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Highly pathogenic (HP) PRRSV isolates have been discovered within both PRRSV-1 and PRRSV-2 genotypes and investigated in recent years especially for their ability to cause extremely severe disease in conventional pig herds. The exacerbation of general and respiratory clinical signs has been attributed not only to an efficient replication (virulence) but also to the ability to dysregulate viral recognition and induce mechanisms of immune evasion or immune enhancement of humoral and cellular anti-viral responses differently from non-HP PRRSV isolates in terms of intensity and temporal onset. Thus, the understanding of the immunopathogenesis of HP PRRSV is a major concern for the study of virus biology and development of efficacious vaccines. The present study aims at addressing the modulation of relevant immune cell subsets by flow cytometry in the blood of 4-week-old pigs experimentally infected with the recently discovered PR40/2014 HP PRRSV-1.1 strain phenotypically characterized in Canelli et al. (2017) compared to pigs infected with a non-HP PRRSV isolate (PR11/2014) and uninfected controls. PR40 infected animals showed an early and marked reduction of pro-inflammatory CD172α+ CD14+CD16+ and CD14+CD163+ monocytes and TCRγδ+CD8α+/CD8α- lymphocytes when pigs were most infected, possibly due to a recruitment sustaining an acute inflammatory response in target tissues. The prolonged increased CD3+CD16+ NKT cell levels may sustain peripheral inflammation and/or the anti-viral response. The late reduction (potential depletion) of γ/δ T lymphocytes and CD3+CD4+CD8α- naïve Th lymphocytes paralleled with the delayed increase of CD3+CD4+CD8α+ memory and CD3+CD4-CD8α/β + cytotoxic T lymphocytes. In addition, PR40 infection showed an early depletion of activated CD4+CD25+ T lymphocytes and Tregs together with an intense and lasting depletion of CD21+ B lymphocytes. Overall, these features demonstrate that the more severe clinical signs observed upon infection with the HP PR40 strain are sustained by remarkable changes in the peripheral blood distribution of immune cells and provide further insights into the immune regulation/immunopathogenesis induced by PRRSV-1 subtype 1 European isolates.

An engineered killer peptide (KP) based on a recombinant anti-idiotypic antibody representing the functional image of a yeast killer toxin (KT) was demonstrated to mediate antimicrobial effects against fungi and viruses. KP binds to murine dendritic cells and macrophages and up-regulate co-receptor expression, thus sustaining CD4+ lymphocyte activation. No immunological data are available in domestic animals thus KP-induced immunomodulation was evaluated in porcine monocyte and lymphocyte subsets. PBMC from healthy adult pigs were stimulated with KP or a scramble peptide (SP), or kept unstimulated for 24, 48 and 72 h, and subsequently analyzed by flow cytometry. In monocytes, KP induced a strong dose-dependent shift from a major fraction of CD172α+CD14+ low cells to a predominant fraction of CD172α+CD14+ high cells, known to sustain leukocyte activation/differentiation and inflammatory responses. The CD16+ cell percentages, specifically the CD3+CD16+ natural killer T (NKT) cell fraction and CD16 expression showed an intense and stable dose-dependent increase while the CD3−CD16+ NK cell fraction decreased. CD4+ and CD8+ T cells increased and CD8α and CD8β expression were up-regulated. CD8β+ cytotoxic T cells and CD16+ cells comparably increased. A marked stimulation of activated CD16+CD25+ and CD8β+CD25+ cells was observed at 24 h. The increase of CD8α+ cells and CD8α expression were due to increased CD4+CD8α+ (memory T helper) cells, also showing a CD8α+ high phenotype. Concomitantly, the CD4+CD8α- T helper lymphocyte fraction significantly decreased. Overall, KP induced a wide modulation of innate immune and T cells that can exert regulatory and cytotoxic functions, which are fundamental for an efficient Th1 response.

PRRS is one of the main viral diseases in pig production, causing huge economic losses to the swine industry worldwide. The virus shows an intrinsic genomic instability and is able to change continuously, with the emergence of new strains, with different pathogenicity patterns. Commercially available vaccines only partially prevent or counteract the disease and the correlated losses. Moreover, the emergence of highly virulent and pathogenetic isolates represents a particular concern for PRRS control and diagnosis. The purpose of this study was to evaluate the efficacy of a modified-live virus (MLV) PRRSV-1 commercial vaccine in reducing the severity of the disease and minimizing losses upon challenge with a highly pathogenic PRRSV-1.1 Italian isolate (PRRSV-1_PR40/2014). Four different groups were compared: C (unvaccinated-uninfected), VAC-C (vaccinated-uninfected), PR40 (unvaccinated-infected) and VAC-PR40 (vaccinated-infected). The tested vaccine provided partial, but statistically significant clinical, virological and pathological protection after challenge under experimental conditions. In particular, vaccinated animals showed reduced viremia in terms of duration and magnitude, reduced respiratory signs and pathological lesions. Vaccination was able to trigger adaptive immunity able to respond efficiently also against the HP PR40 isolate. Vaccinated animals showed higher average daily weight gain, even during the viremic period, compared to non-vaccinated challenged pigs.

Highly pathogenic (HP) isolates of the PRRS virus started emerging in North America and Asia in the late 1990s. More recently, they have emerged in Europe. These isolates are characterized by high viral loads, severe general clinical signs and high mortality, in sows, weaners and growers. Their genome shows a discontinuous aminoacids deletion in the non-structural protein 2 (NSP2). The present study was aimed at characterizing the clinical, pathological and immunological features of a highly pathogenetic, Italian PRRSV-1 subtype 1 isolate (PRRSV1_PR40/2014), following experimental infection in conventional 4-weeks-old pigs. The PRRSV1_PR40/2014 infected group showed severe clinical signs (high fever and dispnoea). Pathological lesions, including severe lymphocytopenia in bronchial lymph-nodes and thymus were also recorded. Higher serum PRRSV genome copies and lower virus neutralizing antibody titer were observed in the PR40 group, when compared to the group infected with a conventional PRRSV strain. The genetic analysis of the strain, and the phenotypic features observed in the field and reproduced in the experimental study, confirmed the high pathogenicity of the Italian PRRSV-1 subtype 1 PR40 isolate.

Astroviruses (AstVs) are nonenveloped RNA small round viruses (SRVs) with a genome of 6.8-7.9 kb. Known avian AstVs are spread worldwide; they have been associated with poult enteritis and mortality syndrome in the United States and reported in Italy in intensive turkey and guinea fowl flocks. Nevertheless, their real prevalence and their pathogenic role in avian enteritis affecting Italian flocks is far from clear. Negative staining electron microscopy (nsEM) is used for the routine diagnosis of avian enteric SRVs, although it cannot distinguish morphologically similar particles. Enzyme-linked immunosorbent assay (ELISA), reverse-transcription PCR (RT-PCR), and genomic sequencing are now used for this specific purpose. We analyzed 329 samples of chicken, turkey, and guinea fowl intestinal contents from Italian poultry flocks. Most samples were from enteritis outbreaks, but we also included samples from three longitudinal studies (one on 11 broiler flocks and the other two on a guinea fowl flock). We first examined the samples with nsEM. SRVs, including AstVs, are often associated with rotaviruses and were the most commonly detected morphotypes in avian enteric diseases. We then analyzed 124 of the samples with an RT-PCR targeting the open reading frame (ORF)-1b of AstV. This gene codes for an RNA-dependent polymerase. We then sequenced and genetically analyzed the RT-PCR positive samples. Phylogenetic analysis distinguished three defined clusters: the first included guinea fowl AstVs and turkey AstVs-2; the second, chicken AstVs; and the third was formed by avian nephritis viruses (ANVs). No strains clustered with turkey AstVs-1. The results indicate that ORF-1b presents certain genetic variability, even among AstVs from the same species. In longitudinal studies, samples retrieved from the same shed were homogeneous, with some exceptions suggesting possible coexistence of different genetic types in the same unit. The finding of ANV-like viruses in commercial guinea fowls underlines the genetic variability of AstVs and strengthens the hypothesis of a varied intraherd situation.

In the last decade, the impact of the porcine circovirus type 2 (PCV-2)-associated diseases in pigs has consistently decreased due to the extensive application of PCV-2 vaccines in sows and piglets. However, clinical outbreaks of PCV-2 systemic diseases are still reported. For this reason, optimisation of vaccination schemes, which also evaluates vaccination of both sows and piglets, has been studied (Opriessnig and others 2010, Fraile and others 2012). A one-dose vaccine administered prior to mating in gilts and sows elicited a strong and homogeneous humoral response, and consequently, an increased colostral PCV-2 antibody concentration (Sibila and others 2013). The present paper aims at evaluating both the humoral and, for the first time, the cellular PCV-2-specific immune response in gilts and sows repeatedly vaccinated at each mating in order to contribute to a better knowledge on vaccination schemes in sows and in their offspring. In a randomised controlled field trial, two cohorts, each comprised by 46 gilts, were investigated. The study protocol was approved by the University of Parma Institutional Animal Care Committee. Animals, seven months old, originated from the same batch of replacements, were randomly allocated by lot either to group ‘VAC’ or to group ‘NV’. On the day of first mating, VAC sows were injected intramuscularly with 2 ml of a PCV-2 vaccine (Porcilis PCV, MSD Animal Health) (off-label use) and NV sows were injected with the adjuvant only. The same treatment was repeated on the day of mating after the weaning of their first and second litter. Blood samples were taken on the day of enrolment to the study, that is, …

The study aims at evaluating gene expression of pro-inflammatory (IL-1β, IL-8, TNF-α), pro-immune (IFN-γ), anti-inflammatory (IL-10) cytokines and of the immunoregulatory signal FoxP3 in association with PRRSV-specific IFN-γ secreting cell (SC) responsiveness upon PRRSV natural infection. Forty PRRSV-negative pigs were assigned to two groups: 20 pigs were vaccinated at 3 weeks of age (weaning) against PRRSV (V-PRRSV) with a modified live virus vaccine (MLV) and 20 pigs were kept non-vaccinated (NV) as controls. Blood samples were collected at 3 (vaccination), 6, 8, 10, 12, 14, and 16 weeks of age. Natural infection occurred from 8 weeks of age onward in both groups and viremia lasted 8 weeks. In the early phase of infection, pro-inflammatory cytokines (IL-1β, IL-8, TNF-α) showed a delayed increase concomitant with the peak of viremia in both groups. In both groups, IL-10 peaked at 12 weeks in association with the increase of pro-inflammatory cytokines. Conversely, in vaccinated pigs (V-PRRSV), IFN-γ showed higher gene expression during the early phase of infection and a more intense secreting cell (SC) response in the late phase. Differently, gene expression of the transcription factor FoxP3, expressed by T regulatory lymphocytes (Tregs), increased significantly in controls only and was associated with the rise of the viral load. Moreover, FoxP3 levels remained significantly higher during the late phase of infection and paralleled with lower levels of IFN-γ SC detected by ELISPOT. The expression/production of immunoregulatory signals involved in Treg activation could be a promising marker to study the immunobiology of PRRSV infection.

In this study, we undertook the genomic characterization of 54 pseudorabies virus (PRV) strains isolated in Italy during 1984-2010. The characterization was based on partial sequencing of the UL44 (gC) and US8 (gE) genes; 44 strains (38 for gene gE and 36 for gC) were isolated on pig farms; 9 originated from dogs and 1 from cattle. These porcine PRV strains, which were closely related to those isolated in Europe and America in the last 20 years, and the bovine strain bovine/It/2441/1992 belong to cluster B in both phylogenetic trees. Six porcine strains that do not belong to cluster B are related in both gE and gC phylogenetic trees to the 'old' porcine PRV strains isolated in the 1970s and 1980s. In the last two decades, the presence of these strains in domestic pig populations has been reduced drastically, whereas they are prevalent in wild boar. The two remaining strains have an interesting genomic profile, characterized by the gC gene being closely related to the old porcine PRV strains, and the gE gene being similar to that of recently isolated strains. Three strains originating from working dogs on pig farms are located in cluster B in both phylogenetic trees. Five strains isolated from hunting dogs have a high degree of correlation with PRV strains circulating in wild boar. The last isolate has a gC gene similar to that in the two porcine strains mentioned previously, and the gE gene is correlated with the strains isolated from hunting dogs. These results provide interesting insight into the genomic characterization of PRV strains and reveal a clear differentiation between the strains isolated from hunting dogs that are related to the wild boar strains and those originating from domestic pigs.

To the Editor: Canine distemper virus (CDV) is an enveloped, single-stranded, negative RNA virus of the family Paramyxoviridae, genus Morbillivirus (1). The host range for CDV is broad, and infection has been found in several mammalian species of the families Canidae, Mustelidae, Procyonidae, Ursidae, and Viverridae (2). Stelvio National Park (SNP) encompasses 1,333 km2 of protected land in Italy and covers 2 regions (Lombardia and Trentino Alto Adige); the Lombardia section of the park covers the northern part of Sondrio Province (Valtellina). SNP is surrounded by other parks (Schweitzer National Park, Adamello, and Adamello-Brenta) to form a large protected area (2,500 km2) in the heart of the Alps mountains. Within SNP, the terrestrial mammals that are susceptible to CDV include red foxes (Vulpes vulpes), stoats (Mustela erminea), weasels (Mustela nivalis), pine martens (Martes martes), beech martens (Martes foina), badgers (Meles meles), and bears (Ursus arctos). In August 2009, three young red foxes were captured in Valtellina (Sondrio), Lombardia, Italy, within the southwestern borders of SNP. The animals showed canine distemper–like signs (e.g., prostration, altered behavior, and conjunctivitis), and CDV infection was confirmed by quantitative reverse transcription–PCR of pooled organs (3). In September and October 2009, another 2 young foxes were captured and found to be positive for CDV. From September on, at least 30 foxes with altered behavior were seen near human habitations and facilities in SNP; 10 were captured. In the same period, infected foxes were also reported from Engadina, Switzerland, at the northern and western borders of SNP. In February 2010, two symptomatic foxes were euthanized in Grosotto, 50 km south of where the initial cases were identified. The epizootic appeared to have originated from the eastern regions of Italy (Trentino Alto Adige, and Veneto), where CDV infection had been reported in red foxes and badgers since August 2006 (4) (Figure). A large CDV epidemic in foxes in southern Bavaria in 2008 has also been described, thus suggesting spread of the virus throughout the Alps area (5). Figure Phylogenetic tree showing the genetic relationships among selected canine distemper virus strains of various lineages and generated by using the full-length nucleotide sequence of the hemagglutinin gene. The tree branches including viruses not from Europe ... Reverse transcription–PCR genotyping of the hemagglutinin (H) gene (6) identified 15 CDV strains, which were analyzed and characterized as European genotypes. The full-length H gene of the CDV strains was determined (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"HM120874","term_id":"298364439","term_text":"HM120874"}}HM120874). Sequence analysis of the H gene indicated that the fox CDV strains were highly related to each other (>99.9% nt and 100% aa identity), to the CDV strains identified in foxes in southern Bavaria 2008 (>99.7% nt and 99.3% aa identity; accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"FI416336-FI416338","start_term":"FI416336","end_term":"FI416338","start_term_id":"220831209","end_term_id":"220831835"}}FI416336-FI416338), and to a canine strain identified in Hungary during 2005–2006 (99.4% nt and 99.2% aa identity; accession no. {"type":"entrez-nucleotide","attrs":{"text":"DQ889177","term_id":"121486035","term_text":"DQ889177"}}DQ889177). During the CDV epizootic in SNP in 2009, cases of CDV in 3 domestic dogs living within the borders of the park were also reported. Because vaccination against CDV is a core recommendation for dogs, most dogs are expected to be vaccinated and protected; population immunity is high enough to keep CDV infection under control, and only sporadic cases occur (7). Accordingly, the reported CDV cases in dogs were more likely a spillover event caused by the high pressure of CDV infection in SNP wildlife. In addition to foxes, badgers in the same area were also reported to have canine distemper–like disease. These findings are consistent with spread of a multihost epizootic, in which foxes likely played a major role in CDV amplification and diffusion because of their social behavior during reproductive season and because of the wide geographic range over which juveniles migrate during the dispersion period. Serologic investigations for CDV in some fox populations in Europe have identified antibody prevalence rates of 4%–26.4% (8), suggesting that CDV circulates in foxes in Europe, but these investigations did not examine spatial and temporal variations in CDV activity. Clues for understanding the pattern of CDV disease in wildlife have been provided by structured surveillance of wild canids living in Yellowstone National Park, USA. Yearly fluctuations in CDV seroprevalence with evidence of multihost outbreaks in distinct years, contemporaneously affecting different animal species, have been noted. Cycles of CDV epizootics that swept through the animals in the park were associated with low pup survival rates (9). In SNP, most foxes captured during the epizootic were juveniles. We have no information on the prevalence rates of CDV-specific antibodies in SNP foxes before the epizootic. However, CDV disease had not been reported in the SNP for at least 10 years, and no animal with CDV infection had been identified in a 2004–2005 survey of red foxes in SNP (10). Similarly, no evidence for CDV infection had been found in carnivores in Trentino Alto Adige during 2001–2002 (10). Accordingly, one can assume that the population immunity in SNP foxes (and in other susceptible hosts) was low. Adequately controlling CDV infection in wildlife in Europe is difficult. It requires concerted transnational actions, including effective surveillance and prompt gathering and dissemination of information.

PORCINE circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) are the two most important pathogens currently responsible for major economic losses to the swine industry worldwide. PCV2 vaccines are extensively used and are able to reduce the clinical signs of PCV2-associated disease. The efficacy of PCV2 vaccination likely depends on both humoral and cellular immunity that can either be maternally acquired (sow vaccination) or actively induced (piglets vaccination) (Fort and others 2008, 2009, Opriessnig and others 2010, Martelli and others 2011). Taking into account that PRRSV suppresses innate immunity and influences the development and the activation of the specific immune response (Butler and others 2014), this study is aimed at evaluating the effects of PRRSV viraemia on the immune response to PCV2 vaccination in four-week-old piglets. Indeed, PCV2 vaccination in two- to four-week-old piglets is extensively applied where PCV2 infection and associated disease are present. Two hundred piglets from a 1000 farrow-to-wean PRRSV infected and unstable sows herd were divided into two treatment groups (100 animals each); one group was vaccinated at four weeks of age with a PCV2 commercial vaccine (Porcilis PCV – MSD) and the second group was not vaccinated. The animals were assigned to the different groups as they came to hand …