Haemophilus parasuis is the etiological agent of Glässer’s disease in swine. H. parasuis comprises strains with heterogeneous virulence capacity, from non-virulent to highly virulent. Determination of the pathogenic potential of the strains is important for diagnosis and disease control. The virulence-associated trimeric autotransporters (vtaA) genes have been used to predict H. parasuis virulence by PCR amplification of their translocator domains. Here, we report a new and improved PCR designed to detect a different domain of the vtaA genes, the leader sequence (LS) as a diagnostic tool to predict virulence. A collection of 360 H. parasuis strains was tested by PCR with LS specific primers. Results of the PCR were compared with the clinical origin of the strains and, for a subset of strains, with their phagocytosis and serum resistance using a Chi-square test. LS-PCR was specific to H. parasuis, and allowed the differential detection of the leader sequences found in clinical and non-clinical isolates. Significant correlation was observed between the results of the LS-PCR and the clinical origin (organ of isolation) of the strains, as well as with their phagocytosis and serum susceptibility, indicating that this PCR is a good predictor of the virulence of the strains. In addition, this new PCR showed a full correlation with the previously validated PCR based on the translocator domain. LS-PCR could be performed in a wide range of annealing temperatures without losing specificity. This newly described PCR based on the leader sequence of the vtaA genes, LS-PCR, is a robust test for the prediction of the virulence potential of H. parasuis strains.

Neonatal diarrhoea represents a major disease problem in the early stages of animal production, increasing significantly pre-weaning mortality and piglets weaned below the target weight. Enteric diseases in newborn piglets are often of endemic presentation, but may also occur as outbreaks with high morbidity and mortality. The objective of this study was to assess the frequency of different pathogens involved in cases of recurrent neonatal diarrhoea in Spain. A total of 327 litters from 109 sow farms located in Spain with neonatal recurrent diarrhoea were sampled to establish a differential diagnosis against the main enteric pathogens in piglets. In total, 105 out of 109 (96.3%) case submissions were positive to one of the examined enteric organisms considered potentially pathogenic (Escherichia coli, Clostridium perfringens types A and C, Transmissible gastroenteritis virus [TGEV], Porcine epidemic diarrhoea virus [PEDV] or Rotavirus A [RVA]). Fifty-eight out of 109 (53.2%) submissions were positive for only one of these pathogens, 47 out of 109 (43.1%) were positive for more than one pathogen and, finally, 4 out of 109 (3.7%) were negative for all these agents. Escherichia coli strains were isolated from all submissions tested, but only 11 of them were classified into defined pathotypes. Clostridium perfringens type A was detected in 98 submissions (89.9%) and no C. perfringens type C was found. Regarding viruses, 47 (43.1%) submissions were positive for RVA, 4 (3.7%) for PEDV and none of them for TGEV. In conclusion, C. perfringens type A, E. coli and RVA were the main pathogens found in faeces of neonatal diarrheic piglets in Spain.

Background: Solely in Europoe, Salmonella Typhimurium causes more than 100,000 infections per year. Improved detection of livestock colonised with S. Typhimurium is necessary to prevent foodborne diseases. Currently, commercially available ELISA assays are based on a mixture of O-antigens (LPS) or total cell lysate of Salmonella and are hampered by cross-reaction. The identification of novel immunogenic proteins would be useful to develop ELISA based diagnostic assays with a higher specificity. Results: A phage display library of the entire Salmonella Typhimurium genome was constructed and 47 immunogenic oligopeptides were identified using a pool of convalescent sera from pigs infected with Salmonella Typhimurium. The corresponding complete genes of seven of the identified oligopeptids were cloned. Five of them were produced in E. coli. The immunogenic character of these antigens was validated with sera from pigs infeced with S. Tyhimurium and control sera from non-infected animals. Finally, human antibody fragments (scFv) against these five antigens were selected using antibody phage display and characterised. Conclusion: In this work, we identified novel immunogenic proteins of Salmonella Typhimurium and generated antibody fragments against these antigens completely based on phage display. Five immunogenic proteins were validated using a panel of positive and negative sera for prospective applications in diagnostics of Salmonela Typhimurium.

As no current data are available on the prevalence of leptospiral infection in swine in Germany, we analysed laboratory data from diagnostic examinations carried out on samples from swine all over Germany from January 2011 to September 2016. A total of 29,829 swine sera were tested by microscopic agglutination test (MAT) for antibodies against strains of eleven Leptospira serovars. Overall, 20.2% (6025) of the total sample collection tested positive for leptospiral infection. Seropositivity ranged between 16.3% (964) in 2011 and 30.9% (941) in 2016 (January to September only). Of all samples, 11.6% (57.3% of the positives) reacted with only one Leptospira serovar, and only 8.6% (42.7% of the positives) reacted simultaneously with two or more serovars. The most frequently detected serovar was Bratislava, which was found in 11.6% (3448) of all samples, followed by the serovars Australis in 7.3% (2185), Icterohaemorrhagiae in 4.0% (1191), Copenhageni in 4.0% (1182), Autumnalis in 3.7% (1054), Canicola in 2.0% (585), and Pomona in 1.2% (368). Modelling shows that both the year and the reason for testing at the laboratory had statistically strong effects on the test results; however, no interactions were determined between those factors. The results support the suggestion that the seropositivities found may be considered to indicate the state of leptospiral infections in the German swine population. Although data from passive surveillance are prone to selection bias, stratified analysis by initial reason for examination and analyses by model approaches may correct for biases. A prevalence of about 20% for a leptospiral infection is most probable for sows with reproductive problems in Germany, with an increasing trend. Swine in Germany are probably a reservoir host for serovar Bratislava, but in contrast to other studies not for Pomona and Tarassovi.

Equine recurrent uveitis (ERU) is considered one of the most important eye diseases in horses and typically appears with relapsing inflammatory episodes without systemic effects. Various disorders have been described as an initial trigger, including infections. Independent of the initiating cause, there are numerous indications that ERU is an immune-mediated disease. We investigated whether neutrophil extracellular traps (NETs) are part of the ERU pathogenesis. Therefore, vitreous body fluids (VBF), sera, and histological sections of the eye from ERU-diseased horses were analyzed for the presence of NET markers and compared with horses with healthy eyes. In addition, NET formation by blood derived neutrophils was investigated in the presence of VBF derived from horses with healthy eyes versus ERU-diseased horses using immunofluorescence microscopy. Interestingly, NET markers like free DNA, histone-complexes, and myeloperoxidase were detected in higher amounts in samples from ERU-diseased horses. Furthermore, in vitro NET formation was higher in neutrophils incubated with VBF from diseased horses compared with those animals with healthy eyes. Finally, we characterized the ability of equine cathelicidins to induce NETs, as potential NET inducing factors in ERU-diseased horses. In summary, our findings lead to the hypothesis that ERU-diseased horses develop more NETs and that these may contribute to the pathogenesis of ERU.

Background: Bacterial lung infections are a major cause of economic losses in the pig industry; they are responsible for approximately 50% of the antibiotics used in pigs and, therefore, also present an increasing concern to consumer protection agencies. In response to this changing market we investigated the feasibility of an old approach aimed at the breeding selection of more resistant pigs. As a first step in this direction we applied a new respiratory health score system to study the susceptibility of four different pig breeding lines (German Landrace, Piétrain, Hampshire, Large White) towards the respiratory tract pathogenActinobacillus(A.)pleuropneumoniae.Results: A controlled experimental aerosol infection with anA. pleuropneumoniaeserotype 7 isolate was performed using 106 weaning pigs of defined breeding lines from the breeds German Landrace, Piétrain, Hamphire, and Large White. Pigs were clinically assessed on days 4 and 20 post infection following a novel scoring system, the Respiratory Health Score (RHS), which combines clinical, sonographic and radiographic examination results. The ranking on day 4 was significantly correlated with the ranking based on the pathomorphological Lung Lesion Score (LLS; Spearman Rank Correlation Coefficient of 0.86 [p < 0.0001]). Based on their RHS pigs were assigned to the different quartiles independent of the breeding line. The RHS-based rankings of pigs on day 4 and on day 20 were highly correlated (Spearman Rank Correlation Coefficient of 0.82 [p < 0.0001]) independent of the breeding line. Pigs of the Hampshire line were predominantly found in the lowest scoring quartile (47.6%) and absent in the highest scoring quartile. In contrast, pigs of the German Landrace and Piétrain breeding lines were predominantly found in the highest scoring quartile (32.3% and 35.7%, respectively).Conclusion: These results demonstrate that the RHS obtained from live pigs shows a highly significant correlation to the lung lesion score considered as a "gold standard". The correlation of the ranking at days 4 and 20 post infection implies that the course of disease is highly dependent on the acute lung damage. The different severity of signs among the tested pig breeding lines clearly suggests a genetic difference in the susceptibility of pigs toA. pleuropneumoniaeinfection.