Pneumococcal colonization and invasive disease studied in a porcine model.

BACKGROUND: Streptococcus pneumoniae, a Gram-positive bacterium carried in the human nasopharynx, is an important human pathogen causing mild diseases such as otitis media and sinusitis as well as severe diseases including pneumonia, meningitis and sepsis. There is a strong resemblance between the anatomy, immunology and physiology of the pig and human species. Furthermore, there are striking similarities between S. suis pathogenesis in piglets and S. pneumoniae pathogenesis in humans. Therefore, we investigated the use of piglets as a model for pneumococcal colonization and invasive disease. RESULTS: Intravenous inoculation of piglets with an invasive pneumococcal isolate led to bacteraemia during 5 days, showing clear bacterial replication in the first two days. Bacteraemia was frequently associated with fever and septic arthritis. Moreover, intranasal inoculation of piglets with a nasopharyngeal isolate led to colonization for at least six consecutive days. CONCLUSIONS: This demonstrates that central aspects of human pneumococcal infections can be modelled in piglets enabling the use of this model for studies on colonization and transmission but also on development of vaccines and host-directed therapies. Moreover this is the first example of an animal model inducing high levels of pneumococcal septic arthritis.

Host-pathogen Interaction at the Intestinal Mucosa Correlates With Zoonotic Potential of Streptococcus suis.

BACKGROUND: Streptococcus suis has emerged as an important cause of bacterial meningitis in adults. The ingestion of undercooked pork is a risk factor for human S. suis serotype 2 (SS2) infection. Here we provide experimental evidence indicating that the gastrointestinal tract is an entry site of SS2 infection. METHODS: We developed a noninvasive in vivo model to study oral SS2 infection in piglets. We compared in vitro interaction of S. suis with human and porcine intestinal epithelial cells (IEC). RESULTS: Two out of 15 piglets showed clinical symptoms compatible with S. suis infection 24-48 hours after ingestion of SS2. SS2 was detected in mesenteric lymph nodes of 40% of challenged piglets. SS2 strains isolated from patients showed significantly higher adhesion to human IEC compared to invasive strains isolated from pigs. In contrast, invasive SS9 strains showed significantly higher adhesion to porcine IEC. Translocation across human IEC, which occurred predominately via a paracellular route, was significantly associated with clonal complex 1, the predominant zoonotic genotype. Adhesion and translocation were dependent on capsular polysaccharide production. CONCLUSIONS: SS2 should be considered a food-borne pathogen. S. suis interaction with human and pig IEC correlates with S. suis serotype and genotype, which can explain the zoonotic potential of SS2.

Schmallenberg virus antibody persistence in adult cattle after natural infection and decay of maternal antibodies in calves.

BACKGROUND: Schmallenberg virus (SBV) has swept through the major part of Europe in the period 2011-2013. A vaccine against SBV has been developed and may be a possible preventive instrument against infection. Presently, there is no data available to refute the assumption that natural SBV infection results in long-term immunity. In that respect, it is of interest to know how long (protecting) virus-neutralizing antibodies are present in naturally infected animals. New-born calves acquire passive immunity from their dams by ingestion and absorption of antibodies present in colostrum, which can block the production of serum antibodies when vaccine is administered to calves with maternally derived antibodies. In that respect, it is useful to know how long it takes for maternal antibodies against SBV to disappear in young animals born from infected dams. RESULTS: Longitudinal whole-herd serological monitoring using virus neutralization test (VNT) indicated that 80% of adult dairy cows still had measurable antibodies against SBV at least 24 months after the estimated introduction of the virus into the herd. Median 2Log VNT titer of the adult dairy cows (≥1 year) dropped from 8.6 to 5.6 in a period of 17 months. Median 2Log VNT maternal antibodies titers of calves sampled within 30 days after birth was 8. Calves lost their maternally-derived antibodies after 5-6 months. There was a definite positive relationship between the VNT titer of the dam and the VNT titer of the corresponding calf (age ≤ 30 days) of dam-calf combinations sampled on the same day: the higher the VNT titer of the dam, the higher the VNT titer (maternal antibodies) of the calf. CONCLUSIONS: Our field data support the assumption that natural SBV infection in adult cows results in persistence of specific antibodies for at least two years. Based on the observed decay of maternally-derived antibodies in calves, it is presumed safe to vaccinate calves against SBV at an age of approximately 6 months.

Corrigendum: Animal-based factors prior to infection predict histological disease outcome in porcine reproductive and respiratory syndrome virus- and Actinobacillus pleuropneumoniae-infected pigs.

[This corrects the article DOI: 10.3389/fvets.2021.742877.].

Rapid emergence of a virulent PB2 E627K variant during adaptation of highly pathogenic avian influenza H7N7 virus to mice.

BACKGROUND: Highly pathogenic avian influenza (HPAI) viruses pose a potential human health threat as they can be transmitted directly from infected poultry to humans. During a large outbreak of HPAI H7N7 virus among poultry in The Netherlands in 2003, bird to human transmission was confirmed in 89 cases, of which one had a fatal outcome. METHODS: To identify genetic determinants of virulence in a mammalian host, we passaged an avian H7N7/03 outbreak isolate in mouse lungs and evaluated the phenotype of the mouse-adapted variant in animal models and in vitro. RESULTS: Three passages in mouse lungs were sufficient to select a variant that was highly virulent in mice. The virus had a MLD50 that was >4.3 logs lower than that of its non-lethal parental virus. Sequence analysis revealed a single mutation at position 627 in PB2, where the glutamic acid was changed to a lysine (E627K). The mouse-adapted virus has this mutation in common with the fatal human case isolate. The virus remained highly pathogenic for chickens after its passage in mice. In ferrets, the mouse-adapted virus induced more severe disease, replicated to higher titers in the lower respiratory tract and spread more efficiently to systemic organs compared with the parental virus. In vitro, the PB2 E627K mutation had a promoting effect on virus propagation in mammalian, but not in avian cells. CONCLUSIONS: Our results show that the E627K mutation in PB2 alone can be sufficient to convert an HPAI H7N7 virus of low virulence to a variant causing severe disease in mice and ferrets. The rapid emergence of the PB2 E627K mutant during mouse adaptation and its pathogenicity in ferrets emphasize the potential risk of HPAI H7N7 viruses for human health.

Early host response in the mammary gland after experimental Streptococcus uberis challenge in heifers.

Streptococcus uberis is a highly prevalent causative agent of bovine mastitis, which leads to large economic losses in the dairy industry. The aim of this study was to examine the host response during acute inflammation after experimental challenge with capsulated Strep. uberis. Gene expression in response to Strep. uberis was compared between infected and control quarters in 3 animals. All quarters (n=16) were sampled at 16 different locations. Microarray data showed that 239 genes were differentially expressed between infected and control quarters. No differences in gene expression were observed between the different locations. Microarray data were confirmed for several genes using quantitative PCR analysis. Genes differentially expressed due to early Strep. uberis mastitis represented several stages of the process of infection: (1) pathogen recognition; (2) chemoattraction of neutrophils; (3) tissue repair mechanisms; and (4) bactericidal activity. Three different pathogen recognition genes were induced: ficolins, lipopolysaccharide binding protein, and toll-like receptor 2. Calgranulins were found to be the most strongly upregulated genes during early inflammation. By histology and immunohistochemistry, we demonstrated that changes in gene expression in response to Strep. uberis were induced both in infiltrating somatic milk cells and in mammary epithelial cells, demonstrating that the latter cell type plays a role in milk production as well as immune responsiveness. Given the rapid development of inflammation or mastitis after infection, early diagnosis of (Strep. uberis) mastitis is required for prevention of disease and spread of the pathogen. Insight into host responses could help to design immunomodulatory therapies to dampen inflammation after (early) diagnosis of Strep. uberis mastitis. Future research should focus on development of these early diagnostics and immunomodulatory components for mastitis treatment.

Effects of hatching system on chick quality, welfare and health of young breeder flock offspring.

Alternative hatching systems have been developed for broiler chickens to provide immediately feed and water after hatch and reduce the number or severity of early life stressors. Besides beneficial effects of these alternative hatching systems on chick quality and performance, broiler health and welfare may be positively affected as well. Especially offspring from young broiler breeder flocks may benefit, as they have been shown to be more sensitive to preturbations than offspring from older breeder flocks. This study evaluated effects of hatching systems on chick quality, health and welfare of young breeder flock offspring, using 3 different hatching systems: conventional hatchery-hatched (HH), hatchery-fed (HF), and on-farm hatching (OH). A total of 24 pens were used in a completely randomized block design, with 8 pens per hatching system and 30 chickens per pen. Chick quality at hatch and performance until 35 d of age was improved in the HF and OH compared to HH treatment, but only minor effects were found on the welfare indicators: footpad dermatitis, hock burn, cleanliness, skin lesion and gait score. No effect was observed on the dynamics of a humoral immune response after NCD vaccination, given at d 0 and 14 of age, as no differences between NCD titers were found at d 18. Animals were vaccinated with a live attenuated infectious bronchitis vaccine virus (IBV) at d 28 to address treatment related differences to disease resilience. The expressions of inflammation and epithelial integrity related genes in the trachea and histo-pathological changes in the trachea were examined at 3 d after vaccine administration. No differences between treatment groups were observed. Although beneficial effects of HF and OH systems were found for young breeder flock offspring on chick quality at hatch and body weight posthatch, only one effect of alternative hatching systems on welfare and health indicators were found. No effect of hatching system on humoral immune response or disease resilience was found.

Potential business model for a European vaccine R&D infrastructure and its estimated socio-economic impact.

Background: Research infrastructures are facilities or resources that have proven fundamental for supporting scientific research and innovation. However, they are also known to be very expensive in their establishment, operation and maintenance. As by far the biggest share of these costs is always borne by public funders, there is a strong interest and indeed a necessity to develop alternative business models for such infrastructures that allow them to function in a more sustainable manner that is less dependent on public financing. Methods: In this article, we describe a feasibility study we have undertaken to develop a potentially sustainable business model for a vaccine research and development (R&D) infrastructure. The model we have developed integrates two different types of business models that would provide the infrastructure with two different types of revenue streams which would facilitate its establishment and would be a measure of risk reduction. For the business model we are proposing, we have undertaken an ex ante impact assessment that estimates the expected impact for a vaccine R&D infrastructure based on the proposed models along three different dimensions: health, society and economy. Results: Our impact assessment demonstrates that such a vaccine R&D infrastructure could achieve a very significant socio-economic impact, and so its establishment is therefore considered worthwhile pursuing. Conclusions: The business model we have developed, the impact assessment and the overall process we have followed might also be of interest to other research infrastructure initiatives in the biomedical field.

Serum protein profiles as potential biomarkers for infectious disease status in pigs.

BACKGROUND: In veterinary medicine and animal husbandry, there is a need for tools allowing the early warning of diseases. Preferably, tests should be available that warn farmers and veterinarians during the incubation periods of disease and before the onset of clinical signs. The objective of this study was to explore the potential of serum protein profiles as an early biomarker for infectious disease status. Serum samples were obtained from an experimental pig model for porcine circovirus-associated disease (PCVAD), consisting of Porcine Circovirus type 2 (PCV2) infection in combination with either Porcine Parvovirus (PPV) or Porcine Reproductive and Respiratory Syndrome virus (PRRSV). Sera were collected before and after onset of clinical signs at day 0, 5 and 19 post infection. Serum protein profiles were evaluated against sera from non-infected control animals. RESULTS: Protein profiles were generated by SELDI-TOF mass spectrometry in combination with the Proteominer™ technology to enrich for low-abundance proteins. Based on these protein profiles, the experimentally infected pigs could be classified according to their infectious disease status. Before the onset of clinical signs 88% of the infected animals could be classified correctly, after the onset of clinical sigs 93%. The sensitivity of the classification appeared to be high. The protein profiles could distinguish between separate infection models, although specificity was moderate to low. Classification of PCV2/PRRSV infected animals was superior compared to PCV2/PPV infected animals. Limiting the number of proteins in the profiles (ranging from 568 to 10) had only minor effects on the classification performance. CONCLUSIONS: This study shows that serum protein profiles have potential for detection and identification of viral infections in pigs before clinical signs of the disease become visible.

Tissue tropism and pathology of highly pathogenic avian influenza H5N6 virus in chickens and Pekin ducks.

The highly pathogenic avian influenza (HPAI) H5N6 virus caused outbreaks on commercial poultry farms in the Netherlands in 2017-2018, holding chickens and Pekin ducks. Intravenous pathogenicity index (IVPI) tests confirmed the high pathogenicity of the virus. Tissues derived from birds from infected farms (natural infection) and IVPI tests (experimental infection) were used to compare histopathology and virus distribution in both poultry species. After natural infection in chickens, histopathologic changes were present in the respiratory tract and several internal organs in both chickens and Pekin ducks. Viral antigen expression in the tissues of chickens varied from that in ducks. Virus expression was found in epithelial, mononuclear and endothelial cells in chickens. In contrast to the major role infected endothelial cells seem to play in systemic infections of chickens, in ducks the number of infected endothelial cells was very limited. Therefore, endothelial cell infection likely does not play a major role in systemic infection and disease progression in HPAI H5N6 virus infected Pekin ducks.

Respiratory health of broilers following chronic exposure to airborne endotoxin.

In and around poultry farms, high concentrations of endotoxins are found that have a negative impact on the health of farmers and local residents. However, little is known about the effects of chronic exposure to endotoxins on the health of poultry. The aim of this study was to identify effects of chronic exposure to airborne endotoxins (E. coli LPS) on the immune system, respiratory tract, disease susceptibility and welfare of broilers. Effects of high (HE) and low endotoxin (LE) concentrations on natural antibody titers (NAb), performance and behavior of broilers were determined. After treatment with a respiratory virus infection, infectious bronchitis virus (IBV), mRNA expression of cytokines and Toll-like receptor (TLR) 4 in the lung, tracheal ciliary activity and lesions in the respiratory tract were determined. Endotoxin affected the immune system and respiratory tract, where HE broilers tended to have lower IgM NAb binding Phosphorylcholine-conjugated to Bovine Serum Albumin, and higher interferon (IFN)-α mRNA expression and more lesions in the nasal tissue compared to LE broilers. Furthermore, HE broilers had higher TLR4 mRNA expression compared to LE broilers. However, endotoxin did not affect NAb levels binding Keyhole Limpet Hemocyanin, IFN-ß and interleukin-10 mRNA expression, IBV replication or lesions in the lung and trachea. HE and LE broilers further had similar body weight, but HE broilers showed numerically more passive behavior compared to LE broilers. In conclusion, chronic exposure to high airborne endotoxin concentrations affects components of the immune system and respiratory tract in broilers and could therefore influence disease susceptibility.

ChAd155-RSV vaccine is immunogenic and efficacious against bovine RSV infection-induced disease in young calves.

Respiratory syncytial virus (RSV) infection causes a substantial lower-respiratory-tract disease burden in infants, constituting a global priority for vaccine development. We evaluated immunogenicity, safety and efficacy of a chimpanzee adenovirus (ChAd)-based vaccine candidate, ChAd155-RSV, in a bovine RSV (bRSV) challenge model. This model closely reproduces the pathogenesis/clinical manifestations of severe pediatric RSV disease. In seronegative calves, ChAd155-RSV elicits robust neutralizing antibody responses against human RSV. Two doses protect calves from clinical symptoms/lung pathological changes, and reduce nasal/lung virus loads after both a short (4-week) and a long (16-week) interval between last immunization and subsequent bRSV challenge. The one-dose regimen confers near-complete or significant protection after short-term or long-term intervals before challenge, respectively. The presence of pre-existing bRSV-antibodies does not affect short-term efficacy of the two-dose regimen. Immunized calves present no clinical signs of enhanced respiratory disease. Collectively, this supports the development of ChAd155-RSV as an RSV vaccine candidate for infants.

Genetic diversity of Streptococcus suis isolates as determined by comparative genome hybridization.

BACKGROUND: Streptococcus suis is a zoonotic pathogen that causes infections in young piglets. S. suis is a heterogeneous species. Thirty-three different capsular serotypes have been described, that differ in virulence between as well as within serotypes. RESULTS: In this study, the correlation between gene content, serotype, phenotype and virulence among 55 S. suis strains was studied using Comparative Genome Hybridization (CGH). Clustering of CGH data divided S. suis isolates into two clusters, A and B. Cluster A isolates could be discriminated from cluster B isolates based on the protein expression of extracellular factor (EF). Cluster A contained serotype 1 and 2 isolates that were correlated with virulence. Cluster B mainly contained serotype 7 and 9 isolates. Genetic similarity was observed between serotype 7 and serotype 2 isolates that do not express muramidase released protein (MRP) and EF (MRP⁻EF⁻), suggesting these isolates originated from a common founder. Profiles of 25 putative virulence-associated genes of S. suis were determined among the 55 isolates. Presence of all 25 genes was shown for cluster A isolates, whereas cluster B isolates lacked one or more putative virulence genes. Divergence of S. suis isolates was further studied based on the presence of 39 regions of difference. Conservation of genes was evaluated by the definition of a core genome that contained 78% of all ORFs in P1/7. CONCLUSIONS: In conclusion, we show that CGH is a valuable method to study distribution of genes or gene clusters among isolates in detail, yielding information on genetic similarity, and virulence traits of S. suis isolates.

Stochastic Assessment of the Economic Impact of Streptococcus suis-Associated Disease in German, Dutch and Spanish Swine Farms.

The economic assessment of animal diseases is essential for decision-making, including the allocation of resources for disease control. However, that assessment is usually hampered by the lack of reliable data on disease incidence, or treatment and control measures, and that is particularly true for swine production diseases, such as infections caused by Streptococcus suis. Therefore, we deployed a questionnaire survey of clinical swine veterinarians to obtain the input data needed for a stochastic model to calculate the costs caused by S. suis, which was implemented in three of the main swine producing countries in Europe: Germany, the Netherlands and Spain. S. suis-associated disease is endemic in those countries in all production phases, though nursery was the phase most severely impacted. In affected nursery units, between 3.3 and 4.0% of pigs had S. suis-associated disease and the mortalities ranged from 0.5 to 0.9%. In Germany, the average cost of S. suis per pig (summed across all production phases) was 1.30 euros (90% CI: 0.53-2.28), in the Netherlands 0.96 euros (90% CI: 0.27-1.54), and in Spain 0.60 euros (90% CI: 0.29-0.96). In Germany, that cost was essentially influenced by the expenditure in early metaphylaxis in nursery and in autogenous vaccines in sows and nursery pigs; in the Netherlands, by expenditure on autogenous vaccines in sows and nursery pigs; and in Spain, by the expenditures in early metaphylaxis and to a lesser extent by the mortality in nursery pigs. Therefore, the differences in costs between countries can be explained to a great extent by the measures to control S. suis implemented in each country. In Spain and in Germany, use of antimicrobials, predominantly beta-lactams, is still crucial for the control of the disease.

Environmentally enriched housing conditions affect pig welfare, immune system and gut microbiota in early life.

BACKGROUND: Conventional pig housing and management conditions are associated with gastrointestinal pathophysiology and disease susceptibility in early life. Developing new strategies to reduce both therapeutic and prophylactic antibiotic use is urgent for the sustainable swine production globally. To this end, housing methodology providing effective environmental enrichment could be a promising alternative approach to reduce antibiotic usage, as it has been proven to positively influence pig welfare and immune status and reduce susceptibility to infections. It is, however, poorly understood how this enriched housing affects systemic and local pulmonary immune status and gut microbiota colonization during early life. In the present study, we compared the effects of two housing conditions, i.e., conventional housing: (CH) versus enriched housing (EH), on immune status and gut microbiota from birth until 61 days of age. RESULTS: The expected benefits of enrichment on pig welfare were confirmed as EH pigs showed more positive behaviour, less aggression behaviour during the weaning transition and better human animal relation during the post weaning phase. Regarding the pigs' immune status, EH pigs had higher values of haemoglobin and mean corpuscular volume in haematological profiles and higher percentages of T cells and cytotoxic T cells in peripheral blood. Furthermore, EH pigs showed higher ex vivo secretion of IL1ß and TNF-α after lipopolysaccharide stimulation of whole blood than CH pigs. The structure of the developing faecal microbiota of CH and EH pigs significantly differed as early as day 12 with an increase in the relative abundance of several bacterial groups known to be involved in the production of short chain fatty acids, such as Prevotella_2, Christensenellaceae_R_7_group and Ruminococcus gauvreauii group. Furthermore, the main difference between both housing conditions post weaning was that on day 61, CH pigs had significantly larger inter-individual variation of ileal and colonic microbiota than EH pigs. In addition to housing, other intrinsic factors (e.g., sex) were associated with gut microbiota development and immune competence. CONCLUSIONS: In addition to the known welfare benefits for pigs, environmentally enriched housing also positively drives important aspects of the development of the immune system and the establishment of gut microbiota in early life. Consequently, EH may contribute to increasing productivity of pigs and reducing antibiotic use.

Animal-Based Factors Prior to Infection Predict Histological Disease Outcome in Porcine Reproductive and Respiratory Syndrome Virus- and Actinobacillus pleuropneumoniae-Infected Pigs.

A large variety of clinical manifestation in individual pigs occurs after infection with pathogens involved in porcine respiratory disease complex (PRDC). Some pigs are less prone to develop respiratory disease symptoms. The variation in clinical impact after infection and the recovery capacity of an individual animal are measures of its resilience. In this paper, we examined which ones of a range of animal-based factors (rectal temperature, body weight, skin lesion scores, behavior, natural antibody serum levels, serum levels of white blood cells, and type of T and granulocyte subsets) when measured prior to infection are related to disease severity. These animal-based factors and the interaction with housing regimen of the piglets (conventional or enriched) were modeled using linear regression to predict disease severity using a dataset acquired from a previous study using a well-established experimental coinfection model of porcine reproductive and respiratory syndrome virus (PRRSV) and Actinobacillus pleuropneumoniae. Both PRRSV and A. pleuropneumoniae are often involved in PRDC. Histological lung lesion score of each animal was used as a measure for PRDC severity after infection. Prior to infection, higher serum levels of lymphocytes (CD3+), naïve T helper (CD3+CD4+CD8-), CD8+ (as well as higher relative levels of CD8+), and memory T helper (CD3+CD4+CD8+) cells and higher relative levels of granulocytes (CD172a) were related to reduced disease severity in both housing systems. Raised serum concentrations of natural IgM antibodies binding to keyhole limpet hemocyanin (KLH) were also related to reduced disease severity after infection. Increased levels of skin lesions at the central body part (after weaning and before infection) were related to increased disease severity in conventional housing systems only. High resisters showed a lower histological lung lesion score, which appeared unrelated to sex. Body temperature, behavior, and growth prior to infections were influenced by housing regimen but could not explain the variation in lung lesion scores after infection. Raised basal lymphocyte counts and lower skin lesion scores are related to reduced disease severity independent of or dependent on housing system, respectively. In conclusion, our study identifies intrinsic animal-based measures using linear regression analysis that predicts resilience to infections in pigs.

Immune responses induced by inactivated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in neonatal pigs using different adjuvants.

Vaccination of neonatal pigs could be supportive to prevent porcine reproductive and respiratory syndrome virus (PRRSV), which is an important porcine pathogen causing worldwide welfare and health problems in pigs of different age classes. However, neonatal immunity substantially differs to adults, thus different vaccines may be required in neonateal pigs. We examined if the immunogenicity and efficacy of inactivated PRRSV (iPRRSV) vaccines in neonatal pigs could be improved with adjuvants containing oil-in water (O/W) emulsions with or without Toll-like receptor (TLR) agonists and by altering the delivery route from intramuscular (i.m.) to the skin. Three-day-old PRRSV-naïve piglets (n = 54, divided in 6 groups) received a prime vaccination and a booster vaccination four weeks later. The vaccine formulations consisted of different O/W emulsions (Montanide™ ISA28RVG (ISA28)), a squalene in water emulsion (SWE) for i.m. or a Stable Emulsion (SE) with squalene for skin vaccination) and/or a mixture of TLR1/2, 7/8 and 9 agonists (TLRa) combined with iPRRSV strain 07V063. These vaccines were delivered either i.m. (ISA28, SWE, TLRa or SWE + TLRa) or into the skin (skiSE + TLRa) with dissolving microneedle (DMN)-patches. All animals received a challenge with homologous PRRSV three weeks after booster vaccination. Specific antibodies, IFN-γ production and viremia were measured at several time-points after vaccination and/or challenge, while lung pathology was studied at necropsy. After booster vaccination, only ISA28 induced a specific antibody response while a specific T-cell IFN-γ response was generated in the SWE group, that was lower for ISA28, and absent in the other groups. This suggests that prime vaccination in neonates induced a specific immune response after booster vaccination, dependent on the emulsion formulation, but not dependent on the presence of the TLRa or delivery route. Despite the measured immune responses none of the vaccines showed any efficacy. Further research focused on the early immune response in draining lymph nodes is needed to elucidate the potential of TLR agonists in vaccines for neonatal pigs.

Early immune responses in skin and lymph node after skin delivery of Toll-like receptor agonists in neonatal and adult pigs.

The skin is potentially an important vaccine delivery route facilitated by a high number of resident antigen presenting cells (APCs), which are known to be stimulated by different Toll-like receptor agonists (TLRa). In this study, neonatal and adult pigs were vaccinated in the skin using dissolving microneedle patches to investigate the immuno-stimulatory potential of different TLRa and possible age-dependent differences early after vaccination. These patches contained TLR1/2a (Pam3Cys), TLR7/8a (R848) or TLR9a (CpG ODN) combined with inactivated porcine reproductive and respiratory syndrome virus (PRRSV) or with an oil-in-water stable emulsion. Vaccinated skin and draining lymph nodes were analysed for immune response genes using microfluidic high-throughput qPCR to evaluate the early immune response and activation of APCs. Skin pathology and immunohistochemistry were used to evaluate the local immune responses and APCs in the vaccinated skin, respectively. In both neonatal and adult pigs, skin vaccination with TLR7/8a induced the most prominent early inflammatory and immune cell responses, particularly in the skin. Skin histopathology and immunohistochemistry of APCs showed comparable results for neonatal and adult pigs after vaccination with the different TLRa vaccines. However, in vaccinated neonatal pigs in the skin and draining lymph node more immune response related genes were upregulated compared to adult pigs. We showed that both neonatal and adult skin could be stimulated to develop an immune response, particularly after TLR7/8a vaccination, with age-dependent differences in regulation of immune genes. Therefore, age-dependent differences in local early immune responses should be considered when developing skin vaccines.

Predictive Value of Precision-Cut Lung Slices for the Susceptibility of Three Animal Species for SARS-CoV-2 and Validation in a Refined Hamster Model.

In assessing species susceptibility for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and in the search for an appropriate animal model, multiple research groups around the world inoculated a broad range of animal species using various SARS-CoV-2 strains, doses and administration routes. Although in silico analyses based on receptor binding and diverse in vitro cell cultures were valuable, exact prediction of species susceptibility based on these tools proved challenging. Here, we assessed whether precision-cut lung slices (PCLS) could facilitate the selection of animal models, thereby reducing animal experimentation. Pig, hamster and cat PCLS were incubated with SARS-CoV-2 and virus replication was followed over time. Virus replicated efficiently in PCLS from hamsters and cats, while no evidence of replication was obtained for pig PCLS. These data corroborate the findings of many research groups that have investigated the susceptibility of hamsters, pigs and cats towards infection with SARS-CoV-2. Our findings suggest that PCLS can be used as convenient tool for the screening of different animal species for sensitivity to newly emerged viruses. To validate our results obtained in PCLS, we employed the hamster model. Hamsters were inoculated with SARS-CoV-2 via the intranasal route. Susceptibility to infection was evaluated by body weight loss, viral loads in oropharyngeal swabs and respiratory tissues and lung pathology. The broadly used hamster model was further refined by including activity tracking of the hamsters by an activity wheel as a very robust and sensitive parameter for clinical health. In addition, to facilitate the quantification of pathology in the lungs, we devised a semi-quantitative scoring system for evaluating the degree of histological changes in the lungs. The inclusion of these additional parameters refined and enriched the hamster model, allowing for the generation of more data from a single experiment.

Age-dependent differences in the pathogenesis of bovine respiratory syncytial virus infections related to the development of natural immunocompetence.

The severity of respiratory syncytial virus (RSV) infections appears to differ with age in both humans and bovines. A primary RSV infection in naïve infants and in young calves runs a more severe course when they are 1-6 months old than in their first month of life. The relative lack of clinical signs in the first month of age may be due to high levels of maternally derived neutralizing antibodies or low exposure to infectious virus. This study examined whether age-dependent differences in the pathogenesis of bovine RSV (bRSV) between neonatal and young calves may be due to differences in age-dependent immunocompetence. To study the effect of age and immune parameters on bRSV disease in neonatal and young calves, neonatal (1-day-old) calves without maternally derived antibodies were infected experimentally with bRSV and the severity of disease and immune responses were evaluated in comparison with disease in similar 6-week-old infected calves. Neonatal calves had more extensive virus replication and lung consolidation, but lower pro-inflammatory [in particular tumour necrosis factor alpha (TNF-α)] responses, specific humoral immune responses, lung neutrophilic infiltration and clinical signs of disease than 6-week-old calves. The lack of correlation between virus replication and clinical signs suggests an important role of pro-inflammatory cytokines, in particular TNF-α, in the disease. The capacity to produce pro-inflammatory TNF-α appeared to increase with age, and may explain the age-dependent differences in RSV pathogenesis.