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.

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.

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.

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.

Transient correlation between viremia levels and IL-10 expression in pigs subclinically infected with porcine circovirus type 2 (PCV2).

Immunological impairment by porcine circovirus type 2 (PCV2) infection is well documented in pigs suffering from postweaning multisystemic wasting syndrome. Nonetheless, little is known about immune status of pigs that remain PCV2 subclinically infected. Thus, seven pigs successfully infected in an experimental inoculation and without developing disease and nine control non-inoculated pigs were examined. Serological, virological and immunological determinations were done throughout ten weeks post-infection (PI). At week 3 PI, inoculated animals presented the peak of viremia and produced higher levels of IL-10 than the controls; correlation between viral load and IL-10 amounts was observed (p<0.05). Also, the ratio IgM/IgG suffered a shift skewing IgM production towards an IgG response. By 10 weeks PI, levels of IL-10 disappeared and the viremia decreased. In summary, subclinically PCV2-infected pigs developed a transient PCV2-specific IL-10 response during the viremic phase of infection which coincided with the inversion of the IgM/IgG ratio.

The challenge of PRRS immunology.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most challenging subjects of research in veterinary viral immunology, and the immune response against PRRS virus (PRRSV) still is poorly understood. Infected pigs develop a strong and rapid humoral response but these initial antibodies do not confer protection and can even be harmful by mediating an antibody-dependent enhancement of disease. In contrast, development of neutralising antibodies (NAs) is delayed and generation of cell-mediated immune responses, such as PRRSV-specific interferon (IFN)-gamma secreting cells, is initially erratic. In spite of this, induction of strong and rapid NAs and IFN-gamma responses seem to be required for effective vaccination. PRRSV strongly modulates the host's immune responses. The virus inhibits key cytokines, such as IFN-alpha, and may induce regulatory cytokines, such as interleukin (IL)-10. Development of NAs seems to be impaired by the existence of a decoy epitope close to the main neutralisation epitope in glycoprotein 5. This ability to modulate the host immune response probably varies among strains or isolates. The genetic diversity of the virus is very high and it has been shown that this diversity can have serious implications for the development of vaccines, since the immunity induced by one strain may be only partial against a different strain, even within the same genotype. With this panorama, the development of newer and universally efficacious PRRSV vaccines is challenging, but the present state of knowledge allows optimism if collaborative efforts are undertaken in the scientific community.

Distribution of hepatitis E virus infection and its prevalence in pigs on commercial farms in Spain.

A survey to detect antibodies against hepatitis E virus (HEV) was undertaken on 41 Spanish pig farms using an indirect enzyme-linked immunosorbent assay (ELISA). Forty of the farms (97.6%) were positive for anti-HEV IgG antibodies, while 34/41 (82.9%) had IgM positive animals. The highest proportion of IgG positive pigs comprised the adult sows (45/74, 60.8%; P=0.01), followed by young piglets aged 3-6 weeks (79/218, 36.2%). IgM positive pigs were more frequently found to be those animals > or = 12 weeks of age (P<0.001). In a second part of the study, the age distribution of the infection on one HEV infected farm was studied by combining the ELISA test with reverse transcription polymerase chain reaction (RT-PCR). On this farm, viraemia was mostly detected in animals aged 8-12 weeks and faecal shedding of HEV was detected in pigs of the same age. All HEV sequences corresponded to genotype 3. The study confirmed that HEV is spread in pigs in Spain and is probably endemic in many farms.

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.

Association of hepatitis E virus (HEV) and postweaning multisystemic wasting syndrome (PMWS) with lesions of hepatitis in pigs.

The aim of the study was to determine the presence of swine hepatitis E virus (HEV) RNA and antibodies in postweaning multisystemic wasting syndrome-affected (n=114) and non-affected (n=46) pigs and the possible association with hepatitis lesions. Forty-four pigs were RT-PCR positive (28.2%); 25 of them were PMWS cases, while 19 were non-PMWS pigs. In both groups, HEV RT-PCR results were associated with hepatitis (OR=5.61 for PMWS-affected pigs and OR=5.17 for non-PMWS affected pigs; p=0.01). No interaction was detected in a logistic regression between PMWS occurrence and HEV infection for the development of hepatitis lesions. Seropositivity to HEV was more likely to occur in pigs with hepatitis (51.9%) compared to pigs without hepatitis (36.1%; p=0.03). Significant differences in optical densities were notices comparing the lesional stage of pigs (p=0.009). While pigs with slight or moderate hepatitis were seropositive, pigs with more severe lesions were seronegative to HEV. These results indicate that swine HEV infection can be a significant contributor to the development of moderate hepatitis in pigs regardless of the PMWS status.

Biosecurity measures on swine farms in Spain: perceptions by farmers and their relationship to current on-farm measures.

A survey was conducted on 172 farms belonging to the same company in order to ascertain the biosecurity measures applied and the importance that farmers attributed to each measure. A questionnaire about general data, the opinions of farmers about biosecurity measures, and a detailed checklist of the biosecurity measures actually applied on the farm was completed for each one of the participating farms. Opinion data were initially analysed by means of a cluster analysis (multidimensional scaling method); then, a logistic regression was used to analyse the relationship between the perceptions of the farmer and the measures applied on the farm. In general, farmers implemented measures to reduce risks of contamination from people and animals other than pigs. In contrast, biosecurity measures related to replacement stock were not applied as often. On average, the farmers scored the biosecurity on their own farms as 6.7 on a scale of 0-10. The most important measures, according to the farmers' perceptions, were the availability of a sanitary ford, a fence around the farm, the restriction of visits and vehicles, using bird-proof nets in windows, having changing facilities, applying quarantines, and the use of other measures related to replacement stock. The perception of a given measure was strongly influenced by the measures actually applied on the farm. Thus, for example, those who did not have a sanitary ford highlighted the importance of disinfecting vehicles while those who had one neglected this type of disinfection. In conclusion, knowledge of the relationships between perceptions and measures taken is important in developing effective biosecurity strategies on pig farms.

Sow porcine circovirus type 2 (PCV2) status effect on litter mortality in postweaning multisystemic wasting syndrome (PMWS).

Previous studies have described a "litter effect" associated with mortality in postweaning multisystemic wasting syndrome (PMWS) affected farms. The main objective of this study was to evaluate litter mortality in different PMWS affected farms and to characterize it in relation to three variables of the sow: parity, porcine circovirus type 2 (PCV2) infectious status and PCV2 antibody titres. The study was performed in seven farms that experienced PMWS in nurseries and/or fattening areas. Fifteen sows from each farm were randomly selected from the same farrowing batch. Serum samples were analyzed for antibodies to PCV2 and for genomic detection of PCV2. Four piglets from each sow (60 piglets per farm) were selected and ear-tagged at birth. Out of 420 initial piglets, 104 (25%) died. Sixty three of them (60%) were necropsied, and 40 (63%) diagnosed as PMWS based on case definition criteria. Our results show that sow PCV2 viremia was significantly related to piglet mortality since more piglets per litter died from viremic than from non-viremic sows. Additionally, a significantly greater proportion of animals died from sows that had low antibody titres against PCV2 (39% vs. 18% from sows with medium to high antibody titres). The present study, of exploratory nature, confirms previous results and further characterizes the so called "litter effect" by establishing that the sow PCV2 status had a significant effect on litter mortality in PMWS affected farms.

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.

Evolution of ORF5 of Spanish porcine reproductive and respiratory syndrome virus strains from 1991 to 2005.

ORF5 sequences of porcine reproductive and respiratory syndrome virus (PRRSV) were analysed to determine genetic diversity, codon usage, positive and negative selection sites and potential changes in the predicted glycoprotein 5 (GP5). A hypothetical GP5 containing all selected sites was constructed to determine its characteristics. These sequences corresponded to isolates obtained 10 years apart (1991-1995, 18 strains) and a second set (n = 46) from 2000 to 2005. Similarity to Lelystad virus (LV) decreased from 95.5% in 1991-1995 to 89.5% in 2000-2005. Three highly variable regions were found in ORF5. Codon usage was different in both sets for leucine, glutamine, serine and proline. Thus, 2000-2005 sequences used codons more similar to those present in highly expressed pig genes compared to the 1991-1995 set. Twenty four sites of positive selection and 20 sites of negative selection were found in GP5, most of them in transmembrane regions. Additional glycosylation in N37 of GP5 was common in 2000-2005 but some sequences lack a glycosylation site in N46. The hypothetical GP5 was only 88.1% similar to LV and was less hydrophobic. Taking together these results suggest that PRRSV is still adapting to pig cells.

Epidemiology of salmonella infections in pig units and antimicrobial susceptibility profiles of the strains of Salmonella species isolated.

One hundred and thirteen finishing pig units and 74 sow units in Catalonia, Spain, were examined to determine the prevalence of salmonella infections and the factors that could be associated with them. Pooled faecal samples were taken from the finishing units, and samples of faeces were collected from individual sows. The Salmonella isolates were serotyped, phage typed and examined for their antimicrobial susceptibility to 18 common antimicrobial drugs. In addition, blood samples from pigs on 141 farms were analysed by ELISA. In both the bacteriological and serological surveys, a questionnaire with 84 questions was completed for each farm. Salmonella species were isolated from 20 per cent of the finishing units and 24 per cent of the sow units; 14 serotypes were detected in the finishing pigs and 11 in the sows. More than 30 per cent of the strains were resistant to tetracycline, sulphonamides, ampicillin or streptomycin, and 69 per cent of the strains were resistant to three or more agents up to 10 compounds. Seventy-seven per cent of the farms had at least one seropositive animal, and 26 per cent of these farms had an individual seroprevalence of 50 per cent or more. The factors associated (P<0.05) with the excretion of Salmonella species in the finishing units were the practice of raising livestock other than pigs (odds ratio [OR]=6.18), the herd size (OR=5.87), and a past history of clinical salmonellosis (OR=4.97). For the sows, the factors associated (P<0.05) with the excretion of Salmonella species were having open-flushed drainage of sewage (OR=34.48), a lack of rodent control measures (OR=0.05) and the number of sows in the unit (OR=9.26). Factors associated with seropositivity in the finishing units were a lack of bird-proof nets (OR=0.30) and the use of water from private wells (OR=3.64).

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.

Order-disorder phenomena in myelinated nerve sheaths: V. Effects of temperature on rat sciatic and optic nerves, and structural differences between the two types of nerve.

We describe in this work X-ray scattering and electron microscope studies of rat sciatic and optic nerves as a function of temperature. The scattering experiments were analyzed as described in the previous papers of this series: a variety of parameters were determined, some of which characterize the lattice disorder, others the structure of the motif. The main results are the following. All the parameters determined by the X-ray scattering study vary with temperature and the temperature-dependence is specific for the type of nerve (sciatic or optic). Most of the disorder-related parameters display a minimum or a maximum in the vicinity of physiological temperature (38 degrees C in rat); this observation, strongly supported by the electron microscope study, shows that the degree of organization of myelin is highest near physiological temperature. The structure of the motif, as revealed by the electron density profile, is fairly different in the two types of nerves (in contrast with the assumption made by previous workers); the structure also varies with temperature and the temperature-induced alterations are nerve-type specific. In the two types of nerve the thickness of the lipid bilayer varies with temperature as expected for a lipid-containing system with hydrocarbon chains in the disordered conformation. In sciatic nerve the thickness of the (thinner) cytoplasmic polar layer, which is also the layer most affected by lattice disorder in this type of nerve, decreases dramatically with increasing temperature. In optic nerve, in which lattice disorder predominantly affects the extracellular layer, the thickness of both the cytoplasmic and the extracellular layer is barely affected by temperature.

Mutations in the quinolone-resistance determining region (QRDR) of Salmonella strains isolated from pigs in Spain.

Quinolone-resistance determining region (QRDR) of Gyrase A gene was sequenced in 54 Salmonella strains of pig origin that have different quinolone-resistance patterns. Those strains accounted for 12 different serotypes. Mutations at Ser83 or Asp87 were predominant in the studied isolates. However, for serotypes Anatum and Virchow, resistance to quinolones seemed to be linked to specific mutations, namely, Ser83-->Tyr and Ser83-->Phe, respectively. Other mutations found in different positions did not seem to have clinical significance except for changes at Asp82.

Comparison of two commercial ELISAs for the serological diagnosis of salmonellosis in pigs.

Serum samples from 361 pigs (194 fattening pigs and 167 sows) were examined by means of two commercial ELISAs (Svanovir; Svanova Biotech and Salmotype; Labor Diagnostik) used for the serological diagnosis of salmonellosis in pigs; 211 of the samples came from farms of known bacteriological status and the other 150 were collected randomly from 60 farms of unknown status. The ELISAs were done according to the manufacturers' directions and the samples were categorised accordingly. The results were compared by using a linear regression analysis and by the calculation of Kappa values. To try to improve the agreement between the tests, the raw optical densities (ODS) were transformed to sample/positive (S/P) ratios by using the positive control as a reference, and cut-off values for these S/P ratios were calculated by means of a receiver operating characteristic (ROC) analysis. All but two of the known infected farms were recognised as such by both tests. However, the correlation of the raw ODS for individual pigs was poor (r=0.546) and had a Kappa value for the results categorised according to the manufacturers' recommendations of 0.191. On some farms the correlation was high (r=0.97) but on others it was low (r=0.05) with no apparent reason for the difference. The S/P ratios did not improve the agreement (Kappa=0.25).