ISRAEL JOURNAL OF
VETERINARY MEDICINE
Vol. 58 (2-3)
2003
BIOCHEMICAL AND SEROLOGICAL
IDENTIFICATION OF MYCOPLASMAS ISOLATED
FROM PNEUMONIC BOVINE LUNGS IN NIGERIA
A.T.P. Ajuwape1, J.O. Ikheloa1, M.O. Ojo1, O.O. Alaka2 and A.I.
Adetosoye1
1. Department of Veterinary Microbiology and Parasitology
2. Department of Veterinary Pathology, University of Ibadan; Ibadan. Nigeria.
Abstract
Thirty-nine strains of Mycoplasma were isolated from pneumonic lung tissues of
cattle observed at the abattoirs. The strains were biochemically characterized and
identified serologically. On the basis of biochemical characterization the strains
were divided into 3 groups. [A] Twenty-two strains which hydrolyzed glucose,
digested serum and reduced tetrazolium chloride. [B] Eight strains which
catabolized arginine only. [C] Nine strains which reduced tetrazolium chloride and
had phosphatase activity. Group A strains were identified serologically as M.
mycoides subsp. Mycoides; group B strains were identified serologically as M.
arginini while the strains in group C were identified as M. bovis. The lung tissue
showed acute fibrino-purulent pleuropneumonia. Isolation of M. bovis showed that
this organism plays an important role is severe pneumonia. Efforts should be made
to produce a vaccine against M. bovis, similar to that for M. mycoides subsp.
Mycoides, to protect calves against severe pneumonia.
Introduction
Mycoplasma mycoides subsp mycoides is the causative agent of contagious
bovine pleuropnuemonia[CBPP]; a highly contagious disease and the most
important respiratory disease in the world [4]; as well as a serious economic
disease of cattle in many parts of the world including tropical Africa, France, India,
Italy, Middle East, Portugal and Spain [18,20]. Contagious bovine
pleuropneumonia has caused greater losses in cattle than any other disease after
Rinderpest [15]. This disease was eradicated from the U. S. A. in 1892 [1]; from
South Africa by launching the JP28 campaign in 1971, but the disease has been on
the increase since 1986 [12]. In Nigeria CBPP outbreaks occurred in Kano,
Katsina, Borno, Sokoto and Kaduna States [14]. The re- emergence of the disease
in Nigeria might have been due to the introduction of chronic carriers from
neighbouring African countries such as Cameroon, Chad and Niger Republic. It
has been reported that outbreaks occurred when healthy and carrier animals gather
at river banks during the dry season [16]. This close contact between the healthy
and carrier animals facilitated the transmission of Mycoplasma responsible for the
disease. This investigation was undertaken to identify Mycoplasma species
associated with pneumonia in cattle in Nigeria.
Materials and Methods
During the rainy season, from May to September 1997, pneumonic lung tissues
and blood samples were collected from cattle with pneumonia at abattoirs in
Maiduguri, Kaduna, Sokoto and Ibadan. The clotted blood was centrifuged and the
sera were stored on ice. Each pneumonic lung was cut into two, one half was put
into 10% formalin while the remaining half was put into a sterile nylon bag, frozen
and transported on ice to Ibadan and kept at -200C at the Department of Veterinary
Microbiology and Parasitology, University of Ibadan.
Bacteriology
The tissues were seeded on blood agar, MacConkey agar and Medium N
Mycoplasma agar [9] containing no glucose, according to standard methods (2,10)
for the isolation of pathogenic bacteria and Mycoplasma.
Characterization of Mycoplasma isolates:
The Mycoplasma isolates were respectively cloned 3 times, and an isolated
colony was inoculated in Mycoplasma broth and incubated at 370C for three days
after which the culture was inoculated on Mycoplasma agar. This procedure was
repeated three times.
Absence of bacterial reversion
The Mycoplasma isolates were subcultured in Medium N [Mycoplasma agar
without penicillin].
Gaseous requirement
All the Mycoplasma isolates were grown in Mycoplasma medium N. 0.01 ml of
10 fold serial dilution from 10-6 was inoculated on blood agar and incubated
aerobically. They were also inoculated on Mycoplasma agar and incubated at 370C
in a candle jar. The plates were examined every 48 hours to record Mycoplasma
growth. The plates were inoculated for 10 days and plates not showing colonies
resembling Mycoplasma were discarded.
Morphological studies
The colonial morphology was studied with a stereomicroscope. All the strains
were grown anaerobically in a candle jar for 10 days. Each colony was examined
for morphological characteristics of Mycoplasma.
Sensitivity to digitonin
The tests were performed as the disc growth inhibition test[5]. Filter paper discs
[6.35mm in diameter] were soaked with 0.02ml of 1.5% [W/V] ethanolic solution
of digitonin [Sigma Chemical Co. St. Louis, U.S.A] and dried overnight at 370C.
Seven milliliter of medium N (Mycoplsma agar) without glucose was dispensed in
5 cm glass petri dishes. The plates were dried before use and inoculated with
0.01ml of culture containing 1x105 CFU per ml. The running drop technique was
employed. The discs were pressed gently on the middle of the inoculated area. The
plates were incubated at 370C in a candle jar. The inhibition zone was measured
after 5 days of incubation at 370C.
Tests for cholesterol requirement
This was carried out as described previously (7). The inoculated plates were
examined for growth every second day during incubation at 370C for 10 days. The
requirement for cholesterol was determined indirectly by a sensitivity test against
digitonin by the method described elsewhere [11]
Tetrazolium reduction:
The test was performed by incorporating 5ml of 2,3,5, triphenyl tetrazolium
hydrochloride [2% solution W/V] to 15ml of medium N containing no glucose in
vacutainer tubes. The pH of the medium was adjusted to 7.5.
Phosphatase activity
The test was performed by inoculating medium N with the test organism. The
medium N contained heated horse serum plus 200,000 I.U/ml penicillin, (0.20ml),
1.0ml of thallium acetate and 1ml of phenolphthalein sodium diphosphate. The pH
was adjusted to 7.8. The organisms under test were incubated for 4 days, after
which a drop of 30-40% potassium hydroxide was added. A positive reaction was
shown by a pink colour, which faded very quickly.
Test for proteolytic activity
Strands of developed black and white film, measuring 90x5mm were used. The
film was sterilized by autoclaving as 1000C for 1 hr. The sterilized films was
dropped into Medium N culture of the Mycoplasma isolate under test. During the
period of incubation the gelatin on the film was hydrolysed. A positive test showed
black granules of gelatin at the bottom of the test tube, which was incubated for 7
days.
Glucose hydrolysis
The medium and the test substrates were prepared as described previously [10].
The test substrates were made by adding 1.0ml of 50% [w/v] stock solution of
glucose to the broth medium. The medium was inoculated with a single colony.
The reaction was read by comparison with inoculated and uninoculated base
medium as well as with uninoculated test substrate. A positive result was recorded
when a colour change (red-yellow) between the inoculated test substrate and each
of the control substrates was present. For negative reaction there was no colour
change. The test was observed for 14 days.
Catabolism of Arginine
The base medium was the same as for glucose fermentation. The pH was
adjusted to 7.3. The test substrate consisted of base medium with the addition of
4.25ml L-arginine[Sigma] of 30% (w/v) stock solution.
Formation of Film and Spots
Medium N was prepared with a high (40ml) concentration of the horse serum.
The plates were inoculated and incubated at 370C for 14 days. They were
examined for film and spots after 3,7 and 14 days incubation.
Growth inhibition test
The test was carried out by the running drop technique. Medium N [9] was
prepared. The agar plated was inoculated with 0.01ml of test containing 105 and
106†CFU/ml. After drying, a 6mm disc soaked with antiserum was gently placed
in the middle of the Mycoplasma streak. The plate was incubated at 370C for
arginine-positive strains while others were incubated at a lower temperature (room
temperature) in an anaerobic, candle jar.
Histopathology
Lung tissues were fixed in 10% phosphate buffered formalin dehydrated in
graded dilutions of ethanol and embedded in paraffin. Thin sections (5µ) of tissues
obtained on the grease-free glass slides were stained with heamotoxylin and eosin.
Stained sections were examined for histological changes.
Results
Morphological studies:- colonies of Mycoplasma strains after incubation at 370C
for four days exhibited the characteristic fried egg appearance with an elevated
central spot.
Reversion experiment
No bacteria-like colonies developed on the media without penicillin during
passage of the strains.
Cholesterol requirement
All strains were digitonin sensitive.
Gaseous requirement
They all grew well aerobically under candle jar.
Growth at 370C:
All the strains grew well at 370C.
Biochemical characteristics
The strains were biochemically studied using glucose, arginine, phosphate, 2, 3,
5-tetrazolium chloride, protein digestion. Table I shows the biochemical reactions
of the isolates.
Table 1. Biochemical characteristics of bovine mycoplasmas.
No. of strains
Sensitivity to
digitonin
(mm)
Catabolism of Reduction of
arginine
tetrazolium
chloride
Phosphatase
activity
Digestion of
serum
Film & Sp
M.mycoides
22
4-5
-
+
-
+
-
M.arginini
8
3-4
+
-
-
-
-
M.bovis
9
4.5 - 6
-
+
+
-
-
Growth inhibition test
The growth of 22 Mycoplasma strains which fermented glucose, was inhibited by
the antiserum to M. mycoides subsp. Mycoides. The growth of 8 Mycoplasma
strains was inhibited by antiserum to M. arginni while the growth of 9 Mycoplasma
strains were inhibited by antiserum to M. bovis.
Film and Spots; None of the Mycoplasma strains showed film or spots.
Histopathology
The pneumonic lung tissues showed typical acute fibrinopurulent pleuropneumonia consistent with those associated
with early stages of contagious bovine pleuropnuemonia.
Figure I shows severe congestion, oedema and fibrin
exudation into thickened interalveolar spaces, subpleural
space and into alveolar lumen. Some alveoli were
confluent and emphysematous, while a few were collapsed.
Numerous neutrophils, macrophages, many of which have
haemosiderin pigments and engulfed erythrocytes, and
epithelioid cells were found in distended interalveolar
septa and around blood vessels. Bronchiolar epithelium
were hyperplastic and folded and considerable quantity of
oedematous fluid and inflammatory cells were found in the
lumen.
No pathogenic bacteria other than Mycoplasma
organisms, of any clinical significance, were isolated from
these lung tissues.
Figure 1b, shows lung tissue with large quantities of
fibrin in interlobular spaces. H & E x 100.
Figure 1a, shows histopathological c
tissue with severe pulmonary oedema and
with infiltration of neutrophils H & E x 100
Figure 1c, shows lung tissue with neutroph
spaces H & E x 100.
Table 2. Summary of growth inhibition tests on mycoplasma strains grouped by biochemical
characterization.
Species group
Antisera
M. mycoides
subsp.mycoides
M. arginini
M. bovis
M.mycoides subsp
mycoides A
22
-
-
M. arginini B
-
8
-
M. bovis
-
-
9
Discussion
In this study, Mycoplasma mycoides subsp mycoides, M. bovis and M. arginini
were isolated from the pneumonic lung tissues of cattle slaughtered at the abattoirs.
In spite of the JP 28 campaign of 1971 to eradicate and control CBPP in Africa and
the annual vaccination of cattle against CBPP in Nigeria, especially in the Northern
States where about 90% of Nigerian cattle live, CBPP, a deadly and the most
important respiratory disease of cattle, is still present in Nigeria.
M.mycoides subsp. mycoides were isolated from pneumonic lungs at slaughter in
Sokoto and Kaduna abattoirs. The prescence of CBPP in these States and the
recovery of this species of Mycoplasma from pneumonic cattle might have been
due to the introduction of carrier animals into Nigeria from the neighbouring
countries of Niger, Chad and Cameroon. The animals may have been missed
during vaccination or they may have been vaccinated but due to poor storage of the
vaccine, the vaccinated animal might not have been protected.
In the study M. bovis was isolated from pneumonic lung tissues of cattle
observered at the abattoirs in Maiduguri. Similar finding was obtained recently [8].
At that time M. bovis, M. dispar, M. ovipneumoniae and Acholeplasma laidlawi
were isolated from lung tissues collected at Maiduguri abattoirs. In this
investigation 31% of the sera collected were positive for M. bovis. Reports from
other parts of the world (3,12,15,19) indicated that M. bovis is an important
pathogenic agent responsible for severe pneumonia in cattle.
Consequent to the isolation of M. mycoides subsp mycoides and M. bovis in this
investigation, it is suggested that efforts should be made to ensure that cattle are
vaccinated regularly against Mycoplasma species causing pneumonia from the
species of Mycoplasma responsible for the disease in such areas. The vaccine
should be used along with TI-44 vaccine. Movement of animals from neighbouring
countries should be properly controlled. Government agencies should ensure that
animals crossing Nigerian borders are vaccinated and free of CBPP to prevent the
introduction of carriers into the herd.
Acknowledgement
This work was financed by the National Agricultural Research Project RGS 024 and Prof. A.I.
Adetosoye. Department of Veterinary Microbiology and Parasitology University of Ibadan; Ibadan.
Nigeria.
LINKS TO OTHER ARTICLES IN THIS ISSUE
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Acknowledgement
This work was financed by the National Agricultural Research Project RGS 024 and Prof. A.I.
Adetosoye. Department of Veterinary Microbiology and Parasitology University of Ibadan; Ibadan.
Nigeria.