Resistive-Based Micro-Kelvin Temperature Resolution for Ultra-Stable Space Experiments.

High precision temperature measurements are a transversal need in a wide area of physical experiments. Space-borne gravitational wave detectors are a particularly challenging case, requiring both high precision and high stability in temperature measurement. In this contribution, we present a design able to reach 1 µK/Hz in most of the measuring band down to 1 mHz, and reaching 20 µK/Hz at 0.1 mHz. The scheme is based on resistive sensors in a Wheatstone bridge configuration which is AC modulated to minimize the 1/f noise. As a part of our study, we include the design of a test bench able to guarantee the high stability environment required for measurements. We show experimental results characterising both the test bench and the read-out, and discuss potential noise sources that may limit our measurement.

Pathobiology of the highly pathogenic avian influenza viruses H7N1 and H5N8 in different chicken breeds and role of Mx 2032 G/A polymorphism in infection outcome.

Chickens are highly susceptible to highly pathogenic avian influenza viruses (HPAIVs). However, the severity of infection varies depending of the viral strain and the genetic background of the host. In this study, we evaluated the pathogenesis of two HPAIVs (H7N1 and H5N8) and assessed the susceptibility to the infection of local and commercial chicken breeds from Spain. Eight chicken breeds were intranasally inoculated with 105 ELD50 of A/Chicken/Italy/5093/1999 (H7N1) or A/Goose/Spain/IA17CR02699/2017 (H5N8 clade 2.3.4.4. B) and monitored during 10 days. Chickens were highly susceptible to both HPAIVs, but H7N1 was considerably more virulent than H5N8 as demonstrated by the highest mortality rates and shortest mean death times (MDT). Both HPAIVs produced severe necrosis and intense viral replication in the central nervous system, heart and pancreas; however, the lesions and replication in other tissues were virus-dependent. High levels of viral RNA were detected by the oral route with both viruses. In contrast, a low number of H5N8-inoculated chickens shed by the cloacal route, demonstrating a different pattern of viral shedding dependent of the HPAIV. We found a high variation in the susceptibility to HPAIVs between the different chicken breeds. The birds carrying the genotype AA and AG at position 2032 in chicken Mx gene presented a slightly higher, but not significant, percentage of survival and a statistically significant longer MDT than GG individuals. Our study demonstrated that the severity of HPAI infection is largely dependent of the viral isolate and host factors, underlining the complexity of HPAI infections.

Retrospective study on transmissible viral proventriculitis and chicken proventricular necrosis virus (CPNV) in the UK.

Chicken proventricular necrosis virus (CPNV) is a recently described birnavirus, which has been proposed to be the cause of transmissible viral proventriculitis (TVP). The understanding of the epidemiology of both the virus and the disease is very limited. A retrospective investigation on TVP and CPNV in broiler chicken submissions from the UK from between 1994 and 2015 was performed with the aims of assessing the longitudinal temporal evolution of TVP and CPNV, and to review the histological proventricular lesions in the studied chickens. Ninety-nine of the 135 included submissions (73.3%) fulfilled the TVP-diagnostic criteria, while the remaining 36 submissions (26.7%) displayed only lymphocytic proventriculitis (LP). The first detection of CPNV by PCR dated from 2009. Results showed a rise in the number of both TVP and positive CPNV RT-PCR submissions from 2009 with a peak in 2013, suggesting that they may be an emerging or re-emerging disease and pathogen, respectively. Twenty-two out of the 99 submissions displaying TVP lesions (22%) and four out of the 36 (11%) submissions with LP gave positive CPNV RT-PCR results, further supporting the association between CPNV and TVP and confirming that CPNV is present in a low proportion of proventriculi that do not fulfil the TVP-diagnostic criteria. In addition, intranuclear inclusion bodies were observed in 22 of the submissions with TVP. The vast majority of these cases (21 of 22, 96%) gave negative CPNV RT-PCR results, raising the question of whether a virus other than CPNV is responsible for some of these TVP-affected cases.RESEARCH HIGHLIGHTSTVP and CPNV have been present in British broilers since at least 1994 and 2009, respectively.TVP and CPNV seem to be an emerging and re-emerging disease and pathogen, respectively.CPNV was detected in proventriculi with both TVP and LP-lesions.Viruses other than CPNV may be responsible for some TVP-affected cases.

Detection of transmissible viral proventriculitis and chicken proventricular necrosis virus in the UK.

Increasing evidence suggests that a new birnavirus, named chicken proventricular necrosis virus (CPNV), is the aetiological agent of transmissible viral proventriculitis (TVP). The present work aimed to explore the possible presence of both TVP and CPNV in the UK. Forty-four chickens showing TVP-compatible gross lesions were classified into three groups based on the histological lesions: (i) TVP-affected chickens: lymphocytic infiltration and glandular necrosis (n = 15); (ii) lymphocytic proventriculitis (LP)-affected chickens: lymphocytic infiltration without necrosis (n = 18); and (iii) without proventriculitis (WP): no lymphocytic infiltration or necrosis (n = 11). Nine proventriculi (seven out of 15 corresponding to TVP, and two out of 11 corresponding to LP) were positive for CPNV by reverse transcriptase polymerase chain reaction (RT-PCR). These results support the previously suggested idea of CPNV as causative agent of TVP. Moreover, these data show that CPNV can also be detected in a number of cases with LP, which do not fulfil the histological TVP criteria. Phylogenetic analysis of partial sequences of gene VP1 showed that British CPNV sequences were closer to other European CPNV sequences and might constitute a different lineage from the American CPNV. TVP cases with negative CPNV PCR results may be due to chronic stages of the disease or to the reduced PCR sensitivity on formalin-fixed paraffin-embedded tissues. However, involvement of other agents in some of the cases cannot totally be ruled out. As far as the authors are aware, this is the first peer-reviewed report of TVP as well as of CPNV in the UK, and the first exploratory CPNV phylogenetic study.

Bile acids induce glucagon-like peptide 2 secretion with limited effects on intestinal adaptation in early weaned pigs.

Early weaning is a stressful event characterized by a transient period of intestinal atrophy that may be mediated by reduced secretion of glucagon-like peptide (GLP) 2. We tested whether enterally fed bile acids or plant sterols could increase nutrient-dependent GLP-2 secretion and improve intestinal adaptation in weanling pigs. During the first 6 d after weaning, piglets were intragastrically infused once daily with either deionized water (control), chenodeoxycholic acid (CDC; 60 mg/kg body weight), or ß-sitoesterol (BSE; 100 mg/kg body weight). Infusing CDC increased plasma GLP-2 (P < 0.05) but did not affect plasma GLP-1 and feed intake. The intestinal expression of glucagon-like peptide 2 receptor, sodium-dependent bile acid transporter, farnesoid X receptor, and guanosine protein-coupled bile acid receptor genes were not affected by CDC treatment. The intragastric administration of CDC did not alter the weight and length of the intestine, yet increased the activation of caspase-3 in ileal villi (P < 0.02) and the expression of interleukin 6 (P < 0.002) in the jejunum. In contrast, infusing BSE did not affect any of the variables that were measured. Our results show that the enteral administration of the bile acid CDC potentiates the nutrient-induced secretion of endogenous GLP-2 in early-weaned pigs. Bile acid-enhanced release of GLP-2, however, did not result in improved intestinal growth, morphology, or inflammation during the postweaning degenerative phase.

Casein glycomacropeptide in the diet may reduce Escherichia coli attachment to the intestinal mucosa and increase the intestinal lactobacilli of early weaned piglets after an enterotoxigenic E. coli K88 challenge.

Casein glycomacropeptide (CGMP), a glycoprotein originating during cheese manufacture, has shown promising effects by promoting the growth of some beneficial bacteria in vitro, although its activity has not been well explored. The present study was designed to evaluate the effects of CGMP against enterotoxigenic Escherichia coli (ETEC) K88 in vitro (Trial 1) and in vivo (Trial 2). In Trial 1, increasing concentrations of CGMP (0, 0.5, 1.5 or 2.5 mg/ml) were tested regarding its ability to block the attachment of ETEC K88 to ileal mucosa tissues obtained from piglets. Increasing the concentration of CGMP resulted in a gradual decrease in ETEC K88 attachment to the epithelial surface. In Trial 2, seventy-two piglets were distributed in a 2 × 2 factorial combination including or omitting CGMP in the diet (control diet v. CGMP) and challenged or not with ETEC K88 (yes v. no). Inclusion of CGMP increased crude protein, ammonia and isoacid concentrations in colon digesta. CGMP also increased lactobacilli numbers in ileum and colon digesta, and reduced enterobacteria counts in mucosa scrapings and the percentage of villi with E. coli adherence measured by fluorescence in situ hybridisation. The inclusion of CGMP in the diets of challenged animals also prevented the increase of enterobacteria in ileal digesta. We can conclude that CGMP may improve gut health by diminishing the adhesion of ETEC K88 to the intestinal mucosa, by increasing the lactobacilli population in the intestine and by reducing the overgrowth of enterobacteria in the digestive tract of piglets after an ETEC K88 challenge.

Persistence of low pathogenic avian influenza virus in artificial streams mimicking natural conditions of waterfowl habitats in the Mediterranean climate.

Avian influenza viruses (AIVs) can affect wildlife, poultry, and humans, so a One Health perspective is needed to optimize mitigation strategies. Migratory waterfowl globally spread AIVs over long distances. Therefore, the study of AIV persistence in waterfowl staging and breeding areas is key to understanding their transmission dynamics and optimizing management strategies. Here, we used artificial streams mimicking natural conditions of waterfowl habitats in the Mediterranean climate (day/night cycles of photosynthetic active radiation and temperature, low water velocity, and similar microbiome to lowland rivers and stagnant water bodies) and then manipulated temperature and sediment presence (i.e., 10-13 °C vs. 16-18 °C, and presence vs. absence of sediments). An H1N1 low pathogenic AIV (LPAIV) strain was spiked in the streams, and water and sediment samples were collected at different time points until 14 days post-spike to quantify viral RNA and detect infectious particles. Viral RNA was detected until the end of the experiment in both water and sediment samples. In water samples, we observed a significant combined effect of temperature and sediments in viral decay, with higher viral genome loads in colder streams without sediments. In sediment samples, we didn't observe any significant effect of temperature. In contrast to prior laboratory-controlled studies that detect longer persistence times, infectious H1N1 LPAIV was isolated in water samples till 2 days post-spike, and none beyond. Infectious H1N1 LPAIV wasn't isolated from any sediment sample. Our results suggest that slow flowing freshwater surface waters may provide conditions facilitating bird-to-bird transmission for a short period when water temperature are between 10 and 18 °C, though persistence for extended periods (e.g., weeks or months) may be less likely. We hypothesize that experiments simulating real environments, like the one described here, provide a more realistic approach for assessing environmental persistence of AIVs.

Effects of vaccination against paratuberculosis on tuberculosis in goats: diagnostic interferences and cross-protection.

BACKGROUND: Most countries carrying out campaigns of bovine tuberculosis (TB) eradication impose a ban on the use of mycobacterial vaccines in cattle. However, vaccination against paratuberculosis (PTB) in goats is often allowed even when its effect on TB diagnosis has not been fully evaluated. To address this issue, goat kids previously vaccinated against PTB were experimentally infected with TB. RESULTS: Evaluation of interferon-γ (IFN-γ) secretion induced by avian and bovine tuberculins (PPD) showed a predominant avian PPD-biased response in the vaccinated group from week 4 post-vaccination onward. Although 60% of the animals were bovine reactors at week 14, avian PPD-biased responses returned at week 16. After challenge with M. caprae, the IFN-γ responses radically changed to show predominant bovine PPD-biased responses from week 18 onward. In addition, cross-reactions with bovine PPD that had been observed in the vaccinated group at week 14 were reduced when using the M. tuberculosis complex-specific antigens ESAT-6/CFP-10 and Rv3615c as new DIVA (differentiation of infected and vaccinated animals) reagents, which further maintained sensitivity post-challenge. Ninety percent of the animals reacted positively to the tuberculin cervical comparative intradermal test performed at 12 weeks post-infection. Furthermore, post-mortem analysis showed reductions in tuberculous lesions and bacterial burden in some vaccinated animals, particularly expressed in terms of the degree of extrapulmonary dissemination of TB infection. CONCLUSIONS: Our results suggest a degree of interference of PTB vaccination with current TB diagnostics that can be fully mitigated when using new DIVA reagents. A partial protective effect associated with vaccination was also observed in some vaccinated animals.

Exploratory study on the influence of climatological parameters on Mycoplasma hyopneumoniae infection dynamics.

The objective of the present work was to elucidate the potential relationship between Mycoplasma hyopneumoniae infection and seroconversion dynamics and climatological conditions in four groups of pigs from the same farm born in different seasons of the year. Nasal swabs and blood samples were taken from 184 pigs at 1, 3, 6, 9, 12, 15, 18, 22 and 25 (slaughter age) weeks of age. Outside climatologic parameters, including temperature (°C), relative humidity (%), precipitation (l/m(2)) and wind speed (m/s) were recorded weekly from January 2003 to June 2004. Percentage of nPCR detection of M. hyopneumoniae in nasal swabs was associated significantly with the weekly precipitation rate [P = 0.0018, OR = 1.31 (IC = 1.11-1.55)]; the higher the precipitation rate, the higher the probability of being M. hyopneumoniae nPCR-positive. On the other hand, the percentage of seropositive pigs had a significant association with mean weekly temperature rate [P = 0.0012, OR = 0.89 [IC = 0.84-0.95]); the lower the temperature, the higher the probability of being M. hyopneumoniae seropositive. Animals born in autumn (when higher precipitations rates were recorded), entering finishing units in winter (when lower temperatures were recorded), and reaching slaughter in spring, had the highest probability of being infected by M. hyopneumoniae and the highest probability of being M. hyopneumoniae seropositive.

Corrigendum: Dual Host and Pathogen RNA-Seq Analysis Unravels Chicken Genes Potentially Involved in Resistance to Highly Pathogenic Avian Influenza Virus Infection.

[This corrects the article DOI: 10.3389/fimmu.2021.800188.].

A case of uterine inclusion cysts in a sow.

BACKGROUND: Serosal inclusion cysts are thin walled-structures located on the peritoneal surface of the uterus, frequently observed as multiple cystic structures in aggregates or grape-like clusters containing a clear, non-viscous fluid. In human and veterinary medicine, they are thought to be developed under hormonal effects, or after manipulation or inflammation of the reproductive tract. However, they have not yet been described in swine. CASE PRESENTATION: A uterus of a 3-year-old crossbreed sow was condemned at slaughter due to the presence of multiples cystic cavities attached to the serosal surface. Microscopically, multiple cystic dilations emerging from the serosa were lined by a simple and flattened epithelium (cytokeratine positive and vimentin negative on immunohistochemistry) supported by a subepithelial layer of collagen. Grossly and histologically, they were diagnosed as serosal inclusion cysts. CONCLUSION: To the authors' knowledge, this report represents the first description of serosal inclusion cysts in sows. These lesions should be taken into consideration within the differential diagnostic list of cystic peritoneal lesions such as cystic neoplasms, congenital cysts, and parasitic diseases.

A 10-Year Retrospective Study of Inclusion Body Hepatitis in Meat-Type Chickens in Spain (2011-2021).

A surge in fowl adenovirus (FAdV) causing inclusion body hepatitis (IBH) outbreaks has occurred in several countries in the last two decades. In Spain, a sharp increase in case numbers in broilers and broiler breeder pullets arose since 2011, which prompted the vaccination of breeders in some regions. Our retrospective study of IBH cases in Spain from 2011 to 2021 revealed that most cases were reported in broilers (92.21%) and were caused by serotypes FAdV-8b and -11, while cases in broiler breeder pullets were caused by serotypes FAdV-2, -11, and -8b. Vertical transmission was the main route of infection, although horizontal transmission likely happened in some broiler cases. Despite the inconsistent and heterogeneous use of vaccines among regions and over time, the number of cases mirrored the use of vaccines in the country. While IBH outbreaks were recorded year-long, significantly more cases occurred during the cooler and rainier months. The geographic distribution suggested a widespread incidence of IBH and revealed the importance of a highly integrated system. Our findings contribute to a better understanding of FAdV infection dynamics under field conditions and reiterate the importance of surveillance, serological monitoring of breeders, and vaccination of breeders against circulating serotypes to protect progenies.

Dual Host and Pathogen RNA-Seq Analysis Unravels Chicken Genes Potentially Involved in Resistance to Highly Pathogenic Avian Influenza Virus Infection.

Highly pathogenic avian influenza viruses (HPAIVs) cause severe systemic disease and high mortality rates in chickens, leading to a huge economic impact in the poultry sector. However, some chickens are resistant to the disease. This study aimed at evaluating the mechanisms behind HPAIV disease resistance. Chickens of different breeds were challenged with H7N1 HPAIV or clade 2.3.4.4b H5N8 HPAIV, euthanized at 3 days post-inoculation (dpi), and classified as resistant or susceptible depending on the following criteria: chickens that presented i) clinical signs, ii) histopathological lesions, and iii) presence of HPAIV antigen in tissues were classified as susceptible, while chickens lacking all these criteria were classified as resistant. Once classified, we performed RNA-Seq from lung and spleen samples in order to compare the transcriptomic signatures between resistant and susceptible chickens. We identified minor transcriptomic changes in resistant chickens in contrast with huge alterations observed in susceptible chickens. Interestingly, six differentially expressed genes were downregulated in resistant birds and upregulated in susceptible birds. Some of these genes belong to the NF-kappa B and/or mitogen-activated protein kinase signaling pathways. Among these six genes, the serine protease-encoding gene PLAU was of particular interest, being the most significantly downregulated gene in resistant chickens. Expression levels of this protease were further validated by RT-qPCR in a larger number of experimentally infected chickens. Furthermore, HPAIV quasi-species populations were constructed using 3 dpi oral swabs. No substantial changes were found in the viral segments that interact with the innate immune response and with the host cell receptors, reinforcing the role of the immune system of the host in the clinical outcome. Altogether, our results suggest that an early inactivation of important host genes could prevent an exaggerated immune response and/or viral replication, conferring resistance to HPAIV in chickens.

Increasing breast milk betaine modulates Akkermansia abundance in mammalian neonates and improves long-term metabolic health.

Accelerated postnatal growth is a potentially modifiable risk factor for future obesity. To study how specific breast milk components contribute to early growth and obesity risk, we quantified one-carbon metabolism-related metabolites in human breast milk and found an inverse association between milk betaine content and infant growth. This association was replicated in an independent and geographically distinct cohort. To determine the potential role of milk betaine in modulating offspring obesity risk, we performed maternal betaine supplementation experiments in mice. Higher betaine intake during lactation increased milk betaine content in dams and led to lower adiposity and improved glucose homeostasis throughout adulthood in mouse offspring. These effects were accompanied by a transient increase in Akkermansia spp. abundance in the gut during early life and a long-lasting increase in intestinal goblet cell number. The link between breast milk betaine and Akkermansia abundance in the gut was also observed in humans, as infants exposed to higher milk betaine content during breastfeeding showed higher fecal Akkermansia muciniphila abundance. Furthermore, administration of A. muciniphila to mouse pups during the lactation period partially replicated the effects of maternal breast milk betaine, including increased intestinal goblet cell number, lower adiposity, and improved glucose homeostasis during adulthood. These data demonstrate a link between breast milk betaine content and long-term metabolic health of offspring.

Coupling characterization and noise studies of the optical metrology system onboard the LISA Pathfinder mission.

We describe the first investigations of the complete engineering model of the optical metrology system (OMS), a key subsystem of the LISA Pathfinder science mission to space. The latter itself is a technological precursor mission to LISA, a spaceborne gravitational wave detector. At its core, the OMS consists of four heterodyne Mach-Zehnder interferometers, a highly stable laser with an external modulator, and a phase meter. It is designed to monitor and track the longitudinal motion and attitude of two floating test masses in the optical reference frame with (relative) precision in the picometer and nanorad range, respectively. We analyze sensor signal correlations and determine a physical sensor noise limit. The coupling parameters between motional degrees of freedom and interferometer signals are analytically derived and compared to measurements. We also measure adverse cross-coupling effects originating from system imperfections and limitations and describe algorithmic mitigation techniques to overcome some of them. Their impact on system performance is analyzed within the context of the Pathfinder mission.

Evaluation of dietary supplementation of a novel microbial muramidase on gastrointestinal functionality and growth performance in broiler chickens.

This study was conducted to assess the effect of dietary supplementation of Muramidase 007 to broiler chickens on gastrointestinal functionality, evaluating growth performance, apparent ileal digestibility, intestinal histomorphology, vitamin A in plasma and cecal microbiota. A total of 480 one-day male chicks (Ross 308) were distributed in 16 pens allocated in 2 experimental diets: the control diet (CTR) without feed enzymes, coccidiostat or growth promoters, and the experimental diet (MUR): CTR supplemented with 35,000 units (LSU(F))/kg of the Muramidase 007. Digesta and tissue samples were obtained on days 9 and 36 of the study. A lower feed conversion ratio was observed in the MUR treatment. Apparent ileal digestibility of DM, organic matter and energy were improved by Muramidase 007. It was also observed that MUR improved digestibility of total fatty acids, mono-unsaturated fatty acids and poly-unsaturated fatty acids, and content of vitamin A in plasma at day 9 (P < 0.05). Histomorphological analysis of jejunum samples revealed no differences in the villus height or crypt depth; but a higher number of goblet cells and intraepithelial lymphocytes at day 36 with MUR. No differences were observed in plate counts of enterobacteria or Lactobacillus along the gastrointestinal tract, neither on the cecal short-chain fatty acids. An statistical trend was observed for reduction of cecal clostridia at day 9 for MUR. Analysis of cecal microbiota structure by 16S rRNA gene sequencing revealed relevant changes correlated to age. At day 9, broilers receiving MUR showed decreased alpha diversity compared to CTR that was not detected at day 36. Changes in specific taxonomic groups with an increase in Lactobacillus genus were identified. In conclusion, evaluation of the variables in this study indicates that dietary Muramidase 007 contributes to improve feed conversation ratio and gastrointestinal function in broiler chickens. Effects could have been mediated by slight shifts observed in the intestinal microbiota. More studies are guaranteed to fully understand the mechanisms involved.

Infection, excretion and seroconversion dynamics of porcine circovirus type 2 (PCV2) in pigs from post-weaning multisystemic wasting syndrome (PMWS) affected farms in Spain and Denmark.

Longitudinal case-control studies were performed in post-weaning multisystemic wasting syndrome (PMWS) affected farms from Denmark and Spain using similar designs. Fourteen independent batches of 100-154 pigs per batch were monitored from birth to PMWS outbreak occurrence. Pigs displaying PMWS-like signs and matched healthy cohorts were euthanized during the clinical outbreak. PMWS was diagnosed according to internationally accepted criteria and pigs were classified as: (i) PMWS cases, (ii) wasted non-PMWS cases and (iii) healthy pigs. Porcine circovirus type 2 (PCV2) quantitative PCR (qPCR) and serology techniques were applied to analyse longitudinally collected sera and/or nasal and rectal swabs. Results showed that PCV2 load increased in parallel to waning maternal antibody levels, reaching the maximum viral load concurrent with development of clinical signs. PMWS affected pigs had higher PCV2 prevalence and/or viral load than healthy pigs in all collected samples at necropsy (p<0.0001-0.05) and even in sera and nasal swabs at the sampling prior to PMWS outbreak (p<0.01-0.05). Danish farms had a higher PCV2 prevalence in young piglets as well as an earlier PMWS presentation compared to Spanish farms. PMWS diagnoses were confirmed by laboratory tests in only half of pigs clinically suspected to suffer from PMWS. Positive and significant correlations were found among PCV2 viral loads present in sera, nasal swabs, rectal swabs and lymphoid tissues (R=0.289-0.827, p<0.0001-0.01), which indicates that nasal and rectal swabs were suitable indicators of PCV2 excretion. Sensitivity and/or specificity values observed from both tests used separately or combined suggested that qPCR and/or serology tests are not apparently able to substitute histopathology plus detection of PCV2 in tissues for the individual PMWS diagnosis within PMWS affected farms. However, qPCR appears to be a potential reliable technique to diagnose PMWS on a population basis.

Development of cell-mediated immunity to porcine circovirus type 2 (PCV2) in caesarean-derived, colostrum-deprived piglets.

The interaction between porcine circovirus type 2 (PCV2) and the pig immune system has been suggested to be a determinant event for the pathogenesis of postweaning multisystemic wasting syndrome (PMWS). To gain insight into the host immune mechanisms developed upon PCV2 infection, early innate and adaptive immune responses were examined in 1-week-old, caesarean-derived, colostrum-deprived piglets using a subclinical infection model of PCV2 in combination with lipopolysaccharide (LPS) as a potential immunostimulation factor. The use of LPS did not show any significant effect on the course of PCV2 infection, nor did in the evolution of the immunological parameters evaluated. Ex vivo responses were detected as early as 1 day post-infection (PI) and consisted of an elevation of the plasmatic levels of interleukin (IL)-8 in PCV2-inoculated pigs followed by an increase on plasmatic IFN-alpha at day 5 PI. Regarding IL-10, only one PCV2-inoculated pig was positive (day 7 PI); this pig was the only one in which viremia persisted until the end of the study. In vitro cytokine determination showed that, regardless of the treatment administrated to the pigs, an IL-10 release was observed when peripheral blood mononuclear cells (PBMC) cultures were stimulated with PCV2. Seroconvertion to PCV2 measured by an immunoperoxidase monolayer assay (IPMA) occurred between 7 and 14 days PI, whereas neutralizing antibodies (NA) did not appear until day 29 PI. PCV2 DNA was first detected in serum at day 7 PI, reaching the peak of viremia between days 14 and 21 PI, followed by a drop in viral load that was found coincident with the appearance of PCV2-specific IFN-gamma-secreting cells (PCV2-IFN-gamma-SC) and NA. Results from the present work suggest that viral clearance might be mediated by the development of PCV2-IFN-gamma-SC in contribution to the PCV2-specific NA.

Development of two Trichoplusia ni larvae-derived ELISAs for the detection of antibodies against replicase and capsid proteins of porcine circovirus type 2 in domestic pigs.

The main aim of the present study was to describe new methods for the identification of antibodies against the PCV2 capsid (Cap) and replicase (Rep) proteins in pig sera. Specifically, two new indirect enzyme-linked immunosorbent assays (ELISA) were developed based on recombinant PCV2 Cap (rCap) and Rep/Rep' (rRep) proteins expressed in baculovirus and produced in Trichoplusia ni insect larvae. Both assays were validated by testing serum samples in a longitudinal study of 107 animals with different clinico-pathological features of PCV2 infection: pigs with postweaning multisystemic wasting syndrome (PMWS), wasted pigs without a diagnosis of PMWS and healthy animals. Longitudinal antibody profiles indicated that healthy animals had significantly higher anti-Cap and anti-Rep antibody levels than the rest of the animal groups at 11 weeks of age. Moreover, PMWS affected pigs could be distinguished from the rest of the pig groups by their lower anti-Rep antibody levels at 11 weeks of age and at necropsy. The results demonstrate the potential of these two ELISAs for large-scale serological studies. This study represents the first longitudinal study of the induction of anti-Cap and anti-Rep antibodies in farms affected by PMWS, from 1 week of age until the occurrence of disease.

Hepatitis E virus infection dynamics and organic distribution in naturally infected pigs in a farrow-to-finish farm.

The objective of the present study was to determine the pattern of Hepatitis E virus (HEV) infection in a naturally infected, farrow-to-finish herd. For that purpose, a prospective study was conducted in randomly selected 19 sows and 45 piglets. Blood samples were collected from sows at 1 week post-farrowing and from piglets at 1, 3, 6, 9, 12, 15, 18 and 22 weeks of age. Furthermore 3 or 5 animals were necropsied at each bleeding day (but at 1 week of age), and serum, bile, liver, mesenteric lymph nodes and faeces taken. HEV IgG, IgM and IgA antibodies were determined in serum and viral RNA was analysed in all collected samples by semi-nested RT-PCR. Histopathological examination of mesenteric lymph nodes and liver was also conducted. From 13 analysed sows, 10 (76.9%) were positive to IgG, one to IgA (7.7%) and two to IgM (15.4%) antibodies specific to HEV. In piglets, IgG and IgA maternal antibodies lasted until 9 and 3 weeks of age, respectively. IgG seroconversion occurred by 15 weeks of age while IgM and IgA at 12. On individual basis, IgG was detectable until the end of the study while IgM and IgA antibody duration was of 4-7 weeks. HEV RNA was detected in serum at all analysed ages with the highest prevalence at 15 weeks of age. HEV was detected in faeces and lymph nodes for the first time at 9 weeks of age and peaked at 12 and 15 weeks of age. This peak coincided with the occurrence of hepatitis as well as with HEV detection in bile, liver, mesenteric lymph nodes and faeces, and also with highest IgG and IgM OD values at 15 weeks. Finally, different HEV sequences from this farm were obtained, which they clustered within 3 different groups, together with other Spanish sequences, all of them of genotype 3. Moreover, the present study also indicates that the same pig can be infected with at least two different strains of HEV during its productive life. This is the first study characterizing HEV infection in naturally infected pigs with chronological virus detection and its relationship with tissue lesions throughout the productive life of the animals.