cultivo vs pcr

8/3/2019 cultivo vs pcr

1/8


2/8

protozoon identification by microscopic examination

[15] have been the most routinely employed diagnostic

test for bovine trichomonosis in bulls [1518]. However,

the sensitivity (Se) of culture for diagnosis ofT. foetus,

although it is considered the gold-standard method,

varied from 84 to 96% under experimental conditions

[17,1921] to low as 70% under field suboptimalconditions [18]. Lack of appropriate specificity (Sp) is

another limitation of culture since Trichomonads,

Tetratrichomonads spp. and Pentatrichomonas hominis,

morphologically similar to T. foetus by light microscopy

were isolated from cultured preputial smegma of bulls

[5,2224]. The AI industry in the USA prescribes for

bovine trichomonosis a rigorous protocol of sixweeklyT.

foetus negative cultures for bulls older than 365 day of

age [13]; although this diagnostic routine hasproven to be

highly effective in controlling disease, the industry is

continually looking for new or improved diagnosticmethods.

We hypothesize that polymerase chain reaction

(PCR)-based diagnosis of bovine trichomonosis may

be at least as sensitive and specific as culture because it

relieson the amplificationof DNA from the organism and

not on the successful culture of the live organism.

Experimentally, PCR detected T. foetus from cultured

isolates, cervico-vaginal mucus, and tissues from female

genitalia [5,20,22,2527]; in vitro, it differentiated T.

foetus from other related trichomonads, such as

Tetratrichomonads spp. [27,28]. However, Se and Spof PCR on smegma taken directly from bulls, where

samples may contain inhibitory factors and DNA may be

easily disrupted, are unknown. The time interval of

smegma samples from the field collection to processing

in the diagnostic laboratory could also affect results of

PCR and culture in the diagnosis of T. foetus. Longer

intervals between sampling and DNA extraction may

decrease the sensitivity of PCR, and improper tempera-

ture storage of samples prior to culture incubation may

kill T. foetus or allow growth of contaminants [17,18].

The objectives of this study were to determine the Se

and Sp of In Pouch TF1 culture and PCR for detectingT. foetus directly from the genital secretions in

experimentally infected bulls and to investigate whether

a 24-h delay affected this diagnosis.

2. Materials and methods

2.1. Strains used for bull inoculums

T. foetus D1 strain, isolated from a cow with

pyometra in a herd outbreak of trichomonosis and

employed to vaginally infect heifers [2931], was used

for experimental T. foetus inoculation of bulls. The D1

strain was grown in Diamonds trypticase yeast extract

maltose media containing 10% fetal calf serum [32] and

incubated at 37 8C. In the development of subcultures

and axenic cultures, penicillin (1000 UI/mL), strepto-

mycin (1 mg/mL) and nystatin (500 UI/mL) were

added.To validate the Sp of the diagnostic tests, some bulls

were inoculated with a Tetratrichomonas spp. strain

isolated from preputial samples of a virgin bull [5] and

cultured at 37 8C in Diamonds tryptycase yeast extract

maltose media containing 10% bovine serum [32].

Simultaneously, some bulls were inoculated with

Campylobacter (C.) fetus venerealis and some with

C. fetus fetus, both occasional inhabitants of the bulls

preputial cavity [33,34]. The C. fetus venerealis strain

was isolated from a C. fetus venerealis- induced

abortion submitted to the California Animal Healthand Food Safety Laboratory; and, the C. fetus fetus

strain was obtained from the American Type Culture

Collection, Manassas, Va. (ATCC 15296). Both C. fetus

venerealis and C. fetus fetus were incubated at 37 8C in

a microaerophilic atmosphere on Skirrow medium

[33,35].

2.2. Inoculation and sampling of bulls

Mature (46-year-old) Holstein or Jersey bulls

(n = 79) negative by T. foetus and C. fetus venerealisculture, were selected and randomly assigned to

treatment groups as follows: 19 bulls were inoculated

with 106 live T. foetus, 13 were inoculated with 107

motile C. fetus venerealis, 11 were inoculated with 106

live T. foetus and 107 motile C. fetus venerealis (dual

infection), 9 were inoculated with 106 live Tetratricho-

monas spp., 8 were inoculated with 107 motile C. fetus

fetus, and 19 were not inoculated. The assignment of

groups determined two populations: bulls exposed to T.

foetus (n = 30) and bulls not exposed to T. foetus

(n = 49). The sample size of 30 or more animals per

group for these two proportions (exposed versus notexposed) with an expected difference of 70% or more

between true infected and negative populations had a

power test of 0.91. The infective doses were based on

previous reports of T. foetus [1,36] and C. fetus

venerealis experimental infections [37,38]. The infec-

tive doses were suspended in 2 mL of phosphate-

buffered saline (PBS) and inoculated into the fornix of

the preputial cavity. Following the infusion, the

preputial orifice was manually held closed while the

operator externally massaged the fornix area for

approximately 1 min. For each bull, genital secretions

E.R. Cobo et al. / Theriogenology 68 (2007) 853860854


3/8

(preputial smegma) were collected weekly from 2 week

before to 6 week post-inoculation. An aliquot was

inoculated into In Pouch TF1 culture media and other

aliquot in PBS for PCR. Samples from 40 of the 49

controls bulls were analyzed by culture and PCR; and,

samples of the remaining nine bulls were arbitrarily

analyzed by PCR only. These nine bulls were alwaysPCR negative. The bulls were inoculated and sampled

by using an insemination/infusion pipette (79 cm

long 0.64 cm o.d. 0.32 cm i.d.; Cassou straws,

IMV, France) covered by a plastic sheath. Inside the

preputial cavity, the pipette was pulled forward through

the sheath to expose the tip and moved back and forward

in short strokes adjacent to the glans penis, especially

near the fornix, while aspirating and massaging the

glans penis to encourage greater amounts of smegma in

to the pipette. A new kit was used for each bull to

prevent cross contamination. All bulls were hygieni-cally sampled by the same qualified operator, and latex

exam gloves were changed between bulls.

2.3. Culture and PCR on preputial samples with

short and long time shipping simulation

Effect of storage of the preputial secretion samples

on the culture and PCR methodology for T. foetus

detection was investigated simulating short (4 h) and

long (24 h) shipping periods. For culture, aliquots of

smegma from 39 of the 79 bulls (seven inoculated withT. foetus, six with C. fetus venerealis, seven with T.

foetus and C. fetus venerealis, six with Tetratrichomo-

nas spp., six with C. fetus fetus, and seven not

inoculated) were immediately inoculated into two

different In Pouch TF1 pouches, which remained at

room temperature (approximately 22 8C) for the short

or long period, before being incubated at 37 8C. For

PCR, aliquots of smegma of the same 39 bulls were

taken into two different containers and immediately

refrigerated at 4 8C for the short or long period, before

DNA extraction.

2.4. Culture and polymerase chain reaction (PCR)

on genital secretion samples

Preputial smegma was tested for T. foetus by culture

utilizing a highly selective medium, In Pouch TF1

(Biomed Diagnostics, San Jose, CA, USA) [19,39].

Samples were incubated at 37 8C and examined on days

1, 3, 5, and 7 after sampling by placing a drop on a glass

slide and observing them at 40100 magnification

using light microscopy [5,19,22,29]. Samples were

considered positive when living trichomonads with size,

shape and a wave-like, rapid and irregular jerky

movement of the protozoan body compatible with T.

foetus were observed [5].

For PCR, DNA was extracted directly from preputial

secretions in PBS using proteinase K digestion (Dneasy

Tissue Kit Qiagen, Germany 69504). Specific primers

(TFR1, TFR2, TFR3 and TFR4) for the 5.8S rRNAgene and the flanking internal transcribed spacer

regions ITS1 and ITS2 of T. foetus were utilized

[28]. Primers TFR1 (TGC TTC AGT TCA GCG GGT

CTT CC) and TFR2 (CGG TAG GTG AAC CTG CCG

TTG G) amplify the rRNA gene derived from

conserved sequences of the 30-end of the 18S subunit

rRNA gene and the 50-end of the 28S subunit rRNA

gene, defining the Trichomonadidae family by yielding

a product from all members of this family of 372-bp

[28]. The TFR3 (CGG GTC TTC CTATAT GAG ACA

GAA CC), complementary to the 50

-end of the 28SrRNA gene; and TFR4 (CCT GCC GTT GGA TCA

GTT TCG TTA A), located at the border of the 18S

rRNA gene and ITS1, specifically target T. foetus,

yielding a product of 347 bp [27]. The PCR reaction

was carried out in a 50 mL final volume with a sample

template volume of 3 mL. The DNA polymerase used

was 2.0 U AmpliTaq Gold, and the final concentrations

of magnesium chloride, dNTPs, and primers were

maintained at 15 mM, 5 mM, and 400 mM, respec-

tively. Amplification was carried out in a Perkin-Elmer

2400 thermocycler with one cycle of 958

C for 10 min,35cycles of 95 8C for 30 s,60 8C for 30 s, and 72 8Cfor

1 min,andafinalextensionstepof72 8C for 7 min[27].

Amplicons were separated by electrophoresis on 1.5%

agarose gel and detected by staining with ethidium

bromide. Extracted DNA from T. foetus and Tricho-

monas vaginalis were used as a positive and negative

control, respectively.

2.5. Statistical analysis

For analysis, the 30 bulls exposed to T. foetus,

including those inoculated only with T. foetus (n = 19)and those inoculated with both T. foetus and C. fetus

venerealis (n = 11) (dual infection), were considered as

the inoculated group. In the same way, the 49 bulls

not exposed to T. foetus, including those bulls

inoculated with other microorganisms (Tetratrichomo-

nas spp. (n = 9), C. fetus fetus (n = 8), only C. fetus

venerealis (n = 13)) and those not inoculated (n = 19)

were considered as the control group. Assuming all

the T. foetus inoculated bulls were potentially

infected, Se was defined as the probability of the

test finding positive results among bulls which were

E.R. Cobo et al. / Theriogenology 68 (2007) 853860 855


4/8

inoculated with T. foetus [Se: true positives/(true

positives + false negatives)]. Furthermore, Sp was

defined as the probability of the test finding no positive

result among bulls which were not inoculated with T.

foetus [Sp: true negatives/(true negatives + false posi-

tives)]. The Se and Sp were calculated at each week

post-inoculation for culture alone, PCR alone, and bothculture and PCR in parallel. The Se and Sp of culture

alone, PCR alone, and both culture and PCR in parallel

on animals sampled in two, three, and six consecutive

weeks were also calculated. Parallel interpretation

schemes (positive if at least one test was positive;

negative only if the sample yielded negative results for

both culture and PCR) were used to maximize

sensitivity of detecting all infected animals [40].

Groups inoculated with T. foetus or not inoculated

were assumed to represent the state of nature.

The effect of storage of the preputial secretionsamples on the PCR and culture methodology for T.

foetus detection was analyzed using Kappa statistics

agreement between results from the 4 and 24 h

simulated shipping time periods.

3. Results

3.1. Culture

For culture, 26 of 30 T. foetus inoculated bulls

(86.7%) yielded at least one culture positive resultduring the 6-week period, whereas four bulls were

negative throughout the study. From 180 smegma

samples of T. foetus inoculated bulls, 122 (67.7%)

yielded culture positive results.

3.2. PCR

For PCR, 27 of 30 T. foetus inoculated bulls (90.0%)

yielded at least one PCR positive result during the 6-week period, whereas three bulls were negative

throughout the study. From 180 smegma samples of

T. foetus inoculated bulls, 119 (66.1%) yielded PCR

positive results.

3.3. In parallel tests

Considering both tests in parallel in the same sample,

28 of 30 T. foetus inoculated bulls (93.3%) yielded at

least one culture and/or PCR positive result during the

6-week period, whereas two bulls were negative forboth methods throughout the study. From 180 smegma

samples of T. foetus inoculated bulls, 141 (78.3%)

yielded culture and/or PCR positive results. Thus, the

estimated proportion of bulls inoculated with T. foetus

that became infected (i.e. positive to at least once

culture and/or PCR) was similar for culture (86.7%),

PCR (90.0%), and both culture and PCR (93.3%). The

proportion of bulls infected (i.e. positive to culture

and/or PCR) inoculated with T. foetus alone or in

combination with C. fetus venerealis (dual infection)

was 89.5% (17 of 19 bulls T. foetus infected) and100.0% (11 of 11 bulls T. foetus infected), respectively

(P = 0.14).


Table 1

Sensitivity (Se) and specificity (Sp) and the 95% precision (margin of error, MOE; expressed as %) for T. foetus culture, PCR, and both (parallel)

methods, on smegma samples from bulls inoculated or not inoculated with T. foetus weekly sampled for 6 week post-inoculation

Week Group Culture PCR Both (parallel)

Pos Neg Se MOE Sp MOE Pos Neg Se MOE Sp MOE Pos Neg Se MOE Sp MOE

1 Inoc 20 10 12 18 22 8

Cont 1 39 67 17 100 5 0 49 40 18 100 0 1 39 73 16 98 5

2 Inoc 20 10 19 10 23 7

Cont 1 39 67 17 100 5 1 47 66 17 98 4 2 38 77 15 95 7

3 Inoc 19 11 21 9 25 5

Cont 1 39 63 17 100 5 1 48 70 16 98 4 1 39 83 13 98 5

4 Inoc 21 9 21 9 23 7

Cont 0 40 70 16 100 0 1 48 70 16 98 4 0 40 77 15 100 0

5 Inoc 22 8 22 8 23 7

Cont 0 40 73 16 100 0 1 48 73 16 98 4 1 39 77 15 98 5

6 Inoc 20 10 23 7 25 5

Cont 0 40 67 17 100 0 1 48 77 15 98 4 0 40 83 13 100 0

Av Inoc 20.3 9.7 19.7 10.2 23.5 6.5

Cont 0.5 3 9.5 68 17 100 2 0.8 48 66 16 98 3 0.8 39.2 78 15 98 4


5/8

3.4. Testing on samples taken on consecutive weeks

When analyzing only one diagnostic test (culture or

PCR) and the combination of both tests on the same

sample (in parallel) per week in a 6-week post-

inoculation period, the average Se for detecting T. foetus

infected bulls was: for culture, 67.8%; for PCR, 65.9%;and for the combination of both, 78.3% (Tables 1 and 2).

For the 6-week period, the Se of culture and/or PCR

were not significantly different by weeks, with the

exception of PCR alone at week 1 where the Se was

particularly low (40%; Table 1). The average Sp for

culture was 99%, for PCR 98%, and for both 98%, with

no significant differences among weeks (Table 1).

The current standard diagnosis of six weekly

consecutive cultures had a Se of 86.7% and a Sp of

97.5% (Table 2). The culture results interpreting two

consecutive weeks together had a Se of 76.0% and Sp of98.5%; and PCR interpreting two consecutive weeks had

a Se of 78.0% and Sp of 96.7% (Table 2). Interpreting

three consecutive weeks, the culture results had a Se of

80.0% and Sp of 98.1%; and, PCR a Se of 85.0% and Sp

of 95.4%. Higher Se and Sp were observed when the

combination of both culture and PCR on the same sample

was interpreted for three consecutive weeks (Se 87.5%

and Sp 95.6%) and six consecutive weeks (Se 93.3% and

Sp 92.5%)(Table 2).Regarding the Sp of themethods, for

culture, one control bull (inoculated with Tetratricho-

monas spp.) and, for PCR, four control bulls (threeinoculated with C. fetus venerealis and one with C. fetus

fetus) yielded positive results for T. foetus at a single or

few intermittent weeks.

3.5. Storage effects

Smegma samples stored 4 and 24 h yielded almost

the same culture result, since only one sample

disagreed, being positive for T. foetus culture at 4 h

but negative at 24 h. Likewise, agreement of PCR on

smegma samples processed at 4 and 24 h afterextraction had no discrepancy (Kappa 1 0.2) as 49

samples were positive and 203 negative to PCR at both 4

and 24 h.

4. Discussion

We investigated alternative strategies for T. foetus

diagnoses by culture or PCR and the parallel

interpretation of the test results of several consecutive

weeks. A single culture or PCR seemed to be equally

sensitive detecting T. foetus (Se of 67.8 and 65.9%,respectively). Also, culture and PCR had similar Se

when analyzing combinations of two (76.0 and 78.0%,

respectively), three (80.0 and 85.0%, respectively), and

six (86.7 and 90.0%, respectively) consecutive weeks.

Sp was greater than 90% for all these testing schemes.

The similar performance of culture and PCR agrees

with in vitro studies where PCR made from 5-day

cultures of male genital secretions agreed 92.9% with

culture [18]. The current gold-standard of six weekly

cultures for diagnosing T. foetus in bulls yielded a Se of

86.7% and Sp of 97.5%. However, the use of PCR forthree consecutive weeks (Se 85.0%, Sp 95.4%) and both

culture and PCR on the same sample for three

consecutive weeks (Se 87.5%, Sp 95.6%) appeared to

be similar to this standard of six weekly cultures.

Culture and PCR tests in parallel on a single sample had

a Se (78.3%) and Sp (98.5%) similar to culture (Se

76.0%, Sp 98.5%) or PCR (Se 78.0%, Sp 96.7%)

applied on two consecutive weeks. As expected, the use

of both methods together for six consecutive weeks had

the highest Se (93.3%) and high Sp (92.5%). These

alternative strategies for T. foetus diagnostics utilizing

culture and PCR may require less time and perhaps lesscost in the surveillance of AI bulls and beef bulls prior to

the breeding season. Combinations of tests to classify

individuals as positive or negative have been used in

many diagnostic, health-certification, disease-surveil-

lance and eradication programs for livestock disease

[40]. Moreover, knowing the Se and Sp of a diagnostic

test (In Pouch TF1 and PCR) the positive and the

negative predictive values can be calculated, but these

values will be influenced by the prevalence of the

disease (bovine trichomonosis) in a specific geographic

area. However, results for tests that measure similar


Table 2

Sensitivity (Se) and specificity (Sp) and the 95% confidence intervals

for T. foetus culture, PCR, and both methods applied alone or

combined for 2, 3, or 6 week

Test(s) Week Se (%) 95% CI Sp (%) 95% CI

Cult 1 67.8 51.1; 84.1 98.8 96.3; 101.2

PCR 1 65.9 49.5; 81.8 98.3 95.0; 101.6

Both 1 78.3 63.7; 93.0 98.5 95.6; 101.4Cult 2 76.0 60.8; 91.3 98.5 95.6; 101.4

PCR 2 78.0 64.0; 92.0 96.7 91.6; 101.9

Both 2 83.3 70.2; 96.5 96.5 90.9; 102.1

Cult 3 80.0 65.8; 94.2 98.1 94.5; 101.8

PCR 3 85.0 72.3; 97.7 95.4 89.6; 101.2

Both 3 87.5 75.9; 99.1 95.6 9.4; 101.9

Cult 6 86.7 74.5; 98.8 97.5 92.7; 102.3

PCR 6 90.0 79.3; 100.7 91.8 84.2; 99.5

Both 6 93.3 84.4; 102.3 92.5 84.3; 100.7

Bulls considered positive had to have a culture and/or PCR positive

results on their smegma samples at least once during the 6-week

period. Bulls considered negative had to have culture and PCR

negative results on all of their samples.


6/8

biological targets (in this case identification of T.

foetus by culture and identification of fragments of

DNA by PCR) may be similarly affected by same

circumstances, such as low organism concentration or

improper sample storage. Thus, combination of tests

that measure different biological processes, such as

detection of the agent by culture/PCR and antibodyresponse, are recommended although preputial and

systemic humoral immune response appears not to be

associated with natural T. foetus infection in bulls

[10,41].

The proportion of bulls inoculated with T. foetus and

infected (i.e. positive to culture and/or PCR at least

once) was high (>85%) in agreement with previous

literature [1,42]. However, some infected bulls yielded

sporadic negative culture or PCR results due likely to

the insensitivity of the method, presence of inhibitors,

variation of the sampling, or fluctuation of the protozoalpopulation in the preputial cavity [16,18]. Conversely,

we were unable to demonstrate infection by either

method at any time in two of 30 T. foetus-inoculated

bulls (6.7%) for the 6-week post-inoculation period,

although they were inoculated with infective doses.

This individual natural resistance to the disease,

reported previously [1,36], could be determined by

factors such as host genetic variations or specific

preputial inhabiting flora, that need to be defined. From

the similar T. foetus infection rate between groups

inoculated alone with T. foetus and in combination withC. fetus venerealis, we inferred that the presence ofC.

fetus venerealis did not alter the colonizing properties of

T. foetus.

We observed that In Pouch TF1 culture had a Se of

67.8% identifying positive samples from T. foetus

inoculated bulls. Similarly, in other work, we have

reported that culture yielded a Se of 72.4% detecting

bulls naturally exposed to T. foetus [7]. However, on

samples from bulls chronically and persistently infected

with T. foetus, In Pouch TF1 (95.8%) [19] and Diamond

(81.6% to 93.2%) [39] culture yielded an apparently

better sensitivity detecting T. foetus than we reportedhere. The lower Se reported in this study may be

attributed to the fact that all T. foetus inoculated bulls

(and their samples) were considered potentially

infected for the purpose of providing a gold standard

for the calculation of the Se and Sp, although we were

unable to demonstrate infection in two bulls by any

means. Conversely, this lower sensitivity may reflect

limitations that could happen while testing bulls under

natural conditions, where some bulls become easily

infected and harbor large quantities of protozoa,

whereas other bulls may be naturally resistant to

the disease or maintain the infection at low levels. The

present study encourages the use of In Pouch TF1 for

bovine trichomonosis diagnosis in bulls because, as

discussed above, it identified T. foetus infected bulls,

especially when applied repeated times, and because it

was comparable to other reliable methods such as PCR.

Conversely, In Pouch TF1 was insensitive for othertrichomonads since only Tetratrichomonas spp. was

capable of only temporary growth in smegma samples

of one bull experimentally inoculated with Tetratricho-

monas spp.

The PCR method we used successfully detected T.

foetus when applied directly to smegma samples of

bulls experimentally inoculated with T. foetus. In

agreement, Felleisen TFR3/4 primers were capable

of detecting T. foetus on: several isolations from

outbreaks; on vaginal mucus of heifers; on formalin-

fixed paraffin-embedded infected endometrial tissues;and, on infected aborted bovine fetuses [26]. The in

vitro biological sensitivity of TFR3/4 was approxi-

mately 90% and the specificity of 98% [18], detecting as

little as 0.03 pg of purified T. foetus DNA [27] or

approximately two organisms per milliliter of sheath-

wash samples under laboratory conditions [18]. Thus,

we confirmed the Felleisen PCR as a sensitive method

to detect T. foetus in genital samples taken directly from

bulls, and we reaffirmed its use as a diagnostic method

in cattle production. Conversely, the finding of sporadic

false negative results in some infected bulls indicatedPCR is still an imperfect method. Degradation of DNA,

due to poor specimen preparation, inadequate sampling,

transportation [43], and presence of blood [18],

DNAase or natural flora in bovine preputial smegma,

could affect those primers with larger target sequences.

Our interpretation is that a low Se of PCR alone in

preputial samples at week 1 may have occurred because

the T. foetus primers that we used in field-collected

samples apparently did not detect small numbers of

organisms or organisms that do not propagate as

expected [27]. This inability of PCR to accurately detect

T. foetus early after inoculation may be overcome by theculture that allows organisms to multiply by incubation.

We inferred that PCR had a high specificity and

accurately distinguished T. foetus from other tricho-

monads, since Tetratrichomonas spp. were difficult to

detect even when we knew exactly which bull was

inoculated and when. The sporadic finding of PCR-

positive results in non-T. foetus inoculated bulls may

indicate that occasionally trichomonads with some

grade of DNA homology with T. foetus could normally

inhabit the preputial cavity. Alternatively, these bulls

could have arrived already T. foetus infected. Though



7/8

errors in our sampling and/or PCR methodology are

possible, they were not reported. In vitro studies

validated the specificity of primers TFR3/4 for

differentiating phylogenetically closely related organ-

isms because no amplification was observed with

genomic DNAs of T. vaginalis, Trichomonas gallinae,

Tetratrichomonas gallinarum, Trichomonas tenax, andP. hominis as well as genomic DNA of bacteria and

bovine cells [27,28,44]. Other ways to distinguish

different trichomonads, such as fixation of smears and

staining [45] or electron microscopy [22,24], may

require further handling and be more expensive. Lastly,

the agreement between culture and PCR in samples

stored up to 24 h indicated that these methods could be

efficiently applied to smegma samples properly stored.

Specifically, smegma samples that are commonly

shipped by overnight bus or couriers should be kept

at room temperature for culture and at 48C for PCR.

Especially important for PCR is that maintaining

samples at 4 8C may limit proliferation of T. foetus

and other organisms from the preputial cavity, which

secrete hydrolytic enzymes that cause rapid DNA

breakdown [46].

5. Conclusion

If sample quality/quantity is adequate and the sample

is handled appropriately, the PCR and culture may offer

similar Se and Sp for detecting T. foetus in smegmafrom infected bulls. Moreover, PCR or both tests

applied in parallel on smegma samples sampled on three

consecutive weeks may be as sensitive and specific as

the current gold-standard of six weekly cultures.

Acknowledgments

We thank the National Association of Animal

Breeders (NAAB) and Certified Semen Services, Inc.,

for their support and the AI companies, ABS-Global,

Accelerated Genetics, Alta-Genetics USA, Andro-

genics/Jerseyland Sires, Genex Cooperative, and SelectSires that provided the bulls for the research.

References

[1] Parsonson IM, Clark BL, Dufty J. The pathogenesis of Tritri-

chomonas foetus infection in the bull. Aust Vet J 1974;50:4213.

[2] Honigberg BM. Trichomonads of veterinary importance. Para-

sitic protozoa, vol. 2. New York: Academic Press; 1978. p. 163

273.

[3] Parsonson IM, Clark BL, Dufty JH. Early pathogenesis and

pathology ofTritrichomonas foetus infection in virgin heifers. J

Comp Pathol 1976;86:5966.

[4] Skirrow SZ, BonDurant RH. Immunoglobulin isotype of specific

antibodies in reproductive tract secretions and sera in Tritricho-

monas foetus-infected heifers. Am J Vet Res 1990;51:64553.

[5] BonDurant RH, Gajadhar A, Campero CM, Johnson E, Lun Z-R,

Nordhausen RW, et al. Preliminary characterization of a Tri-

trichomonas foetus-like protozoan isolated from preputial

smegma of virgin bulls. Bovine Pract 1999;33:1247.

[6] Rae DO, Chenoweth PJ, Genho PC, McIntosh AD, Crosby CE,Moore SA. Prevalence of Tritrichomonas foetus in a bull popu-

lation and effect on production in a large cow-calf enterprise. J

Am Vet Med Assoc 1999;214:10515.

[7] Perez A, Cobo E, Martinez A, Campero C, Spath E. Bayesian

estimation of Tritrichomonas foetus diagnostic test sensitivity

and specificity in range beef bulls. Vet Parasitol 2006;142:159

62.

[8] Guest G. Extra-label update. FDA Vet 1988;1:56.

[9] Clark BL, Dufty JH, Parsonson IM. Immunisation of bulls

against trichomoniasis. Aust Vet J 1983;60:1789.

[10] Campero CM, Hirst RG, Ladds PW, Vaughan JA, Emery DL,

Watson DL. Measurement of antibody in serum and genital

fluids of bulls by ELISA after vaccination and challenge with

Tritrichomonas foetus. Aust Vet J 1990;67:1758.

[11] Ball L, Dargatz DA, Cheney JM, Mortimer RG. Control of

venereal disease in infected herds. Vet Clin North Am Food

Anim Pract 1987;3:56174.

[12] Herrick J. Trichomonosis control. Large Anim Vet 1989;44:11.

[13] CSS. Minimum requirements for disease control of semen

produced for artificial insemination. Columbia, MO, US: Certi-

fied Semen Services (CSS). National Association Animal Bree-

ders (NAAB); 2003.

[14] Borchardt KNB, Thomas M, Harmon W. Evaluation of a new

culture method for diagnosing Tritrichomonas foetus infection.

Vet Med 1992;(February):10412.

[15] Levine N. The trichomonads. Minneapolis, MN: Burgess Pub-

lishing Co.; 1961. p. 82106.[16] Parker S, Campbell J, Ribble C, Gajadhar A. Comparison of two

sampling tools for diagnosis of Tritrichomonas foetus in bulls

andclinicalinterpretation of culture results. J Am Vet MedAssoc

1999;215:2315.

[17] Parker S, Campbell J, Ribble C, Gajadhar A. Sample collection

factors affect the sensitivity of the diagnostic test for Tritricho-

monas foetus in bulls. Can J Vet Res 2003;67:13841.

[18] Mukhufhi N, Irons PC, Michel A, Peta F. Evaluation of a PCR

test for the diagnosis ofTritrichomonas foetus infection in bulls:

effects of sample collection method, storage and transport

medium on the test. Theriogenology 2003;60:126978.

[19] Parker S, Campbell J, Gajadhar A. Comparison of the diagnostic

sensitivity of a commercially available culture kit and a diag-

nostic culture test using Diamonds media for diagnosing Tri-trichomonas foetus in bulls. J Vet Diagn Invest 2003;15:4605.

[20] Hoevers J, Snowden K, Graham S, Barling K. A comparison of

PCRand in vitro culture for detectionofTritrichomonas foetus in

bovine preputial scrapings. J Eukaryot Microbiol 2003;50(Suppl.):

699700.

[21] Kimsey PB, Darien BJ, Kendrick JW, Franti CE. Bovine tricho-

moniasis: diagnosis and treatment. J Am Vet Med Assoc

1980;177:6169.

[22] Campero CM,Rodriguez Dubra C, Bolondi A, Cacciato C, Cobo

E, Perez S, et al. Two-step (culture and PCR) diagnostic

approach for differentiation of non-T. foetus trichomonads from

genitalia of virgin beef bulls in Argentina. Vet Parasitol

2003;112:16775.



8/8

[23] Parker S, Campbell J, McIntosh K, Gajadhar A. Diagnosis of

trichomoniasis in virgin bulls by culture and polymerase chain

reaction. Can Vet J 2003;44:7324.

[24] Cobo ER, Campero CM, Mariante RM, Benchimol M. Ultra-

structural study of a tetratrichomonad species isolated from

prepucial smegma of virgin bulls. Vet Parasitol 2003;117:

195211.

[25] Parker S, Lun ZR, Gajadhar A. Application of a PCR assay toenhance the detection and identification ofTritrichomonas foetus

in cultured preputial samples. J Vet Diagn Invest 2001;13:

50813.

[26] BonDurant RH, Campero CM, Anderson ML, Van Hoosear KA.

Detection ofTritrichomonas foetus by polymerase chain reaction

in cultured isolates, cervicovaginal mucus, and formalin-fixed

tissues from infected heifers and fetuses. J Vet Diagn Invest

2003;15:57984.

[27] Felleisen RS, Lambelet N, Bachmann P, Nicolet J, Muller N,

Gottstein B. Detection of Tritrichomonas foetus by PCR and

DNA enzyme immunoassay based on rRNA gene unit

sequences. J Clin Microbiol 1998;36:5139.

[28] Felleisen RS. Comparative sequence analysis of 5.8S rRNA

genes and internal transcribed spacer (ITS) regions of tricho-

monadid protozoa. Parasitology 1997;115(Pt 2):1119.

[29] BonDurant RH, Corbeil RR, Corbeil LB. Immunization of virgin

cows with surface antigen TF1.17 of Tritrichomonas foetus.

Infect Immun 1993;61:138594.

[30] Corbeil LB, Anderson ML, Corbeil RR, Eddow JM, BonDurant

RH. Female reproductive tract immunity in bovine trichomo-

niasis. Am J Reprod Immunol 1998;39:18998.

[31] Anderson ML, BonDurant RH, Corbeil RR, Corbeil LB.

Immune and inflammatory responses to reproductive tract infec-

tion with Tritrichomonas foetus in immunized and control

heifers. J Parasitol 1996;82:594600.

[32] Diamond L. Lumen dwelling protozoa: entamoeba, trichomo-

nads, and giardia. Boca Raton, FL: CRC Press; 1983. p. 8999.[33] Clark BL,Dufty JH.Isolation ofCampylobacter fetus from bulls.

Aust Vet J 1978;54:2623.

[34] Eaglesome M, Garcia M. Microbial agents associated with

bovine genital tract infections and semen. Part I. Brucella

abortus, Leptospira, Campylobacter fetus and Tritrichomonas

foetus. Vet Bull 1992;62:74375.

[35] Luechtefeld NW, Wang WL, Blaser MJ, Reller LB. Evaluation

of transport and storage techniques for isolation of Campylo-

bacter fetus subsp. jejuni from turkey cecal specimens. J Clin

Microbiol 1981;13:43843.

[36] Clark BL, Parsonson IM, Dufty JH. Experimental infection of

bulls with Tritrichomonas foetus. Aust Vet J 1974;50:18991.

[37] Vasquez LA, Ball L, Bennett BW, Rupp GP, Ellis R, Olson JD,

et al. Bovine genital campylobacteriosis (vibriosis): vaccinationof experimentally infected bulls. Am J Vet Res 1983;44:15537.

[38] Clark BL, Dufty JH, Monsbourgh MJ, Parsonson IM. Immuni-

sation against bovine vibriosis. Vaccination of bulls against

infection with Campylobacter fetus subsp. venerealis. Aust

Vet J 1974;50:4079.

[39] Schonmann MJ, BonDurant RH, Gardner IA, Van Hoosear K,

Baltzer W, Kachulis C. Comparison of sampling and culture

methods for the diagnosis of Tritrichomonas foetus infection in

bulls. Vet Rec 1994;134:6202.

[40] Gardner IA, Stryhn H, Lind P, Collins MT. Conditional depen-

dence between tests affects the diagnosis and surveillance of

animal diseases. Prev Vet Med 2000;45:10722.

[41] Rhyan JC, Wilson KL, Wagner B, Anderson ML, BonDurant

RH, Burgess DE, et al. Demonstration of Tritrichomonas foetus

in the external genitalia and of specific antibodies in preputial

secretions of naturally infected bulls. Vet Pathol 1999;36:406

11.

[42] Clark BL, Parsonson IM, Dufty JH. Infection of bulls with

Tritrichomonas foetus through mating with infected heifers.

Aust Vet J 1974;50:180.

[43] Crucitti T, Van Dyck E, Tehe A, Abdellati S, Vuylsteke B, Buve

A, et al. Comparison of culture and different PCR assays for

detection of Trichomonas vaginalis in self collected vaginal

swab specimens. Sex Transm Infect 2003;79:3938.

[44] Felleisen RS. Comparative genetic analysis of tritrichomonadid

protozoa by the random amplified polymorphic DNA technique.

Parasitol Res 1998;84:1536.[45] Lun ZR, Gajadhar AA. A simple and rapid method for staining

Tritrichomonas foetus and Trichomonas vaginalis. J Vet Diagn

Invest 1999;11:4714.

[46] Wang AL, Wang CC. Isolation and characterization of DNA

from Tritrichomonas foetus and Trichomonas vaginalis. Mol

Biochem Parasitol 1985;14:32335.


Documents

cultivo vs pcr