Occurrence of 'Candidatus Mycoplasma haemosuis' in fattening pigs, sows and piglets in Germany using a novel gap-based quantitative real-time PCR assay.

BACKGROUND: The appearance of the novel porcine haemotrophic mycoplasma (HM) species 'Candidatus Mycoplasma haemosuis' was reported in apparently healthy but also in clinically sick animals in China, Korea and in a case report from Germany. Outside of Asia, however, nothing further is known about the frequency of 'Ca. M. haemosuis' in pigs to date. To investigate the distribution of this novel HM species in Germany, fattening pigs, sows and pre-suckling piglets were examined using a herein developed quantitative real-time PCR assay (qPCR). Because the piglets were sampled before the first colostrum uptake, additional information on a possible vertical transmission from dams to their offspring was obtained. RESULTS: Our novel qPCR assay successfully detected 'Ca. M. haemosuis' in all blood samples from the 'Ca. M. haemosuis'-infected pigs. No cross-reactivity was detected when DNA from non-target Mycoplasma spp. and other bacterial species representing 105 bacteria/reaction were used as a template. The lower limit of detection of the qPCR was thus 10 gap gene copies per reaction and 2.5 x 103 genome equivalents (GE) per mL blood. 'Candidatus M. haemosuis' was detected by this qPCR in blood samples from a total out of 6.25% sows (13/208), 4.50% pre-suckling piglets (28/622) and 17.50% fattening pigs (35/200). On farm level, 3 out of 21 piglet producing farms (14.28%) and 9 out of 20 fattening farms (45.00%) were positive for 'Ca. M. haemosuis'. Co-infections with M. suis were evident in all age groups. CONCLUSION: 'Candidatus M. haemosuis' infection is present in German pig farms and the detection of the novel porcine HM species in piglets immediately after birth before colostrum intake indicates vertical transmission. The novel qPCR assay specific for 'Ca. M. haemosuis' described herein will be a prerequisite for future studies on the prevalence, epidemiology as well as the clinical and economic impact of 'Ca. M. haemosuis' infections.

Discriminating the eight genotypes of the porcine circovirus type 2 with TaqMan-based real-time PCR.

BACKGROUND: The porcine circovirus type 2 (PCV2) is divided into eight genotypes including the previously described genotypes PCV2a to PCV2f and the two new genotypes PCV2g and PCV2h. PCV2 genotyping has become an important task in molecular epidemiology and to advance research on the prophylaxis and pathogenesis of PCV2 associated diseases. Standard genotyping of PCV2 is based on the sequencing of the viral genome or at least of the open reading frame 2. Although, the circovirus genome is small, classical sequencing is time consuming, expensive, less sensitive and less compatible with mass testing compared with modern real-time PCR assays. Here we report about a new PCV2 genotyping method using qPCR. METHODS: Based on the analysis of several hundred PCV2 full genome sequences, we identified PCV2 genotype specific sequences or single-nucleotide polymorphisms. We designed six TaqMan PCR assays that are specific for single genotypes PCV2a to PCV2f and two qPCRs targeting two genotypes simultaneously (PCV2g/PCV2d and PCV2h/PCV2c). To improve specific binding of oligonucleotide primers and TaqMan probes, we used locked nucleic acid technology. We evaluated amplification efficiency, diagnostic sensitivity and tested assay specificity for the respective genotypes. RESULTS: All eight PCV2 genotype specific qPCRs demonstrated appropriate amplification efficiencies between 91 and 97%. Testing samples from an epidemiological field study demonstrated a diagnostic sensitivity of the respective genotype specific qPCR that was comparable to a highly sensitive pan-PCV2 qPCR system. Genotype specificity of most qPCRs was excellent. Limited unspecific signals were obtained when a high viral load of PCV2b was tested with qPCRs targeting PCV2d or PCV2g. The same was true for the PCV2a specific qPCR when high copy numbers of PCV2d were tested. The qPCR targeting PCV2h/PCV2c showed some minor cross-reaction with PCV2d, PCV2f and PCV2g. CONCLUSION: Genotyping of PCV2 is important for routine diagnosis as well as for epidemiological studies. The introduced genotyping qPCR system is ideal for mass testing and should be a valuable complement to PCV2 sequencing, especially in the case of simultaneous infections with multiple PCV2 genotypes, subclinically infected animals or research studies that require large sample numbers.

Clinical, haematological and pathomorphological findings in Mycoplasma suis infected pigs.

BACKGROUND: Mycoplasma suis (M. suis) belongs to the group of haemotrophic mycoplasmas and is known as the causative agent of infectious anaemia in pigs. In the last few years valuable insights into the mechanism of adhesion and invasion, shedding patterns and cell tropism of M. suis were gained by the use of new molecular techniques. However, details on M. suis induced lesions as well as the distribution of M. suis in different organs are still lacking. Therefore, seven splenectomised pigs were experimentally infected and clinical and laboratory investigations as well as a detailed histopathological examination were performed. Detection and quantification of M. suis DNA in blood and various tissue samples was done using a quantitative real-time PCR. RESULTS: During the course of experimental infection, periodically occurring signs of infectious anaemia of pigs including severe icteroanaemia, fever, apathy and anorexia were observed. In addition, dermatological manifestations such as haemorrhagic diathesis presenting as petechiae occurred. The most important haematological alterations were normochromic, normocytic anaemia, hypoglycaemia as well as increased bilirubin and urea concentrations. Necropsy revealed predominant evidence of haemolysis with consecutive anaemia, as well as disseminated intravascular coagulation. M. suis was found in all investigated tissues with the highest copy numbers found in the kidneys. In Giemsa stained sections M. suis was only detected red blood cell (RBC)-associated. CONCLUSION: In the present study, no RBC independent sequestration of M. suis was detected in organs of experimentally infected pigs. Pathological findings are most likely resulting from haemolysis, consecutive anaemia as well as from disseminated intravascular coagulation and subsequent organ impairments.

Detection of Mycoplasma suis in pre-suckling piglets indicates a vertical transmission.

BACKGROUND: Transmission of Mycoplasma (M.) suis mainly occurs via iatrogenic or zootechnical manipulations or due to ranking fights. Other transmission routes including ingestion of secretes/excretes; blood-sucking arthropods and intra-uterine transmission have thought to play an epidemiological role without being experimentally proven. To investigate a vertical transmission of M. suis under field conditions blood samples from pre-suckling piglets and their corresponding dam were examined for M. suis by quantitative polymerase chain reaction (qPCR) in 21 farms in Southern Germany. RESULTS: A total of 14.35% of the 474 blood samples from pre-suckling piglets reacted qPCR positive. Additionally, M. suis was detected in 65 (31.25%) of the 208 sows at farrowing. On farm level, 16 (76.2%) of the 21 farms had at least one M. suis positive animal. M. suis positive farms had an average of 0.41 more stillborn piglets per litter than M. suis negative farms (p = 0.007). CONCLUSION: The present study provides further insights into M. suis infection dynamics as it is the first detection of M. suis in piglets immediately after birth prior to colostrum intake and the first large scale investigation of M. suis in sows at farrowing.

Full genome characterization of porcine circovirus type 3 isolates reveals the existence of two distinct groups of virus strains.

BACKGROUND: The occurrence of the novel porcine circovirus type 3 (PCV3) was reported from the Americas, Asia and Europe. Although this virus was detected in association with various clinical syndromes in pigs, its role as possible swine pathogen remains unclear. PCV3 was detected with high prevalence in Polish farms, but to date no genome sequences were available from European PCV3 strains. METHODS: We collected 1060 serum samples from piglets at the age of 20-24 weeks from 53 farms distributed all over Germany. PCV3 DNA was detected using a real-time PCR and subsequently complete PCV3 genome sequences were obtained after multiply primed rolling circle amplification and sequencing of overlapping PCR products. Phylogenetic analysis was performed by neighbor-joining method and maximum likelihood method. RESULTS: We obtained 15 complete PCV3 genome sequences as well as nine partial sequences including the putative ORFs 1, 2 and 3 from PCV3 viremic animals in German pig farms. Phylogenetic analysis of these German as well as 30 full genome sequences received from GenBank divided the PCV3 strains into two main groups and several subclusters. Furthermore, we were able to define group specific amino acid patterns in open reading frame 1 and 2. CONCLUSION: PCV3 is distributed with high prevalence in German pig industry. Phylogenetic analysis revealed two clearly separated groups of PCV3 strains, which might be considered as PCV3 genotypes. Specific nucleotide and amino acid marker positions may serve for easy and fast intraspecies classification and genotyping of PCV3 strains. No correlation between PCV3 variants with their geographical origin was evident. We found the same diversity of PCV3 strains in Germany as in other countries. We hypothesize that PCV3 is not a newly emerging virus in the German pig population. Future studies will have to show, if PCV3 genotype specific biological properties are evident.

Updating the proteome of the uncultivable hemotrophic Mycoplasma suis in experimentally infected pigs.

Mycoplasma suis belongs to the hemotrophic mycoplasmas that are associated with acute and chronic anemia in a wide range of livestock and wild animals. The inability to culture M. suis in vitro has hindered its characterization at the molecular level. Since the publication of M. suis genome sequences in 2011 only one proteome study has been published. Aim of the presented study was to significantly extend the proteome coverage of M. suis strain KI_3806 during acute infection by applying three different protein extraction methods followed by 1D SDS-PAGE and LC-MS/MS. A total of 404 of 795 M. suis KI_3806 proteins (50.8%) were identified. Data analysis revealed the expression of 83.7% of the predicted ORFs with assigned functions but also highlights the expression of 179 of 523 (34.2%) hypothetical proteins with unknown functions. Computational analyses identified expressed membrane-associated hypothetical proteins that might be involved in adhesion or host-pathogen interaction. Furthermore, analyses of the expressed proteins indicated the existence of a hexose-6-phosphate-transporter and an ECF transporter. In conclusion, our proteome study provides a further step toward the elucidation of the unique life cycle of M. suis and the establishment of an in vitro culture. All MS data have been deposited in the ProteomeXchange with identifier PXD002294 (http://proteomecentral.proteomexchange.org/dataset/PXD002294).

Comparison of porcine epidemic diarrhea viruses from Germany and the United States, 2014.

Since 2013, highly virulent porcine epidemic diarrhea virus has caused considerable economic losses in the United States. To determine the relation of US strains to those recently causing disease in Germany, we compared genomes and found that the strain from Germany is closely related to variants in the United States.

Effects of the glucagon-like peptide-1 receptor agonist liraglutide in juvenile transgenic pigs modeling a pre-diabetic condition.

BACKGROUND: The glucagon-like peptide-1 receptor (GLP1R) agonist liraglutide improves glycemic control and reduces body weight of adult type 2 diabetic patients. However, efficacy and safety of liraglutide in adolescents has not been systematically investigated. Furthermore, possible pro-proliferative effects of GLP1R agonists on the endocrine and exocrine pancreas need to be further evaluated. We studied effects of liraglutide in adolescent pigs expressing a dominant-negative glucose-dependent insulinotropic polypeptide receptor (GIPR(dn)) in the beta-cells, leading to a pre-diabetic condition including disturbed glucose tolerance, reduced insulin secretion and progressive reduction of functional beta-cell mass. METHODS: Two-month-old GIPR(dn) transgenic pigs were treated daily with liraglutide (0.6-1.2 mg per day) or placebo for 90 days. Glucose homeostasis was evaluated prior to and at the end of the treatment period by performing mixed meal and intravenous glucose tolerance tests (MMGTT and IVGTT). Finally animals were subjected to necropsy and quantitative-stereological analyses were performed for evaluation of alpha- and beta-cell mass, beta-cell proliferation as well as acinus-cell proliferation. RESULTS: MMGTT at the end of the study revealed 23% smaller area under the curve (AUC) for glucose, a 36% smaller AUC insulin, and improved insulin sensitivity, while IVGTT showed a 15% smaller AUC glucose but unchanged AUC insulin in liraglutide- vs. placebo-treated animals. Liraglutide led to marked reductions in body weight gain (-31%) and food intake (-30%) compared to placebo treatment, associated with reduced phosphorylation of insulin receptor beta (INSRB)/insulin-like growth factor-1 receptor beta (IGF1RB) and protein kinase B (AKT) in skeletal muscle. Absolute alpha- and beta-cell mass was reduced in liraglutide-treated animals, but alpha- and beta-cell mass-to-body weight ratios were unchanged. Liraglutide neither stimulated beta-cell proliferation in the endocrine pancreas nor acinus-cell proliferation in the exocrine pancreas, excluding both beneficial and detrimental effects on the pig pancreas. CONCLUSIONS: Although plasma liraglutide levels of adolescent transgenic pigs treated in our study were higher compared to human trials, pro-proliferative effects on the endocrine or exocrine pancreas or other liraglutide-related side-effects were not observed.

PCV2 vaccination induces IFN-γ/TNF-α co-producing T cells with a potential role in protection.

Porcine circovirus type 2 (PCV2) is one of the economically most important pathogens for swine production worldwide. Vaccination is a powerful tool to control porcine circovirus diseases (PCVD). However, it is not fully understood how PCV2 vaccination interacts with the porcine immune system. Especially knowledge on the cellular immune response against PCV2 is sparse. In this study we analysed antigen-specific T cell responses against PCV2 in a controlled vaccination and infection experiment. We focused on the ability of CD4(+) T cells to produce cytokines using multicolour flow cytometry (FCM). Vaccination with a PCV2 subunit vaccine (Ingelvac CircoFLEX®) induced PCV2-specific antibodies only in five out of 12 animals. Conversely, vaccine-antigen specific CD4(+) T cells which simultaneously produced IFN-γ and TNF-α and had a phenotype of central and effector memory T cells were detected in all vaccinated piglets. After challenge, seroconversion occurred earlier in vaccinated and infected pigs compared to the non-vaccinated, infected group. Vaccinated pigs were fully protected against viremia after subsequent challenge. Therefore, our data suggests that the induction of IFN-γ/TNF-α co-producing T cells by PCV2 vaccination may serve as a potential correlate of protection for this type of vaccine.

Magnitude and kinetics of multifunctional CD4+ and CD8ß+ T cells in pigs infected with swine influenza A virus.

Although swine are natural hosts for influenza A viruses, the porcine T-cell response to swine influenza A virus (FLUAVsw) infection has been poorly characterized so far. We have studied Ki-67 expression and FLUAVsw-specific production of IFN-γ, TNF-α and IL-2 in CD4(+) and CD8ß(+) T cells isolated from piglets that had been intratracheally infected with a H1N2 FLUAVsw isolate. IFN-γ(+)TNF-α(+)IL-2(+) multifunctional CD4(+) T cells were present in the blood of all infected animals at one or two weeks after primary infection and their frequency increased in four out of six animals after homologous secondary infection. These cells produced higher amounts of IFN-γ, TNF-α and IL-2 than did CD4(+) T cells that only produced a single cytokine. The vast majority of cytokine-producing CD4(+) T cells expressed CD8α, a marker associated with activation and memory formation in porcine CD4(+) T cells. Analysis of CD27 expression suggested that FLUAVsw-specific CD4(+) T cells included both central memory and effector memory populations. Three out of six animals showed a strong increase of Ki-67(+)perforin(+) CD8ß(+) T cells in blood one week post infection. Blood-derived FLUAVsw-specific CD8ß(+) T cells could be identified after an in vitro expansion phase and were multifunctional in terms of CD107a expression and co-production of IFN-γ and TNF-α. These data show that multifunctional T cells are generated in response to FLUAVsw infection of pigs, supporting the idea that T cells contribute to the efficient control of infection.

Dietary calcium concentration and cereals differentially affect mineral balance and tight junction proteins expression in jejunum of weaned pigs.

Ca plays an essential role in bone development; however, little is known about its effect on intestinal gene expression in juvenile animals. In the present study, thirty-two weaned pigs (9·5 (SEM 0·11) kg) were assigned to four diets that differed in Ca concentration (adequate v. high) and cereal composition (wheat-barley v. maize) to assess the jejunal and colonic gene expression of nutrient transporters, tight junction proteins, cytokines and pathogen-associated molecular patterns, nutrient digestibility, Ca balance and serum acute-phase response. To estimate the impact of mucosal bacteria on colonic gene expression, Spearman's correlations between colonic gene expression and bacterial abundance were computed. Faecal Ca excretion indicated that more Ca was available along the intestinal tract of the pigs fed high Ca diets as compared to the pigs fed adequate Ca diets (P> 0.05). High Ca diets decreased jejunal zonula occludens 1 (ZO1) and occludin (OCLN) expression, up-regulated jejunal expression of toll-like receptor 2 (TLR2) and down-regulated colonic GLUT2 expression as compared to the adequate Ca diets (P< 0.05). Dietary cereal composition up-regulated jejunal TLR2 expression and interacted (P= 0.021) with dietary Ca on colonic IL1B expression; high Ca concentration up-regulated IL1B expression with wheat-barley diets and down-regulated it with maize diets. Spearman's correlations (r> 0·35; P< 0·05) indicated an association between operational taxonomic units assigned to the phyla Bacteroidetes, Firmicutes and Proteobacteria and bacterial metabolites and mucosal gene expression in the colon. The present results indicate that high Ca diets have the potential to modify the jejunal and colonic mucosal gene expression response which, in turn, interacts with the composition of the basal diet and mucosa-associated bacteria in weaned pigs.

Emergence of porcine epidemic diarrhea virus in southern Germany.

BACKGROUND: Over the last years, porcine epidemic diarrhea virus (PEDV) has caused devastating enteric diseases in the US and several countries in Asia, while outbreaks in Europe have only been reported sporadically since the 1980s. At present, only insufficient information is available on currently circulating PEDV strains in Europe and their impact on the European swine industry. In this case report, we present epidemic outbreaks of porcine epidemic diarrhea in three farms in South-Western Germany. CASE PRESENTATION: Epidemic outbreaks of diarrhea affecting pigs of all age groups were reported in three farms, one fattening farm and two piglet producing farms, in South-Western Germany between May and November 2014. In the fattening farm yellowish, watery diarrhea without evidence of mucus or blood was associated with a massive reduction of feed consumption. Severity of clinical signs and mortality in young suckling pigs varied significantly between the two affected sow farms. While mortality in suckling piglets reached almost 70 % in one sow herd, no increase in suckling piglet mortality was observed in the second sow farm. In all three cases, PEDV was confirmed in feces and small intestines by RT-qPCR. Phylogenetic analyses based on full-length PEDV genomes revealed high identity among strains from all three herds. Moreover, the German strains showed very high nucleotide identity (99.4 %) with a variant of PEDV (OH851) that was isolated in the United States in January 2014. This strain with insertions and deletions in the S-gene (so called INDEL strains) was reported to show lower virulence. Slightly lower identities were found with other strains from the US and Asia. CONCLUSION: Phylogenetic information on the distribution of PEDV strains in Europe is severely lacking. In this case report we demonstrate that acute outbreaks of PEDV occurred in southern Germany in 2014. Current strains were clearly different from isolates found in the 1980s and were closely related to a PEDV variant found in the US in 2014. Moreover, the present case report indicates that variant strains of PEDV, containing insertions and deletions in the S gene, which were reported to be of lower virulence, might be able to cause high mortality in suckling piglets.

Associations among Haemophilus parasuis, Mycoplasma hyorhinis, and porcine reproductive and respiratory syndrome virus infections in pigs with polyserositis.

The objective of this study was to determine the associations among Haemophilus parasuis, Mycoplasma hyorhinis, and porcine reproductive and respiratory syndrome (PRRS) virus (EU-field strain) infections in 95 pigs with polyserositis. A significant association between H. parasuis and M. hyorhinis was identified. H. parasuis and M. hyorhinis were significantly more often detected in PRRS virus positive pigs.

Association entre infections avecHaemophilus parasuis, Mycoplasma hyorhinis,et le virus du syndrome dysgénésique et respiratoire du porc chez les porcs atteints de polysérosite. L'objectif de l'étude était d'étudier l'association de Haemophilus parasuis, Mycoplasma hyorhinis, et du SDRP (souche européenne-sauvage) chez 95 porcs atteints de polysérosite. Une association significative a été mise en évidence entre H. parasuis et M. hyorhinis. H. parasuis et M. hyorhinis ont été significativement plus fréquemment détectés chez SDRP positif porcs.(Traduit par les auteurs).

Different sample types in pigs challenged with Haemophilus parasuis following two treatment schemes with tulathromycin.

This study aimed to test the efficacy of samplings for the detection of Haemophilus parasuis after metaphylactic treatment and subsequent challenge using an established model for Glässer's disease. In this model, 36 piglets were equally assigned to a negative control, a positive control, and two trial groups receiving tulathromycin 7 or 4 days prior to challenge. The piglets of three groups were challenged intratracheally with H. parasuis serovar 5. As a result, four pigs in each challenged group died or had to be euthanised within 10 days post challenge. The remaining 15 pigs of these challenged groups survived until termination of the experiment (days 14-15). All pigs were necropsied and collective swabs of serosal surfaces were tested by bacterial culture and PCR. Samples of tarsal synovial fluid and joint capsule, cerebrospinal fluid (CSF) and brain swabs were tested by PCR. A total of 22 out of the 27 challenged animals had macroscopically detectable polyserositis and all of them tested positive in the collective swab samples. Haemophilus parasuis was more frequently detected in pigs that died within the first 10 days compared to those surviving until days 14-15 (P < 0.001), and those that succumbed within 10 days showed higher positivity rates in the brain and CSF. All pigs which were positive in the CSF had detectable meningitis. At days 14-15, joint samples from 5 of the remaining 15 pigs tested positive for H. parasuis. Four of these five animals did not show any macroscopic or histological lesions in the joints. In conclusion, collective swabs were the best sample material in acute cases, whereas samples from the joints gave the best results in chronic cases. In this challenge model it was not possible to prove the metaphylactic effect of tulathromycin administered 4 and 7 days prior to infection with H. parasuis.

Haemotrophic Mycoplasmas Infecting Pigs: A Review of the Current Knowledge.

Haemotrophic mycoplasmas (haemoplasmas) are a group of highly specific and adapted bacteria. Three different haemoplasma species in pigs are known to date: Mycoplasma (M.) suis, M. parvum and 'Candidatus (Ca.) M. haemosuis'. Even though these bacteria have been known in pig farming for a long time, it is difficult to draw general conclusions about the relevance of their infections in pigs. This review summarizes the current knowledge on the three porcine haemoplasma species with regards to clinical and pathological descriptions, pathobiology, epidemiology and diagnostics as well as prevention and therapy. Overall, it is clear that considerably more data are available for M. suis than for the other two species, but generally, porcine haemoplasmas were found to be highly prevalent all over the world. Mycoplasma suis is the most virulent species, causing acute infectious anaemia in pigs (IAP), whereas M. parvum usually results in chronic and subclinical infections associated with performance losses. Little is known about the clinical significance of the recently discovered third porcine species 'Ca. M. haemosuis'. So far, the described pathogenic mechanisms mainly include direct destruction of erythrocytes via adhesion, invasion, eryptosis and nutrient scavenging, indirect erythrocyte lysis due to immune-mediated events and immune dysregulation processes. A review of published diagnostic data confirms PCR assays as the current standard method, with various cross-species and species-specific protocols. Overall, there is a need for further examination to obtain valuable insights for practical application, specifically regarding the importance of subclinical infections in naturally infected animals. An essential requirement for this will be to gain a more comprehensive understanding of the mechanisms operating between the host and the pathogen.

Hemotrophic Mycoplasmas-Vector Transmission in Livestock.

Hemotrophic mycoplasmas (HMs) are highly host-adapted and specialized pathogens infecting a wide range of mammals including farm animals, i.e., pigs, cattle, sheep, and goats. Although HMs have been known for over 90 years, we still do not know much about the natural transmission routes within herds. Recently, it has been repeatedly discussed in publications that arthropod vectors may play a role in the transmission of HMs from animal to animal. This is mainly since several HM species could be detected in different potential arthropod vectors by PCR. This review summarizes the available literature about the transmission of bovine, porcine, ovine, and caprine HM species by different hematophagous arthropod vectors. Since most studies are only based on the detection of HMs in potential vectors, there are rare data about the actual vector competence of arthropods. Furthermore, there is a need for additional studies to investigate, whether there are biological vectors in which HMs can multiply and be delivered to new hosts.

Influenza surveillance in pigs: balancing act between broad diagnostic coverage and specific virus characterization.

BACKGROUND: Monitoring of infectious diseases on swine farms requires a high diagnostic sensitivity and specificity of the test system. Moreover, particularly in cases of swine influenza A virus (swIAV) it is desirable to include characterization of the virus as precisely as possible. This is indispensable for strategies concerning prophylaxis of swIAV and furthermore, to meet the requirements of a purposeful monitoring of newly emerging swIAV strains in terms of vaccine design and public health. Within the present cross-sectional study, we compared the diagnostic value of group samples (wipes of surfaces with direct contact to mouth/nose, dust wipes, udder skin wipes, oral fluids) to individual samples (nasal swabs, tracheobronchial swabs) for both swIAV identification and characterization. Sampling included different stages of pig production on 25 sow farms with attached nursery considered as enzootically infected with swIAV. Firstly, samples were analyzed for IAV genome and subsequently samples with Ct-values < 32 were subtyped by multiplex RT-qPCR. RESULTS: Nasal swabs of suckling piglets and nursery pigs resulted in a higher odds to detect swIAV (p < 0.001) and to identify swIAV subtypes by RT-qPCR (p < 0.05) compared to nasal swabs of sows. In suckling piglets, significant higher rates of swIAV detection could be observed for nasal swabs (p = 0.007) and sow udder skin wipes (p = 0.036) compared to contact wipes. In the nursery, group sampling specimens were significantly more often swIAV positive compared to individual samples (p < 0.01), with exception of the comparison between contact wipes and nasal swabs (p = 0.181). However, in general nasal swabs were more likely to have Ct-value < 32 and thus, to be suitable for subtyping by RT-qPCR compared to dust wipes, contact wipes, udder skin wipes and tracheobronchial swabs (p < 0.05). Interestingly, different subtypes were found in different age groups as well as in different specimens in the same holding. CONCLUSION: Although population-based specimens are highly effective for swIAV monitoring, nasal swabs are still the preferable sampling material for the surveillance of on-farm circulating strains due to significantly higher virus loads. Remarkably, sampling strategies should incorporate suckling piglets and different age groups within the nursery to cover as many as possible of the on-farm circulating strains.

Changing dietary calcium-phosphorus level and cereal source selectively alters abundance of bacteria and metabolites in the upper gastrointestinal tracts of weaned pigs.

Several dietary ingredients may affect the bacterial community structure and metabolism in the porcine gut and may therefore influence animals' health and performance. This study investigated the effects of cereal source and calcium-phosphorus (CaP) level in the diet on bacterial microbiota and metabolites, nutrient intake, and gut environment in weaned pigs. Pigs (n=8/treatment) were fed wheat-barley- or corn-based diets with an adequate or high CaP level for 14 days. Effects on microbiota in the stomach, ileum, and midcolon were assessed using quantitative PCR. Data showed that Enterobacteriaceae, Campylobacter spp., and Helicobacter spp., which all contain highly immune reactive lipopolysaccharide (LPS), were abundant at all gut sites. Diet effects on bacteria and metabolites were moderate and occurred mainly in the upper gut, whereas no effects on bacteria, fermentation products, and LPS could be observed in the colon. Differences in carbohydrate intake with corn versus wheat-barley diets selectively stimulated Bifidobacterium in the stomach and ileum. There was a growth advantage for a few bacterial groups in the stomach and ileum of pigs fed the high versus adequate CaP level (i.e., gastric Enterobacteriaceae and ileal Enterococcus, Bacteroides-Prevotella-Porphyromonas, and Campylobacter). Interestingly, gastrointestinal pH was not affected by dietary CaP level. The present findings demonstrate the stability of the bacterial community and gut environment toward dietary changes even in young pigs. The results on stimulation of gastric and ileal Bifidobacterium by corn diets may be employed in nutritional strategies to support gut health after weaning.

CD27 expression discriminates porcine T helper cells with functionally distinct properties.

Differentiation of porcine T helper cells is still poorly investigated, partly due to a lack of monoclonal antibodies (mAbs) specific for molecules involved in this process. Recently, we identified a mAb specific for porcine CD27 and showed that CD27 is expressed by all naïve CD8α- T helper cells but divides CD8α+ T helper cells into a CD27+ and a CD27- subset. In the present study, detailed phenotypical and functional analyses of these T-helper cell subpopulations were performed. Naïve CD8α-CD27+ T helper cells predominantly resided in various lymph nodes, whereas higher proportions of CD8α+CD27+ and CD8α+CD27- T helper cells were found in blood, spleen and liver. Both CD8α+CD27+ and CD8α+CD27- T helper cells were capable of producing IFN-γ upon in vitro polyclonal stimulation and antigen-specific restimulation. Experiments with sorted CD8α-CD27+, CD8α+CD27+ and CD8α+CD27- T-helper cell subsets following polyclonal stimulation revealed the lowest proliferative response but the highest ability for IFN-γ and TNF-α production in the CD8α+CD27- subset. Therefore, these cells resembled terminally differentiated effector memory cells as described in human. This was supported by analyses of CCR7 and CD62L expression. CD8α+CD27- T helper cells were mostly CCR7- and had considerably reduced CD62L mRNA levels. In contrast, expression of both homing-receptors was increased on CD8α+CD27+ T helper cells, which also had a proliferation rate similar to naïve CD8α-CD27+ T helper cells and showed intermediate levels of cytokine production. Therefore, similar to human, CD8α+CD27+ T helper cells displayed a phenotype and functional properties of central memory cells.

Antimicrobial Susceptibility from a One Health Perspective Regarding Porcine Escherichia coli from Bavaria, Germany.

Antimicrobial resistance is one of the most crucial One Health topics worldwide. Consequently, various national and international surveillance programs collect data and report trends regularly. Ceftiofur, colistin and enrofloxacin belong to the most important and critical class of anti-infective medications in both human and veterinary medicine. In the present study, antimicrobial resistance was analyzed using the epidemiological cut-off (ECOFF) value on 6569 Escherichia coli isolated from pigs in Bavaria, Germany, during five years, from 2016 to 2020. The statistically relevant results regarding antimicrobial resistance revealed a decrease for colistin, an increase for enrofloxacin, and a constant level for ceftiofur. In Germany, the usage of all three antimicrobial substances in livestock has fallen by 43.6% for polypeptides, 59.0% for fluoroquinolones and 57.8% for the 3rd + 4th generation cephalosporines during this time. Despite the decline in antimicrobial usage, a reduction regarding antimicrobial resistance was solely observed for colistin. This finding illustrates that in addition to the restriction of pharmaceutical consumption, further measures should be considered. Improved biosecurity concepts, a reduction in crowding, and controlled animal movements on farms may play a key role in finally containing the resistance mechanisms of bacteria in farm animals.