Analysis of Johne’s disease ELISA status and associated performance parameters in Irish dairy cows

peer-reviewedBackground Infection with Mycobacterium avium subspecies paratuberculosis (MAP) has been associated with reductions in milk production in dairy cows and sub optimal fertility. The aim of this study was to highlight the production losses associated with testing MAP ELISA positive in Irish dairy cows. Secondary objectives included investigation of risk factors associated with testing MAP ELISA positive. A survey of management practices on study farms was also conducted, with examination of associations between management practices and herd MAP status. Blood samples were collected from 4188 breeding animals on 22 farms. Samples were ELISA tested using the ID Screen Paratuberculosis Indirect Screening Test. Production parameters examined included milk yield, milk fat, milk protein, somatic cell count, and calving interval. The association between MAP ELISA status and production data was investigated using multi-level mixed models. Logistic regression was used to identify risk factors for testing JD blood ELISA positive at individual cow level and to identify associations between farm management practices and herd MAP status. Results Data were available for 3528 cows. The apparent prevalence recorded was 7.4 %. Mixed model analysis revealed no statistically significant association between testing MAP ELISA positive and dairy cow production parameters. Risk factors associated with testing positive included larger sized herds being over twice more likely to test positive than smaller herds (OR 2.4 P = <0.001). Friesians were less likely to test positive relative to other breeds. A number of study farmers were engaged in management practices that have previously been identified as high risk for MAP transmission e.g., 73.1 % pooled colostrum and 84.6 % of study farmers used the calving area to house sick animals throughout the year. No significant associations however, were identified between farm management practices and herd MAP status. Conclusion No production losses were identified; however an apparent prevalence of 7.4 % was recorded. With the abolition of EU milk quotas herd size in Ireland is expanding, as herds included in this study were larger than the national average, results may be indicative of future JD levels if no JD control programmes are implemented to minimise transmission

Non-invasive methods of identifying and tracking wild squid

The ability to identify individual free-living animals in the field is an important method for studying their behavior. Apart from invasive external or internal tags, which may cause injury or abnormal behavior, most cephalopods cannot be tagged, as their skin is too soft and delicate for tag retention. Additionally, cephalopods remove many types of tags. However, body markings have been successfully used as a non invasive method to identify individuals of many different species of animals, including whale sharks, grey whales, seals, and zebras. We developed methods to sex and individually identify Caribbean reef squid, Sepiotheuthis sepioidea. Males showed distinct bright dots on their fins on a Basic Brown background and have a light line at the fin edge while the females had a gradual transition from Brown to Pale towards the edge of their fins without showing distinct fin-dots or lines. In the field we used four characters to distinguish individual S. sepioidea from each other – sex, relative size to each other, scars, and patterns of light-colored dots on their mantles and fins. These dot patterns are individually unique and constant in location through time. Observations in the field were backed up by an image database using illustrations and photography

Non-invasive methods of identifying and tracking wild squid

The ability to identify individual free-living animals in the field is an important method for studying their behavior. Apart from invasive external or internal tags, which may cause injury or abnormal behavior, most cephalopods cannot be tagged, as their skin is too soft and delicate for tag retention. Additionally, cephalopods remove many types of tags. However, body markings have been successfully used as a non invasive method to identify individuals of many different species of animals, including whale sharks, grey whales, seals, and zebras. We developed methods to sex and individually identify Caribbean reef squid, Sepiotheuthis sepioidea. Males showed distinct bright dots on their fins on a Basic Brown background and have a light line at the fin edge while the females had a gradual transition from Brown to Pale towards the edge of their fins without showing distinct fin-dots or lines. In the field we used four characters to distinguish individual S. sepioidea from each other – sex, relative size to each other, scars, and patterns of light-colored dots on their mantles and fins. These dot patterns are individually unique and constant in location through time. Observations in the field were backed up by an image database using illustrations and photography

Factors influencing radon concentration during energy retrofitting in domestic buildings: a computational evaluation

The findings of recent research signalled increased radon levels following energy retrofitting of dwellings but to date, there have been limited quantitative data to support this observation. A modelling framework was developed that incorporates a dynamic radon entry rate, capturing changes in pressure differentials, to investigate changes in radon concentration following different energy-efficient retrofit measures in naturally-ventilated dwellings. Simulations examined a range of input criteria: dwelling type, air permeabilities, radon flow exponents, pre and post thermal retrofit characteristics, outdoor weather locations and corresponding wind profiles, as well as different ventilation guidelines. A total of 3,780 simulations were carried out. The air permeability of the building had the greatest impact on radon concentration with increases of up to 107%. Non-linear increases were observed arising from the impacts on pressure differentials due to changes in air permeability. The application of representative weather profiles associated with different locations (e.g. coastal, inland) resulted in differences of up to 37%. To a lesser extent, increased indoor temperature due to thermally retrofitting the building fabric, without changes in air permeability, resulted in radon levels increasing by 7%. Additionally, it was shown that the radon flow exponent was not a significant influence on radon concentrations following a retrofit. The addition of ventilation measures means that it is possible to achieve increased airtightness without impacting on the radon concentration. Overall, the simulations provide quantitative information that explains increased airtightness and elevated radon levels, highlighting the potential for radon concentrations to either increase or decrease following an energy retrofit

Investigating the functionality of an OCT4-short response element in human induced pluripotent stem cells.

Pluripotent stem cells offer great therapeutic promise for personalized treatment platforms for numerous injuries, disorders, and diseases. Octamer-binding transcription factor 4 (OCT4) is a key regulatory gene maintaining pluripotency and self-renewal of mammalian cells. With site-specific integration for gene correction in cellular therapeutics, use of the OCT4 promoter may have advantages when expressing a suicide gene if pluripotency remains. However, the human OCT4 promoter region is 4 kb in size, limiting the capacity of therapeutic genes and other regulatory components for viral vectors, and decreasing the efficiency of homologous recombination. The purpose of this investigation was to characterize the functionality of a novel 967bp OCT4-short response element during pluripotency and to examine the OCT4 titer-dependent response during differentiation to human derivatives not expressing OCT4. Our findings demonstrate that the OCT4-short response element is active in pluripotency and this activity is in high correlation with transgene expression in vitro, and the OCT4-short response element is inactivated when pluripotent cells differentiate. These studies demonstrate that this shortened OCT4 regulatory element is functional and may be useful as part of an optimized safety component in a site-specific gene transferring system that could be used as an efficient and clinically applicable safety platform for gene transfer in cellular therapeutics

Impact of reactive surfaces on the abiotic reaction between nitrite and ferrous iron and associated nitrogen and oxygen isotope dynamics

Anaerobic nitrate-dependent Fe(II) oxidation (NDFeO) is widespread in various aquatic environments and plays a major role in iron and nitrogen redox dynamics. However, evidence for truly enzymatic, autotrophic NDFeO remains limited, with alternative explanations involving the coupling of heterotrophic denitrification with the abiotic oxidation of structurally bound or aqueous Fe(II) by reactive intermediate nitrogen (N) species (chemodenitrification). The extent to which chemodenitrification is caused (or enhanced) by ex vivo surface catalytic effects has not been directly tested to date. To determine whether the presence of either an Fe(II)-bearing mineral or dead biomass (DB) catalyses chemodenitrification, two different sets of anoxic batch experiments were conducted: 2 mM Fe(II) was added to a low-phosphate medium, resulting in the precipitation of vivianite (Fe3(PO4)2), to which 2 mM nitrite (NO−2) was later added, with or without an autoclaved cell suspension (∼1.96×108 cells mL−1) of Shewanella oneidensis MR-1. Concentrations of nitrite (NO−2), nitrous oxide (N2O), and iron (Fe2+, Fetot) were monitored over time in both set-ups to assess the impact of Fe(II) minerals and/or DB as catalysts of chemodenitrification. In addition, the natural-abundance isotope ratios of NO−2 and N2O (δ15N and δ18O) were analysed to constrain the associated isotope effects. Up to 90 % of the Fe(II) was oxidized in the presence of DB, whereas only ∼65 % of the Fe(II) was oxidized under mineral-only conditions, suggesting an overall lower reactivity of the mineral-only set-up. Similarly, the average NO−2 reduction rate in the mineral-only experiments (0.004±0.003 mmol L−1 d−1) was much lower than in the experiments with both mineral and DB (0.053±0.013 mmol L−1 d−1), as was N2O production (204.02±60.29 nmol L−1 d−1). The N2O yield per mole NO−2 reduced was higher in the mineral-only set-ups (4 %) than in the experiments with DB (1 %), suggesting the catalysis-dependent differential formation of NO. N-NO−2 isotope ratio measurements indicated a clear difference between both experimental conditions: in contrast to the marked 15N isotope enrichment during active NO−2 reduction (15εNO2=+10.3 ‰) observed in the presence of DB, NO−2 loss in the mineral-only experiments exhibited only a small N isotope effect (<+1 ‰). The NO−2-O isotope effect was very low in both set-ups (18εNO2 <1 ‰), which was most likely due to substantial O isotope exchange with ambient water. Moreover, under low-turnover conditions (i.e. in the mineral-only experiments as well as initially in experiments with DB), the observed NO−2 isotope systematics suggest, transiently, a small inverse isotope effect (i.e. decreasing NO−2 δ15N and δ18O with decreasing concentrations), which was possibly related to transitory surface complexation mechanisms. Site preference (SP) of the 15N isotopes in the linear N2O molecule for both set-ups ranged between 0 ‰ and 14 ‰, which was notably lower than the values previously reported for chemodenitrification. Our results imply that chemodenitrification is dependent on the available reactive surfaces and that the NO−2 (rather than the N2O) isotope signatures may be useful for distinguishing between chemodenitrification catalysed by minerals, chemodenitrification catalysed by dead microbial biomass, and possibly true enzymatic NDFeO

Impact of reactive surfaces on the abiotic reaction between nitrite and ferrous iron and associated nitrogen and oxygen isotope dynamics

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Visser, A., Wankel, S. D., Niklaus, P. A., Byrne, J. M., Kappler, A. A., & Lehmann, M. F. Impact of reactive surfaces on the abiotic reaction between nitrite and ferrous iron and associated nitrogen and oxygen isotope dynamics. Biogeosciences, 17(16), (2020): 4355-4374, doi:10.5194/bg-17-4355-2020.Anaerobic nitrate-dependent Fe(II) oxidation (NDFeO) is widespread in various aquatic environments and plays a major role in iron and nitrogen redox dynamics. However, evidence for truly enzymatic, autotrophic NDFeO remains limited, with alternative explanations involving the coupling of heterotrophic denitrification with the abiotic oxidation of structurally bound or aqueous Fe(II) by reactive intermediate nitrogen (N) species (chemodenitrification). The extent to which chemodenitrification is caused (or enhanced) by ex vivo surface catalytic effects has not been directly tested to date. To determine whether the presence of either an Fe(II)-bearing mineral or dead biomass (DB) catalyses chemodenitrification, two different sets of anoxic batch experiments were conducted: 2 mM Fe(II) was added to a low-phosphate medium, resulting in the precipitation of vivianite (Fe3(PO4)2), to which 2 mM nitrite (NO−2) was later added, with or without an autoclaved cell suspension (∼1.96×108 cells mL−1) of Shewanella oneidensis MR-1. Concentrations of nitrite (NO−2), nitrous oxide (N2O), and iron (Fe2+, Fetot) were monitored over time in both set-ups to assess the impact of Fe(II) minerals and/or DB as catalysts of chemodenitrification. In addition, the natural-abundance isotope ratios of NO−2 and N2O (δ15N and δ18O) were analysed to constrain the associated isotope effects. Up to 90 % of the Fe(II) was oxidized in the presence of DB, whereas only ∼65 % of the Fe(II) was oxidized under mineral-only conditions, suggesting an overall lower reactivity of the mineral-only set-up. Similarly, the average NO−2 reduction rate in the mineral-only experiments (0.004±0.003 mmol L−1 d−1) was much lower than in the experiments with both mineral and DB (0.053±0.013 mmol L−1 d−1), as was N2O production (204.02±60.29 nmol L−1 d−1). The N2O yield per mole NO−2 reduced was higher in the mineral-only set-ups (4 %) than in the experiments with DB (1 %), suggesting the catalysis-dependent differential formation of NO. N-NO−2 isotope ratio measurements indicated a clear difference between both experimental conditions: in contrast to the marked 15N isotope enrichment during active NO−2 reduction (15εNO2=+10.3 ‰) observed in the presence of DB, NO−2 loss in the mineral-only experiments exhibited only a small N isotope effect (<+1 ‰). The NO−2-O isotope effect was very low in both set-ups (18εNO2 <1 ‰), which was most likely due to substantial O isotope exchange with ambient water. Moreover, under low-turnover conditions (i.e. in the mineral-only experiments as well as initially in experiments with DB), the observed NO−2 isotope systematics suggest, transiently, a small inverse isotope effect (i.e. decreasing NO−2 δ15N and δ18O with decreasing concentrations), which was possibly related to transitory surface complexation mechanisms. Site preference (SP) of the 15N isotopes in the linear N2O molecule for both set-ups ranged between 0 ‰ and 14 ‰, which was notably lower than the values previously reported for chemodenitrification. Our results imply that chemodenitrification is dependent on the available reactive surfaces and that the NO−2 (rather than the N2O) isotope signatures may be useful for distinguishing between chemodenitrification catalysed by minerals, chemodenitrification catalysed by dead microbial biomass, and possibly true enzymatic NDFeO.This research has been supported by the Deutsche Forschungsgemeinschaft (DFG; grant no. GRK 1708, “Molecular principles of bacterial survival strategies”) and the University of Basel, Switzerland

AI For Sheep Using Frozen-thawed Semen.

End of Project ReportInternational experience has been that cervical insemination of sheep with frozen-thawed semen usually yields unacceptably low pregnancy rates (10 to 30%). An exceptional case has been Norway where non-return rates in on-farm usage are around 60%. The objective of the work described in this report was to develop an AI procedure for Irish conditions, based initially on Norwegian protocols, using semen from individual rams. Such a procedure would greatly facilitate and enhance genetic improvement programmes for sheep. The work undertaken had two separate aspects:- (i) studies on semen, including processing and freezing methods, laboratory evaluation of semen quality post thawing and the relationship of in vitro evaluation to fertilisation rate in vivo (ii) studies on pregnancy rate following AI in relation to issues such as ram breed effects, effects of synchronisation, operator differences and the role of ewe breed inducing the timing of ovulation and various physical and physiological assessments of the cervix at AI. The main results in relation to semen studies were that, while a range of differential staining procedures could be used to objectively evaluate semen with respect to proportion of live speramatozoa and the integrity of sperm cells after thawing, these results were not useful as indicators of fertilisation capacity in vivo. The in vitro fertilisation (IVF) of sheep oocytes recovered from abattoir material gave promising results as a method for evaluating the fertilisation capacity of frozen-thawed semen. The technique requires further validation.Department of Agriculture, Food and the Marin