Exudative epidermitis of piglets caused by non-toxigenic Staphylococcus sciuri

Accepted Manuscript Title: Exudative epidermitis of piglets caused by non-toxigenic Staphylococcus sciuri Author: Lixin Lu Kongwang He Yanxiu Ni Zhengyu Yu Aihua Mao PII: DOI: Reference:

S0378-1135(16)30383-2 http://dx.doi.org/doi:10.1016/j.vetmic.2016.12.016 VETMIC 7474

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VETMIC

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22-9-2016 7-12-2016 8-12-2016

Please cite this article as: Lu, Lixin, He, Kongwang, Ni, Yanxiu, Yu, Zhengyu, Mao, Aihua, Exudative epidermitis of piglets caused by non-toxigenic Staphylococcus sciuri.Veterinary Microbiology http://dx.doi.org/10.1016/j.vetmic.2016.12.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Exudative Epidermitis of Piglets Caused by Non-toxigenic Staphylococcus sciuri Lixin Lu, Kongwang He, Yanxiu Ni, Zhengyu Yu, Aihua Mao Author affiliations: Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences·Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture·National Center for Engineering Research of Veterinary Bio-products, Nanjing, 210014, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, China

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Highlights Infections with strains of Staphylococcus sciuri are a potential threat to animal and public health, and a cause for considerable concern. S. sciuri as one of the real pathogen from an acute case of EE in piglets was isolated and has been first identified in genus and using experimental infections. Clinical cases of lesions of lungs and endocarditis in piglets due to S. sciuri was first reported. Methicillin-resistant staphylococci must be really careful in public and animal health. Non-toxigenic S. sciuri is fatal to mice and newly bom piglets the reason for sudden death which may be septic shock.

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Abstract Infections with strains of Staphylococcus sciuri are a potential threat to animal and public health, and a cause for considerable concern. We isolated and identified S. sciuri as a pathogen from an acute outbreak of exudative epidermitis in piglets for further genetic identification using experimental infections. The results of this study showed that S. sciuri strain NJ1306 reproduced exudative epidermitis in experimentally infected 5-day-old piglets. The isolated bacteria also caused sudden death in BALB/c mice following intraperitoneal injection with 5 ×108 CFU of the isolate. The data indicated that strain NJ1306 of S. sciuri was pathogenic to piglets and mice, and the study provided the first known report of clinical lung lesions and endocarditis in piglets due to S. sciuri. Keywords: Staphylococcus sciuri; Exudative epidermitis; Experimental infection; Virulence-associated gene 1. Introduction Exudative epidermitis (EE) is a generalized skin infection that affects suckling and newly weaned piglets. It may cause death due to dehydration within a few days of infection (Wegener and Skov-Jensen, 2006). The disease occurs sporadically, and can be an economically significant cause of mortality, as well as causing poor growth rates (Wegener and Skov-Jensen, 2006). Staphylococcus hyicus is recognized as the pathogenic agent of EE in pigs. The production of secretory exfoliative toxins during infection with EE is considered a major factor in causing skin lesions. (Andresen et al., 1993) and certain strains of S. hyicus cause the characteristic histopathological signs of 3

EE from these toxins (Andresen et al., 1997). Other staphylococci able to produce exfoliative toxins, including S. aureus (van Duijkeren et al., 2007), S. chromogenes (Andresen et al., 2005), and S. sciuri (Chen et al., 2007), have been isolated, although rarely, from cases of EE. However, the research of Chen et al. did not identify a specific S. sciuri strain performed by 16S rRNA sequencing. In the present study, a pathogenic isolate from an acute case of EE in piglets was isolated and identified as a member of the S. sciuri group. The isolate was pathogenic to both 5-day-old piglets and mice in experimental infection. A clinical case of lung lesions and endocarditis in piglets, caused by S. sciuri, is reported here. 2. Materials and methods 2.1. Case and sample selection All procedures were approved by the animal care and use committee of the Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences. Piglets at a conventional pig-rearing farm in the Jiangsu province of China were found to have the clinical symptoms of EE, including gross skin lesions. Euthanasia of the diseased pigs was carried out, to enable necropsy, pathological examination, and isolation of pathogens. 2.2. Etiological isolation and inoculum preparation Swab samples were collected from the diseased dermatitic layer and pericardial fluid of the dead piglets with EE. These swabs were grown aerobically on BactoTM Todd Hewitt Broth (Becton Dickinson Medical Devices Co Ltd, Shanghai) nutrient agar plate at 37 °C for 17 – 24 hours. Bacterial strains were transferred to 5 ml of fresh 4

liquid THB medium and incubated for 24 hours at 37 °C with shaking at 200 rpm, then washed twice with phosphate-buffered saline (PBS) and stored in physiological saline with addition of 30% glycerol at -70 °C until use. 2.3. Species identification The isolate was tested for cellular identification, biochemical characteristics, and antibiotic sensitivity tests were performed on the staphylococcal isolate using colony morphology and pigmentation, Gram staining, and catalase testing. It was further tested for biochemical traits including oxidase and coagulase production and novobiocin sensitivity using a TH-16S bacteria coding system (Microbial Regent Co Ltd, Hangzhou). This coding system is used for a rough to testing for Staphylococcus-Specific according to biochemical traits of bacterium. Species confirmation of isolates was carried out using PCR as follows. DNA was isolated using a commercial DNA extraction kit according to the manufacturer’s instructions (TianGen Biotech Co Ltd, Beijing). Multiplex PCR assay of the Staphylococcus genus-specific 16S rRNA gene, pvl and the mecA gene was performed as previously described (Zhang et al., 2004 and Lina et al., 1999). The primers used are listed in Table 2. PCR products were detected by multiplex PCR as previously described (McClure et al., 2006). Amplification products were resolved in 2% agarose gels (Gene Co Ltd, Hong Kong). The gel figures were photographed with AlphaImager (Alpha Innotech, Beijing). The amplicons were sequenced using primers specific for the Staph 16S rRNA indicated in Table 2 by Thermo Fisher Scientific Inc. 2.4. Examination of the genes encoding staphylococcal toxins 5

PCR of the genes encoding ExhA to ExhD was performed using the relevant primers (Table 2). The full extension of the PCR products was partially sequenced as previously described (Andresen and Ahrens 2004). ShetB was detected by simple PCR as described by Keiko and colleagues (Keiko Futagawa-Saito et al., 2007). Staphylococcal enterotoxin genes (sea to sej) and the toxic shock syndrome toxin gene (tst) were described by previous literature (Monday, Bohach 1999 and Løvseth et al., 2004). The amplification products were resolved in 2% agarose gel. 2.5. Experimental infection in pigs Experimental infections with S. sciuri strain NJ1306 were performed on 6 cross-breed (Danish Landrace, Danish Yorkshire, and Duroc) 5-day-old piglets in the same litter from a pig herd with no history of EE infections. Cultures were grown in 10-ml tubes containing 5 ml of fresh liquid THB medium inoculated with a single colony from an overnight aerobically culture on THB nutrient agar plate at 37 °C. Four piglets were experimentally infected with 1 × 1010 CFU/piglet of the clinical isolate in 2 ml of sterile PBS; the other 2 were injected with 2 ml of sterile PBS as controls. The infections were subcutaneously injected into the neck just behind the right ear. The piglets were observed once each day over a 12-day period and clinical signs recorded. Cotton swab samples from the skin behind the right ear and blood samples from the cranial vena cava were collected from each of the experimental and control animals for microbiological examination. The piglets were clinically observed throughout the experimental period. Samples were replicated in liquid THB medium and incubated for 24 hours at 37 °C with shaking at 200 rpm. Following this, 100 µl of suspension was 6

inoculated onto THB nutrient agar plate at 37 °C for 17 – 24 hours. Bacteria were identified using biochemical tests and the Staph 16S rRNA gene identified by PCR as described above. 2.6. Experimental infection in mice Twenty-five 7-week-old male BALB/c mice were divided into 5 groups of 5 mice each. Four groups of mice were infected with the bacterial isolate culture at a dose of 1 × 109 CFU, 5 × 108 CFU, 2.5 × 108 CFU or 1.25 × 108 CFU/mouse in 0.2 ml of sterile PBS per mouse via intraperitoneal (ip) injection, respectively. The remaining 5 mice were injected ip with 0.2 ml of sterile PBS per mouse as controls. The animals were clinically observed throughout the experiment period. 3. Results 3.1. Fatal EE occurred in piglets EE occurred in suckling piglets on a conventional pig farm with 10 sows, in the Jiangsu province of China in June 2013. The piglets, which were born healthy, began to show clinical signs as early as 3 days after birth. The typical signs included skin reddening, exfoliation, exudation and crusting, and shortness of breath, with the animals dying within 2–7 days. The diseased pigs were treated with amoxicillin and lincomycin for 3 days with no effect. Surviving pigs exhibited skin lesions covered with brownish greasy layers (Fig.1); these pigs grew very slowly and would have had no market value as food, and had to be eliminated. Observed pathological changes at post mortem were lesions of lung (Fig. 2, Panel a, b) and endocarditis (Fig. 3). 3.2. A strain of S. sciuri carrying the methicillin resistance (mecA) gene was isolated 7

from diseased piglets with EE A bacterium was isolated from the diseased dermatitic layer and pericardial fluid of the moribund piglets, and was identified as a Staphylococcus by morphological and biochemical examinations, further confirmed by Gram staining. The biochemical characteristics of the isolate were up to 99.9% similar to standard S. sciuri and the isolate was named NJ1306. More importantly, this isolate was coagulase-negative and resistant to novobiocin (Table 1). To confirm the results of biochemical examination, the strain was also analyzed for gene compositions using PCR assays and the exfoliative toxins were examined simultaneously. As shown in Fig. 4, NJ1306 demonstrated 2 bands, 756 bp and 310 bp for 16S rRNA and mecA gene, which suggested that S. sciuri strain NJ1306 was a methicillin-resistant (mecA) Staphylococcus without Panton-Valentine leukocidin (PVL) gene. Using BLAST (http://www.ncbi.nlm.nih.gov/nucleotide), the Staphylococcus 16S rRNA gene fragment

sequence was shown to be 99.6% homologous in GenBank compared with S. sciuri DSM20345. Using specific primers for the amplification of exfoliative toxins, NJ1306 was found not to carry ExhA–D and ShetB in its genome, and did not harbor encoding genes such as sea to see, seg to sej and tst. 3.3. S. sciuri NJ1306 was pathogenic to piglets and mice To determine whether the newly-isolated stain of S. sciuri NJ1306 was a causative agent for EE, 5-day-old piglets were experimentally infected with it. The infected animals presented with shortness of breath on the second day after infection. Breathing reverted to normal after 6 days. During the following days (days 3–6 p.i.) the lesions of 8

all 4 pigs expanded, and exfoliations developed behind both ears and on other parts of the body. The clinical signs of disease progressed with erythema of the skin over the entire body. As expected, these gross skin alterations in the piglets, caused by infection with S. sciuri strain NJ1306, were typical for EE (Fig. 5). During the experimental infection of pigs, bacterial isolates were also isolated from the skin and blood of the 4 infected animals. These isolates were further confirmed to be the same bacterial strain, with similar biochemical traits and performed by 16S rRNA sequencing. To determine whether the pathogenicity of S. sciuri strain NJ1306 could be generalized to other animals, 7-week-old BALB/c mice were infected with the isolate via ip injection. As shown in Fig. 6, the mice that received doses of 1 × 109 CFU/mouse and 5 × 108 CFU/mouse via ip injection died within 48 hours. Mice that received doses of 2.5 × 108 CFU/mouse, 1.25 × 108 CFU /mouse and the PBS control all survived. We did not observe any sign of exudative dermatitis in the mice. 4. Discussion S. sciuri, first reported by Kloos and colleagues (Kloos et al., 1976), is a coagulase-negative staphylococcal species. Considered one of the most ancestral and dispersed staphylococcal species, the bacterium is one of the important pathogens responsible for endocarditis (Hedin and Widerstrom 1998), peritonitis (Wallet et al., 2000), septic shock (Horii et al., 2001), urinary tract infection (Stepanovic et al., 2003), pelvic inflammatory disease (Stepanovic et al., 2005) and, most frequently, wound infections (Stepanovic et al., 2002). But infections with S.sciuri in humans are very rarely. 9

S. sciuri is an opportunistic pathogen of controversial clinical significance. Chen et al. experimentally infected newborn piglets with the isolate HBXX06 to reproduce EE. In that report, it was determined that S. sciuri could cause fatal EE in piglets both in the field and under experimental conditions (Chen et al., 2007). However, the study did not identify the bacteria as members of S. sciuri genus. We have shown that the S. sciuri strain NJ1306 could be originally isolated from the diseased dermatitic layer and the pericardial fluid of diseased piglets with EE, and that the newly-isolated stain was a causative agent for EE under experimental conditions. These data greatly contribute to a further understanding of staphylococci as pathogens responsible for EE in pigs. It is generally agreed that in addition to the presence of the causative bacteria, the presence of skin wounds is necessary to allow bacteria to invade the epidermis. In addition, certain important environmental and host factors determine whether disease occurs (Wegener and Skov-Jensen 2006). Chen et al. experimentally infected newborn piglets with the isolate HBXX06 by oral feeding, successfully inducing EE, which suggests that oral intake of the pathogen by animals is a major route of exposure (Chen et al., 2007). However, we observed that diseased piglets presented with shortness of breath and lung lesions in both field and experimental conditions, leading to a suspicion that EE infection may occur from airborne transmission. Nevertheless, there is a paucity of reports of respiratory infection caused directly by S. sciuri, and further understanding of the mechanism of the S. sciuri infection route is required. Hedin and Widerstrom (Hedin and Widerstrom, 1998) demonstrated that S. sciuri infection was responsible for a case of endocarditis; endocarditis was also found 10

post-mortem in a diseased piglet in our clinical survey (Fig. 3). But, what are their exact roles in the pathogenesis of acute EE in piglets? General opinion is that the characteristic histopathological signs of EE are caused by certain strains of S. hyicus, which produced exfoliative toxins. Chen and his colleagues isolated a highly pathogenic strain of S. sciuri from the pericardial fluid of a diseased piglet with EE. This strain harbored the gene encoding ExhC (Chen et al., 2007), but did not fit the ITS-PCR pattern of any coagulase-negative staphylococci. Dakic et al. suggested that the production of staphylococcal exotoxins by members of the S. sciuri group is highly unlikely (Dakic et al. 2005). Thus, the isolate HBXX06 is also needed to performe by 16S rRNA sequencing for attribution to S.sciuri. In our studies, S. sciuri strain NJ1306 did not harbor the genes encoding ExhA – D and ShetB. Our analysis of the encoding of the genes of the isolate NJ1306 did not find any of these genes related to the pathogenic factors. However, the isolated bacteria caused disease in the experimental pigs, as well as the sudden death of BALB/c mice infected via 5 × 108 CFU ip injection. The majority of the diseased mice exhibited signs of shivering and tousled fur, with death occurring within 24 hours. The dead mice showed characteristic pathological changes such as deep red blood, pleural effusion, pulmonary hemorrhage, congestion, spleen enlargement and a considerable amount of bleeding with renal engorgement, as well as thinning of the duodenum and jejunum walls. No signs of exudative dermatitis were observed in the mice. Newborn piglets that received 1 × 108CFU/piglet intramuscularly died within 12 hours with no signs of EE (Chen et al., 2007). These results indicate that the isolate is fatal to mice and newborn piglets, and the reason for sudden death may be 11

septic shock. Highly lethal necrotizing pneumonia appeared in the diseased pig clinical cases (Fig. 2. Panel a, b). Possible virulence factors were detected in S. sciuri strain NJ1306, but, relevent virulence-associated genes were not detected in NJ1306. PVL gene has been found responsible for severe infections of the skin and soft tissues (Lina et al., 1999), as well as highly lethal necrotizing pneumonia (Gillet et al., 2002). PVL toxin has been associated with CA-MRSA (Rankin et al., 2005, Giudice et al., 2009). However, whether S. sciuri strains can be isolated from pigs with necrotizing pneumonia was unclear. The S. sciuri NJ1306 strain was negative for pvl gene according to the PCR results, meaning it does not contain the pvl gene. At present the pathogenicity of this bacterium in animals remains unknown. Our data indicated that the S. sciuri NJ1306 strain carries the mecA gene. Not only may mecA-positive strains act as potential donors for emerging MRSA clones, but mecA-positive strains are a potential reservoir for this resistant gene, which may be transferred to other staphylococci (Garza-Gonzalez et al., 2010). Methicillin-resistant staphylococci must be considered carefully in public and animal health. According to previous reports, treatment failure in cases of EE in pigs is very common, partly due to the widespread presence of multidrug resistance in staphylococci (Park et al., 2013). 5. Conclusions An emerging strain of S. sciuri, NJ1306, was isolated from the diseased dermatitic layer and pericardial fluid of piglets affected with EE. The finding that the strain carries the mecA gene suggests that S. sciuri strain NJ1306 may carry plasmids with antibiotic 12

resistance markers. S. sciuri was isolated from acute cases of EE in piglets, identified as a pathogen and primarily identified within the genus and in experimental infections. We reported the first clinical cases of lung lesions and endocarditis in piglets caused by S. sciuri. Non-toxigenic S. sciuri is fatal to mice and neonatal pigs, and this may be due to septic shock. S. sciuri pose a considerable threat to both animal and human health. Ethics statement The study was approved by the Animal Ethics Committee of Jiangsu Institute of Veterinary Medicine. Laboratory animal experimentation was performed in compliance with the Jiangsu Administration Guidelines for the Use of Experimental Animals. All procedures were approved by the Animal Ethics Committee of Jiangsu Institute of Veterinary Medicine (SYXK20131002). All experimental procedures were conducted in conformity with institutional guidelines for the care and use of laboratory animals, and all efforts were made to minimize suffering. Competing interests The authors declare that they have no competing interests. Authors’ contributions Conceived and designed all the experiments: LL KH. Performed the experiments: LL. Participated in the overall planning of the animal experiment, as well as analyzed the data: LL. YN and ZY. Performed PCR and analyzed the data: LL.AM. Wrote the paper: LL. All authors commented and approved the final manuscript. Acknowledgements: We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. The authors thank Dr. Jieyuan Jiang for 13

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Supporting Information Figures Fig.1. Exudative epidermitis occurred in the suckling piglets on a conventional pig farm. Fig.2. Pathological changes were lesion of lungs (panel A, B) in post-mortem pigs. Fig.3. Pathological change was endocarditis of the piglet in the post-mortem pig (indicated by arrows). Fig.4. Demonstration of Staph 16S rRNA, pvl and mecA gene in Staphylococci isolate. ( Lane 1, multiplex PCR assay for simultaneous detection of Staph 16S rRNA, pvl and mecA genes. Lane M, 2 Kb Plus DNA ladder.) Fig.5. The two major clinical manifestations include shortness of breath and skin lesions by infection with S. sciuri strain NJ1306 in piglets. Fig.6. Survival rate of BALB/c mice post infection with S. sciuri NJ1306. Tables Table 1 Biochemical traits of S. sciuri NJ1306 isolated from pigs. Table 2 Primers used in this study.

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Table 1. Biochemical traits of S. sciuri NJ1306 isolated from pigs, Test

Result

Coagulase negative

N

Oxidase positive

P

Novobiocin resistance

R

Urease

-

Sucrose

+

Arginine dihydrolase

-

D-xylose

-

Lactose

+

D-mannose

+

D-xylitol

-

Maltose

+

Trehalose

+

D-mannitol

+

Acetylglucosamine

+

Reduction of nitrate

+

Melibiose

-

D-fructose

+

v-p

-

Sorbitol

+

Abbreviations and symbols: +, positive culture; -, negative culture; N, not; P, positive culture; R, resistance.

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Table 2. Primers used in this study. Genes

Sequence (5′–3′)

Products (bp)

References

16S

AAC TCT GTT ATT AGG GAA GAA CA

756

Zhang et al.

rRNA

CCA CCT TCC TCC GGT TTG TCA CC

pvl

ATC ATT AGG TAA AAT GTC TGG ACA TGA TCC

433

Lina et al.

310

Zhang et al.

316

Andresen

A GCA TCA AGT GTA TTG GAT AGC AAA AGC mecA

GTA GAA ATG ACT GAA CGT CCG ATA A CCA ATT CCA CAT TGT TTC GGT CTA A

ExhA

GCT ACT GGT TTT GTA GTT TCA C

and Ahrens

GTA ACC TAC AAC TCT TAG AAC C ExhB

AAC ACG CCA ATA GAG AAT GTA TCA C

717

and Ahrens

TAT CAA ATC TTA TAC CAG TTA GAA TAT CTC C ExhC

GAA TAA ATA TTA TGG AGT CTC TCC TGA TC

525

GAA CAA ATA TAA TGG AAG AAA CCC AC

588

TGC ACA ATT TCA GTC CCT ATG

Andresen and Ahrens

GAT TTC CCT ACG TGA ATA CCT ACA ATA C ShetB

Andresen and Ahrens

CCA TAG TAT TTC AAT CCA AAA TCA GTA C ExhD

Andresen

463

Keiko et al.

520

Monday and

TGG CTG GAG TTA TTA GGT CAC sea

GCA GGG AAC AGC TTT AGG C

Bohach

GTT CTG TAG AAG TAT GAA ACA CG seb-sec

ACA TGT AAT TTT GAT ATT CGC ACT G

667

Løvseth et al.

284

Monday and

TGC AGG CAT CAT GTC ATA CCA sec

CTT GTA TGT ATG GAG GAA TAA CAA

Bohach

TGC AGG CAT CAT ATC ATA CCA sed

GTG GTG AAA TAG ATA GGA CTG C

385

Bohach

ATA TGA AGG TGC TCT GTG G see

Monday and

TAC CAA TTA ACT TGT GGA TAG AC 21

171

Monday and

Bohach

CTC TTT GCA CCT TAC CGC seg

CGT CTC CAC CTG TTG AAG G

328

Bohach

CCA AGT GAT TGT CTA TTG TCG seh

CAA CTG CTG ATT TAG CTC AG

359

CAA CTC GAA TTT TCA ACA GGT ACC

466

CAT CAG AAC TGT TGT TCC GCT AG

142

GCT TGC GAC AAC TGC TAC AG TGG ATC CGT CAT TCA TTG TTA T

22

Monday and Bohach

CTG AAT TTT ACC ATC AAA GGT AC tst

Monday and Bohach

CAG GCA GTC CAT CTC CTG sej

Monday and Bohach

GTC GAA TGA GTA ATC TCT AGG sei

Monday and

559

Løvseth et al.

Fig.6

Survival of Data 1:Survival proportions Percent survival(%)

100

1× 109 CFU/mouse 5× 108 CFU/mouse 2.5× 108 CFU/mouse 1.25× 108 CFU/mouse

80 60 40 20 0 0

8

16

24

32

40

48

56

Time(hours post-infection)

Fig.6. Survival rate of BALB/c mice post infection with S. sciuri NJ1306.