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Use of a real-time polymerase chain

reaction-based fluorogenic 5' nuclease assay
to evaluate insect vectors of Corynebacterium
pseudotuberculosis infections in horses
Sharon J. Spier, DVM, PhD; Christian M. Leutenegger, Dr Med Vet, PhD; Scott P. Carroll, PhD;
Jenella E. Loye, PhD; Jeannine Berger Pusterla, Dr Med Vet; Tim E. Carpenter, DVM, PhD;
Judy E. Mihalyi, BS; John E. Madigan, DVM, MS

Objective—To develop and use a sensitive molecular been identified on the basis of differences in nitrate
assay for detecting the phospholipase D (PLD) exo- reduction, results on restriction endonuclease analysis,
toxin gene of Corynebacterium pseudotuberculosis in and restriction fragment length polymorphism.2-5 In
an attempt to identify insect vectors that may be contrast to ovine and caprine isolates, equine strains
important in transmission of clinical disease in hors- are capable of reducing nitrate to nitrite. Natural cross-
es. species infection by the specific biotypes is not known
Sample Population—2,621 flies of various species. to occur.6 The 3 forms of disease in horses include limb
Procedure—A real-time polymerase chain reaction infection, known as ulcerative lymphangitis; external
(PCR)-based fluorogenic 5’ nuclease (TaqMan) sys- abscesses; and infection of internal organs. The most
tem (ie, TaqMan PCR assay) was developed for the common form of C pseudotuberculosis infection, char-
detection of the PLD gene in insects. Flies were col- acterized by formation of deep abscesses primarily in
lected monthly (May to November 2002) from 5 farms the pectoral area and ventral abdominal area, is
in northern California where C pseudotuberculosis referred to as “pigeon fever” or “dryland distemper.” 7-10
infection in horses is endemic. Three of the 5 farms Two major virulence factors have been implicated
(which housed a total of 358 horses) had diseased
horses during the study period. A total of 2,621 flies in the pathogenesis of C pseudotuberculosis infections, a
of various species were tested for the PLD gene of C cytotoxic surface lipid and phospholipase D (PLD)
pseudotuberculosis. exotoxin.11,12 The lipid coat appears to facilitate intra-
Results—Evidence of bacterial DNA for the PLD cellular survival of the organism and abscess forma-
gene was detected in skin biopsy specimens from tion. A PLD toxin of approximately 31.5 kd is pro-
clinically affected horses and from 3 fly species col- duced by all C pseudotuberculosis isolates and may pro-
lected from farms where affected horses were mote spread of the infection through increases in vas-
housed. Farms with a high incidence of diseased cular permeability.13-17 Furthermore, PLD toxin may
horses had a high proportion of insects carrying the enhance survival and multiplication of the organism
organism. High percentages of flies with positive via complement depletion and inhibitory effects on
results for the PLD gene were observed in October, phagocytic cells.18,19 Complications including abortion,
when most clinically affected horses were observed. lengthy course of disease, and abscess formation in
Conclusions and Clinical Relevance—Our results internal organs have been reported.20-23 Mortality rates
are consistent with the hypothesis that C pseudotu- for external abscesses are low, but fatalities resulting
berculosis may be vectored to horses by flies. Three from organ involvement occur if diagnosis and appro-
potential vectors were identified, including
Haematobia irritans, Stomoxys calcitrans, and Musca priate antimicrobial treatment is delayed.20-23
domestica. The organism can be identified in up to The mode of infection remains unproven, but it is
20% of house flies (Musca domestica) in the vicinity speculated that the organism is soil-borne and enters
of diseased horses. (Am J Vet Res 2004;65:829–834) the equine host through skin abrasions as experimen-
tally confirmed for caseous lymphadenitis in sheep.24,25
Flies and other insects are potential vectors for the dis-
C orynebacterium pseudotuberculosis is a pleomor-
phic, facultative intracellular, gram-positive rod
that causes 3 distinct disease syndromes in horses,
ease, as indicated by its peak incidence in late summer
and fall and increased incidence in years following
heavy winter rainfall that favor insect populations.20,22,a
caseous lymphadenitis in sheep and goats, and sporad-
Recent studies26,27 in cattle have implicated Musca
ically infects other species including cattle and
domestica as a vector for disease in dairy cattle in Israel.
humans.1 Two biotypes of C pseudotuberculosis have
Insect vectors for disease in horses have not been
Received September 5, 2003. determined to date, and attempts to culture the bacte-
Accepted October 17, 2003. ria from flies, ticks, or soil samples from farms with
From the Departments of Medicine and Epidemiology (Spier, diseased horses have been unsuccessful.8,20,b Several
Leutenegger, Pusterla, Carpenter, Mihalyi, Madigan), School of authors have observed that the seasonal pattern of ven-
Veterinary Medicine, and the Department of Entomology (Carroll,
Loye), University of California, Davis, CA 95616.
tral midline dermatitis, caused by the feeding habits of
Supported by a grant from the T.S. Glide Foundation. the horn fly (Haematobia irritans) or biting midge
The authors thank Rosemarie Ramirez for technical assistance. (Culicoides sp), coincides with the seasonal incidence
Address correspondence to Dr. Spier. of abscesses in horses. This could be the result of either

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mechanical transmission of C pseudotuberculosis by related but clinically irrelevant isolate, C ulcerans, was
horn flies or midges or contamination of ventral mid- excluded by designing the TaqMan PCR system for C pseudo-
line dermatitis lesions from other sources such as soil tuberculosis against a sequence stretch with low sequence
or other fly species.10,22 identity between C pseudotuberculosis and C ulcerans. The
TaqMan PCR system for the detection of the C pseudotuber-
Although C pseudotuberculosis infections in horses culosis PLD gene (CpPld) is designated as the CpPld TaqMan
have been recognized for several decades, few epidemi- PCR system.
ologic and immunologic data are available and most
discussion is subjective or extrapolative from the dis- Fly sample processing and DNA extraction—Collected
ease in sheep and goats. The purpose of the study flies were identified immediately after capture with diagnos-
reported here was to develop and use a sensitive mole- tic characters described by Loomis28 and then transferred into
lysis bufferc for DNA extraction in 96-well, deep-well plates
cular assay for detecting the PLD exotoxin gene of and stored at –20oC until sample processing. Before DNA
C pseudotuberculosis in an attempt to identify insect extraction, two 4-mm-diameter grinding beadse were placed
vectors that may be important in propagation of clini- in each position of a 96-well, deep-well plate and the flies
cal disease. were homogenized by use of a tissue grinderf for 2 minutes at
1,000 strokes/min. After a 30-minute period at 4oC, genomic
Materials and Methods DNA was extracted from the tissue lysates by use of an auto-
Horses and fly collection—Five farms that housed 358 mated nucleic acid workstationg according to the instructions
horses of variable breed, sex, and age, located in Yolo and of the manufacturer. Five microliters of the DNA was used to
Solano Counties of northern California were included in this detect C pseudotuberculosis by TaqMan PCR assay.
study. Farms, numbered 1 to 5, housed 30, 150, 8, 146, and C pseudotuberculosis and C ulcerans microbial cultures
24 horses, respectively. All horses were kept in pasture at and DNA preparation—Lyophilized field isolates of
least half of the time, and they were monitored for clinical C pseudotuberculosis biovar equi and biovar ovis and C ulcerans
signs of naturally occurring C pseudotuberculosis infection. were grown aerobically for 48 hours on Luria-Bertani broth
Farms were visited monthly from May through November agar plates and cultured overnight at 37oC. Single colonies
2002, at which time flies were trapped and information and were picked from cultures with visible colony growth after 24
samples from clinically affected horses with C pseudotubercu- hours, resuspended in 200 µL PBS solution, and digested with
losis infection were collected. Flies were collected from indi- 20 µL of proteinase K and lysis buffer.h The DNA was extract-
vidual horses (clinically affected horses and exposed herd ed from the bacteria according to the recommendation of the
mates) by use of nets and from the environment by use of manufacturer. The DNA was eluted in 200 µL of water, and 5
drift nets and light traps. An attempt was made to collect at µL were used for the TaqMan PCR assay.
least 100 flies from each farm, but this was not always possi-
ble as a result of weather conditions or the use of pesticides Real-time TaqMan PCR assay—Each PCR assay con-
on horses or in the environment. Flies were placed on dry ice tained 400nM of each primer; 80nM of the TaqMan probe;
immediately following capture, identified, then placed in a and commercially available PCR mastermixi containing
lysis bufferc for DNA analysis. 10mM Tris-HCl buffer (pH, 8.3), 50mM KCl, 5mM MgCl2,
Horses were grouped as clinically affected horses with 2.5mM deoxynucleotide triphosphates, 0.625 U DNA poly-
natural infection (n = 41) and as exposed but not infected merasej/reaction, 0.25 U of uracil-N-glycosylasek/reaction,
horses (317). The diagnosis of C pseudotuberculosis infection and 5 µL of the DNA sample in a final volume of 25 µL.
was established on the basis of clinical appearance of horses, Samples were placed in 96-well plates and amplified in an
seasonality of disease occurrence, and typical location of automated fluorometer.l Amplification conditions were 2
lesions on the pectoral area or ventral area of the abdomen. minutes at 50oC, 10 minutes at 95oC, 40 cycles of 15 seconds
Diagnoses were confirmed by culture of nitrate-positive at 95oC, and 60 seconds at 60oC.
C pseudotuberculosis from the lesions. Diagnosis of internal Statistical analysis—The incidence of disease was
abscesses caused by C pseudotuberculosis was made on the defined as the number of affected horses at the farm on the
basis of typical clinical and laboratory findings for internal date of fly collection. The relationship between the incidence
abscesses in combination with a synergistic hemolysis inhi- of disease (%) and PLD gene frequency in fly DNA (percent-
bition titer of ≥ 512 or culture of the organism at necropsy.21,23 age of flies with positive results for the PLD gene that were
Tissue specimens from 10 clinically affected horses were trapped at the farm on that date) was analyzed with the non-
obtained at the time of surgical drainage of external abscess- parametric Spearman rank correlation method by use of a
es. One 3- to 4-mm-diameter biopsy specimen was obtained computer software package.m Values of P < 0.05 were consid-
from the skin overlying the abscess, and tissues were placed ered significant.
immediately into lysis buffer.c A biopsy was not performed on
herd mates that were exposed to the same environment.
Results
Real-time quantitative polymerase chain reaction Analytic specificity and sensitivity of the
assay—A real-time polymerase chain reaction (PCR)-based C pseudotuberculosis TaqMan PCR system—Analytic
fluorogenic 5' nuclease assay (TaqMan) was developed to specificity was tested by use of 8 characterized field
perform quantitative PCR assays (ie, TaqMan PCR assay). A isolates of C pseudotuberculosis and C ulcerans
TaqMan PCR system was designed that targeted the C pseudo- (Table 1). Use of the TaqMan PCR system resulted in a
tuberculosis PLD gene (GenBank accession No. L16586). Two positive signal for colonies of C pseudotuberculosis cul-
primers (CpPLP-98f: 5'-CTACAGCAAATCGGCCAGTCT-3';
and CpPLP-163r: 5'-CGTCATCCACGCCTTGAGT-3’) and
tures with detectable growth. The DNA extracted from
an internal, fluorescent labeled TaqMan probe (CpPLP-120p: colonies obtained from C ulcerans did not yield a posi-
5’-6-FAM-TGCGATTGCCCACCGCGTTTTA-TAMRA-3’) tive signal in TaqMan PCR assay confirming analytic
were designed by use of a commercial software program.d The specificity for C pseudotuberculosis from ovine and
TaqMan PCR system was designed after a multiple sequence equine origin. Analytic sensitivity was indirectly
analysis of ovine and equine C pseudotuberculosis isolates to assessed with a standard curve calculated from dilu-
ensure detection of organisms from both species. A closely tions of DNA obtained from cultured C pseudotubercu-

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Table 1—Corynebacterium isolates, culture results, and real-time TaqMan PCR assay results
Identification Culture TaqMan
Sample No. Isolate Species/origin growth PCR single
1 79-448-12A C pseudotuberculosis Bovine Colonies Strongly positive
2 80-490-9A C pseudotuberculosis Bovine Colonies Strongly positive
3 79-389-11 C pseudotuberculosis Equine Colonies Strongly positive
4 80-434-11 C pseudotuberculosis Equine/abscess Colonies Strongly positive
5 80-395-12 C pseudotuberculosis Equine/abscess Colonies Strongly positive
6 80-379-3 C pseudotuberculosis Ovine/herd infection Colonies Strongly positive
7 6127B C pseudotuberculosis Ovine Colonies Strongly positive
8 41-5 C ulcerans – Colonies Negative

Real-time TaqMan PCR assay = Real-time polymerase chain reaction-based fluorogenic 5' nuclease assay.

Table 2—Results of inhibition studies

CpPld detection (TaqMan signal)
Gain (+) or loss (-)
DNA combination Replicate 1 Replicate 2 Mean SD of single (CT)
Fly DNA alone Neg Neg NA NA NA
Fly DNA alone Neg Neg NA NA NA
Fly DNA alone Neg Neg NA NA NA
Fly DNA alone Neg Neg NA NA NA

Cp DNA alone 20.33 20.42 20.375 0.06 NA

Cp DNA alone 19.61 19.67 19.64 0.04 NA
Cp DNA alone 22.44 22.6 22.52 0.11 NA
Cp DNA alone 20.65 20.08 20.365 0.40 NA

Fly DNA + Cp DNA 20.65 20.15 20.4 0.35 + 2.5%

Fly DNA + Cp DNA 19.93 19.51 19.72 0.30 + 8.0%
Fly DNA + Cp DNA 22.68 22.43 22.555 0.18 + 3.5%
Fly DNA + Cp DNA 20.25 19.91 20.08 0.24 -28.5%
CpPld = C pseudotuberculosis phospholipase D gene. Cp = C pseudotuberculosis. CT = Cycle threshold.
Neg = Negative. NA = Not applicable.

losis isolates. On the basis of the slope of the standard infections, whereas the remaining 3 farms (with 188
curve, the amplification efficiency was calculated by horses) had diseased horses. Among the latter 3 farms,
use of the formula E = 10–1/slope, where E is the percent farm 1 (with 30 resident horses) had a 10% incidence
efficiency and the slope is that of the standard curve. of diseased horses, farm 2 (with 150 resident horses)
The amplification efficiency of the CpPld TaqMan PCR had a 23% incidence of diseased horses, and on the
systems was 97.4%. TaqMan PCR systems with ampli- third farm 4 of 8 resident horses were diseased. Forty
fication efficiencies > 95% have an analytic sensitivity clinically affected horses were observed in October
of 10 molecules/assay as previously shown.29,30 2002 and 1 in May 2002. Of these 41 clinically affect-
ed horses, 38 had external abscesses and 3 (7.3%) had
PCR assay inhibition by extracted fly DNA—The both external and internal abscesses. Of 10 skin biop-
potential problem of PCR assay inhibition mediated by sy specimens collected from affected horses, 5 had pos-
components of the flies was addressed in a separate itive results for C pseudotuberculosis on PCR assay.
experiment in which the DNA extracted from the A total of 2,621 single flies or pooled flies (4
C pseudotuberculosis field isolates was used to spike DNA flies/pool), which were collected either by trapping or
extracted from flies testing negative for C pseudotubercu- netting directly from horses over a 6-month period
losis. Whether fly DNA influenced the signal of C pseudo- (May 6, 2002 to November 11, 2002) were analyzed.
tuberculosis DNA was assessed by comparing the 2 sig- Fly species tested include horn flies (Haematobia irri-
nals obtained on C pseudotuberculosis DNA with or with- tans), stable flies (Stomoxys calcitrans), face flies
out DNA obtained from flies (Table 2). Three of the 4 (M autumnalis), house flies (M domestica), biting
samples tested had no inhibition, whereas 1 sample had midges (Culicoides sp), blackflies (Simulium sp), and
a reduction in the signal by 28.5%, indicating low-grade deer flies (Tabanus sp) and a variety of other insects
inhibition of the PCR assay by fly DNA. To address this that typically do not feed on horses, including
problem in the DNA extracted from the flies, the DNA Drosophila sp, Fannia sp, and the green-bottle cal-
was pretested by use of a universal 18S rRNA TaqMan liphorid. The quality of the DNA extracted from flies
PCR systemn for the presence or absence of inhibition. with negative results for the PLD gene was tested by
For those DNA samples with detectable presence of PCR use of a universal 18S TaqMan PCR system and was not
inhibition, a 1:5 dilution in water eliminated the inhibi- significantly different from DNA tested from flies with
tion and was used for CpPld TaqMan PCR testing. positive results for the PLD gene.
Incidence of C pseudotuberculosis in extracted Overall, 64 samples tested positive, all on DNA
DNA—Of the 5 farms studied, 2 farms (with a total of obtained from single flies with an overall incidence of
270 horses) had no horses with C pseudotuberculosis 2.44% (range, 0% to 20%) for C pseudotuberculosis on

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Table 3—Overview of samples with positive real-time TaqMan PCR assay results

Collection Total No. No. positive Species of flies with

Farm No. Date method flies tested (%) positive results (No.)
1 10/17/02 From horses 85 2 (2.4) Haematobia irritans (2)
1 10/17/02 Fly trap 57 0 (0.0) None

2 10/28/03 Fly trap 145 29 (20.0) Musca domestica (22);

Stomoxys calcitrans (7)
2 10/28/03 From horses 17 0 (0.0) None
3 10/28/03 Fly trap 166 32 (19.3) M domestica (31); S calcitrans (1)
2 11/26/03 Fly trap 320 1 (0.3) M domestica
3 11/26/03 Fly trap 35 0 (0.0) None

PCR assay. The first flies with positive results for the al incidence has been reported to follow winters with
PLD gene were detected in mid-October on farm 1, at greater than average rainfall and temperatures.22 Such
which time 2 of 85 flies collected from horses had pos- an increase in incidence might result from enhanced
itive results (Table 3; incidence of 2.4% for C pseudo- breeding, hatching, and survival of various insects sus-
tuberculosis on PCR assay; flies collected from 4 hors- pected of mechanically transmitting the bacterium.8,20,22
es). None of the 57 flies collected at the same time in Pronounced monthly variations in incidence manifests
traps at that farm had positive results for the PLD gene. as a minimum incidence in winter and spring and a
Much higher frequencies of flies with positive results maximum incidence in fall, peaking in October and
for the PLD gene were detected in late October at farms November.23 Although infection occurs throughout the
2 and 3. Of 145 and 166 flies trapped from these year, of 637 affected horses at the University of
respective farms on October 28, 2002, 29 (20.0%) and California–Davis Veterinary Medical Teaching Hospital
32 (19.3%), respectively, had positive results. No flies between 1982 and 1994, 82% of affected horses were
with positive results for the PLD gene were found in reported after June 30.a The disease is most prevalent in
the comparatively modest sample of flies (n = 17) col- young horses (> 1 and < 5 years of age), but horses of
lected from horses on that date. all ages can be affected when the disease is epidemic on
Notably, high frequencies of positive signals from nonendemic farms. No breed or sex predilection has
flies were coincident with high incidence of diseased been found for disease.23 Horses kept in summer pas-
horses on those farms (ie, 23% at farms 2 and 4 out of ture and horses in increased contact with conspecifics
8 [50%] horses at farm 3). One month later, when most had an increased risk of disease in 1 study.a Results of
horses were recovering from disease (incidence of dis- our study indicate that a large variation exists in the
ease decreased from 23% to 4% at farm 2 and from 50% incidence of disease among farms (from no affected
to 25% at farm 3), significantly fewer flies had positive horses to an incidence of 50%), with a predominance
results for the PLD gene (Table 3). A positive correla- of affected horses in the autumn. All 5 farms we stud-
tion was found between the incidence of horses with ied had large numbers of flying insects (eg, M domesti-
disease on the date of sample collection and the per- ca, M autumnalis, and S calcitrans) during the study
centage of flies that tested positive on that date period, and horses at all farms were observed with ven-
(Spearman rank correlation coefficient = 0.61). tral midline dermatitis, indicative of active populations
Fly species with positive results on PCR assay for of horn flies and Culicoides spp. For all farms, the prox-
the PLD gene were M domestica, S calcitrans, and imity to cattle was < 1 mile. Cattle are the principal lar-
H irritans. None of the other hematophagous or live- val host for H irritans and M autumnalis.28
stock-associated fly species had positive results, In our study, the incidence of flies with positive
although their sample numbers were much smaller. results for the PLD gene increased with an increase in
The absence of positive results for any of the species incidence of diseased horses on the farms and was
that are not livestock associated suggests that it is the highest in farms with the most clinically affected hors-
fly-horse interaction itself that led to the presence of es. It is interesting that the incidence of flies with pos-
C pseudotuberculosis in the 3 species of flies with posi- itive results for the PLD gene was highest in October
tive results. when most of the clinically affected horses were first
observed, and then declined by November when most
Discussion horses were recovering or had recovered from disease.
The incidence of C pseudotuberculosis infection in This pattern suggests that flies may be acquiring the
horses varies markedly between years and seasons.7,23,a bacteria though direct contact with the draining
The disease occurs sporadically at endemic farms and abscesses of diseased horses. However, because of the
can result in epidemics of disease in naïve horses.8,31,b small number of farms in our study and limited sample
On the basis of our observations, the prevalence of dis- collection dates, this evaluation is preliminary and
ease on endemic farms is estimated at 5% to 10%.31 additional data are needed to confirm a relationship
Results of an epidemiologic study32 of 29 infected herds between the incidence of disease and incidence of flies
of dairy cattle in Israel revealed a similar prevalence of carrying the bacteria.
5% in sporadically affected herds, with epidemics of It should be mentioned that the finding of the PLD
infection affecting up to 35% of the herd. A high annu- gene in flies does not provide direct evidence that viable

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C pseudotuberculosis organisms are present, although References

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