Mapping of quantitative trait loci for immune response traits and expression patterns of Toll-like receptors in lymphoid tissues in pigs

The aim of this research was to identify the quantitative trait loci (QTL) affecting antibody and innate immune response traits. For this purpose, Duroc-Pietrain (DUPI) pigs (n = 319) were genotyped with 122 genetic markers and phenotypes of serum antibodies for Mycoplasma hyopneumoniae (Mh) and tetanus toxoid (TT), and interferon-gamma (IFNg) levels were measured following vaccinations (Mh, TT, Porcine Reproductive and Respiratory Syndrome Virus [PRRSV]). Line-cross and imprinting QTL analysis were performed using QTL Express. A total of 30 QTL (12, 6, and 12 QTL for Mh, TT antibody, and IFNg, respectively) were identified, of which 28 QTL were detected by line-cross and 2 QTL by imprinting model. The serum concentration of interleukin 2 (IL2), IL10, IFNg, Toll-like receptor (TLR2) and TLR9 were measured in another group of DUPI population (n = 334) following vaccinations that were genotyped with 82 genetic markers. A total of 33 single QTL were detected, of which eight, twelve and thirteen QTL were identified for immune traits in response to Mh, TT and PRRSV vaccine, respectively. All immune traits are influenced by multiple chromosomal regions implying multiple gene action. Furthermore, expression stability of nine commonly used housekeeping genes (HKG) was studied using qRT-PCR in most lymphoid tissues at different ages (newborn, young and adult) of pigs. This study found that HKG becomes heterogeneous with age and the geometric mean of the RPL4, PPIA and YWHAZ seem to be the most appropriate combination of HKG for accurate normalization of gene expression data in pigs. Moreover, the expression patterns of ten TLRs (1-10) were studied in the same tissues used for HKG study. This study revealed that TLRs mRNA expressions were affected by age and organs. Most of the TLRs expression was higher at young pigs compared to adult and newborn pigs. TLR3 gene was the highest abundant among all TLRs in most tissues. The western blot results of TLR2, 3 and 9 in selected tissues appeared to be consistent with the mRNA expression. The protein localization showed that TLRs expressing cells were abundant in lamina propria, Peyer’s patches in intestine, around and within the lymphoid follicles in the mesenteric and cervical lymph node, within the white pulp in spleen and on the lining cells in bronchioles in lungs. This expressions study first shed light on the expression patterns of all TLR genes in important lymphoid tissues including gut-associated lymphoid tissues in different ages of pigs.QTL-Kartierung von Immunreaktionsmerkmalen und Expressionsmuster des Toll-like Rezeptors in lymphatischen Gewebe beim Schwein Das Ziel dieser Studie war Quantitative Trait Loci (QTL), die Einfluss auf Antikörper und Merkmale der angeborenen Immunreaktion haben, zu identifizieren. Zu diesem Zweck wurden Duroc×Pietrain Schweine (DUPI) (n = 319) mittels 122 genetischen Markern genotypisiert. Die Phänotypen der Antikörperspiegel von Mycoplasma hyopneumoniae (Mh), Tetanus Toxoid (TT) sowie von Interferon-gamma (IFNg) wurden nach der Impfung (Mh, TT, Porcine Reproductive and Respiratory Syndrome Virus [PRRSV]) im Serum gemessen. Die QTL-Analysen wurden mit Hilfe der Software QTL-Express ausgeführt. Insgesamt wurden 30 QTL (jeweils 12, 6 und 12 QTL für Mh, TT-Antikörper und IFNg) identifiziert, wobei bei 2 QTL der Einfluss von Imprintingeffekten nachgewiesen wurde. In einer weiteren Gruppe der DUPI-Population (n = 334), welche mittels 82 Markern genetisch erfasst wurden, wurden die Serumkonzentrationen des Interleukins 2 (IL2), IL10, IFNg, Toll-like Rezeptor 2 (TLR2) und TLR9 nach der Impfung gemessen. Dabei wurden insgesamt 33 QTL detektiert, von denen jeweils 8, 12 und 13 QTL mit der Immunreaktion auf Mh, TT und PRRSV Impfungen assoziierten. Alle Immunmerkmale wurden durch mehrere chromosomale Regionen beeinflusst, was multiple Genaktionen impliziert. Darüber hinaus wurde die Expressionsstabilität von 9 häufig verwendeten ‚house keeping’ Genes (HKG) mit Hilfe der qRT-PCR inngerhalb von lymphatischen Geweben von Tieren unterschiedlichen Alters (Neugeborene, Jungtiere, Adulte) untersucht. Die Ergebnisse dieser Studie zeigten, dass die Expression der HKG mit dem Alter heterogen werden. Somit scheint das geometrische Mittel von RPL4, PPIA und YWHAZ am geeignetsten für die Normalisierung von Genexpressionsdaten beim Schwein zu sein. Dasselbe Gewebe der Referenzgenanalyse wurde für die Expressionsanalyse von zehn TLRs (1-10) verwendet. Diese Analyse zeigte, dass die TLRs mRNA-Expression vom Alter und Organen abhängig war. Dabei konnte eine höhere TLRs Expression bei Jungtieren und eine geringere bei Adulten und Neugeborenen detektiert werden. Das Gen TLR3 hatte das höchste Expressionsniveau in der Mehrzahl an Geweben von allen TRL Genen. Die Ergebnisse des Western Blot von TLR2, 3 und 9 in ausgewählten Geweben stimmten mit den Genexpressions-Analysen überein. Die Protein-Lokalisierung zeigte, dass TLRs in Zellen von lamina propria, Peyer’s Drüsen im Darm, in und um die lymphoiden Folikel des mesenterial und zervikalen Lymphknotens, im weißen Zellengwebe der Milz und in den Bronchialepithelien der Lunge exprimiert werden. Diese Expressionsstudie lieferte die ersten Erkenntnisse über die Expressionsmuster aller TLR in Lymphgeweben einschließlich der Lymphgewebe des Darms bei verschiedenen Alterstufen beim Schwein

Design and application of radio frequency identification systems

The recent world, development of effective technologies for linking the object wireless information is being prompted in various fields. Radio frequency identification (RFID) is the latest technology for automatic identification which allows the transmission of a unique serial number wirelessly. The purpose of this paper is to review RFID systems and its various components infrastructure. The components and features are still under research and being integrated in existing systems to create a marketable and potential new system. To achieve higher performance; low cost, low power RFID tag with efficient anticollision technique which provides a large throughput and flexible security mechanism is required. The review has shown different types of readers, antennas and tags which would becomes a bottleneck to reduce the RFID cost. The paper has shown details the entire components where RFID researchers will get benefit for the development of future technology. The challenges of RFID system design with the entire components (Reader, Tag and Antenna) and its advantages, disadvantages are briefly explained

Transcriptome profile of lung dendritic cells after in vitro porcine reproductive and respiratory syndrome virus (PRRSV) infection

The porcine reproductive and respiratory syndrome (PRRS) is an infectious disease that leads to high financial and production losses in the global swine industry. The pathogenesis of this disease is dependent on a multitude of factors, and its control remains problematic. The immune system generally defends against infectious diseases, especially dendritic cells (DCs), which play a crucial role in the activation of the immune response after viral infections. However, the understanding of the immune response and the genetic impact on the immune response to PRRS virus (PRRSV) remains incomplete. In light of this, we investigated the regulation of the host immune response to PRRSV in porcine lung DCs using RNA-sequencing (RNA-Seq). Lung DCs from two different pig breeds (Pietrain and Duroc) were collected before (0 hours) and during various periods of infection (3, 6, 9, 12, and 24 hours post infection (hpi)). RNA-Seq analysis revealed a total of 20,396 predicted porcine genes, which included breed-specific differentially expressed immune genes. Pietrain and Duroc infected lung DCs showed opposite gene expression courses during the first time points post infection. Duroc lung DCs reacted more strongly and distinctly than Pietrain lung DCs during these periods (3, 6, 9, 12 hpi). Additionally, cluster analysis revealed time-dependent co-expressed groups of genes that were involved in immune-relevant pathways. Key clusters and pathways were identified, which help to explain the biological and functional background of lung DCs post PRRSV infection and suggest IL-1β1 as an important candidate gene. RNA-Seq was also used to characterize the viral replication of PRRSV for each breed. PRRSV was able to infect and to replicate differently in lung DCs between the two mentioned breeds. These results could be useful in investigations on immunity traits in pig breeding and enhancing the health of pigs

Evaluation of suitable reference genes for gene expression studies in porcine alveolar macrophages in response to LPS and LTA

<p>Abstract</p> <p>Background</p> <p>To obtain reliable quantitative real-time PCR data, normalization relative to stable housekeeping genes (HKGs) is required. However, in practice, expression levels of 'typical' housekeeping genes have been found to vary between tissues and under different experimental conditions. To date, validation studies of reference genes in pigs are relatively rare and have never been performed in porcine alveolar macrophages (AMs). In this study, expression stability of putative housekeeping genes were identified in the porcine AMs in response to the stimulation with two pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Three different algorithms (geNorm, Normfinder and BestKeeper) were applied to assess the stability of HKGs.</p> <p>Results</p> <p>The mRNA expression stability of nine commonly used reference genes (<it>B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP </it>and <it>YWHAZ</it>) was determined by qRT-PCR in AMs that were stimulated by LPS and LTA <it>in vitro</it>. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (<it>P </it>< 0.0001). geNorm software revealed that <it>SDHA, B2M </it>and <it>RPL4 </it>showed a high expression stability in the irrespective to the stimulation group, while <it>SDHA, YWHAZ </it>and <it>RPL4 </it>showed high stability in non-stimulated control group. In all cases, <it>GAPDH </it>showed the least stability in geNorm. NormFinder revealed that <it>SDHA </it>was the most stable gene in all the groups. Moreover, geNorm software suggested that the geometric mean of the three most stable genes would be the suitable combination for accurate normalization of gene expression study.</p> <p>Conclusions</p> <p>There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms. In conclusion, the geometric mean of the <it>SDHA, YWHAZ </it>and <it>RPL4 </it>seemed to be the most appropriate combination of HKGs for accurate normalization of gene expression data in porcine AMs without knowing the type of bacterial pathogenic status of the animals.</p

Detection of quantitative trait loci affecting serum cholesterol, LDL, HDL, and triglyceride in pigs

<p>Abstract</p> <p>Background</p> <p>Serum lipids are associated with many serious cardiovascular diseases and obesity problems. Many quantitative trait loci (QTL) have been reported in the pig mostly for performance traits but very few for the serum lipid traits. In contrast, remarkable numbers of QTL are mapped for serum lipids in humans and mice. Therefore, the objective of this research was to investigate the chromosomal regions influencing the serum level of the total cholesterol (CT), triglyceride (TG), high density protein cholesterol (HDL) and low density protein cholesterol (LDL) in pigs. For this purpose, a total of 330 animals from a Duroc × Pietrain F2 resource population were phenotyped for serum lipids using ELISA and were genotyped by using 122 microsatellite markers covering all porcine autosomes for QTL study in QTL Express. Blood sampling was performed at approximately 175 days before slaughter of the pig.</p> <p>Results</p> <p>Most of the traits were correlated with each other and were influenced by average daily gain, slaughter date and age. A total of 18 QTL including three QTL with imprinting effect were identified on 11 different porcine autosomes. Most of the QTL reached to 5% chromosome-wide (CW) level significance including a QTL at 5% experiment-wide (GW) and a QTL at 1% GW level significance. Of these QTL four were identified for both the CT and LDL and two QTL were identified for both the TG and LDL. Moreover, three chromosomal regions were detected for the HDL/LDL ratio in this study. One QTL for HDL on SSC2 and two QTL for TG on SSC11 and 17 were detected with imprinting effect. The highly significant QTL (1% GW) was detected for LDL at 82 cM on SSC1, whereas significant QTL (5% GW) was identified for HDL/LDL on SSC1 at 87 cM. Chromosomal regions with pleiotropic effects were detected for correlated traits on SSC1, 7 and 12. Most of the QTL identified for serum lipid traits correspond with the previously reported QTL for similar traits in other mammals. Two novel QTL on SSC16 for HDL and HDL/LDL ratio and an imprinted QTL on SSS17 for TG were detected in the pig for the first time.</p> <p>Conclusion</p> <p>The newly identified QTL are potentially involved in lipid metabolism. The results of this work shed new light on the genetic background of serum lipid concentrations and these findings will be helpful to identify candidate genes in these QTL regions related to lipid metabolism and serum lipid concentrations in pigs.</p

Improvement of disease resistance in livestock: application of immunogenomics and CRISPR/Cas9 technology

Disease occurrence adversely affects livestock production and animal welfare, and havean impact on both human health and public perception of food-animals production. Combinedefforts from farmers, animal scientists, and veterinarians have been continuing to explore theeffective disease control approaches for the production of safe animal-originated food. Implementing the immunogenomics, along with genome editing technology, has been considering as the key approach for safe food-animal production through the improvement of the host genetic resistance. Next-generation sequencing, as a cutting-edge technique, enables the production of high throughput transcriptomic and genomic profiles resulted from host-pathogen interactions. Immunogenomics combine the transcriptomic and genomic data that links to host resistance to disease, and predict the potential candidate genes and their genomic locations. Genome editing, which involves insertion, deletion, or modification of one or more genes in the DNA sequence, is advancing rapidly and may be poised to become a commercial reality faster than it has thought. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) [CRISPR/Cas9] system has recently emerged as a powerful tool for genome editing in agricultural food production including livestock disease management. CRISPR/Cas9 mediated insertion of NRAMP1 gene for producing tuberculosis resistant cattle, and deletion of CD163 gene for producing porcine reproductive and respiratory syndrome (PRRS) resistant pigs are two groundbreaking applications of genome editing in livestock. In this review, we have highlighted the technological advances of livestock immunogenomics and the principles and scopes of application of CRISPR/Cas9-mediated targeted genome editing in animal breeding for disease resistance.Fil: Islam, Md Aminul. Tohoku University; Japón. Bangladesh Agricultural University; BangladeshFil: Sharmin, Aqter Rony. Bangladesh Agricultural University; BangladeshFil: Rahman, Mohammad Bozlur. Department of Livestock Services; BangladeshFil: Cinar, Mehmet Ulas. Erciyes University; Turquía. Washington State University; Estados UnidosFil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; JapónFil: Uddin, Muhammad Jasim. Bangladesh Agricultural University; Bangladesh. University of Queensland; AustraliaFil: Kitazawa, Haruki. Tohoku University; Japó

Brucellosis among ruminants in some districts of Bangladesh using four conventional serological assays

Brucellosis causes a great economic loss to the livestock industries through abortion, infertility, birth of weak and dead offspring, increased calving interval and reduction of milk yield and it is endemic in Bangladesh. The present study was performed to know the seroprevalence of brucellosis for 1000 ruminants (135 Buffaloes, 465 cattle, 230 goats and 170 sheep) in five different districts of Bangladesh by four conventional serological tests such as: Rose Bengal Plate Test (RBT), tube agglutination test (TAT), competitive enzyme-linked immunosorbent assay (C-ELISA), and Fluorescent polarization assay (FPA). Sheep has the highest prevalence (8.24%) of brucellosis. The seroprevalence of brucellosis was significantly higher in animals with previous abortion record in case of buffaloes, cattle, goats and sheep than that with no abortion record. C-ELISA can be the most suitable choice for extensive use in many kinds of livestocks and accurate estimation of Brucella antibodies in ruminants in Bangladesh