Constant Hepatitis E Virus (HEV) Circulation in Wild Boar and Red Deer in Spain: An Increasing Concern Source of HEV Zoonotic Transmission.

Hepatitis E is a viral zoonosis that affects multiple hosts. The complete dynamics of infection in wildlife are still unknown, but the previous fact facilitates the maintenance and circulation of the virus, posing a risk to human health in the case of meat consumption from susceptible animals. In Spain, it has been shown how domestic pigs, cattle and wildlife (i.e. wild boar and red deer) clearly interact in hunting farms, generating a complex epidemiological situation in terms of interspecies pathogen transmission. Therefore, in this study, we aimed to (i) evaluate the circulation of the virus in geographically close domestic (Iberian pigs) and wild animals (wild boar and deer) living in hunting areas from central Spain over an 8-year period (2003-2010) and (ii) to determine whether HEV could be used as a marker of domestic-wildlife contact. For these purposes, a longitudinal analysis of Iberian pig, wild boar and red deer samples (n = 287) through virological and serological tests was conducted to shed light upon the circulation events of HEV. Regarding HEV RNA detection by real-time RT-PCR, 10.12% samples (95% CI: 5.44-14.8) from wild boar and 16.05% samples (95% CI: 8.06-24.04) from red deer were positive. As for the Iberian pigs, none of the 48 samples was positive for HEV RNA detection. In the serological analysis, 43.75% (95% CI: 29.75-57.75) from Iberian pig, 57.40% (95% CI: 48.10-66.70) from wild boar and 12.85% (95% CI: 5.01-20.69) samples from red deer presented anti-HEV antibodies. Positive samples were distributed among all study years (2003-2010). These results depict the urgent need to improve the inspection and surveillance of these species and their products. In the case of HEV, it is clear that the stable and constant presence of the virus in wildlife and its contact with Iberian pigs pose a risk for human health as they are all destined for human consumption.

Natural Immunity of Sheep and Lambs Against the Schmallenberg Virus Infection.

Since the first reports of the Schmallenberg disease (SBD) outbreaks in late 2011, the disease has spread across Europe, affecting cattle and sheep farms. While Schmallenberg virus (SBV) causes a mild clinical disease in adults, infection of pregnant females may lead to the production of typical congenital malformations (CMFs) in their offspring. It is speculated that the immunity acquired after a SBV infection is effective in preventing further infections. However, this has not been proven in naturally infected sheep, especially if they are pregnant when reinfected. The aim of this study was to monitor the natural immunity in SBV-infected sheep. Twenty-four ewes from the only Spanish farm with a SBV OIE-notified outbreak were sampled. Subsequently, nine pregnant ewes were inoculated with SBV infectious plasma under controlled conditions. Six of them were euthanized before delivery, and their fetuses were inspected for lesions indicative for the SBV infection. The three remaining ewes were allowed to deliver one lamb each. Inoculation of the lambs was scheduled at approx. 3 months after birth. All samples were analyzed for viral RNA by RT-PCR, and for antibodies by an indirect ELISA and a virus neutralization test (VNT). The majority of the 24 ewes showed a serological reaction against SBV. The three ewes that were allowed to lamb down demonstrated variable degrees of seroconversion which corresponded to the levels of immune reaction observed in their lambs. Moreover, no viral RNA was detected, no lesions were observed in the fetuses, and no clinical signs were detected in the inoculated animals. These findings suggest that the immunity acquired by sheep following a natural SBV infection could be sufficient to stop SBV reinfection. However, vaccination could be a valuable tool to control SBV infections and associated economic losses as it affords a more uniform and predictable protection at the flock/herd level.

Development of a Microsphere-based Immunoassay for Serological Detection of African Horse Sickness Virus and Comparison with Other Diagnostic Techniques.

African horse sickness (AHS) is a viral disease that causes high morbidity and mortality rates in susceptible Equidae and therefore significant economic losses. More rapid, sensitive and specific assays are required by diagnostic laboratories to support effective surveillance programmes. A novel microsphere-based immunoassay (Luminex assay) in which beads are coated with recombinant AHS virus (AHSV) structural protein 7 (VP7) has been developed for serological detection of antibodies against VP7 of any AHSV serotype. The performance of this assay was compared with that of a commercial enzyme-linked immunosorbent assay (ELISA) and commercial lateral flow assay (LFA) on a large panel of serum samples from uninfected horses (n = 92), from a reference library of all AHSV serotypes (n = 9), on samples from horses experimentally infected with AHSV (n = 114), and on samples from West African horses suspected of having AHS (n = 85). The Luminex assay gave the same negative results as ELISA when used to test the samples from uninfected horses. Both assays detected antibodies to all nine AHSV serotypes. In contrast, the Luminex assay detected a higher rate of anti-VP7 positivity in the West African field samples than did ELISA or LFA. The Luminex assay detected anti-VP7 positivity in experimentally infected horses at 7 days post-infection, compared to 13 days for ELISA. This novel immunoassay provides a platform for developing multiplex assays, in which the presence of antibodies against multiple ASHV antigens can be detected simultaneously. This would be useful for serotyping or for differentiating infected from vaccinated animals.

Development and evaluation of a SYBR Green real-time RT-PCR assay for evaluation of cytokine gene expression in horse.

Cytokine secretion is one of the main mechanisms by which the immune system is regulated in response to pathogens. Therefore, the measurement of cytokine expression is fundamental to characterizing the immune response to infections. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) is widely used to measure cytokine mRNA levels, but assay conditions should be properly evaluated before analyzing important equine infections through relative quantification of gene expression. The aim of this study was to develop and evaluate a set of RT-qPCR assays for a panel of the most common cytokines in horses involved in innate and adaptive immune responses. Eight cytokines (interleukin (IL)-1ß, IL-2, IL-4, IL-10, IL-12, TNFα, IFNß and IFNγ) and a housekeeping gene (ß-actin) were detected and amplified with the same annealing temperature in a SYBR Green RT-qPCR assay of samples of mitogen-stimulated peripheral blood mononuclear cells from a healthy horse and whole blood from a horse infected with African horse sickness virus. The method gave good efficiency for all genes tested, allowing quantification of relative expression levels. These SYBR Green RT-qPCR assays may be useful for examining cytokine gene expression in horses in response to exposure to economically important pathogens.

The effects of acute and chronic alcohol ingestion on outcome following multiple episodes of mild traumatic brain injury in rats.

OBJECTIVE: Recent studies suggest that in some circumstances, alcohol intoxication at the time of severe head injury may be neuroprotective. The objective of this study was to determine the effect of acute and chronic alcohol ingestion on outcome in rodents sustaining multiple episodes of mild traumatic brain injury while intoxicated. METHOD: For two weeks before experimentation, adult male Sprague-Dawley rats received intoxicating levels of 95% ethanol (3 g/kg) or normal saline (NS) every other day by orogastric instillation. On the day of experimentation, the animals were randomized to receive alcohol or NS. Two hours later, the animals received either mild (1.2 +/- 0.4 ATA) fluid percussion injury (FPI) or no injury. The injured animals received a total of three episodes of FPI (once every four days). Mean reflex recovery time (RRT) was determined (seconds +/- SEM) immediately after each episode. Mean latency time (seconds +/- SEM) for Morris Water Maze (MWM) performance was assessed at post-trauma days 11-19. RESULTS: The chronic alcohol-exposed (CA) and the non-alcohol-exposed (NA) animals intoxicated when injured had prolonged escape, righting, and corneal RRTs after each FPI compared with the nonintoxicated injured animals and the non-injured shams. However, the CA animals had significantly shorter RRTs when compared with the NA rats. All the injured animals had MWM deficits on testing days 1-6 compared with the noninjured controls. On the last two MWM testing days, the injured NA animals had significantly better MWM performance than the injured CA rats. CONCLUSIONS: The injured intoxicated CA animals had a more rapid recovery of reflexes compared with the injured intoxicated NA animals. Despite initial MWM deficits, the injured NA rodents eventually began to learn the MWM. The injured CA rats never learned the maze. Under the conditions of this study, acute alcohol intoxication at the time of multiple episodes of minor head trauma did not provide neuroprotection for NA or CA rodents.

Lactose digestion from yogurt: influence of a meal and additional lactose.

Lactose in yogurt is better digested than lactose in other dairy foods by lactase-deficient individuals, in part because of intraintestinal activity of yogurt microbial beta-galactosidase (beta-gal). The survival and activity of yogurt beta-gal depend on gastrointestinal transit, pH, and viability of the yogurt culture. To evaluate the ability of yogurt beta-gal to digest lactose when yogurt is consumed with food or with additional lactose, 22 healthy lactose-maldigesting individuals were fed 10 test meals. Results of breath-hydrogen expiration, incidence of symptoms, and enzyme and lactose content of gastric aspirates indicate that the consumption of a meal with yogurt does not inhibit, and may slightly improve, lactose digestion from yogurt. However, yogurt beta-gal appears unable to assist in the digestion of additional lactose beyond that normally present in yogurt.