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Antibodies to M. hyopneumoniae in serum diluted 1/40 were detected by a commercial ELISA kit (IDEXX M. hyo. Ab test, IDEXX, Westbrook, USA) according the instructions of the manufacturer. The absorbance value in serum diluted 1/40 (A450 = 0.4) was used as the limit for defining a positive reaction.
The samples of the first 15 pigs out of the 20 collected with paired serum samples available from both herd visits available were analysed from each herd. All blood samples were tested with influenza A antibody ELISA (ID Screen® Influenza A Antibody Competition, IdVet, Grabels, France) according to manufacturer instructions. A sample was considered unclear when the competition percentage (S/N%) was 46–49% and positive when the competition percentage was ≤45%. If a herd had at least one unclear or positive blood sample (pig) in the ELISA test in either of the samplings (first or second), blood samples of that herd were further analysed using a hemagglutination inhibition (HI) test according to European Surveillance Network for Influenza in Pigs. This was done with the antigens H1N1 (SW/Best/96), H1N2 (SW/Gent/7625/99) and H3N2 (SW/St. Oedenrode/96). All antigens were provided by GD Animal Health Service (Deventer, NL). A sample was considered HI positive if the HI titer was ≥1:20. Seroconversion at the individual pig level was defined as an increase in the HI titer between the first and second samples. A herd was considered to have an acute SIV infection if at least one individual pig seroconverted based on the HI test between herd visits.
Ten serum samples collected from the case herds during the second herd visit were examined for antibodies against MHyo. The antibodies were detected using a blocking ELISA commercial kit (MHyo ELISA, Oxoid, Basingstoke, UK), following manufacturer’s instructions. Samples with an optical density value less than 50% of the OD buffer control were interpreted as positive. The sensitivity and specificity of the ELISA test was 100% and 98%, respectively.
Antibodies to P. multocida in serum diluted 1/1000 was detected by a previously described indirect ELISA system based on a sonicated whole cell antigen. The absorbance value in serum diluted 1/1000 (A450 = 0.5) was used as the limit for defining a positive reaction.
Number of colony-forming units (CFU) of B. bronchiseptica per ml of swab fluid or nasal wash, tracheal wash, and BALF were determined as previously described (18, 25). Briefly, samples were serially diluted in phosphate-buffered saline (PBS) and plated on blood agar (trachea wash and BALF) or blood agar supplemented with 2 ug/ml amikacin, 4 ug/ml vancomycin, and 4 ug/ml amphotericin B (nasal swab and nasal wash). An aliquot of BALF was also plated on brain-heart infusion agar supplemented with 0.01 % NAD (w/v) and 5 % horse serum to rule out aerobic bacterial infection (other than B. bronchiseptica in respective groups). For determining viral load, nasal swab, trachea wash and BALF were thawed, filtered with a 0.45 um syringe filter, and fluid was used as previously described for virus isolation and titration (7), with immunohistochemistry staining used for evaluation (8) and the log10-transformed number of TCID50/ml of each sample calculated by the method of Reed and Muench (28). Samples that were negative by virus isolation were assigned a value of zero. Samples that were negative on virus titration but positive by virus isolation were assigned a value of 0.5 (log10) TCID50/ml.
BALF was collected at necropsy (dpi 5) and an aliquot centrifuged at 500 x g for 10 min. The cell-free supernatant was collected and used to evaluate cytokine levels in the lung. The amount of CCL2 (MCP-1), and IFN-α, IL-8, IL-1β, IL-10, and IFN-γ in the cell-free lung lavage was determined by single-plex (KingFisher Biotech) or multiplex cytokine ELISA (Aushon Biosystems), respectively, as previously described (29).
A two-factor repeated measures mixed effects model analysis of Log (CFU) differences between the treatment groups Bb/LAIV and Bb/NV and a dpi repeated measures factor using pigs as subjects was used to evaluate statistical differences in Bb nasal colonization (SAS, v9.2). A weighted regression equation of Log (TCID50) as a function of dpi for Bb/LAIV/Ch, Bb/NV/Ch, LAIV/Ch, and NV/Ch groups using mean Log (TCID50) values and standard weights of 1/variance with 95% confidence limits was used for statistical comparisons of nasal shedding (SAS, v9.2). A one-way ANOVA with a Tukey's post-test was used to evaluate differences in lung lesions, respiratory tract Bb colonization, and respiratory tract IAV burden (GraphPad Prism, v6). An unpaired students t-test was used for analysis of IAV-specific IgA levels, and a Kruskal-Wallis test with a Dunn's multiple comparisons post-test was used to determine statistical differences in lung cytokine levels (GraphPad Prism, v6).
The SEM (standard error of the mean) was calculated for each value. The data from all four treatment groups (H1N1, H1N2, H3N2, and control) were analyzed by the nonparametric Kruskal-Wallis test using Statistical Analysis Systems software (SAS Institute Inc., Cary, NC). A value of P < 0.05 was considered statistically significant.
This report underscores the need to investigate, by laboratory diagnostic methods, all cases presenting with respiratory distress and drop in egg production for IB. In the present case prior to further laboratory, most clinical diagnoses are based on signs and pathological lesions. Future observation and investigation should be designed to investigate the different IBV serotypes and genotypes in circulation across the country with the aim of producing vaccine (s), based on the identified serotypes, for combating the menace of IB in the Nigerian poultry population.
To detect the influenza virus antigens in the lungs, immunohistochemistry (IHC) was performed on all the tissue samples using a previously described protocol. Briefly, tissue slides were incubated with goat polyclonal antibody against the whole anti-influenza A virus (1:100; Chemicon, CA, USA) conjugated with streptavidin-alkaline phosphatase (1:200; Dako, CA). Each section was then counterstained with Mayer’s hematoxylin. Influenza A virus antigen-positive scores were calculated by estimating the number of antigen-positive cells in the lung per microscopic area at 200× magnification. This was based on the following criteria; 0 = no positive cells, 1 = minor (less than 10 positive cells), 2 = moderate (from 11 to 25 positive cells), and 3 = high (more than 26 positive cells).
Whole blood samples were analyzed for different leukocyte proportions and concentrations on a Abacus Junior Vet 5 hematology analyzer (Diatron, Hungary). Proportions of lymphocytes, monocytes and granulocytes were calculated as a percentage of leukocyte concentration.
The general swine influenza A real time RT-PCR method was used for detection of SIV in swabs and tissues, as described previously. Samples with Ct value <30 were considered to be M gene positive, samples having Ct value 30 to 35 with sigmoidal/logarithmic appearance were considered to be weak positive, samples with Ct value >35 were considered to be negative.
The standard bacteriological methods were used for detection of Pm in the nasal swabs and lung samples, as described previously. For Pm isolation collected samples were streaked onto agar containing 5% horse blood and incubated for 24 h at 37°C in 7.5% CO2 atmosphere. Strains with characteristic colony morphology were identified by Api 32E tests (BioMerieux, France). Additionally, for detection of genes encoding DNT, the PCR test was performed according to the previously described procedure.
To determine the prevalence of IBV serotype(s) in the flock, 32 blood samples were obtained via the brachial vein of the chickens. Briefly, 3–5 ml of blood was collected from each bird in syringes. The samples were left to clot in a slanted position and separated sera were decanted into sterile tubes afterward. The sera were transported in a cool box to the laboratory and stored at −20oC until tested. To determine the serotypes of IBV in the flock, the sera were screened using a panel of IBV antigens and antisera [Arkansas (Ark), Connecticut (Conn) and Mass (Charles Rivers Laboratories, CT)] in a hemagglutination-inhibition (HI) test according to the standard methods (OIE, 2008).
To compute the geometric mean titre (GMT), the individual serum sample titres from the same serotype were added up and averaged. The values obtained were then reported as the GMT after cross-checking against GMT values given in the Brugh’s table (Villegas, 2008).
In goats Muellerius capillaris is the most common lung worm. There is diffused pneumonia in affected goats without the presence of any nodular lesion. The parasite predisposes animals to secondary infections thereby compromising with the health in general. A rapid as well as inexpensive method for assessment of herd exposure to lung worm in cattle is the bulk milk ELISA. It is a useful tool for the veterinary practitioners as a herd health monitoring programme component or in the perspective of investigation of herd health. Over the past 15 years, studies have been conducted to prove that sequences of the internal transcribed spacers of ribosomal DNA provide useful genetic markers. This makes the basis for the molecular diagnosis of parasitic pneumonia in sheep and goat using PCR. DNA probes as well as assays based on PCR are used for identification and detection of Dictyocaulus as well as Protostrongylus. The sensitivity of most of the PCR-based assays is more than DNA probe assays. Multiple steps are required for the development of assays based on PCR, which follows the selection of oligonucleotide primers at the initial stage along with reporter probe. It has been found that usually PCR detects the parasitic DNA but certainly advances have been made in preparing samples. For this purpose, it is required to extract the DNA while removing the PCR inhibitors. This helps in achieving greater sensitivity.
Caprine arthritis encephalitis virus (CAEV) is a member of the lentivirus family (in small ruminants) leading to chronic disease of the joints and rarely encephalitis in goat kids under the age of six months. The virus is in close intimation with white blood cells. Thus, any kinds of body secretions containing blood cells are potential sources for virus spread to other animals in the herd [141, 142]. In goats,in order to detect caprine arthritis encephalitis virus (CAEV), serological tests or cell cultures are mainly used. Besides, PCR has also been developed for detection of CAEV sequences from peripheral blood mononuclear cells (PBMC), synovial fluid cells (SFC), and milk cells (MC) from the infected goats. This type of PCR assay especially provides a useful method to detect CAEV infection in goats [66–68]. A two-step TaqMan quantitative (q) PCR, which is specific as well as sensitive for the detection of infection due to CAEV by the use of a set of primers (specific), and a TaqMan probe that targets a region which is highly conserved within the gene that encodes the capsid protein of the virus have been developed. In the total deoxyribonucleotide (DNA) extracts, the proviral DNA can be detected successfully by this assay. The TaqMan qPCR assay provides a fast as well as specific and sensitive means for detection of proviral DNA of the virus and thereby proves to be useful for detection in large scale for eradication programs as well as epidemiological studies.
PCR techniques have been standardizedin several laboratories for the detection of proviral DNA. Other molecular techniques such as cloning and sequencing are also used to provide knowledge on a country or region's specific strain of CAEV. Phylogenetic analyses of the proviral DNAs of CAEV throughout the world have given the suggestion that in certain areas CAEV causes natural infection not only in goats but also in sheep. In order to track the transmission of the disease in near future, phylogenetic analyses may be used [66, 69, 70]. Molecular techniques such as cloning and sequencing are also used to provide knowledge on the prevalence of specific strain of CAEV in a country or a region which may have influence on serological assay as well as corresponding CAEV antigen [33, 71].
We tested 479 human sera for anti-MERS-CoV S1 IgG antibodies by ELISA. Using the ELISA kit recommended cut-offs, 20 sera (4.2%) were reactive with OD ratio of ≥ 1.1 and 21 (4.4%) were borderline reactive with OD ratio of ≥ 0.8 to < 1.1 (Table 2). Using the lower screening cut-off recommended by Muller et al., 173 additional sera would be regarded as suspected positives requiring testing by a neutralisation test. We tested all 479 sera irrespective of ELISA results for MERS-CoV neutralising activity using the ppNT assay; three (0.6%) were positive at a ppNT titre of ≥ 1:20 while one (0.2%) was positive at a ppNT titre of 1:10 (Table 3). Two of these were positive by ELISA (OD ratio cut-off ≥ 1.1), one borderline (OD ratio 0.83) and the other negative (OD ratio of 0.47) as per kit instructions, but would be recommended for confirmatory testing in the algorithm used by Muller et al.. Of these four ppNT-positive sera at a dilution of 1:10, three (0.6%) were confirmed with PRNT90 reactivity, regarded as confirmed neutralising sera, while the one with a ppNT 1:10 and ELISA OD ratio of 0.83 reduced plaque numbers by between 50% to 90%, i.e. positive in PRNT50 but not PRNT90, and regarded as borderline neutralising in the confirmatory test (Table 2).
The 200 sera from Hong Kong serving as negative controls were all negative in PRNT50 and PRNT90 assays. Validation of the ppNT test with 528 negative control sera has been previously reported.
Of the 41 sera that were positive or borderline by ELISA, i.e had an OD ratio ≥ 0.8, 38 were negative by ppNT. The other 438 sera were negative by both tests. There was a significant association between the results of the two tests (chi-squared with Yates correction: 14.9; p = 0.0001). However, the scatterplot between the ELISA and ppNT assays did not reveal a high level of correlation (correlation coefficient R-value: 0.13; 95% CI: 0.039–0.22) (Figure). If we consider that sera positive in the screening ppNT assay and confirmed by PRNT90 as true MERS-CoV positive sera, ELISA with the cut off recommended by the manufacturer had a sensitivity of 66.7% and a positive predictive value (PPV) of 10%. The serum with borderline PRNT activity was also borderline in ELISA reactivity. If the lower OD ratio of ≥ 0.3 is used as the cut-off for selecting sera for confirmatory testing as recommended by Mueller et al., then the sensitivity of the ELISA for screening for sera subsequently confirmed as positive or borderline neutralising positive was 100% but the PPV was only 0.19%.
Nine (6.6%) of 137 slaughterhouse workers, three (1.9%) of 156 camel herders and eight (4.3%) of 186 people from the general population were MERS-CoV antibody-positive in the ELISA test, using the ELISA kit recommended cut-off values (Table 2). Three (2.2%) of 137 slaughterhouse workers, none of 156 camel herders and one (0.5%) of 186 people from the general population were MERS-CoV neutralising antibody positive by ppNT assay. All four MERS-CoV neutralising antibody-positive sera also reduced virus plaque numbers by ≥ 50%; three of them reduced plaque counts by ≥ 90% (Table 2). There was no statistically significant association between exposure groups and MERS-CoV seropositivity by either ELISA or neutralisation tests. It should be noted all groups were resident in camel herding areas likely had some exposures to camels or camel products.
At necropsy, lung affected by pneumonia was recorded for each pig. Lesions were photographed, sketched on a standard diagram, and the proportion of affected lung assessed. In addition, tissues from lung lobes of each animal were collected for viral detection by RT-PCR.
For histopathology, the tissues were fixed in 10% buffered formalin and paraffin-embedded by standard techniques. Tissue sections were stained with hematoxylin and eosin and examined microscopically for histopathologic changes. Lung sections were examined for bronchiolar epithelial changes, and peribronchiolar and alveolar inflammation. Lung sections were given a score from 0-3 to reflect the severity of bronchial epithelial injury based on previously described methods. The lung sections were scored according to the following criteria: 0, no significant lesions; 1, a few airways affected with bronchiolar epithelial damage and light peribronchiolar lymphocytic cuffing often accompanied by mild focal interstitial pneumonia; 1.5, more than a few airways affected (up to 25%) often with mild focal interstitial pneumonia; 2, 50% airways affected often with interstitial pneumonia; 2.5, approximately 75% airways affected, usually with significant interstitial pneumonia; 3, greater than 75% airways affected, usually with interstitial pneumonia. Additionally, the proportion of airways affected in ten microscopic fields at 20 X magnification was also recorded. A single pathologist examined all slides and was blinded to the treatment groups.
Blood samples were collected using venipucture of the jugular vein. After collection serum was separated and stored at -20°C. Sera were subsequently analyzed for the detection of influenza virus antibodies by HI and influenza A Multiscreen ELISA. HI tests were performed following standard procedures. Samples were tested by HI against the challenge strain (IA04), the licensed commercial vaccine isolates γ and δ (A/Sw/IA/110600/00 and A/Sw/NC/031/2005, respectively), and H3N2 (A/Sw/MO/069/2005 H3N2) at arrival, thirteen days after the second vaccine, and at necropsy. Additionally, all sera were tested using the Influenza A Multiscreen ELISA following manufacturer's protocols. The Influenza A Multiscreen ELISA measures antibodies directed against the nucleoprotein (NP) of influenza A viruses.
Inference statistics were done by calculating the 95% confidence interval (CI) of the binomial proportions, except when numbers were too small for statistical significance, in which case only descriptive statistics are presented.
The study was designed as a case-control study. A case (positive herd) was defined as a herd with at least one rRT-PCR-positive sample, or if three or more of at least 20 blood samples were positive for antibodies against influenza A virus. If only one or two of the first 20 samples from a herd were positive with ELISA, the herd was retested with blood samples from 20 previously untested pigs and concluded positive if at least one of these samples were positive.
A questionnaire of 137 questions (123 were closed) was created to record demographics, husbandry information on the herds, and variables of interest. All farmers, irrespective of whether they represented case or control herds, were asked to report if they had observed signs of coughing, sneezing, depression, decrease in feed intake, or increase in reproductive disturbances in their pigs. Farmers who reported clinical signs were asked to estimate the proportion of affected pigs in different age groups. In Norway all nucleus and multiplier herds must keep written records (herd health cards) of all treatments irrespective of whether they were performed by a veterinarian or herd personnel. Farmers were asked to review the herd health cards for all veterinary or farmer treatments initiated during the study period.
The animals in the herds were grouped into four age groups: piglets (suckling piglets before weaning at approximately 5 weeks/10 kg), weaners (piglets after weaning, until approximately 30 kg), growers/finishers/recruit sows (from approximately 30 kg to slaughter weight or breeding age/weight), and sows. This age grouping was chosen because this is the way pigs are most commonly grouped and housed in the Norwegian herds. The transition from one group to the next is in most cases synonymous with a change in the pigs environment.
The interviewees were asked to indicate the occurrence of all observed clinical signs. It was emphasized by the interviewer that the occurrence of clinical signs should be reported as a deviation from the herds' normal clinical situation to lessen the risk of attributing regularly occurring clinical signs to the outbreak of pandemic influenza A (2009) virus. Farmers were asked to indicate the severity of observed clinical signs, but difficulties precisely defining degrees of severity between mild, moderate, or severe signs based on farmers subjective observation led to a simplified binomial classification where signs were classified as either present or absent. In addition, they were asked to estimate the duration of clinical signs in the different age groups of animals. The answer alternatives for duration of clinical signs were less than one week, one to two weeks, or more than two weeks.
The questionnaire was distributed by surface mail in the middle of November 2010. A letter was enclosed with the questionnaire encouraging the farmers to familiarize themselves with the questions and informing them that they would be asked to answer the questionnaire in a telephone interview within the following weeks. The interviews were performed by telephone over a period of 7 weeks between November 2010 and January 2011 by the first author. A paper copy of the questionnaire was used to register the answers for each interview. The data collected were later entered into a purpose-built form using Microsoft Excel 2010 (Microsoft Corporation, Redmond, WA, USA). Basic data analyses were performed in this database.
The initial diagnostic routine was limited to testing for the influenza A virus matrix gene, without subtyping. In view of the severe course of illness, the child was resampled for repeated testing including typing of the haemagglutinin (HA) gene by quantitative real-time PCR for H1 (seasonal and pdm2009), H3, H5, H7 and H9. All typing PCRs were negative.
We determined the full virus genome sequences of a cell culture isolate derived from a respiratory tract sample using Illumina MiSeq. All gene segments (GenBank accession numbers KY250316-KY250323) were 97–98% and 98–100% identical at, respectively, nucleotide and amino acid level to publicly available SIV sequences from the Netherlands (GISAID accession numbers EPI639351, EPI639914, EPI639917, EPI639930, EPI640657, EPI640912, EPI641210, EPI641215). The gene segments were all of the Eurasian avian A(H1N1) SIV lineage that has been circulating in pigs since 1979. Pigs at the farm visited by the patient tested positive for the same SIV (curation of full genome sequence data is in progress). The virus isolate from the patient, A/Netherlands/3315/2016, was sensitive to oseltamivir and zanamivir by NA-star neuraminidase inhibitor resistance detection assay (Applied Biosystems, Nieuwerkerk aan den IJssel, The Netherlands).
Initially, serum samples were analyzed for specific antibodies against A/Sw/Belgium/1/98(H1N1) and A/Sw/Flanders/1/98(H3N2) using hemagglutination-inhibition (HI) assays according to the method described in the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Following introduction of pandemic (H1N1) 2009 virus in the Norwegian pig population, serum samples were tested for influenza-A-specific NP antibodies using an ELISA kit (ID Screen Influenza A Antibody Competition test, ID VET, Montpellier, France). A herd was considered seropositive if three or more blood samples from the herd were positive for influenza-A-specific NP antibodies. In case one or two of the 20 blood samples from a herd were ELISA positive, the herd was retested with samples from another 20 pigs, and the herd concluded positive if at least one of these samples was seropositive. Positive and/or inconclusive samples were retested for hemagglutinin-specific antibodies using HI assay with A/Sw/Belgium/1/98(H1N1), A/Sw/Flanders/1/98(H3N2), and A/California/07/2009 (pandemic (H1N1) 2009) as antigens. The antigens for the HI tests were produced in chicken eggs at the Norwegian Veterinary Institute in Oslo. Titers ≥40 were considered positive.
Nasal swabs (Copan Innovation LTD, Brescia, Italy), individual or in pools of two, were placed in 1 mL of transport medium (EMEM 2% IBFS/Tris) before they were shipped to the laboratory. Total RNA was extracted from 200 μL of the sample material using the automatic extraction instrument NucliSens easyMag (bioMérieux, Norge AS, Oslo, Norway) according to the manufacturer's instruction. Detection of influenza A virus was performed as described by the World Health Organization, The WHO Collaborating Centre for influenza at CDC Atlanta. Specific detection of the HA gene of the pandemic (H1N1) 2009 subtype was carried out on influenza-A-positive samples as described by Robert Koch Institute. Amplification was performed on a Stratagene Mx3500P (LaJolla, CA, USA) with Superscript III Platinum One-Step Quantitative RT-PCR system (Invitrogen, Paisley, UK). In addition, some samples were analyzed at DTU Veterinary (National Veterinary Institute, Technical University of Denmark) using an inhouse real-time RT-PCR for detection of pandemic (H1N1) 2009 virus (ref Statens Serum institut, Denmark, unpublished). One RT-PCR-positive sample was sufficient to confirm a herd as positive for pandemic (H1N1) 2009 virus.
Full genome sequencing of isolate A/sw/Norway/02_11342/2009(H1N1) was carried out through a modified version of the WHO sequencing primers and protocol provided by the WHO Collaborating Centre for influenza in CDC, Atlanta, USA (28 April 2009). Briefly, 1 μL extracted RNA was amplified using 46 primer pairs and a one-step RT-PCR Kit (QIAGEN, Oslo, Norway). The resulting overlapping amplicons, carrying primer-derived M13 terminal sequences, were sequenced with M13 primers using the ABI BigDye Terminator v1.1 Cycle Sequencing Kit and the ABI 3130 Genetic Analyzer (Applied Biosystems, Warrington, UK). Sequences were assembled using Sequencher v.4.9 (GeneCodes Corporation, Ann Arbor, MI, USA) and compared to related H1N1 virus sequences using BioEdit software.
Human sera were tested for MERS-CoV IgG antibodies using a MERS-CoV S1 spike ELISA (EI 2604–9601 G kit, Euroimmun, Lübeck, Germany) according to the manufacturer’s instructions, at the Medical Virology and BSL-3 Laboratory at Institut Pasteur du Maroc, Casablanca, Morocco. The extinction value of the calibrator included in the test defines the upper limit of the reference range in non-infected humans and this value was set as the cut-off. The ELISA was made semi-quantitative by calculating the ratio of the extinction value of the serum sample over the extinction value of the calibrator. The manufacturer recommends cut-off ratios of < 0.8 be interpreted as negative, ≥ 0.8 and < 1.1 as borderline, and ≥ 1.1 as positive. Because subsequent publications suggested a lower ELISA cut-off of ratio ≥ 0.3 for screening purposes for the selection of sera to be confirmed by neutralisation tests, we have also included ELISA optical density (OD) ratios of ≥ 0.3 in our analysis.
All sera were also screened in triplicate in a MERS-CoV pseudoparticle neutralisation test (ppNT) as described previously. All sera positive at a ppNT screening dilution of 1:10 were titrated to end-point in the ppNT assay, as well as in a plaque reduction neutralisation test (PRNT) conducted in BSL-3 containment. The end-point for the ppNT assay was the highest serum dilution giving a ≥ 90% reduction in the luciferase signal compared with negative control. The end-point in the PRNT was the highest serum dilution that gave ≥ 50% (PRNT50) or ≥ 90% (PRNT90) reduction of virus plaques compared with control. The methods have been described elsewhere.
Sera-positive at a titre of ≥ 1:20 in ppNT and ≥ 1:10 in PRNT90 assays were regarded as positive. Sera-positive at a titre of ≥ 1:20 in ppNT and ≥ 1:10 in PRNT50, but negative in PRNT90 assays were regarded as a borderline positive neutralisation result. All other sera were regarded as negative.
For investigation of the presence of virus in general, selected samples were analysed with TEM, pan-viral microarray and de novo NGS. Eight samples from case animals with histological lesions typical of viral infections (villus atrophy) representing all four herds were analysed by TEM with negative results. A total of 18 samples from 13 case and five control animals were tested by microarray. None of the samples tested conclusively positive for any of the viruses present on the pan-viral microarray (data not shown). Samples from five case animals from each herd were pooled and tested by de novo NGS. Endogenous retrovirus was present in all four pools (data not shown). PKV-1 was also present in all four pools as evident by several reads (Table 5). In addition, the pools of samples from herd 1 were positive for rotavirus C, herd 2 were positive for rotavirus A and herd 3 were positive for PTV. One of the samples included in the pool from herd 2 was from the animal that also tested positive for rotavirus A by RT-qPCR and by ELISA.
The experimental unit for analysis consisted of data collected from each individual piglet. One-way analysis of variance (ANOVA) and Kruskal-Wallis test were used in this study. ANOVA is a parametric statistical test to analyze the difference between group means, while the Kruskal Wallis test is a non-parametric analogue of ANOVA. ANOVA was used with variables that showed the following: normal distribution such as ADWG, PRRSV RNA, M. hyopneumoniae DNA, PCV2 DNA, PRRSV antibody titer, M. hyopneumoniae antibody titer, PCV2 antibody titer, and number of IFN-γ-SC. The Kruskal-Wallis test was performed for variables without a normal distribution such as clinical signs, macroscopic lung lesion scores, and microscopic lung lesion scores. When a significant difference existed between the groups, post hoc multiple comparison tests with Tukey’s adjustment was conducted (t-test for ANOVA and Man-Whitney test for Krustal-Wallis analysis) to determine the significant differences between the pairwise groups. A value of P < 0.05 was considered significant.
Clinical and epidemiological information recorded by The Food Safety Authority was available from 43 pig herds suspected to be infected. The information was collected, while the herd was being sampled during the early phase of the outbreak between 10th of October 2009 and 31st of December 2009. The selection of herds was based on the same criteria as described previously. The size of these herds ranged in numbers from 5 to 2000 pigs (average 550 pigs). The investigation used a structured questionnaire consisting of questions on epidemiological information including clinical picture within the herd, likely source of infection, contact with people diagnosed with pandemic influenza or had ILI symptoms and being in contact with pigs while sick, movement of animals in or out of the farm, and handling and biosecurity measures.