We reported on a new species of intestinal spirochete bacterium, Serpulina pilosicoli/, associated with a diarrheal disease of grower-finisher swine, called porcine colonic spirochetosis (PCS). In this report we show that Serpulina pilosicoli, associated with outbreaks of PCS in the United States, attached to the cecal surface of chicks while Serpulina innocens, a non-pathogenic intestinal spirochete, did not. These findings support a role for Serpulina pilosicoli as a cause of diarrhea and reduced feed efficiency in swine

Duhmel, Gerald

Mathiesen, Michelle R.

Muniappa, Nagaraja

English

UNL | Libraries

University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
Nebraska Swine Reports Animal Science Department 
1996 
Pathogenicity of Intestinal Spirochetes Associated with Porcine 
Colonic Spirochetosis 
Gerald Duhmel 
University of Nebraska-Lincoln, gduhamel1@unl.edu 
Nagaraja Muniappa 
University of Nebraska-Lincoln 
Michelle R. Mathiesen 
University of Nebraska-Lincoln, mmathiesen2@unl.edu 
Follow this and additional works at: https://digitalcommons.unl.edu/coopext_swine 
 Part of the Animal Sciences Commons 
Duhmel, Gerald; Muniappa, Nagaraja; and Mathiesen, Michelle R., "Pathogenicity of Intestinal Spirochetes 
Associated with Porcine Colonic Spirochetosis" (1996). Nebraska Swine Reports. 196. 
https://digitalcommons.unl.edu/coopext_swine/196 
This Article is brought to you for free and open access by the Animal Science Department at 
DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Nebraska Swine Reports by 
an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 
20
Gerald E. Duhamel
Nagaraja Muniappa
Michelle R. Mathiesen1
Summary and Implications
We reported on a new species of
intestinal spirochete bacterium,
Serpulina pilosicoli, associated
with a diarrheal disease of grower-
finisher swine, called porcine col-
onic spirochetosis (PCS). In this
report we show that Serpulina pilosi-
coli, associated with outbreaks of
PCS in the United States, attached
to the cecal surface of chicks while
Serpulina innocens, a non-patho-
genic intestinal spirochete, did not.
These findings support a role for
Serpulina pilosicoli as a cause of
diarrhea and reduced feed efficiency
in swine.
Introduction
Because 80 percent of the total
feed cost is associated with the grower-
finisher phase of pork production, im-
proved feed efficiency during that pe-
riod can generate significant cost sav-
ings and increase profits. It is known
that approximately 76 percent of the
basal energy requirement of the pig is
derived from absorption of volatile fatty
acids (VFA) by the large intestine. In
fact, the concentration of VFA pro-
duced from microbial fermentation in
the lower intestine of swine ranges
between 180 and 200 mmoles/L, a
range similar to that found in the ru-
men contents of cattle. In contrast to
cattle where most of the VFA absorp-
tion takes place in the abomasum, VFA
are absorbed from the large intestine of
swine. The quantity of VFA absorbed
from the pig’s lower intestine depends
primarily upon the surface area avail-
able.
Table 1. Differentiating features of Serpulina spp. isolated from the intestine of swine.
Characteristic S. hyodysenteriae S. innocens S. pilosicoli
Hemolysis Strong Weak Weak
Number of
periplasmic flagella 8 to 12 10 to 13 4 to 7
Indole production Positive Negative Negative
Hippurate hydrolysis Negative Negative Positive
16S rDNA PCR† Negative Negative Positive
Associated hosts Swine Swine Swine
Dogs Dogs Dogs
Mice Mice
Birds Guinea pigs
Guinea pigs Non-human primates
Humans
Birds
Associated condition Swine Not Colonic
dysentery pathogenic spirochetosis
†PCR = polymerase chain reaction.
Presently, all weakly β-hemolytic
intestinal spirochetes (WBHIS) inhab-
iting the lower intestine of swine are
assigned to the non-pathogenic spiro-
chete species, Serpulina innocens.
However, certain WBHIS are clearly
associated with a non-fatal wasting
diarrheal disease of growing swine,
called porcine colonic spirochetosis
(PCS). We found that the WBHIS as-
sociated with PCS are distinct but re-
lated to Serpulina innocens and we
proposed the name     Serpulina pilosicoli
to describe these spirochete bacteria
(Table 1). Comparative analyses of
Serpulina pilosicoli isolated from swine
in the United States, Canada, the United
Kingdom, and Australia indicated the
worldwide distribution of PCS. Be-
cause the newly identified Serpulina
pilosicoli can attach and invade the
wall of the lower intestine, we hypoth-
esized that it has the potential to re-
duce feed efficiency by disrupting VFA
absorption. In the present study, we
examined several isolates of Serpulina
pilosicoli for attachment to the surface
of chicks’ ceca. Attachment of Serpulina
pilosicoli to the ceca of chicks is in-
dicative of pathogenicity associated
with reduced surface area available for
absorption of nutrients.
Materials and Methods
Serpulina pilosicoli were obtained
from swine with clinical signs or
lesions of PCS in Nebraska (n=2),
Iowa (n=2), and California (n=3).
After confirming the identity of
Serpulina pilosicoli, using structural,
biochemical, and genotypic analyses
(Table 1), the spirochetes were com-
pared with Serpulina innocens for
attachment to the ceca of chicks. One-
day-old chicks were inoculated by
crop gavage with either sterile
medium or medium containing either
Serpulina innocens or Serpulina
pilosicoli. On day 7, 14 and 21 post-
inoculation, the ceca of control chicks
and Serpulina-inoculated chicks
were collected for bacteriologic and
histopathologic examinations.
Pathogenicity of Intestinal Spirochetes
Associated with Porcine Colonic Spirochetosis
21
Table 2. Attachment of spirochetes to the ceca of chicks challenge-exposed with porcine weakly
β-hemolytic intestinal spirochetes.
Isolate Number of Days post-inoculation†
isolates tested 7 14 21 Total
Serpulina pilosicoli 7 13/20 10/13 8/9 31/42
Infection rate (%) 65 77 89 74
Serpulina innocens 1 0/3 0/5 0/6 0/14
Infection rate (%) 0 0 0 0
Sterile medium Not applicable 0/7 0/3 0/6 0/16
†Number positive for attachment/number challenge-exposed, as determined by histologic examination.
pilosicoli causes inflammation of the
wall of the lower intestine, a lesion
referred to as “non-specific colitis”.
Based on the changes observed in the
ceca of the chicks, this change must
take longer than 21 days to develop.
Because of the diversity of WBHIS
normally present in the intestinal tract
of swine and the widespread occur-
rence of non-pathogenic WBHIS, iso-
lation of WBHIS from swine is only
suggestive of PCS. Demonstration of
spirochetal attachment to the surface
of the lower intestine confirms the
diagnosis of PCS; however, necropsy
of pigs is not routinely performed in
uncomplicated cases of PCS. Assess-
ment of several laboratory procedures
for rapid and accurate identification of
Serpulina pilosicoli indicates that a
preliminary identification of the
spirochete can be made on the basis
of positive hydrolysis of hippurate.
Definitive identification of Ser-
pulina pilosicoli requires amplifi-
cation of a specific 16S rDNA gene
sequence by polymerase chain reac-
tion. Why is it then that Serpulina
pilosicoli is only reported sporadi-
cally as a cause of diarrhea and colitis
in swine? This may be attributable to
either (1) failure to use appropriate
techniques for primary isolation of
the spirochete from intestinal speci-
mens, (2) failure to identify the spiro-
chete from diagnostic specimens, or
(3) failure to identify lesions of PCS
in tissues submitted for diagnostic
evaluation. Serpulina pilosicoli also
has been isolated from stool samples
obtained from children with diarrhea
in developing countries and from
immunocompromized adult individ-
uals infected with the human immun-
odeficiency virus in more devel-
oped countries. Because Serpulina
pilosicoli are isolated from humans
and swine affected with PCS, it raises
the possibility that these spirochetes
may be zoonotic and have a public
health significance.
1Gerald E. Duhamel is an Associate Professor,
Nagaraja Muniappa is a PhD candidate, and Michelle
R. Mathiesen is a laboratory technician in the
Department of Veterinary and Biomedical Sciences
at the University of Nebraska-Lincoln.
Results
Grossly, the ceca of chicks inocu-
lated with either sterile medium or
medium containing either Serpulina
innocens or Serpulina pilosicoli had
no notable changes; the cecal contents
were yellowish brown, foamy and
semisolid. Histologically the ceca of
chicks inoculated with sterile medium
or medium containing Serpulina
innocens had tall columnar epithe-
lium without spirochetes. In contrast,
the ceca of chicks inoculated Ser-
pulina pilosicoli  had spirochetes
attached along the surface (Table 2).
The infection rate of the Serpulina
pilosicoli-inoculated chicks increased
from 65 percent after 7 days, to 77
percent after 14 days and 89 percent
after 21 days. No spirochetes were
found by culture of ceca from chicks
given either sterile medium or medi-
um containing Serpulina innocens
at any time post-inoculation. In con-
trast, large numbers of WBHIS
were isolated from the ceca of chicks
challenge-exposed with Serpulina
pilosicoli.
Discussion
Detailed phenotypic and genotypic
characterization of intestinal spirochetes
has lead to major advances in our
understanding of the molecular epide-
miology of spirochetal diarrhea in hu-
mans and animals. Complete agree-
ment was found between structural,
biochemical, and genotypic analyses
and the results of attachment to the
ceca of chicks. On the bases of these
results, we concluded that Serpulina
pilosicoli has the potential to cause
reduced surface area for absorption of
nutrients from the gut lumen. We found
that the infection rates of chicks in-
creased over time, such that it took 21
days to reach 89 percent of the chicks
with spirochetes attached to the sur-
face of the ceca.
Porcine colonic spirochetosis is
characterized clinically by watery to
mucoid diarrhea without blood, or so
called “cow-pie scours”. Although
diarrhea can affect up to 20 percent of
swine in the grower-finisher phase of
production, depression of weight gain
is the most significant finding with
PCS and this can result in a significant
delay in reaching market weight. Ad-
ditional animal care is a major prob-
lem associated with PCS in all in/all
out management systems because of
the uneven sizes of the pigs causing
disruption of pig flow.
The pathologic changes in chicks
inoculated with Serpulina pilosicoli
were similar to those present in the
early stages of colonic spirochetosis in
humans, swine and other animals. In
swine, the large intestine contains
abundant watery-green or yellow
mucoid materials and variable degree
of exudation and surface erosions are
sometimes visible. Early in the infec-
tion spirochetes attach by one of their
ends along the surface of the lower
intestine producing what appears as a
dark fringe when examined histologi-
cally. The loss of surface area in the
large intestine of pigs accounts for the
reduced feed efficiency and increased
numbers of days to market. Over longer
periods of time, infection with  Serpulina


Pathogenicity of Intestinal Spirochetes Associated with Porcine Colonic Spirochetosis

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Nebraska Swine Reports Animal Science Department 1996 Pathogenicity of Intestinal Spirochetes Associated with Porcine Colonic Spirochetosis Gerald Duhmel University of Nebraska-Lincoln, gduhamel1@unl.edu Nagaraja Muniappa University of Nebraska-Lincoln Michelle R. Mathiesen University of Nebraska-Lincoln, mmathiesen2@unl.edu Follow this and additional works at: https://digitalcommons.unl.edu/coopext_swine  Part of the Animal Sciences Commons Duhmel, Gerald; Muniappa, Nagaraja; and Mathiesen, Michelle R., "Pathogenicity of Intestinal Spirochetes Associated with Porcine Colonic Spirochetosis" (1996). Nebraska Swine Reports. 196. https://digitalcommons.unl.edu/coopext_swine/196 This Article is brought to you for free and open access by the Animal Science Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Nebraska Swine Reports by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 20Gerald E. DuhamelNagaraja MuniappaMichelle R. Mathiesen1Summary and ImplicationsWe reported on a new species ofintestinal spirochete bacterium,Serpulina pilosicoli, associatedwith a diarrheal disease of grower-finisher swine, called porcine col-onic spirochetosis (PCS). In thisreport we show that Serpulina pilosi-coli, associated with outbreaks ofPCS in the United States, attachedto the cecal surface of chicks whileSerpulina innocens, a non-patho-genic intestinal spirochete, did not.These findings support a role forSerpulina pilosicoli as a cause ofdiarrhea and reduced feed efficiencyin swine.IntroductionBecause 80 percent of the totalfeed cost is associated with the grower-finisher phase of pork production, im-proved feed efficiency during that pe-riod can generate significant cost sav-ings and increase profits. It is knownthat approximately 76 percent of thebasal energy requirement of the pig isderived from absorption of volatile fattyacids (VFA) by the large intestine. Infact, the concentration of VFA pro-duced from microbial fermentation inthe lower intestine of swine rangesbetween 180 and 200 mmoles/L, arange similar to that found in the ru-men contents of cattle. In contrast tocattle where most of the VFA absorp-tion takes place in the abomasum, VFAare absorbed from the large intestine ofswine. The quantity of VFA absorbedfrom the pig’s lower intestine dependsprimarily upon the surface area avail-able.Table 1. Differentiating features of Serpulina spp. isolated from the intestine of swine.Characteristic S. hyodysenteriae S. innocens S. pilosicoliHemolysis Strong Weak WeakNumber ofperiplasmic flagella 8 to 12 10 to 13 4 to 7Indole production Positive Negative NegativeHippurate hydrolysis Negative Negative Positive16S rDNA PCR† Negative Negative PositiveAssociated hosts Swine Swine SwineDogs Dogs DogsMice MiceBirds Guinea pigsGuinea pigs Non-human primatesHumansBirdsAssociated condition Swine Not Colonicdysentery pathogenic spirochetosis†PCR = polymerase chain reaction.Presently, all weakly β-hemolyticintestinal spirochetes (WBHIS) inhab-iting the lower intestine of swine areassigned to the non-pathogenic spiro-chete species, Serpulina innocens.However, certain WBHIS are clearlyassociated with a non-fatal wastingdiarrheal disease of growing swine,called porcine colonic spirochetosis(PCS). We found that the WBHIS as-sociated with PCS are distinct but re-lated to Serpulina innocens and weproposed the name     Serpulina pilosicolito describe these spirochete bacteria(Table 1). Comparative analyses ofSerpulina pilosicoli isolated from swinein the United States, Canada, the UnitedKingdom, and Australia indicated theworldwide distribution of PCS. Be-cause the newly identified Serpulinapilosicoli can attach and invade thewall of the lower intestine, we hypoth-esized that it has the potential to re-duce feed efficiency by disrupting VFAabsorption. In the present study, weexamined several isolates of Serpulinapilosicoli for attachment to the surfaceof chicks’ ceca. Attachment of Serpulinapilosicoli to the ceca of chicks is in-dicative of pathogenicity associatedwith reduced surface area available forabsorption of nutrients.Materials and MethodsSerpulina pilosicoli were obtainedfrom swine with clinical signs orlesions of PCS in Nebraska (n=2),Iowa (n=2), and California (n=3).After confirming the identity ofSerpulina pilosicoli, using structural,biochemical, and genotypic analyses(Table 1), the spirochetes were com-pared with Serpulina innocens forattachment to the ceca of chicks. One-day-old chicks were inoculated bycrop gavage with either sterilemedium or medium containing eitherSerpulina innocens or Serpulinapilosicoli. On day 7, 14 and 21 post-inoculation, the ceca of control chicksand Serpulina-inoculated chickswere collected for bacteriologic andhistopathologic examinations.Pathogenicity of Intestinal SpirochetesAssociated with Porcine Colonic Spirochetosis21Table 2. Attachment of spirochetes to the ceca of chicks challenge-exposed with porcine weaklyβ-hemolytic intestinal spirochetes.Isolate Number of Days post-inoculation†isolates tested 7 14 21 TotalSerpulina pilosicoli 7 13/20 10/13 8/9 31/42Infection rate (%) 65 77 89 74Serpulina innocens 1 0/3 0/5 0/6 0/14Infection rate (%) 0 0 0 0Sterile medium Not applicable 0/7 0/3 0/6 0/16†Number positive for attachment/number challenge-exposed, as determined by histologic examination.pilosicoli causes inflammation of thewall of the lower intestine, a lesionreferred to as “non-specific colitis”.Based on the changes observed in thececa of the chicks, this change musttake longer than 21 days to develop.Because of the diversity of WBHISnormally present in the intestinal tractof swine and the widespread occur-rence of non-pathogenic WBHIS, iso-lation of WBHIS from swine is onlysuggestive of PCS. Demonstration ofspirochetal attachment to the surfaceof the lower intestine confirms thediagnosis of PCS; however, necropsyof pigs is not routinely performed inuncomplicated cases of PCS. Assess-ment of several laboratory proceduresfor rapid and accurate identification ofSerpulina pilosicoli indicates that apreliminary identification of thespirochete can be made on the basisof positive hydrolysis of hippurate.Definitive identification of Ser-pulina pilosicoli requires amplifi-cation of a specific 16S rDNA genesequence by polymerase chain reac-tion. Why is it then that Serpulinapilosicoli is only reported sporadi-cally as a cause of diarrhea and colitisin swine? This may be attributable toeither (1) failure to use appropriatetechniques for primary isolation ofthe spirochete from intestinal speci-mens, (2) failure to identify the spiro-chete from diagnostic specimens, or(3) failure to identify lesions of PCSin tissues submitted for diagnosticevaluation. Serpulina pilosicoli alsohas been isolated from stool samplesobtained from children with diarrheain developing countries and fromimmunocompromized adult individ-uals infected with the human immun-odeficiency virus in more devel-oped countries. Because Serpulinapilosicoli are isolated from humansand swine affected with PCS, it raisesthe possibility that these spirochetesmay be zoonotic and have a publichealth significance.1Gerald E. Duhamel is an Associate Professor,Nagaraja Muniappa is a PhD candidate, and MichelleR. Mathiesen is a laboratory technician in theDepartment of Veterinary and Biomedical Sciencesat the University of Nebraska-Lincoln.ResultsGrossly, the ceca of chicks inocu-lated with either sterile medium ormedium containing either Serpulinainnocens or Serpulina pilosicoli hadno notable changes; the cecal contentswere yellowish brown, foamy andsemisolid. Histologically the ceca ofchicks inoculated with sterile mediumor medium containing Serpulinainnocens had tall columnar epithe-lium without spirochetes. In contrast,the ceca of chicks inoculated Ser-pulina pilosicoli  had spirochetesattached along the surface (Table 2).The infection rate of the Serpulinapilosicoli-inoculated chicks increasedfrom 65 percent after 7 days, to 77percent after 14 days and 89 percentafter 21 days. No spirochetes werefound by culture of ceca from chicksgiven either sterile medium or medi-um containing Serpulina innocensat any time post-inoculation. In con-trast, large numbers of WBHISwere isolated from the ceca of chickschallenge-exposed with Serpulinapilosicoli.DiscussionDetailed phenotypic and genotypiccharacterization of intestinal spirocheteshas lead to major advances in ourunderstanding of the molecular epide-miology of spirochetal diarrhea in hu-mans and animals. Complete agree-ment was found between structural,biochemical, and genotypic analysesand the results of attachment to thececa of chicks. On the bases of theseresults, we concluded that Serpulinapilosicoli has the potential to causereduced surface area for absorption ofnutrients from the gut lumen. We foundthat the infection rates of chicks in-creased over time, such that it took 21days to reach 89 percent of the chickswith spirochetes attached to the sur-face of the ceca.Porcine colonic spirochetosis ischaracterized clinically by watery tomucoid diarrhea without blood, or socalled “cow-pie scours”. Althoughdiarrhea can affect up to 20 percent ofswine in the grower-finisher phase ofproduction, depression of weight gainis the most significant finding withPCS and this can result in a significantdelay in reaching market weight. Ad-ditional animal care is a major prob-lem associated with PCS in all in/allout management systems because ofthe uneven sizes of the pigs causingdisruption of pig flow.The pathologic changes in chicksinoculated with Serpulina pilosicoliwere similar to those present in theearly stages of colonic spirochetosis inhumans, swine and other animals. Inswine, the large intestine containsabundant watery-green or yellowmucoid materials and variable degreeof exudation and surface erosions aresometimes visible. Early in the infec-tion spirochetes attach by one of theirends along the surface of the lowerintestine producing what appears as adark fringe when examined histologi-cally. The loss of surface area in thelarge intestine of pigs accounts for thereduced feed efficiency and increasednumbers of days to market. Over longerperiods of time, infection with  Serpulina

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Pathogenicity of Intestinal Spirochetes Associated with Porcine Colonic Spirochetosis

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