Medycyna Weterynaryjna - Summary Med. Weter. 70 (7), 413-416, 2014

Med. Weter. 2014, 70 (7)

413

Praca oryginalna

Original paper

Mycoplasma species, from the bacterial class Mol-

licutes, are the smallest free-living prokaryotes

ca-pable of self-replication. Their main characteristics are

a permanent lack of the cell wall, a low G+C content,

and a small genome size (5). They are fastidious and

therefore difficult to culture in vitro; they generally

form centred colonies on a solid growth medium. As

a possible consequence of their limited biosynthetic

capacity, mycoplasmas usually form a very close

asso-ciation with host cells to receive the growth and

nutri-tional factors essential for their survival. Mycoplasmas

typically colonize mucosal surfaces (25). They are

pathogens, opportunistic pathogens or commensals of

many host species, including man, and occur

world-wide. It is a little known fact that mycoplasmas cause

some of the most severe and economically most

im-portant diseases of cattle (18).

Among the Mycoplasma species that have been

iso-lated from cattle, M. mycoides subsp. mycoides small

colony type (MmmSC) is recognised as the most

signifi-cant, as it causes contagious bovine pleuropneumonia.

It is listed by the Office International des Epizooties

and previously was the only bacterial disease included

in their list A category of economically important

dis-eases. Other Mycoplasma species affecting cattle are

detailed in Table 1.

Currently in the Czech Republic, the presence and

possible role of Mycoplasma species in cattle with

clinical signs of disease is limited. Therefore this study

investigated clinical samples from some clinically

af-fected cattle to detect and identify any Mycoplasma

species present. This information will help veterinary

practitioners in the Czech Republic consider

mycoplas-moses as a diagnosis and help facilitate the

implemen-tation of appropriate control measures.

Material and methods

Clinical samples. Samples were collected from 35 cattle

herds from across the Czech Republic that had reported

*) _{The study was funded from Research Project no. 6215712403 of the}

Ministry of Education, Youth and Sports of the Czech Republic (2007-2009) and from Research Project no. 60/2010/FVL (2010) of the Internal Grant Agency of the University of Veterinary and Pharmaceutical Sciences Brno. We wish to acknowledge Defra’s continued support for Mycoplasma work in AHVLA, Weybridge, Great Britain.

Report on investigations into the Mycoplasma

species present in some clinically sick cattle

in the Czech Republic*

)

JANA JOZEFOVÁ, ROGER D. AYLING*, DAGMAR ZENDULKOVÁ

Department of Infectious Diseases and Microbiology, Veterinary and Pharmaceutical University Brno,

Palackeho tr. 1/3, 612 42 Brno, Czech Republic

*Mycoplasma Group, Department of Bacteriology and Food Safety,

Animal Health and Veterinary Laboratories Agency (Weybridge), Surrey, United Kingdom

Received 14.10.2013

Accepted 10.04.2014

Jozefová J., Ayling R. D., Zendulková D.

Report on investigations into the Mycoplasma species present in some clinically sick cattle

in the Czech Republic

Summary

Between 2007 and 2010, 330 samples from 169 cattle with clinical signs of respiratory diseases, arthritis,

otitis or mastitis were examined for Mycoplasma species in the Czech Republic. Samples were collected and

cultured before being identified by polymerase chain reaction and denaturating gradient gel electrophoresis. In

53 samples, 63 mycoplasmas were detected and identified. M. dispar was the most frequently identified pathogen

with 36 per cent of positive samples, whilst M. bovirhinis was the most common mycoplasma, with 45 per cent.

Other species identified were M. bovoculi, M. bovis, M. bovigenitalium, M. canadense, M. alkalescens, and

M. arginini. Perhaps unusually, M. canadense was the only organism isolated from the joint fluid of an animal

that had arthritis. M. mycoides subsp. mycoides small colony type was not detected. Different Mycoplasma

species were identified in lung lavage and nasal swab samples from the same animals. Mycoplasma species

were present in 22 of the 35 herds tested.

Med. Weter. 2014, 70 (7)

414

problems with calf respiratory disease and/or high somatic

cell counts in milk. Samples were also collected from 145

calves with clinical signs of respiratory infection, arthritis,

and/or otitis, some of which had been refractory to antibiotic

treatment for more than three weeks. In addition, samples

were collected from 24 cows with subclinical or clinical

mastitis. In total, 330 samples were examined between

January 2007 and December 2010, and these were either

nasal, conjunctival and external ear canal swabs,

transtra-cheal lavage, lung, joint fluid, or milk samples

– details are given in Table 2.

Growth conditions, DNA extraction, 16S

rDNA PCR/DGGE analysis and specific PCR

for M. bovis. All samples were cultured in

Hay-flick’s liquid medium in three modifications

with addition of 0.5 per cent glucose, arginine

or urea and Hayflick’s solid medium (9) at

37°C and 5 per cent CO

. Mycoplasmas were

recovered when growth was observed over

a period of 28 days. DNA was extracted from

193 samples by means of NucleoSpin

_Tissue

(Macherey-Nagel) according to the

manufac-turer’s instructions and further examined by

16S rDNA PCR and denaturing gradient gel

electrophoresis (DGGE) (16). M. bovis

posi-tive samples were confirmed by specific PCR

analysis (27).

Results and discussion

By PCR/DGGE testing, eight Mycoplasma

species were identified from 53 of the 193

samples tested (27 per cent). The results

are shown in Figure 1, which also gives

the sampling year and sample type.

Sixty-three mycoplasmas were detected in the

53 samples that yielded Mycoplasma

spe-cies. The most frequent species identified

were M. bovirhinis (24/53 – 45 per cent),

M. dispar (19/53 – 36 per cent), M. bovoculi

(8/53 – 15 per cent), M. bovis (4/53 – 8 per

cent), M. bovigenitalium (3/53 – 6 per cent),

M. canadense and M. alkalescens (both

with 2/53 – 4 per cent), and M. arginini

(1/53 – 2 per cent). Despite the wide range

of Mycoplasma species identified, MmmSC

was not detected. Most mycoplasmas were isolated

from nasal swabs and lavage fluid (both 24/53 – 45

per cent), then from conjunctival swabs and joint fluid

(4/53 – 8 per cent and 1/53 – 2 per cent, respectively).

No mycoplasmas were isolated from 63 milk samples

and 11 ear swabs tested in this study.

Several Mycoplasma species were identified from

different clinical sampling sites. Four calves had

myco-plasma-positive nasal swabs and transtracheal lavages.

Tab. 1. Mycoplasma species detected in cattle

Etiological agent Disease or site of isolation References

M. bovis pneumonia mastitis arthritis reproductive disorders keratoconjunctivitis otitis neurological signs/brain

Pfützner and Sachse, 1996 (22) |

|

↓

Kirby and Nicholas, 1996 (10) Walz et al., 1997 (30) Ayling et al., 2005 (2)

M. dispar pneumonia

mastitis Nicholas, 2004 (18)Jasper, 1981 (8)

M. canis pneumonia ter Laak et al., 1993 (11)

M. alkalescens arthritis mastitis ear canal respiratory disease Whithear, 1983 (31) Manfrin et al., 1998 (14) Lamm et al., 2004 (12) Thomas et al., 2002 (28)

M. bovigenitalium pneumonic lungs

reproductive disorders arthritis granulopapular vulvovaginitis Ruhnke, 1994 (26) |

↓

M. bovoculi keratoconjunctivitis Rosenbusch and Knudtson, 1980 (24)

M. californicum pneumonia

mastitis Baumgartner, 1999 (3)

M. bovirhinis respiratory tract

arthritis conjunctiva ear canal Gourlay et al., 1979 (7) Reeve-Johnson, 1999 (23) Naglic et al., 1996 (17) Lamm et al., 2004 (12)

M. arginini lungs Chazel, 2010 (4)

M. canadense granulopapular vulvovaginitis Lysnyansky et al., 2009 (13)

M. leachii Previously known as Mycoplasma bovine group 7 polyarthritis mastitis respiratory disease Hum et al., 2000 (6)

↓

M. alvi unknown Nicholas et al., 2006 (20)

M. verecundum unknown Nicholas et al., 2006 (20)

Other opportunist Mycoplasma infections may occur in cattle, such as Mycoplasma mycoides subsp. capri which is usually associated with agalactia in goats

Tab. 2. Details of the samples examined during the four year period from 2007 to 2010 (ns – nasal swab, cs – conjunctival

swab, ec – external ear canal, ms – mixed swabs, lf – lavage fluid, jf – joint fluid)

Year Category Number of herds Number of animals ns cs ec ms* lf milk lung jf

2007 calves 6 60 35 40 – – 58 – 1 – 2008 calves 3 15 15 5 – – 10 – – – 2009 calves 14 1** 45 20 11 4 16 – – – 2 dairy cows – 1 – – – – – 1 – – 2010 calves 6 2** 25 16 8 7 1 2 – – – dairy cows 3 23 6 6 – – 4 62 – –

Explanations: * More than one swab delivered in a test-tube with a mycoplasma liquid medium, a mixed sample obtained from more

than one animal; ** Herds in which samples from both calves and cows were taken

Med. Weter. 2014, 70 (7)

415

M. dispar was recovered only from lavage samples,

and M. bovirhinis from the nasal cavities of all four

calves. In addition, M. arginini and M. bovoculi were

isolated from a nasal swab from one of these calves.

Coinfections with multiple Mycoplasma species were

also demonstrated in six other samples. On three

occa-sions, M. bovirihinis and M. bovoculi were identified

from nasal and/or conjunctival swabs; M. bovis with

M. bovigenitalium from lavage fluid twice; and once

with M. bovis, M. bovigenitalium, and M. dispar.

The mycoplasma positivity rate among calves and

young cattle was 32 per cent (47/145), which was four

times higher than in adult cattle (2/24 – 8 per cent). From

the two positive cow samples, one was M. bovirhinis

isolated from a nasal cavity, and the other was M. alka

-lescens isolated from a transtracheal lavage.

Mycoplasmas were detected in 22/35 (63 per cent)

of the herds examined, and more than one quarter

(9/35) had M. bovis and/or M. dispar, which are known

pathogens. A seasonal influence on the incidence of

mycoplasmas in samples was noted: an increased

oc-currence was recorded between November and March

(Figure 2). During this time of the year we received 150

samples, of which 45 were positive, and 55

mycoplasmas were identified in them. By

contrast, in warmer months we received

180 samples, of which only 8 were positive.

A number of Mycoplasma species that

cause disease in cattle have an adverse

af-fect on the welfare of farm animals and the

economics of farming (19). Mycoplasmas

were detected in clinical samples

submit-ted from across the Czech Republic and

identified by molecular methods in 27 per

cent of the samples tested by PCR/DGGE.

These results are similar to those reported

in a French study in which 25 per cent of

samples from respiratory disease had

myco-plasmas (4). The authors reported M. bovis,

but no cases of M. dispar, whereas we found

both pathogens, with M. dispar and M. bovis

at 10 and 2 per cent, respectively. M. dispar

is one of the most fastidious Mycoplasma

species, and therefore its detection at

a higher level than M. bovis is probably

a reflection on its occurrence in the Czech Republic,

although other factors, such as the site and timing of

sampling, may have influenced the species detected.

It is known that M. bovis may be shed intermittently

(22). Some samples were taken more than two weeks

after the onset of clinical signs, and sometimes after

antibiotic treatment.

Some mycoplasmas are thought to be commensals,

or possibly opportunist pathogens, and have been

re-ported to occur in Western Europe at slightly different

levels than those reported here. Ayling et al. (1), Chazel

et al. (4) reported M. bovirhinis and M. arginini at 34

and 14 per cent, respectively, whereas we report their

occurrence at 38 per cent and less than 2 per cent of

the identified mycoplasmas. Ayling et al. (1) reported

M. canadense, M. bovigenitalium and M. bovoculi

at around 1 per cent of cases, whereas in the present

study these mycoplasmas occurred at 3 to 13 per cent.

Comparison with previous reports of mycoplasmas in

the Czech cattle herds shows almost no change in the

percentage of M. bovirhinis and M. bovigenitalium, but

a considerable decrease in the identification of M. argi

-nini (32). Other mycoplasmas detected and identified

in this study were not mentioned, except

for M. bovis, in the 1970’s and 1980’s (33).

Previously, M. canadense has been

as-sociated with outbreaks of granulopapular

vulvovaginitis (13). Perhaps unusually,

M. canadense was the only organism

iso-lated from the joint fluid of an animal with

clinical signs of arthritis. It would be

inter-esting to determine this organism’s role in

this clinical condition.

Obtaining clinical samples by

transtra-cheal lavage is a more invasive process

than taking nasal swabs, but almost half of

positive samples were recovered by lavage.

M. arginini M. alkalescens M. canadense M. bovigenitalium M. bovis M. bovolculi M. dispar M. bovirhinis Year 2007 2008 2009 2010 0 2 4 6 8 10 12 Number of positive samples nasal swab conjunctival swab transtracheal lavage joint fluid

Fig. 1. Mycoplasma species found in the Czech Republic between 2007 and

2010

Fig. 2. Number of mycoplasmas identified each month between 2007 and

2010, which shows seasonal variation

January February March April May June July August September October November December

Med. Weter. 2014, 70 (7)

416

This method also reduces the possible contamination of

the sample from the upper respiratory tract and clearly

demonstrates if the lung of an individual calf or cow

is infected. This was shown here with the four calves

which had M. bovirhinis, M. arginini and M. bovoculi in

the nasal cavity, but the recognised pathogen M. dispar

was isolated from the lung lavage. This supports the

findings of Thomas et al. (29).

This study has identified the presence and

impor-tance of mycoplasma infections in cattle in the Czech

Republic, but with more than 1,300,000 cattle in the

country this small survey is probably an underestimate

of the true incidence of mycoplasmoses.

Conclusions

In the Czech Republic, the monitoring of animal

health status is mainly focused on diseases listed by the

European Commission (EC) and the OIE, to prevent

their reintroduction into the country. These include

a mycoplasma disease known as contagious bovine

pleuropneumonia. The awareness of other bovine

my-coplasmoses and non-notifiable diseases in the cattle

industry is generally low. Cattle health and economic

productivity could be improved by increasing the

aware-ness of these diseases and of the importance of early

laboratory diagnosis.

Veterinary practitioners should be made more aware

of the importance of diagnosing mycoplasma infections.

M. bovis and M. dispar, which are recognized cattle

pathogens, are of particular importance. Transtracheal

lavage is the clinical sample of choice to submit to the

laboratory for the most effective diagnosis of pathogenic

Mycoplasma species.

Further large scale studies of the prevalence of cattle

mycoplamoses in the Czech Republic would provide

more information about the extent of disease and about

the economic and welfare impact of these organisms on

cattle and farming.

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Corresponding author: MVDr. Jana Jozefová, Palackého tř. 1/3, 612 42 Brno; e-mail: mvdr.pospichalova@seznam.cz