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Occurrence of Treponema spp. in porcine skin ulcers and gingiva
Veterinary Microbiology 165 (2013) 402–409
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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Occurrence of Treponema spp. in porcine skin ulcers and gingiva Frida Karlsson a,*, Olov Svartstro¨m b, Katinka Bela´k c, Claes Fellstro¨m a, Ma¨rit Pringle d a
Department of Clinical Sciences, Swedish University of Agricultural Sciences, SE-75007 Uppsala, Sweden Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, SE-75007 Uppsala, Sweden Department of Pathology and Wildlife Diseases, National Veterinary Institute, SE-75189 Uppsala, Sweden d Department of Animal Health and Antimicrobial Strategies, National Veterinary Institute, SE-75189 Uppsala, Sweden b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 January 2013 Received in revised form 7 March 2013 Accepted 27 March 2013
Porcine shoulder ulcers and ear necrosis are a significant animal welfare concern and impair efficient livestock production. Although spirochetes have been detected in both types of lesions the potential role of these bacteria in lesion propagation has received little attention. The objective of this study was to investigate the occurrence of spirochetes of the genus Treponema in shoulder ulcers or ear necrosis in pigs and compare these with treponemes from porcine gingiva. Samples were collected from gingiva and necrotic ulcers in 169 pigs. Presence of spirochetes was observed in silver stained histological sections and by phase contrast microscopy in scrapings from the necrotic lesions. Additionally, PCR of the 16SrRNA–tRNAIle intergenic spacer region (ISR2) was used to detect Treponema spp. in all samples. Combined analysis showed that 73% of the shoulder ulcers and 53% of the ear necroses were positive for spirochetes. Treponema spp. were detected in 9.7% of the gingival samples. Comparative DNA sequence analysis of the ISR2 sequences revealed the presence of three distinct genetic phylotypes of Treponema spp. corresponding to Treponema pedis, and as yet two unnamed phylotypes represented by GenBank sequences C1UD1 (Acc. No. AY342041) and C1BT2–8 (Acc. No. AY342046). Detection of identical ISR2 sequences from gingiva and ulcer samples indicates that oral Treponema spp. are spread from mouth to ulcer. We conclude that Treponema spp. frequently occur in shoulder ulcers and ear necrosis in pigs, and suggest a possible infection route through biting and licking. ß 2013 Elsevier B.V. All rights reserved.
Keywords: Treponema Spirochete Pig Ear necrosis Ear biting Shoulder ulcer
1. Introduction Spirochetes of the genus Treponema are associated with a number of diseases in the skin or mucus membranes in several mammals. Examples are Treponema pallidum subsp. pallidum, the causative agent of syphilis in humans (Ho and Lukehart, 2011), Treponema denticola, a human
* Corresponding author at: Department of Clinical Sciences, Division of Diagnostics and Large Animal Clinical Sciences, Swedish University of Agricultural Sciences (SLU), Box 7054, SE-75007 Uppsala, Sweden. Tel.: +46 18 671466; fax: +46 18 672919. E-mail address: [email protected] (F. Karlsson). 0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.03.031
oral pathogen (Visser and Ellen, 2011), and Treponema phagedenis-like and Treponema pedis, isolated from bovine digital dermatitis lesions (Walker et al., 1995). It has long been known that spirochetes occur in a variety of necrotic ulcers in pigs (Blandford et al., 1972; Cleland, 1908; Dodd, 1906; Gilruth, 1910; Harcourt, 1973; Hindmarsch, 1937; Neitz and Canham, 1930; Osborne and Ensor, 1955). Until recently, these spirochetes had only been described microscopically and not further characterized because of difficulties in culturing this type of organisms. However, in 2008 our group was able to isolate and characterize spirochetes of the genus Treponema from shoulder ulcers, ear necrosis and gingiva of pigs (Pringle et al., 2009; Pringle and Fellstro¨m, 2010).
F. Karlsson et al. / Veterinary Microbiology 165 (2013) 402–409
Shoulder ulcers in sows cause economic losses due to early slaughter. Thin sows and rough bedding are often discussed as contributing factors, but sometimes ulcers occur even in well-fed sows (Zurbrigg, 2006). Shoulder ulcers in sows are considered as an important animal welfare issue and pre-disposing factors have therefore been broadly studied (Cleveland-Nielsen et al., 2004; Davies et al., 1996; Zurbrigg, 2006). However, the literature on microbiological findings in sow shoulder ulcers is sparse. Various ear lesions related to biting are frequently observed in pigs (Cameron, 2012). The cause of ear necrosis, a more severe syndrome, is unknown, but biting as well as microbial and toxicological causes have been discussed (Harcourt, 1973; Richardson et al., 1984; Taylor, 2012; Weissenbacher-Lang et al., 2012). Cocci and spirochetes have been microscopically confirmed in smears from these ulcers (Harcourt, 1973) and both staphylococci and streptococci have been isolated (Hansen and Busch, 2008; Mirt, 1999; Richardson et al., 1984; Weissenbacher-Lang et al., 2012), but culturing for spirochetes was not performed in these studies. In a recent study from Canada the presence of both staphylococci and Treponema spp. in ear necrosis was investigated (Park, 2011). Although cultivation of Treponema spp. did not succeed, 8.6% of the samples were positive for spirochetes in Warthin–Starry silver staining. In this study three culture independent methods were used to detect Treponema spp.; phase contrast microscopy, Warthin–Starry silver staining and ISR2-based PCR. Our aims were to study the occurrence of different Treponema spp. in shoulder ulcers and ear necrosis in pigs, to visualize the density and location of spirochetes in these ulcers through histopathology, and to investigate a possible association between treponemes from gingiva and from porcine skin ulcers.
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2.2. Sampling procedure All work involving animals was approved by the ethical committee of animal experiments in Uppsala (C261/8, C259/11). Each pig was restrained and the ulcer assessed and photographed. The lesions were measured and graded into two classes, mild or severe, based on the appearance of the lesions on the date of sampling. The age of the each lesion was not known. The shoulder ulcers were classified according to a system described by Jensen et al. (2011). In this system a severe shoulder ulcer is equal to or larger than 5 cm in diameter and surrounded by a thickened margin. A mild shoulder ulcer is every ulcer equal to or larger than 2 cm in diameter but not qualifying as a severe shoulder ulcer. Lesions with a diameter less than 2 cm were not included as shoulder ulcers. To classify the cases of ear necrosis, severe ear necrosis was defined as lesions involving 1/3 or more of the ventral margin of the ear. All other ear lesions were classified as mild. For consistency, all lesions were described and classified by the same veterinarian (F. Karlsson) for all herds. In cases where shoulder ulcers or ear lesions were bilateral, only one side was sampled. Local anesthesia was achieved by subcutaneous injection of 2–5 ml of lidocain 20 mg/ml. The ulcer was cleaned with water and a sample was taken by scraping with a sterile scalpel. The scalpel and the material were immediately put in a plastic tube containing 5 ml FABG (fastidious anaerobe broth, LAB 71, LabM, with 2.0 g Dglucose per liter). Two cotton swab samples were taken from the gingiva and put in a plastic tube containing 5 ml of PBS (phosphate buffered saline). In cases where a sow with piglets was sampled, cotton swab samples were also taken from the gingiva of two piglets and pooled into one sample. From the margin of the lesion one or two punch biopsies with a diameter of 4–8 mm were collected. The biopsies were immediately fixated in 10% buffered formalin.
2. Materials and methods 2.3. Preparation of samples 2.1. Sampled animals and herds Eighteen Swedish pig herds (A–R) with a history of shoulder ulcers or ear necrosis were visited. These herds were chosen based on information from the herd veterinarians. Three to ten pigs from each herd were sampled both from ulcers and gingiva, except for one herd (K), where no gingival samples were taken. In all, 109 pigs with either shoulder ulcers (n = 52) or ear necrosis (n = 57) were sampled from April 2010 until December 2011. Gingival samples were also collected from 60 piglets from 30 of the sows with shoulder ulcers (two piglets from each sow), and pooled as pairs into 30 samples for PCR analysis. The total number of analyzed gingival samples from sows, weaners and piglets was 134, of in total 169 pigs. (Detailed information about the herds and the sampled animals is presented in supplementary file 1. Locations of the sampled herds are shown in supplementary file 2.) Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic. 2013.03.031.
2.3.1. Biopsies After fixation in formalin the biopsies were embedded in paraffin according to routine histological procedures and thin sections (5–7 mm) cut. The sections were stained with hematoxylin and eosin (HE) for histopathological evaluation and with Warthin–Starry silver staining (W–S) to detect spirochetes. The biopsies were interpreted as positive or negative for spirochetes by an experienced pathologist. Sections with thin, spirally shaped, dark brown bacteria were noted as positive. Each section was screened for spirochetes, and based on the screening, the field with the highest focal density of spirochetes was chosen. The number of spirochetes in that area was used to classify the section. The density of spirochetes was classified as abundant, moderate or sparse according to the approximate number of spirochetes seen in the chosen field viewed at 25 magnification. Abundant occurrence was defined as >100 spirochetes, moderate as 10–100 spirochetes and sparse as <10 spirochetes per field.
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2.3.2. Scrapings and gingival samples On arrival to the laboratory the tubes with the scraping samples were centrifuged and decanted. A loop of 10 ml was taken from the material and examined by phase contrast microscopy for the presence of spirochetes. Thin, helically shaped bacteria approximately 10 mm in length were interpreted as treponemes. The remaining material was stored at 20 8C. The cotton swabs from the sampled gingivae were removed from the tubes. The tubes and the material suspended in the PBS were centrifuged, decanted and stored at 20 8C. 2.3.3. DNA preparation All samples were subsequently washed twice with PBS, centrifuged, suspended in 200 ml PBS and 200 mg proteinase K and incubated 90 min at 54 8C. The samples were centrifuged, decanted and the pellets were suspended in 50 ml sterile water, then boiled for 10 min, chilled on ice for 10 min and centrifuged. Supernatants were stored at 20 8C until used for PCR. 2.4. Amplification and sequencing of ISR2 A PCR protocol developed by Stamm et al. (2002) was followed to amplify the 16SrRNA–tRNAIle intergenic spacer region (ISR2). Amplicons were separated by agarose gel electrophoresis and DNA bands approximately 200–400 bp in length were excised from the gel and purified using Gel
Band Purification Kit (GE Healthcare). The purified PCR amplicons were cloned into the pCR14-TOPO1 vector and chemically transformed into TOP10 E. coli cells using the One Shot1 Chemical Transformation Protocol of the TOPO TA Cloning1 kit for Sequencing (Invitrogen). Three to six clones were analyzed with either of two methods: (A) The ISR2 was amplified from single transformed colonies using PuRe TaqTM ReadytoGoTM PCR Beads (IllustraTM), analyzed by electrophoresis in a 1.5% agarose gel, purified using Gel Band Purification Kit (GE Healthcare) and sequenced directly (ABI 3730XL, Macrogen Inc.) or (B) Single colonies were cultured over-night in tubes with 5 ml LB-medium and ampicillin (50 mg/ml) in a 37 8C shake-incubator, the tubes were centrifuged, the pellets purified using Gene Jet Plasmid Miniprep Kit (Fermentas) and plasmids were sent for sequencing (ABI 3730XL, Macrogen Inc.). Sequences were edited and trimmed in CLC Main Workbench 6.2 (CLC Bio) to fragments of 131–211 bp covering the entire 16SrRNA–tRNAIle intergenic spacer region. Homology searches were performed with the megablast algorithm BLAST1 (Altschul et al., 1990) at the National Center for Biotechnology Information (NCBI). A progressive alignment was made in CLC Main Workbench 6.2 with default gap cost parameters and the ‘‘very accurate (slow)’’ algorithm and a phylogenetic tree was constructed using the neighbor-joining method. One sequence representing each branch of the phylogenetic tree was deposited in GenBank (Acc. Nos. KC494406–KC494480).
Fig. 1. (a) Severe ear necrosis of a weaned pig in herd O. Photo: F. Karlsson. (b) Silver stained section of a shoulder skin ulcer from herd E. Spirochetes are visualized in the necrotic cell debris (arrows). Photo: F. Karlsson. Warthin–Starry (original magnification 1.25 100). (c) Histological section of a shoulder skin ulcer from herd E. The epidermis (EP) shows severe thickening (acanthosis and hyperkeratosis) and is covered by crusts. Below the crusts, an ulcer can be seen with abundant debris (arrow). Photo: K. Bela´k and R. Feinstein. Hematoxylin and eosin (original magnification 0.63 2.5). (d) A severe shoulder ulcer of a sow in herd A. Photo: F. Karlsson.
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3. Results
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corresponding numbers were 7 (27%) in the mild cases and 5 (18%) in the severe cases.
3.1. Gross pathology 3.3. Phase contrast microscopy 3.1.1. Ear necrosis The majorities of the ear necroses were bilateral and situated on the lower margin of the ear, starting from the junction of the ear and neck. The length of the lesions varied from 1 cm to the entire lower margin of the ear (Fig. 1a) and the width was typically half a centimeter to one centimeter, but sometimes most of the ear was affected. Some of the lesions were covered with a crust, usually 1–2 ml thick; others had exuding or bleeding surfaces. The color varied from red to brown/gray. Although the lesions were in different stages at the time of sampling, signs of inflammation such as redness, swelling, warmth and pain were generally prominent. In total, 29 pigs were classified as having mild, and 28 as having severe ear necrosis. 3.1.2. Shoulder ulcers The shoulder ulcers sampled differed in severity and size. The majority of the ulcers were bilateral. Mild ulcers were superficial and small, characterized by a pink, red or brown color and covered with a thin crust. Severe ulcers extended deep into the skin and underlying tissues (Fig. 1d) and were sometimes covered by a crust. On closer examination extensive damage and necrosis was discovered underneath. The skin around the lesion was hard and thick. Occasionally a characteristic, foul smell was recognized and on palpation gray pus sometimes exuded from the lesion. In total, 15 sows had mild shoulder ulcers and 37 sows had severe shoulder ulcers. 3.2. Histopathology 3.2.1. HE-staining The epidermis was completely necrotized in 31% of all samples. When present, it was hyperplastically thickened with hyperkeratosis and acanthosis (Fig. 1c). Variable hemorrhages, erosions and ulcerations, as well as epidermal pustulation and crusting were observed, and these lesions contained colonies of coccoid bacteria. Dermal inflammation was most prominent beneath areas of ulceration, pustulation and crusting and was perivascular to interstitial in most cases with a highly variable inflammatory infiltrate, dominated by neutrophils in acute and plasma cells and lymphocytes in chronic cases. In the pronounced chronic cases there were signs of granulation tissue with vascularization and fibrosis. 3.2.2. Warthin–Starry silver staining Silver stained spirochetes were observed in 34 (67%) of the shoulder ulcers and 12 (22%) of the ear necroses. In general, spirochetes were located on the necrotic dermal surface and were in many cases focally distributed. A W–S stained section from a shoulder ulcer is shown in Fig. 1b. The results from the W–S staining compared to the other two detection methods are shown in Table 1. Spirochetes were detected in 8 (53%) of the mild shoulder ulcers and 26 (72%) of the severe type (Table 2). In ear necrosis the
At examination by phase contrast microscopy 22 (46%) shoulder ulcers and 12 (21%) ear necroses were positive for treponemes. The results from the phase contrast microscopy are compared with the results from the W–S staining and the PCR in Table 1. Four (27%) of the mild and 18 (55%) of the severe shoulder ulcers were positive compared to 7 (24%) of the mild and 5 (18%) of the severe ear necroses. 3.4. ISR2 PCR Twenty seven shoulder ulcers (52%) and 26 ear necroses (46%) were positive for Treponema spp. using ISR2-based PCR as a detection method. These results are compared to microscopic detection methods in Table 1. Five (33%) of the mild type and 22 (59%) of the severe type of shoulder ulcers were positive, while the corresponding results from
Table 1 No. of positive samples from ulcers and gingiva. Herd
Shoulder ulcers
Gingiva
Piglets’ gingiva
WSa PCMb PCRc Alld Tot.e PCRc Tot.e PCRc Tot.e A B C D E F G H I J Tot. pos. Tot.e Herd
6 1 3 2 3 5 3 4 4 3
7 0 Ntf 0 3 2 4 4 2 0
6 1 3 4 2 4 2 1 3 1
7 1 3 4 3 5 4 4 4 3
34 51g
22 48h
27 52
38 52
Tot. pos. Tot.e a
2 0 0 0 2 3 0 0 1 0
7 3 4 6 3 5 6 6 5 7
PCMb
Nt 0 2 0 2 0 0 Nt Nt 0
8
Nt 2 4 6 2 5 4 Nt Nt 7
4
52
52
Ear necrosis WSa
K L M N O P Q R
7 3 4g,h 6 3 5 6 6 5 7
30 Gingiva
PCRc
Alld
Tot.e i
0 3 1 0 2 1 3 2
3 2 0 2 2 0 0 3
4 2 4 9 3 0 2 2
4 3 4 9 3 1 3 3
5 10 4 10 5 9 9 5
12 54i
12 57
26 57
30 57
57
PCRc
Tot.e
Nt 1 0 0 0 0 0 0
Nt 10 4 10 5 9 9 5
1 52j
Warthin–Starry silver staining. b Phase contrast microscopy. c ISR2-based PCR. d Positive in at least one of three methods. e Tot., total sampled. f Not tested. g Four sows were sampled from herd C, but only three were investigated by W–S. h No PCM investigation was done on the samples from herd C. i Five pigs were sampled from herd K, but only two were investigated by W–S. j No gingival swabs for PCR were taken from herd K.
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Table 2 Density of spirochetes in shoulder ulcers and ear necrosis as detected by WS-staining (N = 105). Classification of ulcersa
Total
Mild SU Severe SU Mild EN Severe EN
15 36c 26d 28
Occurrence of spirochetes
Densityb
Not detected
Detected
Abundant
Moderate
Sparse
7 10 19 23
8 26 7 5
1 9 1 0
4 11 3 4
3 6 3 1
a Shoulder ulcers (SU) classified as severe or mild, according to Jensen et al. (2011). Ear necroses (EN) classified as severe (lesions involving 1/3 or more of the ventral margin of the ear, alternatively both ventral and dorsal parts of the ear) or mild necrosis (all other lesions). b The density of spirochetes classified as abundant (>100), moderate (10–100), sparse (<10) or not detected according to the approximate number of spirochetes seen in the field with the highest focal density of spirochetes, viewed at 25 magnification. c The total number of severe SU were 37, but from one of these no biopsy was taken. d The total number of mild EN were 29, but from three of these no biopsies were taken.
pigs with ear necrosis were 11 (38%) positive mild lesions and 15 (54%) positive severe lesions. The PCR for the gingival samples resulted in 13 (9.7%) positive samples. Of these, eight samples were from adult pigs, one from a weaner and four from piglets. Treponema spp. was confirmed by PCR in necrotic lesions from all except one herd (an ear necrosis herd). 3.5. All three methods When combining the results and defining a positive sample as one positive in at least one of three methods, the occurrence of spirochetes was 73% in shoulder ulcers, 53% in ear necrosis and 9.7% in gingiva. Spirochetes were detected in porcine skin ulcers in all herds. 3.6. ISR2 sequences Only unique sequences from each animal and sampling site were included in the phylogenetic tree (Fig. 2). These were in total 132 treponemal ISR2 sequences. Hence, some of these were identical, but did not origin from the same animal and sampling site (ulcer or gingiva). Of the 132 sequences 51 originated from shoulder ulcers, 41 from ear necrosis and 40 from gingiva. Sequences were derived from all except two herds. The maximum number of unique sequences found in one sampling site was six from a gingiva, five from a shoulder ulcer and five from an ear lesion. The majority of the sequences formed three groups (A–C) in the phylogenetic tree, see Fig. 2. These three groups clustered with previously deposited ISR2 sequences, group A with C1UD1 (Acc. No. AY342041), group B with T. pedis, and group C with C1BT2–8 (Acc. No. AY342046). Sequences from ear necrosis, shoulder ulcers and gingiva were represented in all clusters. In cluster A (35 sequences) 18 were from ear necrosis, 8 from shoulder ulcers and 9 from gingiva. Cluster B consisted of 31 sequences including 9 from ear necrosis, 21 from shoulder ulcers and only one from gingiva. In cluster C (29 sequences) 11 originated from ear necrosis, 9 from shoulder ulcers and 9 from gingiva. The variation among the ISR2 sequences was high but identical sequences of different origin (ear necrosis, shoulder ulcers and gingiva) were found, both within and between herds. Two of the sequences from sows with shoulder ulcers
(B1134b and B1152b) were identical to sequences from the gingiva of their piglets (K1134c and K1152b). 4. Discussion This study is a continuation of our work on Treponema spp. in necrotic skin ulcers in pigs, adding to the findings in previous pilot studies (Pringle et al., 2009; Pringle and Fellstro¨m, 2010). Here, three culture independent methods were used to investigate the occurrence of Treponema spp. in porcine shoulder ulcers and ear necrosis. Treponemal ISR2 sequences from gingiva and ulcers were compared, in order to detect a possible transmission route from mouth to ulcer. The main reason for using three methods in parallel was not to compare the different methods but rather to increase the detection rate of treponemes. The appearances of the ear lesions in this study were in agreement with earlier descriptions of the disease (Harcourt, 1973; Richardson et al., 1984). The majority of the lesions were located at the ventral margin of the ear. Several stages of lesions were observed, but the early vesicles preceding mild lesions described by Richardson et al. (1984) were however not observed, and the progression of lesions was not followed in this crosssectional study. In HE-staining of the sections from ear necrosis the findings of hyperkeratosis, acanthosis and intraepidermal abscesses were in accordance with earlier findings (Harcourt, 1973; Mirt, 1999; Richardson et al., 1984). In contrast to our results, Harcourt noted that there was little inflammation in the sections from the investigated ear lesions, and Blandford et al. (1972), found the sections in their study less purulent than expected. It should be noted, however, that the studies of Harcourt (1973) and Blandford et al. (1972) were limited to very few cases, as most early studies on necrotic skin ulcers in pigs. In a study from 2012 on 36 piglets, necrosis, coccoid bacteria, various inflammatory cells and granulation tissue were seen in the histological sections (Weissenbacher-Lang et al., 2012). This was in accordance with our findings. They also noted thrombus formation and re-epithelization, which was not observed in this study. The main histopathological findings in the HE-stained sections from shoulder ulcers such as various inflammatory cell types, accumulations of bacteria, necrosis, fibrosis and granulation tissue were in accordance with the
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findings of Jensen (2009), although this author more thoroughly describes the pathological findings of different stages of shoulder ulcers. In our study, punch biopsies were taken on live sows, and therefore the underlying bone was not sampled or investigated. Warthin–Starry silver staining has long been used to detect spirochetes in histological tissue samples. In a report from 1973 on two pigs with ear necrosis, Harcourt found numerous spirochetes in one sample, and a small number in another. In a similar case report from 1972 Blandford et al. (1972) identified spirochetes in one investigated sample Blandford et al. (1972). Richardson et al. (1984) detected spirochetes in only one of 38 cases. To the best of our knowledge, the only recent study where W–S staining was used to screen for spirochetes in ear necrosis is from Canada (Park, 2011), where spirochetes were detected in only 8.6% of the investigated cases, compared to our results of 22%. The difference between our and the Canadian results could either be a true variation in occurrence or a variation in interpretation of the sections. Furthermore the Canadian study mainly included pigs with ear tip necrosis (Park, 2011), while our study consisted to the very main part of pigs with the necrosis located ventrally. To the best of our knowledge, silver staining to detect spirochetes in shoulder ulcers has not been performed before and the literature on microbial occurrence in these ulcers is in general sparse. In one study from 2003, aerobic culturing from shoulder ulcers was successful in 85% of the cases (Lund, 2003). Trueperella pyogenes (Arcanobacterium pyogenes) was the most common bacterial species detected. Culturing for anaerobes was not performed. A rapid method to detect spirochetes is through direct microscopy. Identification of spirochetes in skin ulcers by microscopic examination of smears has been performed since the beginning of the 1900s (Cleland, 1908; Dodd, 1906). Both this method and W–S staining can be used to confirm spirochetes in tissue, but cannot affiliate the spirochetes into a specific genus. In the gingival samples, the detection of treponemes was only performed by PCR. The detection rate was lower in gingiva (9.7%) compared to ulcers (46–52%). As we expect Treponema spp. to be a part of the pigs’ normal mouth flora as in humans, dogs and cats (Valdez et al., 2000; Visser and Ellen, 2011) (although in low numbers in a healthy gingiva), the low number of positive samples was surprising, but may be explained by the difficulties in getting enough material when sampling gingiva with a cotton swab. One might also argue that a major part (61%) of the gingival samples belonged to young pigs (piglets or weaners) and there are probably a lower proportion of treponemes in young individuals. This has not been shown in pigs, but there is information available that spirochetes are observed less frequently in plaque from young children (3–5 years) than in older children (6–12 years) (Loesche, 1988). When looking exclusively at adult pigs, the detection rate of treponemes in gingiva was 15%. The low detection rate compared to ulcers does not reject the hypothesis of a possible infection route through biting and licking. A very small proportion of treponemes originating from gingiva may well infect an ulcer. An interesting
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finding was the higher occurrence of treponemes in the shoulder ulcers compared to ear necrosis. The ulcers on the shoulders sometimes develop into deep lesions with an environment more anaerobic than the ear necrosis lesions. In this study, the presence of genus Treponema was confirmed by ISR2-based PCR, a method used by others to detect and genetically characterize treponemes in digital dermatitis and ulcerative mammary dermatitis in cattle (Stamm et al., 2002, 2009). This region is short and variable and sequence analysis of ISR2 has a higher discriminatory resolution than analysis of the 16SrRNA-region (Stamm et al., 2002). The sequenced PCR products in the current study created an insight of the genetic diversity of treponemes in the ulcers and gingiva. In total, 132 ISR2 sequences were selected to be included in the phylogenetic tree (Fig. 2). Of these, 75 sequences representing each branch of the phylogenetic tree were deposited in GenBank (Acc. Nos. KC494406–KC494480). Names of all the sequences, accession numbers and sources of origin are available in supplementary file 3. Three main clusters were revealed in the phylogenetic tree. Sequences from ear necrosis, shoulder ulcers and gingiva could be found in all these clusters and were in some cases identical. This shows that treponemes of the three main treponemal phylotypes detected in this study can reside both in ulcers and/or gingiva. In two cases we found identical sequences from sows’ shoulder ulcers (B1134b and B1152b) and from gingiva of their piglets (K1134c and K1152b). As the variation of the ISR2 sequences was substantial this 100% identity indicates spreading from mouth to ulcer. This is not surprising as there are many anecdotal reports describing licking and biting behavior of the piglets in shoulder ulcers of sows. From pigs with ear necrosis, residing in the same pen, we did not find a corresponding identity between sequences from gingiva and ulcers. However, there were several examples of identical sequences found in both ear necrosis and gingiva of pigs from separate pens and herds. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2013.03.031. In this study we could not identify the treponemal phylotype solely by interpreting the length of the PCRproducts on the gel, as the variations in sequence length were not discriminating. The phylogenetic tree revealed three distinct main groups (A–C) where sequences from this study clustered with C1UD1 (Acc. No. AY342041) (A), T. pedis (B), and C1BT2–8 (Acc. No. AY342046) (C). Both C1UD1 (Acc. No. AY342041) and C1BT2–8 (Acc. No. AY342046) are ISR2 sequences acquired from ulcerative mammary dermatitis (UMD) in cattle, described in a study from California (Stamm et al., 2009). The T. pedis group (B) was represented by sequences from UMD; C1BT2–3 (Acc. No. AY342044), and digital dermatitis; 1-9185 (Acc. No. AF179255) in cattle (Stamm et al., 2002, 2009), and shoulder ulcers; B683 (Acc. No. GQ916628), 653S (Acc. No. GQ916626), ear necrosis; TA4 (Acc. No. GQ916625), and gingiva; TM1 (Acc. No. GQ916627) of pigs (Pringle et al., 2009; Pringle and Fellstro¨m, 2010). The most frequently detected phylotype was C1UD1 (Acc. No. AY342041). One single sequence (B1184c) was identical to C1BF-3 (Acc. No.
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Fig. 2. Phylogenetic tree based on distance matrix analysis of the 16SrRNA–tRNAIle intergenic spacer region (ISR2) (fragment length 131–211 nucleotides) from Treponema spp. Three distinct clusters (A–C) are revealed, corresponding to T. pedis (B), and two unnamed phylotypes represented by GenBank sequences C1UD1 (Acc. No. AY342041) (A) and C1BT2–8 (Acc. No. AY342046) (C). Sequences acquired from GenBank are marked with accession numbers. Accession numbers and origin for all the sequences from this study are found in supplementary file 3.
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AY342051) and another one (M1180e) identical to C1UD2 (Acc. No. AY342042), both originating from UMD (Stamm et al., 2009). In pilot studies we only identified one species, T. pedis (Pringle et al., 2009; Pringle and Fellstro¨m, 2010), but as shown in the current study the number of phylotypes increased with the increasing number of samples. Although this study points out three main phylotypes of treponemes occurring in porcine ulcers and gingiva, additionally 37 sequences occurred as small clusters or single sequences in the phylogenetic tree. Therefore the number of detected phylotypes of Treponema spp. in pigs is similar to the number found in digital dermatitis in cattle (Rasmussen et al., 2012). A fair assumption is that additional phylotypes will be identified as research continues on treponemes in ulcers and gingiva. 5. Conclusion In conclusion, Treponema spp. are frequently present in shoulder ulcers and ear necrosis in pigs and the number of phylotypes occurring in the ulcers are higher than previously reported. The three main treponemal phylotypes detected in this study can reside both in ulcers and/ or gingiva and identical ISR2 sequences were found from both sites. This suggests a possible infection route through biting and licking. The role of Treponema spp. in the progression of ear necrosis and shoulder ulcers is still unknown. Acknowledgements We would like to thank all the veterinarians who helped us identify herds; Axel Sanno¨, Carl-Johan Ehlorsson, Erik Gammelga˚rd, Gunnar Johansson, Jan Fischerstro¨m, Johanna Fjelkner, Karolina Dahlqvist, Lena Eliasson-Selling, Margareta Wallgren and Maria Lindberg. We would also like to thank Ricardo Feinstein for help with photograpy and Annette Backhans, Anna Rosander, Dolores GavierWide´n, Jenny Larsson, Sa´ndor Bela´k and Richard Zuerner for valuable comments regarding the manuscript. The project was financed by The Swedish Research Council Formas. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Blandford, T.B., Bygrave, A.C., Harding, J.D., Little, T.W., 1972. Suspected porcine ulcerative spirochaetosis in England. Vet. Rec. 90, 15. Cameron, R., 2012. Ear necrosis, (necrotic ear syndrome and ulcerative spirochetosis of the ear). In: Zimmerman, J.J., Karriker, L.A., Ramirez, A., Schwartz, K.J., Stevenson, G.W. (Eds.), Diseases of Swine. WileyBlackwell, Chichester, pp. 256–257. Cleland, J.B., 1908. Note on spirochaetes in castration tumours of pigs. Parasitol. Suppl. J. Hyg. 1, 218–219. Cleveland-Nielsen, A., Baekbo, P., Ersbøll, A.K., 2004. Herd-related risk factors for decubital ulcers present at post-mortem meat-inspection of Danish sows. Prev. Vet. Med. 64, 113–122. Davies, P.R., Morrow, W.E., Miller, D.C., Deen, J., 1996. Epidemiologic study of decubital ulcers in sows. J. Am. Vet. Med. Assoc. 208, 1058–1062.
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Dodd, S., 1906. A disease of the pig, due to a spirochaeta. J. Comp. Pathol. Ther. 19, 216–222. Gilruth, J.A., 1910. Spirochaetae in lesions affecting the pig. Proc. R. Soc. Victoria 23, 105–109. Hansen, K.K., Busch, M.E., 2008. Antibiotic treatment as an intervention against ear necrosis in one herd. In: Proceedings of the 20th International Pig Veterinary Society Congress. Durban, South Africa, June 22– 26, (P512.015), p. 594. Harcourt, R.A., 1973. Porcine ulcerative spirochaetosis. Vet. Rec. 92, 647–648. Hindmarsch, W.L., 1937, Ulcerative Granuloma of Pigs (spirochaetal tumours of pigs). New South Wales Department of Agriculture, Veterinary Research Report, 64–70. Ho, E.L., Lukehart, S.A., 2011. Syphilis: using modern approaches to understand an old disease. J. Clin. Invest. 121, 4584–4592. Jensen, H.E., 2009. Investigation into the pathology of shoulder ulcerations in sows. Vet. Rec. 165, 171–174. Jensen, H.E., Bonde, M.K., Ba˚dsgaard, N.P., Dahl-Pedersen, K., HaubroAndersen, P., Herskin, M.S., Jørgensen, E., Kaiser, M., Lindahl, J., Nielsen, J.P., Rhymer-Friis, C., Stege, H., Hjelholt Jensen, K., 2011. En enkel og valideret skala for klinisk vurdering af skuldersa˚r. Dansk veterinaertidsskrift 09, 6–12 (in Danish). Loesche, W.J., 1988. The role of spirochetes in periodontal disease. Adv. Dent. Res. 2, 275–283. Lund, M., 2003. Skuldersa˚r hos søer- patoanatomisk karakteristik, kødkontrolmæssige og dyreetiske aspekter. Master thesis. The University of Copenhagen, Copenhagen (in Danish). Mirt, D., 1999. Lesions of so-called flank biting and necrotic ear syndrome in pigs. Vet. Rec. 144, 92–96. Neitz, W.O., Canham, A.S., 1930. A short note on the spirochaetal wound infection of pigs. Report of the Director of Veterinary Services and Animal Industry, 69–80. Osborne, H.G., Ensor, C.R., 1955. Some aspects of the pathology, aetiology, and theurapeutics of foot-rot in pigs. N. Z. Vet. J. 3, 91–99. Park, J., 2011. Investigation of Exudative Epidermitis and Ear Necrosis in Pigs. PhD thesis. The University of Guelph, Guelph. Pringle, M., Backhans, A., Otman, F., Sjo¨lund, M., Fellstro¨m, C., 2009. Isolation of spirochetes of genus Treponema from pigs with ear necrosis. Vet. Microbiol. 139, 279–283. Pringle, M., Fellstro¨m, C., 2010. Treponema pedis isolated from a sow shoulder ulcer. Vet. Microbiol. 142, 461–463. Rasmussen, M., Capion, N., Klitgaard, K., Rogdo, T., Fjeldaas, T., Boye, M., Jensen, T.K., 2012. Bovine digital dermatitis: possible pathogenic consortium consisting of Dichelobacter nodosus and multiple Treponema species. Vet. Microbiol. 160, 151–161. Richardson, J.A., Morter, R.L., Rebar, A.H., Olander, H.J., 1984. Lesions of porcine necrotic ear syndrome. Vet. Pathol. 21, 152–157. Stamm, L.V., Bergen, H.L., Walker, R.L., 2002. Molecular typing of papillomatous digital dermatitis-associated Treponema isolates based on analysis of 16S-23S ribosomal DNA intergenic spacer regions. J. Clin. Microbiol. 40, 3463–3469. Stamm, L.V., Walker, R.L., Read, D.H., 2009. Genetic diversity of bovine ulcerative mammary dermatitis-associated Treponema. Vet. Microbiol. 136, 192–196. Taylor, D.J., 2012. Treponema pedis: cutaneous spirochetosis. In: Zimmerman, J.J., Karriker, L.A., Ramirez, A., Schwartz, K.J., Stevenson, G.W. (Eds.), Diseases of Swine. Wiley-Blackwell, Chichester, pp. 878–879. Valdez, M., Haines, R., Riviere, K.H., Riviere, G.R., Thomas, D.D., 2000. Isolation of oral spirochetes from dogs and cats and provisional identification using polymerase chain reaction (PCR) analysis specific for human plaque Treponema spp. J. Vet. Dent. 17, 23–26. Walker, R.L., Read, D.H., Loretz, K.J., Nordhausen, R.W., 1995. Spirochetes isolated from dairy cattle with papillomatous digital dermatitis and interdigital dermatitis. Vet. Microbiol. 47, 343–355. Weissenbacher-Lang, C., Voglmayr, T., Waxenecker, F., Hofstetter, U., Weissenbo¨ck, H., Hoelzle, K., Hoelzle, L.E., Welle, M., Ogris, M., Bruns, G., Ritzmann, M., 2012. Porcine ear necrosis syndrome: a preliminary investigation of putative infectious agents in piglets and mycotoxins in feed. Vet. J. 194, 392–397. Visser, M.B., Ellen, R.P., 2011. New insights into the emerging role of oral spirochaetes in periodontal disease. Clin. Microbiol. Infect. 17, 502–512. Zurbrigg, K., 2006. Sow shoulder lesions: risk factors and treatment effects on an Ontario farm. J. Anim. Sci. 84, 2509–2514.
Contents lists available at SciVerse ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Occurrence of Treponema spp. in porcine skin ulcers and gingiva Frida Karlsson a,*, Olov Svartstro¨m b, Katinka Bela´k c, Claes Fellstro¨m a, Ma¨rit Pringle d a
Department of Clinical Sciences, Swedish University of Agricultural Sciences, SE-75007 Uppsala, Sweden Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, SE-75007 Uppsala, Sweden Department of Pathology and Wildlife Diseases, National Veterinary Institute, SE-75189 Uppsala, Sweden d Department of Animal Health and Antimicrobial Strategies, National Veterinary Institute, SE-75189 Uppsala, Sweden b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 January 2013 Received in revised form 7 March 2013 Accepted 27 March 2013
Porcine shoulder ulcers and ear necrosis are a significant animal welfare concern and impair efficient livestock production. Although spirochetes have been detected in both types of lesions the potential role of these bacteria in lesion propagation has received little attention. The objective of this study was to investigate the occurrence of spirochetes of the genus Treponema in shoulder ulcers or ear necrosis in pigs and compare these with treponemes from porcine gingiva. Samples were collected from gingiva and necrotic ulcers in 169 pigs. Presence of spirochetes was observed in silver stained histological sections and by phase contrast microscopy in scrapings from the necrotic lesions. Additionally, PCR of the 16SrRNA–tRNAIle intergenic spacer region (ISR2) was used to detect Treponema spp. in all samples. Combined analysis showed that 73% of the shoulder ulcers and 53% of the ear necroses were positive for spirochetes. Treponema spp. were detected in 9.7% of the gingival samples. Comparative DNA sequence analysis of the ISR2 sequences revealed the presence of three distinct genetic phylotypes of Treponema spp. corresponding to Treponema pedis, and as yet two unnamed phylotypes represented by GenBank sequences C1UD1 (Acc. No. AY342041) and C1BT2–8 (Acc. No. AY342046). Detection of identical ISR2 sequences from gingiva and ulcer samples indicates that oral Treponema spp. are spread from mouth to ulcer. We conclude that Treponema spp. frequently occur in shoulder ulcers and ear necrosis in pigs, and suggest a possible infection route through biting and licking. ß 2013 Elsevier B.V. All rights reserved.
Keywords: Treponema Spirochete Pig Ear necrosis Ear biting Shoulder ulcer
1. Introduction Spirochetes of the genus Treponema are associated with a number of diseases in the skin or mucus membranes in several mammals. Examples are Treponema pallidum subsp. pallidum, the causative agent of syphilis in humans (Ho and Lukehart, 2011), Treponema denticola, a human
* Corresponding author at: Department of Clinical Sciences, Division of Diagnostics and Large Animal Clinical Sciences, Swedish University of Agricultural Sciences (SLU), Box 7054, SE-75007 Uppsala, Sweden. Tel.: +46 18 671466; fax: +46 18 672919. E-mail address: [email protected] (F. Karlsson). 0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.03.031
oral pathogen (Visser and Ellen, 2011), and Treponema phagedenis-like and Treponema pedis, isolated from bovine digital dermatitis lesions (Walker et al., 1995). It has long been known that spirochetes occur in a variety of necrotic ulcers in pigs (Blandford et al., 1972; Cleland, 1908; Dodd, 1906; Gilruth, 1910; Harcourt, 1973; Hindmarsch, 1937; Neitz and Canham, 1930; Osborne and Ensor, 1955). Until recently, these spirochetes had only been described microscopically and not further characterized because of difficulties in culturing this type of organisms. However, in 2008 our group was able to isolate and characterize spirochetes of the genus Treponema from shoulder ulcers, ear necrosis and gingiva of pigs (Pringle et al., 2009; Pringle and Fellstro¨m, 2010).
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Shoulder ulcers in sows cause economic losses due to early slaughter. Thin sows and rough bedding are often discussed as contributing factors, but sometimes ulcers occur even in well-fed sows (Zurbrigg, 2006). Shoulder ulcers in sows are considered as an important animal welfare issue and pre-disposing factors have therefore been broadly studied (Cleveland-Nielsen et al., 2004; Davies et al., 1996; Zurbrigg, 2006). However, the literature on microbiological findings in sow shoulder ulcers is sparse. Various ear lesions related to biting are frequently observed in pigs (Cameron, 2012). The cause of ear necrosis, a more severe syndrome, is unknown, but biting as well as microbial and toxicological causes have been discussed (Harcourt, 1973; Richardson et al., 1984; Taylor, 2012; Weissenbacher-Lang et al., 2012). Cocci and spirochetes have been microscopically confirmed in smears from these ulcers (Harcourt, 1973) and both staphylococci and streptococci have been isolated (Hansen and Busch, 2008; Mirt, 1999; Richardson et al., 1984; Weissenbacher-Lang et al., 2012), but culturing for spirochetes was not performed in these studies. In a recent study from Canada the presence of both staphylococci and Treponema spp. in ear necrosis was investigated (Park, 2011). Although cultivation of Treponema spp. did not succeed, 8.6% of the samples were positive for spirochetes in Warthin–Starry silver staining. In this study three culture independent methods were used to detect Treponema spp.; phase contrast microscopy, Warthin–Starry silver staining and ISR2-based PCR. Our aims were to study the occurrence of different Treponema spp. in shoulder ulcers and ear necrosis in pigs, to visualize the density and location of spirochetes in these ulcers through histopathology, and to investigate a possible association between treponemes from gingiva and from porcine skin ulcers.
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2.2. Sampling procedure All work involving animals was approved by the ethical committee of animal experiments in Uppsala (C261/8, C259/11). Each pig was restrained and the ulcer assessed and photographed. The lesions were measured and graded into two classes, mild or severe, based on the appearance of the lesions on the date of sampling. The age of the each lesion was not known. The shoulder ulcers were classified according to a system described by Jensen et al. (2011). In this system a severe shoulder ulcer is equal to or larger than 5 cm in diameter and surrounded by a thickened margin. A mild shoulder ulcer is every ulcer equal to or larger than 2 cm in diameter but not qualifying as a severe shoulder ulcer. Lesions with a diameter less than 2 cm were not included as shoulder ulcers. To classify the cases of ear necrosis, severe ear necrosis was defined as lesions involving 1/3 or more of the ventral margin of the ear. All other ear lesions were classified as mild. For consistency, all lesions were described and classified by the same veterinarian (F. Karlsson) for all herds. In cases where shoulder ulcers or ear lesions were bilateral, only one side was sampled. Local anesthesia was achieved by subcutaneous injection of 2–5 ml of lidocain 20 mg/ml. The ulcer was cleaned with water and a sample was taken by scraping with a sterile scalpel. The scalpel and the material were immediately put in a plastic tube containing 5 ml FABG (fastidious anaerobe broth, LAB 71, LabM, with 2.0 g Dglucose per liter). Two cotton swab samples were taken from the gingiva and put in a plastic tube containing 5 ml of PBS (phosphate buffered saline). In cases where a sow with piglets was sampled, cotton swab samples were also taken from the gingiva of two piglets and pooled into one sample. From the margin of the lesion one or two punch biopsies with a diameter of 4–8 mm were collected. The biopsies were immediately fixated in 10% buffered formalin.
2. Materials and methods 2.3. Preparation of samples 2.1. Sampled animals and herds Eighteen Swedish pig herds (A–R) with a history of shoulder ulcers or ear necrosis were visited. These herds were chosen based on information from the herd veterinarians. Three to ten pigs from each herd were sampled both from ulcers and gingiva, except for one herd (K), where no gingival samples were taken. In all, 109 pigs with either shoulder ulcers (n = 52) or ear necrosis (n = 57) were sampled from April 2010 until December 2011. Gingival samples were also collected from 60 piglets from 30 of the sows with shoulder ulcers (two piglets from each sow), and pooled as pairs into 30 samples for PCR analysis. The total number of analyzed gingival samples from sows, weaners and piglets was 134, of in total 169 pigs. (Detailed information about the herds and the sampled animals is presented in supplementary file 1. Locations of the sampled herds are shown in supplementary file 2.) Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic. 2013.03.031.
2.3.1. Biopsies After fixation in formalin the biopsies were embedded in paraffin according to routine histological procedures and thin sections (5–7 mm) cut. The sections were stained with hematoxylin and eosin (HE) for histopathological evaluation and with Warthin–Starry silver staining (W–S) to detect spirochetes. The biopsies were interpreted as positive or negative for spirochetes by an experienced pathologist. Sections with thin, spirally shaped, dark brown bacteria were noted as positive. Each section was screened for spirochetes, and based on the screening, the field with the highest focal density of spirochetes was chosen. The number of spirochetes in that area was used to classify the section. The density of spirochetes was classified as abundant, moderate or sparse according to the approximate number of spirochetes seen in the chosen field viewed at 25 magnification. Abundant occurrence was defined as >100 spirochetes, moderate as 10–100 spirochetes and sparse as <10 spirochetes per field.
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2.3.2. Scrapings and gingival samples On arrival to the laboratory the tubes with the scraping samples were centrifuged and decanted. A loop of 10 ml was taken from the material and examined by phase contrast microscopy for the presence of spirochetes. Thin, helically shaped bacteria approximately 10 mm in length were interpreted as treponemes. The remaining material was stored at 20 8C. The cotton swabs from the sampled gingivae were removed from the tubes. The tubes and the material suspended in the PBS were centrifuged, decanted and stored at 20 8C. 2.3.3. DNA preparation All samples were subsequently washed twice with PBS, centrifuged, suspended in 200 ml PBS and 200 mg proteinase K and incubated 90 min at 54 8C. The samples were centrifuged, decanted and the pellets were suspended in 50 ml sterile water, then boiled for 10 min, chilled on ice for 10 min and centrifuged. Supernatants were stored at 20 8C until used for PCR. 2.4. Amplification and sequencing of ISR2 A PCR protocol developed by Stamm et al. (2002) was followed to amplify the 16SrRNA–tRNAIle intergenic spacer region (ISR2). Amplicons were separated by agarose gel electrophoresis and DNA bands approximately 200–400 bp in length were excised from the gel and purified using Gel
Band Purification Kit (GE Healthcare). The purified PCR amplicons were cloned into the pCR14-TOPO1 vector and chemically transformed into TOP10 E. coli cells using the One Shot1 Chemical Transformation Protocol of the TOPO TA Cloning1 kit for Sequencing (Invitrogen). Three to six clones were analyzed with either of two methods: (A) The ISR2 was amplified from single transformed colonies using PuRe TaqTM ReadytoGoTM PCR Beads (IllustraTM), analyzed by electrophoresis in a 1.5% agarose gel, purified using Gel Band Purification Kit (GE Healthcare) and sequenced directly (ABI 3730XL, Macrogen Inc.) or (B) Single colonies were cultured over-night in tubes with 5 ml LB-medium and ampicillin (50 mg/ml) in a 37 8C shake-incubator, the tubes were centrifuged, the pellets purified using Gene Jet Plasmid Miniprep Kit (Fermentas) and plasmids were sent for sequencing (ABI 3730XL, Macrogen Inc.). Sequences were edited and trimmed in CLC Main Workbench 6.2 (CLC Bio) to fragments of 131–211 bp covering the entire 16SrRNA–tRNAIle intergenic spacer region. Homology searches were performed with the megablast algorithm BLAST1 (Altschul et al., 1990) at the National Center for Biotechnology Information (NCBI). A progressive alignment was made in CLC Main Workbench 6.2 with default gap cost parameters and the ‘‘very accurate (slow)’’ algorithm and a phylogenetic tree was constructed using the neighbor-joining method. One sequence representing each branch of the phylogenetic tree was deposited in GenBank (Acc. Nos. KC494406–KC494480).
Fig. 1. (a) Severe ear necrosis of a weaned pig in herd O. Photo: F. Karlsson. (b) Silver stained section of a shoulder skin ulcer from herd E. Spirochetes are visualized in the necrotic cell debris (arrows). Photo: F. Karlsson. Warthin–Starry (original magnification 1.25 100). (c) Histological section of a shoulder skin ulcer from herd E. The epidermis (EP) shows severe thickening (acanthosis and hyperkeratosis) and is covered by crusts. Below the crusts, an ulcer can be seen with abundant debris (arrow). Photo: K. Bela´k and R. Feinstein. Hematoxylin and eosin (original magnification 0.63 2.5). (d) A severe shoulder ulcer of a sow in herd A. Photo: F. Karlsson.
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3. Results
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corresponding numbers were 7 (27%) in the mild cases and 5 (18%) in the severe cases.
3.1. Gross pathology 3.3. Phase contrast microscopy 3.1.1. Ear necrosis The majorities of the ear necroses were bilateral and situated on the lower margin of the ear, starting from the junction of the ear and neck. The length of the lesions varied from 1 cm to the entire lower margin of the ear (Fig. 1a) and the width was typically half a centimeter to one centimeter, but sometimes most of the ear was affected. Some of the lesions were covered with a crust, usually 1–2 ml thick; others had exuding or bleeding surfaces. The color varied from red to brown/gray. Although the lesions were in different stages at the time of sampling, signs of inflammation such as redness, swelling, warmth and pain were generally prominent. In total, 29 pigs were classified as having mild, and 28 as having severe ear necrosis. 3.1.2. Shoulder ulcers The shoulder ulcers sampled differed in severity and size. The majority of the ulcers were bilateral. Mild ulcers were superficial and small, characterized by a pink, red or brown color and covered with a thin crust. Severe ulcers extended deep into the skin and underlying tissues (Fig. 1d) and were sometimes covered by a crust. On closer examination extensive damage and necrosis was discovered underneath. The skin around the lesion was hard and thick. Occasionally a characteristic, foul smell was recognized and on palpation gray pus sometimes exuded from the lesion. In total, 15 sows had mild shoulder ulcers and 37 sows had severe shoulder ulcers. 3.2. Histopathology 3.2.1. HE-staining The epidermis was completely necrotized in 31% of all samples. When present, it was hyperplastically thickened with hyperkeratosis and acanthosis (Fig. 1c). Variable hemorrhages, erosions and ulcerations, as well as epidermal pustulation and crusting were observed, and these lesions contained colonies of coccoid bacteria. Dermal inflammation was most prominent beneath areas of ulceration, pustulation and crusting and was perivascular to interstitial in most cases with a highly variable inflammatory infiltrate, dominated by neutrophils in acute and plasma cells and lymphocytes in chronic cases. In the pronounced chronic cases there were signs of granulation tissue with vascularization and fibrosis. 3.2.2. Warthin–Starry silver staining Silver stained spirochetes were observed in 34 (67%) of the shoulder ulcers and 12 (22%) of the ear necroses. In general, spirochetes were located on the necrotic dermal surface and were in many cases focally distributed. A W–S stained section from a shoulder ulcer is shown in Fig. 1b. The results from the W–S staining compared to the other two detection methods are shown in Table 1. Spirochetes were detected in 8 (53%) of the mild shoulder ulcers and 26 (72%) of the severe type (Table 2). In ear necrosis the
At examination by phase contrast microscopy 22 (46%) shoulder ulcers and 12 (21%) ear necroses were positive for treponemes. The results from the phase contrast microscopy are compared with the results from the W–S staining and the PCR in Table 1. Four (27%) of the mild and 18 (55%) of the severe shoulder ulcers were positive compared to 7 (24%) of the mild and 5 (18%) of the severe ear necroses. 3.4. ISR2 PCR Twenty seven shoulder ulcers (52%) and 26 ear necroses (46%) were positive for Treponema spp. using ISR2-based PCR as a detection method. These results are compared to microscopic detection methods in Table 1. Five (33%) of the mild type and 22 (59%) of the severe type of shoulder ulcers were positive, while the corresponding results from
Table 1 No. of positive samples from ulcers and gingiva. Herd
Shoulder ulcers
Gingiva
Piglets’ gingiva
WSa PCMb PCRc Alld Tot.e PCRc Tot.e PCRc Tot.e A B C D E F G H I J Tot. pos. Tot.e Herd
6 1 3 2 3 5 3 4 4 3
7 0 Ntf 0 3 2 4 4 2 0
6 1 3 4 2 4 2 1 3 1
7 1 3 4 3 5 4 4 4 3
34 51g
22 48h
27 52
38 52
Tot. pos. Tot.e a
2 0 0 0 2 3 0 0 1 0
7 3 4 6 3 5 6 6 5 7
PCMb
Nt 0 2 0 2 0 0 Nt Nt 0
8
Nt 2 4 6 2 5 4 Nt Nt 7
4
52
52
Ear necrosis WSa
K L M N O P Q R
7 3 4g,h 6 3 5 6 6 5 7
30 Gingiva
PCRc
Alld
Tot.e i
0 3 1 0 2 1 3 2
3 2 0 2 2 0 0 3
4 2 4 9 3 0 2 2
4 3 4 9 3 1 3 3
5 10 4 10 5 9 9 5
12 54i
12 57
26 57
30 57
57
PCRc
Tot.e
Nt 1 0 0 0 0 0 0
Nt 10 4 10 5 9 9 5
1 52j
Warthin–Starry silver staining. b Phase contrast microscopy. c ISR2-based PCR. d Positive in at least one of three methods. e Tot., total sampled. f Not tested. g Four sows were sampled from herd C, but only three were investigated by W–S. h No PCM investigation was done on the samples from herd C. i Five pigs were sampled from herd K, but only two were investigated by W–S. j No gingival swabs for PCR were taken from herd K.
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Table 2 Density of spirochetes in shoulder ulcers and ear necrosis as detected by WS-staining (N = 105). Classification of ulcersa
Total
Mild SU Severe SU Mild EN Severe EN
15 36c 26d 28
Occurrence of spirochetes
Densityb
Not detected
Detected
Abundant
Moderate
Sparse
7 10 19 23
8 26 7 5
1 9 1 0
4 11 3 4
3 6 3 1
a Shoulder ulcers (SU) classified as severe or mild, according to Jensen et al. (2011). Ear necroses (EN) classified as severe (lesions involving 1/3 or more of the ventral margin of the ear, alternatively both ventral and dorsal parts of the ear) or mild necrosis (all other lesions). b The density of spirochetes classified as abundant (>100), moderate (10–100), sparse (<10) or not detected according to the approximate number of spirochetes seen in the field with the highest focal density of spirochetes, viewed at 25 magnification. c The total number of severe SU were 37, but from one of these no biopsy was taken. d The total number of mild EN were 29, but from three of these no biopsies were taken.
pigs with ear necrosis were 11 (38%) positive mild lesions and 15 (54%) positive severe lesions. The PCR for the gingival samples resulted in 13 (9.7%) positive samples. Of these, eight samples were from adult pigs, one from a weaner and four from piglets. Treponema spp. was confirmed by PCR in necrotic lesions from all except one herd (an ear necrosis herd). 3.5. All three methods When combining the results and defining a positive sample as one positive in at least one of three methods, the occurrence of spirochetes was 73% in shoulder ulcers, 53% in ear necrosis and 9.7% in gingiva. Spirochetes were detected in porcine skin ulcers in all herds. 3.6. ISR2 sequences Only unique sequences from each animal and sampling site were included in the phylogenetic tree (Fig. 2). These were in total 132 treponemal ISR2 sequences. Hence, some of these were identical, but did not origin from the same animal and sampling site (ulcer or gingiva). Of the 132 sequences 51 originated from shoulder ulcers, 41 from ear necrosis and 40 from gingiva. Sequences were derived from all except two herds. The maximum number of unique sequences found in one sampling site was six from a gingiva, five from a shoulder ulcer and five from an ear lesion. The majority of the sequences formed three groups (A–C) in the phylogenetic tree, see Fig. 2. These three groups clustered with previously deposited ISR2 sequences, group A with C1UD1 (Acc. No. AY342041), group B with T. pedis, and group C with C1BT2–8 (Acc. No. AY342046). Sequences from ear necrosis, shoulder ulcers and gingiva were represented in all clusters. In cluster A (35 sequences) 18 were from ear necrosis, 8 from shoulder ulcers and 9 from gingiva. Cluster B consisted of 31 sequences including 9 from ear necrosis, 21 from shoulder ulcers and only one from gingiva. In cluster C (29 sequences) 11 originated from ear necrosis, 9 from shoulder ulcers and 9 from gingiva. The variation among the ISR2 sequences was high but identical sequences of different origin (ear necrosis, shoulder ulcers and gingiva) were found, both within and between herds. Two of the sequences from sows with shoulder ulcers
(B1134b and B1152b) were identical to sequences from the gingiva of their piglets (K1134c and K1152b). 4. Discussion This study is a continuation of our work on Treponema spp. in necrotic skin ulcers in pigs, adding to the findings in previous pilot studies (Pringle et al., 2009; Pringle and Fellstro¨m, 2010). Here, three culture independent methods were used to investigate the occurrence of Treponema spp. in porcine shoulder ulcers and ear necrosis. Treponemal ISR2 sequences from gingiva and ulcers were compared, in order to detect a possible transmission route from mouth to ulcer. The main reason for using three methods in parallel was not to compare the different methods but rather to increase the detection rate of treponemes. The appearances of the ear lesions in this study were in agreement with earlier descriptions of the disease (Harcourt, 1973; Richardson et al., 1984). The majority of the lesions were located at the ventral margin of the ear. Several stages of lesions were observed, but the early vesicles preceding mild lesions described by Richardson et al. (1984) were however not observed, and the progression of lesions was not followed in this crosssectional study. In HE-staining of the sections from ear necrosis the findings of hyperkeratosis, acanthosis and intraepidermal abscesses were in accordance with earlier findings (Harcourt, 1973; Mirt, 1999; Richardson et al., 1984). In contrast to our results, Harcourt noted that there was little inflammation in the sections from the investigated ear lesions, and Blandford et al. (1972), found the sections in their study less purulent than expected. It should be noted, however, that the studies of Harcourt (1973) and Blandford et al. (1972) were limited to very few cases, as most early studies on necrotic skin ulcers in pigs. In a study from 2012 on 36 piglets, necrosis, coccoid bacteria, various inflammatory cells and granulation tissue were seen in the histological sections (Weissenbacher-Lang et al., 2012). This was in accordance with our findings. They also noted thrombus formation and re-epithelization, which was not observed in this study. The main histopathological findings in the HE-stained sections from shoulder ulcers such as various inflammatory cell types, accumulations of bacteria, necrosis, fibrosis and granulation tissue were in accordance with the
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findings of Jensen (2009), although this author more thoroughly describes the pathological findings of different stages of shoulder ulcers. In our study, punch biopsies were taken on live sows, and therefore the underlying bone was not sampled or investigated. Warthin–Starry silver staining has long been used to detect spirochetes in histological tissue samples. In a report from 1973 on two pigs with ear necrosis, Harcourt found numerous spirochetes in one sample, and a small number in another. In a similar case report from 1972 Blandford et al. (1972) identified spirochetes in one investigated sample Blandford et al. (1972). Richardson et al. (1984) detected spirochetes in only one of 38 cases. To the best of our knowledge, the only recent study where W–S staining was used to screen for spirochetes in ear necrosis is from Canada (Park, 2011), where spirochetes were detected in only 8.6% of the investigated cases, compared to our results of 22%. The difference between our and the Canadian results could either be a true variation in occurrence or a variation in interpretation of the sections. Furthermore the Canadian study mainly included pigs with ear tip necrosis (Park, 2011), while our study consisted to the very main part of pigs with the necrosis located ventrally. To the best of our knowledge, silver staining to detect spirochetes in shoulder ulcers has not been performed before and the literature on microbial occurrence in these ulcers is in general sparse. In one study from 2003, aerobic culturing from shoulder ulcers was successful in 85% of the cases (Lund, 2003). Trueperella pyogenes (Arcanobacterium pyogenes) was the most common bacterial species detected. Culturing for anaerobes was not performed. A rapid method to detect spirochetes is through direct microscopy. Identification of spirochetes in skin ulcers by microscopic examination of smears has been performed since the beginning of the 1900s (Cleland, 1908; Dodd, 1906). Both this method and W–S staining can be used to confirm spirochetes in tissue, but cannot affiliate the spirochetes into a specific genus. In the gingival samples, the detection of treponemes was only performed by PCR. The detection rate was lower in gingiva (9.7%) compared to ulcers (46–52%). As we expect Treponema spp. to be a part of the pigs’ normal mouth flora as in humans, dogs and cats (Valdez et al., 2000; Visser and Ellen, 2011) (although in low numbers in a healthy gingiva), the low number of positive samples was surprising, but may be explained by the difficulties in getting enough material when sampling gingiva with a cotton swab. One might also argue that a major part (61%) of the gingival samples belonged to young pigs (piglets or weaners) and there are probably a lower proportion of treponemes in young individuals. This has not been shown in pigs, but there is information available that spirochetes are observed less frequently in plaque from young children (3–5 years) than in older children (6–12 years) (Loesche, 1988). When looking exclusively at adult pigs, the detection rate of treponemes in gingiva was 15%. The low detection rate compared to ulcers does not reject the hypothesis of a possible infection route through biting and licking. A very small proportion of treponemes originating from gingiva may well infect an ulcer. An interesting
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finding was the higher occurrence of treponemes in the shoulder ulcers compared to ear necrosis. The ulcers on the shoulders sometimes develop into deep lesions with an environment more anaerobic than the ear necrosis lesions. In this study, the presence of genus Treponema was confirmed by ISR2-based PCR, a method used by others to detect and genetically characterize treponemes in digital dermatitis and ulcerative mammary dermatitis in cattle (Stamm et al., 2002, 2009). This region is short and variable and sequence analysis of ISR2 has a higher discriminatory resolution than analysis of the 16SrRNA-region (Stamm et al., 2002). The sequenced PCR products in the current study created an insight of the genetic diversity of treponemes in the ulcers and gingiva. In total, 132 ISR2 sequences were selected to be included in the phylogenetic tree (Fig. 2). Of these, 75 sequences representing each branch of the phylogenetic tree were deposited in GenBank (Acc. Nos. KC494406–KC494480). Names of all the sequences, accession numbers and sources of origin are available in supplementary file 3. Three main clusters were revealed in the phylogenetic tree. Sequences from ear necrosis, shoulder ulcers and gingiva could be found in all these clusters and were in some cases identical. This shows that treponemes of the three main treponemal phylotypes detected in this study can reside both in ulcers and/or gingiva. In two cases we found identical sequences from sows’ shoulder ulcers (B1134b and B1152b) and from gingiva of their piglets (K1134c and K1152b). As the variation of the ISR2 sequences was substantial this 100% identity indicates spreading from mouth to ulcer. This is not surprising as there are many anecdotal reports describing licking and biting behavior of the piglets in shoulder ulcers of sows. From pigs with ear necrosis, residing in the same pen, we did not find a corresponding identity between sequences from gingiva and ulcers. However, there were several examples of identical sequences found in both ear necrosis and gingiva of pigs from separate pens and herds. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2013.03.031. In this study we could not identify the treponemal phylotype solely by interpreting the length of the PCRproducts on the gel, as the variations in sequence length were not discriminating. The phylogenetic tree revealed three distinct main groups (A–C) where sequences from this study clustered with C1UD1 (Acc. No. AY342041) (A), T. pedis (B), and C1BT2–8 (Acc. No. AY342046) (C). Both C1UD1 (Acc. No. AY342041) and C1BT2–8 (Acc. No. AY342046) are ISR2 sequences acquired from ulcerative mammary dermatitis (UMD) in cattle, described in a study from California (Stamm et al., 2009). The T. pedis group (B) was represented by sequences from UMD; C1BT2–3 (Acc. No. AY342044), and digital dermatitis; 1-9185 (Acc. No. AF179255) in cattle (Stamm et al., 2002, 2009), and shoulder ulcers; B683 (Acc. No. GQ916628), 653S (Acc. No. GQ916626), ear necrosis; TA4 (Acc. No. GQ916625), and gingiva; TM1 (Acc. No. GQ916627) of pigs (Pringle et al., 2009; Pringle and Fellstro¨m, 2010). The most frequently detected phylotype was C1UD1 (Acc. No. AY342041). One single sequence (B1184c) was identical to C1BF-3 (Acc. No.
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Fig. 2. Phylogenetic tree based on distance matrix analysis of the 16SrRNA–tRNAIle intergenic spacer region (ISR2) (fragment length 131–211 nucleotides) from Treponema spp. Three distinct clusters (A–C) are revealed, corresponding to T. pedis (B), and two unnamed phylotypes represented by GenBank sequences C1UD1 (Acc. No. AY342041) (A) and C1BT2–8 (Acc. No. AY342046) (C). Sequences acquired from GenBank are marked with accession numbers. Accession numbers and origin for all the sequences from this study are found in supplementary file 3.
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AY342051) and another one (M1180e) identical to C1UD2 (Acc. No. AY342042), both originating from UMD (Stamm et al., 2009). In pilot studies we only identified one species, T. pedis (Pringle et al., 2009; Pringle and Fellstro¨m, 2010), but as shown in the current study the number of phylotypes increased with the increasing number of samples. Although this study points out three main phylotypes of treponemes occurring in porcine ulcers and gingiva, additionally 37 sequences occurred as small clusters or single sequences in the phylogenetic tree. Therefore the number of detected phylotypes of Treponema spp. in pigs is similar to the number found in digital dermatitis in cattle (Rasmussen et al., 2012). A fair assumption is that additional phylotypes will be identified as research continues on treponemes in ulcers and gingiva. 5. Conclusion In conclusion, Treponema spp. are frequently present in shoulder ulcers and ear necrosis in pigs and the number of phylotypes occurring in the ulcers are higher than previously reported. The three main treponemal phylotypes detected in this study can reside both in ulcers and/ or gingiva and identical ISR2 sequences were found from both sites. This suggests a possible infection route through biting and licking. The role of Treponema spp. in the progression of ear necrosis and shoulder ulcers is still unknown. Acknowledgements We would like to thank all the veterinarians who helped us identify herds; Axel Sanno¨, Carl-Johan Ehlorsson, Erik Gammelga˚rd, Gunnar Johansson, Jan Fischerstro¨m, Johanna Fjelkner, Karolina Dahlqvist, Lena Eliasson-Selling, Margareta Wallgren and Maria Lindberg. We would also like to thank Ricardo Feinstein for help with photograpy and Annette Backhans, Anna Rosander, Dolores GavierWide´n, Jenny Larsson, Sa´ndor Bela´k and Richard Zuerner for valuable comments regarding the manuscript. The project was financed by The Swedish Research Council Formas. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Blandford, T.B., Bygrave, A.C., Harding, J.D., Little, T.W., 1972. Suspected porcine ulcerative spirochaetosis in England. Vet. Rec. 90, 15. Cameron, R., 2012. Ear necrosis, (necrotic ear syndrome and ulcerative spirochetosis of the ear). In: Zimmerman, J.J., Karriker, L.A., Ramirez, A., Schwartz, K.J., Stevenson, G.W. (Eds.), Diseases of Swine. WileyBlackwell, Chichester, pp. 256–257. Cleland, J.B., 1908. Note on spirochaetes in castration tumours of pigs. Parasitol. Suppl. J. Hyg. 1, 218–219. Cleveland-Nielsen, A., Baekbo, P., Ersbøll, A.K., 2004. Herd-related risk factors for decubital ulcers present at post-mortem meat-inspection of Danish sows. Prev. Vet. Med. 64, 113–122. Davies, P.R., Morrow, W.E., Miller, D.C., Deen, J., 1996. Epidemiologic study of decubital ulcers in sows. J. Am. Vet. Med. Assoc. 208, 1058–1062.
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