Isolation and prevalence of Mycoplasma agalactiae in Kurdish

Aim: Ruminant Mycoplasmosis are important diseases worldwide and several are listed by the World Organization for Animal Health (OIE) to be of major economic significant. The aim of this study was to isolation mycoplasmas from sheep presenting contagious agalactiae (CA) in Kurdistan in the West of Iran. Materials and Methods: Sixty-nine samples included (milk, conjuctiva swabs, synovial fluid and ear canal swabs) were examined by PCR assay during 2011-2012. DNA was extracted from enriched samples. Two primers (forward and reverse) amplify a 163bp region of 16S rRNA gene of Mycoplasma genus and two primers amplify 375bp region of 16S rRNA gene of Mycoplasma agalactiae (M. agalactiae) species were used. Results: This proved that 46 samples (66.7%) were infected with Mycoplasma in culture and PCR test, respectively. On the PCR test, 15 isolates (32.6%) examined were positive for M. agalactiae that showed specific amplicon at 375bp. All Mycoplasma positive samples were analyzed for M. agalactiae infection by PCR method and 31 isolates (67.4%) examined were negative for M. agalactiae. The finding of other mycoplasmas with significant epidemiology challenges existing plans for the control of CA in sheep population in Iran. Conclusion: The results of the present study show that M. agalactiae in CA disease in Kurdistan Province, 32.6% involved. In Iran, only M. agalactiae vaccines are commercially available, thus, the animals are not protected against these other species.


Introduction
urine, feces, and nasal or ocular liquids of infected animals.Ewes can be infected through the udder, and Mycoplasma agalactiae (M.agalactiae) is the lambs can be infected by the consumption of principal etiological agent of contagious agalactiae colostrums.Thus, it is recommended that newborn (CA) in small ruminants.In male and female sheep, animals be removed from the dam immediately after this syndrome is produced mostly by Mycoplasma birth and fed only by using pasteurized colostrums.agalactiae subsp.agalactiae, but other mycoplasmas The period of incubation of this syndrome varies are also implicated (M.capricolum subsp.capricolum, between 7 and 56 days.Most cases of infection occur M. mycoides subsp.capri, M. mycoides subsp. in the summer, during birth and peak lactation periods.mycoides LC, and M. putrefaciens).CA is a serious Several mycoplasmas can be isolated from milk and infectious process characterized by three major signs: blood for a short period during the infectious process [4].mastitis, arthritis, and keratoconjunctivitis [1].It Nowadays, this syndrome affects animals in causes the reduction and even the suppression of milk most countries that have a high production of sheep production and occasionally causes abortion and death and goats.It is found across several continents, including in up to 70% of young animals, resulting in serious mainly North America, western Asia, North Africa, economic loss [2].M. agalactiae is one of the principal and Europe, and is endemic in most Mediterranean mycoplasmas of ovine and caprine ruminants weeks while by PCR due to no need of purification and precipitated, and tubes were placed in 56°C bath for cloning, pathogenic Mycoplasma are easy to detected 4h.Then 200µl saturated phenol was added and tubes even within the mixed organisms in a sample, without was centrifuged at 13000 rpm for 20min.Upper phase spending long times (It requires at least 2 to 3h) [6].was transferred to another tube and equal volume of Several different methods of PCR have been described mixed phenol/Cholorophorm (1:1) was added.After and used on the bases of different gene by researchers centrifuged at 13000 rpm for 20min the aqueous phase [7,8].The polymerase chain reaction (PCR) with was transferred to another tube and added equal mycoplasmal 16S rRNA and specific primers have volume of pure cholorophorm and was centrifuged at been applied for detection of a variety of Mycoplasma 13000 rpm for 5min.Upper phase was transferred to a species [9][10][11].The rRNA is naturally present in high new tube and mixed with 1/10 volume of acetate copy numbers (up to 10,000 molecules per cell) [12].sodium (3M) and were precipitated in -20°C The aim of this study was to isolation and prevalence refrigerator with 2 fold volume of cool and pure of M. agalactiae from sheep presenting CA in Kurdistan ethanol (20min), then the tube was centrifuged at in the West of Iran.
13,000 rpm for 15min.200µl of 70% ethanol was added and the tube was centrifuged at 13,000 rpm for

Materials and Methods
5min, the DNA was dried and resuspended in DDW at Samples collection and processing: A total, 69 4°C and used for PCR [10].In this study two primers samples included (Milk, Conjunctiva swabs, Synovial (forward and reverse) which have been already fluid, and Ear canal swabs) were examined by PCR designed by Kojima et al. [10] and amplify a 163bp assay during 2011-2012.All sheep tested had been region of 16S rRNA gene of Mycoplasma genus and previously examined to confirm they had clinical two primers have been already designed by Tola et al. signs of CA.On average, flocks selected to participate [14] and amplify 375bp region of 16S rRNA gene of in the study constituted about 5% of all herds in the M. agalactiae species were used.The primers study area.The samples were immediately placed in sequence and their corresponding genes are shown in test tube with transport Mycoplasma culture medium Table 1.DNA amplification were carried out in a total and were kept at 4°C until they have been transported volume of 35.25µl containing 17.5µl DNA, 0.1µl of to the Razi Vaccine and Serum Research Institute each primers, 0.5µl dNTP mix (10mM) {Cinnagen laboratory in 24h.Transport media contain thallous Inc.}, 4µl Mgcl2 (25mM) {Cinnagen Inc.}, 2.5µl PCR acetate (250mg/liter), which is toxic and inhibitory to buffer (10x) {Cinnagen Inc.}, and 0.25µl Tag DNA some mycoplasmas but not those causing CA and polymerase (5unit/µl) {Cinnagen Inc.}.Reaction reduce bacterial contamination from clinical sample mixture were thermocycled 30 times beginning with [13].The collected samples in transport culture an initial denaturation step of minute at 94°C.The medium were then incubated on PPLO broth in order temperature and time profile of each cycle was as for the primary Mycoplasma to be propagated and following: 94°C for 1min (Annealing) and 72°C for enriched in a humid air with 5% CO2 at 37°C over 1min (Extension), PCRs were finished with a final night in laboratory.According to Pharmacopoeia extension step at 72°C for 5min.PCR products were (2005) negative and positive controls were PPLO stored at 4°C.PCRs were carried out using two broth and standard strain of M. agalactiae (NCTC, programmable thermal cycler (Primus and Master 10123) respectively.gradient).Positive and negative controls were included in all tests.Each micro liter aliquot of each PCR method: The enriched samples were used in PCR products was mixed with 2µl loading buffer (6×).PCR method.DNA was extracted from enriched The PCR products and 100bp DNA ladder were then samples using a previously described method [10].In separated by electrophoresis on 1% Agarose gel and this method 500µl of samples were placed in 1 × 5µl stained with 0.5µl/ml Ethidium bromide (100 volts for Eppendorf tube, micro centrifuged at 13000 rpm for 1h) following UV Translluminator.15min.100µl of lyses buffer was added to 100µl of

Results
and are extremely sensitive.It can provide a rapid early warning system when carried out on clinical At first, 69 samples were enriched in PPLO broth samples, enabling a full investigation to take place media.After culture in 24 samples (34.8%), fried egg when results are positive.However, negative results colony appeared on the agar media and detected should not be considered definitive.PCR has been Mycoplasma species (Figure -1).PCR identification of accepted as a valuable tool for the diagnosis of Mycoplasma species in total enriched samples was Mycoplasma infections, has the advantage of easy use, successful in 31 strains and showed specific amplicon rapid availability of results, and standardization, and is at 163bp (Figure -2).This proved that 46 samples more suitable for processing of a large number of (66.7%) were infected with Mycoplasma in PCR test.specimens [15].In this study, 15 isolates were positive Then all Mycoplasma positive samples were analyzed for M. agalactiae that showed specific amplicon at for M. agalactiae infection by PCR method.On the 375bp.In one study, using the PCR method, the PCR test, 15 isolates (32.6%) examined were positive presence of M. agalactiae was detected in 20 out of for M. agalactiae that showed specific amplicon at 101 animals examined (19.80%).In 12 animals out of 375bp (Figure -3).This method established that 3 out 54 (22.22%) the conjunctival swabs samples were of 17 (17.64%) of conjunctiva swab, zero percent of positive for M. agalactiae and in 8 out of 47 animals ear canal swab, 10 out of 14 (71.43%) of milk and 2 out (17%) milk samples were positive in west central, Iran of 8 (25%) synovial fluid samples were positive.Our [16].In Jordan, Mycoplasma was isolated from eight results suggested that milk secretion and conjunctiva (13%) of the 62 milk samples and seven (2.3%) of the samples are suitable for PCR detection of M. agalactia.
310 nasal swabs collected from sheep, unusually M. agalactiae was not isolated of the sheep samples [17].

Discussion
In our study showed that 3 out of 17 (17.64%) of PCR assay is routinely used in many laboratories Conjunctiva swab, zero percent of ear canal swab, 10  out of 14 (71.43%) of milk and 2 out of 8 (25%) [22] and healthy carriers are important in Iran, where synovial fluid samples were positive.Result of PCR the interchange of animals is a common practice.The with 367 milk samples with M. agalactiae primers peculiarity and the risk of this carrier state is that it may showed that 11(3%) of them were positive in west of occur independently of any clinical expression, and Iran [18].A total of 240 random raw milk samples the most frequent location of infection identified to were collected from cows, buffaloes, sheep and goats date is the external ear canal [5].In Kurdistan, carrier in Assiut and Qena cities, Egypt (30 samples each).sheep introduced the disease from the other areas.
Fifteen strains of Mycoplasma were isolated from raw These high percentages could explain the fast difmilk.The strains were biochemically characterized fusion of contagious agalactiae, and we recommend followed by PCR assay for confirmation.On the basis that practicing veterinarians should include the of biochemical characterization the strains were external ear as a primary culture site for determining divided into 5 species as follow M.

Table - 1
. Nucleotide sequences and primers used for identification of M. agalactiae by PCR

Table - 2
. Distribution of samples for culture, Mycoplasma-PCR, and M. agalactiae-PCR results arginini, M. the presence or absence of mycoplasmas in sheep.The bovirhinis, M. bovis, M. species group 7, and M. results reported in this paper confirmed the detection dispar, but M. bovis could not be detected [19].In the of M. agalactiae modest agent of CA in sheep in Mediterranean region, CA alone is estimated annually Kurdistan province in the West of Iran. to cost at least US$ 30 million mainly as the result of