Advanced Manufacturing, Formulation and Microencapsulation of Therapeutic Phages.

Manufacturing and formulation of stable, high purity, and high dose bacteriophage drug products (DPs) suitable for clinical usage would benefit from improved process monitoring and control of critical process parameters that affect product quality attributes. Chemistry, Manufacturing, and Controls (CMC) for both upstream (USP) and downstream processes (DSP) need mapping of critical process parameters (CPP) and linking these to critical quality attributes (CQA) to ensure quality and consistency of phage drug substance (DS) and DPs development. Single-use technologies are increasingly becoming the go-to manufacturing option with benefits both for phage bioprocess development at the engineering run research stage and for final manufacture of the phage DS. Future phage DPs under clinical development will benefit from implementation of process analytical technologies (PAT) for better process monitoring and control. These are increasingly being used to improve process robustness (to reduce batch-to-batch variability) and productivity (yielding high phage titers). Precise delivery of stable phage DPs that are suitably formulated as liquids, gels, solid-oral dosage forms, and so forth, could significantly enhance efficacy of phage therapy outcomes. Pre-clinical development of phage DPs must include at an early stage of development, considerations for their formulation including their characterization of physiochemical properties (size, charge, etc.), buffer pH and osmolality, compatibility with regulatory approved excipients, storage stability (packaging, temperature, humidity, etc.), ease of application, patient compliance, ease of manufacturability using scalable manufacturing unit operations, cost, and regulatory requirements.

Genome characterization of the novel lytic phage vB_AbaAut_ChT04 and the antimicrobial activity of its lysin peptide against Acinetobacter baumannii isolates from different time periods.

Acinetobacter baumannii is an important opportunistic pathogen, usually associated with immunocompromised individuals with a prolonged period of stay in a hospital. Currently, the incidence of multi-drug resistant A. baumannii (MDR-AB) and extensively drug-resistant A. baumannii (XDR-AB) is increasing rapidly in Thailand, mirroring the trend worldwide. Novel therapeutic approaches for the treatment of A. baumannii infection using bacteriophages are being evaluated, and the use of phage-derived peptides is being tested as alternative approach to fighting infection. In this study, we isolated and determined the biological features of a lytic A. baumannii phage called vB_AbaAut_ChT04 (vChT04). The vChT04 phage was classified as a member of the family Autographiviridae of the class Caudoviricetes. It showed a short latent period (10 min) and a large burst size (280 PFU cell-1), and it was able to infect 52 out of 150 clinical MDR-AB strains tested (34.67%). Most of the phage-sensitive strains were A. baumannii strains that had been isolated during the same year that the phage was isolated. The phage showed activity across a broad pH (pH 5.0-8.0) and temperature (4-37°C) range. Whole-genome analysis revealed that the vChT04 genome comprises 41,158 bp with a 39.3% GC content and contains 48 open reading frames (ORFs), 28 of which were assigned putative functions based on homology to previously identified phage genes. Comparative genomic analysis demonstrated that vChT04 had the highest similarity to phage vB_AbaP_WU2001, which was isolated in the southern part of Thailand. An endolysin gene found in the vChT04 genome was used to synthesize an antimicrobial peptide (designated as PLysChT04) and its antimicrobial activity was evaluated using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. The MIC and MBC values of peptide PLysChT04 against MDR-AB and XDR-AB were 312.5-625 µg/mL, and it was able to inhibit both phage-susceptible and phage-resistant isolates collected over different time periods. PLysChT04 showed good efficacy in killing drug-resistant A. baumannii and other bacterial strains, and it is a promising candidate for development as an alternative therapeutic agent targeting A. baumannii infections.

Diagnosis of sub-clinical coccidiosis in fast growing broiler chickens by MicroRNA profiling.

Coccidiosis in broiler chickens, caused by infection with Eimeria spp. remains one of the most economically important production diseases. Development of a genetic biomarker panel of sub-clinical infection would be an important biological tool for the management of broiler flocks. We analysed expression of MicroRNAs (miRNAs) to determine the potential for these in diagnosing coccidiosis in broiler flocks. miRNA expression, in the ilea of Ross 308 broilers, was compared between chickens naturally clinically or sub-clinically infected with Eimeria maxima and Eimeria acervulina using NextSeq 500 sequencing. 50 miRNAs with greatest coefficient of variance were determined and principal component analysis showed that these miRNAs clustered within the clinical and sub-clinical groups much more closely than uninfected controls. Following false detection rate analysis and quantitative PCR we validated 3 miRNAs; Gallus gallus (gga)-miR-122-5p, gga-miR-205b and gga-miR-144-3p, which may be used to diagnose sub-clinical coccidiosis.

Sex pilus specific bacteriophage to drive bacterial population towards antibiotic sensitivity.

Antimicrobial resistance (AMR) is now a major global problem largely resulting from the overuse of antibiotics in humans and livestock. In some AMR bacteria, resistance is encoded by conjugative plasmids expressing sex-pili that can readily spread resistance through bacterial populations. The aim of this study was to use sex pilus-specific (SPS) phage to reduce the carriage of AMR plasmids. Here, we demonstrate that SPS phage can kill AMR Escherichia coli and select for AMR plasmid loss in vitro. For the first time, we also demonstrate that SPS phage can both prevent the spread of AMR Salmonella Enteritidis infection in chickens and shift the bacterial population towards antibiotic sensitivity.

Differential immune response to Eimeria maxima infection in fast- and slow-growing broiler genotypes.

Very little has been reported comparing resistance to coccidiosis in fast or slow growing broilers, the latter of which are becoming more prevalent in the broiler industry. We examined mRNA expression in the intestines of fast and slow growing broilers following Eimeria infection. We show that by day 13 post-infection (d pi) with 2500 or 7000 oocysts of Eimeria maxima, slower-growing (Ranger Classic) broilers significantly (P < 0.01) upregulated expression of proinflammatory cyclooxygenase genes (LTB4DH, PTSG1 and PTSG2) above that detected in fast growing (Ross 308) broilers. Expression of CD8α mRNA was downregulated in Ross 308 at day 6d pi with either 2500 or 7000 oocysts of E maxima (P < 0.05), compared to uninfected controls, but was not differentially expressed in Ranger Classic. CD4 genes were not differentially expressed in either chicken line infected with either infectious oocyst dose at d6 pi, compared to uninfected controls. However, at d13 pi, CD4 expression was significantly upregulated in both chicken lines infected with either infectious oocyst dose, compared to uninfected controls (P < 0.05) but this was significantly greater in Ranger Classic broilers compared to Ross 308 (P < 0.05). At d13 pi, expression of CD3 chains (required for T lymphocyte activation) was significantly increased in Ranger Classic compared to Ross 308, infected with either oocyst dose (P < 0.05-0.01). Expression of IL-2 and IL-15 mRNA, required for T lymphocyte proliferation was also significantly upregulated, or maintained longer, in Ranger Classic broilers compared to Ross 308. These differences in immune response to E maxima corresponded with a reduction in E maxima genome detected in the intestines of Ranger Classic compared to Ross 308.

Differential gene response to coccidiosis in modern fast growing and slow growing broiler genotypes.

We analysed intestinal tissues from groups of fast growing (Ross 308) broilers with natural or experimental coccidiosis, by genomic microarray. We identified genes that were differentially expressed (DE) in all groups and analysed expression of a panel of these, by qPCR, in Ross 308 and slow growing (Ranger classic) broilers, infected with 2500 or 7000 oocysts of Eimeria maxima for 6 or 13 days post-infection (dpi). Four genes (ADD3, MLLT10, NAV2 and PLXNA2) were upregulated (P <0.05) in Ross 308 but were not DE in Ranger Classic at 6 dpi with 2500 oocysts. Six genes (PTPRF, NCOR1, CSF3, SGK1, CROR and CD1B) were upregulated (P <0.05) in both Ross 308 and Ranger Classic infected with 2500 oocysts at 6 dpi but were not DE at 6 dpi with 7000 oocysts. At 13 dpi with 7000 oocysts, NAV2 and NCOR1 were upregulated in Ross 308 (P <0.05) and PTPRF was upregulated in both genotypes (P <0.05). DE of immune genes within the biomarker panel also occurred, with CSF3 upregulated in both genotypes infected with 2500 oocysts at 6 dpi and in Ranger Classic infected with 7000 oocysts, at 6 and 13 dpi (P <0.05). IL-22 was down-regulated in Ranger Classic infected with 2500 or 7000 oocysts at 6 dpi (P <0.05) but upregulated in both genotypes at 13 dpi (P <0.05). CD72 was down-regulated in Ranger Classic infected with 2500 oocysts at 6 dpi and with 7000 oocysts at 6 and 13 dpi (P <0.05). CD72 was upregulated in Ross 308 infected with 2500 oocysts at 6 dpi but was down-regulated following infection with 7000 oocysts at 13 dpi (P <0.05). In conclusion, differential gene expression occurs in fast and slow growing broiler genotypes with coccidiosis. In addition, we highlight a potential genetic biomarker panel for early diagnosis of coccidiosis.

Cryptosporidium-associated diarrhoea in neonatal calves in Algeria.

Neonatal calf diarrhoea triggered by the enteric protozoan parasite Cryptosporidium is a leading cause of morbidity and mortality in calves aged 1-month-old or younger globally. Infected cattle in general and calves in particular have also been demonstrated as major contributors of zoonotic C. parvum oocysts in the environment and have been linked to a number of waterborne outbreaks of human cryptosporidiosis. Little is known on the occurrence, geographical distribution, and molecular diversity of Cryptosporidium infections affecting bovine populations in Algeria. In this study faecal specimens were randomly collected from 460 cattle aged between two days and 18 months on 10 farms located in the provinces of Aïn Defla, Blida, Sétif, and Tizi Ouzou between the autumn of 2015 and the spring of 2016. Faecal samples were microscopically examined using the modified Ziehl-Neelsen acid-fast technique as screening method. Microscopy-positive samples were confirmed by a commercial coproantigen enzyme-linked immunosorbent assay (Bio-X Diagnostics). The identification of Cryptosporidium species and sub-genotypes in confirmed samples was conducted by PCR and sequence analyses of the small subunit ribosomal RNA (ssu rRNA) and the 60 kDa glycoprotein (gp60) genes of the parasite. Overall, 52.2% (240/460) of the investigated cattle tested positive to Cryptosporidium by microscopy. The infection was widespread in all 10 farms surveyed, but was significantly more prevalent in those from Blida in the central part of the country. Bovine cryptosporidiosis affected cattle of all age groups but with different outcomes. Pre-weaned (up to one month old) calves typically presented with diarrhoea, whereas older animals mostly harboured sub-clinical infections. The commercial ELISA used only detected 15.8% (38/240) of the samples that previously tested positive by microscopy, demonstrating a poor performance in field epidemiological surveys. Sequence analysis of the 29 isolates generated at the ssu rRNA loci confirmed the presence of four Cryptosporidium species including C. parvum (72.4%), C. bovis (13.8%), C. andersoni, (3.4%), and C. ryanae (3.4%). Two additional isolates (7.0%) could only be identified at the genus level. Eight out of the 21 isolates assigned to C. parvum were identified as sub-genotype IIaA16G2R1 at the gp60 locus. C. parvum was almost exclusively found infecting pre-weaned calves, whereas C. ryanae and C. andersoni were only detected in asymptomatic animals. Bovine cryptosporidiosis is highly endemic in the surveyed area and represents a veterinary public health concern that should be adequately tackled by Algerian veterinary health authorities and policy makers.

Molecular approaches to the diagnosis and monitoring of production diseases in pigs.

Production disease in pigs is caused by a variety of different pathogens, mainly enteric and respiratory and can result in significant economic loss. Other factors such as stress, poor husbandry and nutrition can also contribute to an animal's susceptibility to disease. Molecular biomarkers of production disease could be of immense value by improving diagnosis and risk analysis to determine best practice with an impact on increased economic output and animal welfare. In addition to the use of multiplex PCR or microarrays to detect individual or mixed pathogens during infection, these technologies can also be used to monitor the host response to infection via gene expression. The patterns of gene expression associated with cellular damage or initiation of the early immune response may indicate the type of pathology and, by extension the types of pathogen involved. Molecular methods can therefore be used to monitor both the presence of a pathogen and the host response to it during production disease. The field of biomarker discovery and implementation is expanding as technologies such as microarrays and next generation sequencing become more common. Whilst a large number of studies have been carried out in human medicine, further work is needed to identify molecular biomarkers in veterinary medicine and in particular those associated with production disease in the pig industry. The pig transcriptome is highly complex and still not fully understood. Further gene expression studies are needed to identify molecular biomarkers which may have predictive value in identifying the environmental, nutritional and other risk factors which are associated with production diseases in pigs.