Antimicrobial resistance in Escherichia coli isolated from pigs and associations with aggregated antimicrobial usage in Ireland: A herd-level exploration.

AIMS: Antimicrobial resistance (AMR) is of significant global concern and is a major One Health issue. There is evidence to suggest that increased antimicrobial usage (AMU) can be associated with AMR patterns, and therefore, there have been efforts to reduce AMU in anticipation of reducing AMR emergence risk. The aim of this study was to investigate whether there were any associations between AMU and AMR patterns of commensal Escherichia coli isolated from pig herds in Ireland. METHODS AND RESULTS: Data on AMR from a panel of antimicrobials (AMDs) were gathered as part of national surveillance activities. These data were associated with reported usage of AMDs, on a year-quarter basis, measured in mg/kg at a herd-level using generalized estimating equation regression analysis. Associations were tested with AMR presence or multi-drug resistance (MDR; ≥3 classes) profiles and total AMU during the contemporaneous quarter and previous quarter, respectively. Furthermore, individual and AMD class-based associations were tested. The final dataset contained 218 observations (herd-quarter usage and AMR resistance profile) from 122 herds during 2019-2021. Apparent resistance prevalence varied according to AMD type, with the highest mean prevalence found with tetracycline at 51.57% (95% CI: 45.06%-58.09%). There were significant associations between a herd obtaining a positive AMR result for any AMDs and the overall levels of AMU during the year-quarter. Furthermore, there were significant positive associations between MDR and total AMU. At the compound level, chloramphenicol resistance was significantly associated with increased usage of trimethoprim/sulfadiazine and chlortetracycline, respectively (p < 0.010). Tetracycline resistance was associated with increased use of chlortetracycline (p = 0.008). At the antimicrobial class level, there was a significant positive relationship between the usage of phenicol and the probability of a resistance for chloramphenicol (p = 0.026) and between the usage of tetracycline and tetracycline resistance probability (p = 0.018). CONCLUSIONS: Our data provide evidence of associations between overall AMU and AMR or MDR risk at the herd-quarter level. There was also evidence of associations between specific AMDs and patterns of resistance. Associations varied depending on whether time lags in usage were modelled or how usage was modelled (e.g. dichotomized or continuous). Associations with rarely used AMDs (e.g. critically important AMDs) were precluded due to a lack of statistical power. Continued monitoring of both AMU and AMR is crucial to assess the impacts of policy changes aimed at reducing AMU.

Invited review: Antimicrobial resistance in bovine mastitis pathogens: A review of genetic determinants and prevalence of resistance in European countries.

Antimicrobial resistance is an urgent and growing problem worldwide, both for human and animal health. In the animal health sector actions have been taken as concerns grow regarding the development and spread of antimicrobial resistance. Mastitis is the most common infection in dairy cattle. We aimed to summarize the genetic determinants found in staphylococci, streptococci, and Enterobacteriaceae isolated from mastitic milk samples and provide a comparison of percentage resistance to a variety of antimicrobials in European countries.

Timing of Transcriptomic Peripheral Blood Mononuclear Cell Responses of Sheep to Fasciola hepatica Infection Differs From Those of Cattle, Reflecting Different Disease Phenotypes.

Infection with the zoonotic trematode Fasciola hepatica, common in many regions with a temperate climate, leads to delayed growth and loss of productivity in cattle, while infection in sheep can have more severe effects, potentially leading to death. Previous transcriptomic analyses revealed upregulation of TGFB1, cell death and Toll-like receptor signalling, T-cell activation, and inhibition of nitric oxide production in macrophages in response to infection. However, the differences between ovine and bovine responses have not yet been explored. The objective of this study was to further investigate the transcriptomic response of ovine peripheral blood mononuclear cells (PBMC) to F. hepatica infection, and to elucidate the differences between ovine and bovine PBMC responses. Sixteen male Merino sheep were randomly assigned to infected or control groups (n = 8 per group) and orally infected with 120 F. hepatica metacercariae. Transcriptomic data was generated from PBMC at 0, 2 and 16 weeks post-infection (wpi), and analysed for differentially expressed (DE) genes between infected and control animals at each time point (analysis 1), and for each group relative to time 0 (analysis 2). Analysis 2 was then compared to a similar study performed previously on bovine PBMC. A total of 453 DE genes were found at 2 wpi, and 2 DE genes at 16 wpi (FDR < 0.1, analysis 1). Significantly overrepresented biological pathways at 2 wpi included role of PKR in interferon induction and anti-viral response, death receptor signalling and RIG-I-like receptor signalling, which suggested that an activation of innate response to intracellular nucleic acids and inhibition of cellular apoptosis were taking place. Comparison of analysis 2 with the previous bovine transcriptomic study revealed that anti-inflammatory response pathways which were significantly overrepresented in the acute phase in cattle, including IL-10 signalling, Th2 pathway, and Th1 and Th2 activation were upregulated only in the chronic phase in sheep. We propose that the earlier activation of anti-inflammatory responses in cattle, as compared with sheep, may be related to the general absence of acute clinical signs in cattle. These findings offer scope for "smart vaccination" strategies for this important livestock parasite.

Transcriptomic Analysis of Ovine Hepatic Lymph Node Following Fasciola hepatica Infection - Inhibition of NK Cell and IgE-Mediated Signaling.

Fasciola hepatica is a trematode parasite responsible for major economic losses in livestock production, and is also a food-borne zoonotic agent in developing rural regions. For years, the immunoregulatory mechanisms employed by the parasite have hampered efforts to develop a successful vaccine candidate. Given that a comprehensive understanding of the immune response to infection is needed, we investigated the gene expression changes in ovine hepatic lymph nodes after experimental infection with F. hepatica. Lymph nodes from uninfected and infected animals were processed for RNA sequencing (RNA-seq) at 16 weeks post-infection. Comparison of groups revealed 5,132 differentially-expressed genes (DEGs). An inhibition of pro-inflammatory pathways, which has previously been described during fasciolosis, was evident in our data. However, other signals previously identified in ruminant peripheral blood mononuclear cells (PBMC) or liver tissue, such as activation of TGF-ß or apoptosis-related pathways were not detected. We found inhibition of some key immunological pathways, including natural killer (NK) cell activity and IgE-mediated signaling. These may point to additional some as yet unrecognized mechanisms employed by the parasite to evade the host immune response. Understanding these, and leveraging information from this and other omics studies, will be important for the development of future vaccine prototypes against this parasite.

Update on the presence of Ixodes ricinus at the western limit of its range and the prevalence of Borrelia burgdorferi sensu lato.

It is often suggested that due to climate and environmental policy changes, the risk from tick-borne disease is increasing, particularly at the geographical limits of the vector distribution. Our project aimed to determine whether this was true for the risk of Lyme borreliosis in Ireland which is the western-most limit of Ixodes ricinus, the European vector of Borrelia burgdorferi sensu lato. The availability of a historical data set of tick infection rates compiled in the 1990s represented a unique opportunity as it provided a baseline against which current data could be compared. Following construction of a spatial predictive model for the presence and absence of I. ricinus based on data from 491 GPS locations visited between 2016 and 2019, 1404 questing nymphs from 27 sites were screened for the presence of Borrelia spp. using a TaqMan PCR aimed at the 23S rRNA gene sequence. All positive ticks were further analysed by nested PCR amplification and sequence analysis of the 5 S-23 S intergenic spacer. The model indicated that areas with the highest probability of tick presence were mostly located along the western seaboard and the Shannon and Erne river catchments, coinciding with historical high incidence areas of bovine babesiosis, while the infection rate of questing nymphs with B. burgdorferi s.l. and the prevalence of the various genospecies have remained surprisingly stable over the last 3 decades. Clear communication of the potential disease risk arising from a tick bite is essential in order to allay undue concerns over tick-borne diseases among the general public.

Fasciola hepatica products can alter the response of bovine immune cells to Mycobacterium avium subsp. paratuberculosis.

BACKGROUND: Fasciola hepatica causes economically important disease in livestock worldwide. The relevance of this parasitic infection extends beyond its direct consequences due to its immunoregulatory properties. OBJECTIVES: Given the importance of the T helper 1 (Th1) immune response in controlling infections with Mycobacterium avium subspecies paratuberculosis (MAP) in cattle, we aimed to establish the immunological consequences that co-infection with F. hepatica might have on the course of Johne's disease (JD). METHODS: This study compared the in vitro response of bovine immune cells to infection with MAP or exposure to MAP antigens following F. hepatica infection or stimulation with F. hepatica products. RESULTS: We found a decreased proliferation of peripheral blood mononuclear cells (PBMCs) after infection with F. hepatica. This reduction was inversely correlated with fluke burden. Pre-stimulation with F. hepatica molecules produced a significant reduction of ileocaecal lymph node leucocyte proliferation in response to MAP antigens. Additionally,F. hepatica products reduced expression of the CD14 receptor by macrophages and increased levels of apoptosis and bacterial (MAP) uptake. CONCLUSIONS: Overall, F. hepatica infection had little impact on the in vitro response of immune cells to MAP, whereas in vitro co-stimulation with F. hepatica molecules had a measurable effect. Whether this is likely to affect JD progression during in vivo chronic conditions remains unclear.

Fasciola hepatica Infection in Cattle: Analyzing Responses of Peripheral Blood Mononuclear Cells (PBMC) Using a Transcriptomics Approach.

The parasitic helminth Fasciola hepatica (liver fluke) causes economic loss to the livestock industry globally and also causes zoonotic disease. New control strategies such as vaccines are urgently needed, due to the rise of drug resistance in parasite populations. Vaccine development requires a comprehensive understanding of the immunological events during infection. Previous in vivo studies by our group have investigated global differentially expressed genes (DEGs) in ovine peripheral blood mononuclear cells (PBMC) in response to both acute and chronic F. hepatica infection. This work demonstrated that pathways involved in the pathogenesis of ovine fasciolosis included fibrosis, inhibition of macrophage nitric oxide production, and antibody isotype switching, among others. Transcriptomic changes in PBMC populations following F. hepatica infection in cattle, in which the disease phenotype is quite different, have not yet been examined. Using RNA sequencing we investigated gene expression changes in PBMC isolated from 9 non-infected and 11 F. hepatica-experimentally-infected calves immediately before infection, at 1 and at 14 weeks post-infection. Longitudinal time-course comparisons between groups revealed 21 and 1,624 DEGs driven exclusively by F. hepatica infection in cattle at acute and chronic stages, respectively. These results show that fewer DEGs at the acute stage of infection can be identified in cattle, as compared with sheep. In addition, the log2 fold-changes of these DEGs were relatively low (-1 to 3) reflecting the different clinical presentation of F. hepatica infection in cattle. Gene pathways for hepatic fibrosis and hepatic cholestasis along with apoptosis of antigen-presenting cells were enriched at chronic stages. Our results reflect the major differences in the disease phenotype between cattle and sheep and may indicate pathways to target in vaccine development.

Spatial patterns of Fasciola hepatica and Calicophoron daubneyi infections in ruminants in Ireland and modelling of C. daubneyi infection.

BACKGROUND: Fasciola hepatica has always represented a threat to Irish livestock because the Irish climate is highly suitable for the main local intermediate host of the parasite, the snail Galba truncatula. The recent clinical emergence of infections due to Calicophoron daubneyi has raised the question of whether the two parasites, which share a niche during part of their life-cycles, interact in some way. Here, we used geographical information systems (GIS) to analyse the distribution of both parasites in cattle and sheep. We also developed the first predictive model of paramphistomosis in Ireland. RESULTS: Our results indicated that, in cattle, liver fluke infection is less common than rumen fluke infection and does not exhibit the same seasonal fluctuations. Overall, we found that cattle had a higher likelihood of being infected with rumen fluke than sheep (OR = 3.134, P < 0.01). In addition, infection with one parasite increased the odds of infection with the other in both host species. Rumen fluke in cattle showed the highest spatial density of infection. Environmental variables such as soil drainage, land cover and habitat appeared to be the most important risk factors for C. daubneyi infection, followed by rainfall and vegetation. Overall the risk of infection with this parasite was predicted to be higher in the west of the country. CONCLUSIONS: This study shows differences between the infection rates and spatial patterns of bovine and ovine infections with F. hepatica and C. daubneyi in Ireland. Whether the reasons for this are due to susceptibility, exposure and/or management factors is yet to be determined. Furthermore, the rumen fluke model indicates distinct risk factors and predicted distribution to those of F. hepatica, suggesting potential biological differences between both parasite species.

Validation of a spatial liver fluke model under field conditions in Ireland.

Fasciola hepatica is the causative agent of fasciolosis, a global disease of a wide range of mammals, particularly sheep and cattle. Liver fluke infection causes annual losses estimated at around €2.5 billion to livestock and food industries worldwide. Various models have been developed to define risk factors and predict exposure to this liver fluke in ruminants in European countries, most of them based exclusively on data from dairy herds. The aim of this study was to validate a published theoretical baseline risk map of liver fluke exposure and cluster maps in Ireland, by including further explanatory variables and additional herd types that are spatially more widespread. Three approaches were employed: i) comparison of predicted and actual exposure; ii) comparison of cluster distribution of hotspots and coldspots; and iii) development of a new model to compare predicted spatial distribution and risk factors. Based on new survey data, the published baseline predictive map was found to have a sensitivity of 94.7%, a specificity of 5%, a positive predictive value of 60% and a negative predictive value of 38.2%. In agreement with the original model, our validation highlighted temperature and rainfall among the main risk factors. In addition, we identified vegetation indices as important risk factors. Both the previously published and our new model predict that exposure to Fasciola is higher in the western parts of Ireland. However, foci of high probability do not match completely, nor do the location of clusters of hotspots and coldspots.

The immunoregulatory effects of co-infection with Fasciola hepatica: From bovine tuberculosis to Johne's disease.

Fasciola hepatica (liver fluke) is a parasite prevalent in much of the world that causes the economically-important disease of fasciolosis in livestock. The threat that this disease poses extends beyond its direct effects due to the parasite's immunomodulatory effects. Research at this laboratory is focusing on whether this immunoregulation can, in animals infected with liver fluke, exert a bystander effect on concurrent infections in the host. It has already been established that F. hepatica infection reduces cell mediated immune responses to Mycobacterium bovis in cattle, and that the interaction between the two pathogens can be detected on an epidemiological scale. This review explores the immunological consequences of co-infection between F. hepatica and other bacterial infections. Arguments are presented suggesting that immunity of cattle to Mycobacterium avium subsp. paratuberculosis is also likely to be affected.