Assessment of three commercial ELISAs for the detection of antibodies against Porcine epidemic diarrhea virus at different stages of the immune response.

Three commercial ELISAs -two based on spike (E1 and E3) and one on nucleocapsid protein (E2)-were used to analyze the development and persistence of antibodies against Porcine epidemic diarrhea virus (PEDV). Seventy-five four-week-old PEDV-negative piglets were inoculated orally with a European G1b PEDV (INOC) and fourteen were kept as controls (CTRL). After the inoculation, E3 detected positive animals as soon as 7 days post inoculation (dpi), while the earliest detection with E1 and E2 was at 14 dpi. All samples were positive at 21 and 28 dpi using E1 and E3, respectively, while E2 failed to detect 23.3 % of the inoculated pigs at any time point. The percentages of positive samples were different through the study: E1 and E3 > E2 from 14 to 56 dpi; and E3 > E1 > E2 from 56 to 154 dpi (P < 0.05). Five months after the inoculation, E3 still detected 92.0 % (IC95 % = 85.1-98.8 %) of pigs as positive, while E1 and E2 detected only 27.0 % (IC95 % = 16.0-37.9 %) and 0%, respectively. The sensitivity for E2 never exceeded 0.62. Specificity was 1 for all ELISAs. These different outcomes could be related to the ELISA strategies (indirect versus competition), the antigens used, the cut-off, or to other intrinsic factors of each test. The observed differences could be of importance when assessing whether older animals, such as fatteners or gilts, had previously been in contact with PEDV.

Activation of pro- and anti-inflammatory responses in lung tissue injury during the acute phase of PRRSV-1 infection with the virulent strain Lena.

Porcine reproductive and respiratory syndrome virus (PRRSV) plays a key role in porcine respiratory disease complex modulating the host immune response and favouring secondary bacterial infections. Pulmonary alveolar macrophages (PAMs) are the main cells supporting PRRSV replication, with CD163 as the essential receptor for viral infection. Although interstitial pneumonia is by far the representative lung lesion, suppurative bronchopneumonia is described for PRRSV virulent strains. This research explores the role of several immune markers potentially involved in the regulation of the inflammatory response and sensitisation of lung to secondary bacterial infections by PRRSV-1 strains of different virulence. Conventional pigs were intranasally inoculated with the virulent subtype 3 Lena strain or the low virulent subtype 1 3249 strain and euthanised at 1, 3, 6 and 8 dpi. Lena-infected pigs exhibited more severe clinical signs, macroscopic lung score and viraemia associated with an increase of IL-6 and IFN-γ in sera compared to 3249-infected pigs. Extensive areas of lung consolidation corresponding with suppurative bronchopneumonia were observed in Lena-infected pigs. Lung viral load and PRRSV-N-protein+ cells were always higher in Lena-infected animals. PRRSV-N-protein+ cells were linked to a marked drop of CD163+ macrophages. The number of CD14+ and iNOS+ cells gradually increased along PRRSV-1 infection, being more evident in Lena-infected pigs. The frequency of CD200R1+ and FoxP3+ cells peaked late in both PRRSV-1 strains, with a strong correlation between CD200R1+ cells and lung injury in Lena-infected pigs. These results highlight the role of molecules involved in the earlier and higher extent of lung lesions in piglets infected with the virulent Lena strain, pointing out the activation of routes potentially involved in the restraint of the local inflammatory response.

Biosecurity assessment of Argentinian pig farms.

The pig industry is growing very fast in Argentina with an increasing need for replacement animals, feedstuff and transportation of animals. One of the main competitive advantages of the Argentinian pig industry is its being free of most major pig diseases. Within this context, applying measures aimed to reduce the risk of introduction and spread of pathogens is critical. The aim of the present study was to assess the biosecurity of Argentinian pig farms. Two types of farms were assessed: firstly, all official suppliers of high-genetic-value (n = 110) and secondly, a sample from commercial farms (n = 192). Data on the external and internal biosecurity practices applied on the farms was collected with a questionnaire. Data was analysed using a correspondence analysis and a hierarchical clustering analysis, which allowed identification of types of farms with regard to the biosecurity measures applied. Key variables characterizing the clusters were identified through an indicator value analysis. In addition, the external biosecurity of the farms was evaluated by using risk assessment tools with respect to the potential introduction of porcine epidemic diarrhoea virus. Results made evident three clusters: the first one which, amongst other measures, applied several barriers to prevent the entry of people, trucks and other vehicles, and could be considered as a group of high biosecurity, and the two other groups which applied a lower number of external and internal biosecurity measures. The results of the risk assessment showed that the routes with the highest risk of disease introduction were: replacement animals, vehicles transporting feed or animals, and visitors. The assessment of the external biosecurity showed that most Argentinian farms were not prepared for the contingency of a pathogen such as porcine epidemic diarrhoea virus. Special efforts should be made in official suppliers of high-genetic-value farms with poor biosecurity scores since they are at the top of the pig production chain and can be key for the spread of diseases.

Development of a risk assessment tool for improving biosecurity on pig farms.

In the present study a risk assessment tool was developed for improving biosecurity on pig farms as part of a voluntary program for PRRS control on farms located in NE Spain. The arrival of the PRRS virus through different routes was estimated based on their likelihood of harbouring the virus and the estimation of a score for the probability of introduction. For each possible route of introduction or spread within the herds, single or combined biosecurity measures aimed at reducing the probability of PRRS virus transmission were selected. Results showed that the scores for the probability of introduction and spreading were related to a highly variable application of biosecurity measures. The tool developed in the present study may be suitable for identifying where efforts should be focused in biosecurity actions forming part of disease control programs.

Transmission of Porcine reproductive and respiratory syndrome virus 1 to and from vaccinated pigs in a one-to-one model.

The present study examined transmission by contact of Porcine reproductive and respiratory syndrome virus (PRRSV) 1 in a one-to-one model to vaccinated and unvaccinated pigs and from vaccinated infected pigs to other vaccinated pigs. The experiment started by randomly assigning weaned pigs to groups V (n=24) and U (n=26). V pigs were vaccinated with a commercial live attenuated PRRSV vaccine and the U animals were kept as unvaccinated controls. Twenty-eight days later, 6U pigs were separated and allocated in individual boxes. The remaining 20U pigs were intranasally inoculated with PRRSV isolate 3267 (from now on designated as seeder (S) pigs) and 48h later were distributed in boxes where they were commingled with either V or U pigs in 1:1 groups (first contact phase), resulting in 6S:U and 14S:V pairs. As soon as a V pig was detected to be viremic because of contact with a S, the infected V (from now on designated as Vinf) was transferred (<24h after detection) to a new pen where it was comingled with a new V pig (designated as V2) in a second contact phase. For the first contact phase, pigs were maintained 21days at maximum and for the second contact phase the maximum exposure period was 14days. Two V pigs tested positive for the vaccine virus (>99.5% similarity) when they were relocated with the corresponding V2 pigs and they were removed; thus, only 12Vinf were finally considered. All V pigs (12/12) exposed to S animals became infected although the first detection of viremia occurred at 13.6±3.6days, one week later than in U (p<0.05). Also, duration of viremia was shorter for Vinf compared to U, (5.5±4.3days versus 12.5±2.7days). The Vinf group showed remarkable individual variability: eight animals had a viremic period of 5 or less days (3.0±1.4) while the remaining four had a longer viremic period of more than one week (10.8±2.9). This situation was not observed in U. In the second contact phase, transmission from Vinf to V2 pigs occurred in 7/8 cases (87.5%). The mean duration of viremia for V2 was 4.8±3.4 and two different patterns were again observed: two animals had viremias of 9-10days and the rest averaged 3.0±1.4days (range: 2-5days). Vaccinated groups Vinf and V2 had a significantly lower PRRSV shedding in oral fluids for at least the first 9days after the onset of the viremia compared to U, and shedding for V2 was even significantly lower (p<0.05) than shedding for Vinf. Our experimental design reproduced the worst-case scenario for evaluating the effect of vaccination and, under such conditions; it was still efficacious in slowering PRRSV transmission and decreasing the global viral load and particularly oral shedding.

Genetic Analysis of Human Preimplantation Embryos.

Preimplantation development comprises the initial stages of mammalian development, before the embryo implants into the mother's uterus. In normal conditions, after fertilization the embryo grows until reaching blastocyst stage. The blastocyst grows as the cells divide and the cavity expands, until it arrives at the uterus, where it "hatches" from the zona pellucida to implant into the uterine wall. Nevertheless, embryo quality and viability can be affected by chromosomal abnormalities, most of which occur during gametogenesis and early embryo development; human embryos produced in vitro are especially vulnerable. Therefore, the selection of chromosomally normal embryos for transfer in assisted reproduction can improve outcomes in poor-prognosis patients. Additionally, in couples with an inherited disorder, early diagnosis could prevent pregnancy with an affected child and would, thereby, avoid the therapeutic interruption of pregnancy. These concerns have prompted advancements in the use of preimplantation genetic diagnosis (PGD). Genetic testing is applied in two different scenarios: in couples with an inherited genetic disorder or carriers of a structural chromosomal abnormality, it is termed PGD; in infertile couples with increased risk of generating embryos with de novo chromosome abnormalities, it is termed preimplantation genetic screening, or PGS.

Confirmation rates of array-CGH in day-3 embryo and blastocyst biopsies for preimplantation genetic screening.

PURPOSE: The purpose of this study was to compare the confirmation rate of day-3 embryo biopsy (blastomere) and trophectoderm biopsy using array-comparative genomic hybridization (array-CGH) technology. METHODS: A blinded study was conducted to re-analyse 109 embryos previously diagnosed as chromosomally abnormal by array-CGH. Preimplantation genetic screening (PGS) was performed using array-CGH on day 3 (n = 50) or day 5 (n = 59). Partial chromosome gains or losses were excluded (n=6), and only whole chromosome aneuploidies were considered. Re-analysis of whole blastocysts was carried out following the same array-CGH protocol used for PGS. RESULTS: The PGS result was confirmed in the whole blastocyst in (a) 49/50 (98 %) abnormal embryos after day-3 biopsy and (b) 57/59 (96.6 %) abnormal embryos after trophectoderm biopsy. One embryo (1/50; 2 %) was diagnosed as abnormal, with monosomy 18, on day 3, and software analysis of the whole blastocyst gave a euploid result; however, a mosaic pattern was observed for monosomy 18 in the whole blastocyst. Two trophectoderm biopsy cases (3.4 %) did not have the abnormalities (trisomy 7, and trisomy 1 and 4, respectively) verified in the whole embryo. Concordance rates for both biopsy strategies and for individual chromosomes were evaluated by Fisher's exact test and showed no significant differences. CONCLUSIONS: Both types of biopsies showed similar high concordance rates with whole blastocyst results. Therefore, regarding the confirmation rates shown in this work, day-3 embryo biopsies can be representative of the whole embryo and both types of biopsy can be used for clinical analysis in PGS following the described array-CGH protocol.

Analysis of ORF5 and full-length genome sequences of porcine reproductive and respiratory syndrome virus isolates of genotypes 1 and 2 retrieved worldwide provides evidence that recombination is a common phenomenon and may produce mosaic isolates.

UNLABELLED: Recombination is currently recognized as a factor for high genetic diversity, but the frequency of such recombination events and the genome segments involved are not well known. In the present study, we initially focused on the detection of recombinant porcine reproductive and respiratory syndrome virus (PRRSV) isolates by examining previously published data sets of ORF5 sequences (genotypes 1 and 2) obtained worldwide. We then examined full-length genome sequences in order to determine potential recombination breakpoints along the viral genome. For ORF5, 11 sets of genotype 1 sequences from different geographical areas, including 2 Asian, 1 American, and 7 European regions, and three sets of genotype 2, including sets from China, Mexico, and the United States, were analyzed separately. Potential recombination breakpoints were detected in 10/11 genotype 1 sets, including 9 cases in which the clustering of at least one isolate was different before and after the breakpoints. In genotype 2, potential breakpoints and different tree clustering of at least one strain before and after the breakpoint were observed in 2 out of 3 sets. The results indicated that most of the ORF5 data sets contained at least one recombinant sequence. When the full-length genome sequences were examined, both genotype 1 and 2 sets presented breakpoints (10 and 9, respectively), resulting in significantly different topologies before and after the breakpoints. Mosaic genomes were detected in genotype 1 sequences. These results may have significant implications for the understanding of the molecular epidemiology of PRRSV. IMPORTANCE: PRRSV is one of the most important viruses affecting swine production worldwide, causing big economic losses and sanitary problems. One of the key questions on PRRSV arises from its genetic diversity, which is thought to have a direct impact on immunobiology, epidemiology, diagnosis, and vaccine efficacy. One of the causes of this genetic diversity is recombination among strains. This study provides evidence that recombinant PRRSV isolates are common in most of the countries with significant swine production, especially PRRSV genotype 1. This observation has implications in the proper characterization of PRRSV strains, in the future development of phylogenetic studies, and in the development of new PRRSV control strategies. Moreover, the present paper emphasizes the need for a deeper understanding of the mechanisms and circumstances involved in the generation of genetic diversity of PRRSV.

Comparison of different vaccination schedules for sustaining the immune response against porcine reproductive and respiratory syndrome virus.

In order to better understand how immunization against porcine reproductive and respiratory syndrome virus (PRRSV) can be improved using commercial vaccines, different strategies of immunization were applied in the field using an inactivated vaccine (INV), a modified live vaccine (MLV) or a combination of the two and the responses compared. In experiment 1 (E1), 21 piglets were distributed in three groups. Group A was vaccinated with a commercial INV at 2.5, 3.5 and 6.5 months old; group B pigs received the INV at 1.5, 2.5, 5.5 and 6.5 months old, while pigs in group C were kept as unvaccinated controls. At 7.5 months of age all pigs were challenged with PRRSV and followed for 21 days. In experiment 2 (E2), 32 piglets were distributed evenly in four groups. Groups A, B and C were vaccinated with a commercial MLV at 1.5 months old, while group D pigs were kept as controls. At 4.5 months old, groups A and C received the INV while B received a second MLV, 1 month later group C pigs received a third INV. At 6.5 months old all pigs were challenged as in E1. In both experiments, total antibodies, neutralizing antibodies (NA) and cell-mediated immunity (CMI) were evaluated, and viraemia was determined after challenge. In E1, immunization with an INV induced high interferon-γ responses after the second and subsequent vaccinations. Development of NA after challenge was faster in INV vaccinated pigs compared to unvaccinated controls. In E2, re-vaccination with INV induced NA responses similar to re-vaccination with MLV; however, a significant increase in NA titres after challenge was only detected in group C pigs. The use of combined protocols (MLV+INV) was superior to the use of MLV alone in inducing cell mediated immunity. In conclusion, the highest immune responses against PRRSV after a single shot were achieved with MLV; after that, INV re-vaccination should be considered as the best strategy to induce significant boosters.

Biosecurity practices in Spanish pig herds: perceptions of farmers and veterinarians of the most important biosecurity measures.

One hundred Spanish pig farms were surveyed to determine the biosecurity measures currently applied, as reported by farmers, and to investigate the importance awarded by farmers and veterinarians to each of these measures. Data was gathered by means of a questionnaire administered to farmers and veterinarians. Biosecurity measures were reported based on two scenarios: in the presence and in the absence of a highly contagious disease. Multiple-correspondence and two-step cluster analyses were performed to investigate the effect of farm type on the biosecurity level. Farmers awarded significantly higher scores to their farms' level of biosecurity than the veterinarians servicing said farms. According to the farmers and veterinarians, the most important biosecurity measures were those aimed at minimising the risk of disease introduction by visits and vehicles. Biosecurity practices seeking to reduce the risk of disease introduction by breeding stock were not applied on a considerable number of farms. The findings also revealed that medium-sized to large farms located in high pig density regions reported higher biosecurity measures than small herds located in low pig density areas.

Simultaneous porcine circovirus type 2 and Mycoplasma hyopneumoniae co-inoculation does not potentiate disease in conventional pigs.

The aim of this study was to assess the effect of simultaneous experimental inoculation of porcine circovirus type 2 (PCV2; intranasal delivery) and Mycoplasma hyopneumoniae (Mhyo; transtracheal delivery) into conventional, seropositive 6-week-old piglets. Thirty-six male piglets were assigned randomly to four groups: control (n=6), PCV2 (n=6), Mhyo (n=12) and PCV2+Mhyo (n=12). Blood samples and faecal and nasal swabs were collected at 0, 7, 14 and 21 days post inoculation (dpi). No significant clinical signs attributable to PCV2 infection were observed during the experiment. Coughing was recorded in three pigs from the Mhyo group and six from the PCV2+Mhyo group. No significant differences in mean body weight and rectal temperature were observed between the groups. Mild microscopical lesions similar to those reported for post-weaning multisystemic wasting syndrome were observed in two PCV2 pigs and in one PCV2+Mhyo animal. Mhyo-compatible lung lesions were observed in 21/24 pigs inoculated with Mhyo (10 from the Mhyo group and 11 from the PCV2+Mhyo group). PCV2 was detected by in-situ hybridization in 3/12 PCV2 and in 4/12 PCV2+Mhyo animals. No significant differences in PCV2 load (serum and nasal and faecal swabs), duration of viraemia or antibody titre were detected between PCV2-inoculated groups. No significant differences in Mhyo load in nasal swabs, percentage of Mhyo-seropositive pigs and mean lung score was detected between Mhyo-inoculated groups. Under the conditions of the present study, concurrent inoculation of PCV2 and Mhyo did not result in potentiation of clinical signs and lesions attributed to either infection.

Effects of challenge with a virulent genotype II strain of porcine reproductive and respiratory syndrome virus on piglets vaccinated with an attenuated genotype I strain vaccine.

Porcine reproductive and respiratory syndrome virus (PRRSV) is endemic in most parts of Asia, where genotype I and II strains of diverse virulence may coexist. This study evaluated the outcome of infection with a highly virulent Asian genotype II PRRSV isolate in piglets vaccinated with a genotype I vaccine. Twenty-one 3-week-old piglets were divided in three groups: Pigs in group V (n=8) were vaccinated with an attenuated genotype I commercial PRRSV vaccine, while pigs in group U (n=8) and a control group (group C; n=5) were unvaccinated; 6 weeks later, pigs in groups V and U were challenged intranasally with a highly virulent strain of genotype II PRRSV (1×10(5) 50% tissue culture infectious doses/mL), while pigs in group C received a placebo. Over a period of 21 days after challenge, vaccinated pigs had significantly lower mortality (0/8 versus 2/8), fewer days of fever, a lower frequency of catarrhal bronchopneumonia, higher weight gains (13.4 versus 6.6 kg) and lower levels of viraemia compared to unvaccinated challenged pigs. Immunisation with a genotype I attenuated PRRSV vaccine provided partial protection against challenge with a highly virulent genotype II strain.

Seroprevalence and risk factors of swine influenza in Spain.

Swine influenza is caused by type A influenza virus. Pigs can be infected by both avian and human influenza viruses; therefore, the influenza virus infection in pigs is considered an important public health concern. The aims of present study were to asses the seroprevalence of swine influenza subtypes in Spain and explore the risk factors associated with the spread of those infections. Serum samples from 2151 pigs of 98 randomly selected farms were analyzed by an indirect ELISA for detection of antibodies against nucleoprotein A of influenza viruses and by the hemagglutination inhibition (HI) using H1N1, H1N2 and H3N2 swine influenza viruses (SIV) as antigens. Data gathered in questionnaires filled for each farm were used to explore risk factors associated with swine influenza. For that purpose, data were analyzed using the generalized estimating equations method and, in parallel by means of a logistic regression. By ELISA, 92 farms (93.9%; CI(95%): 89.1-98.7%) had at least one positive animal and, in total, 1340/2151 animals (62.3%; CI(95%): 60.2-64.3%) were seropositive. A total of 1622 animals (75.4%; CI(95%): 73.6-77.2%) were positive in at least one of the HI tests. Of the 98 farms, 91 (92.9%; CI(95%): 87.7-98.1%) had H1N1 seropositive animals; 63 (64.3%; CI(95%): 54.6-73.9%) had H1N2 seropositive pigs and 91 (92.9%; CI(95%): 87.7-98.1%) were positive to H3N2. Mixed infections were detected in 88 farms (89.8; CI(95%): 83.7-95.9%). Three risk factors were associated with seroprevalences of SIV: increased replacement rates in pregnancy units and, for fatteners, existence of open partitions between pens and uncontrolled entrance to the farm.

Improving FISH diagnosis for preimplantation genetic aneuploidy screening.

BACKGROUND: In our routine programme of preimplantation genetic aneuploidy screening (PGS) by fluorescence in situ hybridization (FISH), nine chromosomes (13, 15, 16, 17, 18, 21, 22, X and Y) are analysed in two consecutive hybridization rounds. We also perform additional hybridization rounds for these chromosomes, using probes that bind to different loci, for non-conclusive results and for confirmation of certain aneuploidies. The aim of this study was to evaluate the impact of additional hybridization rounds on FISH accuracy. METHODS: This is a retrospective analysis of our FISH data from 1000 PGS cycles performed from December 2007 to December 2008 for various indications. In addition to the hybridization rounds described above, 132 of the embryos diagnosed as chromosomally abnormal were re-analysed on Day 5. RESULTS: A total of 2477 embryos were re-hybridized, 1496 due to non-conclusive results and 981 to confirm observed aneuploidies. After re-hybridization, 882 embryos (59%) were then diagnosed as normal, 600 embryos (40.1%) had a clear abnormality and only 14 embryos (0.9%) remained non-informative. From the 981 embryos in the latter group, 890 embryos had monosomies and, after re-hybridization 174 embryos (19.6%) were normal and 716 (80.5%) had confirmed monosomies. In contrast, re-hybridization confirmed 90 (98.9%) of the 91 observed trisomies. In addition, Day-5 re-analysis of abnormal embryos showed a higher rate of concordant diagnosis between Day 3 and Day 5 when re-hybridizations had been included on Day-3 (95 versus 82.7%; P= 0.0443), especially for the confirmation of monosomies (82.8 versus 61.0%; P = 0.0087). CONCLUSIONS: Our data indicate that additional hybridization rounds improve the accuracy of the diagnosis, increasing the number of chromosomally normal embryos available for transfer. Re-hybridization with additional probes as a standard approach to PGS could enhance the potential benefits of the technique.

One dose of a porcine circovirus 2 (PCV2) sub-unit vaccine administered to 3-week-old conventional piglets elicits cell-mediated immunity and significantly reduces PCV2 viremia in an experimental model.

The immunogenicity and efficacy generated by one dose of a PCV2 sub-unit vaccine (Porcilis PCV) were evaluated in 3-week-old conventional piglets. Vaccination induced both humoral and cell-mediated responses against PCV2, which were increased after the challenge with a PCV2 genotype "b" isolate. High levels of maternally derived antibodies (IPMA >or= 10 log(2)) at the time of vaccination were found to interfere with the active seroconversion, whereas titres below 8 log(2) allowed the development of a proper antibody response. Nevertheless, the immunity induced by one dose of the product was partly protective against PCV2 infection, since viremia, shedding and viral load in tissues were significantly reduced in vaccinated pigs compared to controls.

Porcine circovirus type 2 (PCV2) vaccination of conventional pigs prevents viremia against PCV2 isolates of different genotypes and geographic origins.

The efficacy of recently developed porcine circovirus type 2 (PCV2) vaccines has not been tested yet against PCV2 isolates of the two proposed genotypes. In the present work, the efficacy of a subunit vaccine containing PCV2 capsid protein was evaluated by using a challenge model with four different PCV2 isolates of different genotype and geographic origin. The vaccine prevented the development of viremia in all cases as well as significantly decreased nasal and faecal shedding of the virus. Also, the vaccine elicited PCV2-specific neutralizing antibodies to PCV2 even in the presence of maternally derived immunity.