Multidrug resistant staphylococci isolated from pigs with exudative epidermitis in North eastern Region of India.

Exudative epidermatitis or greasy pig disease (GPD) is a contagious disease of pig and endemic worldwide caused by toxigenic strains under genus Staphylococcus. The present study reported an outbreak of GPD in Champhai district of Mizoram adjoining to the southern border of Myanmar. A total of 60 samples were collected from 22 clinically affected animals and processed for isolation and identification of Staphylococcus spp. All the isolates were subjected to antimicrobial sensitivity assay, biofilm production assay and detection of virulence genes, biofilm genes and mec genes followed by cloning and sequencing for phylogenetic analysis. A total of 44 staphylococci belonged to four species (S. sciuri, S. aureus,S. lentus, and S. hyicus) were isolated. Majority of the isolates were multidrug resistant with maximum resistance against ampicillin, penicillin including vancomycin. None of the S. hyicus isolates was methicillin resistant (MRSH) but 66·67% isolates were MRSA. By PCR, mecA gene was detected in S. aureus (n = 2), S. sciuri (n = 4) and S. lentus (n = 3). Biofilm associated gene icaD was detected in S. aureus (n = 3), S. sciuri (n = 5), S. hyicus (n = 4) and S. lentus (n = 6). The exfoliative toxin genes (ehxB, shetA and tsst1) were detected in S. hyicus (n = 3) and S. aureus (n = 1) isolates. All the isolates were closely related with the isolates from pigs of China, Germany, Japan and USA. The pathogens might be transmitted through illegal migration of pigs from Myanmar to India.

Autogenous vaccination reduces antimicrobial usage and mortality rates in a herd facing severe exudative epidermitis outbreaks in weaned pigs.

This study was conducted in a commercial 1000-sow herd facing recurrent exudative epidermitis (EE) outbreaks during the nursery period and assessed the efficacy of autogenous vaccination in controlling such outbreaks. The vaccine was produced using three Staphylococcus hyicus isolates recovered from affected pigs shortly before the onset of the study. All of those isolates were positive for the exhB gene, which encodes the exfoliative toxin type B (ExhB). From four consecutive farrowing batches of sows, two batches were vaccinated (V) against S hyicus at five and two weeks before farrowing, and two sow batches remained non-vaccinated (NV). Vaccination efficacy was primarily determined by the levels of metaphylactic antimicrobial usage, and the morbidity and mortality data for the pigs of the V and NV sows. The total amount of antimicrobials used metaphylactically against EE in pigs among the V and NV farrowing batches was 39,600 and 88,550 mg, respectively. The used daily dose pig to animal daily dose pig ratio for the V and NV batches were 1.31 and 1.79, respectively (a ratio of 0.8 to 1.2 is indicative of correct dosing). The morbidity and mortality rates were V=6.50 and NV=14.36 (P=0.008), and V=2.59 and NV=5.02 (P=0.000), respectively. To conclude, autogenous vaccination of the sows with a vaccine based on exhB-positive S hyicus isolates reduced metaphylactic treatment with antimicrobials as well as the morbidity and mortality rates in weaned pigs compared with pigs from NV sow batches.

Detection and molecular characterization of porcine circovirus type 2 (PCV2) from piglets with exudative epidermitis in Uruguay.

Porcine circovirus type 2 (PCV2) is an economically important emerging pathogen associated with distinct syndromes and diseases in swine, collectively known as porcine circovirus associated diseases (PCVAD). The main purpose of this study was to investigate the presence of PCV2 in piglets affected with exudative epidermitis (EE) in Uruguay. In addition we aimed to analyze the phylogenetic relationships of the isolated strains. In June 2011 an outbreak of EE detected in a small herd was reported. Piglets presented skin lesions compatible with EE and symptoms associated with postweaning multisystemic wasting syndrome (PMWS) were also observed. Sera from affected and healthy animals were tested for the presence of viral DNA. Exclusively, diseased piglets were infected with PCV2. Phylogenetic analysis showed that PCV2 isolates belonged to PCV2b genotype. We report the detection and molecular characterization of PCV2 strains for the first time in Uruguay.

Distribution of the putative virulence factor encoding gene sheta in Staphylococcus hyicus strains of various origins.

In the present study, Staphylococcus (S.) hyicus strains isolated in Russia (n = 23) and Germany (n = 17) were investigated for the prevalence of the previously described genes sheta and shetb. Sheta was detected in 16 S. hyicus strains. Sheta-positive strains were mainly found among strains isolated from exudative epidermitis, and frequently together with the exfoliative toxin-encoding genes exhD and exhC. Partial sequencing of sheta in a single S. hyicus strain revealed an almost complete match with the sheta sequence obtained from GenBank. None of the S. hyicus strains displayed a positive reaction with the shetb-specific oligonucleotide primer used in the present study. According to the present results, the exotoxin encoding gene sheta seems to be distributed among S. hyicus strains in Russia and Germany. The toxigenic potential of this exotoxin, which does not have the classical structure of a staphylococcal exfoliative toxin, remains to be elucidated.

Nationwide molecular surveillance of exfoliative toxigenic Staphylococcus hyicus on pig farms across Japan.

We investigated the carriage of Staphylococcus hyicus and their exfoliative toxin genes (exhA, exhB, exhC, exhD, and shetb) among 424 pigs from 38 Japanese pig farms by PCR and characterized the isolates by 16S-23S intergenic spacer region polymorphism analysis. S. hyicus (n=207) were isolated from 17.9% of pigs and 72.5% of them were toxigenic. The isolation rate of toxigenic S. hyicus was four times higher in the pigs with exudative epidermitis than the healthy pigs (87.6% versus 19.6%; p<0.01). Among the toxins, exhA was the most prevalent, being detectable in 49.3% of toxin gene-positive strains. S. hyicus isolates were distributed into nine ITS-PCR types (a-i), with ITS-PCR types d, h, and e being predominant accounting for 70% (145/207) of total isolates or 76% (114/150) of toxin gene-positive strains. This study denotes high prevalence of toxigenic strains among S. hyicus circulating on pig farms in Japan.

Production of exfoliative toxin by isolates of Staphylococcus hyicus from different countries.

A total of 218 isolates of Staphylococcus hyicus from pigs in eight countries (Belgium, Croatia, Germany, Japan, Korea, Slovenia, the uk and the usa) and 44 isolates from other animals in Belgium, India, Japan and the usa were examined for the genes encoding the exfoliative toxins ExhA, ExhB, ExhC and ExhD by multiplex pcr. The expression of the toxins was confirmed by immunoblot analysis, using monoclonal or polyclonal antibodies specific for each of the toxins. The porcine isolates were from pigs with exudative epidermitis, pigs with other lesions and from healthy pigs, and one or more of the toxins could be found among the isolates from the pigs in all the countries. Toxigenic strains of S hyicus were isolated from both healthy and diseased pigs, but the chance of isolating toxigenic strains from pigs with exudative epidermitis was greater than from pigs with other lesions or healthy pigs. Of the 44 isolates from other animal species, only one isolate, from a hare from Belgium, produced ExhB, and one isolate, from a cow with mastitis from Japan, produced ExhA.

Exudative epidermitis and porcine circovirus-2 infection in a Swedish SPF-herd.

An outbreak of exudative epidermitis (EE) among piglets in a Swedish SPF-herd initiated a survey for indications as to the cause of disease. The herd was established by caesarean section and has been closed to all new animal material, with the exception of semen for artificial insemination (AI). The study comprised serum samples from the SPF-herd over a 10-year period (n=109) and a close monitoring of animals in the herd during the period after the EE outbreak. Serum samples from conventional boars at the AI-station servicing the herd were also included (n=9). All serum samples were tested for antibodies to porcine circovirus-2 (PCV-2). In addition, 3-week-old piglets from three litters (n=24) farrowed close after the initial EE outbreak were closely monitored for clinical signs of skin disease, sampled for Staphylococcus hyicus, tested for antibodies to porcine parvovirus and in sequentially collected serum samples tested for interferon-alpha (IFN-alpha) and interleukin-6. The PVC-2 serology showed that animals in the herd were sero-negative at least until 2 months prior to the EE outbreak. During the period close after the EE outbreak the animals showed varying levels of antibodies to PCV-2 but all the tested animals had sero-converted 4 months later. The AI boars were also sero-positive to PCV-2 at the time of the EE outbreak. Animals in the SPF-herd remained sero-positive to PCV-2 during the following 7 years. In the monitored litters, one piglet had clinical EE and 15 piglets displayed defined erythemas on the abdomen. Fourteen of the piglets also had IFN-alpha in serum on one or more occasions during the study, indicating viral activity among the animals. S. hyicus was isolated from all of the piglets from the earliest sampling point (3 days of age) and onwards, irrespective of clinical signs. PCV-2 was isolated from lymph node tissue collected from one of the EE affected pigs.Further, increases in the number of stillborn piglets, small litters (<6 piglets) and repeat breeders could be correlated to the time of PCV-2 sero-conversion. Coincidence of active viral infection and sero-conversion to PCV-2 points to the virus as the cause of the EE outbreak and reproductive disturbances.

Isolation of Staphylococcus hyicus subsp. hyicus from pigs affected with exudative epidermitis and experimental infection of piglets with isolates.

Five strains of Staphylococcus hyicus subsp. hyicus were isolated: three strains (P-1, P-2 and P-3) from the crust on the body surface of 6-month-old pigs on a farm in Aomori prefecture, and two (P-5 and P-6) from both the crust on the body surface and the joint of a 1-month-old piglet with exudative epidermitis (EE) on another farm. The characterization of the isolates and the experimental infection of the piglets with strain P-1 were carried out. Subcutaneous inoculation with the bacterial suspension (10(10) CFU) produced EE to all nine piglets. Eight of them had exudation and exfoliation within 24 hr of infection. Histopathologically, disappearance of stratum corneum and necrosis with vacuolar degeneration of prickle cells were remarkable in the epidermis. Infiltration of neutrophils and lymphocytes were observed in the dermis. The results clearly indicate that S. hyicus is responsible for incrustation of the body surface of weanling pigs and exudative epidermitis in young piglets.