A Sensitive and Rapid Wastewater Test for SARS-COV-2 and Its Use for the Early Detection of a Cluster of Cases in a Remote Community.

Throughout the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has been used to monitor trends in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prevalence in the community. A major challenge in establishing wastewater surveillance programs, especially in remote areas, is the need for a well-equipped laboratory for sample analysis. Currently, no options exist for rapid, sensitive, mobile, and easy-to-use wastewater tests for SARS-CoV-2. The performance of the GeneXpert system, which offers cartridge-based, rapid molecular clinical testing for SARS-CoV-2 in a portable platform, was evaluated using wastewater as the input. The GeneXpert demonstrated a SARS-CoV-2 limit of detection in wastewater below 32 copies/mL with a sample processing time of less than an hour. Using wastewater samples collected from multiple sites across Canada during February and March 2021, a high overall agreement (97.8%) was observed between the GeneXpert assay and laboratory-developed tests regarding the presence or absence of SARS-CoV-2. Additionally, with the use of centrifugal filters, the detection threshold of the GeneXpert system was improved to <10 copies/mL in wastewater. Finally, to support on-site wastewater surveillance, GeneXpert testing was implemented in Yellowknife, a remote community in Northern Canada, where its use successfully alerted public health authorities to undetected transmission of COVID-19. The identification of SARS-CoV-2 in wastewater triggered clinical testing of recent travelers and identification of new COVID-19 cases/clusters. Taken together, these results suggest that GeneXpert is a viable option for surveillance of SARS-CoV-2 in wastewater in locations that do not have access to established testing laboratories. IMPORTANCE Wastewater-based surveillance is a powerful tool that provides an unbiased measure of COVID-19 prevalence in a community. This work describes a sensitive wastewater rapid test for SARS-CoV-2 based on a widely distributed technology, the GeneXpert. The advantages of an easy-to-use wastewater test for SARS-CoV-2 are clear: it supports surveillance in remote communities, improves access to testing, and provides faster results allowing for an immediate public health response. The application of wastewater rapid testing in a remote community facilitated the detection of a COVID-19 cluster and triggered public health action, clearly demonstrating the utility of this technology. Wastewater surveillance will become increasingly important in the postvaccination pandemic landscape as individuals with asymptomatic/mild infections continue transmitting SARS-CoV-2 but are unlikely to be tested.

Detection of mpox virus in wastewater provides forewarning of clinical cases in Canadian cities.

Wastewater-based surveillance (WBS) has shown to be an effective tool in monitoring the spread of SARS-CoV-2 and has helped guide public health actions. Consequently, WBS has expanded to now include the monitoring of mpox virus (MPXV) to contribute to its mitigation efforts. In this study, we demonstrate a unique sample processing and a molecular diagnostic strategy for MPXV detection that can inform on the epidemiological situation of mpox outbreaks through WBS. We conducted WBS for MPXV in 22 Canadian wastewater treatment plants (WWTPs) for 14 weeks. Three MPXV qPCR assays were assessed in this study for the detection of MPXV which include the G2R assays (G2R_WA and G2R_G) developed by the Centers for Disease Control and Prevention (CDC) in 2010, and an in-house-developed assay that we have termed G2R_NML. The G2R_NML assay was designed using reference genomes from the 2022 MPXV outbreak and provides a larger qPCR amplicon size to facilitate Sanger sequencing. Results show that all three assays have similar limits of detection and are able to detect the presence of MPXV in wastewater. The G2R_NML assay produced a significantly greater number of Sanger sequence-confirmed MPXV results compared to the CDC G2R assays. Detection of MPXV was possible where provincial surveillance indicated overall low caseloads, and in some sites forewarning of up to several weeks was observed. Overall, this study proposes that WBS of MPXV provides additional information to help fill knowledge gaps in clinical case-surveillance and is potentially an essential component to the management of mpox.

Coupling wastewater-based epidemiological surveillance and modelling of SARS-COV-2/COVID-19: Practical applications at the Public Health Agency of Canada.

Wastewater-based surveillance (WBS) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) offers a complementary tool for clinical surveillance to detect and monitor coronavirus disease 2019 (COVID-19). Since both symptomatic and asymptomatic individuals infected with SARS-CoV-2 can shed the virus through the fecal route, WBS has the potential to measure community prevalence of COVID-19 without restrictions from healthcare-seeking behaviours and clinical testing capacity. During the Omicron wave, the limited capacity of clinical testing to identify COVID-19 cases in many jurisdictions highlighted the utility of WBS to estimate disease prevalence and inform public health strategies; however, there is a plethora of in-sewage, environmental and laboratory factors that can influence WBS outcomes. The implementation of WBS, therefore, requires a comprehensive framework to outline a pipeline that accounts for these complex and nuanced factors. This article reviews the framework of the national WBS conducted at the Public Health Agency of Canada to present WBS methods used in Canada to track and monitor SARS-CoV-2. In particular, we focus on five Canadian cities-Vancouver, Edmonton, Toronto, Montréal and Halifax-whose wastewater signals are analyzed by a mathematical model to provide case forecasts and reproduction number estimates. The goal of this work is to share our insights on approaches to implement WBS. Importantly, the national WBS system has implications beyond COVID-19, as a similar framework can be applied to monitor other infectious disease pathogens or antimicrobial resistance in the community.

Overview of Canada's Antimicrobial Resistance Network (AMRNet): A data-driven One Health approach to antimicrobial resistance surveillance.

The Antimicrobial Resistance Network (AMRNet) is a laboratory-based antimicrobial resistance (AMR) surveillance system under development at the Public Health Agency of Canada's (PHAC's) National Microbiology Laboratory. The AMRNet surveillance system captures information on antimicrobial susceptibility testing from clinical and veterinary laboratories including both public and private facilities. In the future, the AMRNet system will also capture relevant data from existing PHAC surveillance systems for AMR including the Canadian Integrated Program for Antimicrobial Resistance Surveillance, the Canadian Nosocomial Infection Surveillance Program and the Enhanced Surveillance of Antimicrobial-Resistant Gonorrhea program, and contribute to the Canadian Antimicrobial Resistance Surveillance System. AMRNet's integrated "One Health" approach will allow health professionals and researchers to take a multi-dimensional perspective of AMR in both human and animal health in Canada and will make Canada a leader in AMR surveillance. AMRNet is a collaboration between PHAC, provincial and territorial public health organizations as well as clinical and veterinary laboratories across the country. As part of a phased rollout, AMRNet is now collecting human clinical data from three provinces, from both inpatients and outpatients. Ultimately, AMRNet aims to capture all antimicrobial susceptibility testing results from all bacterial and fungal pathogens across Canada. This article describes the AMRNet surveillance system, including program objectives, system structure and the data collected. The integration of human and animal data in AMRNet will inform One Health responses to AMR issues. The capacity to collect and to disseminate data to stakeholders in real time is a critical step to addressing emerging AMR issues in Canada.

ECTyper: in silico Escherichia coli serotype and species prediction from raw and assembled whole-genome sequence data.

Escherichia coli is a priority foodborne pathogen of public health concern and phenotypic serotyping provides critical information for surveillance and outbreak detection activities. Public health and food safety laboratories are increasingly adopting whole-genome sequencing (WGS) for characterizing pathogens, but it is imperative to maintain serotype designations in order to minimize disruptions to existing public health workflows. Multiple in silico tools have been developed for predicting serotypes from WGS data, including SRST2, SerotypeFinder and EToKi EBEis, but these tools were not designed with the specific requirements of diagnostic laboratories, which include: speciation, input data flexibility (fasta/fastq), quality control information and easily interpretable results. To address these specific requirements, we developed ECTyper (https://github.com/phac-nml/ecoli_serotyping) for performing both speciation within Escherichia and Shigella, and in silico serotype prediction. We compared the serotype prediction performance of each tool on a newly sequenced panel of 185 isolates with confirmed phenotypic serotype information. We found that all tools were highly concordant, with 92-97 % for O-antigens and 98-100 % for H-antigens, and ECTyper having the highest rate of concordance. We extended the benchmarking to a large panel of 6954 publicly available E. coli genomes to assess the performance of the tools on a more diverse dataset. On the public data, there was a considerable drop in concordance, with 75-91 % for O-antigens and 62-90 % for H-antigens, and ECTyper and SerotypeFinder being the most concordant. This study highlights that in silico predictions show high concordance with phenotypic serotyping results, but there are notable differences in tool performance. ECTyper provides highly accurate and sensitive in silico serotype predictions, in addition to speciation, and is designed to be easily incorporated into bioinformatic workflows.

Rapid and accurate SNP genotyping of clonal bacterial pathogens with BioHansel.

Hierarchical genotyping approaches can provide insights into the source, geography and temporal distribution of bacterial pathogens. Multiple hierarchical SNP genotyping schemes have previously been developed so that new isolates can rapidly be placed within pre-computed population structures, without the need to rebuild phylogenetic trees for the entire dataset. This classification approach has, however, seen limited uptake in routine public health settings due to analytical complexity and the lack of standardized tools that provide clear and easy ways to interpret results. The BioHansel tool was developed to provide an organism-agnostic tool for hierarchical SNP-based genotyping. The tool identifies split k-mers that distinguish predefined lineages in whole genome sequencing (WGS) data using SNP-based genotyping schemes. BioHansel uses the Aho-Corasick algorithm to type isolates from assembled genomes or raw read sequence data in a matter of seconds, with limited computational resources. This makes BioHansel ideal for use by public health agencies that rely on WGS methods for surveillance of bacterial pathogens. Genotyping results are evaluated using a quality assurance module which identifies problematic samples, such as low-quality or contaminated datasets. Using existing hierarchical SNP schemes for Mycobacterium tuberculosis and Salmonella Typhi, we compare the genotyping results obtained with the k-mer-based tools BioHansel and SKA, with those of the organism-specific tools TBProfiler and genotyphi, which use gold-standard reference-mapping approaches. We show that the genotyping results are fully concordant across these different methods, and that the k-mer-based tools are significantly faster. We also test the ability of the BioHansel quality assurance module to detect intra-lineage contamination and demonstrate that it is effective, even in populations with low genetic diversity. We demonstrate the scalability of the tool using a dataset of ~8100 S. Typhi public genomes and provide the aggregated results of geographical distributions as part of the tool's output. BioHansel is an open source Python 3 application available on PyPI and Conda repositories and as a Galaxy tool from the public Galaxy Toolshed. In a public health context, BioHansel enables rapid and high-resolution classification of bacterial pathogens with low genetic diversity.

Attenuated cytokine responses in porcine lymph node cells stimulated with CpG DNA are associated with low frequency of IFNalpha-producing cells and TLR9 mRNA expression.

The immune stimulatory effects of synthetic CpG DNA, on porcine peripheral blood mononuclear cells (PBMC) have been reported, but little is known about CpG-induced responses in other lymphoid tissues of pigs. We investigated innate immune responses induced by CpG DNA in cells from blood, lymph nodes (LN) and spleens of pigs. Porcine PBMC and lymph node cells (LNC) were stimulated in vitro with three classes (A-, B- and C-class) of CpG oligodeoxynucleotides (ODNs), and a non-CpG control ODN. All three classes of CpG ODNs induced significant production of IFNalpha, TNFalpha, IL-1, IL-6 and IL-12 in PBMC. In contrast, in LNC, only IL-12 was stimulated by all three classes of CpG ODNs, while IFNalpha, and IL-6 were induced by A- and C-class ODNs. No TNFalpha was induced in LNC by any of the ODNs. Significant lymphocyte proliferation was induced in PBMC by all three classes of CpG ODNs and non-CpG control. However, in LNC, B- and C-class ODNs induced significant proliferation, while no proliferation was seen with A-class and non-CpG control ODN. All three classes of ODNs induced NK-like cytotoxicity in PBMC and spleen cells, but were less effective in inducing NK cytotoxicity in LNC. We then investigated the reasons for the relatively poor CpG-induced responses in LNC. Our investigations revealed that LNC had a lower frequency of IFNalpha-secreting cells and expressed low levels of TLR9 mRNA compared to PBMC. We conclude that the lower number of IFNalpha-secreting cells and receptor expression may contribute to the attenuated responses in LNC following stimulation with CpG ODN.

Comprehensive assessment of the quality of Salmonella whole genome sequence data available in public sequence databases using the Salmonella in silico Typing Resource (SISTR).

Public health and food safety institutions around the world are adopting whole genome sequencing (WGS) to replace conventional methods for characterizing Salmonella for use in surveillance and outbreak response. Falling costs and increased throughput of WGS have resulted in an explosion of data, but questions remain as to the reliability and robustness of the data. Due to the critical importance of serovar information to public health, it is essential to have reliable serovar assignments available for all of the Salmonella records. The current study used a systematic assessment and curation of all Salmonella in the sequence read archive (SRA) to assess the state of the data and their utility. A total of 67 758 genomes were assembled de novo and quality-assessed for their assembly metrics as well as species and serovar assignments. A total of 42 400 genomes passed all of the quality criteria but 30.16 % of genomes were deposited without serotype information. These data were used to compare the concordance of reported and predicted serovars for two in silico prediction tools, multi-locus sequence typing (MLST) and the Salmonella in silico Typing Resource (SISTR), which produced predictions that were fully concordant with 87.51 and 91.91 % of the tested isolates, respectively. Concordance of in silico predictions increased when serovar variants were grouped together, 89.25 % for MLST and 94.98 % for SISTR. This study represents the first large-scale validation of serovar information in public genomes and provides a large validated set of genomes, which can be used to benchmark new bioinformatics tools.

An improved DNA array-based classification method for the identification of Salmonella serotypes shows high concordance between traditional and genotypic testing.

Previously we developed and tested the Salmonella GenoSerotyping Array (SGSA), which utilized oligonucleotide probes for O- and H- antigen biomarkers to perform accurate molecular serotyping of 57 Salmonella serotypes. Here we describe the development and validation of the ISO 17025 accredited second version of the SGSA (SGSA v. 2) with reliable and unambiguous molecular serotyping results for 112 serotypes of Salmonella which were verified both in silico and in vitro. Improvements included an expansion of the probe sets along with a new classifier tool for prediction of individual antigens and overall serotype from the array probe intensity results. The array classifier and probe sequences were validated in silico to high concordance using 36,153 draft genomes of diverse Salmonella serotypes assembled from public repositories. We obtained correct and unambiguous serotype assignments for 31,924 (88.30%) of the tested samples and a further 3,916 (10.83%) had fully concordant antigen predictions but could not be assigned to a single serotype. The SGSA v. 2 can directly use bacterial colonies with a limit of detection of 860 CFU/mL or purified DNA template at a concentration of 1.0 x 10-1 ng/µl. The SGSA v. 2 was also validated in the wet laboratory and certified using panel of 406 samples representing 185 different serotypes with correct antigen and serotype determinations for 60.89% of the panel and 18.31% correctly identified but an ambiguous overall serotype determination.

Innate immune responses induced by classes of CpG oligodeoxynucleotides in ovine lymph node and blood mononuclear cells.

CpG ODN signal through Toll-like receptor 9 (TLR9) and trigger a cascade of events that lead to activation of innate and adaptive immune responses. Our current understanding of the immunobiology of host responses to CpG is based largely on studies on peripheral blood mononuclear cells (PBMC) and splenocytes. Little is known regarding CpG-induced responses in other lymphoid tissues. In the present study, we investigated responses induced by CpG in both PBMC and lymph nodes. Cells were isolated from the superficial cervical lymph node (LNC) and blood and then stimulated with CpG ODN (either A-, or B- or C-class ODN). Cytokine production was assayed by ELISA, and lymphocyte proliferation was determined by (3)H-thymidine incorporation. NK-like cytotoxicity was analyzed by lysis of (51)Cr-labelled target cells. All three classes of CpG induced IFNalpha and IFNgamma in LNC. In contrast, only A and C-class ODN induced IFNalpha and IFNgamma in PBMC. Moreover, the IFN levels in LNC were 20-40-fold higher than in PBMC. Furthermore, all classes of ODN induced higher IL-12 levels in LNC (five- to six-fold) than in PBMC. Both B and C-class ODN induced good proliferative responses in PBMC and LNC, but the A-class ODN did not induce proliferation of PBMC and only induced moderate proliferation of LNC. A-class ODN induced significant NK-like activity in LNC. Thus, all three classes of CpG ODN induced similar responses in LNC, and these responses were consistently higher than in PBMC. These observations indicate that CpG ODN-induced responses differ between blood and lymph nodes, and suggest that the functional classification of CpG ODN based on PBMC responses may not be directly applicable to cells from other immune tissues.

Systemic innate immune responses following intrapulmonary delivery of CpG oligodeoxynucleotides in sheep.

Mucosal delivery of CpG oligodeoxynucleotide (ODN) in mice has been shown to induce potent innate immunostimulatory responses and protection against infection. We evaluated the efficacy of CpG ODN in stimulating systemic innate immune responses in sheep following delivery to the pulmonary mucosa. Intrapulmonary (IPM) administration of B-Class CpG ODN in saline induced transient systemic responses which included increased rectal temperatures, elevated serum 2'5'-A synthetase and haptoglobin concentrations. The ODN dose required to induce detectable systemic responses following IPM delivery could be reduced by approximately 80% if the CpG ODN was administered in 30% emulsigen instead of saline. Intrapulmonary B-Class CpG ODN formulated in 30% emulsigen produced similar effects when compared to those seen following SC injection. These responses were CpG ODN-specific since control GpC ODN did not induce any detectable response. Intrapulmonary administration of both B-Class and the newly described C-Class CpG ODN produced similar effects indicating that both classes of CpG ODN were comparably effective in stimulating innate immune system following mucosal delivery. Administration of CpG ODN directly into the lungs or delivery of CpG ODN via an intratracheal (IT) infusion also produced similar systemic responses. These observations support the conclusion that mucosal delivery of CpG ODN is an effective route for induction of systemic acute phase responses and antiviral effector molecules in large animals, and may be helpful in controlling systemic infections.

The Validation and Implications of Using Whole Genome Sequencing as a Replacement for Traditional Serotyping for a National Salmonella Reference Laboratory.

Salmonella serotyping remains the gold-standard tool for the classification of Salmonella isolates and forms the basis of Canada's national surveillance program for this priority foodborne pathogen. Public health officials have been increasingly looking toward whole genome sequencing (WGS) to provide a large set of data from which all the relevant information about an isolate can be mined. However, rigorous validation and careful consideration of potential implications in the replacement of traditional surveillance methodologies with WGS data analysis tools is needed. Two in silico tools for Salmonella serotyping have been developed, the Salmonella in silico Typing Resource (SISTR) and SeqSero, while seven gene MLST for serovar prediction can be adapted for in silico analysis. All three analysis methods were assessed and compared to traditional serotyping techniques using a set of 813 verified clinical and laboratory isolates, including 492 Canadian clinical isolates and 321 isolates of human and non-human sources. Successful results were obtained for 94.8, 88.2, and 88.3% of the isolates tested using SISTR, SeqSero, and MLST, respectively, indicating all would be suitable for maintaining historical records, surveillance systems, and communication structures currently in place and the choice of the platform used will ultimately depend on the users need. Results also pointed to the need to reframe serotyping in the genomic era as a test to understand the genes that are carried by an isolate, one which is not necessarily congruent with what is antigenically expressed. The adoption of WGS for serotyping will provide the simultaneous collection of information that can be used by multiple programs within the current surveillance paradigm; however, this does not negate the importance of the various programs or the role of serotyping going forward.

Stimulation of innate immune responses by CpG oligodeoxynucleotide in newborn lambs can reduce bovine herpesvirus-1 shedding.

Stimulation of the innate immune system is potentially very important in neonates who have an immature adaptive immune system and vaccination cannot be used to reduce the risk of infection. CpG oligodeoxynucleotide (ODN) can stimulate innate immune responses in newborn chickens and mice, but similar studies are lacking in other mammalian species. We have shown previously that CpG ODN can both stimulate an acute-phase immune response and induce the antiviral effector molecule, 2'5'-A synthetase, in adult sheep. Therefore, the immunostimulatory activity of A class and B class CpG ODN was evaluated in newborn lambs, and the capacity of CpG ODN-induced responses to reduce viral shedding was evaluated following aerosol challenge with the respiratory pathogen, bovine herpesvirus-1 (BHV-1). In vitro CpG ODN stimulation of peripheral blood mononuclear cells (PBMC) isolated from newborn lambs (3-5 days old) and adult sheep induced equivalent CpG-specific proliferative responses and interferon-alpha (IFN-alpha) secretion. CpG ODN-induced IFN-alpha secretion by neonatal PBMCs was, however, significantly (p < 0.01) enhanced 6 days after subcutaneous (s.c.) injection of 100 microg/kg CpG ODN 2007. Newborn lambs injected s.c. with B class CpG ODN 2007 or the inverted GpC control ODN formulated in 30% Emulsigen (MVP Laboratories, Ralston, NE) displayed CpG ODN-specific increases in body temperature (p < 0.0001), serum 2'5'-A synthetase activity (p = 0.0015), and serum haptoglobin (p = 0.07). CpG ODN-treated lambs also displayed a transient reduction in viral shedding on day 2 postinfection (p < 0.05), which correlated (p < 0.03) with serum 2'5'-A synthetase levels on the day of viral challenge. These observations confirmed that CpG ODNs effectively activate innate immune responses in newborn lambs and CpG ODN-induced antiviral responses correlated with a reduction in viral shedding.

Bovine toll-like receptor 9: a comparative analysis of molecular structure, function and expression.

Non-methylated CpG motifs, present in viral and bacterial DNA, are one of many pathogen-associated molecular patterns (PAMP) recognized by the mammalian innate immune system. Recognition of this PAMP occurs through a specific interaction with toll-like receptor 9 (TLR9) and this interaction can induce cytokine responses that influence both innate and adaptive immune responses. Previous investigations determined that both the flanking sequences in synthetic CpG oligodeoxynucleotides (CpG ODN) and the cellular pattern of TLR9 expression can influence species-specific responses to CpG ODN. Therefore, the structure, function and cellular distribution of bovine TLR9 were compared with what is known for mice and human. Analysis of the bovine TLR9 gene revealed greater sequence homology between cattle and humans than cattle and mice Similar CpG motifs induced optimal activation of both human and bovine leukocytes and these motifs were distinct from those which activated mouse leukocytes. Functional analyses with CpG ODN stimulated bovine blood leukocytes revealed that class A CpG ODN were more potent inducers of interferon-alpha (IFN-alpha) than class B CpG ODN. Furthermore, magnetic activated cell sorting of bovine blood leukocyte subpopulations implicated dendritic cells but not monocytes in the regulation of CpG ODN-induced IFN secretion. Thus, the cellular pattern of CpG ODN-induced responses in cattle shared many similarities with human leukocytes. Collectively, these analyses revealed substantial conservation of TLR9 structure and TLR9 function in blood leukocytes of humans, cattle and other domestic species.

Bovine and ovine blood mononuclear leukocytes differ markedly in innate immune responses induced by Class A and Class B CpG-oligodeoxynucleotide.

Cytosine-phosphate-guanosine (CpG)-DNA can induce an impressive array of innate immune responses that may directly or indirectly contribute to the clearance of infectious agents. Assays, such as lymphocyte proliferative responses, have been used to demonstrate that the immunostimulatory activity of CpG-DNA is conserved among a broad range of vertebrate species, but no studies have been completed to determine if qualitative differences exist among species for CpG-oligodeoxynucleotide (ODN)-induced innate immune responses. In this study, we assessed the capacity of a Class A (ODN 2216) and a Class B (ODN 2007) CpG-ODN to induce innate immune responses in two closely related species, ovine (n = 28) and bovine (n = 29). The secretion of interferon (IFN)-alpha and IFN-gamma and non-major histocompatability complex (MHC)-restricted cytotoxic activity were assayed with CpG-ODN-stimulated peripheral blood mononuclear cells (PBMC). These investigations revealed significant interspecies and intraspecies variation in the responses. As expected, ODN 2216 was a potent inducer of IFN-alpha secretion by both bovine and ovine PBMC, but ODN 2007 also induced dose-dependent, CpG-specific IFN-alpha secretion by ovine PBMC. In contrast, a significant dose-dependent, CpG-specific IFN-gamma secretion response was only observed following ODN 2216 stimulation of bovine PBMC. Furthermore, both ODN 2216 and ODN 2007 induced CpG-specific non-MHC-restricted cytotoxicity with ovine but not bovine PBMC. Finally, there was not a single assay in which PBMC from all sheep or cattle responded at a detectable level. A striking aspect of these results is that such marked differences in CpG-ODN induced innate responses existed both between and within two closely related species.

Strategies for enhancing the immunostimulatory effects of CpG oligodeoxynucleotides.

Synthetic oligodeoxynucleotides (ODN) containing CpG sequences are recognized as a "danger" signal by the immune system of mammals. As a consequence, CpG ODN stimulate innate and adaptive immune responses in humans and a variety of animal species. Indeed, the potential of CpG ODN as therapeutic agents and vaccine adjuvants has been demonstrated in animal models of infectious diseases, allergy and cancer and are currently undergoing clinical trials in humans. While CpG ODN are potent activators of the immune system, their biologic activity is often transient, subsequently limiting their therapeutic application. Modifications in the CpG ODN backbone chemistry, various delivery methods including mixing or cross-linking of ODN to other carrier compounds have been shown to significantly enhance the biologic activity of ODN. However, the exact mechanisms that mediate this enhancement of activity are not well understood and may include local cell recruitment and activation, cytokine production, upregulation of receptor expression and increasing the half-life of ODN through creation of a depot. We will review the various approaches that have been used in enhancing the immunostimulatory effects of CpG ODN in vivo and also discuss the possible mechanisms that may be involved in this enhancement.

Quantitative analysis of pro-inflammatory cytokine mRNA expression in Theileria annulata-infected cell lines derived from resistant and susceptible cattle.

The pathogenic mechanisms involved in tropical theileriosis, caused by the tick-borne protozoan parasite Theileria annulata, are unclear. Pathology is associated with the schizont stage of the parasite, which resides within bovine macrophages. Breed-specific differences in pathology have been observed in cattle, several Bos indicus breeds are relatively resistant to tropical theileriosis whilst Bos taurus cattle are highly susceptible. Infected cells express pro-inflammatory cytokines and it has been hypothesized that these cytokines play a major role in the pathology of the disease. Therefore, using quantitative RT-PCR we investigated the expression of the key candidates, interleukin 1 beta (IL-1beta), IL-6 and tumour necrosis factor alpha (TNF-alpha), in T. annulata low passage infected cell lines derived ex vivo from experimental infection of resistant and susceptible cattle. mRNA for each cytokine was detected in all cell lines investigated at levels higher than those observed in resting monocytes. However, the analyses did not identify any breed-specific differences. Therefore, these results are not consistent with the hypothesis that differential regulation of infected cell derived pro-inflammatory cytokines (IL-1beta, IL-6 and TNF-alpha) accounts for the breed-related differences in resistance and susceptibility to T. annulata infection. Other, currently unknown mechanisms may be of greater importance.

All three classes of CpG ODNs up-regulate IP-10 gene in pigs.

The analysis of CpG ODN induced innate immune responses in different animal species has shown substantial similarities and differences in levels and types of induced cytokines profile. The objectives of these studies were to identify innate immune biomarkers activated by three classes of CpG ODNs in pigs. For this purpose, we investigated the kinetics of innate immune responses in immune cells from pigs following in vitro and in vivo stimulation with CpG ODNs. The mRNA expression of cytokine and chemokine genes were assayed by SYBR green based quantitative real time PCR. A-class CpG ODN induced significant but transient levels of IFN-gamma, IL-12 (P40), IL-6, IL-4 and TNF-alpha mRNA, C-class CpG ODN induced significant level of IFN-gamma, IFN-alpha and IL-12 mRNA and the lowest level of IL-4 (Th-2 type) mRNA. A very low level of some cytokines stimulation was observed by GC ODNs. It is noteworthy, that IL-12 (P35) mRNA was significantly stimulated by B-class GpC ODN 7909. Interestingly, all classes of CpG ODNs induced significant level of IP-10 at 12h post stimulation. These in vitro and in vivo observations suggest that interferon-gamma inducible protein 10 (IP-10) may be a reliable biomarker for immune activity induced by CpG ODNs in pigs.

Subcutaneous, but not intratracheal administration of the TLR9 agonist, CpG DNA transiently reduces parainfluenza-3 virus shedding in newborn lambs.

Synthetic oligodeoxynucleotides (ODN) containing CpG motifs signal through TLR9 and activate innate immunity resulting in protection against a variety of parasitic, bacterial and viral pathogens in mouse models. However, few studies have demonstrated protection in humans and large animals. In the present investigations, we evaluated protection by CpG ODN in a parainfluenza-3 (PI-3) virus infection in neonatal lambs. Subcutaneous (SC) injection of CpG ODN induced high levels of 2'5'-A synthetase and significantly reduced PI-3 virus shedding in newborn lambs. Furthermore, pre-treatment of newborn lambs with SC CpG ODN 2 days, but not 6 days prior to the virus challenge was protective. In contrast, intratracheal (IT) administration of CpG ODN induced 2'5'-A synthetase but had no significant impact on PI-3 virus shedding in nasal secretions. We conclude that a systemic administration of CpG ODN and the timing of the treatment are critical for the protection of neonatal lambs against a respiratory viral infection.

Standardisation and comparison of serial dilution and single dilution enzyme linked immunosorbent assay (ELISA) using different antigenic preparations of the Babesia (Theileria) equi parasite.

Serial dilution and single dilution enzyme linked immunosorbent assays (ELISA) were standardised and their sensitivity and specificity were compared for serodiagnosis of Babesia equi infection. The antibody titres of 24 donkey sera of known identity were determined separately by serial dilution ELISA using three different B. equi antigens namely whole merozoite (WM), cell membrane (CM) and high speed supernatant (HSS). The ratios of the optical density (OD) of known positive and known negative sera at different serum dilutions were calculated and termed as the positive/negative (P/N) ratio. The coefficients of correlation (r) were calculated between the P/N ratios at different dilutions of sera and the log10 antibody titres of the same sera were ascertained by serial dilution ELISA. The highest value of 'r' was obtained at a serum dilution of 1:200. From log10 antibody titre of sera (y) and their P/N ratio at a dilution of 1:200 (x), regression equations (y = a + bx) were calculated separately for the three antigens. Test sera were diluted to 1:200, their OD were read in duplicate wells and were converted to the P/N ratio. Antibody titres were predicted from the P/N ratio using a regression equation separately for the three antigens. Titres obtained by both ELISAs were not significantly different from each other, thus confirming that single dilution ELISA could be successfully used to replace conventional serial dilution ELISA. The sensitivity, specificity and predictive value of single dilution ELISA was validated statistically using 42 B. equi disease-positive sera and 106 B. equi disease-negative sera. The WM antigen was found to be the most sensitive with a higher predictive value for negative test sera as compared to the CM or HSS antigens. Sera positive for other equine infections including Babesia caballi showed no cross-reaction with the three B. equi antigens in ELISA, thus the test was immunologically specific. Antibody titres of 109 unknown field donkey/horse sera obtained by serial and single dilution ELISA using the WM antigen did not show any significant difference. Since the single dilution ELISA was found to be more economical, convenient, sensitive, specific than the serial dilution ELISA and has a high predictive value, it is suitable for use in sero-epidemiological studies on B. equi infections in the field.