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Local immunization impacts the response of dairy cows to Escherichia coli mastitis

Current vaccines to Escherichia coli mastitis have shown some albeit limited efficacy. Their mode of action has not been documented, and immune responses protecting the mammary gland against E. coli are not completely understood. To improve our knowledge of mammary gland immune protection, cows immunized either intramuscularly or intramammarily with the E. coli P4 were submitted to a homologous mastitis challenge. A third group of mock-immunized cows serve as challenge controls. Local immunization modified favorably the course of infection, by improving bacterial clearance while limiting inflammation. Systemic clinical signs and reduction in milk secretion were also contained. This occurred with a modification of the cytokine profile, such as an increase in IFN-γ and a reduction in TNF-α concentrations in milk. Concentrations of IL-17A and IL-22 increased in milk at the onset of the inflammatory response and remained high up to the elimination of bacteria, but concentrations did not differ between groups. Accelerated bacteriological cure was not linked to an increase in the initial efficiency of phagocytosis in milk. Results support the idea that antibodies did not play a major role in the improvement, and that cell-mediated immunity is the key to understanding E. coli vaccine-induced protection of the mammary gland.

Aerosol Route to Administer Teicoplanin in Mechanical Ventilation: In Vitro Study, Lung Deposition and Pharmacokinetic Analyses in Pigs

BACKROUND Glycopeptides given intravenously achieve low airway concentrations. Nebulization of teicoplanin may be an efficient way of delivering a high concentration of this antibiotic to the lung. This multistep study assessed the feasibility of teicoplanin nebulization during mechanical ventilation by evaluating: the stability of its antibiotic effect; epithelial tolerance; lung deposition and systemic absorption in ventilated pigs. METHODS Nebulized and non-nebulized teicoplanin activity was tested on Staphylococcus aureus cultures. The cytotoxic effect of teicoplanin on human respiratory epithelial cells was assessed by measuring lactate dehydrogenase activity released, cell viability, and transepithelial electrical resistance. Volume median diameter of particles of nebulized teicoplanin was measured by laser diffraction during mechanical ventilation. The deposited mass of teicoplanin nebulized with a vibrating mesh nebulizer in ventilated piglets was assessed by scintigraphy. Blood pharmacokinetics of teicoplanin administered either intravenously or by nebulization was compared. RESULTS No decrease of antibiotic activity was observed after nebulization. In vitro cytotoxicity of teicoplanin was only observed with 1000 times the dose recommended for intravenous administration. Volume median diameter of particles was 2.5±0.1 μm. Of the initial nebulizer charge of teicoplanin, 24±7% was present in the lungs of ventilated pigs after the nebulization. Amount absorbed in blood was low (3.4%±0.9%) after nebulization, and blood stream elimination half-life value was 25.4 h. CONCLUSIONS Teicoplanin was administered efficiently by nebulization during mechanical ventilation, without any effect on its pharmacological properties or any cytotoxicity. The pharmacokinetic parameters are promising in view of its time-dependent killing process. All the results of our multi-step study highlighted the potential of teicoplanin to be nebulized during mechanical ventilation.

Semen from scrapie-infected rams does not transmit prion infection to transgenic mice

Scrapie is the most common transmissible spongiform encephalopathy (TSE) in livestock. Natural contamination in sheep flocks is presumed to occur by maternal transmission to offspring. However, horizontal prion transmission from animal to animal exists and may be significant in sustaining and spreading contagion in the field. Artificial insemination is widely used in modern farming, and as large amounts of prion protein have been found in sheep sperm membrane, epididymal fluid and seminal plasma, horizontal transmission by this route was hypothesized since no clear information has been obtained on possible sexual transmission of TSE. We therefore tested the contamination levels of semen from scrapie-infected rams at different stages of incubation, including the clinical phase of the disease. We report here that under our experimental conditions ram semen did not transmit infectivity to scrapie-susceptible transgenic mice overexpressing the V(136)R(154)Q(171) allele of the sheep prion (PRNP) gene. These results suggest that artificial insemination and natural mating have a very low or negligible potential for the transmission of scrapie in sheep flocks.

A Universal Influenza Vaccine Can Lead to Disease Exacerbation or Viral Control Depending on Delivery Strategies

The development of influenza A virus (IAV) vaccines, which elicits cross-strain immunity against seasonal and pandemic viruses is a major public health goal. As pigs are susceptible to human, avian, and swine-adapted IAV, they would be key targets of so called universal IAV vaccines, for reducing both the zoonotic risk and the economic burden in the swine industry. They also are relevant preclinical models. However, vaccination with conserved IAV antigens (AGs) in pigs was reported to elicit disease exacerbation. In this study, we assessed whether delivery strategies, i.e., dendritic cell (DC) targeting by the intradermal (ID) or intramuscular (IM) routes, impact on the outcome of the vaccination with three conserved IAV AGs (M2e, NP, and HA2) in pigs. The AGs were addressed to CD11c by non-covalent binding to biotinylated anti-CD11c monoclonal antibody. The CD11c-targeted AGs given by the ID route exacerbated disease. Conversely, CD11c-targeted NP injected by the IM route promoted T cell response compared to non-targeted NP. Furthermore, the conserved IAV AGs injected by the IM route, independently of DC targeting, induced both a reduction of viral shedding and a broader IgG response as compared to the ID route. Our findings highlight in a relevant animal species that the route of vaccine delivery impacts on the protection induced by conserved IAV AGs and on vaccine adverse effects. Finally, our results indicate that HA2 stands as the most promising conserved IAV AG for universal vaccine development.

The anti-influenza M2e antibody response is promoted by XCR1 targeting in pig skin

XCR1 is selectively expressed on a conventional dendritic cell subset, the cDC1 subset, through phylogenetically distant species. The outcome of antigen-targeting to XCR1 may therefore be similar across species, permitting the translation of results from experimental models to human and veterinary applications. Here we evaluated in pigs the immunogenicity of bivalent protein structures made of XCL1 fused to the external portion of the influenza virus M2 proton pump, which is conserved through strains and a candidate for universal influenza vaccines. Pigs represent a relevant target of such universal vaccines as pigs can be infected by swine, human and avian strains. We found that cDC1 were the only cell type labeled by XCR1-targeted mCherry upon intradermal injection in pig skin. XCR1-targeted M2e induced higher IgG responses in seronegative and seropositive pigs as compared to non-targeted M2e. The IgG response was less significantly enhanced by CpG than by XCR1 targeting, and CpG did not further increase the response elicited by XCR1 targeting. Monophosphoryl lipid A with neutral liposomes did not have significant effect. Thus altogether M2e-targeting to XCR1 shows promises for a trans-species universal influenza vaccine strategy, possibly avoiding the use of classical adjuvants.

A Rift Valley fever virus Gn ectodomain-based DNA vaccine induces a partial protection not improved by APC targeting

Rift Valley fever virus, a phlebovirus endemic in Africa, causes serious diseases in ruminants and humans. Due to the high probability of new outbreaks and spread to other continents where competent vectors are present, vaccine development is an urgent priority as no licensed vaccines are available outside areas of endemicity. In this study, we evaluated in sheep the protective immunity induced by DNA vaccines encoding the extracellular portion of the Gn antigen which was either or not targeted to antigen-presenting cells. The DNA encoding untargeted antigen was the most potent at inducing IgG responses, although not neutralizing, and conferred a significant clinical and virological protection upon infectious challenge, superior to DNA vaccines encoding the targeted antigen. A statistical analysis of the challenge parameters supported that the anti-eGn IgG, rather than the T-cell response, was instrumental in protection. Altogether, this work shows that a DNA vaccine encoding the extracellular portion of the Gn antigen confers substantial—although incomplete—protective immunity in sheep, a natural host with high preclinical relevance, and provides some insights into key immune correlates useful for further vaccine improvements against the Rift Valley fever virus.Rift Valley fever: DNA vaccines hold promise, but need workA vaccine made from the genome of Rift Valley fever virus (RVFV) offers partial protection, but pieces of the puzzle are missing, say scientists. French and Spanish researchers, led by the French National Institute for Agricultural Research’s Isabelle Schwartz-Cornil, tested in sheep three slightly-differing vaccine candidates using RVFV genes. Such DNA vaccines are designed to generate proteins which a host’s immune system can use to arm itself against a genuine viral infection. Two of the candidates, designed to target cells that would present the viral proteins to the host’s immune system, provided some benefit to the vaccinated sheep. However, the third untargeted candidate, was the most efficient at protecting sheep, although not completely, and at boosting antibody levels despite not neutralizing the virus. These results provide hope for DNA vaccines against RVFV, and offer direction for future research effort.

Fetopathic effects of experimental Schmallenberg virus infection in pregnant goats

Schmallenberg virus (SBV) is an emerging virus responsible for congenital malformations in the offspring of domestic ruminants. It is speculated that infection of pregnant dams may also lead to a significant number of unrecognized fetal losses during the early period of gestation. To assess the pathogenic effects of SBV infection of goats in early pregnancy, we inoculated dams at day 28 or 42 of gestation and followed the animals until day 55 of gestation. Viremia in the absence of clinical signs was detected in all virus-inoculated goats. Fetal deaths were observed in several goats infected at day 28 or 42 of gestation and were invariably associated with the presence of viral genomic RNA in the affected fetuses. Among the viable fetuses, two displayed lesions in the central nervous system (porencephaly) in the presence of viral genome and antigen. All fetuses from goats infected at day 42 and the majority of fetuses from goats infected at day 28 of gestation contained viral genomic RNA. Viral genome was widely distributed in these fetuses and their respective placentas, and infectious virus could be isolated from several organs and placentomes of the viable fetuses. Our results show that fetuses of pregnant goats are susceptible to vertical SBV infection during early pregnancy spanning at least the period between day 28 and 42 of gestation. The outcomes of experimental SBV infection assessed at day 55 of gestation include fetal mortalities, viable fetuses displaying lesions of the central nervous system, as well as viable fetuses without any detectable lesion.

Computed Tomography (CT) Scanning Facilitates Early Identification of Neonatal Cystic Fibrosis Piglets

Background Cystic Fibrosis (CF) is the most prevalent autosomal recessive disease in the Caucasian population. A cystic fibrosis transmembrane conductance regulator knockout (CFTR-/-) pig that displays most of the features of the human CF disease has been recently developed. However, CFTR -/- pigs presents a 100% prevalence of meconium ileus that leads to death in the first hours after birth, requiring a rapid diagnosis and surgical intervention to relieve intestinal obstruction. Identification of CFTR -/- piglets is usually performed by PCR genotyping, a procedure that lasts between 4 to 6 h. Here, we aimed to develop a procedure for rapid identification of CFTR -/- piglets that will allow placing them under intensive care soon after birth and immediately proceeding with the surgical correction. Methods and Principal Findings Male and female CFTR +/- pigs were crossed and the progeny was examined by computed tomography (CT) scan to detect the presence of meconium ileus and facilitate a rapid post-natal surgical intervention. Genotype was confirmed by PCR. CT scan presented a 94.4% sensitivity to diagnose CFTR -/- piglets. Diagnosis by CT scan reduced the birth-to-surgery time from a minimum of 10 h down to a minimum of 2.5 h and increased the survival of CFTR -/- piglets to a maximum of 13 days post-surgery as opposed to just 66 h after later surgery. Conclusion CT scan imaging of meconium ileus is an accurate method for rapid identification of CFTR -/- piglets. Early CT detection of meconium ileus may help to extend the lifespan of CFTR -/- piglets and, thus, improve experimental research on CF, still an incurable disease.

99mTc-N-IFC: a new isoniazid derivative for Mycobacterium diagnostic

Abstract We present in this work a new technetium-99m-labeled derivative from isoniazid. The labeling was achieved with a double-ligand transfer through the use of a ferrocene derivative. A further referred to as 99mTc-N-IFC (N-Isonicotinamide ferrocene carboxamide labeled with 99mTc), targeting infections in experimental animals, has been synthesized. The N-IFC was chemically synthesized and then labeled with technetium-99m. It has been confirmed through this work that it was obtained with high radiolabelling yield (95%). Radiochemical analyses of 99mTc-N-IFC revealed that the molecule was efficiently labeled with a little free pertechnetate in the preparations containing purified compound. Only 1–2% of the tracer was leached out from the complex at 24 h when incubated in serum at 37 ºC confirmed its high stability. The radiolabeled complex was found to be 10% bound to blood protein, which corresponds to a fast retention advantage. Biodistribution study showed the renal route of excretion and has also demonstrated that our radiolabeled compound is rapidly and significantly accumulated (P<0.5) at infection sites. Thigh model of localized infection was prepared in mice by injecting of BCG (pGFM-11) (fluorexcente BCG) live bacteria in growing phase. The confirmation of the bacteria presence in infection sites has been established through its fluorescence characteristic. The comparison of the 99mTc-N-IFC accumulation at sites of BCG (pGFM-11) infected animals, which is expressed as target-to-non-target ratio, (3.14) with other radiotracers was discussed. This allowed us to consider that 99mTc-N-IFC could be a good radiotracer for mycobacterial infections. Obtained results were in good and encourage to undergo a similar labeling for the Mycobacterium tuberculosis as perspective of this work.

The Pig: A Relevant Model for Evaluating the Neutrophil Serine Protease Activities during Acute Pseudomonas aeruginosa Lung Infection.

The main features of lung infection and inflammation are a massive recruitment of neutrophils and the subsequent release of neutrophil serine proteases (NSPs). Anti-infectious and/or anti-inflammatory treatments must be tested on a suitable animal model. Mice models do not replicate several aspects of human lung disease. This is particularly true for cystic fibrosis (CF), which has led the scientific community to a search for new animal models. We have shown that mice are not appropriate for characterizing drugs targeting neutrophil-dependent inflammation and that pig neutrophils and their NSPs are similar to their human homologues. We induced acute neutrophilic inflammatory responses in pig lungs using Pseudomonas aeruginosa, an opportunistic respiratory pathogen. Blood samples, nasal swabs and bronchoalveolar lavage fluids (BALFs) were collected at 0, 3, 6 and 24 h post-insfection (p.i.) and biochemical parameters, serum and BAL cytokines, bacterial cultures and neutrophil activity were evaluated. The release of proinflammatory mediators, biochemical and hematological blood parameters, cell recruitment and bronchial reactivity, peaked at 6h p.i.. We also used synthetic substrates specific for human neutrophil proteases to show that the activity of pig NSPs in BALFs increased. These proteases were also detected at the surface of lung neutrophils using anti-human NSP antibodies. Pseudomonas aeruginosa-induced lung infection in pigs results in a neutrophilic response similar to that described for cystic fibrosis and ventilator-associated pneumonia in humans. Altogether, this indicates that the pig is an appropriate model for testing anti-infectious and/or anti-inflammatory drugs to combat adverse proteolytic effects of neutrophil in human lung diseases.