The Borrelia 4 ก B. burgdorferi lyme disease B. anserina goose septicemia The Leptospira L. interrogans (200 serovars) In cattle: milk drop syndrome I

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1 Swine Spirochetal Diarrhea: An Update.... ก ก ก ก ก ก Colitis non specific Epidemic Diarrhea Gastric Ulceration TGE E. coli Coccidiosis ( ) Trichuris suis ( ) Clostridium difficile Ascaris suum ( ) Clostridium perfringens Salmonellosis Intestinal Spirochetosis Swine Dysentery Ileitis PIA PHE PPE Haemorrhagic bowel disease ก ก ก ก ก ก Aerobic, microaerophilic or anaerobic ก ก ก ก rifampin ( กก ก ก ก ก ) ก (protoplasmic cylinders, PC) ก 3 Trilayered membrane outer sheath (OS) Periplasmic flagella (PF) ก (rings, hook & basal body) PF OS & PC ก 2 3 PC PF PF ก 2 ก 1

2 The Borrelia 4 ก B. burgdorferi lyme disease B. anserina goose septicemia The Leptospira L. interrogans (200 serovars) In cattle: milk drop syndrome In swine: Swine herds s disease In dogs: Interstitial nephritis In humans: Weil s disease The Treponema T. cuniculi: : rabbit syphilis (vent disease) T. pallidum: : human syphilis The Brachyspira (former Serpulina) B. hyodysenteriae: Swine dysentery B. pilosicoli: Colonic spirochetosis B. innocens: commensal B. alvinipulli: Egg drop syndrome B. alborgii:?? B. murdochii: commensal B. intermedia: Egg drop syndrome B. canis (provisionally):?? ก ก ก ก Brachyspira ก ก (anaerobic requirements) (α1-globulin) ก ก ก Swine Dysentery: ก : Brachyspira hyodysenteriae ( : Serpulina hyodysenteriae). ก 12 serogroups, 9 serotypes. ก : ก ก kg. (starter-finisher) ก ก ก ก ก ก ก ก 2

3 ก ก : ก ก ก ก ก ก : ก ก ก ก ก ก 1-2 ก ก ก ก ก Brachyspira ก ก ก 50% FCR 0.58 ก ก 20 ก ก ก ก ก ก 3-4 ก ก ก ก ก ก : ก???? ก 3

4 : ก - cecum, colon and rectum กก ก ก : ก ก (hyperemia and edema) Colonic sub-mucosal glands ก ก ก ก ก ก ก (soft to watery and contain exudates) 4

5 ก ก ก : fresh smear ก ก ก 5

6 1. ก ก 138 กก Microscopic Agglutination Agglutination antigen no. Agglutination titers > <50 D6b/ b/6b/ b/15 (NOX1) D3a/ a/8/31(nox1) D7a/ a/10/25 (NOX1) D4a/ a/11/9 (NOX1) D 1d/ d/6/15 (NOX4) D5a/ a/11/9 ( S rdna of B. pilosicoli) Titers show reciprocal of maximum dilution of the serum giving 50% microscopic agglutination of organism. 2. ก ELISA ELISA antigens No. ELISA TITERS > <200 D6b/ b/6b/ b/15 (NOX1) D3a/ a/8/31(nox1) D7a/ a/10/25 (NOX1) D4a/ a/11/9 (NOX1) D 1d/ d/6/15 (NOX4) D5a/ a/11/9 (16S rdna of B. pilosicoli) The titers show reciprocal of maximum titer giving more than 10% colorimetric value. Serological detection concepts Indirect fluorescent antibody test, Passive hemolysis test, Complement fixation test, Microtitration agglutination test, ELISA Whole cell antigen: false-positive Lipopolysaccharide (LPS): fewer false positive : false negative ก ก BmpB (30kD: outer membrane lipoprotein) B. hyodysenteriae ก ก ก ก ก ก ก ก : Brachyspira hyodysenteriae ก Note: ก ก ก ก ก ก ก ก ก SDS-PAGE 2D-PAGE ก ก ก 6

7 PCR Products ก NOX1, NOX2 and NOX4. Lane 1, 100 bp DNA ladder; Lane 2, negative control test; Lane 3, DNA from S. intermedia strain amplified using the NOX2 primers (1004 bp); Lanes 4 and 5, PCR product from S. murdochii strain and S. innocens strain B256T respectively, amplified with the NOX4 primers (729 bp); Lane 6, DNA from S. hyodysenteriae strain B78T amplified using the NOX1 primers (821 bp). ก ก ก ก Brachyspira ก ก ก ก Lanes 1 and 10,, molecular size markers; lane 2, Brachyspira group IV strain P43; lane 3, Brachyspira group I strain B78; lane 4, Brachyspira group II strain AN26:93; lane 5, Brachyspira group III strain C301; lane 6, Brachyspira group IV strain AN497:93; lane 7, E. coli; ; lane 8, Salmonella typhimurium; lane 9, Brachyspira group IV field isolate (Scotland) P99/7/93; lane 11,, negative control. The samples were analyzed after PCR at one dilution only. Result of PCR/RFLP using 23 rrna distinguishable for B. pilosicoli (a), B. hyodysenteriae, B. pilosicoli, and B. alvinipulli (b). ก D-PCR products of B. hyodysenteriae and B. pilosicoli ก column-extracted feces. Lanes: 1, 100-bp molecular size ladder; 2, no-template PCR control; 3, B. hyodysenteriae B78T- spiked sample; 4, farm 2 sample containing B. hyodysenteriae; 5, B. pilosicoli P43/6/78T- spiked sample; 6, farm 3 sample containing B. pilosicoli; 7 and 8, farm 2 samples containing mixed cultures of B. hyodysenteriae and B. pilosicoli. Arrows: p, B. pilosicoli D-PCR product; h, B. hyodysenteriae D-PCR product. 7

8 ก ก Brachyspira M ก ก multiplex PCR B. hyodysenteriae tlya and L. intracellularis 16S rrna genes Lane M: 100 bp DNA ladder; lane 1: : M-PCR test for identification of B. hyodysenteriae and L. intracellularis (amplicons 658 bp and 319 bp, respectively); lanes 2-7: : field strains of B. hyodysenteriae positive for the tlya sequences (amplicon 658 bp); lane 8: : DNA-negative PCR mix control Lanes 1, B. hyodysenteriae ATCC27164; 2, B. pilosicoli P43/6/78; 3, B. innocens ATCC29796; 4, B.aalborgi NCTC11492;5, B. avinipulli C1; 6, D3a/8/31; 7, D1a/10/25; 8, D2a/10/259, D1d/6/15 ก : Porcine Intestinal Spirochetosis : Brachyspira (Serpulina) pilosicoli ก (growers)/ )/ kg ( finishers ) ก ก - ก ก ก ก ก ก ก ก ก ก ก ก host range ก : ก ก ก ก watery green or yellow mucoid and frothy contents ก ก 8

9 ก ก ก ก : Non Specific Colitis Salmonellosis PIA PHE Haemorrhagic Porcine Intestinal Adenomatosis Parasitic infestation Other Brachyspira spp. can cause very similar signs, including the mortality. ก colitis: (%) EU/2001 PL/2004 Brachyspira pilosicoli 39% 0.8% Atypical Brachyspira 8% - B. hyodysenteriae 8% 10% Lawsonia intracellularis 12% 25% Salmonella choleraesuis 11% - Yersinia pseudotuberculosis 15% - E. coli 5% - Clostridium perfringens 2% - ก ก Bh. Li. Healthy pigs 7.3% 6.2% Diarrheic pigs 19.4% 17.9% Growing pigs 16.4% 14.2% Fattening pigs 35.8% 28.2% Sows 21.2% 35.6% Thomson and others,

10 ก ก ก : ก ก ก ก ก B. hyodysenteriae 61 5 C 18 4 C C < C 4 กก 180 ก 13 Acute outbreak ก ก ก ก : ก ก ก ก ก ก ก ก ก ก ก ก ก ก ก ก ก ก Herd eradication: (Typical procedures without total depopulation) ก ก ก ก ก ก ก ก ก ก ก ก ก ก กก ก ก 1 ก ก ก ก ก ก ก 10

11 : ก ก ก ก B. pilosicoli ก (Fossi et al., 2000) ก ก 200 ppm tiamulin ก ก ก ก ก ก ก ก ก 3 : ก ก ก : B. pilosicoli 7 : ก B. innocens High incidence in September to February Sero-survey 69% of pig population in endemic area 23% of pigs without typical symptom (Sanyasuthcharee et al., 1984, Kramomthong et al.,1996) First Isolation of B. pilosicoli in Thailand Table 3. Prevalence of Brachyspira spp. devided into diarrheal pigs and healthy pigs. Status Diarrhea Number Positive (%) - pig 74 15( ) - herd 19 9(47.3) Healthy - pig 52 0(0) - herd 4 0(0) 11

12 Table 4. Prevalence of Brachyspira spp. in bloody diarrhea pigs in 5 provinces. Provinces Rachaburi Number Positive (%) Rachaburi 35 6(8.1) Nakornpathom 29 9(12.2) Saraburi 5 0(0) Rayong 4 0(0) Nakornsawan 1 0(0) Total (20.3) Table 5. Biochemical characterisation of reference strains and 15 field isolates of Brachyspira spp. Type strain Hemolysis Indole Hippurate B. hyodysenteriae ATCC strong + - B.innocens ATCC weak - - B. pilosicoli ATCC weak - + P1/16/7 strong isolates P2/19/9 strong + - P15/1/4 strong isolate P24/1/1010 strong - - (Fellström and Gunnarsson, 1995) Table 6. Comparison of the isolation and observation rates of canine intestinal spirochete between adults and young dogs AGE No. of samples used No. of positive sample (rates%) Observation a) Number in parentheses indicate the percentage of positive sample. Isolation Adult 91 7 (11.7) a) 4 (6.7) Puppy (28.6) 12 (13.2) Total (21.8) 16 (10.6) Table 7. Comparison of biochemical properties among 29 canine isolates Biochemical test Indole Hippulate Esculin Hemolytic Gram staining No. of Isolates B. hyodysenteriae SB - B. pilosicoli WB - B. innocens WB - D5/4/ WB - D7/3/ WB - D10/4/ WB - D4/24/ WB - D1/8/ WB - 12

13 Fig. 9 Comparison of protein profiles of unidentified canine isolates divided into three group. Pilo type A type B type MK Escherichia coli Leptonema illini Leptospira interrogans Tryponema bryantii Borrelia burgdorferi 780 Spirochaeta isovalerica Spirochaeta halophila Brachyspira aalborgi Brachyspira pilosicoli Brachyspira hyodysenteriae Pilo type Brachyspira murdochii Brachyspira innocens 772 D7b/6/15 B type Brachyspira intermedia 102 D8d/10/ D5a/11/9 829 D4d/6/ D6b/6/ D1d/6/15 A type Mk, Standard marker Pilo type : lanes, 1, D5/4/12; 2, D7/3/2 A type: lanes, 3, D4/24/10; 4, D1/8/8. B type lanes 5, 6, D10/4/12. Fig. 10 Phylogenetic tree based on matrix analysis from 16S rdna gene sequences of spirochetes including Brachyspira spp. and canine intestinal spirochetes. E. coli sequence is used as an outgroup. Conclusions Prevalence of Brachyspira spp. was 20.3% among the diarrhea pig. The pig that found the spirochetes was common between weeks of age. Biochemically, all isolates was identified as B. hyodysenteriae. Most of B. hyodysenteriae are strong hemolysis, indole postive and hipurate negative. Atypical indole - negative B. hyodysenteriae was reported firstly in Thailand. The provinces that found B. hyodysenteriae were Nakornpathom and Ratchaburi. Most of isolates were obtained from the farm with recovered of swine dysentery. Negative isolation of bloody diarrhea pig might cause by - Lawsonia intracellularis,salmonellosis, Trichuris suis,, Hemolytic. E. coli, Gastric ulcer. The protein profiles of 14 canine isolates were different from those of the references whereas that of the other 2 isolates were similar to that of B. pilosicoli. 13

14 Minimal Inhibitory Concentration (MIC) Comparative MICs (µg/ml) of various antibiotics against B. hyodysenteriae Karlsson and others, Karlsson and others, 2002 Routine susceptibility methods include breakpoint agar dilution, Kirby-Bauer disk diffusion and NCCLS broth microdilution. The MIC is defined as the lowest concentration of a range of antibiotic dilutions (expressed in mg/l, µg/ml), which inhibits all detectable bacterial growth. Interpretation: Breakpoint Standard Susceptible, Intermediate, Resistance The value in agar method being on average on step lower in broth dilution method. MIC B. hyodysenteriae ก ก ก 6 (Prapasarakul et al., 2004) Fig. 1 MIC values of 6 antimicrobial agents against B. hyodysenteriae isolates from guilt and sow in Thailand Actylisovaleryltyl Strains Tylosin osin Lincomycin Doxycycline Tiamulin Valnemulin P1/16/7 > P2/27/7 > P1/21/8 > ,5 P1/19/9 > P2/19/9 > P1/6/10 > ,25 P4/6/10 > P1/24/10 > P2/24/10 > P1/29/10 > Sero E ,25 <0.25 No of isolate ,25 0, Valnemulin Tiamulin Lincomycin Ty losin Doxycycline Avilosin Avilosin Doxy cy cline Tylosin Lincomy cin Tiamulin Valnemulin Concentration (mg/ml) 14

15 Table 9. Comparison of MIC50 of the antimicrobials for B. hyodysenteriae isolated between 2001 and Antimicrobial agents MIC50 (µg/ml) Year Tylosin 512 >512 Lincomycin Valnemulin Doxycycline - 8 Actylisovaleryltylosin - 32 Tiamulin 2 32 Mechanism of Macrolide Resistance Macrolides: inhibiting protein synthesis through binding to 50S ribosomal subunit. Macrolide resistance: post-transcriptional transcriptional modification of the 23S rrna (Weisblum, 1999). Mutation of A2058 to G, C, or U (Karlsson et al., 1999 and Sigmund et al., 1984). All isolates recovered from recurrent pig with bloody dysentery in 2003, 2058 Partial sequence alignments (corresponding to the nucleotide 1854 to 2257 in 23S rdna numbering of E coli ) of 23S rdna for B. hyodysenteriae ATCC and 6 representative of intestinal spirochetes ATCC :ATTACCCGCAACAGGACGGTAAGACCCCGTGAACCTTTACTATAACTTGACATTGAGTTT AAGACCCCGTGAACCTTTACTATAACTTGACATTGAGTTT 239 D4a/ a/6/15/ 181:ATTACCCGCAACAGGACGG :ATTACCCGCAACAGGACGGAAAGACCCCGTGAACCTTTACTATAACTTGACATTGAGTTT AAGACCCCGTGAACCTTTACTATAACTTGACATTGAGTTT 240 D6b/ b/6/15/ 180:ATTACCCGCAACAGGACGG :ATTACCCGCAACAGGACGGAAAGACCCCGTGAACCTTTACTATAACTTGACATTGAGTTT AAGACCCCGTGAACCTTTACTATAACTTGACATTGAGTTT 239 D1a/ a/10/5/ 181:ATTACCCGCAACAGGACGG :ATTACCCGCAACAGGACGGAAAGACCCCGTGAACCTTTACTATAACTTGACATTGAGTTT AAGACCCCGTGAACCTTTACTATAACTTGACATTGAGTTT 240 D2a/ a/11/9/ 181:ATTACCCGCAACAGGACGG :ATTACCCGCAACAGGACGGAAAGACCCCGTGAACCTTTACTATAACTTGACATTGAGTTT AAGACCCCGTGAACCTTTACTATAACTTGACATTGAGTTT 240 D3a/ a/11/9/ 181:ATTACCCGCAACAGGACGG :ATTACCCGCAACAGGACGGAAAGACCCCGTGAACCTTTACTATAACTTGACATTGAGTTT AAGACCCCGTGAACCTTTACTATAACTTGACATTGAGTTT 240 D4a/ a/11/9/ 181:ATTACCCGCAACAGGACGG :ATTACCCGCAACAGGACGGAAAGACCCCGTGAACCTTTACTATAACTTGACATTGAGTTT AAGACCCCGTGAACCTTTACTATAACTTGACATTGAGTTT 240 ********************* ******************************************** Fig. 2 Secondary structural model of the central loop region of domain V of E. coli 23S rrna (J01695) (Brosius et al., 1980, Gutell et al., 1994). 15

16 Table 10. The 23S rdna partial sequence of acquired tylosin resistants of intestinal spirochetes. Antigenicity of micro-organismsorganisms Isolation no. MIC values (mg/ml) Base positions of 23SrDNA at ATCC CAGGACGGAAAGACCCCGTGA 100 CAGGACGGGAAGACCCCGTGAAAGACCCCGTGA ATCC CAGGACGGAAAGACCCCGTGA 100 CAGGACGGGAAGACCCCGTGAAAGACCCCGTGA D1a/8/ CAGGACGGAAAGACCCCGTGA 100 CAGGACGGAAAGCCCCCGTGACCCCGTGA D2a/11/ CAGGACGGAAAGACCCCGTGA 100 CAGGACGGAAAGCCCCCGTGACCCCGTGA D5a/10/ CAGGACGGAAAGACCCCGTGA 100 CAGGACGGAAAGCCCCCGTGACCCCGTGA G, adenine changed to guanine at base position C, adenine changed to cytosine at base position Microbial components that stimulate CMI Gram negative bacteria LPS Gram positive bacteria Teichoic acids Spirochetes lipoprotein Fungi beta glucan Vaccine for mucosal immunity to combat emerging infection Autogenous Bacterin: 1 st, 2 nd serological response, reduce clinical sign and lesion but not reduce colonization. Cooked rice-based diet: retarded establishment and shedding The systemic vaccine has no effect in terms of providing against protective colon. B. pilosicoli: booster vaccine and degree of colonization do not relate to antibody titers. Lack of more antibiotics Lack of vaccine available Comparative diagnosis between SD and PPE Causal agent B. hyodysenteriae L. intracellularis Age group kg Breeding, Growing pig Target organ colon caecum ileum, proximal colon Infectivity 90 days 70 days Incubation period days 13 days Antigenicity Exogenous Endogenous Vector transmission Various Rat Lesion Hyperemia edema Thickening edema necrosis Vaccine Not available Available SD PPE 16

17 PED TGE Round worm Larva migration Salmonellosis Oocyst Coccidiosis Ileitis PIA Clostridium difficile What is your diagnosis? ก 3 ก ก kg ก LAWSONIA INTRACELLULARIS ก 10% ก 5% ก ก ก ก Dysentery Swine dysentery Gastric ulcer Thank You Questions? 17