Veterinary Microbiology 111 (2005) 159–169

www.elsevier.com/locate/vetmic

Identification of three novel mycoplasma species from

ostriches in South Africa
A. Botes a, B.M. Peyrot b, A.J. Olivier b, W.P. Burger b, D.U. Bellstedt a,*
a
Department of Biochemistry, University of Stellenbosch, Stellenbosch, South Africa
b
Research and Development Division, Klein Karoo Group, Oudtshoorn, South Africa

Received 27 May 2005; received in revised form 30 September 2005; accepted 4 October 2005

Abstract

Mycoplasmas have been implicated in certain clinical syndromes in ostriches and are associated with upper respiratory tract
infections. As these infections result in production losses, they are of considerable economic importance to the South African
ostrich industry. Although poultry mycoplasmas have been shown to infect ostriches, the existence of unique ostrich-specific
mycoplasmas has been suggested. In this study, mycoplasmas were isolated from ostriches in the Klein Karoo, Central Karoo
and Garden Route areas of the Western and Northern Cape Provinces of South Africa and identified using 16S rRNA gene
sequencing. These sequences indicated that ostriches in these areas carry three unique mycoplasmas and were not infected with
chicken mycoplasmas. Phylogenetic analysis of the 16S rRNA sequences of the three isolated ostrich mycoplasmas showed
them to be quite divergent and to fall into two distinct phylogenetic groupings. Unique sequences within the 16S rRNA gene of
the ostrich mycoplasmas were subsequently used for the development of specific primers for the detection and diagnosis of
mycoplasma infections in ostriches. Chickens kept in close proximity to infected ostriches were not infected with these ostrich
mycoplasmas.
# 2005 Elsevier B.V. All rights reserved.

Keywords: Ostrich; Mycoplasma; Specific PCR; Ostrich disease

1. Introduction come to the fore. In these syndromes, mycoplasmas

In South Africa, increased commercial ostrich
production has necessitated a system of intensive
rearing, during which certain clinical syndromes have
* Corresponding author. Present address: Department of Biochem-
istry, University of Stellenbosch, Private Bag X1, Matieland 7602,
South Africa. Tel.: +27 21 8085840; fax: +27 21 8085863.
E-mail address: dub@sun.ac.za (D.U. Bellstedt).
have been implicated together with other pathogens
such as Escherichia coli, Pseudomonas aeruginosa,
Pasteurella species and occasionally Avibacterium
paragallinarum (Verwoerd, 2000; Blackall et al.,
2005). As this results in stock losses, reduced produc-
tion and, when secondary infections are present,
downgrading of carcasses, mycoplasmas are viewed to
be of considerable economic importance to the South
African ostrich industry.

0378-1135/$ – see front matter # 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2005.10.017
160 A. Botes et al. / Veterinary Microbiology 111 (2005) 159–169
Mycoplasmas in ostriches are usually associated
with respiratory disease and cause an inflammation
of the upper respiratory tract (Huchzermeyer, 1994;
Verwoerd, 2000). Poultry mycoplasmas have long
been thought to be responsible for these infections.
Verwoerd (2000) reported that M. synoviae had been
isolated from the respiratory tract of ostriches with
respiratory symptoms. Peccati et al. (1996) found
that ostriches showed immune responses to poultry
mycoplasmas (M. gallisepticum, M. synoviae and M.
meleagridis) when kept together with poultry in
northern Italy, although infection could not be
determined. Cadman et al. (1994) also documented
antibody responses to M. gallisepticum and/or M.
synoviae in slaughter ostriches in Zimbabwe. Cline
et al. (1997) found that ostriches could be infected
experimentally with M. gallisepticum and show
clinical signs of disease. Cline et al. (1997) were,
however, not successful in experimentally infecting
ostriches with M. synoviae. Shivaprasad (1993), on
the other hand, reported that mycoplasmas occur in
ostriches, but established that these mycoplasmas
were not M. gallisepticum, M. synoviae, M.
meleagridis or M. iowae, which are typically
associated with respiratory infections in poultry.
This indicates that ostriches may harbour unique
ostrich-specific mycoplasmas, which have also been
suggested by other authors (Smith, 1993; Shane,
1998).
The identification and classification of mycoplas-
mas is hampered by the relative scarcity of useful
morphological and biochemical characters. This has
prompted the much wider use of molecular genetic
techniques in mycoplasma identification and classi-
fication (Weisburg et al., 1989). The 16S rRNA gene is
unique to prokaryotes and is routinely used for
identification purposes. In addition, sequence analysis
of conserved genes such as the 16S rRNA gene, allows
phylogenetic relationships to be established between
mycoplasmas thereby making the identification of
novel species possible without laboratory cultivation
(Wang and Wang, 1996).
In this study, mycoplasmas were isolated from
ostriches in the main ostrich producing areas in South
Africa, identified using 16S rRNA gene sequencing
and their phylogenetic relationships determined.
Unique sequences within this gene were subsequently
used for the development of PCR assays for the
detection and diagnosis of mycoplasma infections in
ostriches. Chickens held in close proximity to
ostriches were tested for mycoplasma infections with
a view to establishing whether they could be infected
with ostrich mycoplasmas.
2. Materials and methods
2.1. Sample collection and processing
A total of 206 (3 eye, 17 sinus, 4 choana, 161
trachea, 14 air sac, 2 jejenum, 2 ileum, 1 caecum, 1
colon and 1 yolk) swabs were collected from ostriches
on ostrich farms in the Northern and Western Cape
Provinces, South Africa. These farms were situated in
the Klein Karoo, the Central Karoo and Garden Route
areas (see Fig. 1). Samples were collected from
ostriches showing symptoms of respiratory infection,
either from live birds or during post-mortem
examinations.
Thirty-four trachea swabs were taken from back-
yard chickens on farms in the Oudtshoorn district.
These chickens had unrestricted access to ostrich pens
whereby they came into direct physical contact with
ostriches.
In most cases, samples collected were immediately
used for mycoplasma cultivation. If immediate
processing of samples was not possible, swabs were
placed into Amies transport medium without charcoal
and stored at 4 8C for up to 24 h. Modified Chanock’s
broth medium could also be used as an effective
transport medium. Penicillin (Novapen, Novo Nor-
disk, Denmark) at 500–1000 units/ml was added to the
medium to suppress bacterial contaminants.
2.2. Cultivation of mycoplasmas
Mycoplasmas were cultivated at the Klein Karoo
Research and Diagnostic Laboratory in Oudtshoorn.
Swabs were plated onto sterile modified Chanock’s
agar medium (OIE, 2000) and the plates incubated in
candle jars at 37 8C for 2–21 days. The plates were
examined every two days using a stereomicroscope at
10–40
magnification with obliquely transmitted
light. A single colony of primary cultures of each
of the isolates was subsequently used for DNA
extraction and mycoplasma identification.
 A. Botes et al. / Veterinary Microbiology 111 (2005) 159–169
Fig. 1. A map showing the different districts from where ostrich mycoplasma positive samples were obtained in the Western Cape and Northern
Cape Province, South Africa.
2.3. Isolation of genomic DNA
DNA was extracted from the mycoplasma-contain-
ing solid agar using the N-cetyl-N,N,N-trimethyl
ammonium bromide (CTAB) method of Doyle and
Doyle (1987). The CTAB method was originally
developed for the extraction of genomic DNA from
fresh plant tissue. Briefly, 500 ml of a 2
CTAB buffer
(100 mM Tris–HCl, pH 8.0; 1.4 M NaCl; 20 mM
EDTA, pH 8.0; 2%, v/v, CTAB; 0.2%, v/v, 2-
mercaptoethanol) was added to the mycoplasma-
containing agar and incubated at 60 8C for 1 h. To this,
500 ml chloroform-isoamylalcohol (24:1, v/v) was
added and gently mixed for 10 min followed by
centrifugation for 5 min at 7000
g. The upper
aqueous phase was removed and to this, a 2/3 volume
of cold isopropanol was added and mixed gently. This
161
sample was incubated overnight at 20 8C to allow for
the precipitation of nucleic acids. The sample was
subsequently centrifuged for 2 min at 3000
g. The
supernatant was decanted and 1.5 ml wash buffer
(40 mM ammonium acetate:absolute ethanol, 1:3)
added to the pellet. The pellet was resuspended in the
wash buffer and incubated at room temperature for
20 min. This was followed by centrifugation for 1 min
at 3000
g, after which the supernatant was once
again decanted and the pellet air-dried to remove any
remaining ethanol. The DNA pellet was finally
redissolved overnight at 4 8C in 250 ml TE-buffer
(10 mM Tris–HCl, pH 8.0; 1 mM EDTA, pH 8.0).
A negative control sample was obtained by
performing DNA isolation on the modified Chanock’s
agar medium, which had not been inoculated with
mycoplasmas.
162 A. Botes et al. / Veterinary Microbiology 111 (2005) 159–169
2.4. Primer choice for amplification of the 16S
rRNA gene
The presence of mycoplasmas was verified by PCR
based on the use of a 16S rRNA general primer pair
(GPO3 and MGSO), used for the identification of
mycoplasmas at both genus and species level (Van
Kuppeveld et al., 1992). The GPO3 primer sequence
was, however, slightly modified by moving the
position of the primer on the 16S rRNA sequence
two basepairs upstream. This was done to avoid the
possibility of secondary structure formation of the
GPO3 primer (as indicated by the computer program
Primer Designer, version 2.0), and the primer was
subsequently renamed GPO3F.
The complete 16S rRNA gene of mycoplasmas was
amplified using the 16S rRNA primers developed for
E. coli (Hauben et al., 1997) in combination with the
general mycoplasma primers. The primer combina-
tions of 16F27 and MGSO as well as GPO3F and
16R1541, were used to amplify the entire 16S rRNA
gene as two overlapping fragments as illustrated in
Fig. 2. All primers were synthesized by the DNA
Synthesis Laboratory, Department of Molecular and
Cellular Biology, University of Cape Town. Primer
sequences are shown in Table 1.
2.5. PCR amplification of the mycoplasma 16S
rRNA gene
Amplification reactions using primers GPO3F and
MGSO were carried out in 20 ml volumes. Each
Fig. 2. Primer strategy used for the PCR detection, amplification and sequencing of the mycoplasma 16S rRNA gene. The 16F27, MGSO and
16R1541 primers were subsequently used for the sequencing of the entire mycoplasma 16S rRNA gene.
reaction mixture contained 2 ml of 10
reaction buffer
(RB, JMR-Holdings, USA), 0.8 ml of 5 mM of each
deoxynucleotide (dATP, dCTP, dGTP and dTTP;
Advanced Biotechnologies Ltd., UK), 0.4 ml of each
primer (20 pmol/ml), 1.6 ml of 25 mM MgCl2, 0.04 ml
of Taq DNA polymerase (0.2 units; Super-Therm Taq
polymerase, JMR-Holdings, USA), 12.76 ml deio-
nized and sterile filtered water and 2 ml of purified
DNA.
Amplification of the 16S rRNA gene using primer
pairs 16F27 and MGSO as well as GPO3F and
16R1541 was carried out in 50 ml reaction volumes.
Each reaction mixture contained 5 ml of 10
RB, 2 ml
of 5 mM of each deoxynucleotide (dATP, dCTP, dGTP
and dTTP), 1 ml of each primer (20 pmol/ml), 4 ml of
25 mM MgCl2, 0.1 ml of Taq DNA polymerase (0.2
units), 31.90 ml deionized and sterile filtered water
and 5 ml of purified DNA.
All the above DNA amplifications were performed
in a Hybaid PCR Express Thermal Cycler. The
thermal cycler was programmed to perform 35 cycles
of 94 8C (45 s), 55 8C (45 s) and 72 8C (1 min 30 s),
followed by a final extension reaction for 6 min at
72 8C. A negative control sample was included in each
PCR reaction.
2.6. Detection of PCR products
Amplified DNA was analyzed by gel electrophor-
esis. Ten microliters of the PCR product, mixed with
a 0.1 volume of gel loading buffer (50% glycerol;
0.1%, v/v, bromophenol blue; 50 mM EDTA; 100 mM
 A. Botes et al. / Veterinary Microbiology 111 (2005) 159–169 163

Table 1
Primers used for amplification, sequencing and detection of ostrich mycoplasmas
Primer Sequence bp-position Tm (8C)a
General mycoplasma primers (Van Kuppeveld et al., 1992)
GPO3F (F) 50 -TGGGGAGCAAACAGGATTAGATACC-30 745 78
MGSO (R) 50 -TGCACCATCTGTCACTCTGTTAACCTC-30 1017 79
16S rRNA primers developed for E. coli (Hauben et al., 1997)
16F27 (F) 50 -AGAGTTTGATC(A/C)TGGCTCAG-30 8b 59
16F530 (F) 50 -T(C/T)(C/T)GTGCCAGCAGCCGCGG-30 512b 66
16R1541 (R) 50 -GGTTGGATCACCTCCTT-30 1525b 52
Ms01-specific primers
MS012 (F) 50 -AACATTAGTTAATGCCGGATACGC-30 114 75
MS01D (R) 50 -GCCAGTATCCAAAGCGAGCC-30 613 75
Ms02-specific primers
MS02H (F) 50 -AATATAAAAGGAGCGTTTGC-30 160 70
MS02A (R) 50 -AAGGCAATAGCATTTCCTCTACT-30 447 70
Ms03-specific primers
MS03A (F) 50 -AGTGCTAATGCCGGATACTTATAC-30 118 70
MS03C (R) 50 -CGTTAACCTCTATACAATTCTAGCG-30 639 70
(F) Forward primer; (R) reverse primer.
a
Tm as calculated by the computer program Primer Designer, version 2.0.
b
Numbering of target position based on that of the E. coli 16S rRNA sequence.

Tris–base, pH 8.0) was separated on a 2% agarose gel
(Molecular Grade Agarose D1-LE, Whitehead Scien-
tific) in 1
TAE buffer (Tris–base; glacial acetic acid;
0.5 M EDTA, pH 8.0). Ethidium bromide (0.175 mg/
ml) was included in the gel for ultraviolet (UV)
visualization of the DNA.
2.7. Sequencing of the mycoplasma 16S rRNA
gene
The PCR products of primer pairs 16F27 and
MGSO (medium fragment, 1048 bp) as well as
GPO3F and 16R1541 (small fragment, 769 bp) were
purified as templates for sequencing reactions (see
Fig. 2). Firstly, PCR products were electrophoresed on
a 0.5% agarose gel in 1
TAE buffer containing
ethidium bromide (0.15 mg/ml) as previously
described. DNA containing bands were excised under
a UV light and purified using the Promega Wizard
PCR Prep Kit according to the manufacturers’
instructions.
The purified DNA samples were each concentrated
by centrifugal evaporation to 20 ml on a Savant
Speedvac and subsequently analyzed by gel electro-
phoresis. Two microliters of the PCR product, mixed
with a 0.1 ml volume of gel loading buffer (50%
glycerol; 0.1% bromophenol blue; 50 mM EDTA;
100 mM Tris–base, pH 8.0) was separated on a 2%
agarose gel in 1
TAE buffer. Ethidium bromide
(0.175 mg/ml) was included in the gel for UV
visualization of the DNA.
Sequencing reactions on the purified DNA tem-
plates were prepared using the ABI PRISM1
BigDyeTM Terminator v3.0 Cycle Sequencing Ready
Reaction Kit. The medium fragment was sequenced
with primers 16F27 and MGSO, and the small
fragment was sequenced with primer 16R1541 (see
Fig. 2). Amplifications were performed in a Hybaid
PCR Express Thermal Cycler, programmed to per-
form 35 cycles of 96 8C (10 s), 52 8C (30 s) and 60 8C
(4 min), followed by a final extension reaction of
10 min at 60 8C. Sequencing PCR reaction products
were analyzed with an ABI PRISM1 373 DNA
Sequencer at the DNA sequencing facility of the
University of Stellenbosch.
2.8. Phylogenetic analysis
Sequence data generated using each of the three
primers were combined to form a contiguous 16S
rRNA gene sequence. These sequences were then
aligned to and compared with other avian 16S rRNA
164 A. Botes et al. / Veterinary Microbiology 111 (2005) 159–169
gene sequences available on the NCBI nucleotide
sequence database as well as sequences of other related
Mollicutes and bacteria. Sequences were initially
aligned using the automatic alignment function of
the DNA and Protein Sequence Alignment (DAPSA)
program (Harley, 1998) and final alignments were made
manually. Three D loop regions, of which the alignment
was often ambiguous, were excluded from the final
analysis. The final length of the aligned data set was
1417 bp of which 707 were constant, 147 parsimony
uninformative and 563 parsimony informative. The
aligned sequence matrix was subsequently imported
into the BioEdit program and used to generate a Nexus
file for phylogenetic analysis.
The data matrix was analysed by parsimony using
the programme PAUP* version 4.0b10 (Swofford,
1993) which treats missing and gap character states as
missing data. The most parsimonious trees were
sought using 1000 replicates of random addition
sequence with TBR branch swapping, holding a
maximum of 10 trees during random addition and with
MulTrees on. Clade support was calculated with 1000
bootstrap replicates using TBR swapping with random
addition and MulTrees on.
2.9. Development for specific ostrich mycoplasma
PCRs
Based on computer alignments (DAPSA) of the
16S rRNA gene sequences of Ms01, Ms02 and Ms03,
primer pairs were selected for each from an area in
which the sequence of each mycoplasma was unique
(Neefs et al., 1990). Primers were designed using the
computer software package, Primer Designer Version
2.0. Primer sequences are shown in Table 1. Optimal
cycling times and annealing temperatures were
determined in order to yield optimum specificity.
Negative control samples were included in all
analyses. The 34 samples obtained from chickens in
the Oudtshoorn district were also included in these
analyses.
Amplification reactions were carried out in 20 ml
volumes as described before for mycoplasma detec-
tion using the respective ostrich mycoplasma-specific
primers. DNA amplifications were performed using 30
cycles of 94 8C (30 s), 57 8C (15 s) and 72 8C (1 min),
followed by a final extension reaction for 6 min at
72 8C. PCR products were separated by gel electro-
phoresis and visualized as previously described. The
identity of PCR products was confirmed by sequen-
cing as previously described.
3. Results
3.1. Cultivation of mycoplasmas
Mycoplasma colonies were usually visible by 48–
96 h after inoculation of the plates. Colony growth was
typical of mycoplasmas, exhibiting the classic fried-
egg morphology particularly in well-developed
colonies. This morphology was, however, not always
clearly seen in young cultures. The mycoplasmas
tended to penetrate the surface of solid media,
resulting in a central nucleus that remained embedded
in the medium while the rest of the colony (peripheral
zone) could be removed by gently scraping over the
agar.
3.2. Preparation of genomic DNA
The CTAB method, commonly used for the
isolation of plant DNA, was chosen for the extraction
of mycoplasma DNA from agar-containing media
because agar is of plant origin. Mycoplasma DNA
could be isolated successfully from the solid agar
medium using the CTAB method as evidenced by
spectroscopy (absorption at 260 and 280 nm) and the
numerous positive PCRs subsequently performed on
these samples.
3.3. Identification of mycoplasma-containing
samples
Mycoplasma-containing samples were identified
by PCR using the general mycoplasma primers
(GPO3F and MGSO), which produced an amplifica-
tion product of 270 bp. Such amplification products
were obtained from 185 samples. These positive
samples were from the eyes (2), sinuses (12), choana
(3), trachea (154), air sacs (13) and caecum (1), while
none of the samples from the jejunum, ileum, colon or
yolk gave PCR products. The clinical origin of the
mycoplasma positive samples is shown in Table 2. The
mycoplasma positive isolates from these samples were
subsequently subjected to sequencing analysis.
 A. Botes et al. / Veterinary Microbiology 111 (2005) 159–169 165

Table 2 fragment with 16R1541) routinely gave sequencing

Clinical origin of mycoplasma positive samples
runs of 600–700 bp in length. These sequences
Tissue Ms01 Ms02 Ms03 Ms01/3 Ms02/3 Total overlapped extensively and a contiguous 16S rRNA
Trachea 20 16 99 15 4 154 sequence was generated for each mycoplasma-con-
Sinus 11 1 12 taining sample.
Air sac 4 2 7 13
Three unique mycoplasma sequences were identi-
Choana 1 2 3
Eye 2 2 fied by sequencing of the 16S rRNA gene of the
Caecum 1 1 mycoplasma isolates obtained from the ostrich
Jejunum samples. The three mycoplasmas species were named
Ileum Ms01, Ms02 and Ms03. The 16S rRNA sequences of
Colon
these mycoplasmas were deposited in GenBank
Yolk
(GenBank Accession Numbers DQ223545, DQ22
Total 35 20 111 15 4 185 3546 and DQ223547 for Ms01, Ms02 and Ms03,
respectively). The different ostrich mycoplasmas
isolated in each of the districts from which samples
3.4. Amplication and sequencing of 16S rRNA were collected are shown in Table 3. No other known
genes of mycoplasma DNA poultry or avian mycoplasmas were isolated from any
of the ostrich samples. None of 34 mycoplasma-
Amplification of the entire 16S rRNA gene of containing samples from chickens was found to
isolated mycoplasma DNA was achieved by using the contain any of the three ostrich mycoplasmas, but
primer combinations 16F27 and MGSO as well as instead contained M. gallinaceum (8), M. gallinarum
GPO3F and 16R1541 which produced overlapping (13) and M. pullorum (9). Two samples had mixed
amplification products of 1048 and 769 bp, respec- infection of M. gallinaceum and M. gallinarum, one of
tively. Sequencing of these fragments (the 1048 bp M. gallinaceum and M. pullorum, and one of M.
fragment with 16F27 and MGSO, and the 769 bp gallinarum and M. pullorum.

Table 3
Incidence of Ms01, Ms02 and Ms03 in each of the districts from where samples were obtained
District Mycoplasma
Ms01 Ms02 Ms03 Ms01 and Ms03 Ms02 and Ms03
Klein Karoo
Ladismith 2/22 2/22 13/22 5/22 –
Calitzdorp 11/19 2/19 4/19 2/19 –
Oudtshoorn 14/76 9/76 39/76 – 3/76
Volmoed 2/27 5/27 19/27 – 1/27
De Rust 5/14 – 6/14 3/14 –
Uniondale – – 2/2 – –
Noll – – 1/1 – –
Schoemanshoek – – 7/7 – –
Herold – – 2/2 – –
Lategansvlei 1/12 – 11/12 – –
Garden Route
Heidelberg – 1/2 1/2 – –
Riversdale – – 2/2 – –
Albertinia – – 1/1 – –
Mossel Bay – 1/7 1/7 5/7 –
Central Karoo
Victoria-West – – 2/2 – –
166 A. Botes et al. / Veterinary Microbiology 111 (2005) 159–169
3.5. Phylogenetic analysis
Alignment data showed 88.4% sequence similarity
between Ms01 and Ms02, 88.7% sequence similarity
between Ms01 and Ms03 and 93.1% sequence
similarity between Ms02 and Ms03, respectively.
Tree searches produced a single tree of a length of
2855 steps, with a consistency index of 0.412 and a
retention index of 0.697 (Fig. 3). Nodal support was
only regarded as significant if bootstrap values
exceeded 70%. The phylogeny retrieved is in
agreement with previously obtained phylogenies of
the Mollicutes (Weisburg et al., 1989). Using
Clostridium perfringens as outgroup, the genus
Aneuroplasma was basal to the rest of the Mollicutes.
The genus Mycoplasma was found not to be
monophyletic. A basal lineage, including a clade
containing the genus Spiroplasma and M. mycoides,
and the genus Ureaplasma and some other members of
Mycoplasma, was retrieved with strong bootstrap
support. The remaining members of Mycoplasma were
retrieved in a second strongly supported clade. In this
clade, two further strongly supported clades were
retrieved. Ms02 and Ms03 were found in one clade in
which Ms03 was found to be closely related to M.
gallinaceum and Ms02 occupied a basal position close
to M. synoviae. Ms01 falls into a separate clade with
its closest relative being M. falconis. According to the
alignment data, Ms01 and M. falconis have 97.8%,
Ms02 and M. synoviae have 92.2% and Ms03 and M.
gallinaceum have 94.6% sequence similarity, respec-
tively.
3.6. Development for specific ostrich mycoplasma
PCRs
Alignment of the 16S rRNA sequences of Ms01,
Ms02 and Ms03 with other mycoplasma species
allowed the identification of regions with conserved
and variable sequences. A number of primers
potentially specific for each of the ostrich mycoplas-
mas were designed based on these variable areas.
Primers that showed the required specificity for each
ostrich mycoplasma, were MS012 and MS01D for
Ms01, MS02H and MS02A for Ms02 and MS03A and
MS03C for Ms03, respectively (Table 1). The specific
primer pairs for Ms01, Ms02 and Ms03 produced PCR
products of 494, 288 and 517 bp, respectively. These
were sequenced to confirm that the correct fragment
had been amplified. Each of the 185 mycoplasma
positive ostrich samples, negative control samples and
chicken mycoplasma-containing samples were tested
separately by PCR using each of the ostrich-specific
mycoplasma primer pairs. The specific PCRs were
able to successfully differentiate between the three
different ostrich mycoplasmas. The specific primers
were also able to identify mixed infections (as
indicated in Figs. 2 and 3). Negative controls and
poultry mycoplasma-containing samples obtained
from chickens gave no amplification products in
any of the ostrich mycoplasma-specific PCRs.
4. Discussion
By sequencing the 16S rRNA gene of the
mycoplasma positive samples it was found that
ostriches carry three unique mycoplasmas in the main
ostrich producing areas in South Africa. This confirms
the existence of unique mycoplasma species in
ostriches, which has previously been suggested by
other authors (Shivaprasad, 1993; Smith, 1993; Shane,
1998). The identification of unique mycoplasma
sequences alone does not meet with the requirements
for the formal description of new Mollicutes species.
These also include a variety of biochemical tests as laid
down by the Mollicutes Taxonomy Committee (Whit-
comb et al., 1995). However, due to the difficulties
experienced in the cultivation of all three ostrich
mycoplasmas, these tests have not yet been performed,
and for this reason cannot be formally described here.
The three mycoplasmas were therefore provisionally
named Ms01, Ms02 and Ms03 until formally described.
Mycoplasmas are usually named according to their host
or disease pathology and ostrich mycoplasmas should
therefore be named ‘Mycoplasma struthiolus’ (Ms)
after their host, Struthio camelus.
No specific geographic trends in the distribution of
these mycoplasmas could be established. Ms03 was
widely distributed in all of the districts sampled in the
Central Karoo, Klein Karoo and Garden Route area of
the Northern and Western Cape Province, South Africa,
while Ms01 and Ms02 were found in fewer districts.
The most likely reason for this is that ostriches are often
moved from one farm to another, which would disturb
any natural distribution patterns. Mixed infections of
 A. Botes et al. / Veterinary Microbiology 111 (2005) 159–169 167

Fig. 3. The single most parsimonius 16S rRNA gene tree of avian mycoplasmas found in the phylogenetic analysis. Branch lengths and bootstrap
values are indicated above and below the line, respectively. Bootstrap values below 50 are not shown.
168 A. Botes et al. / Veterinary Microbiology 111 (2005) 159–169
more than one ostrich mycoplasma were also found in a
number of cases. None of the chickens that were tested
were infected with any of the three ostrich mycoplas-
mas but instead were infected with the typical poultry
mycoplasmas, M. gallinaceum, M. gallinarum and M.
pullorum. The evidence presented here therefore
indicates that the Ms01, Ms02 and Ms03 possess
distinct host specificity for ostriches, and that poultry is
not infected with these ostrich mycoplasmas in the
South African farming situation. Our results showed
that no ostriches were infected with chicken myco-
plasmas in the areas that we investigated, even where
ostriches were kept in close proximity to poultry. This
may, however, be because no commercial poultry
flocks, in accordance with the recommendations made
by Verwoerd (2000), were kept on ostrich farms.
The 16S rRNA gene has previously been found to
be a convenient genetic marker to compare the
phylogenetic relationships among avian mycoplasmas
(Weisburg et al., 1989) and the phylogeny established
in this study was fully congruent with that established
by these authors. Phylogenetic analysis of the 16S
rRNA gene sequences of the three isolated ostrich
mycoplasmas, showed them to be quite divergent and
fall into two distinct mycoplasma groupings. Ms01
appears to be phylogenetically distinct from the other
ostrich mycoplasmas and falls in a different clade with
M. falconis being its closest relative. The analysis
indicated that Ms02 and Ms03 grouped in the same
clade as M. synoviae and M. meleagridis. Due to this
close relationship, Ms02 and Ms03 may have been
previously misidentified as M. synoviae in ostriches
(Verwoerd, 2000). As antibodies to Ms02 and Ms03
may possess cross-reactivity to M. synoviae and M.
meleagridis, this may also explain why Peccati et al.
(1996) found that ostriches possessed antibodies to M.
synoviae and M. meleagridis, although an infection
with these mycoplasmas cannot be excluded in their
study as they were kept with poultry. The fact that
Cline et al. (1997) were not successful in experimen-
tally infecting ostriches with M. synoviae, may also
indicate that this mycoplasma does not possess the
ability to infect ostriches. As Ms03 is closely related to
M. gallinaceum, a typical poultry mycoplasma, one
might have expected that it may have infected poultry,
and reciprocally that ostriches would be susceptible to
M. gallinaceum. The evidence presented in this study
indicates that neither occurs and that these myco-
plasmas possess distinct host specificity in the field
situation in these areas in South Africa.
In this study, samples were taken from ostriches with
upper respiratory tract infections. A high incidence of
mycoplasma positive samples was obtained, primarily
from the upper respiratory system and eyes and only one
positive sample from the alimentary tract. Although
further studies will have to be undertaken to confirm
whether these organisms are primarily responsible for
respiratory disease or only cause secondary infections,
these results do indicate that mycoplasmas are often
associated with respiratory disease in ostriches. As this
study was heavily biased to sampling from the res-
piratory tract, further studies in which greater numbers
of samples from the alimentary tract are analysed, will
have to be undertaken to assess if mycoplasmas can also
occur in the alimentary tract of the ostrich.
Differences between the 16S rRNA sequences of the
ostrich mycoplasmas were used for the development of
ostrich mycoplasma-specific primers. PCR is an ideal
method for the diagnosis of diseases where the
infectious agents, such as mycoplasmas, are time-
consuming to isolate and difficult to identify (Kiss et al.,
1997). It has the further advantage of being able to
detect infections before symptoms are visible (Moalic
et al., 1997). For this reason, these specific PCR assays
are now being used routinely in the research laboratory
of the Klein Karoo Co-operative in Oudtshoorn to
detect mycoplasmas in ostriches. The ostrich myco-
plasma-specific PCR assays developed in this study will
be very valuable in determining whether mycoplasmas
are responsible for respiratory and alimentary tract
infections in ostriches in South Africa in future studies.
Acknowledgements
The financial assistance of the Klein Karoo Group
is gratefully acknowledged. C.A. Mutlow, S. Abra-
hams and C.A. de Villiers are thanked for their expert
technical assistance.
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