Ivan Leyva-Baca DVM., MSc., PhD.
Animal Health Product Applications
Life Sciences Solutions
Austin, Texas, USA
The world leader in serving science

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

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• 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.
Vilela RC, Benchimol M. IL-10 release by bovine epithelial cells cultured with Trichomonas vaginalis and Tritrichomonas foetus . Memórias do Instituto Oswaldo Cruz . 2013;108(1):110-112. doi:10.1590/S0074-02762013000100018. http://openi.nlm.nih.gov/ https://commons.wikimedia.org/wiki/File%3ATritrichomonas_foetus.jpg https://commons.wikimedia.org/wiki/File%3ATritrichomonas_foetus_(259_26)_Cultured.jpg

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•
T. foetus genome is divided in five haploid chromosomes
• Genome size ~160 MB

Transmission: is mainly venereal
“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
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 5 th month of gestation)
• Temporary infertility (2-6 months)
• Early return to estrus (Most common clinical sign of T. foetus infection in cows)
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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)

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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)

• 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:
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• 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

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Prevalence of Tritrichomonas foetus in beef bulls in the State of Chihuahua, Mexico Tested with MagMAX ™ Sample Preparation System and VetMAX TM -Gold Trich Detection Kit
*González R.E
1 ., Ramírez-Godinez.J.A
1 ., Leal T.B.A
1 ., Lastra G.C
1 ., Ordoñez B.P.L
1 .,
Álvarez G.J.A
1 ., Santellano E.E
1 ., Esparza V.M.E
1 ., Baxter J 2 ., Leyva-Baca. I .
2
1 Facultad de Zootecnia y Ecología. Universidad Autónoma de Chihuahua.
2 Animal 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

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Prevalence of Tritrichomonas foetus in beef bulls in the State of Chihuahua, Mexico Tested with MagMAX ™ Sample Preparation System and VetMAX TM -Gold Trich Detection Kit
Sample size to estimate true prevalence
Inputs
Assumed true prevalence
Sensitivity
Specificity
Population size
Confidence
0.25
0.98
0.98
60000
0.95
Desired precision 0.05
Results
Sample size required
Population = 60000
Sample size
320
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:
Sp = 0.7
Sp = 0.8
Sp = 0.9
Sp = 0.95
Sp = 0.99
Sp = 0.999
Se = 0.7
2220
Se = 0.8 Se = 0.9 Se = 0.95 Se = 0.99 Se = 0.999
1467 1039 892 795 775
1320
791
603
479
453
957
620
491
403
385
727
501
410
346
332
643
454
377
321
309
585
422
353
303
293
573
415
348
300
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

Prevalence of Tritrichomonas foetus in beef bulls in the State of Chihuahua, Mexico Tested with MagMAX ™ Sample Preparation System and VetMAX TM -Gold Trich Detection Kit
Materials and Methods:
In years 2014, 2015 & 2016 (n=880) Bulls were sampled and tested as follows:
Day 1: Sample Collection & Culture Day 2: Nucleic Acid Extraction and Real-time PCR
37ºC x 24h
TrichIT (Morris Livestock
Products, Delavan, WI) &
InPouch (Biomed)
Incubation
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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7500 Fast real-time PCR
MagMAX Sample preparation system
•
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

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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
(LeyvaBaca, O’Connell) JVDI, 2014, Vol. 26(1) 72-87
Objectives:
1.
Compare different sample preparation systems and various real-time PCR (feeder lab workflows) with the 5X MagMAX TM -pathogen RNA/DNA purification kit and amplification with VetMAX TM 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

Prevalence of Tritrichomonas foetus in beef bulls in the State of Chihuahua, Mexico Tested with MagMAX ™ Sample Preparation System and VetMAX TM -Gold Trich Detection Kit
15.79% (3)
37.5% (12)
32
19
104
31.73% (33)
74
10.81% (8)
24
26.23% (48)
183
16.67% (4)
Brangus
Angus
Razas
Brangus
Angus
Hererford
Charolais
Salers
Hereford
Charolais
Salers
Criollo
Criollo
Brahaman
Beef Master
Brahman
Beefmaster
Pardo Suizo
Simmental
Pardo Suizo
Simmental
Limousin
Limousin TOTAL
1
1
6
1
5
450
Total Infectados
183
24
48
4
74
104
19
32
8
33
3
12
0
1
3
0
1
113
%
26.23
16.67
10.81
31.73
15.79
37.5
0
20
0
100
50
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Prevalence of Tritrichomonas foetus in beef bulls in the State of Chihuahua, Mexico Tested with MagMAX ™ Sample Preparation System and VetMAX TM -Gold Trich Detection Kit
• Age as risk factor for BovineTrichomoniasis:
• 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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Prevalence of Tritrichomonas foetus in beef bulls in the State of Chihuahua, Mexico Tested with MagMAX ™ Sample Preparation System and VetMAX TM -Gold Trich Detection Kit
• Distribution of Sampling and Positive Bulls 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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Table 1. Economic opportunity for Mexico & Chihuahua increassing calving rate by 1%
Entity
Mexico
Chihuahua
Cow population
8,000,000.00
120,000.00
% Calving rate
50.00%
50.00%
Calves born annually
4,000,000.00
60,000.00
Improvement in calving rate by 1%
(extra calves)
40,000.00
600.00
Average calf weight (Lbs) for exportation to
USA
400.00
400.00
$CWT
Calf value once exported in USA
Economic impact with 1% Increase in Calving Rate
$ 300.00
$ 300.00
$ 1,200.00
$ 1,200.00
$ 48,000,000.00
$ 720,000.00
Table 2. Economic impact by increasing the calving rate in Mexico
Number of calves born
50%
4,000,000.00
60%
4,800,000.00
Inclease in calving rate
70%
5,600,000.00
80%
6,400,000.00
90%
7,200,000.00
Economic impact by increasing in calving rate in Mexico
$ 4,800,000,000 $ 5,760,000,000 $ 6,720,000,000 $ 7,680,000,000 $ 8,640,000,000
Table 3. Economic impact by increasing the calving rate in Chihuahua
50% 60%
Inclease in calving rate
70% 80%
Number of calves born 60,000.00
72,000.00
84,000.00
96,000.00
90%
108,000.00
Economic impact by increasing in calving rate in Chihuahua
$ 72,000,000 $ 86,400,000 $ 100,800,000 $ 115,200,000 $ 129,600,000
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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.
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