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.

Efficacy of one dose vaccination against experimental infection with two Mycoplasma hyopneumoniae strains.

BACKGROUND: Mycoplasma hyopneumoniae (M. hyopneumoniae) is the primary agent of enzootic pneumonia in pigs. Pigs are often infected with different M. hyopneumoniae strains. This study assessed the efficacy of vaccination against experimental infection with two genetically different M. hyopneumoniae strains in weaned piglets. At 33 days of age (D0), 45 M. hyopneumoniae-free piglets were randomly assigned to three different groups: 1) negative control group (NCG; n = 5): not vaccinated, not infected, 2) positive control group (PCG; n = 20): not vaccinated, infected, and 3) vaccination group (VG; n = 20): single vaccination with an inactivated whole-cell M. hyopneumoniae vaccine (Hyogen®, Ceva) (D1), infected. The PCG and VG were endotracheally inoculated with 7 × 107 CCU in 7 ml of the highly virulent M. hyopneumoniae strain F7.2C (D24) and 7 × 107 CCU in 7 ml low virulent strain F1.12A (D25). A respiratory disease score (RDS) was assessed from D24 until D53. At D53 (euthanasia), macroscopic lung lesions (MLL) were scored, log copies of M. hyopneumoniae DNA (qPCR) and IL-1 and IL-6-concentrations (ELISA) on bronchoalveolar lavage fluid were determined. RESULTS: The RDS and MLL at euthanasia were respectively 0, 1.20 and 0.55 (P < 0.001) and 0, 7.56 and 0.68 (P < 0.001) for NCG, PCG and VG, respectively. The qPCR results for PCG and VG were 3.99 and 1.78 log copies (P < 0.001), respectively, with a significant difference between PCG and VG. The IL-1 and IL-6 results at euthanasia for NCG, PCG and VG were 17.61, 1283.39 and 53.04 pg/ml (P < 0.001) and 148.10, 493.35 and 259.80 pg/ml (P = 0.004), respectively with a significant difference between PCG and VG. CONCLUSIONS: Vaccination with Hyogen® in pigs was efficacious against an experimental challenge with both a low and highly virulent M. hyopneumoniae strain as the vaccinated pigs coughed significantly less, and showed significantly less lung lesions compared to the non-vaccinated challenged pigs: the vaccinated animals showed a 52.9% lower RDS and 91.0% lower MLL compared to the PCG. In the bronchoalveolar lavage fluid collected at the necropsy of the vaccinated pigs, a significantly lower amount of M. hyopneumoniae-DNA and a significantly lower IL-1 and IL-6 concentration was found compared to the pigs of the PCG.

Cross-sectional study on the in-herd prevalence of Mycoplasma hyopneumoniae at different stages of pig production.

BACKGROUND: A cross-sectional study was carried out to assess the prevalence of Mycoplasma hyopneumoniae infections before vaccination in 3-week-old piglets and to gain information about infection dynamics. METHODS: In 13 German and three Austrian farms with a known history of enzootic pneumonia, 790 piglets and 158 sows were sampled (blood samples, tracheobronchial swabs [TBS] [piglets], laryngeal swabs [LS] [sows]), and 525 pen-based oral fluids (OFs) were collected in growing and fattening pigs. Laboratory diagnostics included enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR) analyses. RESULTS: Antibodies to M. hyopneumoniae were present in 87.5 per cent of all herds. Seroprevalence ranged from 0.0 to 100.0 per cent and 0.0 to 88.0 per cent in sows and piglets, respectively. M. hyopneumoniae-deoxyribonucleic acid (DNA) was present in 3.8 and 0.4 per cent of LS and TBS, respectively. Gilts had a 10.9 times higher chance being M. hyopneumoniae PCR-positive than older sows. In 75.0 per cent of all farms, M. hyopneumoniae-DNA was present in OFs. Detection rate was significantly higher in OFs of 20-week-old than in younger pigs (p < 0.001). CONCLUSION: Results indicate that M. hyopneumoniae infections of the lower respiratory tract in piglets are rare but highlight the role of gilts in maintaining infection in the herd. Collecting OFs seems promising for surveillance, if coughing occurs simultaneously.

Different local, innate and adaptive immune responses are induced by two commercial Mycoplasma hyopneumoniae bacterins and an adjuvant alone.

Introduction: Enzootic pneumonia still causes major economic losses to the intensive pig production. Vaccination against its primary pathogen, Mycoplasma hyopneumoniae, is carried out worldwide to control the disease and minimize clinical signs and performance losses. Nonetheless, the effects of both infection with, and vaccination against Mycoplasma hyopneumoniae on the innate and adaptive immune responses remain largely unknown. Therefore, we conducted a study in which piglets were injected once with a commercial bacterin V1 or V2, or the adjuvant of V1 (A) to investigate their effect on local, innate and adaptive immune responses. Methods: Three weeks after vaccination, piglets were challenge infected with M. hyopneumoniae and euthanized four weeks later to assess vaccine efficacy via macroscopic and microscopic evaluation of lung lesions. Blood and broncho-alveolar lavage fluid (BAL) samples were collected to measure antibody responses, cellular immunity, BAL cytokine levels and BAL M. hyopneumoniae DNA load as well as cytokine secretion by monocytes. Results: After vaccination, proliferation of antigen-specific CD3+ T cells and a higher percentage of TNF-α+ CD8+, and TNF-α+ and TNF-α+IFN-γ+ CD4+CD8+ T cells was seen in V1, while proliferation of or a significant increase in cytokine production by different T cell subsets could not be observed for animals from V2. Interestingly, LPS-stimulated blood monocytes from V1 and A secreted less IL-10 on D7. After challenge, higher levels of IgA, more IL-10 and less IL-1ß was detected in BAL from V1, which was not observed in V2. Animals from A had significantly more IL-17A in BAL. The macroscopic lung lesion score and the M. hyopneumoniae DNA load at euthanasia was lower in V1, but the microscopic lung lesion score was lower in both vaccinated groups. Discussion: In conclusion, these results indicate that the two commercial bacterins induced different local and adaptive immune responses, that the adjuvant alone can reduce anti-inflammatory innate immune responses, and that both vaccines had a different efficacy to reduce Mycoplasma-like lung lesions and M. hyopneumoniae DNA load in the lung.

Cross-Sectional Study on the Prevalence of PCV Types 2 and 3 DNA in Suckling Piglets Compared to Grow-Finish Pigs in Downstream Production.

Vertical transmission is a consistently discussed pathway of porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3) transmission in pigs. To evaluate the presence of PCV2 and PCV3 in piglets, we collected tissue samples from 185 piglets that were crushed within the first week of life from 16 farms located in Germany and Austria. Pooled samples consisting of thymus, inguinal lymph node, myocardium, lung and spleen were examined for PCV2 and PCV3 by qPCR. Furthermore, oral fluid samples (OFS) from grow−finish pigs were collected and examined the same way. In piglets, PCV2 was highly prevalent (litters: 69.4%; piglets: 61.6%), whereas PCV3 prevalence was low (litters: 13.4%; piglets: 13.0%). In total, 72.6% and 67.2% of all collected OFS were PCV2 or PCV3 positive, respectively. Sow vaccination against PCV2 was identified as a protective factor concerning PCV2 in piglets (OR: 0.279; CI: 0.134−0.578; p < 0.001), whereas the porcine reproductive and respiratory syndrome virus (PRRSV) vaccination of sows was identified as a protective factor concerning PCV3 in piglets (OR: 0.252 CI: 0.104−0.610; p = 0.002). Our results show that PCV2, but not PCV3, is ubiquitous in suckling piglets and that early PCV3 infections might be modulated by PRRSV−PCV3 interaction. However, the ubiquitous nature of both viruses in older pigs could be confirmed.

Maternally Derived Antibody Levels Influence on Vaccine Protection against PCV2d Challenge.

Piglets from a porcine circovirus type 2 (PCV2) stable farm of low and high levels of maternally derived antibodies (MDA) against PCV2 were vaccinated either with a whole virus type or a PCV2 ORF2 antigen-based commercial subunit vaccine at three weeks of age. Two non-vaccinated groups served as low and high MDA positive controls. At four weeks post vaccination, all piglets were challenged with a PCV2d-2 type virus strain and were checked for parameters related to vaccine protection over a four-week observation period. MDA levels evidently impacted the outcome of the PCV2d-2 challenge in non-vaccinated animals, while it did not have a significant effect on vaccine-induced protection levels. The humoral immune response developed faster in the whole virus vaccinates than in the subunit vaccinated pigs in the low MDA groups. Further, high MDA levels elicited a stronger negative effect on the vaccine-induced humoral immune response for the subunit vaccine than for the whole virus vaccine. The group-based oral fluid samples and the group mean viraemia and faecal shedding data correlated well, enabling this simple, and animal welfare-friendly sampling method for the evaluation of the PCV2 viral load status of these nursery piglets.

Effectiveness of two intramuscular combined vaccines for the control of Mycoplasma hyopneumoniae and porcine circovirus type 2 in growing pigs: a randomized field trial.

BACKGROUND: Mycoplasma hyopneumoniae and Porcine circovirus type 2 are two economically important pathogens affecting growing pigs. Control and prevention of both diseases can be accomplished by vaccination, together with biosecurity and good management practices. Many commercial vaccines are available. The aim of this study was to assess the efficacy of Hyogen® and Circovac® administered mixed at weaning and to compare this protocol with a competitor ready-to-use (RTU) vaccine. CASE PRESENTATION: A randomised field trial was designed in a commercial farrow-to-finish farm located in France. A total of 641 pigs born from 54 different sows were included in this study. Piglets at weaning were allocated into three groups: the first one vaccinated with Hyogen® and Circovac® combined (group A), the second one vaccinated with a competitor RTU vaccine (group B) and the last one unvaccinated. Only minor local reactions for both vaccination groups could be observed which revealed a good safety of both protocols. Both vaccination schemes in this trial didn't improve wean-to-slaughter growth performances but significantly reduced lung lesions, lung fissures and pleurisy at slaughter, produced a seroconversion for both M. hyopneumoniae and PCV-2 and significantly reduced the PCV-2 viral load in blood. When we compared groups A and B, we observed no significant differences in growth performances, mortality, clinical signs, percentages of affected lungs at slaughter, lung fissures and pleurisy, and no difference in pathogens detection. However, two statistical differences were observed between both vaccines: the mean lung lesion score and the percentage of extensive lung lesions were lower in group A. This is consistent with lower M. hyopneumoniae loads in the lower respiratory tract in pigs from group A but this difference was not statistically significant. CONCLUSIONS: Results reported in this case study must be considered with caution since it was done in only one farm. In this trial, Hyogen® and Circovac® mixed together under field conditions offered a successful protection of growing pigs and significantly decreased the extension of lung lesions during a natural field challenge when compared with a competitor RTU vaccine.

HOX gene expression in phenotypic and genotypic subgroups and low HOXA gene expression as an adverse prognostic factor in pediatric ALL.

BACKGROUND: HOX genes play an important role in both normal lymphopoiesis and leukemogenesis. However, HOX expression patterns in leukemia cells compared to normal lymphoid progenitors have not been systematically studied in acute lymphoblastic leukemia (ALL) subtypes. PROCEDURE: The RNA expression levels of HOXA, HOXB, and CDX1/2 genes were analyzed by qRT-PCR in a cohort of 61 diagnostic pediatric ALL samples and FACS-sorted subpopulations of normal lymphoid progenitors. RESULTS: The RNA expression of HOXA7-10, HOXA13, and HOXB2-4 genes was exclusively detected in leukemic cells and immature progenitors. The RNA expression of HOXB6 and CDX2 genes was exclusively detected in leukemic cells but not in B-lineage cells at any of the studied developmental stages. HOXA3-4, HOXA7, and HOXB3-4 genes were differentially expressed between BCP-ALL and T-ALL subgroups, and among genotypically defined MLL/AF4, TEL/AML1, BCR/ABL, hyperdiploid and normal karyotype subgroups. However, this differential expression did not define specific clusters in hierarchical cluster analysis. HOXA7 gene was low expressed at the RNA level in patients with hyperdiploid leukemia, whereas HOXB7 and CDX2 genes were low expressed in TEL/AML1-positive and BCR/ABL-positive cases, respectively. In contrast to previous findings in acute myeloid leukemia, high HOXA RNA expression was associated with an excellent prognosis in Cox's regression model (P = 0.03). In MLL/AF4-positive ALL, lower HOXA RNA expression correlated with the methylation status of their promoters. CONCLUSIONS: HOX gene RNA expression cannot discriminate leukemia subgroups or relative maturity of leukemic cells. However, HOXA RNA expression correlates with prognosis, and particular HOX genes are expressed in specific genotypically characterized subgroups.

Comparison of vaccination protocols against Mycoplasma hyopneumoniae during the gilt acclimation period.

This study evaluated different gilt vaccination protocols against Mycoplasma (M.) hyopneumoniae at acclimation and their effect on the genetic diversity. A total of 180 M. hyopneumoniae naïve gilts were selected 1 week post-entry (wpe) at the acclimation barn in a clinically affected M. hyopneumoniae farm. Gilts were distributed according to the M. hyopneumoniae antibodies levels into three different vaccination schedules: A) four doses of a M. hyopneumoniae commercial vaccine at 2, 4, 6 and 8 wpe; B) two vaccine doses at 2 and 6 wpe and PBS at 4 and 8 wpe; and C) four PBS doses at the same wpe. Detection of M. hyopneumoniae (rt-PCR) and antibodies (ELISA) were assessed in gilts at 1, 14, 27 and 34 wpe and in 6 of their piglets at weaning. Rt-PCR positive gilts were detected at 14 wpe, being the proportion significantly lower in groups A and B (3/120, 3%) than C (27/60, 45%). Seroconversion was detected at 14 wpe, showing significant differences in percentage of inhibition (PI) between groups A (median 4.9, range 3.1-19.9) and B (5.5, 3.7-13.5), and C (14.3, 3.3-53.2). Gilts remained seropositive over the study and significant differences in PI were detected between groups A and B versus C. All piglets were rt-PCR negative, but the proportion of seropositive piglets coming from vaccinated gilts was significantly higher than the non-vaccinated group. M. hyopneumoniae characterization showed high variability. Hence, gilt vaccination with 2 or 4 doses significantly decreased the pathogen infectious pressure, variability, and provided high antibody levels to gilts and their offspring.

Characterization of Mycoplasma hyopneumoniae strains in vaccinated and non-vaccinated pigs from Spanish slaughterhouses.

This study aimed to describe Mycoplasma hyopneumoniae (M. hyopneumoniae) genetic variability in vaccinated (V) and non-vaccinated (NV) slaughtered pigs showing cranio-ventral pulmonary consolidation (CVPC). Ten V and 10 NV fattening farms with respiratory problems associated to M. hyopneumoniae were selected. Lung lesions of one batch per farm were scored at slaughterhouse and the enzootic pneumonia (EP)-index was calculated. Moreover, three lungs showing the most extensive CVPC per farm were sampled and tested for M. hyopneumoniae detection by real-time (rt)-PCR. Positive samples with cycle threshold ≤30 were selected to be genotyped by sequencing of four loci (P97, P146, H1 and H5). Typing profiles (TP) were assigned considering four or two (P97, P146) loci. Five commercial vaccines for M. hyopneumoniae (VS) and two reference strains (RF) were also genotyped. The EP-index (mean ± SD) in NV farms (3.8 ± 1.9) was not significantly different from V ones (2.2 ± 1.3). From the 60 selected lungs, 46 (76.7%) were M. hyopneumoniae positive by rt-PCR (25/30 and 21/30 from NV and V farms, respectively), and 43 (93.5%) of those were successfully genotyped. A total of 24 different TP(12 in V and 12 in NV farms) or 17 TP(9 in V and 9 in NV farms, being one TP in both farm types) were identified by analyzing four or two loci, respectively. One to three TP per farm were detected, being different from VS and RF. Interestingly, farms with same breeding origin had the same TP using two loci, but such link was not found using four loci. Therefore, high inter-farm and limited intra-farm M. hyopneumoniae genetic variability were detected, but variability depended on the number of studied loci.

Effect of different cyanobacterial biomasses and their fractions with variable microcystin content on embryonal development of carp (Cyprinus carpio L.).

While numerous studies focused on the effects of microcystins, the role of other components of complex cyanobacterial water blooms in toxicity is poorly understood. In this study we have evaluated effects of various fractions of cyanobacterial biomass with different composition and microcystin content on embryolarval development of carp (Cyprinus carpio). The following samples (fractions) of four natural water blooms were prepared and tested: complex cyanobacterial biomass, crude aqueous extract of biomass, cellular pellet remaining from aqueous extract, permeate (i.e. microcystin-free fraction prepared during C-18 solid-phase extraction; SPE), and eluate (i.e. fraction prepared by SPE containing mostly microcystins). Complex biomass and the crude aqueous extract (regardless of microcystin content and/or microcystin variants present) in the sample were the most toxic. On the other hand, eluate fractions of all samples containing microcystins in concentrations 8-255 microgL(-1) induced no or only weak toxic effects. Exposures of fish to permeate fractions (with removed microcystins) of two samples dominated by Aphanizomenon sp. and Planktothrix sp. resulted in significant mortality, while other two samples dominated by Microcystis spp. induced minor effects. We have also observed significant inhibition of glutathione S-transferases (GST) at most fractions of the Aphanizomenon sp. and Planktothrix sp. dominated samples. Our data indicate that cyanobacterial water blooms as well complex biomass extracts induce significant embryolarval toxicity in common carp. However, these effects were independent of microcystin content, and the most pronounced effects were observed with the non-Microcystis dominated samples. Therefore, a critical examination of microcystin role in overall ecotoxicology of complex cyanobacterial blooms is needed.

Topology of chromosomes 18 and X in human blastomeres from 3- to 4-day-old embryos.

The positions of chromosomes 18 and X fluorescence in situ hybridization signals were analyzed in blastomeres generated from human in vitro fertilization 3- to 4-day-old embryos after preimplantation screening of aneuploidy of chromosomes 13, 16, 18, 21, 22, X, and Y. Fluorescent signal localization compared with a three-dimensional sphere model of random signal distribution revealed significant differences, providing evidence of peripheral localization of chromosome 18 in aneuploid (p=0.0013) and aneuploid/euploid blastomeres (p=0.0011). No differences were found in localization of chromosome 18 in euploid and in chromosome X in euploid and aneuploid blastomeres.

QF-PCR examination of parental and meiotic origin of trisomy 21 in Central and Eastern Europe.

Study of parental/meiotic origin of free trisomy 21 in nuclear families from Russia (70 cases), Ukraine (32 cases), and 22 from Germany revealed maternal nondisjunction in 77.3% (Germany), 93.8% (Ukraine), and 91.4% (Russia), paternal origin in 13.6%, 6.2%, and 8.6%, respectively. Maternal meiosis I errors were found in 84.4% (Ukraine), 77.1% (Russia), paternal origin in 3.1% (Ukraine), 2.9% (Russia). Maternal meiosis II errors occurred in 9.4% and 14.3% and paternal in 3.1% and 5.7% in Ukraine and Russia, respectively. No significant differences were found in maternal/paternal origin among Ukraine, Russia, Germany, and published data from other European regions.

Chromosome topology in normal and aneuploid blastomeres from human embryos.

OBJECTIVES: To find whether chromosomes 13, 16, 18, 21, 22, X and Y in blastomeres of human embryos are nonrandomly localized, whether their aneuploidy affects their localization and if eventual early inactivation of chromosome X with peripheral localization is present. METHODS: Relative distances from the nucleus center and edge of 1,198 fluorescence in situ hybridization signals in 98 human blastomeres were measured in digital images for comparison with a mathematical model of random distribution in spherical nucleus. RESULTS: Comparison with the mathematical model revealed that localization of chromosomes 13, 16, 21, 22, X and Y in normal and aneuploid blastomeres and that of chromosome 18 in normal blastomeres was not significantly different from random distribution. Similarly, chromosome X in blastomeres with more than one X did not appear to have a preferential localization. Only chromosome 18 in aneuploid blastomeres was differently distributed (p < 0.0001) with a shift to the nuclear periphery (p =or < 0.0001). CONCLUSIONS: Peripheral localization of chromosome 18 in aneuploid blastomeres is related to embryo aneuploidy. Conversely, a peripheral localization of the inactive X chromosome was not found in blastomeres from 3-4 day old embryos. These results open the possibility to improve embryo selection after pre-implantation diagnosis.