mycoplasma spp. in colombian dairy herds, an epidemiological … · 2017. 5. 3. · bovine mastitis...

Facultad de Ciencias Agropecuarias

Programa de Doctorado en Ciencias Agrarias

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1. GENERAL INFORMATION

Project title: Mycoplasma spp. in Colombian dairy herds, an epidemiological approach

Name of the research groups involved in the project

Name of the research group: Calidad de Leche y Epidemiología Veterinaria

Faculty/Department: Facultad de Ciencias Agropecuarias / Departamento de

Sistemas de Produccion

Clasificación _____

Name of the research group: Biología de la Producción Pecuaria

Faculty/Department: Facultad de Ciencias Agropecuarias / Departamento de

Sistemas de Produccion

Clasificación _A2__

Project Type: Basic research: ☐

Applied research: ☒

Creation: ☐

Technological innovation1: ☐

Innovation type: Product technological innovation: ☐

Process technological innovation: ☐

Organizational innovation: ☐

Strategic area of the development plan

Biotechnology ☒ Art, culture, humanism ☐ Social problems ☐

Health ☐ Environmental ☐ Does not belong to any of these

strategical areas ☐

MEMBERS OF THE RESEARCH TEAM

Name / link to the Caldas

University Department or Program

Knowledge area

(according to annex 1) Juan Felipe Velasco Bolaños* Doctorado en Ciencias Agrarias Agronomy, Veterinary and related

Alejandro Ceballos Márquezǂ Sistemas de Producción Agropecuaria Agronomy, Veterinary and related

Sofie Piepers§ External advisor Agronomy, Veterinary and related

Simon Dufour§ External advisor Agronomy, Veterinary and related

Place of Project Execution: (Municipality/Department)

Municipality: Manizales Department: Caldas

Budget

Total value of the project: $ 518,200,000

financial sources: COLCIENCIAS &

UNIVERSIDAD DE CALDAS

Value requested: $

Length of execution (months): 36 months

* Student, ǂ Professor, § External

1 Se refiere a aquellos proyectos que tienen como objetivo el desarrollo de nuevos productos o procesos, así como las

modificaciones tecnológicas importantes en productos o procesos



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2. ABSTRACT

Bovine mastitis nowadays is one of the most expensive disease in dairy herds affecting the milk

yield and milk quality. Several contagious mastitis pathogens are described, one of the most

important is Mycoplasma spp. with a prevalence around 5% in bulk tank milk and 21% at cow level

(Fox, 2011). Mycoplasma spp. is related not only to clinical or subclinical mastitis in cows but also

to several diseases in calves and cows, and its importance is that this pathogen is highly contagious,

persistent and the fact that antibiotic therapy is not effective, therefore, the only way to control it

is with preventive measures, identification, segregation and culling of infected animals (Fox et al.,

2005; Aebi et al., 2012). Nevertheless, the diagnostic needs specific conditions and materials that

difficult the identification of the pathogen (Hoggan 1999). There are also several species of

mycoplasma affecting cattle, and three of them are the most common in mastitis milk, M. bovis,

M. californicum, and M. Bovigenitalum, in that order. There are studies that suggest that these

pathogens induce different affections and response in the host, and even a same specie genotype

may induce a disease presentation in one host quite different from that seen in another (González

and Wilson, 2003; Register et al., 2015). The Mycoplasma spp is been reported all over the world,

but in Colombia there is only one study that demonstrate its presence in dairy herds, but

epidemiological information is inexistent in the country, for that reason the aim of these project is

to establish the epidemiology of Mycoplasma spp., and its classification and characterization, also

to determine its prevalence at intra-herd at cow and calf level, and the among herds prevalence.

Additionally, evaluate the dynamics of the pathogen in the region and the risk factors associated to

it. The last will bring an improvement of the health status and milk quality of the farms also

reducing the milk loss that can be caused by these bacteria.



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3. CONFORMATION AND BACKGROUND OF THE RESEARCHER’S TEAM

The research group Biología de la Producción Pecuaria of the Caldas University, trough the

research team of Milk Quality and Veterinary Epidemiology had strengthened the agricultural and

livestock sector of the Coffee Triangle Region, in Colombia through the formulation and

development of projects that aims to improve productivity and competitivity providing solutions

to the main problems. Most of the research projects developed by the group includes milk quality

and bovine mastitis.

This Project is located inside the research line Milk Quality and Veterinary Epidemiology, in which

14 people participate, including two professors, four PhD students, three master students, three

laboratory and field technicians and two undergraduate veterinary students.

The Milk Quality and Veterinary Epidemiology group during the last years is been working on the

molecular diagnosis, classification and characterization of mastitis-causing pathogens and udder

health trying to establish and develop strategies that contribute to the prevention and control of

mastitis in the Coffee Triangle Region.

The director of the project will be Dr. Alejandro Ceballos-Marquez, which is associate professor

of the Caldas University, international advisor on udder health, with 10 years of experience on this

field. He is also an expert on extension service programs and milk quality, also is a founder member

of the Latin American Mastitis Research Network (RELIM).

The PhD student will be Juan Felipe Velasco, professional in veterinary and animal sciences, also

with a master in veterinary sciences working on milk quality with the epidemiological aspects of

Streptococcus agalactiae in Colombian dairy herds. He has been selected twice to develop a

Colciencias research grant, also in milk quality.

As advisors, Dr. Sofie Piepers, which is a professor of the Department of Obstetrics, Reproduction

and Herd Health of the Faculty of Veterinary Medicine in Ghent University, Belgium. She is also

a founder member of the M-Team, which mission is to provide advice, service and training related

to udder health and milk quality, tailored to the needs of the farmers.

Dr. Simon Dufour is a professor at the University of Montreal in Canada. He has extensive

experience in milk quality, microbiology, mastitis and veterinary epidemiology. He is currently the

director of the Canadian Bovine Mastitis and Milk Quality Research Network (CBMMQRN).



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4. PROJECT DESCRIPTION

4.1 Justification

Bovine mastitis is one of the most expensive diseases affecting dairy farms around the world, it is

reported that one single case can cost 250 USD, the last because of the cost of the antibiotic

treatments, the discard of treated milk, the veterinary services, the loss on the milk yield, and in the

worst scenario the early culling of the cows (Huijps et al., 2008, 2009). Mastitis affects milk quality

in many ways, one of those is the reduction in the useful components of milk and increases the

level of undesirable elements, during mastitis milk protein fraction varies, increasing the

immunoactive fractions in relation with nutritious one. Lactoferrin levels in lacteal secretions may

increase 30 times, and also transferrin and the complement proteins, but the level of casein is

decreased by up to 20%, which represents lower nutritional value, yield and useful live (Keefe,

1997; Blowey and Edmondson, 2010; Alnakip et al., 2014).

Different species of bacteria had the ability to cause infection of the mammary gland (MG),

including Mycoplasma spp., classified as a high contagious pathogen that can cause a severe

inflammatory reaction in the udder (Gröhn et al., 2004; Schukken et al., 2009), Mycoplasma spp.

can also be located in several body sites of in cattle, and according to the site it can generate

different diseases like arthritis, otitis, keratoconjuctivitis, pneumonia and reproductive disorders

(González and Wilson, 2003).

Is also known that calves can get infected with the pathogen, by vertical transmission through the

uterus or via feed milk, or by horizontal transmission by contact with infected cows or calves. In

any case, the pathogen can remain infective in those calves, that growing as infected heifers could

get into the milking and infect more cows from the herd or transmit the infection to the next

generation (Pfützner and Sachse, 1996; Tenk, 2005).

The frequency of isolation of Mycoplasma spp. is lower in comparison with other pathogens in

milk. Is described that the transmission of mycoplasma mastitis has different epidemiology and a

different set of risk factors than other contagious mastitis pathogens (Fox et al., 2003). The

Mycoplasma spp. diagnosis had special requirements that made difficult to culture it, reason why

this pathogen is described as under-reported (Fox, 2012).

The Mycoplasma spp. mastitis responds poorly to antimicrobial treatment, and is usually

unsuccessful due to the high antibiotic resistance of the mycoplasmas. There is no effective therapy

reported for mastitis, by the contrary the antibiotic treatment is not contemplated in the control of

the disease; the protocol described to control mycoplasma mastitis is to identify the infected cows,

segregate them and finally culling them (Bushnell, 1984; González and Wilson, 2003; Aebi et al.,

2015; Barberio et al., 2016).

Commonly, there are five species of mycoplasma (M) related to mastitis in bovine MG,



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Mycoplasma bovis, M. bovigenitalium, M. californicum, M. canadense and M. alkalescens; of those

five, three are the most frequently related to mastitis: M. bovis, M. bovigenitalium and M.

californicum (Boonyayatra et al., 2012a). In Latin American countries such as Mexico, Brazil,

Chile, Argentina, and also in Colombia those pathogens had been reported (Infante-Martínez et al.,

1999; Ulloa, 2013; Andrade-Becerra et al., 2014; Tamiozzo et al., 2014).

In Colombia, there is only one research that aim for Mycoplasma spp. isolates in bovine milk at a

quarter level (Andrade-Becerra et al., 2014), the findings of that study evidence that the pathogen

is in the Colombian dairy herds but don give enough information to establish a prevalence at intra-

herd and among herds level. Once is known that there are cattle infected with this pathogen is

needed to implement strategies that lead to the correct diagnosis of the pathogen, that means

microbiological and molecular analysis for reduce misclassification of the pathogen species. Is also

need to stablish the risk factors at the individual level in the herd, and at herd level to determine

risk factors and implement control measures and to determine the spatial patterns of the bacteria.

In the Coffee Triangle Region, the Caldas Univeristy had executed two research projects trying to

determine the prevalence, dynamics and the effect of milk quality of several bacteria like

Staphylococus aureus and Streptococcus agalactiae, but the search for mycoplasma was not

performed on those research’s, and no other projects in Colombia had reported the occurrence of

mastitis caused by mycoplasma in the Coffee Triangle Region.

Because of the above, the present project aims to establish the epidemiology of Mycoplasma spp.,

and its classification and characterization to stablish the prevalence at intra-herd at cow and calf

level, and the prevalence among herds. Additionally, to evaluate the dynamics of the pathogen in

the region and the risk factors associated to it. The last will bring an improvement of the health

status and milk quality of the farms also reducing the milk loss that can be caused by these bacteria.

4.2 Theoretical framework

4.2.1 Mastitis

Bovine mastitis is a MG infect-contagious disease, in which inflammation occurs as a response to

the invasive, via teat canal, of different types of bacteria, fungi, yeasts and even algae and viruses.

However, bacteria are responsible of the 90% of the mastitis cases (Bradley, 2002).

When the bacteria surpass the natural defense mechanisms of the MG, a series of immunological

events occur. In that order, neutrophils constitute the first defense line against the bacteria that

penetrates trough the teat canal, likewise, cellular defense factors are released (lymphocytes,

macrophages and more neutrophils) with humoral factors (immunoglobulins, complement factors,

lactoferrin, lactoperoxidase and lysozyme), and different cytokines favor inflammatory reactions.



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Therefore, the number of immunoactive cells will represent an increase in the SCC in the milk as

a response to bacteria (Sordillo et al., 1997; Sordillo and Streichner, 2002).

Mastitis can be classified by the clinical signs in subclinical or clinical, and the etiologic agent

classify the mastitis in contagious or environmental. The contagious mastitis is caused by bacteria

that can be found in the udder or teat skin, mainly Streptococcus agalactiae, Staphylococcus aureus

and Mycoplasma spp., microorganisms that have potential to spread from infected cows to

numerous healthy cows, usually during milking (Pieterse and Todorov, 2010; Britten, 2012).

Environmental mastitis is caused by microorganisims from fecal origin mainly, but can also heavily

contaminate organic materials such as bedding, feed, wather, and soil in the cows’ surroundings

such as Escherichia coli, Kelbsiella spp., Enterobacter spp., and environmental streptococci as

Streptococcus uberis and Streptococcus dysgalactiae (Hogan and Smith, 2012).

The clinical mastitis is easier to diagnose than subclinical mastitis, because observable changes in

the udder and milk appearance are visible, such as inflammation, hardness, redness, edema,

swelling, clots in the milk, or serous or bloody secretions in the milk. It also can appear with

increase on body temperature, heart rate, decay and anorexia (Blowey and Edmondson, 2010;

Calderón et al., 2011). By the contrary, subclinical mastitis does not show evident signs, and milk

aspect remain normal but an increase in SCC and reduction of casein, calcium and total solids

occur. Because of the last, subclinical mastitis is responsible of the highest economic losses around

the globe (Calderón et al., 2011).

4.2.2 Mycoplasma mastitis

The Mycoplasma spp., are simple pleomorphic bacteria that lack a cell wall, they attaches to the

host cells to obtain their nutriments (Kirk and Mellenberger, 2011; Kruze, 2015). The products of

metabolism and growth of the mycoplasmas irritates the MG tissue, leading to an increase in the

immunological response of the host (Jasper, 1982).

The mycoplasmas are considered as contagious pathogens that can cause clinical or subclinical

mastitis, at the same time Mycoplasma spp., is associated with several diseases in the dairy cattle,

such as arthritis, otitis, keratoconjuctivitis, pneumonia and reproductive disorders (González and

Wilson, 2003; Olde Riekerink et al., 2006). Except form New Zealand, which is possibly free of

the pathogen, mastitis caused by Mycoplasma spp. is been reported in all the world (McDonald et

al., 2009; Nicholas et al., 2016).

Mastitis caused by Mycoplasma spp., do not have an effective antimicrobial therapy, and the only

way to control this mastitis is achieved through the identification and segregation or culling of

infected cows (Bushnell, 1984; González and Wilson, 2003; Fox et al., 2005; Aebi et al., 2015).

As a contagious pathogen, its transmission from cow to cow occurs during the milking through the

hands of the milker, contaminated liners or by the contact with fomites, or drying paper towels



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previously used in infected cows (Fox, 2012). Additionally, there are other infection sources like

nose to nose contact, airborne transmission, or in calves by vertical transmission through the uterus

or via feed milk, in those calves the pathogen can remain infective and cause mastitis in the first

lactation (Pfützner and Sachse, 1996; Tenk, 2005).

There are nine species of mycoplasma isolated from bovine milk, but only five are related to

mastitis, those are M. bovis, M. claiformicum, M. bovigenitalium, M. alkalescens, and M.

canadense, all of them produces similar mastitis but may differ in the severity (González and

Wilson, 2003). Of the five listed before, the first three are the more prevalent, the M. bovis, is the

most common Mycoplasma spp. associated with mastitis and accounts from 78 to 93%, of the

isolates, the followed by, M. californicum with a prevalence from 3 to 5% and M. bovigenitalium

with 1 to 10% of the mycoplasmas isolated (Boonyayatra et al., 2012a; Gioia et al., 2016).

4.2.3 Mycoplasma diagnosis

The diagnostic of Mycoplasma spp., is carried out by bacteriological culture of an aseptic milk

sample, and whose growth would be confirmatory for the presence of mastitis due to mycoplasma.

However, a negative growth does not mean the absence of the pathogen (Biddle et al., 2004; Ulloa,

2013).

The culture of Mycoplasma requires special conditions to its growth, it needs special culture media,

and a long period of incubation (3 to 10 d) under 37°C and 10% of CO2 concentration atmosphere

(Hogan et al., 1999); reason why routine microbiological analysis for this pathogen are not

performed and is considered under-diagnosed (Fox, 2012).

Another difficulty for the Mycoplasma spp. diagnosis is that infected cows could shed mycoplasma

intermittently, 29% of the times with concentrations lower than 100 colony former units

(CFU)/mL, which is the below the detection limit for traditional culture(Biddle et al., 2003;

Boonyayatra et al., 2010). Additionally, storage and thawing of milk samples is harmful to

mycoplasma, then culturing fresh milk samples maximize the detection of Mycoplasma spp.,

suggesting the importance of proper milk sample handling before culture to avoid false negative

results (Biddle et al., 2004; Boonyayatra et al., 2010)

Most of mycoplasmas isolated from the udder are pathogenic and growth typically as “fried egg”

colonies under traditional microbiological procedures. Nonetheless, misdiagnosis with

Acholeplasma spp., another Mollicute, that growth in similar conditions and colonies can occur,

because it cannot be distinguished from mycoplasma by conventional microbiological diagnosis

(Hogan et al., 1999). But in contrast, is a common nonpathogenic saprophytic contaminant in the

dairy environment and sometimes a contaminant of BTM and cow milk samples (Bushnell, 1984;

Jasper, 1981).

For the differentiation of these two bacteria there are complementary tests like the inhibition of

sterols with digitonin discs, method supported by the need of sterols to form the cytoplasmic



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membrane of mollicutes. Generally, sterols are taken from the environment, but Acholeplasma spp.

had the ability to synthesize sterols by metabolic paths, whereas Mycoplasma spp. cannot. In that

order, adding digitonin discs to the growth media lead to an inhibition of the development of

mycoplasma colonies, without affecting Acholeplasma spp. growth (Boonyayatra et al., 2012b).

Another method for the differentiation are the nucleic acids-based methods, using specific primers

that target specific genes of both bacteria (Zadoks, 2004; Stakenborg et al., 2005; Boonyayatra et

al., 2012b; Gioia et al., 2016). The nucleic acids-based methods are highly specific and can also

discriminate between closely related organisms, showing a greater potential than the

microbiological culture (Riffon et al., 2001).

The nucleic acids-based methods can also be used to classify the different species of mycoplasma

into M. bovis, M. bovigenitalium, M californicum and others, using PCR, Real Time PCR or

denaturing gradient gel electrophoresis (DGGE) (Hirose et al., 2001; McAuliffe et al., 2003;

Higuchi et al., 2011; Boonyayatra et al., 2012a). The nucleic acids-based methods advantages are

sensitivity, high specificity, speed, cost efficiency and potential to screen a large amount of samples

(Cree, 2011). Also, had detection levels as low as 5 CFU/mL in milk samples have been reported.

Likewise, these molecular methods are used to determine whether mastitis is predominantly caused

by one strain of a pathogen or by a multitude of strains (Zadoks, 2004; Zadoks and Schukken,

2006).

According to the above, the Multi Locus Sequence Typing (MLST) method is been described as a

robust, scalable and highly standardized method usefully to differentiate among strains allowing

an unambiguous classification of specific bacteria based on the sequencing and comparison of

housekeeping genes (Pavón and Maiden, 2009). Also, is been described that the MLST method

developed for M. bovis had a high discriminatory power (Register et al., 2015)

Since 2012 several MLST schemes for M. bovis had been developed with results linked to private

and public databases (Bürki et al., 2016), which facilitate the new data entry, dissemination and

comparison of the M. bovis around the world, activity that contributes to the understanding of the

population structure of the pathogen, a useful tool in epidemiologic studies, that can help with the

prevention and reduction of disease impact, also can help to clarify if different genotypes of a

pathogen induces different presentations of the disease In one host and another (Register et al.,

2015).

4.3 Objectives

General Objective:



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To evaluate the epidemiology of Mycoplasma spp. based on the analysis of risk factors in dairy

farms positive to this pathogen in Colombia (Caldas, Quindío, Risaralda).

Specific objectives:

1. To establish the intra-herd prevalence at cow and calf level, and the prevalence among

herds, and to determine the association between the bulk tank milk results and their somatic

cell count

2. To determine the molecular characteristics of Mycoplasma bovis isolated of the different

sources.

3. To analyze spatial and clustering data of dairy herds positive to Mycoplasma spp. and

Mycoplasma bovis sequence types

4.4 Materials and Methods

4.4.1 Study Design and Sample Size

A hybrid study with two-stage sampling design will be carried out at herd level and at intra-herd

level on 115 bulk tanks from farms located in the Coffee Triangle Region composed by three

departments, Caldas, Quindío, and Risaralda in Colombia.

The sample size was calculated with a desired precision of 95% and estimate proportions from 3

to 8%, based on the prevalence reports of Mycoplasma spp. in bulk tank milk from Canada

(Francoz et al., 2012). The formulae showed below give as a result a sample size that varies from

44 to 113 bulk tanks, the higher number is taken as the final sample size (115 farms) (Dohoo et al.,

2009).

𝑛 =𝑧𝑎2𝑝𝑞

𝑑2

Where: n = Sample size

Z = The value of Za required for confidence = 1-a (Z0,05 = 1,96)

p = estimate of the proportion (3 to 8%)

q = 1- p

d = allowable error (5%)

The farms will be selected randomly from a database gathered from the six milk processors

(Celema, Alival, Normandy, Alpina, Parmalat and Colanta) in the region and the ones registered

in the Comité de Ganaderos del Centro (Risaralda). The farm database will be composed from the



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farms in the region that meets the two inclusion criteria (a & b), and the single exclusion criteria;

the first one is that a) the farm must have a bulk tank, also b) the facility for sample collection, and

it will be excluded if c) the bulk tank receives milk from more than one producer (Communitarian

bulk tanks).

Farms selected that decide not to participate of the research will be replaced for another one until

the sample size is reached, not without asking basic information of the non-participant farms

including: number of lactating cows, average of milk liters delivered daily, and if its available, the

average for the SCC.

4.4.2 Sampling

The first sampling stage will be carried out at herd level, taking one sample (100 mL) of the bulk

tank monthly up to 3 samplings. the sample will be divided in two aliquots, one will include a

preservative (Bronopol; Broad Spectrum Microtabs II, Advanced Instruments Inc) for SCC and the

second one in a sterile vial for microbiological analysis.

The second stage is at intra-herd level, and only carried on for Mycoplasma bovis positive farms

of stage one. In the farm one composite milk sample of each lactating cow will be taken (50 mL)

in a sterile vial for microbiological analysis. Likewise, of all the calves fed with milk will be taken

a throat swab using a sterile swab moistened with saline solution, later the swab will be introduced

in 2 mL of modified Hayflick broth (Oxoid).

All the stage one and two samples will be carried to the Milk Quality and Veterinary Epidemiology

Laboratory under refrigeration conditions for the respective analysis.

4.4.3 Data Collection

During the visit to the farms the X,Y coordinates will be recorded using a GPS (Oregon 650,

Garmin), also the application of a survey designed to collect information about mycoplasma risk

factors at herd level will be done. The survey includes the following aspects: general information

of the herd, biosecurity, milking procedures, milking machine, treatment procedures and nutritional

aspects of cows and calves. Additional information related to the last six months SCC will be

recorded too.

Also, at the cow and calf sampling information at that level will be collected too. That includes for

the cow: age, parity, stage of lactation, breed, milk yield, previous antibiotic therapy in the current

lactation, and if its available the last six months SCC. And for the calves: age, breed, sex and if

joints inflammation, lameness or respiratory symptoms are visible.

4.4.4 Laboratory analysis



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The milk sample with bronopol will be sent to the Milk Quality and Milk Safety Laboratory in the

Antioquia University for the SCC determination by infrared spectroscopy by automatized methods

(CombiFossTM, Foss).

Sterile milk samples will be processed in the shortest possible time after the admission into the

laboratory, due to the detrimental effects of long periods storage or freezing in the bacteria

(Punyapornwithaya et al., 2009).

For the microbiological analysis of bulk tank milk and cows, a 4 steps protocol is going to be used.

a) an aliquot of 100 µL of milk will be inoculated into 2 mL of modified Hayflick broth (Oxoid)

for its incubation under a 37°C with 10% of CO2 conditions for 48 h (Hogan et al., 1999). After

that time, b) homogenization of the vial will be made using a vortex stirrer for 5 s (Vortex, Labnet)

c) then, 100 µL of the broth will be cultured on modified Hayflick solid media. Again, the plates

will be incubated under the mentioned conditions during 10 days, d) checking the plates in search

for suspicious “fried egg” colonies during the days 3, 7 and 10 of incubation using a stereoscopic

microscope at 40x (Betscope). The solid media surrounding the suspicious colonies will be cut and

introduced into modified Hayflick broth for obtaining pure colonies after 2 d of incubation for the

molecular analyzes (Ulloa, 2013).

The pure colonies will be cryo-preserved under freezing temperatures (-80°C) in vials with

cryobeads.

4.4.5 Mycoplasma spp: molecular confirmation

Starting with a suspicious colony on the solid media, or with the pure suspicions colony on the

broth, the first step for molecular confirmation will be the inoculation of the suspicious colony on

solid media or 100 µL of broth with pure colonies into a new 2 mL vial with modified Hayflick

broth, the vial will be incubated for 4 days under the same temperature and CO2 conditions

mentioned above. After that period, the vials will be centrifugated under 12,000 g for 30 s, then the

supernatant will be eliminated and the pellet in the bottom of the vial will be used for the DNA

extraction.

The DNA extraction will be carried on with a commercial kit designed for the extraction of Gram-

negative bacteria following the manufacturer instructions (Axygen Bacterial genomic DNA

miniprep kit, Axygen Biosciences). The DNA cuality and quantity will be determined by a

Nanodrop 1000 (Thermo Fisher Scientific), samples within 30 and 50 ng/µL will be included in

the molecular analysis without dilution, if higher concentrations are found, these will be diluted to

50 ng/µL using ultra-pure water for PCR (Bioline). Finally, The DNA will be stored on 0,2 mL

vials under freezing temperatures of -20°C until use.



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The PCR reaction will follows the methodology suggested by Boonyayatra et al., (2012b) with a

reaction of 50 µL of final volume, using 25 µL of a commercial master mix (MangoMix, Bioline),

5 µL of template (50 ng/µL), 1 µL the primers shown in Table 1. And 17 µL of ultra-pure water

(Bioline). The PCR program will be done as follows: initial denaturation at 94°C for 30 s, followed

by 35 cycles of denaturation at 94°C for 30 s, primer annealing at 55°C for 2 min, and extension at

72°C for 2 min. A final extension will be performed at 72°C for 5 min. The final PCR products

will be electrophoresed on a 2% agarose gel stained with RedGelTM (Biotium), with 100 volts for

90 min, and DNA bands will be visualized with a transilluminator (GelDoc, Biorad). A single band

of PCR product indicate the presence of a Mycoplasma spp., and the presence of more than one

band indicate Acholeplasma spp.

Table 1. Primers used for the PCR targeting the 16S-23S rARN intergenic spacer regions (IGSR)

of Mycoplasma spp. and Acholeplasma spp. (Boonyayatra et al., 2012b).

Primer Oligonucleotide sequence

Size (pb) Target

F2 5'-GTG(C/G)GG(A/C)TGGATCACCTCCT-3'

236 to 365

16S-23S rARN

IGSR

Mycoplasma

spp. R2 5'-GCATCCACCA(A/T)A(A/T)AC(C/T)CTT-3'

R34 5'-CCACTGTGTGCCCTTTGTTCCT-3' 219 & 426

16S-23S rARN

IGSR

Acholeplasma

spp.

4.4.6 Mycoplasma spp: molecular classification

With the DNA used for the molecular confirmation of Mycoplasma spp., the molecular

classification will be performed using a PCR modified protocol with a 50 µL final volume reaction

using 25 µL of a commercial master mix (MangoMix, Bioline), 3 µL of template (50 ng/µL), 1 µL

the primers shown in Table 2, and 16 µL of ultra-pure water (Bioline) (Karahan et al., 2010). The

PCR program will be done as follows: initial denaturation at 94°C for 30 s, followed by 30 cycles

of denaturation at 94°C for 1 min, annealing at 54°C for 1 min, and extension at 72°C for 1 min,

followed by a final extension at 72°C for 10 min. The amplified products will be electrophoresed

on a 2% agarose gel stained with RedGelTM (Biotium) at 80 volts for two hours. The DNA bands

will be visualized with a transilluminator (GelDoc, Biorad).

Table 2. Primers for the molecular classification of Mycoplasma spp.

Primer Oligonucleotide sequence Target Size

(pb)

Reference

M. bovis 5'-TATTGGATCAACTGCTGGAT-3' mb-mp81 447



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5-AGATGCTCCACTTATCTTAG-3 Foddai et al.,

2005.

M. californicum 5-GCACTTAGACGAAAGAGGGATT-3 rpoB

100 Boonyayatra et al., 2012b. 5-GGATTATCATCACCTTTGGGACT-3

M.

bovigenitalium

5-CTTTCTACGGAGTACAAAGCTAAT-3 16S-23S

rRNA

310 Hirose et al., 2001. 5-GAGAGAATTGTTCYCTCAAAACTA-3

4.4.7 Mycoplasma bovis: molecular characterization

The molecular characterization of M. bovis will be carried out following the protocols described

by Register et al., (2015) for MLST, the MLST will use the same PCR protocol for the seven primer

sets (Table 3), a 50 µL final volume reaction including 25 µL of a commercial master mix

(MangoMix, Bioline), 0.5 mM of each primer, 1 µL of DNA template and the remaining amount

will be filled with ultra-pure water (Bioline). The PCR will be carried out on a thermocycler (c1000,

BioRad) with the following cycling conditions: 94°C for 5 min and 35 cycles of 94°C for 10 s,

55°C for 30 s and 72°C for 1 min, followed by a final elongation step of 72°C for 5 min.

The PCR amplicons will be sequenced in the (LABORATORY OF RUUD) by sanger method

using the primers described above. Later the MLST scheme developed by Register et al., (2015)

will be employed by assigning an allele number for individual genes using alignments of each gene

sequence against the database available at http://pubmlst.org/mbovis/ until the sequence type (ST)

is gathered (Bürki et al., 2016). Concatenated sequences of the seven genes will be investigated in

order to assemble a phylogenetic tree using maximum-likelihood, including 1000 bootstrap

replications and software the method of neighbor joining using DNAStar (Bürki et al., 2016).

Finally, the application of the Simpsons diversity index will be used to calculate the dominance

index and also, to calculate diversity index (1-l), at intra herd level and among herd-level using the

following equation (Simpson, 1949; Hunter and Gaston, 1988):

𝑙 =∑ 𝑛𝑖(𝑛𝑖 − 1)𝑅𝑖=1

𝑁(𝑁 − 1)

Where: R = number of species

ni = number of individuals belonging to the ith type

N = total number of individuals

Table 3. Primers used for the MLST PCR targeting the seven housekeeping genes of Mycoplasma

bovis (Register et al., 2015).

Primer Oligonucleotide sequence Amplicon

size (bp)

http://pubmlst.org/mbovis/



14

adh-1-1 5’- GGA GTA ACT AGT TAC AAA GCA CTT A -3' 546

adh-1-2 5’- TGC TAG TTG TTC AAA CAC GT -3’

gltx-3 5’- TGG TGA GTA TTC AAT AAG GT -3’ 530

gltX-4 5’- GTT TTG AGA ATC ATT GCA -3’

gpsA-3 5’- AAA ATG TGA GGA ATT GAT CA -3’ 521

gpsA-2 5’- CCA ATT CCA ATT GCT AAA AC -3’

GyrB-1 5’- AGC TTG CTA ATT GCA CCA -3’ 678

GyrB-2 5’- TAT TTT GAA CAA ATT TTG CAT -3’

pta-2-1 5’- AAT TCG TAA TGG CAA AGA AG -3’ 490

pta-2-2 5’- CTT AGC TTT TCT TAC ATT TAG GT -3’

tdk-3 5’ –ATG TAT TTA AAA AGT GGA TTA GG -3’ 572

tdk-4 5’- TAT CTC ATA GCT TTT TTA GC -3’

tkt-1 5’- CCA ACT TAT ATT ATG GTG CA -3’ 533

tkt-2 5’- CCA CCA TAT AAA TTA ATG CC -3’

4.4.8 Spatial data analysis

Spatial data analysis will be done to the farms positive to different microbiological findings (M.

bovis, M. californicum and M. bovigenitalium) and also to the STs of M. bovis via geographic

information systems (GIS). using a GPS x,y (Latitude, longitude) coordinates of the farms to

generate the spatial visualization point data and the density of the bacteria in sub-regions, and at

the intra-herd and among-herds level using EPSG:4236 and WGS 84 coordinates systems in a GIS

software (QGIS, OSGeo).

The information of the movement of cattle among farms will be collected from the Instituto

Colombiano Agropecuario (ICA) or from qualified personnel of the farm so the dynamic dispersion

of the mycoplasmas can be stablished by a spatial cluster analysis of the positive dairy farms in

SaTScanTM software (Information Management Services Inc.) where the null hypothesis will be a

random dispersion of the mycoplasmas versus a specific dispersion pattern.

4.4.9 Statistical analysis

A descriptive analysis will be done to summarize the database, with information about general

aspects of the farms and production, also frequency tables will be used to describe the findings of

microbiological and molecular procedures and the intra-herd and among-herd prevalence of the

mycoplasmas found in the study will be calculated. All the variables of the database with missing

data greater to 30% or with unbalanced data, groups with less than 10% of the observations will be

excluded from the analysis.



15

The farms included in the study will be compared with those that choose not to participate of the

research in aspects like average of SCC, lactating cows, milk yield by a Student’s t-test to determine

if they were similar or not.

The association between the SCC and the and the BTM microbiological results will be established

by a simple linear regression model with the previous transformation of the SCC into a logarithmic

scale to fit a normal distribution.

The risk factors at cow level will be done using the information in the survey with the probability

of being a positive mycoplasma cow determined by the Odds Ratio (OR) obtained by a fixed

logistic regression model clustered by farm with backward elimination process. The same

procedures will be carried out to establish the risk factors at calve level.

Additionally, risk factors at herd level will be determined by a logistic regression model with

backwards elimination process including as the outcome the probability of being a positive farm

to the bacteria included in the study individually and as response variables the solutions of a

previous principal factor analysis method (Reyes et al., 2017).

The databases required for the analysis will be filled using a specialized software (Epidata 3.1,

Epidata Association). Also, all the procedures described in this section will be performed using the

statistical software Stata 14 (StataCorp) (except for softwares mentioned in 4.4.8) and the

significance level will be set at 95% with a type I error probability of 5%.

4.5 Expected Results

4.5.1 Generation of new knowledge

The findings of this study will contribute to the knowledge of which Mycoplasma spp. pathogens

are on the farms of the Coffee Triangle Region. It will also reveal the presence for the first time of

this pathogens in the region.

With the establishment of risk factors at herd, cow and calve level control measurements and

eradication mycoplasma plans can be designed accounting the uniqueness conditions of each farm.

At the same time, the implementation of the diagnosis techniques in the Milk Quality and

Veterinary Epidemiology Laboratory, will allow the producers to access the microbiological

analysis, in order to facilitate decision-making in the farms and obtain better milk quality.

The acknowledgment of the Mycoplasma bovis STs will make possible to clarify the relationship

among the strains and the epidemiological aspects related to them. Also, it will complement the



16

pubMLST database of the bacteria, allowing another researcher to use and compare their findings

with ours.

4.5.2 Strengthening of the Colombian scientific or artistic community

The Colombian scientific community will count, at the end of the project, with one PhD graduate

student who would be able to develop new research projects in the future at the highest scientific

level and the training of new magisters or PhD students.

All the research group members will be trained in aspects related with the epidemiological aspects

and diagnosis of Mycoplasma. Who at the end of the study will be suitable referents with the ability

to discern and evaluate the presence of Mycoplasma causing mastitis in the field.

The conformation of an international committee for the assessment and advisory of the project will

strengthen the collaborative work and the development of strategies for prevention and control of

mastitis.

4.5.3 Social / public appropriation of knowledge

The divulgation of the results of the study to the scientific community will be done by two ways:

a. The presentation of three abstracts in international scientific conference proceedings related

to milk quality and mastitis.

b. The submission of three papers to scientific journals indexed by the SJR.

The social divulgation will be done by the socialization of the results with the milk producers of

the regions involved in the study and the field veterinarians with the support of the milk processors.

4.6 Impacts from the use of results (max 1p.)

The identification of risk factors and association with milk quality for each species of Mycoplasma

will define the control, segregation or culling strategies in the farms, with the aim of reduce new

infections and maintain an appropriate health status of the cows in the farm which lead to a better

income of the producers.

The diagnosis methodology will be able to determine animals acting as a reservoir of the pathogen

in the herd, so identification of those animals will be the first step of control programs in the farms,

and also may lead to a preventive strategy for reduce the mycoplasma infection when trading

animals between farms.

The Milk Quality and Veterinary Epidemiology Laboratory could offer the service of mycoplasma

microbiological and molecular diagnosis at national level, leading to new research’s in these field.



17

4.7 Chronogram

ITEM

SEMESTER

2017 2018 2019 2020

I II I II I II I

Literature review

Project writing / planning

Objective 1

Objective 2

Objective 3

Manuscripts and thesis preparation

Thesis defense



18

5. COMPROMISES

When the project is completed, there will be a PhD graduate student, three abstracts in international

proceedings, related to de updates of the project, and finally three papers submitted to scientific

journals indexed by Scimago Journal and Country Rank (SJR).



19

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23

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24

7. BUDGET

See annex 2.



25

8. ANNEXES

Annex 1. Knowledge areas according to Colciencias:

Spanish English

Agronomía, veterinaria y afines Agronomy, Veterinary and related

Bellas artes Arts

Ciencias de la educación Educational sciences

Ciencias de la salud Health Sciences

Ciencias sociales y humanas Human and social sciences

Economía, Administración, Contaduría y afines Economy, administration and related

Arquitectura, Urbanismo y afines Architecture, urbanism and related

Ingeniería Civil Civil engineering

Ingeniería Química Chemical engineering

Ingeniería Eléctrica Electrical engineering

Ingeniería de Sistemas Systems engineering

Ingeniería de Sistemas y telecomunicaciones Systems and telecommunication engineering

Ingeniería Electrónica Electronic engineering

Ingeniería Industrial Industrial engineering

Ingeniería Mecánica Mechanical engineering

Ingeniería Ambiental Environmental engineering

Otras ingenierías Other engineering’s

Matemáticas Mathematical

Ciencias Naturales Natural sciences

Humanidades y Ciencias Religiosas Humanities and Religious Sciences

mycoplasma spp. in colombian dairy herds, an epidemiological … · 2017. 5. 3. · bovine mastitis...

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