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									                                                         Brief Communications                                                           379


utilized to prescreen samples prior to more conventional vi-           6. Katz JB, Gustafson GA, Alstad AD, et al.: 1993, Colorimetric
rus isolation assays, again reducing costs.11                             diagnosis of prolonged bluetongue viremia in sheep, using an
   Acknowledgements. I thank Drs. C. Brown and N. J.                      enzyme-linked oligonucleotide sorbent assay of amplified viral
MacLachlan for critical review of this manuscript and Ms.                 nucleic acids. Am J Vet Res 54:2021–2026.
                                                                       7. Melville LF, Kirkland P: 0000, Evaluation of bluetongue virus
Kristen Howery for technical assistance. This work was sup-
                                                                          excretion in the germplasm of cattle. Final report, project NTA
ported in part by funds from the National Animal Breeders                 018. Northern Territory Department of Primary Industry and
Association.                                                              Fisheries, Berrimah, NT, Australia.
                                                                       8. Melville LF, Weir R, Harmsen M, et al.: 0000, Characteristics
                Sources and manufacturers                                 of naturally-occurring bluetongue viral infections of cattle. Proc
a.   Dynal, Great Neck, NY.                                               Southeast Asian Reg Conf Bluetongue 1:245–250, 1996.
b.   AESAR, Ward Hill, MA.                                             9. Pearson JE, Gustafson GA, Carbrey EA: 1984, Bluetongue vi-
c.   Boehringer Mannheim, Indianapolis, IN.                               rus—recommended procedures to qualify semen and embryos
d.   National Association of Animal Breeders, Columbia, MO.               free of bluetongue virus. Proc Int Symp Microbiol 1984:23–30.
                                                                      10. Phillips RM, Carnahan DL, Rademacher DJ: 1986, Virus iso-
                          References                                      lation from semen of bulls serologically positive for bluetongue
                                                                          virus. Am J Vet Res 47:84–85.
 1. Akita GY, Glenn J, Castro AE, Osburn BI: 1993, Detection of       11. Shad G, Wilson WC, Mecham JO, Evermann JF: 1997, Blue-
    bluetongue virus in clinical samples by polymerase chain reac-        tongue virus detection: a safer reverse transcriptase polymerase
    tion. J Vet Diagn Invest 5:154–158.                                   chain reaction for prediction of viremia in sheep. J Vet Diagn
 2. Aradaib IE, Akita GY, Osburn BI: 1994, Detection of epizootic         Invest 9:118–124.
    hemorrhagic disease virus serotypes 1 and 2 in cell culture and   12. Van Engelenburg FAC, Maes RK, Vanoirschot JT, Rijsewijk
    clinical samples using polymerase chain reaction. J Vet Diagn         FAM: 1993, Development of a rapid and sensitive polymerase
    Invest 6:143–147.                                                     chain reaction assay for detection of bovine herpesvirus type-1
 3. Dangler CA, de Mattos CA, de Mattos CC, Osburn BI: 1990,              in bovine semen. J Clin Microbiol 31:3129–3135.
    Identifying bluetongue virus ribonucleic acid sequences by the    13. Wiedmann M, Brandon R, Wagner P, et al.: 1993, Detection of
    polymerase chain reaction. J Virol Meth 28:281–292.                   bovine herpesvirus-1 in bovine semen by a nested PCR assay.
 4. Howard T, Bowen RA, Pickett B: 1985, Isolation of bluetongue          J Virol Methods 44:129–139.
    virus from bull semen. Prog Clin Biol Res 178:127–134.            14. Wilson WC: 1994, Development of nested-PCR tests based on
 5. Kahrs RF, Gibbs EPJ, Larsen RE: 1980, The search for viruses          sequence analysis of epizootic hemorrhagic disease viruses non-
    in bovine semen, a review. Theriogenology 14:151–165.                 structural protein 1 (NS1). Virus Res 31:357–365.




J Vet Diagn Invest 11:379–382 (1999)


      Minimum inhibitory concentration of selected antimicrobial agents for Serpulina
                           isolated from chickens and rheas

                               Darrell W. Trampel, Joann M. Kinyon, Neil S. Jensen

   Chicken intestinal spirochetes are a heterogeneous popu-           is characterized by end-on attachment of spirochetes to the
lation of bacteria that differ in morphology, hemolytic pat-          apical cell membrane of cecal enterocytes to form a dense
tern, indole production, enzymatic activity, relationship to          layer of bacteria aligned parallel to each other and perpen-
the mucosal surface, and genetics.10,19,20 On the basis of mul-       dicular to the mucosal surface.22 Clinical signs associated
tilocus enzyme electrophoresis and 16S ribosomal RNA se-              with cecal spirochetosis in chickens include a reduced
quence analysis, intestinal spirochetes pathogenic for chick-         growth rate, diarrhea, wet feces on feathers around the vent,
ens have been assigned to 1 of 3 genetically distinct species:        feces stained eggs, delayed onset of egg production in pul-
Serpulina intermedia, S. pilosicoli, or S. alvinipulli.10,16,19,23    lets, and reduced egg production in laying hens.1,5,18,22 Spi-
Serpulina intermedia and S. alvinipulli are found in the ceca         rochetes isolated from hens with diarrhea have been orally
as tangled masses in the lumen, lying unattached on the mu-           administered to specific-pathogen-free chickens, resulting in
cosal surface, and in the crypts.3,18 Infection with S. pilosicoli    watery feces and slight retardation of growth.1,19 Epidemio-
                                                                      logic studies from The Netherlands and Western Australia
                                                                      have shown that the prevalence of intestinal spirochetes in
  From the Veterinary Extension (Trampel) and the Veterinary Di-
agnostic Laboratory (Kinyon), College of Veterinary Medicine, Iowa
                                                                      layer and broiler breeder flocks experiencing diarrhea or re-
State University, Ames, IA 50011, and the Enteric Diseases and        duced egg production is significantly greater than their prev-
Food Safety Research Unit, National Animal Disease Center, Agri-      alence in clinically normal flocks.4,9
cultural Research Service, US Department of Agriculture, Ames, IA        Necrotizing typhlocolitis in juvenile common rheas (Rhea
50010 (Jensen).                                                       americana) from Ohio farms and a zoo was initially de-
  Received for publication March 16, 1998.                            scribed in 1992.15 Affected birds in 3 flocks experienced de-
380                                                            Brief Communications


   Table 1. Estimated effectiveness of selected antimicrobial compounds against Serpulina hyodysenteriae and S. pilosicoli based upon
in vitro minimum inhibitory concentration.

                                                                                                      MIC ( g/ml)

      Bacterium                     Antimicrobial                    Susceptible                        Intermediate           Resistant

S. hyodysenteriae                   lincomycin                         40                              40–80                       80
                                    carbadox                            0.125                           0.125 to       1            1
                                    tiamulin                            2                               2–4                         4
S. pilosicoli                       lincomycin                         25                              50                          75
                                    carbadox                            0.125                           0.125 to       1            1
                                    tiamulin                            1                               1 to 2                      2



pression, lethargy, anorexia, loose stools, and 25–80% mor-                Columbus (isolates OhC-1, OhC-2, OhR-1, OhR303), and
tality. Spirochetes characterized by strong -hemolysis were                the University of Nebraska, Lincoln (isolate Ne12551).
isolated from the ceca. Sequence analysis of 16S ribosomal                 Ia42167 and Ia308 have been identified as S. pilosicoli on
RNA showed that spirochetes from this outbreak and those                   the basis of multilocus enzyme electrophoresis.10 OhC-1 and
later isolated from sick rheas in Iowa are strains of S. hy-               OhC-2 belong to a new species of avian intestinal spiro-
odysenteriae, the etiologic agent of swine dysentery.7 A ret-              chetes recently named S. alvinipulli.10,17 Ia35829, OhR-1, and
rospective review of rhea submissions to the California Vet-               OhR303 from rheas are strains of S. hyodysenteriae.7 The
erinary Diagnostic Laboratory System from 1990 to 1993                     remaining rhea isolate, Ne12551, has not been genetically
revealed that 41% (13 of 31) of rhea chicks submitted be-                  identified.
tween 6 and 52 weeks of age had necrotizing typhlocolitis.6                   Antibacterial agents included in this study are commonly
   Antimicrobial agents are used for prevention and treat-                 available to commercial poultry producers or have been suc-
ment of bacterial diseases of poultry when therapy is likely               cessfully used to treat intestinal spirochete infections of
to be cost effective.21 Under ideal circumstances, selection               swine. The following antimicrobial agents were tested at se-
of an appropriate antimicrobial agent begins with isolation                rial dilutions ranging from 0.0005 to 200 g/ml: lincomycin,
and identification of the causative bacteria and determining                carbadox, tiamulin, bacitracin, chlortetracycline, oxytetra-
the minimal inhibitory concentration (MIC) for a panel of                  cycline, erythromycin, neomycin, and tylosin. Penicillin and
potential therapeutic drugs.14 Descriptions of antibiotic sus-             streptomycin were tested at serial dilutions ranging from 7.5
ceptibility patterns of intestinal spirochetes from chickens or            to 17,000 IU/ml. Previously established MICs of lincomycin,
rheas have not been published. This study was undertaken                   carbadox, and tiamulin for treatment of infections by S. hy-
to determine MICs for 11 antibacterial compounds against 4                 odysenteriae and S. pilosicoli in swine are listed in Table
spirochetes isolated from the ceca of chickens and 4 spiro-                1.2,11
chetes cultured from the lower intestinal tract of rheas. The                 Antimicrobial compounds in phosphate-buffered saline
MIC method chosen was agar dilution, which has been wide-                  (PBS) stock solutions were diluted to desired concentrations
ly used for susceptibility testing of Serpulina sp.2,8,12,13               first in PBS and then in molten trypticase soy bovine blood
   Spirochetes used in this study were originally cultured                 agar (TSBBA) 24 hours prior to the test. PBS without an
from the ceca of chickens or rheas submitted to the veteri-                antimicrobial agent was used as a growth control. Plates
nary diagnostic laboratories at Iowa State University, Ames                were solidified, stored overnight at 4 C, and dried to elimi-
(isolates Ia42167, Ia308, Ia35829), Ohio State University,                 nate surface moisture immediately before use.


   Table 2. Minimum inhibitory concentrations ( g/ml) for 11 antimicrobial agents against 2 S. pilosicoli isolates and 2 S. alvinipulli
isolates obtained from chicken ceca.

                                       S. pilosicoli                         A. alvinipulli

  Antimicrobial              Ia42167                   Ia308          OhC-1                   OhC-2                    Range      Median

Lincomycin                    25.0                  25.0             12.5                 12.5                   12.5–25.0        25.0
Carbadox                       0.005                 0.005            0.0005               0.005               0.0005–0.005        0.005
Tiamulin                       0.1                   0.1              0.01                 0.01                  0.01–0.1          0.1
Chlortetracycline            100.0                 100.0             10.0                 10.0                   10.0–100.0      100.0
Oxytetracycline                1.0                  10.0              0.01                 0.1                   0.01–10.0         1.0
Tylosin                       10.0                  10.0              0.1                 10.0                    0.1–10.0        10.0
Bacitracin                    25.0                  25.0              7.0                  7.0                    7.0–25.0        25.0
Erythromycin                 100.0                 100.0              1.0                 10.0                    1.0–100.0      100.0
Neomycin                      10.0                 100.0              0.01                10.0                   0.01–100.0       10.0
Penicillin*                  170.0                 170.0             17.0                 17.0                   17.0–170.0      170.0
Streptomycin*                 75.0                  75.0             75.0                 75.0                     75.0           75.0
  * Tested at serial dilutions ranging from 7.5 to 17,000 IU/ml.
                                                         Brief Communications                                                              381


   Table 3. Minimum inhibitory concentrations ( g/ml) for 11 antimicrobial agents against 4 isolates of Serpulina hyodysenteriae cultured
from rhea intestinal tracts.

                                                        Isolates

  Antimicrobial             Ia35829          Ne12551               OhR-1                OhR303               Range               Median

Lincomycin                   25.0             25.0               12.5                   12.5               12.5–25.0             25.0
Carbadox                      0.005            0.005              0.005                  0.005                0.005               0.005
Tiamulin                      0.01             0.01               0.01                   0.01                 0.01                0.01
Chlortetracycline            10.0             10.0              100.0                   10.0               10.0–100.0            10.0
Oxytetracycline            100.00              ND*              100.0                  100.0                100.0               100.0
Tylosin                     100.0             10.0               10.0                   10.0               10.0–100.0            10.0
Bacitracin                   35.0              ND                35.0                   35.0                 35.0                35.0
Erythromycin                100.0            100.0              100.0                  100.0                100.0               100.0
Neomycin                    100.0            100.0              100.0                  100.0                100.0               100.0
Penicillin†               1,700.0              ND             1,700.0                1,700.0              1,700.0             1,700.0
Streptomycin†                75.0             75.0               75.0                   75.0                 75.0                75.0
 * ND not done.
 † Tested at serial dilutions ranging from 7.5 to 17,000 IU/ml.



   Spirochete isolates were streaked on TSBBA plates 96                for all remaining antimicrobial agents in this study were
hours prior to the test and incubated anaerobically at 42 C.           identical for all 4 of the rhea isolates. MICs for oxytetra-
Approximately 0.2–0.3 ml of hemolyzed agar was suspended               cycline, neomycin, and penicillin tended to be higher for
in a 3-ml trypticase soy broth/bead tube and vortexed to               rhea than for chicken isolates. Each of the 4 rhea isolates
render agar chunks into a slurry. Inocula prepared in this             had identical MICs for 8 of the 11 antimicrobial agents test-
manner ranged from 3          107 to 1     108 colony-forming          ed, which may reflect similar genetic patterns.
units/ml per ml. Drops (10 l) of each diluted isolate were                Lincomycin is approved for use in broiler chickens and
applied in a replicate pattern to antimicrobial MIC plates,            probably represents the drug of choice for treatment of in-
each of which contained a different level of antimicrobial             testinal infections caused by Serpulina sp. in chickens and
agent within the agar.                                                 rheas. Carbadox and tiamulin are approved for treatment of
   Inoculated plates were inverted, placed in anaerobic jars           swine dysentery but have not been cleared by the Food and
at 42 C, and incubated for 96 hours. The MIC was the lowest            Drug Administration’s Center for Veterinary Medicine for
antimicrobial concentration (highest dilution) that prevented          use in poultry.
growth. Each MIC represents the median value of 5 tests,
with each test conducted in duplicate.                                                               References
   All chicken spirochetes, consisting of 2 S. pilosicoli iso-
lates and 2 S. alvinipulli isolates, were highly susceptible to            1. Davelaar FG, Hovind-Hougen K, Dwars RM, et al.: 1986, In-
lincomycin, carbadox, and tiamulin according to criteria es-                  fectious typhlitis in chickens caused by spirochetes. Avian
tablished for swine S. pilosicoli (Table 2). MICs were highly                 Pathol 15:247–258.
                                                                           2. Duhamel GE, Kinyon JM, Mathiesen MR, et al.: 1998, In vitro
variable for chlortetracycline, oxytetracycline, tylosin, baci-
                                                                              activity of four antimicrobial agents against North American
tracin, erythromycin, neomycin, and penicillin, suggesting
                                                                              isolates of porcine Serpulina pilosicoli. J Vet Diagn Invest 10:
that these compounds may be effective against some isolates                   350–356.
but not against others. The MIC for streptomycin was con-                  3. Dwars RM, Smit HF, Davelaar FG: 1990, Observations on avian
sistently high, and this antimicrobial agent probably would                   intestinal spirochaetosis. Vet Q 12:51–55.
not be effective against any of the spirochetes isolated from              4. Dwars RM, Smit HF, Davelaar FG, Van’T Veer W: 1989, Inci-
chickens. Antibiograms for Ia42167 and Ia308 were identi-                     dence of spirochaetal infections in cases of intestinal disorders
cal except for neomycin and oxytetracycline. OhC-1 and                        in chickens. Avian Pathol 18:591–595.
OhC-2 had the same sensitivity to 7 of 11 compounds tested.                5. Griffiths IB, Hunt BW, Lister SA, Lamont MH: 1987, Retarded
With the exception of carbadox and streptomycin, higher                       growth rate and delayed onset of egg production associated with
antimicrobial concentrations were required to inhibit strains                 spirochete infection in pullets. Vet Rec 121:35–37.
of S. pilosicoli from Iowa. Differences in antimicrobial sus-              6. Hanley RS, Woods LW, Stillian DJ, Dumonceaux GA: 1994,
ceptibility patterns observed between S. pilosicoli and S. al-                Serpulina-like spirochetes and flagellated protozoa associated
                                                                              with a necrotizing typhlitis in the rhea (Rhea americana). Proc
vinipulli from Ohio may be due to dissimilar genetics or to
                                                                              Annu Meet Am Assoc Avian Vet 1994:157–162.
development of resistance by S. pilosicoli following previous
                                                                           7. Jensen NS, Stanton TB, Swayne DE: 1996, Identification of the
exposure to antimicrobial agents in the field.                                 swine pathogen Serpulina hyodysenteriae in rheas (Rhea amer-
   All rhea spirochetes, consisting of 3 S. hyodysenteriae iso-               icana). Vet Microbiol 52:259–269.
lates and 1 unidentified spirochete, were susceptible to lin-               8. Kinyon JM, Harris DL: 1980, In vitro susceptibility of Trepo-
comycin, carbadox, and tiamulin if MICs determined for                        nema hyodysenteriae and Treponema innocens by the agar di-
swine S. hyodysenteriae are used as the standard (Table 3).                   lution method. Proc Int Symp Vet Lab Diagn 2:125–128.
Chlortetracycline and tylosin had variable MICs. The MICs                  9. McClaren AJ, Hampson DJ: 1996, The prevalence of intestinal
382                                                          Brief Communications


      spirochaetes in poultry flocks in Western Australia. Aust Vet J            ognition of two new species of intestinal spirochetes: Serpulina
      74:319–321.                                                               intermedia sp. nov. and Serpulina murdochii sp. nov. Int J Syst
10.   McClaren AJ, Trott DJ, Swayne DE, et al.: 1997, Genetic and               Bacteriol 47:1007–1012.
      phenotypic characterization of intestinal spirochetes colonizing    17.   Stanton TB, Postic D, Jensen NS: 1998, Serpulina alvinipulli,
      chickens and allocation of known pathogenic isolates to three             sp. nov., a new Serpulina species enteropathogenic for chickens.
      distinct genetic groups. J Clin Microbiol 35:412–417.                     Int J Syst Bacteriol 48:669–676.
11.   Messier S: 1992, Sensibilite in vitro d’isolats quebecois de Ser-
                                   ´                     ´ ´              18.   Swayne DE, Bermudez AJ, Sagartz JE, et al.: 1992, Association
      pulina (Treponema) hyodysenteriae envers differents agents an-
                                                       ´                        of cecal spirochetes with pasty vents and dirty eggshells in lay-
      tibacteriens. Med Vet Que 22:32–34.
            ´               ´    ´                                              ers. Avian Dis 36:776–781.
12.   Messier S, Higgins R, Moore C: 1990, Minimal inhibitory con-        19.   Swayne DE, Eaton KA, Stoutenberg J, et al.: 1995, Identifica-
      centrations of five antimicrobials against Treponema hyodys-               tion of a new intestinal spirochete with pathogenicity for chick-
                                                                                ens. Infect Immun 63:430–436.
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                                                                          20.   Swayne DE, McClaren AJ: 1997, Avian intestinal spirochaetes
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                                                                                and avian intestinal spirochaetosis. In: Intestinal spirochaetes in
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      bacteria, approved standard, 3rd ed. vol. 13 (6). NCCLS, Vil-       21.   Tanner AC: 1993, Antimicrobial drug use in poultry. In: Anti-
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J Vet Diagn Invest 11:382–384 (1999)


                            Ehrlichia platys infection and disease in dogs in Spain

                                            A. Sainz, I. Amusategui, M. A. Tesouro

   Ehrlichia platys is the causative agent of infectious cyclic              Hematologic and biochemical parameters before (day 0)
thrombocytopenia in the dog.3,4 The implicated vector in E.               and after (days 15–240) treatment are shown in Table 1.
platys transmission apparently is the tick Rhipicephalus san-             Prothrombin time, activated partial thromboplastin time, fi-
guineus. This disease was initially diagnosed in the USA in               brinogen, and antithrombin III were within the normal phys-
1978.4 Although the majority of the cases have occurred in                iologic range. Bleeding time was 4.67 minutes. Additional
the USA, the presence of an E. platys-like agent in Greece                parameters before treatment were as follows: 35 mg/dl urea,
and France has been recently reported.1,9 Reports of E. platys            0.9 mg/dl creatinine, and 448     103 neutrophils/ l. Urinal-
in Spain are limited to the direct observation of Ehrlichia-              ysis revealed a specific gravity of 1,040 g/ml, a slight pro-
like inclusion bodies within platelets in blood smears from               teinuria, and a large number of renal pelvic cells and tubular
dogs attended by the authors and by several veterinarians in              cells and a few bladder cells. Antibody to Leishmania
different areas of Spain (Central area, Cataluna, Valencia and            infantum was not detected by immunofluorescent assay
Andalucia) (J. Cario, A. Fisac, M. Calvo, personal commu-                 (IFA). Antibody to E. canis was detected by IFA (titer-2,560;
nication).                                                                positive titer is considered 40). Babesia sp. inside eryth-
   A 3-year-old male Drahthaar mix dog was presented at                   rocytes (Fig. 1) and Ehrlichia-like inclusion bodies within
the clinic of the Veterinary School of Madrid with a history              platelets (Fig. 2) were observed on peripheral blood (or buf-
of massive tick infestation for 1 month. The owners had                   fy coat) smears. Antibody to E. platys was detected by IFA
noted 2 episodes of slight epistaxis (unilateral from the right           (titer-640). Western blot analysis indicated that the serum
nostril). Clinically, the dog had poor general condition, leth-           from the patient contained the antibody found in serum of
argy, anorexia, respiratory distress, mucous membrane pal-                dogs infected with the Louisiana isolate of E. platys (Fig.
lor, fever (39.8 C), purulent ocular discharge, splenomegaly,             3).10
and muzzle hyperkeratosis. Radiography revealed no para-                     The dog was treated with doxycycline (10 mg/kg/day per
nasal sinus alterations.                                                  os for 28 days) and imidocarb dipropionate (5 mg/kg sub-
                                                                          cutaneously on days 1 and 14). General condition improved,
   From the Departamento de Patologia Animal II, Facultad de Ve-          and weight gain was evident 15 days after the treatment
terinaria, 28040 Madrid, Spain.                                           began. Muzzle hyperkeratosis and fever had abated. Two
   Received for publication February 18, 1998.                            additional episodes of epistaxis with small amounts of blood

								
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