Triggering Toll-Like Receptor 5 signaling during pneumococcal superinfection prevents the selection of antibiotic resistance.

Toll-like receptor 5 (TLR5) signaling plays a key role in antibacterial defenses. We previously showed that respiratory administration of flagellin, a potent TLR5 agonist, in combination with amoxicillin improves the treatment of primary pneumonia or superinfection caused by amoxicillin-sensitive or -resistant Streptococcus pneumoniae. Here, the impact of adjunct flagellin therapy on antibiotic dose/regimen and the selection of antibiotic-resistant S. pneumoniae was investigated using superinfection with isogenic antibiotic-sensitive and -resistant bacteria and population dynamics analysis. Our findings demonstrate that flagellin allows for a 200-fold reduction in the antibiotic dose, achieving the same therapeutic effect observed with antibiotic alone. Adjunct treatment also reduced the selection of antibiotic-resistant bacteria in contrast to the antibiotic monotherapy. Finally, we developed a mathematical model that captured the population dynamics and estimated a 20-fold enhancement immune-modulatory factor on bacterial clearance. This work paves the way for the development of host-directed therapy and refinement of treatment by modeling.

Porcine ear necrosis is associated with social behaviours in weaned piglets.

BACKGROUND: Porcine ear necrosis (PEN) is a worldwide health issue and its aetiology is still unclear. The aim of this study was to describe the prevalence and the severity of PEN in a commercial farm, associated with pig behaviour and health biomarkers measures. On two consecutive batches, PEN prevalence was determined at the pen level. PEN scores, blood haptoglobin concentration and oxidative status were measured on two pigs per pen (n = 48 pens) 9, 30 and 50 days (D) after arrival to the post-weaning unit. Social nosing, oral manipulation and aggression of pen mates and exploration of enrichment materials were observed on two to three pigs per pen twice a week from D9 to D50. RESULTS: At the pen level, the higher the time spent nosing pen mates, the lower the percentage of pigs affected by PEN during both the early and the late post-weaning periods (P < 0.002) and, in the opposite, the higher the time spent orally manipulating pen mates during the late post-weaning period, the higher the percentage of affected pigs (P = 0.03). At the pig level, the higher the increase in hydroperoxides and haptoglobin during the early post-weaning period, the higher the PEN scores on D30 (P < 0.001). CONCLUSIONS: This study suggests that a high incidence of social nosing, which can be an indicator of good social cohesion in a group, was significantly associated with less frequent lesions of PEN. In opposite, high incidence of oral manipulation of pen mates may increase the percentage of PEN-affected pigs. According to these observations, PEN is a multifactorial condition which may have social causes among others.

Alternative Samples for Porcine Reproductive and Respiratory Syndrome Surveillance in an Endemic PRRSV-1-Infected Breeding Herd: A Descriptive Study.

Knowing porcine reproductive and respiratory syndrome (PRRS) status is essential for designing herd management protocols. For this, weaning-age pigs are a key subpopulation. Recently, different alternatives to blood sampling have been introduced because they are easier, welfare-friendly and cost-saving tools. Moreover, most of them allow the testing of more animals and seem to be more sensitive in low-prevalence scenarios. However, these studies were implemented mainly in PRRSV-2-infected herds. The first objective of our study was to compare the rate of detection of PRRSV-1 by RT-qPCR in individual serum samples, family oral fluid samples (FOF) and udder wipes (UW) collected the day before weaning. The second objective was to evaluate the suitability of pooling. The study was performed on a 210-sow farrow-to-finish farm which was PRRSV-1 infected and unstable. A total of 119 litters were sampled. The rate of detection of PRRSV-1 in blood samples, FOF and UW was 10.9%, 7.6% and 0.8%, respectively. The agreement between sera and FOF was almost perfect even if the detection capacity of sera was numerically superior to FOF. The Ct values of positive sera were statistically lower than those of FOF. Two modalities of pooling (1:3 and 1:5) were tested for sera and FOF. For sera, both modalities did not impact the PRRSV-1 status either at the litter level or at the batch one. On the other hand, whatever the modality (pooled by 3 or 5), most of the pools of FOF gave negative results, misclassifying many litters and batches.

PRRSV-1 Stabilization Programs in French Farrow-to-Finish Farms: A Way to Reduce Antibiotic Usage.

Infection with the porcine reproductive and respiratory syndrome virus type 1 (PRRSV-1) has serious economic consequences for the pig industry. Swine practitioners and other agricultural advisors often describe an increase in antibiotic use when PRRSV-1 is circulating. Our objective was to assess the impact of PRRSV-1 stabilization programs on reducing antibiotic use in 19 French farrow-to-finish farms that successfully implemented such a protocol between 2007 and 2019. For each farm, we compared the global antibiotic consumption, including all physiological stages (expressed in mg/PCU and ALEA) one year before (P1) and one year after (P2) the implementation of the protocol, and the change between P1 and P2 was calculated in percentages. The data were also analyzed by level of consumption. We showed that antibiotic use decreased significantly between P1 and P2 if expressed in mg/PCU and showed a decreased tendency in terms of exposure (ALEA) after PRRSV-1 stabilization. Concerning the change from P1 to P2, depending on the level of consumption in P1, our results showed that the higher the consumption levels were in P1, the greater the antibiotic reduction in P2. This study highlights the ability of a stabilization protocol against PRRSV-1 to reduce antibiotic use, especially on farms that have high consumption rates. These hopeful results show that further investigations about the relationship between PRRSV-1 and antibiotic usage could be beneficial.

PRRSV Detection by qPCR on Serum Samples Collected in Due-to-Wean Piglets in Five Positive Stable Breeding Herds Following a Sow Mass Vaccination with a Modified Live Vaccine: A Descriptive Study.

Data concerning PRRSV-1 vaccine virus strains dissemination within vaccinated sow herds are scarce. However, it is a big concern for swine practitioners when designing the PRRSV diagnostics strategy in vaccinated farms. At the same time, the possibility of vaccine virus transmission from sows to their offspring is important to have in mind in order to limit the risk of recombination between different PPRSV-1 modified live virus vaccine (MLV1) when both sows and piglets have to be vaccinated. This study was conducted in five PRRSV-stable breeding herds. The selected farms presented different characteristics regarding production parameters and biosecurity management practices in order to be, as much as possible, representative of French swine production herds. In four different batches following a sow mass vaccination with a PRRSV-1 modified live virus vaccine (ReproCyc® PRRS EU, Boehringer Ingelheim, Ingelheim, Germany), we failed to detect the vaccine virus in due-to-wean piglets in all of the herds. This should mean that the dissemination of the vaccinal strain is a rare event, even just after a sow vaccination, at least for the vaccine tested in our study.

Microbiological Findings and Associated Histopathological Lesions in Neonatal Diarrhoea Cases between 2020 and 2022 in a French Veterinary Pig Practice.

This retrospective study described the aetiologies of neonatal diarrhoea cases and their associations with histological findings. A total of 106 diarrhoeic neonatal piglets were selected. Cultures, MALDI typings, PCRs and evaluation of intestinal lesions were performed. A total of 51 cases (48.1%) were positive for only one pathogen and 54 (50.9%) were positive for more than one pathogen. Clostridium perfringens type A was the most frequently detected pathogen (61.3%), followed by Enterococcus hirae (43.4%), rotavirus type A (38.7%), rotavirus type C (11.3%) and enterotoxigenic Escherichia coli (3.8%). Only lesions in the small intestine were correlated with detected pathogens. The detection of rotavirus was associated with an increased probability of observing villous atrophy (p < 0.001), crypt hyperplasia (p = 0.01) and leucocyte necrosis in the lamina propria (p = 0.05). The detection of Clostridium perfringens type A was associated with an increased probability of observing bacilli in close proximity to the mucosa (p < 0.001) and a decreased probability of observing epithelial necrosis (p = 0.04). Detection of Enterococcus hirae was associated with an increased probability of observing enteroadherent cocci (p < 0.001). Multivariate regression logistic models revealed that epithelial necrosis was more likely to occur in Enterococcus hirae-positive piglets (p < 0.02) and neutrophilic infiltrate was more likely to occur in Clostridium perfringens type A- and Enterococcus hirae-positive piglets (p = 0.04 and p = 0.02, respectively).

Competence remodels the pneumococcal cell wall exposing key surface virulence factors that mediate increased host adherence.

Competence development in the human pathogen Streptococcus pneumoniae controls several features such as genetic transformation, biofilm formation, and virulence. Competent bacteria produce so-called "fratricins" such as CbpD that kill noncompetent siblings by cleaving peptidoglycan (PGN). CbpD is a choline-binding protein (CBP) that binds to phosphorylcholine residues found on wall and lipoteichoic acids (WTA and LTA) that together with PGN are major constituents of the pneumococcal cell wall. Competent pneumococci are protected against fratricide by producing the immunity protein ComM. How competence and fratricide contribute to virulence is unknown. Here, using a genome-wide CRISPRi-seq screen, we show that genes involved in teichoic acid (TA) biosynthesis are essential during competence. We demonstrate that LytR is the major enzyme mediating the final step in WTA formation, and that, together with ComM, is essential for immunity against CbpD. Importantly, we show that key virulence factors PspA and PspC become more surface-exposed at midcell during competence, in a CbpD-dependent manner. Together, our work supports a model in which activation of competence is crucial for host adherence by increased surface exposure of its various CBPs.

Amoxicillin-resistant Streptococcus pneumoniae can be resensitized by targeting the mevalonate pathway as indicated by sCRilecs-seq.

Antibiotic resistance in the important opportunistic human pathogen Streptococcus pneumoniae is on the rise. This is particularly problematic in the case of the ß-lactam antibiotic amoxicillin, which is the first-line therapy. It is therefore crucial to uncover targets that would kill or resensitize amoxicillin-resistant pneumococci. To do so, we developed a genome-wide, single-cell based, gene silencing screen using CRISPR interference called sCRilecs-seq (subsets of CRISPR interference libraries extracted by fluorescence activated cell sorting coupled to next generation sequencing). Since amoxicillin affects growth and division, sCRilecs-seq was used to identify targets that are responsible for maintaining proper cell size. Our screen revealed that downregulation of the mevalonate pathway leads to extensive cell elongation. Further investigation into this phenotype indicates that it is caused by a reduced availability of cell wall precursors at the site of cell wall synthesis due to a limitation in the production of undecaprenyl phosphate (Und-P), the lipid carrier that is responsible for transporting these precursors across the cell membrane. The data suggest that, whereas peptidoglycan synthesis continues even with reduced Und-P levels, cell constriction is specifically halted. We successfully exploited this knowledge to create a combination treatment strategy where the FDA-approved drug clomiphene, an inhibitor of Und-P synthesis, is paired up with amoxicillin. Our results show that clomiphene potentiates the antimicrobial activity of amoxicillin and that combination therapy resensitizes amoxicillin-resistant S. pneumoniae. These findings could provide a starting point to develop a solution for the increasing amount of hard-to-treat amoxicillin-resistant pneumococcal infections.

Streptococcus pneumoniae is a bacterium that can cause pneumonia, meningitis and other life-threatening illnesses in humans. Currently, many S. pneumoniae infections are treated with the antibiotic amoxicillin, which kills the bacteria by weakening a structure known as the cell wall that surrounds each bacterium. However, more and more S. pneumoniae cells are becoming resistant to amoxicillin, making it harder to treat such infections. We need new ways to effectively treat S. pneumoniae infections in humans. One potential strategy would be to combine amoxicillin with another drug that boosts the activity of amoxicillin so that it is able to kill the resistant bacteria. Two drugs that both target the same process in cells are more likely to boost each other's activity. Therefore, Dewachter et al. decided to search for another drug that also weakens the cell wall of S. pneumoniae. The team first developed a new screening approach called sCRilecs-seq to silence individual genes in single S. pneumoniae cells. By looking at many cells that each had a different gene that was no longer active, the team were able to identify several genes that when silenced resulted in the cells becoming longer than normal cells (a sign the bacteria may have weak cell walls). Further experiments revealed that the cell walls of these bacteria were weaker than normal cells due to a shortage in a cell wall building material known as undecaprenyl phosphate. Dewachter et al. then demonstrated that combining an existing drug known as clomiphene ­ which is known to inhibit undecaprenyl phosphate production and is currently used to treat infertility in humans ­ together with amoxicillin is able to effectively kill S. pneumoniae that are resistant to amoxicillin alone. Clomiphene also boosted the activity of amoxicillin against S. pneumoniae that remain sensitive to the antibiotic. Before this new drug combination may be used to help treat S. pneumoniae infections in human patients, further experiments will be needed to find out the optimum dose of clomiphene to use with amoxicillin. In the future, the new screening approach developed by Dewachter et al. may also prove useful to other researchers studying a wide range of biological questions.

Microbial content of non-fermented liquid feed consumed by sows affects the occurrence of neonatal diarrhoea in their piglets: A case-control study.

BACKGROUND: Microbial colonisation of piglets' intestines starts at birth, especially from contact with sow's faeces. Piglet microbiota could therefore be influenced by the sow's diet. The objective of this study was to evaluate whether the microbiological flora of liquid feed for sows can be associated with the development of neonatal diarrhoea. METHODS: This study was carried out on 10 case farms with neonatal diarrhoea and 10 control farms without neonatal diarrhoea. On each farm, a microbiological analysis of gestating and lactating liquid feed was performed. A generalised linear model was used to study the impact of the liquid feed microbiological counts and pH on the probability of neonatal diarrhoea developing. RESULTS: For thermotolerant coliforms, sulphite-reducing bacteria, heterotrophic bacteria and lactic-acid bacteria counts, there was no significant difference between case and control farms. The higher the count of total coliforms, enterococci and yeasts in sow non-fermented liquid feed, the greater the probability of observing neonatal diarrhoea. Moreover, taking into account total coliforms and yeasts counts together is highly predictive of neonatal diarrhoea risk. CONCLUSION: This study offers new perspectives of investigation and understanding of neonatal diarrhoea in breeding farms feeding sows with a non-fermented liquid feed.

Haematological reference intervals of sows at end gestation in ten French herds, the impact of parity on haematological parameters and the consequences on reproductive performance.

BACKGROUND: Changes in haematological values occur during the reproductive cycle. In veterinary swine practice, haematological reference intervals for this period are scarce. Over past decades, there has been a remarkable increase in reproductive prolificacy, possibly making previously established haematological reference intervals for sows outdated. OBJECTIVES: The aim of this study was to provide updated haematological reference intervals for sows at end-gestation, to study the influence of parity on those haematological parameters and to evaluate the impact of haemoglobin levels on production performance. METHODS: The data presented in this article were obtained using blood samples from 198 apparently healthy and conventionally managed group-housed sows at end-gestation from ten breeding herds located in France. The samples were analysed for haematological variables using impedance technique on Horiba ABX analyser (Horiba, Kyoto, Japan). The reference intervals were calculated according to the guidelines of The American Society for Veterinary Clinical Pathology using SUMMARY procedure in R Studio. Analysis of variance (ANOVA) models were used to evaluate the influence of parity on each haematological parameter and the impact of haemoglobin values on production performances at farrowing. Differences were considered as significant if p < 0.05. RESULTS: Reference intervals produced in this study were similar to previously published references but we noticed marked differences in white blood cell values. The study of the impact of parity revealed significant changes for gilts and parity 5 + sows regarding haematological values. Gilts had higher red and white blood cells counts, haemoglobin values and haematocrit values. Regarding haemoglobin values, the higher the number of liveborn and weaned piglets per litter, the lower the haemoglobin value at end-gestation. For sows of fifth or higher gestation, we found that the higher the percentage of stillborn piglets, the lower the haemoglobin value at end-gestation. CONCLUSIONS: This study provides haematological reference intervals for sows at end-gestation. These will be useful for swine veterinarians and researchers for a better understanding of the influence of parity on haematological parameters and haemoglobin values and their relation to reproductive performance.

Effectiveness of two intramuscular combined vaccines for the control of Mycoplasma hyopneumoniae and porcine circovirus type 2 in growing pigs: a randomized field trial.

BACKGROUND: Mycoplasma hyopneumoniae and Porcine circovirus type 2 are two economically important pathogens affecting growing pigs. Control and prevention of both diseases can be accomplished by vaccination, together with biosecurity and good management practices. Many commercial vaccines are available. The aim of this study was to assess the efficacy of Hyogen® and Circovac® administered mixed at weaning and to compare this protocol with a competitor ready-to-use (RTU) vaccine. CASE PRESENTATION: A randomised field trial was designed in a commercial farrow-to-finish farm located in France. A total of 641 pigs born from 54 different sows were included in this study. Piglets at weaning were allocated into three groups: the first one vaccinated with Hyogen® and Circovac® combined (group A), the second one vaccinated with a competitor RTU vaccine (group B) and the last one unvaccinated. Only minor local reactions for both vaccination groups could be observed which revealed a good safety of both protocols. Both vaccination schemes in this trial didn't improve wean-to-slaughter growth performances but significantly reduced lung lesions, lung fissures and pleurisy at slaughter, produced a seroconversion for both M. hyopneumoniae and PCV-2 and significantly reduced the PCV-2 viral load in blood. When we compared groups A and B, we observed no significant differences in growth performances, mortality, clinical signs, percentages of affected lungs at slaughter, lung fissures and pleurisy, and no difference in pathogens detection. However, two statistical differences were observed between both vaccines: the mean lung lesion score and the percentage of extensive lung lesions were lower in group A. This is consistent with lower M. hyopneumoniae loads in the lower respiratory tract in pigs from group A but this difference was not statistically significant. CONCLUSIONS: Results reported in this case study must be considered with caution since it was done in only one farm. In this trial, Hyogen® and Circovac® mixed together under field conditions offered a successful protection of growing pigs and significantly decreased the extension of lung lesions during a natural field challenge when compared with a competitor RTU vaccine.

Controlling hepatitis E virus in the pig production sector: Assessment of the technical and behavioural feasibility of on-farm risk mitigation strategies.

Hepatitis E virus (HEV) is a zoonotic agent with pigs as the main reservoir in industrialised countries. Recent studies conducted on pig farms, in experimental conditions or through modelling approaches, have led to a better understanding of the spread of HEV on pig farms. The findings have also made it possible to define a set of measures to reduce HEV prevalence and the risk of marketing contaminated products. The objective of this study was to assess the feasibility of a set of HEV control strategies on pig farms. Individual semi-structured interviews were conducted with farmers, veterinarians and farming advisors to collect general data, their level of knowledge of HEV, their opinion on the technical feasibility of certain changes in practices, their perception of the respective responsibilities of the different stakeholders, and their feelings about the importance of the issue, following the framework of the Theory of Planned Behaviour. The interviews made it possible to highlight potential barriers and preferred motivators for the implementation of on-farm risk mitigation strategies. Barriers included lack of knowledge, scientific gaps, perceived inability to control HEV, and low perception of the importance of the issue. Motivators included professional satisfaction, family recognition, and the opportunity to achieve higher quality standards. Three clusters of stakeholders were also identified, with a group of leaders who could help unlock reluctance and disseminate innovations. This type of behavioural approach appeared useful to help risk managers facilitate zoonotic control on pig farms.