Pigs Lacking the Scavenger Receptor Cysteine-Rich Domain 5 of CD163 Are Resistant to Porcine Reproductive and Respiratory Syndrome Virus 1 Infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) has a narrow host cell tropism, limited to cells of the monocyte/macrophage lineage. CD163 protein is expressed at high levels on the surface of specific macrophage types, and a soluble form is circulating in blood. CD163 has been described as a fusion receptor for PRRSV, with the scavenger receptor cysteine-rich domain 5 (SRCR5) region having been shown to be the interaction site for the virus. As reported previously, we have generated pigs in which exon 7 of the CD163 gene has been deleted using CRISPR/Cas9 editing in pig zygotes. These pigs express CD163 protein lacking SRCR5 (ΔSRCR5 CD163) and show no adverse effects when maintained under standard husbandry conditions. Not only was ΔSRCR5 CD163 detected on the surface of macrophage subsets, but the secreted, soluble protein can also be detected in the serum of the edited pigs, as shown here by a porcine soluble CD163-specific enzyme-linked immunosorbent assay (ELISA). Previous results showed that primary macrophage cells from ΔSRCR5 CD163 animals are resistant to PRRSV-1 subtype 1, 2, and 3 as well as PRRSV-2 infection in vitro Here, ΔSRCR5 pigs were challenged with a highly virulent PRRSV-1 subtype 2 strain. In contrast to the wild-type control group, ΔSRCR5 pigs showed no signs of infection and no viremia or antibody response indicative of a productive infection. Histopathological analysis of lung and lymph node tissue showed no presence of virus-replicating cells in either tissue. This shows that ΔSRCR5 pigs are fully resistant to infection by the virus.IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) is the etiological agent of PRRS, causing late-term abortions, stillbirths, and respiratory disease in pigs, incurring major economic losses to the worldwide pig industry. The virus is highly mutagenic and can be divided into two species, PRRSV-1 and PRRSV-2, each containing several subtypes. Current control strategies mainly involve biosecurity measures, depopulation, and vaccination. Vaccines are at best only partially protective against infection with heterologous subtypes and sublineages, and modified live vaccines have frequently been reported to revert to virulence. Here, we demonstrate that a genetic-control approach results in complete resistance to PRRSV infection in vivo CD163 is edited so as to remove the viral interaction domain while maintaining protein expression and biological function, averting any potential adverse effect associated with protein knockout. This research demonstrates a genetic-control approach with potential benefits in animal welfare as well as to the pork industry.

Precision engineering for PRRSV resistance in pigs: Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function.

Porcine Reproductive and Respiratory Syndrome (PRRS) is a panzootic infectious disease of pigs, causing major economic losses to the world-wide pig industry. PRRS manifests differently in pigs of all ages but primarily causes late-term abortions and stillbirths in sows and respiratory disease in piglets. The causative agent of the disease is the positive-strand RNA PRRS virus (PRRSV). PRRSV has a narrow host cell tropism, limited to cells of the monocyte/macrophage lineage. CD163 has been described as a fusion receptor for PRRSV, whereby the scavenger receptor cysteine-rich domain 5 (SRCR5) region was shown to be an interaction site for the virus in vitro. CD163 is expressed at high levels on the surface of macrophages, particularly in the respiratory system. Here we describe the application of CRISPR/Cas9 to pig zygotes, resulting in the generation of pigs with a deletion of Exon 7 of the CD163 gene, encoding SRCR5. Deletion of SRCR5 showed no adverse effects in pigs maintained under standard husbandry conditions with normal growth rates and complete blood counts observed. Pulmonary alveolar macrophages (PAMs) and peripheral blood monocytes (PBMCs) were isolated from the animals and assessed in vitro. Both PAMs and macrophages obtained from PBMCs by CSF1 stimulation (PMMs) show the characteristic differentiation and cell surface marker expression of macrophages of the respective origin. Expression and correct folding of the SRCR5 deletion CD163 on the surface of macrophages and biological activity of the protein as hemoglobin-haptoglobin scavenger was confirmed. Challenge of both PAMs and PMMs with PRRSV genotype 1, subtypes 1, 2, and 3 and PMMs with PRRSV genotype 2 showed complete resistance to viral infections assessed by replication. Confocal microscopy revealed the absence of replication structures in the SRCR5 CD163 deletion macrophages, indicating an inhibition of infection prior to gene expression, i.e. at entry/fusion or unpacking stages.

Bacillus pumilus probiotic feed supplementation mitigates Lawsonia intracellularis shedding and lesions.

The causative agent of ileitis, Lawsonia intracellularis, is commonly associated with diarrhea and reduced weight gain in growing pigs. The effect of in-feed probiotics on L. intracellularis infection dynamics was evaluated. In brief, 70 2.5-week-old-pigs were randomly divided into six groups with 10-20 pigs each. All pigs were fed an age appropriate base ration for the duration of the study, which was supplemented with one of three Bacillus strains including B. amyloliquefaciens (T01), B. licheniformis (T02) and B. pumilus (T03). Another group was orally vaccinated with a commercial live L. intracellularis vaccine (VAC) at 3 weeks of age. At 7 weeks of age, T01-LAW, T02-LAW, T03-LAW, VAC-LAW and the POS-CONTROL groups were challenged with L. intracellularis while the NEG-CONTROL pigs were not challenged. All pigs were necropsied 16 days later. By the time of inoculation, all VAC-LAW pigs had seroconverted and at necropsy 10-65% of the pigs in all other challenged groups were also seropositive. The results indicate a successful L. intracellularis challenge with highest bacterial DNA levels in POS-CONTROL pigs, VAC-LAW pigs and T01-LAW pigs. There was a delay in onset of shedding in T02-LAW and T03-LAW groups, which was reflected in less severe macroscopic and microscopic lesions, reduced intralesional L. intracellularis antigen levels and a lower area under the curve for bacterial shedding. Under the study conditions, two of the probiotics tested suppressed L. intracellularis infection. The obtained findings show the potential of probiotics in achieving antibiotic-free control of L. intracellularis.

Analysis of the genetic diversity and mRNA expression level in porcine reproductive and respiratory syndrome virus vaccinated pigs that developed short or long viremias after challenge.

Porcine reproductive and respiratory syndrome virus (PRRSv) infection alters the host's cellular and humoral immune response. Immunity against PRRSv is multigenic and vary between individuals. The aim of the present study was to compare several genes that encode for molecules involved in the immune response between two groups of vaccinated pigs that experienced short or long viremic periods after PRRSv challenge. These analyses include the sequencing of four SLA Class I, two Class II allele groups, and CD163, plus the analysis by quantitative realtime qRT-PCR of the constitutive expression of TLR2, TLR3, TLR4, TLR7, TLR8 and TLR9 mRNA and other molecules in peripheral blood mononuclear cells.

Investigating porcine parvoviruses genogroup 2 infection using in situ polymerase chain reaction.

BACKGROUND: Porcine parvovirus 2 (PPV2) was detected in swine serum without showing any relationship with disease. The emergence of the virus seemed to be a unique event until other genetically highly similar parvoviruses were identified in China and, later in 2012, the presence of the virus was also described in Europe. PPV2 is widely distributed in pig populations where it is suspected to be involved in respiratory conditions, based on its frequent detection in lung samples. In order to investigate the potential pathogenic involvement of PPV2, 60 dead pigs were examined from two farms. They were necropsied and tested for PPV2 and PCV2 (Porcine circovirus type 2) by PCR; by Brown and Brenn (B&B) staining for bacteria; by immunohistochemistry (IHC) to detect CD3, Swine leukocyte antigen class II DQ (SLAIIDQ), lysozyme, porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza (SIV), Mycoplasma hyopneumoniae (Mhyo); and by in situ hybridization (ISH) to detect ssDNA and dsDNA of PCV2. PPV2 positive samples were subjected to in situ polymerase chain reaction (IS-PCR) including double staining method to detect PPV2 and host cell markers. To calculate statistical difference we used GENMOD or LOGISTIC procedures in Statistical Analysis System (SAS®). RESULTS: We found that the PPV2 was localized mostly in lymphocytes in lungs, lymph nodes and liver. Neither CD3 antigen nor lysozyme was expressed by these infected cells. In contrast, low levels of SLAIIDQ were expressed by infected cells, suggesting that PPV2 may have a specific tropism for immature B lymphocytes and/or NK lymphocytes though possibly not T lymphocytes. CONCLUSION: The overall conclusion of this study indicates that PPV2 may contribute to the pathogenesis of pneumonia.

Quasispecies evolution of the prototypical genotype 1 porcine reproductive and respiratory syndrome virus early during in vivo infection is rapid and tissue specific.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major infectious threat to the pig industry worldwide. Increasing evidence suggests that microevolution within a quasispecies population can give rise to high sequence heterogeneity in PRRSV; potentially impacting the pathogenicity of the virus. Here, we report on micro-evolutionary events taking place within the viral quasispecies population in lung and lymph node 3 days post infection (dpi) following experimental in vivo infection with the prototypical Lelystad PRRSV (LV). Sequence analysis revealed 16 high frequency single nucleotide variants (SNV) or differences from the reference LV genome which are assumed to be representative of the consensus inoculum genome. Additionally, 49 other low frequency SNVs were also found in the inoculum population. At 3 dpi, a total of 9 and 10 SNVs of varying frequencies could already be detected in the LV population infecting the lung and lymph nodes, respectively. Interestingly, of these, three and four novel SNVs emerged independently in the two respective tissues when compared to the inoculum. The remaining variants, though already present at lower frequencies in the inoculum, were positively selected and their frequency increased within the quasispecies population. Hence, we were able to determine directly from tissues infected with PRRSV the repertoire of genetic variants within the viral quasispecies population. Our data also suggest that microevolution of these variants is rapid and some may be tissue-specific.

Distinct functional enrichment of transcriptional signatures in pigs with high and low IFN-gamma responses after vaccination with a porcine reproductive and respiratory syndrome virus (PRRSV).

Little is known about the host factor in the response to PRRSV vaccination. For this purpose, piglets were immunized with a commercial PRRSV-live vaccine and classified as high responders (HR) or low responders (LR) as regards to the frequencies of virus-specific IFN-γ-secreting cells. Six weeks post vaccination, PBMCs isolated from three individuals with the most extreme responses in each HR and LR groups and 3 unvaccinated controls, were either stimulated with phytohaemagglutinin, challenged with the vaccine or mock treated for 24 h, prior conducting transcriptional studies, gene ontology and pathway analyses. The LR group had very low neutralizing antibody levels and showed a higher number of down-regulated transcripts compared with the HR group (FDR < 0.2, P < 0.001). Down-regulated genes encoded chemoattractants, proinflammatory cytokines and the interferon-inducible GBP family, and showed enrichment in wounding (FDR < 3.6E-13), inflammation (FDR < 8E-12), defence (FDR < 8.7E-09) and immunity (FDR < 7.6E-08), suggesting immune response impairment. In the HR group, down-regulated genes were involved in protein transport (FDR < 4.77E-03), locomotory behavior (FDR < 5.47E-3), regulation of protein localization (FDR < 1.02E-02), and regulation of TNF superfamily member 15 and miR181. In contrast, the HR group presented up-regulated transcripts associated with wounding (FDR < 4.95). Moreover, IFN-γ was predicted to be an inhibited upstream regulator since IFN-γ pathways were associated with higher number of down-regulated genes in the LR (n = 40) than the HR (n = 10). Divergent responses to PRRSV-vaccination may be the result of the genetic background of the host.

Genomic variation in macrophage-cultured European porcine reproductive and respiratory syndrome virus Olot/91 revealed using ultra-deep next generation sequencing.

BACKGROUND: Porcine Reproductive and Respiratory Syndrome (PRRS) is a disease of major economic impact worldwide. The etiologic agent of this disease is the PRRS virus (PRRSV). Increasing evidence suggest that microevolution within a coexisting quasispecies population can give rise to high sequence heterogeneity in PRRSV. FINDINGS: We developed a pipeline based on the ultra-deep next generation sequencing approach to first construct the complete genome of a European PRRSV, strain Olot/9, cultured on macrophages and then capture the rare variants representative of the mixed quasispecies population. Olot/91 differs from the reference Lelystad strain by about 5% and a total of 88 variants, with frequencies as low as 1%, were detected in the mixed population. These variants included 16 non-synonymous variants concentrated in the genes encoding structural and nonstructural proteins; including Glycoprotein 2a and 5. CONCLUSION: Using an ultra-deep sequencing methodology, the complete genome of Olot/91 was constructed without any prior knowledge of the sequence. Rare variants that constitute minor fractions of the heterogeneous PRRSV population could successfully be detected to allow further exploration of microevolutionary events.

Down-regulation of mechanisms involved in cell transport and maintenance of mucosal integrity in pigs infected with Lawsonia intracellularis.

Lawsonia intracellularis is an obligate intracellular bacterium, responsible for the disease complex known as proliferative enteropathy (PE). L. intracellularis is associated with intestinal crypt epithelial cell proliferation but the mechanisms responsible are yet to be defined. Microarray analysis was used to investigate the host-pathogen interaction in experimentally infected pigs to identify pathways that may be involved. Ileal samples originating from twenty-eight weaner pigs experimentally challenged with a pure culture of L. intracellularis (strain LR189/5/83) were subjected to microarray analysis. Microarray transcriptional signatures were validated using immunohistochemistry and quantitative real time PCR of selected genes at various time points post challenge. At peak of infection (14 days post challenge) 86% of altered transcripts were down regulated, particularly those involved in maintenance of mucosal integrity and regulation of cell transport. Among the up-regulated transcripts, CD163 and CDK1 were novel findings and considered to be important, due to their respective roles in innate immunity and cellular proliferation. Overall, targeted cellular mechanisms included those that are important in epithelial restitution, migration and protection; maintenance of stable inter-epithelial cell relationships; cell transport of nutrients and electrolytes; innate immunity; and cell cycle.

Pig immune response to general stimulus and to porcine reproductive and respiratory syndrome virus infection: a meta-analysis approach.

BACKGROUND: The availability of gene expression data that corresponds to pig immune response challenges provides compelling material for the understanding of the host immune system. Meta-analysis offers the opportunity to confirm and expand our knowledge by combining and studying at one time a vast set of independent studies creating large datasets with increased statistical power. In this study, we performed two meta-analyses of porcine transcriptomic data: i) scrutinized the global immune response to different challenges, and ii) determined the specific response to Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection. To gain an in-depth knowledge of the pig response to PRRSV infection, we used an original approach comparing and eliminating the common genes from both meta-analyses in order to identify genes and pathways specifically involved in the PRRSV immune response. The software Pointillist was used to cope with the highly disparate data, circumventing the biases generated by the specific responses linked to single studies. Next, we used the Ingenuity Pathways Analysis (IPA) software to survey the canonical pathways, biological functions and transcription factors found to be significantly involved in the pig immune response. We used 779 chips corresponding to 29 datasets for the pig global immune response and 279 chips obtained from 6 datasets for the pig response to PRRSV infection, respectively. RESULTS: The pig global immune response analysis showed interconnected canonical pathways involved in the regulation of translation and mitochondrial energy metabolism. Biological functions revealed in this meta-analysis were centred around translation regulation, which included protein synthesis, RNA-post transcriptional gene expression and cellular growth and proliferation. Furthermore, the oxidative phosphorylation and mitochondria dysfunctions, associated with stress signalling, were highly regulated. Transcription factors such as MYCN, MYC and NFE2L2 were found in this analysis to be potentially involved in the regulation of the immune response. The host specific response to PRRSV infection engendered the activation of well-defined canonical pathways in response to pathogen challenge such as TREM1, toll-like receptor and hyper-cytokinemia/ hyper-chemokinemia signalling. Furthermore, this analysis brought forth the central role of the crosstalk between innate and adaptive immune response and the regulation of anti-inflammatory response. The most significant transcription factor potentially involved in this analysis was HMGB1, which is required for the innate recognition of viral nucleic acids. Other transcription factors like interferon regulatory factors IRF1, IRF3, IRF5 and IRF8 were also involved in the pig specific response to PRRSV infection. CONCLUSIONS: This work reveals key genes, canonical pathways and biological functions involved in the pig global immune response to diverse challenges, including PRRSV infection. The powerful statistical approach led us to consolidate previous findings as well as to gain new insights into the pig immune response either to common stimuli or specifically to PRRSV infection.

Structural and functional annotation of the porcine immunome.

BACKGROUND: The domestic pig is known as an excellent model for human immunology and the two species share many pathogens. Susceptibility to infectious disease is one of the major constraints on swine performance, yet the structure and function of genes comprising the pig immunome are not well-characterized. The completion of the pig genome provides the opportunity to annotate the pig immunome, and compare and contrast pig and human immune systems. RESULTS: The Immune Response Annotation Group (IRAG) used computational curation and manual annotation of the swine genome assembly 10.2 (Sscrofa10.2) to refine the currently available automated annotation of 1,369 immunity-related genes through sequence-based comparison to genes in other species. Within these genes, we annotated 3,472 transcripts. Annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type 1 Interferon families. We found gene duplications for 18 genes, including 13 immune response genes and five non-immune response genes discovered in the annotation process. Manual annotation provided evidence for many new alternative splice variants and 8 gene duplications. Over 1,100 transcripts without porcine sequence evidence were detected using cross-species annotation. We used a functional approach to discover and accurately annotate porcine immune response genes. A co-expression clustering analysis of transcriptomic data from selected experimental infections or immune stimulations of blood, macrophages or lymph nodes identified a large cluster of genes that exhibited a correlated positive response upon infection across multiple pathogens or immune stimuli. Interestingly, this gene cluster (cluster 4) is enriched for known general human immune response genes, yet contains many un-annotated porcine genes. A phylogenetic analysis of the encoded proteins of cluster 4 genes showed that 15% exhibited an accelerated evolution as compared to 4.1% across the entire genome. CONCLUSIONS: This extensive annotation dramatically extends the genome-based knowledge of the molecular genetics and structure of a major portion of the porcine immunome. Our complementary functional approach using co-expression during immune response has provided new putative immune response annotation for over 500 porcine genes. Our phylogenetic analysis of this core immunome cluster confirms rapid evolutionary change in this set of genes, and that, as in other species, such genes are important components of the pig's adaptation to pathogen challenge over evolutionary time. These comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response.

Characterisation of autophagy disruption in the ileum of pigs infected with Lawsonia intracellularis.

Lawsonia intracellularis is the aetiological agent of proliferative enteropathy, an enteric disease endemic in swine. Survival in its intracellular niche of the ileum epithelial lining requires the capacity to subvert, repress or exploit the host immune response to create an environment conducive to bacterial propagation. To better understand how L. intracellularis survives in its intracellular niche, we have performed an investigation into the dynamic relationship between infection and the host autophagy response by immunohistochemistry in experimentally infected porcine ileum samples.Beclin1, a protein required early in the autophagy pathway was observed to be distributed with a basal to apical concentration gradient in the crypts of healthy piglets, whilst infected piglets were observed to have no gradient of distribution and an increase in the presence of Beclin1 in crypts with histological characteristics of L. intracellularis residence. Detecting microtubule-associated protein light chain 3 (LC3) is used as a method for monitoring autophagy progression as it associates with mature autophagosomes. For LC3 there was no notable change in signal intensity between crypts with characteristic L. intracellularis infection and healthy crypts of uninfected pigs. Finally, as p62 is degraded with the internal substrate of an autophagosome it was used to measure autophagic flux. There was no observed reduction or redistribution of p62.These preliminary results of the autophagy response in the ileum suggest that L. intracellularis affects autophagy. This disruption to host ileum homeostasis may provide a mechanism that assists in bacterial propagation and contributes to pathogenesis.

Host inhibits replication of European porcine reproductive and respiratory syndrome virus in macrophages by altering differential regulation of type-I interferon transcriptional response.

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease caused by a positive RNA strand arterivirus. PRRS virus (PRRSV) interacts primarily with lung macrophages. Little is known how the virus subverts the innate immune response to initiate its replication in alveolar macrophages. Large-scale transcriptional responses of macrophages with different levels of susceptibility to PRRSV infection were compared over 30 h of infection. This study demonstrates a rapid and intense host transcriptional remodelling during the early phase of the replication of the virus which correlates with transient repression of type-I interferon transcript as early as 8 h post-infection. These results support the suggestion from previous studies that host innate immune response inhibits replication of European porcine reproductive and respiratory syndrome virus in macrophages by altering differential regulation of type-I interferon transcriptional response.

Metagenomic sequencing of clinical samples reveals a single widespread clone of Lawsonia intracellularis responsible for porcine proliferative enteropathy.

Lawsonia intracellularis is a Gram-negative obligate intracellular bacterium that is the aetiological agent of proliferative enteropathy (PE), a common intestinal disease of major economic importance in pigs and other animal species. To date, progress in understanding the biology of L. intracellularis for improved disease control has been hampered by the inability to culture the organism in vitro. In particular, our understanding of the genomic diversity and population structure of clinical L. intercellularis is very limited. Here, we utilized a metagenomic shotgun approach to directly sequence and assemble 21 L. intracellularis genomes from faecal and ileum samples of infected pigs and horses across three continents. Phylogenetic analysis revealed a genetically monomorphic clonal lineage responsible for infections in pigs, with distinct subtypes associated with infections in horses. The genome was highly conserved, with 94 % of genes shared by all isolates and a very small accessory genome made up of only 84 genes across all sequenced strains. In part, the accessory genome was represented by regions with a high density of SNPs, indicative of recombination events importing novel gene alleles. In summary, our analysis provides the first view of the population structure for L. intracellularis, revealing a single major lineage associated with disease of pigs. The limited diversity and broad geographical distribution suggest the recent emergence and clonal expansion of an important livestock pathogen.

Comparative genomics of Toll-like receptor signalling in five species.

BACKGROUND: Over the last decade, several studies have identified quantitative trait loci (QTL) affecting variation of immune related traits in mammals. Recent studies in humans and mice suggest that part of this variation may be caused by polymorphisms in genes involved in Toll-like receptor (TLR) signalling. In this project, we used a comparative approach to investigate the importance of TLR-related genes in comparison with other immunologically relevant genes for resistance traits in five species by associating their genomic location with previously published immune-related QTL regions. RESULTS: We report the genomic localisation of TLR1-10 and ten associated signalling molecules in sheep and pig using in-silico and/or radiation hybrid (RH) mapping techniques and compare their positions with their annotated homologues in the human, cattle and mouse whole genome sequences. We also report medium-density RH maps for porcine chromosomes 8 and 13. A comparative analysis of the positions of previously published relevant QTLs allowed the identification of homologous regions that are associated with similar health traits in several species and which contain TLR related and other immunologically relevant genes. Additional evidence was gathered by examining relevant gene expression and association studies. CONCLUSION: This comparative genomic approach identified eight genes as potentially causative genes for variations of health related traits. These include susceptibility to clinical mastitis in dairy cattle, general disease resistance in sheep, cattle, humans and mice, and tolerance to protozoan infection in cattle and mice. Four TLR-related genes (TLR1, 6, MyD88, IRF3) appear to be the most likely candidate genes underlying QTL regions which control the resistance to the same or similar pathogens in several species. Further studies are required to investigate the potential role of polymorphisms within these genes.

Evidence of CPV2c introgression into Croatia and novel insights into phylogeny and cell tropism.

Canine parvovirus type 2 (CPV2) emerged for the first time in 1978 and evolved into two antigenic variants CPV2a and CPV2b and the third new antigenic variant CPV2c reported in 2000 in Italy. During 2014 unexplained outbreaks of gastroenteritis were observed in kennels where an extensive vaccination program was ongoing and where vaccinated animals showed pathologic lesions consistent with typical parvovirosis. The aim of this study was to investigate whether CPV2 could have played a role in the emergence of these cases and to evaluate genetic or pathological specificities of the virus and the disease. Using PCR and phylogenetic analysis we showed that the CPV2c variant is circulating in Croatia and is in close relationships with isolates from North and South America. Histopathological lesions and cell tropism that are known for CPV2 we are reporting the identification of the virus in glial cells and ovaries. It seems that evolution of CPV and CPV2a-c and adaptation to dogs are two independent events. Croatian isolates had specific and some unique amino acid mutations under positive selection. The effect of the alterations on the immunoglobulin binding cannot be excluded.

Genetic perspectives on host responses to porcine reproductive and respiratory syndrome (PRRS).

Porcine reproductive and respiratory syndrome (PRRS) is the most economically important disease in pig populations, worldwide. Current research, both in vitro and in vivo, has failed to provide industry with a reliable or effective method to combat the disease. In this paper the present knowledge of the genetics of the host response to porcine reproductive and respiratory syndrome virus (PRRSV) is reviewed. Special reference is made to clinical signs of disease, in vitro and in vivo studies, and evidence of genetic variation in host response to the disease. It is concluded that although clinical signs are numerous, and in vitro and in vivo studies often fail to yield comparable results, there is sufficient evidence of genetic variation in host responses to infection to examine the possibility of breeding for enhanced resistance or tolerance. Advances in genomics have allowed examination of changes in gene expression in response to infection to be examined in tandem with genomewide linkage disequilibrium scans. These advances could allow the possibility for commercial breeding programs to be established, selecting for PRRS resistance or tolerance. When breeding for resistance to one disease, such as PRRS, it could be postulated that the viral control mechanism being exploited could have beneficial effects on resistance to other viral diseases in pigs if, for example, the mechanisms act on primary immune pathways associated with viral replication. Conversely, however, selection for disease resistance could facilitate an increase in susceptibility to other diseases or a reduction in overall productivity. Extensive data recording may be required to guard against such possibilities. Overall, breeding for disease control in pigs is an underutilized tool that could have desirable long-term effects in breeding programs. More research is needed to examine the possible pathways of PRRS resistance so that viable control methods can be found to ease the disease burden and thus increase animal welfare and economic viability.

Innate immune responses to replication of porcine reproductive and respiratory syndrome virus in isolated Swine alveolar macrophages.

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease caused by a positive RNA strand arterivirus. PRRS virus (PRRSV) interacts primarily with lung macrophages. Identifying the genetic components involved in host resistance/susceptibility would represent an important step forward in the design of disease control programs. In this study, alveolar macrophages derived from five commercial pig lines were used to study the innate immune response to PRRSV infection in vitro. Analysis by flow cytometry has demonstrated that bronchial alveolar lavage fluid (BALF) preparations were almost exclusively composed of alveolar macrophages and that the pigs tested were free from infection. Macrophages from the Landrace line showed significantly reduced virus replication and poor growth of PRRSV during 30 h of infection. By 72 h, PRRSV viral load was down to 2.5 log(10) TCID(50) compared with an average of 5 log(10) TCID(50) for the other breeds tested. These observations suggest that factors intrinsic to the Landrace breed may be responsible for this reduced or delayed response to PRRSV. Preliminary investigation suggests that the PRRSV coreceptor, sialoadhesin, may not be responsible for the Landrace macrophage phenotype as its abundance and localisation were comparable in all the breeds. Strikingly, we found that the reduced or delayed growth of PRRSV was temporally associated with high levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-8 mRNA accumulation and substantial reduction of secretion of IL-8, suggesting a key contributory role for cytokine synthesis and secretion during the innate immune response to PRRSV infection.

Lawsonia intracellularis exploits ß-catenin/Wnt and Notch signalling pathways during infection of intestinal crypt to alter cell homeostasis and promote cell proliferation.

Lawsonia intracellularis is an obligate intracellular bacterial pathogen that causes proliferative enteropathy (PE) in pigs. L. intracellularis infection causes extensive intestinal crypt cell proliferation and inhibits secretory and absorptive cell differentiation. However, the affected host upstream cellular pathways leading to PE are still unknown. ß-catenin/Wnt signalling is essential in maintaining intestinal stem cell (ISC) proliferation and self-renewal capacity, while Notch signalling governs differentiation of secretory and absorptive lineage specification. Therefore, in this report we used immunofluorescence (IF) and quantitative reverse transcriptase PCR (RTqPCR) to examine ß-catenin/Wnt and Notch-1 signalling levels in uninfected and L. intracellularis infected pig ileums at 3, 7, 14, 21 and 28 days post challenge (dpc). We found that while the significant increase in Ki67+ nuclei in crypts at the peak of L. intracellularis infection suggested enhanced cell proliferation, the expression of c-MYC and ASCL2, promoters of cell growth and ISC proliferation respectively, was down-regulated. Peak infection also coincided with enhanced cytosolic and membrane-associated ß-catenin staining and induction of AXIN2 and SOX9 transcripts, both encoding negative regulators of ß-catenin/Wnt signalling and suggesting a potential alteration to ß-catenin/Wnt signalling levels, with differential regulation of the expression of its target genes. We found that induction of HES1 and OLFM4 and the down-regulation of ATOH1 transcript levels was consistent with the increased Notch-1 signalling in crypts at the peak of infection. Interestingly, the significant down-regulation of ATOH1 transcript levels coincided with the depletion of MUC2 expression at 14 dpc, consistent with the role of ATOH1 in promoting goblet cell maturation. The lack of significant change to LGR5 transcript levels at the peak of infection suggested that the crypt hyperplasia was not due to the expansion of ISC population. Overall, simultaneous induction of Notch-1 signalling and the attenuation of ß-catenin/Wnt pathway appear to be associated with the inhibition of goblet cell maturation and enhanced crypt cell proliferation at the peak of L. intracellularis infection. Moreover, the apparent differential regulation of apoptosis between crypt and lumen cells together with the strong induction of Notch-1 signalling and the enhanced SOX9 expression along crypts 14 dpc suggest an expansion of actively dividing transit amplifying and/or absorptive progenitor cells and provide a potential basis for understanding the development and maintenance of PE.