Research and Innovation Opportunities to Improve Epidemiological Knowledge and Control of Environmentally Driven Zoonoses.

While zoonotic diseases are defined by transmission processes between animals and humans, for many of these diseases the presence of a contaminated environmental source is the cause of transmission. Most zoonoses depend on complex environmentally driven interactions between humans and animals, which occur along an occupational and recreational environmental continuum, including farming and animal marketing systems, environmental management systems, and community leisure environments. Environmentally driven zoonoses (EDZs) are particularly challenging to diagnose and control as their reservoirs are in the natural environment and thus often escape conventional surveillance systems that rely on host monitoring. Changes in the environment as a result of climate change [1], human population density [2], and intensification of agriculture [3] have been linked to increasing transmission events for this group of infections. As such, there is a recognised need to be able to detect the presence of EDZs in the environment as a means to better anticipate transmission events and improve source attribution investigations. Finally, the recognition that a One Health approach is needed to combat these infections is signalling to governments the need to develop policy that optimises trade-offs across human, animal, and environmental health sectors. In this review, we discuss and critically appraise the main challenges relating to the epidemiology, diagnosis, and control of environmental zoonotic disease. Using a set of exemplar diseases, including avian influenza and antimicrobial resistant pathogens, we explore the epidemiological contexts (risk factors) within which these infections not only impact human health but also contribute to animal health and environmental impacts. We then critically appraise the surveillance challenges of monitoring these infections in the environment and examine the policy trade-offs for a more integrated approach to mitigating their impacts.

Survey and Sequence Characterization of Bovine Mastitis-Associated Escherichia coli in Dairy Herds.

Escherichia coli is frequently associated with mastitis in cattle. "Pathogenic" and "commensal" isolates appear to be genetically similar. With a few exceptions, no notable genotypic differences have been found between commensal and mastitis-associated E. coli. In this study, 24 E. coli strains were isolated from dairy cows with clinical mastitis in three geographic regions of Australia (North Queensland, South Queensland, and Victoria), sequenced, then genomically surveyed. There was no observed relationship between sequence type (ST) and region (p = 0.51). The most common Multi Locus Sequence Type was ST10 (38%), then ST4429 (13%). Pangenomic analysis revealed a soft-core genome of 3,463 genes, including genes associated with antibiotic resistance, chemotaxis, motility, adhesion, biofilm formation, and pili. A total of 36 different plasmids were identified and generally found to have local distributions (p = 0.02). Only 2 plasmids contained antibiotic resistance genes, a p1303_5-like plasmid encoding multidrug-resistance (trimethoprim, quaternary ammonium, beta-lactam, streptomycin, sulfonamide, and kanamycin) from two North Queensland isolates on the same farm, while three Victorian isolates from the same farm contained a pCFSAN004177P_01-like plasmid encoding tetracycline-resistance. This pattern is consistent with a local spread of antibiotic resistance through plasmids of bovine mastitis cases. Notably, co-occurrence of plasmids containing virulence factors/antibiotic resistance with putative mobilization was rare, though the multidrug resistant p1303_5-like plasmid was predicted to be conjugative and is of some concern. This survey has provided greater understanding of antibiotic resistance within E. coli-associated bovine mastitis which will allow greater prediction and improved decision making in disease management.

Efficacy of a Lactobacillus-Based Teat Spray on Udder Health in Lactating Dairy Cows.

Teat disinfection is a common pre- and post-milking mastitis prevention practice that is part of a mastitis control program in dairy herds. Commercially available teat disinfectants are generally chemical-based products. The use of these products has occasionally raised concerns about the risk of chemical residues in milk. An alternative treatment or prevention strategy based on probiotics has the potential to circumvent this risk. Two treatments were compared in a cross-over clinical trial in a single herd: a lactobacillus-based, post-milking teat spray (LACT), and a commercial iodine-based post-milking teat disinfectant product as (positive control, PC). The effect of the two treatments on cow somatic cell counts was quantified using a multivariate mixed-effects linear regression model with cow fitted as a random effect. The odds of teat end scores increasing from a low to a high score tended to be lower (OR = 0.74, 95% CI 0.54-1.01, P = 0.06) for cows receiving LACT treatment. On average, there was also a tendency for a lower somatic cell counts in the LACT treated cows (antilog of coefficient = 0.91, 95% CI 0.80-1.03, P = 0.13) compared with the PC treated cows. The application of the lactobacillus-based product to teats could reduce the rate of teat end scores progression from low to higher scores, and potentially improve teat end sphincter functions and udder health. Further, larger scale validation work is required to support the findings of the current study.

Systematic review of an intervention: the use of probiotics to improve health and productivity of calves.

The aims of this study were to undertake a systematic review and meta-analysis of the types of probiotic formulations that are commercially available and to critically appraise the available evidence for the effectiveness of probiotics in improving the health and productivity of calves. Relevant papers were identified to answer the question: 'In calves aged between birth to one year, is the use of probiotics associated with changes in haematological or biochemical parameters, faecal bacteria counts, average daily live weight gain, dry matter intake, or feed conversion ratio?' The search of the literature yielded 67 studies that fit the primary screening criteria. Included studies were assessed for bias and confounding using a predefined risk assessment tool adapted from the Cochrane Collaboration's tool for assessing risk of bias in randomised trials and GRADE guidelines. Meta-analysis was performed using Review Manager and R. Random sequence generation was low in more than 59 % of studies. Risk of allocation concealment and performance bias were largely unclear in over 68 % of studies. Calves fed probiotics had increased average daily live weight gains (ADG) from birth to weaning (mean difference [MD] = 83.14 g/d 95 % CI = 58.36-107.91, P < 0.001) compared with calves on a control diet. Calf age reduced the level of heterogeneity of the effect of probiotics on ADG for calves between one to three weeks of age (τ2 = 73.15; I2 = 4%; P = 0.40) but not for calves older than three weeks of age (τ2 = 2892.91; I2 = 73 %; P < 0.001). Feed conversion ratio (FCR) was lower for calves on probiotics (MD = -0.13 kg of dry matter intake (DMI) to kg of live weight (LW) gain, 95 % CI = -0.17 to -0.09, P < 0.001), and the heterogeneity of effect was large in younger aged calves (τ2 = 0.05; I2 = 78 %; P = 0.03). The risk of bias regarding the methodology in the included studies was high. The quality of evidence for each outcome was categorised as moderate. There is sufficient data to support the effectiveness of probiotic use in some applications such as for the improvement of performance and productivity parameters of calves. However, the evidence is weak for other potential probiotic uses in calves such as improved health and reduced risk of disease. Therefore, the existing data are inconclusive and do not support the use of probiotics as an alternative to antimicrobials to improve calf health and productivity.

COVID-19 Infection Diagnosis: Potential Impact of Isothermal Amplification Technology to Reduce Community Transmission of SARS-CoV-2.

The current coronavirus disease 2019 (COVID-19) pandemic is largely driven by community transmission, after 2019 novel Coronavirus (2019-nCoV or SARS-CoV-2) crosses the borders. To stop the spread, rapid testing is required at community clinics and hospitals. These rapid tests should be comparable with the standard PCR technology. Isothermal amplification technology provides an excellent alternative that is highly amenable to resource limited settings, where expertise and infrastructure to support PCR are not available. In this review, we provide a brief description of isothermal amplification technology, its potential and the gaps that need to be considered for SARS-CoV-2 detection. Among this emerging technology, loop-mediated amplification (LAMP), recombinase polymerase amplification (RPA) and Nicking enzyme-assisted reaction (NEAR) technologies have been identified as potential platforms that could be implemented at community level, without samples referral to a centralized laboratory and prolonged turnaround time associated with the standard COVID-19 RT-PCR test. LAMP, for example, has recently been shown to be comparable with PCR and could be performed in less than 30 min by non-laboratory staff, without RNA extractions commonly associated with PCR. Interestingly, NEAR (ID NOW™ COVID-19 (Abbott, IL, USA) was able to detect the virus in 5 min. More so, isothermal platforms are cost effective and could easily be scaled up to resource limited settings. Diagnostics developers, scientific community and commercial companies could consider this alternative method to help stop the spread of COVID-19.

Ebolavirus diagnosis made simple, comparable and faster than molecular detection methods: preparing for the future.

BACKGROUND: The 2014/2015 Ebolavirus outbreak resulted in more than 28,000 cases and 11,323 reported deaths, as of March 2016. Domestic transmission of the Guinea strain associated with the outbreak occurred mainly in six African countries, and international transmission was reported in four countries. Outbreak management was limited by the inability to rapidly diagnose infected cases. A further fifteen countries in Africa are predicted to be at risk of Ebolavirus outbreaks in the future as a consequence of climate change and urbanization. Early detection of cases and reduction of transmission rates is critical to prevent and manage future severe outbreaks. We designed a rapid assay for detection of Ebolavirus using recombinase polymerase amplification, a rapid isothermal amplification technology that can be combined with portable lateral flow detection technology. The developed rapid assay operates in 30 min and was comparable with real-time TaqMan™ PCR. METHODS: Designed, screened, selected and optimized oligonucleotides using the NP coding region from Ebola Zaire virus (Guinea strain). We determined the analytical sensitivity of our Ebola rapid molecular test by testing selected primers and probe with tenfold serial dilutions (1.34 × 1010- 1.34 × 101 copies/µL) of cloned NP gene from Mayinga strain of Zaire ebolavirus in pCAGGS vector, and serially diluted cultured Ebolavirus as established by real-time TaqMan™ PCR that was performed using ABI7500 in Fast Mode. We tested extracted and reverse transcribed RNA from cultured Zaire ebolavirus strains - Mayinga, Gueckedou C05, Gueckedou C07, Makona, Kissidougou and Kiwit. We determined the analytical specificity of our assay with related viruses: Marburg, Ebola Reston and Ebola Sudan. We further tested for Dengue virus 1-4, Plasmodium falciparum and West Nile Virus (Kunjin strain). RESULTS: The assay had a detection limit of 134 copies per µL of plasmid containing the NP gene of Ebolavirus Mayinga, and cultured Ebolavirus and was highly specific for the Zaire ebolavirus species, including the Guinea strain responsible for the 2014/2015 outbreak. The assay did not detect related viruses like Marburg, Reston, or Sudan viruses, and other pathogens likely to be isolated from clinical samples. CONCLUSIONS: Our assay could be suitable for implementation in district and primary health laboratories, as only a heating block and centrifuge is required for operation. The technique could provide a pathway for rapid screening of patients and animals for improved management of outbreaks.

Virulent gene profile and antibiotic susceptibility pattern of Shiga toxin-producing Escherichia coli (STEC) from cattle and camels in Maiduguri, North-Eastern Nigeria.

Prevalence and distribution of Shiga toxin-producing Escherichia coli (STEC) serogroups from the faecal samples of cattle and camels slaughter in Maiduguri abattoir and their antibiotic resistance profile of the isolates were determined. The highest prevalence (24%) was recorded in the month of September and more STEC isolates came from cattle than the camels. There was significant (P < 0.05) seasonal trend in the prevalence of STEC among cattle and camel with more cases recorded during the wet season. Although, the study did not demonstrate significant influence of sex from the various sources. The serogroups recorded in this study were O157, O26, O91, O103 and O111. There was no significant difference (P < 0.05) between the detection rates of serogroups. The serogroup O26 was significantly (P < 0.05) the most observed serogroup in both camels and cattle. None of the STEC isolates tested positive for the O45 serogroup. PCR assays shows that 50 (63.3%) of the 86 STEC isolates carried the stx2 gene, 34 (43%) possessed the stx1 gene, and 14 (16.3%) carried both stx1 and stx2 genes. Other genes detected include eae and ehlyA. The antimicrobial resistance among the STEC O157 and non-O157 isolates from cattle and camels in Maiduguri abattoir were very high and the STEC isolates were resistant to at least one or more of the antimicrobial agents tested. There was also multidrug resistance with the most frequent occurring patterns been ampicillin/nalidixic acid and tetracycline/trimethoprim. However, all the 79 isolates were sensitive to chloramphenicol, ceftazidime and ceftriaxone; therefore, these drugs could be drugs of choice in the treatment of STEC infections.

Recombinase polymerase amplification: Emergence as a critical molecular technology for rapid, low-resource diagnostics.

Isothermal molecular diagnostics are bridging the technology gap between traditional diagnostics and polymerase chain reaction-based methods. These new techniques enable timely and accurate testing, especially in settings where there is a lack of infrastructure to support polymerase chain reaction facilities. Despite this, there is a significant lack of uptake of these technologies in developing countries where they are highly needed. Among these novel isothermal technologies, recombinase polymerase amplification (RPA) holds particular potential for use in developing countries. This rapid nucleic acid amplification approach is fast, highly sensitive and specific, and amenable to countries with a high burden of infectious diseases. Implementation of RPA technology in developing countries is critically required to assess limitations and potentials of the diagnosis of infectious disease, and may help identify impediments that prevent adoption of new molecular technologies in low resource- and low skill settings. This review focuses on approaching diagnosis of infectious disease with RPA.