Characterization of Antimicrobial Resistance in Escherichia coli Isolated from Diarrheic and Healthy Weaned Pigs in Catalonia.

Postweaning diarrhea (PWD) is a multifactorial concern in the swine industry that leads to high antibiotic consumption, usually without testing susceptibility, increasing the risk of the selection of Escherichia coli-resistant strains. In this study, 251 E. coli strains isolated from fecal samples of diarrheic (n = 148) and apparently healthy piglets (n = 103) in farms in Catalonia were tested against their susceptibility to fourteen different antimicrobials. The phenotypic antimicrobial resistance (AMR) testing revealed high levels of AMR, with 41.4% of the isolates presenting a multidrug-resistant (MDR) profile. More specifically, resistance to class D (prudence) antimicrobials such as erythromycin (99.6%), amoxicillin (95.2%), streptomycin (91.6%), tetracycline (88.8%), lincospectin (64.5%), and sulfamethoxazole/trimethoprim (60%) was very high, as well as to class C (caution) antimicrobials such as florfenicol (45%). A special concern was observed for antimicrobial category B (restrict), like quinolones and colistin, that both presented a high rate of resistance. Colistin use was substantially reduced in Spain, but resistance is still present in weaned pigs, presenting a MIC90 of 4 µg/mL. This suggests that reducing antibiotic use is not enough to eliminate this AMR. Finally, it was found that piglets suffering diarrhea were more commonly carriers of MDR strains than the healthy ones (49.3% vs. 35%, p = 0.031). Therefore, given the high rates of resistance to the most commonly used antimicrobials, especially in diseased pigs, a new non-antibiotic-based approach should be implemented for the management of PWD.

Looking for Possible Benefits of Combining Short-Chain Fructo-Oligosaccharides (scFOS) with Saccharomyces cerevisiae Sc 47 on Weaned Pigs Orally Challenged with Escherichia coli F4.

The objective of this work was to evaluate the effect of supplementing short-chain fructo-oligosaccharides (scFOS) combined or not with live yeast Saccharomyces cerevisiae Sc 47 on weanling pigs challenged with Escherichia coli F4+. We allocated ninety-six piglets to four experimental diets: control (CTR); supplemented with scFOS (5 g/kg Profeed® P95) (scFOS); S. cerevisiae Sc 47 (1 g/kg Actisaf® Sc 47 HR +) (YEA); or both (SYN). Parameters included: performance; E. coli F4+ detection; fermentation activity; inflammatory biomarkers; and ileal histomorphology. Our results showed that supplementing scFOS was able to reduce the incidence of diarrhea, and both supplements were able to lower counts of EHEC along the gut. Supplementing scFOS was mostly associated with changes in the gut ecosystem and increases in the lactobacilli population, while S. cerevisiae Sc 47 registered increases in the numbers of ileal intraepithelial lymphocytes. The synbiotic mixture showed the lowest diarrhea incidence and fecal scores, benefiting from complementary modes of action and possible synergistic effects due to a hypothesized yeast-LAB cross-feeding phenomenon in the foregut. In conclusion, our results evidence that supplementing scFOS or Saccharomyces cerevisiae Sc 47 is efficacious to fight post-weaning colibacillosis, and combining both could be beneficial in high-risk scenarios.

Infection dynamics, transmission, and evolution after an outbreak of porcine reproductive and respiratory syndrome virus.

The present study was aimed at describing the infection dynamics, transmission, and evolution of porcine reproductive and respiratory syndrome virus (PRRSV) after an outbreak in a 300-sow farrow-to-wean farm that was implementing a vaccination program. Three subsequent batches of piglets (9-11 litters/batch) were followed 1.5 (Batch 1), 8 (Batch 2), and 12 months after (Batch 3) from birth to 9 weeks of age. The RT-qPCR analysis showed that shortly after the outbreak (Batch 1), one third of sows were delivering infected piglets and the cumulative incidence reached 80% by 9 weeks of age. In contrast, in Batch 2, only 10% animals in total got infected in the same period. In Batch 3, 60% litters had born-infected animals and cumulative incidence rose to 78%. Higher viral genetic diversity was observed in Batch 1, with 4 viral clades circulating, of which 3 could be traced to vertical transmission events, suggesting the existence of founder viral variants. In Batch 3 though only one variant was found, distinguishable from those circulating previously, suggesting that a selection process had occurred. ELISA antibodies at 2 weeks of age were significantly higher in Batch 1 and 3 compared to Batch 2, while low levels of neutralizing antibodies were detected in either piglets or sows in all batches. In addition, some sows present in Batch 1 and 3 delivered infected piglets twice, and the offspring were devoid of neutralizing antibodies at 2 weeks of age. These results suggest that a high viral diversity was featured at the initial outbreak followed by a phase of limited circulation, but subsequently an escape variant emerged in the population causing a rebound of vertical transmission. The presence of unresponsive sows that had vertical transmission events could have contributed to the transmission. Moreover, the records of contacts between animals and the phylogenetic analyses allowed to trace back 87 and 47% of the transmission chains in Batch 1 and 3, respectively. Most animals transmitted the infection to 1-3 pen-mates, but super-spreaders were also identified. One animal that was born-viremic and persisted as viremic for the whole study period did not contribute to transmission.

In the presence of non-neutralising maternally derived antibodies, intradermal and intramuscular vaccination with a modified live vaccine against porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) induce similar levels of neutralising antibodies or interferon-gamma secreting cells.

The purpose of this study was to compare the immune response generated by the intramuscular and the intradermal vaccination route against the porcine reproductive and respiratory syndrome virus (PRRSV). Piglets from a seronegative and a seropositive farm were selected (n = 28 piglets per farm), and each group was divided into two groups and vaccinated after weaning with modified live vaccine Unistrain® PRRS (Laboratorios Hipra Amer, Spain) by the intramuscular (IM) or the intradermic (ID) route. For the following 6 weeks, animals were weekly bled to assess the humoral response by PRRSV-specific antibody ELISA and viral neutralisation test. At 0-, 3-, 4- and 6 weeks post-vaccination, peripheral mononuclear blood cells (PBMC) from eight animals per group were recovered to analyse cellular response by IFN-γ ELISPOT and lymphoproliferation. Serum IL-12 was also quantified by ELISA. Seroconversion was first detected 14 days post-vaccination (dpv) for both IM and ID routes, and peaked at 35 dpv (both IM groups and ID seropositive) or 42 dpv (ID seronegative). At 3 weeks after vaccination, 6/27 (22.22%) animals from negative origin had not seroconverted, and neutralising titres were significantly lower at 35 dpv compared to the seropositive origin (mean log2 titres of 1.36 and 4.25 respectively) Also, it was 10 times more probable for them to have high levels of IL-12 a week after vaccination than for animals of seropositive origin. Cellular immune response analysed by lymphoproliferation and IFN-γ ELISPOT was already present at 21 dpv and until 42 dpv, with no significant differences between groups except for a higher lymphoproliferation at 35 dpv in the IM seropositive group (Kruskal-Wallis, p < 0.05). These results indicate that the intradermal route induces an immune response equivalent to the classical intramuscular route even in presence of non-neutralising maternal immunity, which in this study has proven to facilitate seroconversion after vaccination with an heterologous strain.

Antimicrobial Resistance in Bacteria Isolated from Exotic Pets: The Situation in the Iberian Peninsula.

Literature related to antimicrobial resistant (AMR) bacteria in exotic pets is minimal, being essential to report objective data on this topic, which represents a therapeutic challenge for veterinary medicine and public health. Between 2016 and 2020, laboratory records of 3156 exotic pet specimens' microbiological diagnoses and antibiotic susceptibility testing (AST) results were examined. The samples were classified into three animal classes: birds (n = 412), mammalia (n = 2399), and reptilian (n = 345). The most prevalent bacteria in birds and mammals were Staphylococcus spp. (15% and 16%), while in reptiles they were Pseudomonas spp. (23%). Pseudomonas was the genus with the highest levels of AMR in all animal groups, followed by Enterococcus spp. By contrast, Gram-positive cocci and Pasteurella spp. were the most sensitive bacteria. Moreover, in reptiles, Stenotrophomonas spp., Morganella spp., and Acinetobacter spp. presented high levels of AMR. Multidrug-resistant (MDR) bacteria were isolates from reptiles (21%), birds (17%), and mammals (15%). The Enterobacterales had the highest MDR levels: S. marcescens (94.4%), C. freundii (50%), M. morganii (47.4%), K. pneumoniae (46.6%), E. cloacae (44%), and E. coli (38.3%). The prevalence of MDR P. aeruginosa strains was 8%, detecting one isolate with an XDR profile. Regarding antimicrobial use, many antibiotics described as critically important for human use had significant AMR prevalence in bacteria isolated from exotic pets. Under the One-Health approach, these results are alarming and of public health concern since potential transmission of AMR bacteria and genes can occur from exotic pets to their owners in both senses. For this reason, the collaboration between veterinarians and public health professionals is crucial.

Immune response does not prevent homologous Porcine epidemic diarrhoea virus reinfection five months after the initial challenge.

The aim of the present study was to evaluate the duration of protective immunity against Porcine epidemic diarrhoea virus (PEDV). To do so, a two phases study was performed. In the first phase, 75 four-week-old pigs (group A) were orally inoculated (0 days post-inoculation; dpi) with a European PEDV G1b strain and 14 were kept as controls (group B). The second phase started five months later (154 dpi), when animals in group A were homologous challenged and animals in group B were challenged for first time. Clinical signs, viral shedding and immune responses were evaluated after each inoculation, including the determination of antibodies (ELISA and viral neutralization test, IgA and IgG ELISPOTs using peripheral blood mononuclear cells and lymph node cells) and the frequency of interferon-gamma (IFN-γ) secreting cells. During the first phase, loose stools/liquid faeces were observed in all group A animals. Faecal shedding of PEDV occurred mostly during the first 14 days but, in some animals, persisted until 42 dpi. All inoculated animals seroconverted for specific-PEDV IgG and IgA, and for neutralizing antibodies (NA). At 154 dpi, 77% of pigs were still positive for NA. After that, the homologous challenge resulted in a booster for IgG, IgA, NA, as well as specific-PEDV IgG, IgA and IFN-γ secreting cells. In spite of that, PEDV was detected in faeces of all pigs from group A, indicating that the immune response did not prevent reinfection, although the duration of the viral shedding and the total load of virus shed were significantly lower for previously challenged pigs (p < .05). Taken together, the results indicated that, potentially, maintenance of PEDV infection within an endemic farm may occur by transmission to and from previously infected animals and also indicates that sterilizing immunity is shorter than the productive life of pigs.

A retrospective study of porcine epidemic diarrhoea virus (PEDV) reveals the presence of swine enteric coronavirus (SeCoV) since 1993 and the recent introduction of a recombinant PEDV-SeCoV in Spain.

A retrospective evaluation of PEDV-positive samples recovered in Spain before and after the re-emergence of this coronavirus in several European countries was carried out. We described for the first time recombinant SeCoV circulating in Spain between 1993 and 2014 and its misidentification as PEDV when diagnostic assays based on the S-protein or S-gene of the PEDV were used. The complete S-gene sequence of 7 Spanish SeCoV and 30 PEDV Spanish isolates was phylogenetically analysed including the S-gene sequences of the three SeCoV and a representative selection of the PEDV strains with complete genome sequences available in the GenBank. The tree showed a common ancestor for the S-gene of the PEDV and SeCoV, but no evolution from any known PEDV clade was shown for the SeCoV strains. Moreover, complete genome sequences were obtained from 23 PEDV strains recovered in Spanish swine farms since 2014. The phylogenetic tree showed the INDEL type genogroup of these Spanish strains, supporting the lower pathogenicity of this genogroup since no significant economic losses were reported in the affected Spanish swine farms. Four subgroups were detected among PEDV strains in Spain, closely related to the recent European strains. Moreover, eight of the most recent Spanish PEDV isolates formed a subclade together with three European strains from 2015, showing a new evolution branch with a recombinant virus.

Antimicrobial Resistance Profiles and Characterization of Escherichia coli Strains from Cases of Neonatal Diarrhea in Spanish Pig Farms.

Escherichia coli is considered one of the most common agents associated with neonatal diarrhea in piglets. The aim of this work was to characterize the pathogenic and antimicrobial resistance (AMR) profiles of 122 E. coli strains isolated from pigs suffering diarrhea (n = 94) and pigs without diarrhea (n = 28) of 24 farms in Spain. Virulence factors, toxins and AMR (ESBL and colistin) genes and AMR phenotypes of E. coli isolates were analyzed. Low prevalence of pathogenic E. coli strains (26%) was found in both groups. However, ETEC and VTEC strains were more frequently isolated from diarrheic piglets. Irrespectively of diarrhea occurrence, 97.5% of the strains showed a multidrug-resistance (MDR) profile to aminopenicillins, sulfonamides and tetracyclines. It was found that 22% of E. coli was CTX-M+, with CTX-M-14 being the principal allelic variant. Remarkably, 81.5% of CTX-M+ strains were isolated from diarrheic animals and presented an extended MDR profile to aminopenicillins, quinolones and aminoglycosides. Finally, low frequencies of colistin resistance genes mcr-1 (4/122) and mcr-4 (1/122) were found. MDR E. coli strains are circulating in pig farms of Spain, representing a serious threat to animal and public health. More appropriate diagnostic approaches (genetic and AMR phenotypic analysis) should be implemented in animal health to optimize antibiotic treatments.

Antimicrobial resistance profile and prevalence of extended-spectrum beta-lactamases (ESBL), AmpC beta-lactamases and colistin resistance (mcr) genes in Escherichia coli from swine between 1999 and 2018.

The frequent usage of antibiotics in livestock has led to the spread of resistant bacteria within animals and their products, with a global warning in public health and veterinarians to monitor such resistances. This study aimed to determine antibiotic resistance patterns and genes in pig farms from Spain during the last twenty years. Susceptibility to six antibiotics commonly used in pig production was tested by qualitative (disk diffusion) and quantitative (minimum inhibitory concentration, MIC) methods in 200 strains of Escherichia coli which had been isolated between 1999 and 2018 from clinical cases of diarrhoea in neonatal and post-weaned piglets. Results showed resistance around 100% for amoxicillin and tetracycline since 1999, and a progressive increase in ceftiofur resistance throughout the studied period. For colistin, it was detected a resistance peak (17.5% of the strains) in the 2011-2014 period. Concerning gentamicin, 11 of 30 strains with intermediate susceptibility by the disk diffusion method were resistant by MIC. Besides, the most frequent antimicrobial resistance genes were the extended-spectrum beta-lactamase (ESBL) bla CTX-M (13.5% of strains, being CTX-M-14, CTX-M-1 and CTX-M-32 the most prevalent genomes, followed by CTX-M-27, CTX-M-9 and CTX-M-3), AmpC-type beta-lactamase (AmpC) bla CMY-2 (3%) and colistin resistance genes mcr-4 (13%), mcr-1 (7%) and in less proportion mcr-5 (3%). Interestingly, these mcr genes were already detected in strains isolated in 2000, more than a decade before their first description. However, poor concordance between the genotypic mcr profile and the phenotypical testing by MIC was found in this study. These results indicate that although being a current concern, resistance genes and therefore antimicrobial resistant phenotypes were already present in pig farms at the beginning of the century.

High levels of unreported intraspecific diversity among RNA viruses in faeces of neonatal piglets with diarrhoea.

BACKGROUND: Diarrhoea is a major cause of death in neonate pigs and most of the viruses that cause it are RNA viruses. Next Generation Sequencing (NGS) deeply characterize the genetic diversity among rapidly mutating virus populations at the interspecific as well as the intraspecific level. The diversity of RNA viruses present in faeces of neonatal piglets suffering from diarrhoea in 47 farms, plus 4 samples from non-diarrhoeic piglets has been evaluated by NGS. Samples were selected among the cases submitted to the Veterinary Diagnostic Laboratories of Infectious Diseases of the Universitat Autònoma de Barcelona (Barcelona, Spain) and Universidad de León (León, Spain). RESULTS: The analyses identified the presence of 12 virus species corresponding to 8 genera of RNA viruses. Most samples were co-infected by several viruses. Kobuvirus and Rotavirus were more commonly reported, with Sapovirus, Astrovirus 3, 4 and 5, Enterovirus G, Porcine epidemic diarrhoea virus, Pasivirus and Posavirus being less frequently detected. Most sequences showed a low identity with the sequences deposited in GenBank, allowing us to propose several new VP4 and VP7 genotypes for Rotavirus B and Rotavirus C. CONCLUSIONS: Among the cases analysed, Rotaviruses were the main aetiological agents of diarrhoea in neonate pigs. Besides, in a small number of cases Kobuvirus and Sapovirus may also have an aetiological role. Even most animals were co-infected in early life, the association with enteric disease among the other examined viruses was unclear. The NGS method applied successfully characterized the RNA virome present in faeces and detected a high level of unreported intraspecific diversity.

Prevalence of enteric pathogens in diarrheic and non-diarrheic samples from pig farms with neonatal diarrhea in the North East of Spain.

Diarrhea is one of the major causes of neonatal mortality in pigs. In the present study, 31 pig farms with outbreaks of neonatal diarrhea were investigated in Catalonia (NE Spain) from February 2017 until June 2018. Two hundred and fifteen diarrheic samples from 1 to 7 days old piglets were tested for a panel of enteric pathogens. In 19 of the studied farms additional fecal samples from apparently healthy pen-mates were collected and tested for the same panel of infectious agents. Samples were bacteriologically cultured and tested by PCR for E. coli virulence factors genes, C. perfringens types A and C toxins (Cpα, Cpß, Cpß2) and C. difficile toxins (TcdA, TcdB). Moreover, Rotavirus A (RVA), Rotavirus B (RVB), Rotavirus C (RVC), porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) were also determined by RT-qPCR. More than one pathogen could be detected in all of the outbreaks. Nevertheless, RVA was the only agent that could be statistically correlated with the outcome of diarrhea. For the other viruses and bacteria analyzed significant differences between the diseased pigs and the controls were not found. In spite of this, the individual analysis of each of the studied farms indicated that other agents such as RVB, RVC, toxigenic C. difficile or pathogenic E. coli could play a relevant role in the outbreak of diarrhea. In conclusion, the large diversity of agent combinations and disease situations detected in neonatal diarrhea outbreaks of this study stand for a more personalized diagnosis and management advice at a farm level.

Full-genome characterization by deep sequencing of rotavirus A isolates from outbreaks of neonatal diarrhoea in pigs in Spain.

Since early 2017, in Spain there was an apparent increase in reports on rotavirus involvement in neonatal diarrhoea outbreaks, affecting also adult sows. In this study, 16 unrelated outbreaks of diarrhoea in suckling pigs and sows, where rotavirus A was the only pathogen detected, were investigated. Deep-sequencing was performed on total RNA from twenty-four positive faecal samples. Genotyping, phylogenetic and bayesian analyses showed that all isolates had a common ancestor of porcine, or human porcine-like, origin. The new strain was introduced in the population shortly before the onset of the outbreaks. Besides, a high diversification of the VP7 and VP4 genes occurred in a short time. Isolates presented a high number of amino acid changes in the neutralizing epitopes compared to vaccine sequences. The present report illustrates how a new rotavirus A strain may disseminate rapidly and the extremely high diversification that this pathogen may undergo in a short period.

Microbiological diagnosis and antimicrobial sensitivity profiles in diseased free-living raptors.

Free-living raptors (birds of prey) can act as reservoirs of potentially zoonotic agents, but they also can be affected by microorganisms as target hosts. In this retrospective study, microbiological results (n = 663) and antibiotic sensitivity profiles (n = 108) of bacterial isolates were analysed from diseased free-living raptors. Sixty-nine percent of cases (n = 457) yielded bacteria: 58% were in pure culture and 42% were of different species. Remarkably, samples from necropsies (47%) had higher percentage of pure isolations than those obtained from clinical (31%) samples (P < 0.001). Among bacterial isolates, Escherichia coli was the most common agent (35%), principally recovered from necropsied birds with clinical signs of septicaemia or respiratory disorders. Pseudomonas aeruginosa (7%) was isolated from birds with systemic infection and from oral lesions, especially in nocturnal raptors (P < 0.001). Staphylococcus spp. (5%), mainly Staphylococcus aureus, was found to be the most prevalent cause of pododermatitis (35%) and Staphylococcus hyicus was isolated from conjunctivitis (18.2%). Interestingly, 8% of samples with lesions compatible with avian tuberculosis were positive to the Mycobacterium avium complex. The most frequent fungi associated with pneumonic lesions and ingluvitis were Aspergillus spp. and Candida spp., respectively. More than 50% of the 108 isolates (34 different bacterial spp.) demonstrated resistance to clindamycin, ampicillin, tetracycline, cefuroxime, enrofloxacin and trimethoprim/sulphamethoxazole. Among the E. coli strains, 71% (27/38) presented a multidrug-resistance pattern to >3 antimicrobials. Detection in wildlife of antimicrobial-resistant pathogens that might be significant at the animal-human-ecosystem interface is of great relevance under the 'One Health' approach.

Multidrug-resistant Salmonella enterica Serovar Typhimurium Monophasic Variant 4,12:i:- Isolated from Asymptomatic Wildlife in a Catalonian Wildlife Rehabilitation Center, Spain.

Wildlife can act as long-term asymptomatic reservoirs for zoonotic bacteria, such as Salmonella. The prevalence and antimicrobial-susceptibility profiles of Salmonella spp. were assessed in 263 cases in wildlife from 22 animal orders from a wildlife rehabilitation center in Catalonia (NE Spain), September 2013-May 2014. Eleven of 263 tested animals were positive for Salmonella spp., representing an overall prevalence of 4.2%. Prevalences by taxonomic categories were 2% in mammals, 4.7% in birds, and 4.5% in reptiles. By species, one each of European hedgehog (Erinaceus europeus; from a sample of n = 26), Eurasian Eagle Owl (Bubo bubo; n = 2), Barn Owl (Tyto alba; n = 3), Tawny Owl (Strix aluco; n = 20), Egyptian Vulture (Neophron percnopterus; n = 1), Griffon Vulture (Gyps fulvus; n = 1), and Hoopoe (Upupa epops; n = 2), and two each Common Kestrels (Falco tinnunculus; n = 16) and pond sliders (Trachemys scripta; n = 25) were positive for Salmonella. By serotyping, seven of eleven isolates were classified as S. enterica subsp. enterica serovar Typhimurium, and five of seven belonged to the monophasic variant 4,12:i:-. All the monophasic variants were isolated from birds (4/5 in raptors) and showed a multidrug-resistance (MDR) profile to at least ampicillin, streptomycin, sulfonamide, and tetracycline (R-type ASSuT), and up to 12 antibiotics. The large proportion of S. Typhimurium monophasic MDR strains detected in wildlife never treated with antibiotics, especially in raptors, adds more complexity to the epidemiologic control of one of the most frequent serovars involved in human and livestock infection.

Vaccination with a genotype 1 modified live vaccine against porcine reproductive and respiratory syndrome virus significantly reduces viremia, viral shedding and transmission of the virus in a quasi-natural experimental model.

The present study assessed the efficacy of vaccination against genotype 1 porcine reproductive and respiratory syndrome virus (PRRSV) in terms of reduction of the transmission. Ninety-eight 3-week-old piglets were divided in two groups: V (n=40) and NV (n=58) that were housed separately. V animals were vaccinated with a commercial genotype 1 PRRSV vaccine while NV were kept as controls. On day 35 post-vaccination, 14 NV pigs were separated and inoculated intranasally with 2 ml of a heterologous genotype 1 PRRSV isolate ("seeder" pigs, SP). The other V and NV animals were distributed in groups of 5 pigs each. Two days later, one SP was introduced into each pen to expose V and NV to PRRSV. Sentinel pigs were allocated in adjacent pens. Follow-up was of 21 days. All NV (30/30) became viremic after contact with SP while only 53% of V pigs were detected so (21/40, p<0.05). Vaccination shortened viremia (12.2±4 versus 3.7±3.4 days in NV and V pigs, respectively, p<0.01). The 50% survival time for becoming infected (Kaplan-Meier) for V was 21 days (CI95%=14.1-27.9) compared to 7 days (CI95%=5.2-8.7) for NV animals (p<0.01). These differences were reflected in the R value as well: 2.78 (CI95%=2.13-3.43) for NV and 0.53 (CI95%=0.19-0.76) for V pigs (p<0.05). All sentinel pigs (10/10) in pens adjacent to NV+SP pens got infected compared to 1/4 sentinel pigs allocated contiguous to a V+SP pen. These data show that vaccination of piglets significantly decrease parameters related to PRRSV transmission.

Cytokine profiles and phenotype regulation of antigen presenting cells by genotype-I porcine reproductive and respiratory syndrome virus isolates.

The present study examined the immunological response of antigen presenting cells (APC) to genotype-I isolates of porcine reproductive and respiratory syndrome virus (PRRSV) infection by analysing the cytokine profile induced and evaluating the changes taking place upon infection on immunologically relevant cell markers (MHCI, MHCII, CD80/86, CD14, CD16, CD163, CD172a, SWC9). Several types of APC were infected with 39 PRRSV isolates. The results show that different isolates were able to induce different patterns of IL-10 and TNF-α. The four possible phenotypes based on the ability to induce IL-10 and/or TNF-α were observed, although different cell types seemed to have different capabilities. In addition, isolates inducing different cytokine-release profiles on APC could induce different expression of cell markers.

Longitudinal study of hepatitis E virus infection in Spanish farrow-to-finish swine herds.

Hepatitis E is a zoonotic disease and is highly prevalent in European swine livestock. There is a need to compare the infection dynamics of hepatitis E virus (HEV) between herds with the same production system and determine the percentage of animals that could arrive infected at slaughter age. Therefore, a longitudinal study was performed in six Spanish farrow-to-finish affected farms. Twenty piglets per farm were monitored from nursery to slaughter. RT-PCR and serology techniques were applied to analyze longitudinally collected sera and/or faecal samples. Liver and bile samples were also taken at the abattoir. Anti-HEV IgM were firstly detected at 7 weeks of age in 5 farms whereas at 13 weeks of age in 1 farm (farm 2). At slaughter age 50-100% of pigs had seroconverted to anti-HEV IgG in the former 5 farms whereas in the other herd only 5% of pigs were IgG seropositive (farm 2). Six out of 96 livers and 5 out of 80 biles analyzed were HEV positive at the abattoir (total percentage of infected animals: 11.5%). All these positive animals had already seroconverted except 2 pigs of farm 2. Hence, pigs can be seronegative at slaughter age being infected during the latest fattening period. Manipulation of HEV-infected livers or other organs from pigs could be considered a possible route of transmission in Spanish abattoirs. This study represents the first longitudinal survey on swine HEV infection dynamics conducted in different herds.

Comparison of muscle fluid and serum for detection of antibodies against hepatitis E virus in slaughter pigs.

Muscle fluid was investigated as an alternative to serum for detecting anti-hepatitis E virus (HEV) antibodies in slaughter pigs. Samples of serum and diaphragmatic muscle juice from 67 pigs were analysed by anti-HEV IgG ELISA. Compared to the serum ELISA, the ELISA on diaphragmatic muscle fluid had a sensitivity of 93%, a specificity of 91.7%, a κ value of 0.84 and a determination coefficient (R(2)) of 0.77; both tests had global agreement of results. Muscle fluid can be used as an alternative to serum for serological detection of HEV antibodies in slaughter pigs.

Increasing contact with hepatitis E virus in red deer, Spain.

To describe the epidemiology of hepatitis E virus (HEV) in red deer in mainland Spain, we tested red deer for HEV RNA and antibodies. Overall, 10.4% and 13.6% of serum samples were positive by ELISA and reverse transcription-PCR, respectively. The increasing prevalence suggests a potential risk for humans.