response or are non-responders. NAP efficacy has been difficult to recapitulate in animal models, with the duck hepatitis B virus (DHBV) model showing some promise but remaining underexplored for NAP efficacy testing. Here we report on an optimized in vivo DHBV duck model and explore several characteristics of NAP treatment. REP2139 was efficacious in reducing DHBV DNA and DHBsAg levels in approximately half of the treated ducks, whether administered intraperitoneally or subcutaneously. Intrahepatic or serum NAP concentrations did not correlate with efficacy, nor did the appearance of anti-DHBsAg antibodies. Furthermore, NAP efficacy was only observed in experimentally infected ducks, not in endogenously infected ducks (vertical transmission). REP2139 add-on to entecavir treatment induced a deeper and more sustained virological response compared to entecavir monotherapy. Destabilized REP2165 showed a different activity profile with a more homogenous antiviral response followed by a faster rebound. In conclusion, subcutaneous administration of NAPs in the DHBV duck model provides a useful tool for in vivo evaluation of NAPs. It recapitulates many aspects of this class of compound's efficacy in CHB patients, most notably the clear division between responders and non-responders.

Viral Protein 1 (VP1) Sequence-Based Genetic Diversity of SAT 2 FMDV Circulating in Ethiopia from 1990 to 2015.

Introduction: Pathogen molecular epidemiology determines the origin of specific outbreaks locality of foot-and-mouth disease virus serotype South African Territories-2 sequence-based analysis of highly variable Viral Protein 1 (VP1), which helps to identify the evolution of this virus through time and space. The objective of this study was to compare the differences between SAT-2 VP1 sequences of FMDV circulated in Ethiopia from 1990 to 2015 at the genetic level. Methods: The nucleotide and amino acid sequences were analyzed using Basic Local Alignment Search Tools (BLAST), Multiple sequence alignment and sequence editing and Phylogenetic tree reconstruction. The nucleotide and amino acid sequences alignment, distance matrix, and phylogenetic tree constructions were done using the MEGA 6.0 software package. Result and Discussion: In this analysis, we found 76% nucleotide identities and amino acid similarities among the sequences. The overall group mean distance at nucleotide level was 19% with a mean intra-population diversity of 2%. The lowest sequence variation was observed among sequences obtained from the years 2007/09/10, 2014/15, and 1990/91 which was less than 5% among them. This analysis revealed that in the last 25 years, four different topotypes of the FMDV SAT-2 were circulating in Ethiopia. The Arg-Gly-Asp (RGD) amino acid (AA) motif at AA position 144-146 within the G-H loop of the VP1 protein of FMDV is conserved, but up- and downstream hyper-variable AA sequences are identified. In this study, it was observed that four topotypes (IV, XIV, XIII, and VII) were circulating in Ethiopia for 25 years. Further, compared with sequences from neighboring countries (Sudan, Kenya) confirmed the presence of these topotypes. Conclusion: Pertinent to this genetic diversity control strategies in Ethiopia should be based on having regular antigenic and genetic vaccine matching tests with the circulating strain within a defined period, space, transboundary nature of the disease and applying biosecurity measures.

Influence of Live Attenuated Salmonella Vaccines on Cecal Microbiome Composition and Microbiota Abundances in Young Broiler Chickens.

Salmonellosis is a global food safety challenge caused by Salmonella, a gram-negative bacterium of zoonotic importance. Poultry is considered a major reservoir for the pathogen, and humans are exposed through consumption of raw or undercooked products derived from them. Prophylaxis of Salmonella in poultry farms generally mainly involves biosecurity measures, flock testing and culling, use of antibiotics, and vaccination programs. For decades, the use of antibiotics has been a common practice to limit poultry contamination with important pathogenic bacteria such as Salmonella at the farm level. However, due to an increasing prevalence of resistance, non-therapeutic use of antibiotics in animal production has been banned in many parts of the world. This has prompted the search for non-antimicrobial alternatives. Live vaccines are among the developed and currently used methods for Salmonella control. However, their mechanism of action, particularly the effect they might have on commensal gut microbiota, is not well understood. In this study, three different commercial live attenuated Salmonella vaccines (AviPro® Salmonella Vac T, AviPro® Salmonella DUO, and AviPro® Salmonella Vac E) were used to orally vaccinate broiler chickens, and cecal contents were collected for microbiomes analysis by 16S rRNA next generation sequencing. Quantitative real-time PCR (qPCR) was used to study the cecal immune-related genes expression in the treatment groups, while Salmonella-specific antibodies were analyzed from sera and cecal extracts by enzyme-linked immunosorbent assay (ELISA). We show that vaccination with live attenuated Salmonella vaccines had a significant influence on the variability of the broiler cecal microbiota (p = 0.016). Furthermore, the vaccines AviPro® Salmonella Vac T and AviPro® Salmonella DUO, but not AviPro® Salmonella Vac E, had a significant effect (p = 0.024) on microbiota composition. This suggests that the live vaccine type used can differently alter the microbiota profiles, driving the gut colonization resistance and immune responses to pathogenic bacteria, and might impact the overall chicken health and productivity. Further investigation is, however, required to confirm this.

The phage-encoded protein PIT2 impacts Pseudomonas aeruginosa quorum sensing by direct interaction with LasR.

In recent decades, there has been a notable increase in antibiotic-resistant Pseudomonas aeruginosa isolates, necessitating the development of innovative treatments to combat this pathogen. This manuscript explores the potential of different phage proteins to attenuate virulence factors of P. aeruginosa, particularly the type II secretion system (T2SS). PIT2, a protein derived from the lytic Pseudomonas phage LMA2 inhibits the T2SS effectors PrpL and LasA and attenuates the bacterial virulence toward HeLa cells and Galleria mellonella. Using RNAseq-based differential gene expression analysis, PIT2's impact on the LasR regulatory network is revealed, which plays a key role in bacterial quorum sensing. This discovery expands our knowledge on phage-encoded modulators of the bacterial metabolism and offers a promising anti-virulence target in P. aeruginosa. As such, it lays the foundation for a new phage-inspired anti-virulence strategy to combat multidrug resistant pathogens and opens the door for SynBio applications.

Humoral Immune Response in Calves Vaccinated with Monovalent Vaccines or a Trivalent Combination Thereof and Matching of These Vaccines to the Selected Circulating Foot-and-Mouth Disease Viruses in Ethiopia.

Foot-and-mouth disease (FMD) is an endemic, highly contagious, and devastating disease of livestock production in Ethiopia. Control of this disease relies mainly on prophylactic vaccination by willing farmers without a countrywide vaccination program. The objectives of this study were to quantify the humoral immune response and evaluation of the serological relationship of the vaccine strain used with representative field strain isolates. This was performed by primo vaccination of 6-9-month-old Holstein Friesian calves (35 treatment and 4 control calves) on day one and booster vaccination on day 28. Calves were vaccinated using the locally available National Veterinary Institute (NVI), Bishoftu, Ethiopia, inactivated aluminum hydroxide adjuvant monovalent (either O, A, SAT-2 alone) or trivalent (combination of A, O, SAT-2) vaccine (A/ETH/6/2000 (G-VII, O/ETH/38/2005(EA-3) and SAT-2/ETH/64/2009(XIII)). A 2 mL or 4 mL dose was used to vaccinate all calves except the animals that served as a control. In the case of the trivalent vaccine, a 4 mL dose was used to vaccinate calves. The serum was collected at 7, 14, 21, 28, and 56 days post-vaccination (d.p.v.). The humoral immune response was quantified by the solid-phase competitive enzyme-linked immunosorbent assay (SPC ELISA) and the virus-neutralization test (VNT). The serological relationship of heterologous and homologous viruses was also evaluated by adjuvant vaccine matching tests. The r1-value was determined using serum collected 21 d.p.v. An increase in immune response was observed from 7 d.p.v. to 28 d.p.v. in calves who received a 4 mL dose containing a 107.24 antigen load of 100 tissue culture infective dose (100TCID50) virus titer in the formulation. Upon receiving a booster dose on day 28, the humoral immune response was checked on the 56th day post-initial vaccination. Amounts of 54%, 72%, 79%, and 72% of inhibition for A, O, SAT-2, and trivalent vaccine in the three serotypes SPCE, respectively, was measured. Here, it was found that the immune response of calves increased from day 7 to 56, as evidenced by SPCE analysis. Likewise, an increase in antibody titer measured by a one-dimensional virus neutralization assay was also in line with SPCE analysis. This indicates that the vaccine is capable of inducing a neutralizing antibody that confers a protective immune response in 70%, 62%, and 100% heterologous field strains of A, O, and SAT-2 isolates, respectively, and has an average antigenic relationship of >0.3 with a standard deviation of +0.05 (N = 3) to the vaccine strains A/ETH/6/2000, O/ETH/38/2005 and SAT-2/ETH/64/2009, respectively, when using the one-dimensional virus neutralization test. The contribution and importance of this study is a confirmation of the vaccine and the field strain serological relationship for serotype SAT-2 strain and further research/change of vaccination strategy/ improvement in the currently used vaccine to cover a wide range of prevailing genotypes/lineages and induction of sound immune response after vaccination for serotype A and O strain. This study suggests that the trivalent vaccine produced by the National Veterinary Institute containing viral isolates from serotype O, A, and SAT-2 has a good serological relationship with the majority of circulating field strains in Ethiopia.

The phage-encoded PIT4 protein affects multiple two-component systems of Pseudomonas aeruginosa.

IMPORTANCE: More and more Pseudomonas aeruginosa isolates have become resistant to antibiotics like carbapenem. As a consequence, P. aeruginosa ranks in the top three of pathogens for which the development of novel antibiotics is the most crucial. The pathogen causes both acute and chronic infections, especially in patients who are the most vulnerable. Therefore, efforts are urgently needed to develop alternative therapies. One path explored in this article is the use of bacteriophages and, more specifically, phage-derived proteins. In this study, a phage-derived protein was studied that impacts key virulence factors of the pathogen via interaction with multiple histidine kinases of TCSs. The fundamental insights gained for this protein can therefore serve as inspiration for the development of an anti-virulence compound that targets the bacterial TCS.

Epidemiological Dynamics of Foot-and-Mouth Disease in the Horn of Africa: The Role of Virus Diversity and Animal Movement.

The Horn of Africa is a large area of arid and semi-arid land, holding about 10% of the global and 40% of the entire African livestock population. The region's livestock production system is mainly extensive and pastoralist. It faces countless problems, such as a shortage of pastures and watering points, poor access to veterinary services, and multiple endemic diseases like foot-and-mouth disease (FMD). Foot-and-mouth disease is one of the most economically important livestock diseases worldwide and is endemic in most developing countries. Within Africa, five of the seven serotypes of the FMD virus (FMDV) are described, but serotype C is not circulating anymore, a burden unseen anywhere in the world. The enormous genetic diversity of FMDV is favored by an error-prone RNA-dependent RNA polymerase, intra-typic and inter-typic recombination, as well as the quasi-species nature of the virus. This paper describes the epidemiological dynamics of foot-and-mouth disease in the Horn of Africa with regard to the serotypes and topotypes distribution of FMDV, the livestock production systems practiced, animal movement, the role of wildlife, and the epidemiological complexity of FMD. Within this review, outbreak investigation data and serological studies confirm the endemicity of the disease in the Horn of Africa. Multiple topotypes of FMDV are described in the literature as circulating in the region, with further evolution of virus diversity predicted. A large susceptible livestock population and the presence of wild ungulates are described as complicating the epidemiology of the disease. Further, the husbandry practices and legal and illegal trading of livestock and their products, coupled with poor biosecurity practices, are also reported to impact the spread of FMDV within and between countries in the region. The porosity of borders for pastoralist herders fuels the unregulated transboundary livestock trade. There are no systematic control strategies in the region except for sporadic vaccination with locally produced vaccines, while literature indicates that effective control measures should also consider virus diversity, livestock movements/biosecurity, transboundary trade, and the reduction of contact with wild, susceptible ungulates.

Toward Renewable-Based Prebiotics from Woody Biomass: Potential of Tailored Xylo-Oligosaccharides Obtained by Enzymatic Hydrolysis of Beechwood Xylan as a Prebiotic Feed Supplement for Young Broilers.

Although antibiotic resistance emerges naturally, this process has been accelerated by the worldwide overuse and misuse of antibiotics. It is essential to find effective alternatives in the broiler industry to improve poultry health while maintaining production efficiency and product safety. In this study, we aimed to evaluate a potential alternative: wood-derived xylo-oligosaccharides (XOS). The objective of this research was to investigate the potential of XOS prepared using enzymatic hydrolysis of beechwood xylan as a prebiotic feed supplement for broilers. A pilot study was conducted to explore the optimal XOS fraction profile by in vitro fermentation. Subsequently, a semi-continuous enzyme membrane reactor was used, allowing for the production of tailored XOS in large quantities. Given the strong bidirectional relationship between intestinal health, nutrition, and intestinal microbiota composition in broilers, an in vivo experiment was performed to explore the potential of XOS as a prebiotic feed supplement by investigating growth performance, feed conversion ratio, caecal short and medium chain fatty acid (SCFA and MCFA) concentration, and microbiological composition of the caecal content. Results from the pilot study indicated that higher enzyme concentrations in the hydrolysis process yield a product that leads to a higher total SCFA and MCFA- and butyric acid production during in vitro fermentation by caecal bacteria. Supplementation of the tailored XOS to the broiler diet (day 1 (d1)-d8 0.13% wt/wt XOS, d9-d15 0.32% XOS) resulted in higher Bifidobacterium counts, beneficial to the health of birds, on d11 and d15.

Human pluripotent stem cell-derived hepatocytes support complete replication of hepatitis C virus.

BACKGROUND & AIMS: Worldwide, about 180 million people are chronically infected with the hepatitis C virus (HCV). Current in vitro culture systems for HCV depend chiefly on human hepatoma cell lines. Although primary human hepatocytes support HCV infection in vitro, and immunodeficient mice repopulated with human hepatocytes support HCV infection in vivo, these models are limited because of shortage of human livers to isolate hepatocytes. Therefore, there is significant interest in the establishment from of a HCV culture system in human stem cell-derived hepatocyte-like cells. METHODS: Human embryonic stem cell (hESC)-derived hepatocytes were infected with HCV in the presence or absence of direct acting antivirals. After inoculation, replication of HCV was analyzed extensively. RESULTS: We demonstrate that hESC-derived hepatocytes can be infected with the HCV JFH1 genotype 2a, resulting in the production of viral RNA in the stem cell progeny. Viral replication is inhibited by a non-nucleoside HCV polymerase-inhibitor (HCV-796), a cyclophilin binding molecule (Debio 025-Alisporivir) and the protease inhibitor VX-950 (Telaprevir). Stem cell-derived hepatocytes produced, for more than 10 days, the HCV core protein as well as virions that were capable of re-infecting hepatoma cells. CONCLUSIONS: Hepatocytes derived from hESC support the complete HCV replication cycle (including the production of infectious virus), and viral replication in these cells is efficiently inhibited by selective inhibitors of HCV replication.

Molecular Characterization of ASFV and Differential Diagnosis of Erysipelothrix in ASFV-Infected Pigs in Pig Production Regions in Cameroon.

African swine fever and swine erysipelas are two devastating diseases with similar manifestations ravaging the domestic pig industry. Only a single phylogenetic study has been carried out in Cameroon, and neither an extensive genotyping aimed at identifying the different serotypes nor has an appropriate differential diagnosis of different species of Erysipelothrix has been effected in ASF-infected animals. Of the 377 blood or tissue samples randomly collected from pig farms and slaughter slabs from January to August 2020, 120 were positive for ASFV (by PCR), giving a prevalence of 31.83%. Intragenomic resolution through sequencing divulged the presence of genotypes I, and Ia, two variants with 19 (ABNAAAACBNABTDBNAFA) and six (ABNAFA) tandem repeat sequences (TRS), serotype IV, and a single GGAATATATA repeat. The sole presence of E. tonsillarum (avirulent species) and not E. rhusiopathiae (virulent species) indicates that the severity observed during the 2020 ASF outbreak in the sampled regions was exclusively due to ASFV genotype I infection. Such characterisations are necessary for designing effective control measures and future potential vaccine candidates.

Temporal and Spatial Patterns and a Space-Time Cluster Analysis of Foot-and-Mouth Disease Outbreaks in Ethiopia from 2010 to 2019.

Foot-and-mouth disease (FMD) is an endemic disease in Ethiopia, although space-time cluster and monthly variation studies have never been assessed at national level. The current study aimed to identify the spatial and temporal distribution of FMD outbreaks in Ethiopia from national outbreak reports over a period of ten years from 1 January 2010 to 31 December 2019. To this end, a total of 376,762 cases and 1302 outbreaks from 704 districts were obtained from the Minister of Agriculture for analyses. In general, the dry periods, i.e., October to March, of the year were recorded as the peak outbreak periods, with the highest prevalence in March 2012. The monthly average and the outbreak trends over ten years show a decrease of outbreaks from 2010 to 2019. Decomposing the FMD outbreak data time series showed that once an outbreak erupted, it continued for up to five years. Only 12% of the reported outbreaks were assigned to a specific serotype. Within these outbreaks, the serotypes O, A, SAT-2, and SAT-1 were identified in decreasing order of prevalence, respectively. When a window of 50% for the maximum temporal/space cluster size was set, a total of seven FMD clusters were identified in space and time. The primary cluster with a radius of 380.95 km was identified in the southern part of Ethiopia, with a likelihood ratio of 7.67 (observed/expected cases). The third cluster, with a radius of 144.14 km, was identified in the northeastern part of the country, and had a likelihood ratio of 5.66. Clusters 1 and 3 occurred from January 2017 to December 2019. The second cluster that occurred had a radius of 294.82 km, a likelihood ratio of 6.20, and was located in the central and western parts of Ethiopia. The sixth cluster, with a radius of 36.04 km and a likelihood ratio of 20.60, was set in southern Tigray, bordering Afar. Clusters 2 and 6 occurred in the same period, from January 2014 to December 2016. The fourth cluster in northern Tigray had a calculated radius of 95.50 km and a likelihood ratio of 1.17. The seventh cluster occurred in the north-central Amhara region, with a radius of 97 km and a likelihood ratio of 1.16. Clusters 4 and 7 occurred between January 2010 and December 2013. The spatiotemporal and cluster analysis of the FMD outbreaks identified in the context of the current study are crucial in implementing control, prevention, and a prophylactic vaccination schedule. This study pointed out October to March as well as the main time of the year during which FMD outbreaks occur. The area that extends from the south to north, following the central highlands, is the main FMD outbreak area in Ethiopia.