Elisabetta Razzuoli

Metabolic stress and inflammatory response in high-yielding, periparturient dairy cows

Increased disease rates are commonly reported among high-yielding dairy cows in the transition period, extending from 3 weeks before to 3 weeks after calving, and characterized by the occurrence of an inflammatory response in terms of both positive and negative acute phase proteins (APP+ and APP-). To determine the above inflammatory response, the authors had developed the Liver Functionality Index (LFI), which defines the above condition on the basis of some APP- responses (albumin, cholesterol sensu stricto+bilirubin) during the first month of lactation. In this respect, low LFI values are associated to a high inflammatory response and vice versa. The relationship between LFI and inflammatory cytokine response was investigated from day -28 to day +28 with respect to calving in 12 periparturient dairy cows showing the six highest and six lowest LFI values within a cohort of 54 high-yielding dairy cows. The hypothesis being tested was that LFI and APP- on the whole could be used as readout of successful vs. non-successful adaptation to the transition period, with a strong association to disease occurrence. In fact, low LFI cows experienced many more disease cases (13 vs. 3 in high LFI Group) and related drug treatments till day +28. Interleukin-6 (IL-6) serum concentrations were always higher in low LFI cows (P<0.05 on day +28). The greater IL-6 levels were correlated with higher ceruloplasmin (APP+) and lower lysozyme serum concentrations (P<0.05 and <0.1, respectively). This latter finding was correlated with a clear role in vitro of lysozyme in a dose-dependent modulation of the inflammatory response of swine intestinal epithelial cells and bovine peripheral blood mononuclear cells. Hematological examinations showed no significant differences between the two groups under study. On the whole, our results indicate that LFI and LFI-related parameters could be used to identify cows at risk in the transition period toward an improved farm management. Also, our study indicates that disease cases in periparturient, high-yielding dairy cows are correlated with signs of accentuated IL-6 response and other markers of inflammatory phenomena. These likely start in the late lactation period or around dry-off, as suggested by our prepartal data, and proceed at much greater levels after calving.

Strategies for reduced antibiotic usage in dairy cattle farms

The need for antibiotic treatments in dairy cattle farms can be reduced by a combined intervention scheme based on: (1) timely clinical inspections, (2) the assessment of animal-based welfare parameters, and (3) the use of predictive laboratory tests. These can provide greater insight into environmental adaptation of dairy cows and define animals at risk of contracting disease. In the long-term, an improved disease control justifies the adoption of such a combined strategy. Many antibiotic treatments for chronic disease cases are often not justified with a cost/benefit analysis, because the repeated drug administration does not give rise to the expected outcome in terms of animal health. In particular, compared with untreated cases, antibiotics may not lead to greater cure rates for some forms of mastitis. Lastly, a substantial reduction of antibiotic usage in dairy farms can be achieved through the proper use of immunomodulators, aimed at increasing immunocompetence and disease resistance of cows.

IPEC-J2 Cells as Reporter System of the Anti-Inflammatory Control Actions of Interferon-Alpha

Interferon-alpha (IFN-α) shows potent immunomodulatory properties, which underlies its use for low-dose oral treatments of diverse viral infections and immunopathological conditions. The studies on oral administration have been hampered by the lack of recognized in vitro models, reproducing the in vivo control action of IFN-α over inflammatory cytokine responses. Owing to these reasons, the aim of our study was to validate IPEC-J2 (a continuous cell line of porcine intestinal epithelial cells) as a reporter system of the properties of IFN-α. Three different experimental conditions (oxidative stress, inflammatory response, and amplification of lymphoid cell signals) were selected to evaluate the effects of porcine recombinant IFN-α1 (rIFN-α) and 2 natural porcine IFN-α preparations (nIFN-α). The IFNs under study showed significantly different control actions in IPEC-J2 cells. In particular, rIFN-α was shown to down-regulate interleukin (IL)-8, IL-1β, tumor necrosis factor (TNF)-α, and β-defensin 1 genes either directly, or indirectly through second messengers released by IFN-α-treated lymphoid cells. With regard to IL-6, only second messengers from IFN-α-treated lymphoid cells could regulate the expression of this cytokine. Our results suggest that IPEC-J2 cells can be a useful tool for investigating the regulatory actions of type I IFNs and the second messengers thereof. The results provided by this model could be conveniently exploited in studies on enteric diseases sustained by infectious or noninfectious stressors.

Characterization of the interferon-α response of pigs to the weaning stress.

The interferon (IFN)-α response of pigs to the stressing event of early weaning was investigated in a field trial. All the animals under study remained healthy and tested negative for common viral infections. However, a low-titered IFN-α response was detected in many sera by a bioassay on Madin-Darby bovine kidney (MDBK) cells on day +6 after weaning. Porcine IFN-α was unambiguously identified by a neutralization assay on a pool of IFN-α-positive sera. By gel filtration chromatography, the antiviral activity of sera on MDBK cells could be traced back to 3 components of apparent molecular mass 27/18/<14 kDa. Additional components of apparent molecular mass 58 and 41 kDa were revealed by ELISA in Nonidet P-40 lysates of peripheral blood mononuclear cells (PBMC). Also, many pigs tested positive in flow cytometry assays on PBMC for intracellular IFN-α. The expression of porcine IFN-α genes was investigated by reverse transcriptase (RT) real-time polymerase chain reaction at days -1, +6, and +12 with regard to weaning in PBMC of 9 piglets. On days -1 and +12, IFN A5, A6, A12, as well as (in fewer pigs) A1, A7, A11, and A2 genes were shown to be expressed. On the contrary, none of the above genes was expressed on day +6, when plenty of pig sera were IFN-α-positive. Our results indicate that weaning causes the release of IFN-α and the transient shut-off of the corresponding gene transcriptions in PBMC. Interestingly, only IFN A9 gene transcription was shown in vitro to be virus induction-dependent.

Cross-Species Infectivity of H3N8 Influenza Virus in an Experimental Infection in Swine

ABSTRACT Avian influenza A viruses have gained increasing attention due to their ability to cross the species barrier and cause severe disease in humans and other mammal species as pigs. H3 and particularly H3N8 viruses, are highly adaptive since they are found in multiple avian and mammal hosts. H3N8 viruses have not been isolated yet from humans; however, a recent report showed that equine influenza A viruses (IAVs) can be isolated from pigs, although an established infection has not been observed thus far in this host. To gain insight into the possibility of H3N8 avian IAVs to cross the species barrier into pigs, in vitro experiments and an experimental infection in pigs with four H3N8 viruses from different origins (equine, canine, avian, and seal) were performed. As a positive control, an H3N2 swine influenza virus A was used. Although equine and canine viruses hardly replicated in the respiratory systems of pigs, avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Interestingly, antibodies against hemagglutinin could not be detected after infection by hemagglutination inhibition (HAI) test with avian and seal viruses. This phenomenon was observed not only in pigs but also in mice immunized with the same virus strains. Our data indicated that H3N8 IAVs from wild aquatic birds have the potential to cross the species barrier and establish successful infections in pigs that might spread unnoticed using the HAI test as diagnostic tool. IMPORTANCE Although natural infection of humans with an avian H3N8 influenza A virus has not yet been reported, this influenza A virus subtype has already crossed the species barrier. Therefore, we have examined the potential of H3N8 from canine, equine, avian, and seal origin to productively infect pigs. Our results demonstrated that avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Surprisingly, we could not detect specific antibodies against hemagglutinin in any H3N8-infected pigs. Therefore, special attention should be focused toward viruses of the H3N8 subtype since they could behave as stealth viruses in pigs.

Immune Control of PRRS: Lessons to be Learned and Possible Ways Forward

Porcine reproductive and respiratory syndrome (PRRS) is an elusive model of host/virus relationship in which disease is determined by virus pathogenicity, pig breed susceptibility and phenotype, microbial infectious pressure, and environmental conditions. The disease can be controlled by farm management programs, which can be supported by vaccination or conditioning of animals to circulating PRRS virus (PRRSV) strains. Yet, PRRS still represents a cause of heavy losses for the pig industry worldwide. Immunological control strategies are often compounded by poor and late development of adaptive immunity in both vaccinated and infected animals. Also, there is evidence that results of field trials can be worse than those of experimental studies in isolation facilities. Neutralizing antibody (NA) was shown to prevent PRRSV infection. Instead, the role of NA and adaptive immunity on the whole in virus clearance after established PRRSV infections is still contentious. Pigs eventually eliminate PRRSV infection, which may be correlated with an “educated,” innate immune response, which may also develop following vaccination. In addition to vaccination, an immunomodulation strategy for PRRS can be reasonably advocated in pig “problem” farms, where a substantial control of disease prevalence and disease-related losses is badly needed. This is not at odds with vaccination, which should be preferably restricted to PRRSV-free animals bound for PRRSV-infected farm units. Oral, low-dose, interferon-α treatments proved effective on farm for the control of respiratory and reproductive disease outbreaks, whereas the results were less clear in isolation facilities. Having in mind the crucial interaction between PRRSV and bacterial lipopolysaccharides for occurrence of respiratory disease, the strong control actions of low-dose type I interferons on the inflammatory response observed in vitro and in vivo probably underlie the rapid clinical responses observed in field trials.

Time-course of antibody and cell-mediated immune responses to Porcine Reproductive and Respiratory Syndrome virus under field conditions

Major discrepancies are observed between experimental trials of PRRS-virus (PRRSV) infection in isolation facilities and observations made in the field on farm. Owing to the above, a cohort study was carried out in a farrow-to-finish, PRRSV-infected pig farm to characterize the time-course of the virus-specific immune response in two groups of replacement gilts. Despite the occurrence of three and two distinct waves of infection in groups 1 and 2, respectively, the large majority of animals showed little if any PRRSV-specific response in an interferon-gamma release assay on whole blood, whereas non-specific responses were consistently observed. To rule out any possible bias of our test procedure, this was used along with an ELISPOT assay for interferon-gamma-secreting cells with the same reagents on a group of PRRS-virus infected pigs in isolation facilities. A very good agreement was shown between the two sets of results. Also, as opposed to the PRRS model, plenty of Pseudorabies virus-vaccinated pigs under field conditions scored positive in another experiment in the interferon-gamma release assay, ad hoc modified for the Pseudorabies virus. Our results indicate that under field conditions poor or no development rather than delayed development of the PRRS virus-specific interferon-gamma response could be the rule for a long time in non-adult pigs after PRRS virus infection. Housing and hygiene conditions, as well as heavy exposure to environmental microbial payloads in intensive pig farms could adversely affect the hosts immune response to PRRS virus and partly account for the discrepancies between experimental and field studies.

Isolation and culture of pig tonsil lymphocytes.

Tonsils are secondary lymphoid organs that play an important role in host defense. The aim of our study was to develop reliable procedures for isolation and culture of pig tonsil cells, and to validate their possible use in functional immunoassays. Using our isolation procedure, we recovered on average 238.7 ± 107.1 × 10(6) cells per tonsil couple with a mean vitality of 89.8 ± 2.7%. These values significantly decreased 8 months after freezing at -80°C along with the subsequent spontaneous release of both IgA and IgG in culture. These results suggest to use pig tonsil cells within 2 months from thawing to maintain suitable conditions in terms of recovery, vitality and release of antibody in vitro. Tonsil mononuclear cells also showed the ability to secrete antimicrobial peptides and to respond in vitro to immunological stimuli. On the whole, our study has defined operating conditions for tonsil processing, control of bacterial contaminations, time limits of storage at -80°C, as well as for evaluating polyclonal Ig production in vitro. Such procedures are likely to be of some importance in studies on regional immunity and in the development of large animal models for biomedical sciences.

Differential Biological Activities of Swine Interferon-α Subtypes.

Interferons (IFNs) play a crucial role in the hosts immune response and other homeostatic control actions. Three IFN types and several IFN families within the types allow for a plethora of regulatory actions. The number of distinct IFN molecules is highest among type I IFNs and, in particular, within the IFN-α family. In pigs, there are 17 IFN-α subtypes with different antiviral activities and different expression profiles; however, no data are available about biological properties other than the antiviral effector activities. Therefore, 16 porcine IFN-α genes were cloned, expressed in mammalian Chinese hamster ovary cells, and characterized for antiviral, anti-inflammatory, and MHC-modulating activities at a pre-established level of 10 IU/mL. Antiviral activity: IFN-α2, -α5, -α9, and -α10 showed the highest level of activity in a pseudorabies virus yield reduction assay. On the contrary, little, if any, activity was shown by IFN-α3, -α7, -α13, -α4, and -α15. Anti-inflammatory activity: With the exception of IFNs-α2, -α7, -α9, and -α11, all IFN-α subtypes had significant anti-inflammatory control activity in an interleukin-8 (IL-8) yield reduction assay. Gene expression analyses showed that some IFN-α subtypes can significantly downregulate the expression of IL-8, tumor necrosis factor α (TNF-α), IL-6, Toll-like receptor 4 (TLR4), βD1, and nuclear factor-κB (NF-kB) genes, while maintaining or upregulating the expression of βD4. Immunomodulation: A significant upregulation of class I and/or class II MHC was induced by all the IFNs under study, with the exception of IFNs-α11, -α15, and -α16, which instead significantly downregulated class I MHC. Our results indicate that gene duplications in the porcine IFN-α family underlie diverse effector and regulatory activities, being therefore instrumental in host survival and environmental adaptation. This role of IFN-α could be founded on fine-tuning and regulation of pro- and anti-inflammatory control actions after exposure to both infectious and noninfectious environmental stressors.

Preliminary Study on the Relationship Between Skin Temperature of Piglets Measured by Infrared Thermography and Environmental Temperature in a Vehicle in Transit

During two 14-h journeys, carried out in July and September 2009, respectively, the variation in skin temperature measured by infrared thermography was examined on a total of 12 piglets. A thermal camera was placed in front of the pen during the first journey and above the pen during the second. The temperature inside the vehicle was registered throughout the journeys. A positive linear relationship was observed between skin and internal vehicle temperatures with an R 2 of 0.44 and 0.57 in July and September, respectively. The results obtained in this preliminary experiment showed the possibility of recording thermal images of piglets in transit. Thus, thermography, coupled with other body-temperature-recording techniques, could be valuable for assessing the adaptive efforts of pigs to environmental conditions experienced during transport.