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Mexico Perspective: Prevalence of Tritrichomonas foetus in Breeding Bulls from the State of Chihuahua, Mexico - A Real Field Working Experience Ivan Leyva-Baca DVM., MSc., PhD. Animal Health Product Applications Life Sciences Solutions Austin, Texas, USA

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• History: Bovine trichomoniasis first reported as a cause of reproductive failure in cattle by Kunstler in

france1888. The first report of trichomoniasis in The US in the State of Pennsylvania was in 1932

reported by Emmerson and by 1950s it was well established in US Beef Herds

• Prevalence: All prevalence has been mostly estimated with one sampling and culture/microscopy that

varies from 0.18-6% in certain states in the USA. However, an accurate determination of current TF

prevalence in the US has not been reported

• Prevalence in Mexico is not known

• Economic Importance: Using TF prevalence data from a survey of bulls passing through an Oklahoma

auction market estimated reproductive loss in virgin heifers breed to produce their first calf concluded the

cost of Oklahoma cattle industry was from 7.4-20.8 million per year (Williamson, 2009)

Background: Bovine Trichomoniasis History and Distribution

https://commons.wikimedia.org/wiki/File%3AAngus-bull-van-buren-tn1.jpg

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Background: Tritrichomonas foetus Etiology

• Etiology: Tritrichomonas foetus is a spindle to pear-shaped single-celled protozoa with

three anterior flagella, an undulating membrane along the length of its body containing an

accessory filament at its margin, and a single posterior flagellum.

https://commons.wikimedia.org/wiki/File%3ATritrichomonas_foetus.jpg

https://commons.wikimedia.org/wiki/File%3ATritrichomonas_foetus_(259_26)_Cultured.jpg

Vilela RC, Benchimol M. IL-10 release by bovine epithelial cells cultured withTrichomonas

vaginalis and Tritrichomonas foetus . Memórias do Instituto Oswaldo Cruz. 2013;108(1):110-112.

doi:10.1590/S0074-02762013000100018. http://openi.nlm.nih.gov/

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Background: Tritrichomonas foetus Genome

•T. foetus genome is divided in five haploid chromosomes

• Genome size ~160 MB

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Transmission: is mainly venereal

Clinical Signs at Individual Level Bulls: • Bulls don’t exhibit any pathologic lesions

• The immune response is very limited

• Due to the lack of clinical signs the infected bulls tend to

remain actively breeding other cows in untested herds

Cows: • Pyometras (Usually at day 50 post infection) with

prolonged infections

• Purulent debris in the uterine lumen

• Early embryonic death

• Fetal maceration detected by palpation

• Abortion (usually within the 5th month of gestation)

• Temporary infertility (2-6 months)

• Early return to estrus (Most common clinical sign of T.

foetus infection in cows)

Clinical Signs at Herd Level:

• Clinical signs on a herd basis are the

culmination of clinical signs exhibited by

individuals within the herd associated with

the parasite’s impact on female

reproductive efficiency through increased

numbers of non-pregnant cows,

pyometras, abortions, and cows pregnant

but with a later than normal expected

calving date

• The early return to estrus is the main

clinical sign of trich infection during the

breeding period

• Low Calving Rates (30-60%)

• Longer Calving intervals (≤100 days than

non-infected herds)

Background: Transmission and Clinical signs

“This image provided courtesy of Wikimedia Commons & United States Department of Agriculture https://commons.wikimedia.org/wiki/File:Angus_cattle_12.jpg”

https://commons.wikimedia.

org/wiki/File%3AAufreitende

r_Stier_2011.JPG

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• Natural service for breeding

• Extensive range management

• No defined breeding season

• Shared Bulls (Ejidos)

• Ranch/Farm located right next to infected herds

• Commingling of cattle

• Lack of Trich testing

• Low Sensitivity & Specificity Diagnostic techniques (False Negatives, because The

sensitivity of the test is affected by the field conditions (i.e. low number of organisms collected when sampled) handling

conditions (i.e. temperature and transit time to laboratory). Fortunately, the sensitivity has increased with the universal

acceptance of the DNA – based PCR test)

• Not properly testing the entire battery of bulls (Three consecutive testing

of negative in an infected herd must be mandatory)

• Implementing AI from untested bulls (non-reputable source)

• Iatrogenic transfer with improperly sanitized vaginal speculum (very rare)

Background: Risk factors

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• Treatment & Vaccination: There are no legally available drugs or vaccine

to treat affected cattle that are 100% effective

• Control: Given that bulls are persistently infected, the cows are transiently infected and its transmission

is strictly venereal, these factors offer the possibility to manage the disease effectively by

focusing on the identification and removal of infected bulls.

• Diagnostics: is the best tool as one of the pieces for control and eradication of trich from infected herds:

• Surveillance

• Prevalence of the disease in the zone, evaluation of risk factors and identification of undesired events in the ranch will indicate is tested is

required for the entire battery of bulls right after or before breeding season (Pooling is always an option)

• Elimination form infected herds

• Test all bulls 3 times with an interval of two weeks and cull all positive bulls

• Cull all bulls (usually too expensive and depends on the market value at the time)

• Cull all unproductive cows that did not generate a calf at the end od the breeding season (open cows and cows that did not give any calves)

• Allow for at least three estrus cycles for the infected cows to clear out the disease (However, some reports indicate that more cycles are required)

• Use Artificial examination and proven negative bulls to avoid reintroduction of trich into the herd

• Prevention

• Biosecurity (Proper fencing)

• Herd records for reproductive performance

• Replacements (bring only tested negative bulls, virgin bulls and heifers) from a reputable ranch

• Reduce the bull battery to minimize the infection rate

• Surveillance strategy based on risk factors

Management and Control of Bovine Trichomoniasis

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*González R.E1., Ramírez-Godinez.J.A1., Leal T.B.A1., Lastra G.C1., Ordoñez B.P.L1., Álvarez G.J.A1., Santellano E.E1., Esparza V.M.E1., Baxter J2., Leyva-Baca. I. 2

1Facultad de Zootecnia y Ecología. Universidad Autónoma de Chihuahua. 2Animal Health Group at Thermo Fisher Scientific, Austin, TX

The Situation:

In Mexico and the State of Chihuahua the

average calving rate is 55% despite the

proper management strategies such as:

• Usage of high quality vaccines against

abortive diseases

• Improvement of genetics in their herds

• Breeding soundness examinations of bulls

• Cow palpations

• Proper nutrition, mineral and vitamin

supplementations prior breeding season

in some of the ranches

Prevalence of Tritrichomonas foetus in beef bulls in the State of Chihuahua, Mexico Tested with MagMAX™ Sample Preparation System and VetMAXTM-Gold Trich Detection Kit

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Sample size to estimate true prevalence

Inputs

Assumed true prevalence 0.25

Sensitivity 0.98

Specificity 0.98

Population size 60000

Confidence 0.95

Desired precision 0.05

Prevalence of Tritrichomonas foetus in beef bulls in the State of Chihuahua, Mexico Tested with MagMAX™ Sample Preparation System and VetMAXTM-Gold Trich Detection Kit

Sample sizes for varying sensitivity and specificity for population = 60000 Samples sizes required for true prevalence = 0.25, precision = 0.05 and a range of sensitivity and specificity values are shown below:

Se = 0.7 Se = 0.8 Se = 0.9 Se = 0.95 Se = 0.99 Se = 0.999

Sp = 0.7 2220 1467 1039 892 795 775

Sp = 0.8 1320 957 727 643 585 573

Sp = 0.9 791 620 501 454 422 415

Sp = 0.95 603 491 410 377 353 348

Sp = 0.99 479 403 346 321 303 300

Sp = 0.999 453 385 332 309 293 289

Humphry RW, Cameron A, Gunn GJ, 2004. A practical approach to calculate sample size for herd

prevalence surveys. Prev. Vet. Med. 65: 173-188

Sample size to estimate true prevalence based on sensitivity

and specificity of the assay performed:

Results

Sample size required

Sample size

Population = 60000 320

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Prevalence of Tritrichomonas foetus in beef bulls in the State of Chihuahua, Mexico Tested with MagMAX™ Sample Preparation System and VetMAXTM-Gold Trich Detection Kit

• Diagnostics time is reduced to just 24 hours of incubation

time and 2 hours of the sample preparation and real-time

PCR

• Sensitivity and Specificity is dramatically improved

(100/99.4%), respectively

MagMAX Sample preparation system

37ºC x 24h

7500 Fast real-time PCR

TrichIT (Morris Livestock

Products, Delavan, WI) &

InPouch (Biomed)

Incubation

Day 1: Sample Collection & Culture Day 2: Nucleic Acid Extraction and Real-time PCR

Materials and Methods:

In years 2014, 2015 & 2016 (n=880) Bulls were sampled and tested as follows:

If just culture is

implemented, 7 days are

required to reach 60-95%

sensitivity and specificity… https://commons.wikimedia.

org/wiki/File%3ATritrichomo

nas_foetus_(259_26)_Cultu

red.jpg

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Different methodologies of real-time PCR

Objectives:

1. Compare different sample preparation systems and various real-time PCR (feeder lab workflows) with

the 5X MagMAXTM-pathogen RNA/DNA purification kit and amplification with VetMAXTM T. foetus

reagents (Life Technologies workflow)

2. Determine the effect of pooling a single positive sample having various CT ranges with four negative

samples (1:5). If a negative effect was seen, a 1:3 pooling study would then be conducted

Pooling of cultured samples and comparison of multistate laboratory workflows with the MagMAX sample preparation system and VetMAX quantitative polymerase chain reaction reagents for detection of Tritrichomonas foetus–colonized bulls Lee Effinger, Lalitha Peddireddi, Marilyn Simunich, Richard Oberst, Catherine O’Connell & Ivan Leyva-Baca

Oregon Department of Agriculture, Animal Health and Identification Division, Animal Health Laboratory, Salem, OR (Effinger)

Department of Diagnostic Medicine/Pathobiology (Peddireddi), Kansas State University, Manhattan, KS Kansas State

Veterinary Diagnostic Laboratory (Oberst), Kansas State University, Manhattan, KS Animal Health Laboratory, Idaho State

Department of Agriculture, Boise, ID (Simunich) Animal Health and Food Safety Group at Life Technologies, Austin, TX

(Leyva-Baca, O’Connell) JVDI, 2014, Vol. 26(1) 72-87

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Prevalence of Tritrichomonas foetus in beef bulls in the State of Chihuahua, Mexico Tested with MagMAX™ Sample Preparation System and VetMAXTM-Gold Trich Detection Kit

Brangus

Angus

Hererford

Charolais

Salers

Criollo

Brahaman

Beef Master

Pardo Suizo

Simmental

Limousin

Prevalence results: 450 total samples

Razas Total Infectados %

Brangus 183 48 26.23

Angus 24 4 16.67

Hereford 74 8 10.81

Charolais 104 33 31.73

Salers 19 3 15.79

Criollo 32 12 37.5

Brahman 1 0 0

Beefmaster 5 1 20

Pardo Suizo 1 0 0

Simmental 1 1 100

Limousin 6 3 50

TOTAL 450 113

26.23% (48)

16.67% (4) 10.81% (8)

31.73% (33)

15.79% (3)

37.5% (12)

183

24 74

104

19 32

25% prevalence

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• Age as risk factor for BovineTrichomoniasis:

Prevalence of Tritrichomonas foetus in beef bulls in the State of Chihuahua, Mexico Tested with MagMAX™ Sample Preparation System and VetMAXTM-Gold Trich Detection Kit

• One hypothesis for the age to be associated with trich is the development

of crypts in the prepuce

• The other hypothesis is the promiscuity of older bulls (older bulls are

more exposed to more cows vs younger inexperienced bulls)

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• Distribution of Sampling and Positive Bulls

Prevalence of Tritrichomonas foetus in beef bulls in the State of Chihuahua, Mexico Tested with MagMAX™ Sample Preparation System and VetMAXTM-Gold Trich Detection Kit

001 Ahumada

002 Aldama

003 Allende

004 Aquiles Serdán

005 Ascención

006 Bachíniva

007 Balleza

008 Batopilas

009 Bocoyna

010 Buenaventura

011 Camargo

012 Carichí

013 Casas Grandes

014 Coronado

015 Coyame del Sotol

016 La cruz

017 Cuahutémoc

018 Cusihuiriachi

019 Chihuahua

020 Chínipa

021 Delicias

022 Dr. Belisario

Dominguez

023 Galeana

024 Santa Isabel

025 Gómez Farías

026 Gran Morelos

027 Guachochi

028 Guadalupe

029 Guadalupe y Calvo

030 Guazapares

031 Guerrero

032 Hidalgo del Parral

033 Huejotitán

034 Ignacio Zaragoza

035 Janos

036 Jiménez

037 Juárez

038 Julimes

039 López

040 Madera

041 Maguarichi

042 Manuel Benavides

043 Matachí

044 Matamoros

045 Meoqui

046 Morelos

047 Moris

048 Namiquipa

049 Nonoava

050 Nuevo Casas Grandes

051 Ocampo

052 Ojinaga

053 Práxedis G. Guerrero

054 Riva Palacio

055 Rosales

056 Rosario

057 San Francisco Borja

058 San Francisco

Conchos

059 San Francisco del Oro

060 Santa Bárbara

061 Satevó

062 Saucillo

063 Temósachi

064 El Tule

065 Urique

066 Uruachi

067 Valle Zaragoza

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Economic opportunity in Mexico by controlling T. foetus

Entity Cow population % Calving rateCalves born

annually

Improvement in

calving rate by 1%

(extra calves)

Average calf

weight (Lbs) for

exportation to

USA

$CWTCalf value once

exported in USA

Economic impact

with 1% Increase

in Calving Rate

Mexico 8,000,000.00 50.00% 4,000,000.00 40,000.00 400.00 300.00$ 1,200.00$ 48,000,000.00$

Chihuahua 120,000.00 50.00% 60,000.00 600.00 400.00 300.00$ 1,200.00$ 720,000.00$

Table 1. Economic opportunity for Mexico & Chihuahua increassing calving rate by 1%

50% 60% 70% 80% 90%

Number of calves born 4,000,000.00 4,800,000.00 5,600,000.00 6,400,000.00 7,200,000.00

Economic impact by increasing in

calving rate in Mexico4,800,000,000$ 5,760,000,000$ 6,720,000,000$ 7,680,000,000$ 8,640,000,000$

Table 2. Economic impact by increasing the calving rate in Mexico

Inclease in calving rate

50% 60% 70% 80% 90%

Number of calves born 60,000.00 72,000.00 84,000.00 96,000.00 108,000.00

Economic impact by increasing in

calving rate in Chihuahua72,000,000$ 86,400,000$ 100,800,000$ 115,200,000$ 129,600,000$

Table 3. Economic impact by increasing the calving rate in Chihuahua

Inclease in calving rate

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• Herd & Location: Delicias, Chihuahua, Mexico (Name of the operation will remain blinded)

• Overall status of the herd and anamnesis: Big ranch with a lot of open cows every year

despite the feed supplements, minerals, vitamins etc. to sustain proper body condition for

optimal reproduction.

• Breed (s): Milti-Breed predominantly Angus and Hereford in the bull’s battery.

• # of Cows: ~ 2000 cow operation.

• # of Bulls: 60 bulls.

• Vaccination status: Vaccinated for Clostridial and abortive diseases panel (Reputable

source).

• Calving Rate: By the year 2003, they reported 33% calving rate.

• Diagnostics: In year 2003, VetMAX-Gold trich Detection Kit was recommended on the

complete battery of bulls.

• Results: 50% of the Bulls were positive for the test.

• Management strategy: it was recommended to get rid of the entire set of bulls, but the

owner couldn't afford that. Therefore, he just got rid of the infected ones. Two more

sequential smegma samplings on the negative bulls was implemented for Trich testing. All

the open cows were rested at least 3 estrus cycles followed by Artificial Insemination and the

usage of the clean bulls.

• Follow up, by the end of year 2015, the operation reported a calving rate of 72% which is a

~40% increase from previous years.

Clinical case 1:

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Acknowledgments

C .C. Lastra-González, E.

González-Rodríguez, J. A.

Ramírez-Godínez, E.

Santellano-Estrada, J. Baxter y

I. Leyva- Baca

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