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University of Minnesota Collegeof Veterinary Medicine

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Layout and CD-ROM David Brown The University of Minnesota is committed to the policy Logo Design that all personsshall have equal accessto its programs, Ruth Cronje, and Jan Swanson; facilities, and employment without regard to race, color. basedon the original designby Dr. Robert Dunlop creed,religion, nationalorigin, sex. age,marital status. disability,public assistancestatus, or sexualorientation. ll 2006Allen D. LemanSwine Confbrence : an update on genotyping and antibiotic ­susceptibility Simone Oliveira, DVM. MSc, PhD Veterinary Diagnostic Laboratory, University of Minnesota

Introduction Etiology and epidemiology Actinobacillus suis has emerged as a new threat to swine Actinobacillus suis is a Gram-negative bacterium that production. Recent data on isolation of this from colonizes the tonsils, nasal cavity, and vaginal mucosa of clinical samples submitted to the University of Minnesota healthy . This organism is killed within 15 minutes at Veterinary Diagnostic Laboratory has shown an increase a temperature of 60º C, it is sensitive to most disinfectants, of 0.4% or 13,547 cases during the fiscal year (FY) of 2005 and will die out within a few days in clinical specimens.

Advanced Knowledge compared with FY 2002. Although swine veterinarians Affected animals include neonates, weaned, and finish- have made progress in developing prevention and control ing pigs. Historically, high morbidity and mortality due strategies to reduce mortality caused by other respira- to A. suis infection has been observed in high-health tory bacterial such as Haemophilus parasuis, herds, mainly associated with lack of immunity to this Actinobacillus pleuropneumoniae and ­organism. suis, little is know about the epidemiology and control of A. suis. Pathogenesis and virulence factors The main questions are: Actinobacillus suis is a colonizer of the upper respira-

• What are the main clinical presentations and lesions tory tract, and disease has been reproduced by intranasal Advanced Knowledge observed in the field inoculation. Invasion through abrasions in the skin and • What is the technology available for accurate diag- mucous membranes are also likely. One particularity of the nosis of A. suis systemic spread of A. suis is the formation of septic emboli, which will reach various organs forming microcolonies • Which organisms should be included in the differen- surrounded by areas of hemorrhage and necrosis. tial diagnosis of A. suis infection Potential virulence factors that have been reported for • Are there vaccines available in the market A. suis include the expression of capsule, specific lipo- • Is strain variability an issue polysaccharides, and toxins. At least 2 O antigens (LPS) have been described for A. suis: O1 and O2. O2 has been • Is serotyping and serological tests available found to be more prevalent among isolates recovered • Are antibiotic treatments effective? What are the main from lesions or submitted for production of autogenous drugs that can be used for prevention and treatment vaccines compared with O1 strains. Actinobacillus suis produces 2 toxins, which are similar to those produced Although limited information is currently available for by Actinobacillus pleuropneumoniae: Apx I and Apx II. epidemiology and control of A. suis infections, interest These toxins are not identical between the 2 bacterial spe- by field veterinarians and researchers has simultaneously cies, but are very similar, resulting in the development of increased in the past few months. At the University of similar lesions caused by these microorganisms. Minnesota Veterinary Diagnostic Laboratory, we are cur- rently working on the development of new diagnostic tests Clinical signs and lesions for detection and characterization of A. suis. Preliminary results are promising, and somewhat surprising. Actino- Clinical signs and lesions are particularly variable for A. bacillus suis differs considerably from other colonizers of suis. This organism may affect animals from different the upper respiratory tract in their clinical presentation, ages, and clinical signs and lesions may resemble those lesions, and molecular epidemiology. It is certainly a new caused by other organisms, such as Haemophilus parasuis, challenge for the swine industry. A. pleuropneumoniae, and Erysipelothrix rhusiopathiae, for example. Sudden death of suckling pigs may occur in pigs from 2 days to 3 weeks old. Lesions are usually char- acterized by cyanosis, petechial hemorrhages, fever (104 °F), and congestion of the extremities. Weaned pigs may 2006 Allen D. Leman Swine Conference 93 Simone Oliveira develop anorexia, fever, respiratory distress, pneumonia, tory. In this case, PCR can be used as an alternative tool and round/ rhomboid erythematous skin lesions. Sudden to help on the differential diagnosis of A. suis infection. death is also commonly observed. Actinobacillus suis can We have recently standardized PCR tests for detection of colonize the vaginal mucosa, and sows may develop metri- A. pleuropneumoniae and H. parasuis in clinical samples tis and abortion. Finishing pigs may develop pneumonia, at the University of Minnesota VDL. We have also de- arthritis, fatal septicemia, and skin lesions. veloped new PCR tests for detection of A. suis and E. rhusiopathiae. These tests have been especially helpful Petechial to ecchymotic hemorrhages may occur in the in the differential diagnosis of these pathogens. lung, kidneys, heart, liver, spleen, skin, and intestines. Pleurisy and pericarditis may also develop. Miliary ab- Serological tests are not available for diagnosis of A. suis scesses in several organs may result from the systemic infection. Antigens currently used for serotyping (O1/O2) spread of septic emboli. produce cross-reactions with convalescent sera from SPF pigs infected with P. multocida, H. parasuis, and A. pleu- Diagnosis ropneumoniae serotypes 1, 5 and 7. Clinical signs and lesions caused by A. suis are usu- ally non-specific, and differential diagnosis with other Molecular epidemiology pathogens that can cause septicemia is required. Lung Genotyping of A. suis has been previously performed

Advanced Knowledge lesions caused by A. suis and A. pleuropneumoniae may using restriction endonuclease fingerprinting (REF) be indistinguishable, considering that these organism analysis. All A. suis isolates were found to be remarkably produce similar toxins that will contribute to development similar, belonging to one single group. We have recently of necrosis and hemorrhage. Actinobacillus suis may also been working on the standardization and validation of a cause fibrinous pleuritis and pericarditis, and differential repetitive element-based PCR for genotyping of A. suis. diagnosis with H. parasuis should be considered. A recent Preliminary results are in agreement with previously re- survey of bacterial pathogens isolated from 300 tissue ported genotyping features; however, we did find strain samples with fibrinous exudate on the surface demon- variation among A. suis field isolates and reference strains. strated that Haemophilus parasuis was isolated from 37 Another interesting feature of A. suis molecular epide-

samples, whereas A. suis was isolated from 21 samples. miology is the variation observed in antibiotic resistance Advanced Knowledge Other pathogens isolated from fibrinous exudate included: profiles among strains with similar genomic fingerprints. Actinobacillus. indolicus (1), Actinobacillus minor (1), A. Genotyping results for A. suis field isolates will be pre- pleuropneumoniae (8), Arcanobacterium pyogenes (31), sented at the conference. Bordetella bronchiseptica (32), Escherichia coli (19), beta- hemolytic Streptococcus sp. (9), Pasteurella multocida Control and treatment (55), Streptococcus suis (64), and Salmonella sp. (5). Commercial vaccines are not available for A. suis. Data Besides being isolated from lungs with necrosis, hemor- on efficacy of autogenous vaccines is limited, although rhage, and fibrinous pleuritis,A. suis may also be isolated requests to manufacture herd-specific vaccines are com- from systemic sites such as liver, spleen, kidneys, lymph mon. Most pigs usually already have a relatively high titer nodes, uterus and intestines. Isolation from systemic sites against A. suis (which may or may not be specific) prior to is associated with septicemia. Isolation from uterus may vaccination, and response to vaccination will be greater in be associated with abortion and metritis. Isolation from pigs that have lower titers at the time of vaccination. the intestines is more prevalent in neonatal pigs and it is usually associated with septicemia and presence of septic Antibiotics can be used as a preventive measure or to treat emboli. A. suis may also be isolated from the intestines of affected pigs. Recent data for A. suis antibiotic susceptibil- pigs with no lesions. ity profiles for the FI of 2005 (University of Minnesota VDL) is shown in Table 1. Actinobacillus suis isolates Diagnosis of A. suis infection has been traditionally per- sharing similar genomic fingerprints may have completely formed by isolating the microorganism from clinical sam- different susceptibility profiles. One hypothesis is that ples of affected pigs. Gross and histopathological lesions antibiotic resistance genes may be carried in plasmids, (presence of septic emboli in several organs, extensive and strains with similar genetic makeup may carry dif- hemorrhage, and necrosis) are somewhat characteristic ferent plasmids. Genotyping will only characterize the of A. suis infection, however differential diagnosis with genomic DNA. Plasmids are not characterized using this A. pleuropneumoniae, H. parasuis, and E. rhusiopathiae ­technique. for example, should be pursued. Isolation of these pathogens from clinical samples may References be influenced by antibiotic treatments or poor handling Taylor, DJ. (2006) Miscellaneous bacterial infections In: Straw of samples prior to submission to the diagnostic labora- B. E., D’Allaire S., Mengeling W. L., Taylor D.J. (Eds.) 9th Edi-

94 2006 Allen D. Leman Swine Conference

Advanced Knowledge Knowledge Advanced Advanced Conference Swine Allen2006 D. Leman www.vdl.umn.edu. from 1st, 2006 July Retrieved sheet. –Fact by genotyping characterization strain and by PCR tion July) S. (2006, Oliveira www.vdl.umn.edu. from 1st, 2006 July trieved Re –Fact sheet. by genotyping characterization strain and July) S. (2006, Oliveira www.vdl.umn.edu. from 1st, 2006 July Retrieved –Fact sheet. by genotyping characterization strain and by PCR July) S. (2006, Oliveira www.vdl.umn.edu. from 1st, 2006 July trieved Re –Fact sheet. by genotyping characterization strain and July) S. (2006, Oliveira for vaccine autogenous an to response antibody and of pigs cohort of a profile Serologic (2001) M. Gottschalk S. and S., Lacouture D'Allaire L., Lapointe genes. toxin apxIICAvar. and suis suis apxICABDvar. carry and clonal are suis nobacillus (1997) JI. MacInnes S, J, Frey J, Rosendal Van Ostaaijen tion, Table 1: Antibioticsusceptibilityprofiles for Diseases of Swine of Diseases 35 Microbiol. J Clin University ofMinnesota Veterinary thefiscal during year of2005. DiagnosticLaboratory strains isolated from healthy and diseased swine swine diseased and healthy from isolated strains Tylosin (Tartrate) Trimethoprim/Sulphamethoxazole Tilmicosin Tiamulin Tetracycline Sulphathiazole Sulphadimethoxine Sulphachloropyridazine Spectinomycin Oxytetracycline Neomycin Florfenicol Erythromycin Enrofloxacin Danofloxacin Clindamycin Chlortetracycline Cephalothin Ceftiofur Ampicillin Antibiotic . Ames, IA: Iowa State Press. P. Press. Iowa 827-829. State IA: . Ames, Actinobacillus suis Actinobacillus Actinobacillus pleuropneumoniae Actinobacillus suis Actinobacillus rhusiopathiae Erysipelothrix parasuis Haemophilus (5):1131-7. . detection by PCR by PCR detection Vet. 32 Res. detection by PCR by PCR detection Actinobacillus suis Actinobacillus detection detection Actinobacillus suis : 175-183 Acti detec - - - - 99.5 100.0 35.7 6.1 2.5 0.0 0.0 43.6 2.3 3.5 94.1 10.8 1.1 1.5 30.0 0.5 1.0 2.6 50.3 9.4 94.5 93.3 Resistant (%) susceptibility antibiotic and genotyping on update : an isolatedfromclinicalcasessubmittedtothe Susceptible (%) (%) Susceptible 0.0 0.5 56.4 93.9 64.3 97.5 100.0 100.0 96.5 5.9 70.0 89.2 98.5 98.9 97.4 97.7 99.5 49.7 99.0 90.6 6.7 5.5 95

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