Multiclonal human origin and global expansion of an endemic bacterial pathogen of livestock.

Most new pathogens of humans and animals arise via switching events from distinct host species. However, our understanding of the evolutionary and ecological drivers of successful host adaptation, expansion, and dissemination are limited. Staphylococcus aureus is a major bacterial pathogen of humans and a leading cause of mastitis in dairy cows worldwide. Here we trace the evolutionary history of bovine S. aureus using a global dataset of 10,254 S. aureus genomes including 1,896 bovine isolates from 32 countries in 6 continents. We identified 7 major contemporary endemic clones of S. aureus causing bovine mastitis around the world and traced them back to 4 independent host-jump events from humans that occurred up to 2,500 y ago. Individual clones emerged and underwent clonal expansion from the mid-19th to late 20th century coinciding with the commercialization and industrialization of dairy farming, and older lineages have become globally distributed via established cattle trade links. Importantly, we identified lineage-dependent differences in the frequency of host transmission events between humans and cows in both directions revealing high risk clones threatening veterinary and human health. Finally, pangenome network analysis revealed that some bovine S. aureus lineages contained distinct sets of bovine-associated genes, consistent with multiple trajectories to host adaptation via gene acquisition. Taken together, we have dissected the evolutionary history of a major endemic pathogen of livestock providing a comprehensive temporal, geographic, and gene-level perspective of its remarkable success.

Immunoproteomic analysis of the serum IgG response to cell wall-associated proteins of Staphylococcus aureus strains belonging to CC97 and CC151.

CC97 and CC151 are two of the most common Staphylococcus aureus lineages associated with bovine intramammary infection. The genotype of the infecting S. aureus strain influences virulence and the progression of intramammary disease. Strains from CC97 and CC151 encode a distinct array of virulence factors. Identification of proteins elaborated in vivo will provide insights into the molecular mechanism of pathogenesis of these lineages, as well as facilitating the development of tailored treatments and pan-lineage vaccines and diagnostics. The repertoire of genes encoding cell wall-anchored (CWA) proteins was identified for S. aureus strains MOK023 (CC97) and MOK124 (CC151); MOK023 encoded more CWA proteins than MOK124. Serum collected during an in vivo challenge trial was used to investigate whether the humoral response to cell wall proteins was strain-specific. Immunoproteomic analysis demonstrated that the humoral response in MOK023-infected cows predominantly targeted high molecular weight proteins while the response in MOK124-infected cows targeted medium or low molecular weight proteins. Antigenic proteins were identified by two-dimensional serum blotting followed by mass spectometry-based identification of immunoreactive spots, with putative antigens subsequently validated. The CWA proteins ClfB, SdrE/Bbp and IsdA were identified as immunogenic regardless of the infecting strain. In addition, a number of putative strain-specific imunogens were identified. The variation in antigens produced by different strains may indicate that these strains have different strategies for exploiting the intramammary niche. Such variation should be considered when developing novel control strategies including vaccines, therapeutics and diagnostics.

Risk factors for, and genetic association with, intestinal atresia in dairy calves.

Intestinal atresia is an under-diagnosed congenital defect in cattle. It results in complete occlusion of the intestinal lumen and, unless surgically corrected, results in death or euthanasia of the affected calf. There is limited information on the incidence of this condition or on risk factors, including predisposing alleles, associated with the defect. In this study, active surveillance of 39 dairy farms over 8 years identified 197 cases of intestinal atresia among 56 454 calves born, an incidence of 0.35%. The majority of cases (83%) had occlusion of the jejunum, although cases with blockage of the colon (14%) or anus (4%) were also identified. The defect was twice as common in male as in female calves (p < 0.0001), and was more common in progeny of older cows than in progeny of first or second lactation cows (p < 0.001). Year and farm of birth were also significantly associated with incidence (p < 0.05). The incidence of intestinal atresia was highest among the progeny of three related Jersey sires, suggesting that a gene for intestinal atresia was segregating within this family. Linkage analysis utilising 28 affected progeny of two half-sib putative carrier sires identified two putative quantitative trait loci associated with the defect, on chromosomes 14 and 26, although no clear candidate genes were identified. There was no evidence of a sire-effect among the progeny of Holstein-Friesian sires. However, a case-control genome-wide association study involving 91 cases and 375 healthy controls, identified 31 SNP in 18 loci as associated with the defect in this breed. These data suggest that intestinal atresia in dairy calves is not a simple Mendelian trait as previously reported but a complex multigenic disorder.

Bovine milk somatic cell transcriptomic response to Staphylococcus aureus is dependent on strain genotype.

BACKGROUND: Mastitis is an economically important disease of dairy cows with Staphylococcus aureus a major cause worldwide. Challenge of Holstein-Friesian cows demonstrated that S. aureus strain MOK124, which belongs to Clonal Complex (CC)151, caused clinical mastitis, while strain MOK023, belonging to CC97, caused mild or subclinical mastitis. The aim of this study was to elucidate the molecular mechanisms of the host immune response utilising a transcriptomic approach. Milk somatic cells were collected from cows infected with either S. aureus MOK023 or MOK124 at 0, 24, 48, 72 and 168 h post-infection (hpi) and analysed for differentially expressed (DE) genes in response to each strain. RESULTS: In response to MOK023, 1278, 2278, 1986 and 1750 DE genes were found at 24, 48, 72 and 168 hpi, respectively, while 2293, 1979, 1428 and 1544 DE genes were found in response to MOK124 at those time points. Genes involved in milk production (CSN1, CSN10, CSN1S2, CSN2, a-LACTA and PRLR) were downregulated in response to both strains, with a more pronounced decrease in the MOK124 group. Immune response pathways such as NF-κB and TNF signalling were overrepresented in response to both strains at 24 hpi. These immune pathways continued to be overrepresented in the MOK023 group at 48 and 72 hpi, while the Hippo signalling, extracellular matrix interaction (ECM) and tight junction pathways were overrepresented in the MOK124 group between 48 and 168 hpi. Cellular composition analysis demonstrated that a neutrophil response was predominant in response to MOK124, while M1 macrophages were the main milk cell type post-infection in the MOK023 group. CONCLUSIONS: A switch from immune response pathways to pathways involved in maintaining the integrity of the epithelial cell layer was observed in the MOK124 group from 48 hpi, which coincided with the occurrence of clinical signs in the infected animals. The higher proportion of M1 macrophages in the MOK023 group and lack of substantial neutrophil recruitment in response to MOK023 may indicate immune evasion by this strain. The results of this study highlight that the somatic cell transcriptomic response to S. aureus is dependent on the genotype of the infecting strain.

Clinical presentation and immune characteristics in first-lactation Holstein-Friesian cows following intramammary infection with genotypically distinct Staphylococcus aureus strains.

Staphylococcus aureus is an important cause of bovine mastitis, and intramammary infections caused by this pathogen are often characterized as mild, chronic, or persistent. The strains of Staph. aureus associated with mastitis belong to several distinct bovine-adapted bacterial lineages. Studies of host-pathogen interactions have demonstrated that significant differences exist between Staph. aureus strains and lineages in their ability to internalize and to elicit expression of chemokines and pro-inflammatory mediators in bovine cells in vitro. To determine the effect of bacterial strain on the response to intramammary infection in vivo, 14 disease-free, first-lactation cows were randomly allocated to 2 groups and challenged with Staph. aureus strain MOK023 (belonging to CC97) or MOK124 (belonging to CC151). Clinical signs of infection, as well as somatic cell count (SCC), bacterial load, IL-8 and IL-1ß in milk, anti-Staph. aureus IgG in milk and serum, anti-Staph. aureus IgA in milk, and white blood cell populations in milk and blood were monitored for 30 d after the challenge. Cows infected with MOK023 generally developed subclinical mastitis, whereas cows infected with MOK124 generally developed clinical mastitis. Milk yield was reduced to a greater extent in response to infection with MOK124 compared with MOK023 in the first week of the study. Significantly higher SCC, IL-8, and IL-1ß in milk as well as higher anti-Staph. aureus IgG and IgA in milk and anti-Staph. aureus IgG in serum were also observed in response to MOK124 compared with the response to MOK023. Higher proportions of neutrophils were observed in milk of animals infected with MOK124 than in animals infected with MOK023. Higher neutrophil concentration in blood was also observed in the MOK124 group compared with the MOK023 group. Overall, the results indicate that the outcome of mastitis mediated by Staph. aureus is strain dependent.

Anthelmintic resistance among gastrointestinal nematodes of cattle on dairy calf to beef farms in Ireland.

BACKGROUND: The control of gastrointestinal nematodes (GIN) of cattle in pasture-based production systems such as Ireland is highly dependent on the availability of efficacious anthelmintics. There is very little information available on the efficacy of the broad-spectrum anthelmintics against GIN of cattle in Ireland and the aim of this study was to determine the prevalence of anthelmintic resistance on dairy calf to beef farms. RESULTS: GIN burden was monitored on thirty-six recruited farms by performing herd level faecal egg counts (FEC) every 2 weeks. Of these, nine farms were lost from the study as calves were treated with an anthelmintic for Dictyocaulus viviparus, two were lost as they treated for GIN, one dropped out of the study and on one the herd FEC did not reach the threshold for carrying out the Faecal Egg Count Reduction Test (FECRT). On the remaining 23 farms, once the herd FEC reached 100 eggs per gram, a FECRT was carried out. Pre and post-treatment larval cultures were also performed to identify the GIN to genus level. The efficacy of fenbendazole, levamisole, ivermectin and moxidectin was evaluated on 15, 11, 16 and 11 farms respectively. Resistance to fenbendazole was identified on 9 farms (60%) with resistance suspected on a further farm. Resistance to levamisole, ivermectin and moxidectin was detected on 2 (18%), 16 (100%) and 8 (73%) farms respectively. The predominant genera detected pre and post-treatment were Cooperia and Ostertagia with both genera detected post-treatment with fenbendazole and ivermectin. Due to the low proportion of Ostertagia spp. pre-treatment, the efficacy of levamisole or moxidectin against this genus could not be reliably established. CONCLUSIONS: Anthelmintic resistance was widespread on the sampled dairy calf to beef farms in Ireland with resistance to benzimidazole, levamisole, ivermectin and moxidectin detected.

Preservation of genetic and regulatory robustness in ancient gene duplicates of Saccharomyces cerevisiae.

Biological systems remain robust against certain genetic and environmental challenges. Robustness allows the exploration of ecological adaptations. It is unclear what factors contribute to increasing robustness. Gene duplication has been considered to increase genetic robustness through functional redundancy, accelerating the evolution of novel functions. However, recent findings have questioned the link between duplication and robustness. In particular, it remains elusive whether ancient duplicates still bear potential for innovation through preserved redundancy and robustness. Here we have investigated this question by evolving the yeast Saccharomyces cerevisiae for 2200 generations under conditions allowing the accumulation of deleterious mutations, and we put mechanisms of mutational robustness to a test. S. cerevisiae declined in fitness along the evolution experiment, but this decline decelerated in later passages, suggesting functional compensation of mutated genes. We resequenced 28 genomes from experimentally evolved S. cerevisiae lines and found more mutations in duplicates--mainly small-scale duplicates--than in singletons. Genetically interacting duplicates evolved similarly and fixed more amino acid-replacing mutations than expected. Regulatory robustness of the duplicates was supported by a larger enrichment for mutations at the promoters of duplicates than at those of singletons. Analyses of yeast gene expression conditions showed a larger variation in the duplicates' expression than that of singletons under a range of stress conditions, sparking the idea that regulatory robustness allowed a wider range of phenotypic responses to environmental stresses, hence faster adaptations. Our data support the persistence of genetic and regulatory robustness in ancient duplicates and its role in adaptations to stresses.

Genetic basis of benzimidazole resistance in Teladorsagia circumcincta in Ireland.

Resistance to benzimidazole (BZ) anthelmintics is common in ovine nematodes of economic importance. Single nucleotide polymorphisms (SNP) at three positions in the isotype 1 ß- tubulin gene have been associated with BZ resistance and molecular tests for the detection of BZ resistance have been developed. In order to determine if such tests are practicable in Ireland the polymorphisms associated with BZ resistance must be identified. To this end, BZ-resistant nematodes were recovered from four farms in Ireland. Resistant Teladorsagia circumcincta, Cooperia curticei and Trichostrongylus colubriformis were recovered, with resistant T. circumcincta the most common and the only species studied further. Sequencing of the isotype 1 ß-tubulin gene from resistant T. circumcincta identified a T - A transition, resulting in an F200Y substitution known to be responsible for BZ-resistance, on three of the farms. However, on the fourth farm the frequency of the resistant A allele was only 0.33 indicating another BZ resistance mechanism may be present on this farm. An additional polymorphism resulting in a substitution of glutamate for leucine (E198L) was also found on this farm at low frequency (0.17). No polymorphisms at position 167 were identified on any farm. Therefore, molecular tests to detect BZ resistance in T. circumcincta in Ireland could prove useful; however, they may result in some instances of resistance remaining undetected.

A nationwide survey of anthelmintic treatment failure on sheep farms in Ireland.

BACKGROUND: Between 2013 and 2015 the Department of Agriculture, Food and the Marine (DAFM) administered a sheep technology adoption programme (STAP), with the aim of increasing profitability on Irish sheep farms by encouraging the adoption of best management practices. One of the options available to STAP participants was to test the efficacy of the anthelmintic treatment (benzimadazole, levamisole or macrocyclic lactone) used in their flocks by means of a drench test, which is a modification of the faecal egg count reduction test; individual faecal samples were collected from the same group of lambs before and after anthelmintic treatment, the number of eggs present pre and post treatment was subsequently determined from a pooled sample. RESULTS: In total, 4211 drench tests were undertaken by farmers during the 3 years of the programme. Information on the anthelmintic product used was available for 3771 of these tests; anthelmintics from the classes benzimidazole (BZ), levamisole (LV) and macrocyclic lactone (ML) (avermectins (AVM) plus moxidectin (MOX)) were used in 42.0%, 23.4% and 32.5% of tests, respectively. The remaining 2.1% of tests involved an inappropriate product. The efficacy of treatment against 'other trichostrongyles' (excluding Nematodirus spp and Strongyloides papillosus.) could be established for 1446 tests, and 51% of these tests were considered effective (i.e. a reduction of faecal egg count (FEC) ≥ 95%). There was a significant difference among the drug groups in efficacy; 31.5%, 51.9%, 62.5% and 84% of treatments were considered effective for BZ, LV, AVM, MOX, respectively. The efficacy of treatment against Nematodirus spp. could be established for 338 tests and the overall efficacy was 96%. CONCLUSIONS: Due to the significant difference among the anthelmintic classes for efficacy against 'other trichostrongyles' along with the high level of efficacy against Nematodirus spp., a genus for which anthelmintic resistance is rarely reported, it is concluded that anthelmintic resistance was responsible for the majority of the anthelmintic treatment failures observed.

The roles of whole-genome and small-scale duplications in the functional specialization of Saccharomyces cerevisiae genes.

Researchers have long been enthralled with the idea that gene duplication can generate novel functions, crediting this process with great evolutionary importance. Empirical data shows that whole-genome duplications (WGDs) are more likely to be retained than small-scale duplications (SSDs), though their relative contribution to the functional fate of duplicates remains unexplored. Using the map of genetic interactions and the re-sequencing of 27 Saccharomyces cerevisiae genomes evolving for 2,200 generations we show that SSD-duplicates lead to neo-functionalization while WGD-duplicates partition ancestral functions. This conclusion is supported by: (a) SSD-duplicates establish more genetic interactions than singletons and WGD-duplicates; (b) SSD-duplicates copies share more interaction-partners than WGD-duplicates copies; (c) WGD-duplicates interaction partners are more functionally related than SSD-duplicates partners; (d) SSD-duplicates gene copies are more functionally divergent from one another, while keeping more overlapping functions, and diverge in their sub-cellular locations more than WGD-duplicates copies; and (e) SSD-duplicates complement their functions to a greater extent than WGD-duplicates. We propose a novel model that uncovers the complexity of evolution after gene duplication.

High level of treatment failure with commonly used anthelmintics on Irish sheep farms.

BACKGROUND: In 2013 a Technology Adoption Program for sheep farmers was established to encourage the implementation of best management practices on sheep farms in Ireland. There were 4,500 participants in this programme in 2013. As part of this programme, farmers had the option to carry out a drench test to establish the efficacy of their anthelmintic treatment. RESULTS: Flock faecal samples were collected before and after treatment administration and gastrointestinal nematode eggs enumerated. In total there were 1,893 participants in the task, however only 1,585 included both a pre- and post-treatment faecal sample. Of those, 1,308 provided information on the anthelmintic product that they used with 46%, 23% and 28% using a benzimidazole (BZ), levamisole (LEV) and macrocyclic lactone (ML) product respectively. The remaining farmers used a product inapplicable for inclusion in the task such as a flukicide or BZ/LEV combination product. Samples were included for analysis of drench efficacy if the pre-treatment flock egg count was ≥200 eggs per gram and the interval post-sampling was 10-14 days for BZ products, 4-7 days for LEV products and 14-18 days for ML products. These criteria reduced the number of valid tests to 369, 19.5% of all tests conducted. If the reduction post-treatment was ≥95% the treatment was considered effective. Only 51% of treatments were considered effective using this criterion. There was a significant difference in efficacy between the anthelmintic drug classes with BZ effective in only 30% of treatments, LEV effective in 52% of cases and ML effective in 76% of cases. CONCLUSIONS: Gastrointestinal nematode anthelmintic treatments, as practiced on Irish farms, have a high failure rate. There was a significant difference between the efficacies of the anthelmintic classes with BZ the least effective and ML the most effective.

Comparison of traditional copromicroscopy with image analysis devices for detection of gastrointestinal nematode infection in sheep.

Sustainable parasite control practices are necessary to combat the negative effects of gastrointestinal nematodes on animal health and production while reducing the selection pressure for anthelmintic resistance. Parasite diagnostic tests can inform treatment decisions, the timing and effectiveness of treatment and enable livestock breeding programmes. In recent years new diagnostic methods have been developed, some incorporating machine learning (ML), to facilitate the detection and enumeration of parasite eggs. It is important to understand the technical characteristics and performance of such new methods compared to long standing and commonly utilised methods before they are widely implemented. The aim of the present study was to trial three new diagnostic tools relying on image analysis (FECPAKG2, Micron and OvaCyte) and to compare them to traditional manual devices (McMaster and Mini-FLOTAC). Faecal samples were obtained from 41 lambs naturally infected with gastrointestinal nematodes. Samples were mixed and separated into 2 aliquots for examination by each of the 5 methods: McMaster, Mini-FLOTAC, FECPAKG2, Micron and OvaCyte. The techniques were performed according to their respective standard protocols and results were collected by trained staff (McMaster and Mini-FLOTAC) or by the device (FECPAKG2, Micron and OvaCyte). Regarding strongyle worm egg count, McMaster values varied from 0 to 9,000 eggs per gram (EPG). When comparing replicate aliquots, both the Mini-FLOTAC and Micron methods displayed similar repeatability to McMaster. However, we found FECPAKG2 and OvaCyte significantly less precise than McMaster. When comparing parasite egg enumeration, significant positive linear correlations were established between McMaster and all other methods. No difference was observed in EPG between McMaster and Mini-FLOTAC or FECPAKG2; however, Micron and OvaCyte returned significantly higher and lower EPG, respectively, compared to McMaster. The number of eggs ascribed to other parasite species was not sufficient for performing a robust statistical comparison between all methods. However, it was noted that FECPAKG2 generally did not detect Strongyloides papillosus eggs, despite these being detected by other methods. In addition, Moniezia spp and Trichuris spp eggs were detected by OvaCyte and Mini-FLOTAC, respectively, but not by other methods. The observed variation between traditional and new methods for parasite diagnostics highlights the need for continued training and enhancing of ML models used and the importance of developing clear guidelines for validation of newly developed methods.

Exploring Interrelationships between Colour, Composition, and Coagulation Traits of Milk from Cows, Goats, and Sheep.

This study explores the interrelationships between the composition, coagulation, and colour of sheep, goat, and cow milk to identify their similarities and differences and to assess whether the relationships between the variables are common to all species or whether they emerge from species-specific relationships. For this purpose, 2400 individual milk samples were analysed. The differences and similarities between the species were determined using discriminant analysis and cluster analysis. The results show a clear differentiation between species. Sheep milk stands out for its cheesemaking capacity and shows similarities with goat milk in composition and coagulation. Nonetheless, colorimetry highlights a greater similarity between sheep and cow milk. Composition and colorimetry were more discriminating than coagulation, and the variables that differed the most were fat, protein, curd yield, lightness, and red-green balance. Using canonical correlation, the interrelationships between the different sets of variables were explored, revealing patterns of common variation and species-specific relationships. Colorimetric variables were closely related to milk solids in all species, while in sheep milk, an inverse relationship with lactose was also identified. Furthermore, a strong relationship was revealed for all species between colour and curd yield. This could be modelled and applied to estimate the technological value of milk, proving colorimetry as a useful tool for the dairy industry.

The use of cathepsin L1 (FhCL1) serological ELISA in sentinel screening for liver fluke on sheep farms.

Fasciola hepatica is a parasitic helminth (worm) that poses a significant economic threat to the ruminant livestock industry worldwide. The disease, fasciolosis, can result in a range of clinical signs including anaemia, weight loss and death, with the most severe symptoms attributed to early acute infection when the parasite is migrating through the liver. Early diagnosis and intervention are essential for the control and management of the disease to prevent productivity losses. The traditional gold standard method of diagnosis uses faecal egg counts (FEC) that is limited to detecting patent infections from 10 to 12 weeks post infection (WPI). In contrast, serological assays can detect pre-patent infections as we have shown that enzyme-linked immunosorbent assays (ELISA) using the F. hepatica cysteine peptidase cathepsin L1 (FhCL1) can detect liver fluke infections from 3 to 4 WPI. Here, we used FEC and ELISA to monitor liver fluke infections in sentinel lambs from three commercial farms in Ireland from September 2021 to March 2022. All three farms showed a significant increase in FhCL1 antibody levels and FEC over this time, with a substantial rise in positive infection detection between late November and January. However, ELISA screening detected infection at least two months prior to FEC (September). This suggests that the regular screening of sentinel lambs for F. hepatica seroconversion in a "test and treat" approach could mitigate the negative damaging impact of early fasciolosis on flock health, welfare and productivity and inform management strategies. In addition, we show that whole blood samples taken on Whatman® protein saver cards could replace conventional serum blood tubes for blood collection. Cards can be stored at room temperature for long periods of time and samples revisited at any time for re-analysis. The adoption of these cards on farm together with the FhCL1 ELISA would provide a simpler, cost-effective, and eco-friendly method for testing sentinel lambs for liver fluke disease.

Fasciola hepatica antioxidant and protease-inhibitor cocktail recombinant vaccines administered five times elicit potent and sustained immune responses in sheep but do not confer protection.

Our laboratory's vaccine development strategy against the livestock parasite Fasciola hepatica centres around disrupting key biological processes by combining groups of antigens with similar/complementary functional actions into a single vaccine cocktail. In this study the focus was on antioxidant protein vaccines and a protease inhibitor vaccine aimed at disrupting the parasite's ability to defend against oxidative stress and protease-inhibitor balance, respectively. Two combinations of recombinantly expressed antioxidants were assessed, namely peroxiredoxin (rFhPrx), thioredoxin (rFhTrx) and thioredoxin-glutathione reductase (rFhTGR) (Group 1) and rFhPrx, rFhTrx, and two superoxide dismutases (rFhSOD1 and rFhSOD3) (Group 2). The protease inhibitor vaccine cocktail included representatives of each of the key secreted protease inhibitor families, namely a Kunitz-type inhibitor (rFhKT1), a serpin (rFhSrp1) and a stefin, (rFhStf1) (Group 3). The vaccine combinations were formulated in adjuvant Montanide 61VG administered at five timepoints; two before experimental challenge with 60 F. hepatica metacercariae and three after infection. The vaccine combinations did not reduce the liver fluke burden, and only Group 2 displayed a marginal reduction in egg viability (8.2%). Despite previous results showing an effect of liver fluke vaccines on overall weight gain in infected animals, no significant (P value >0.05) impact on weight gain was observed in this study. Antibodies were elicited against all the vaccine antigens within the cocktails and were maintained at high levels to the end of the trial, due to our strategy of continuing vaccine administration after infection. However, these responses were not boosted by the challenge F. hepatica infection. A comparative analysis with previous vaccine data using a protease inhibitor vaccine found no repeat of the promising outcomes associated with this vaccine, indicating that the addition of rFhSrp1 to the vaccine cocktail did not improve vaccine efficacy. Assessment of liver pathology across the two trials using a modified liver enzyme score (glutamate dehydrogenase to platelet ratio) at eight weeks post infection suggests an association with liver fluke burden above 45 flukes, which could be used to predict liver pathology in future trials. The results reported in this study highlight the ambiguousness in liver fluke vaccine development and the difficulty in obtaining consistent and repeatable protection. This work stresses the need for repetition of trials and the use of sufficiently sized groups to assess vaccine efficacy with adequate statistical power.

Immunoproteomic analysis of the secretome of bovine-adapted strains of Staphylococcus aureus demonstrates a strain-specific humoral response.

Staphylococcus aureus is a common pathogen associated with bovine intramammary infection. A number of distinct S. aureus lineages are associated with such infections although there is a dearth of knowledge regarding the major immunogenic antigens associated with each lineage and whether these antigens provide protection against heterologous strains. Identification of the major immunogenic antigens of the predominant bovine-adapted S. aureus lineages would assist in the design of effective vaccines and diagnostic tests to control intramammary infections caused by S. aureus. The aim of this study was to characterise the serum IgG response to S. aureus extracellular proteins in cows infected with strains from different lineages, as well as to identify antigenic proteins produced by these strains. Genotypic characterisation found that strain MOK124 (CC151) encoded more toxins, including the ruminant-specific leukocidin LukMF, compared to strain MOK023 (CC97). In addition, MOK124 secreted more toxins in vitro, compared to MOK023. Immunoproteomic analysis was performed using sera from cows infected with either MOK023 or MOK124. One-dimensional serum blotting revealed that cows infected with MOK023 predominantly generated a humoral response against high molecular weight proteins while cows infected with MOK124 primarily generated a humoral response against low molecular weight proteins. Two-dimensional serum blotting demonstrated that antibodies produced by an MOK023 infected cow could cross react with some of the extracellular proteins produced by MOK124 and vice versa. Mass spectrometry analysis of immunoreactive proteins identified common candidate immunogens produced by both strains, including α-hemolysin and ß-hemolysin. In addition, strain-specific candidate immunogens were also identified. This study demonstrates that genes encoding important S. aureus secreted virulence factors, the production of cognate gene products, and the humoral immune response to infection is, to an extent, strain-dependent. However, the identification of some common candidate immunogens suggests that there are proteins that can be exploited for further vaccine or diagnostic research that targets S. aureus strains from a variety of lineages.

In vitro evaluation of fitness parameters for isolates of Teladorsagia circumcincta resistant and susceptible to multiple anthelmintic classes.

Anthelmintic resistance (AR) is an ever increasing problem for the sheep industry. Several studies worldwide have investigated reversing the trend of increasing AR and documented evidence for reversion toward susceptibility has been found. The hypothesis that resistance mutations compromise parasite fitness was drawn from this evidence. The aim of this study was to assess whether there were measurable differences in the fitness of Teladorsagia circumcincta isolates depending on their AR status. Four isolates were selected for the trial based on their known resistance status; D and M were multi-drug resistant, and T and W were susceptible to the benzimidazole, levamisole, and macrocyclic lactone anthelmintic classes. A secondary aim was to develop a series of in vitro bioassays for assessing fitness characteristics of parasites. The in vitro assays included; the cold stress test measured the number of third stage larvae (L3) developing from eggs stored at 4 °C for different lengths of time. Larval aging measured the locomotory activity of L3 after storage at 30 °C for different lengths of time. The exsheathment assay measured the exsheathment percentage of L3. Larval Length used length as a proxy for fecundity. The egg hatch assay evaluated egg hatch rate in water at room temperature. All isolates exhibited a decrease in the number of L3 recovered after storage of eggs at 4 °C (p < 0.001). Storage of L3 at 30 °C significantly influenced the ability of L3 to migrate through a 20 µm sieve (p < 0.001), however, there were no differences between isolates (p > 0.05). Exsheathment rate was higher for isolate D in comparison to isolates M and W, and for isolate T compared to isolate W. Isolate W was significantly longer than all other isolates (p < 0.05), whilst isolate M was significantly longer than isolate D (p < 0.05). No significant differences were found between isolates in egg hatch (p > 0.05). Overall, the results do not support differences in fitness associated with anthelmintic resistance status, even though differences were seen between the isolates for some assays. This suggests there is considerable variation in fitness parameters between isolates, making it difficult to determine whether resistance genotypes come with lower fitness.

Diagnosis of sheep fasciolosis caused by Fasciola hepatica using cathepsin L enzyme-linked immunosorbent assays (ELISA).

Fasciolosis, a global parasitic disease of agricultural livestock, is caused by the liver fluke Fasciola hepatica. Management and strategic control of fasciolosis on farms depends on early assessment of the extent of disease so that control measures can be implemented quickly. Traditionally, this has relied on the detection of eggs in the faeces of animals, a laborious method that lacks sensitivity, especially for sub-clinical infections, and identifies chronic infections only. Enzyme linked immunosorbent assays (ELISA) offer a quicker and more sensitive serological means of diagnosis that could detect early acute infection before significant liver damage occurs. The performance of three functionally-active recombinant forms of the major F. hepatica secreted cathepsins L, rFhCL1, rFhCL2, rFhCL3, and a cathepsin B, rFhCB3, were evaluated as antigens in an indirect ELISA to serologically diagnose liver fluke infection in experimentally and naturally infected sheep. rFhCL1 and rFhCL3 were the most effective of the four antigens detecting fasciolosis in sheep as early as three weeks after experimental infection, at least five weeks earlier than both coproantigen and faecal egg tests. In addition, the rFhCL1 and rFhCL3 ELISAs had a very low detection limit for liver fluke in lambs exposed to natural infection on pastures and thus could play a major role in the surveillance of farms and a 'test and treat' approach to disease management. Finally, antibodies to all three cathepsin L proteases remain high throughout chronic infection but decline rapidly after drug treatment with the flukicide, triclabendazole, implying that the test may be adapted to trace the effectiveness of drug treatment.

Increasing importance of anthelmintic resistance in European livestock: creation and meta-analysis of an open database.

Helminth infections are ubiquitous in grazing ruminant production systems, and are responsible for significant costs and production losses. Anthelmintic Resistance (AR) in parasites is now widespread throughout Europe, although there are still gaps in our knowledge in some regions and countries. AR is a major threat to the sustainability of modern ruminant livestock production, resulting in reduced productivity, compromised animal health and welfare, and increased greenhouse gas emissions through increased parasitism and farm inputs. A better understanding of the extent of AR in Europe is needed to develop and advocate more sustainable parasite control approaches. A database of European published and unpublished AR research on gastrointestinal nematodes (GIN) and liver fluke (Fasciola hepatica) was collated by members of the European COST Action "COMBAR" (Combatting Anthelmintic Resistance in Ruminants), and combined with data from a previous systematic review of AR in GIN. A total of 197 publications on AR in GIN were available for analysis, representing 535 studies in 22 countries and spanning the period 1980-2020. Reports of AR were present throughout the European continent and some reports indicated high within-country prevalence. Heuristic sample size-weighted estimates of European AR prevalence over the whole study period, stratified by anthelmintic class, varied between 0 and 48%. Estimated regional (country) prevalence was highly heterogeneous, ranging between 0% and 100% depending on livestock sector and anthelmintic class, and generally increased with increasing research effort in a country. In the few countries with adequate longitudinal data, there was a tendency towards increasing AR over time for all anthelmintic classes in GIN: aggregated results in sheep and goats since 2010 reveal an average prevalence of resistance to benzimidazoles (BZ) of 86%, macrocyclic lactones except moxidectin (ML) 52%, levamisole (LEV) 48%, and moxidectin (MOX) 21%. All major GIN genera survived treatment in various studies. In cattle, prevalence of AR varied between anthelmintic classes from 0-100% (BZ and ML), 0-17% (LEV) and 0-73% (MOX), and both Cooperia and Ostertagia survived treatment. Suspected AR in F. hepatica was reported in 21 studies spanning 6 countries. For GIN and particularly F. hepatica, there was a bias towards preferential sampling of individual farms with suspected AR, and research effort was biased towards Western Europe and particularly the United Kingdom. Ongoing capture of future results in the live database, efforts to avoid bias in farm recruitment, more accurate tests for AR, and stronger appreciation of the importance of AR among the agricultural industry and policy makers, will support more sophisticated analyses of factors contributing to AR and effective strategies to slow its spread.

TITLE: Importance croissante de la résistance aux anthelminthiques chez les ruminants européens : création et méta-analyse d'une base de données ouverte. ABSTRACT: Les helminthes sont omniprésents dans les systèmes de production de ruminants au pâturage et sont responsables de coûts et de pertes de production importants. La résistance aux anthelminthiques (RA) des parasites est maintenant répandue dans toute l'Europe, bien qu'il existe encore des lacunes dans nos connaissances dans certaines régions et certains pays. La RA est une menace majeure pour la pérennité de la production moderne de ruminants, en diminuant la productivité, en compromettant la santé et le bien-être des animaux, et en augmentant les émissions de gaz à effet de serre au travers d'une augmentation du parasitisme et des intrants agricoles. Une meilleure compréhension de l'étendue de la RA en Europe est nécessaire pour développer et préconiser des approches de lutte antiparasitaire plus durables. Une base de données intégrant des informations publiées et non publiées en Europe concernant la RA des nématodes gastro-intestinaux (NGI) et de la douve du foie (Fasciola hepatica) a été compilée par les membres de l'action européenne COST « COMBAR ¼ (« Combattre la résistance aux anthelminthiques chez les ruminants ¼) et combinée avec les données d'une précédente étude systématique concernant la RA des NGI. Au total, 197 publications sur la RA des NGI étaient disponibles pour analyse, représentant 535 études dans 22 pays et couvrant la période 1980­2020. Des signalements de RA étaient présents sur tout le continent européen et certains rapports indiquaient une forte prévalence nationale. Les estimations heuristiques pondérées par la taille de l'échantillon de la prévalence de la RA en Europe sur toute la période d'étude, stratifiées par classe d'anthelminthiques, variaient de 0 à 48 %. La prévalence régionale (nationale) estimée était très hétérogène, variant entre 0 % et 100 % selon le secteur de l'élevage et la classe d'anthelminthique, et augmentait généralement avec les efforts de recherche dans le pays. Dans les quelques pays disposant de données longitudinales adéquates, il y avait une tendance à l'augmentation de la RA au fil du temps pour toutes les classes d'anthelminthiques des NGI : les résultats agrégés chez les ovins et caprins depuis 2010 révèlent une prévalence moyenne de résistance aux benzimidazoles (BZ) de 86 %, aux lactones macrocycliques sauf moxidectine (ML) de 52 %, au lévamisole (LEV) de 48 % et à la moxidectine (MOX) de 21 %. Tous les genres principaux de NGI ont survécu au traitement dans diverses études. Chez les bovins, la prévalence de la RA variait selon les classes d'anthelminthiques de 0 à 100 % (BZ et ML), 0 à 17 % (LEV) et 0 à 73 % (MOX), et Cooperia et Ostertagia ont survécu aux traitements. Une RA suspectée chez F. hepatica a été signalée dans 21 études portant sur 6 pays. Pour les NGI, et encore plus pour F. hepatica, il y avait un biais d'échantillonnage en faveur des exploitations individuelles suspectées de RA, et l'effort de recherche était biaisé vers l'Europe occidentale et en particulier le Royaume-Uni. La capture continue des résultats futurs dans la base de données, en direct, les efforts pour éviter les biais dans le recrutement des exploitations, des tests plus précis pour la RA et une meilleure appréciation de l'importance de la RA parmi l'industrie agricole et les décideurs politiques, soutiendront des analyses plus sophistiquées des facteurs contribuant à la RA, et des stratégies efficaces pour ralentir sa propagation.

The in vitro host cell immune response to bovine-adapted Staphylococcus aureus varies according to bacterial lineage.

Mastitis is the most economically important disease affecting dairy cattle worldwide. Staphylococcus aureus is a highly prevalent cause of mastitis, causing infections ranging from sub-clinical to gangrenous. However, the interaction between the genotype of the infecting strain of S. aureus and the host response remains largely uncharacterised. To better understand the variation in presentation and outcomes of S. aureus-mediated bovine mastitis, we studied the interaction of a panel of mastitis isolates from several prominent bovine-associated lineages with bovine mammary epithelial cells (bMEC) and neutrophils. Significant differences in immune gene expression by infected primary or immortalised bMEC, or their elaboration of neutrophil chemoattractants, were observed and were dependent on the lineage of the infecting strain. Differences were also apparent in the invasiveness of S. aureus strains and their ability to survive killing by neutrophils. Our results demonstrate that a range of immune responses occur, suggesting the importance of S. aureus strain in dictating mastitis disease course. S. aureus lineages may therefore have adopted differing strategies for exploitation of the intramammary niche. Consequently, improved diagnosis of infecting lineage may enable better prognosis for S. aureus mastitis and reduce morbidity and economic loss.