Detection of Mycoplasma species in raw milk of lactating animals in Assiut and Qena city of Egypt

The incidence of Mycoplasma Species in raw milk of different animal species was determined. A total of 240 random raw milk samples were collected from cows, buffaloes, sheep and goats in Assiut and Qena cities, Egypt (30 samples each). Fifteen strains of Mycoplasma were isolated from raw milk. The strains were biochemically characterized followed by PCR assay for confirmation. On the basis of biochemical characterization the strains were divided into 5 species as follow M. arginini, M. bovirhinis, M. bovis, M. species group 7, and M. dispar. M. bovis could not be detected. The most prevalent isolated species was M. bovirhinis which was isolated from 6.67% of cow's milk samples collected from Assiut City. Whereas M. arginini, was the most prevalent species in the examined milk samples collected from Qena City. None of the five M. bovirhinis strains that were identified biochemically were confirmed by PCR assay through amplification of 16S rRNA region of the gene that was amplified at 316 bp. Efforts should be made to protect animals against mycoplasma infection.


Introduction
Mycoplasma mycoides subsp.Mycoides, which was described initially by Nocard and Roux in Mycoplasmas belong to the class Mollicutes 1898.The first mycoplasma isolated from and are among the smallest free-living humans was detected by Dienes and Edsall in microorganisms capable of auto-replication.They 1937 which we know now as Mycoplasma hominis.
" are fastidious bacteria.The term "mycoplasma Other human mycoplasmas, Mycoplasma (Greek, mykeys= fungus and plasma= formed) salivarium, Mycoplasma fermentans, in addition emerged in the 1950s (Edward et al., 1956).In the to Mycoplasma pneumoniae which was first 1960s, mycoplasmas were designated members isolated by Eaton et al., (1944). of a class named Mollicutes, which derives from Mycoplasma pneumoniae remains an important Latein words meaning soft (mollis) and skin cause of pneumonia, and is also associated for its (cutis) (Waites et al., 2001).Many species are extra pulmonary manifestation, nausea, vomiting important veterinary pathogens causing and abdominal pain are the most common respiratory infection, mastitis, conjunctivitis, symptoms for extra pulmonary manifestion (Kim arthritis, and abortion (Nicholas, 1998(Nicholas, ). et al., 2005)).Hepatitis, hematuria, skin rash, gastro-Mycoplasmal organisms are nearly enteritis and myepicarditis were reported in some ubiquitous in both the plant and animal kingdoms cases (Lee et al., 1986).Although scientists have as colonizers and pathogens.The first isolated 17 species of Mycoplasma from human, mycoplasma to be isolated in a culture was the 4 species of the organism are responsible for most bovine pleuropneumonia agent now known as clinically significant infection (Waites, 2009).In animals, Mycoplasma is an emerging and surface spreading technique on Mycoplasma agar extremely contagious mastitis pathogen.Several according to Carter and wise, (1995).The plates species have been associated with mastitis, were incubated at 5-10 % Co incubator for 72 2 Mycoplasma bovis, M. califoranicum, M. hours at 35-37°C.Plates are held for 7 days before canadense, M. arginini, M. bovigenitalium, M. reported as negative.Suspected colonies should alkalescens, M. bovirhinis and M. dispar (Kumar be optimized by seeing"fried egg" shaped colonies.and Garg, 1991).The bacterium can be shed in c) Identification of isolates: Digitonin sensitivity milk in large numbers before clinical signs was performed as the disc inhibition test appears with relatively few organisms required to according to Erno and Stipkorits, (1973).The infect a quarter so, 25 to 30 % or more of a dairy biochemical procedures in mycoplasma herd can be become infected during an outbreak identification have been standardaaized by of Mycoplasma (Bayoumi et al., 1988).Aloutto et al., (1970).They involve the hydrolysis Microbiological culture is generally used of arginine, phosphatase activity, film and spots for detection and identification of mycoplasmas.production and tertazolium reduction.However, the former techniques are time consuming

d) DNA extraction and PCR amplification of 16S
and not sensitive enough in some cases.Therefore, rRNA M. bovirhinis: Specific primer of M. PCR assay was originally developed for confirmation bovirhinis was used in the molecular detection of of mycoplasma species using the gene for16S the isolated organism according to the protocol rRNA (Kobayashi et al., 1998).
illustrated by Kobayashi et al., (1998) with some Owing to Mycoplasma is a unique bacterium modification as follow: bacterial DNA was that does not always receive the attention it extracted following grown up in the phosphate merits, considering the number of illness it causes buffered saline (PBS), one millilitre of inoculated and the degree of morbidity associated with it, PBS was centrifuged at 15,000 rpm g for 10 this study was planned to know its prevalence in minutes.The NucleoSpin® Tissue Kit raw milk of cows, buffaloes, sheep and goats.
(Germany) was used to obtain bacterial DNA,

Materials and methods
according to the manufacturer recommendations and stored at -20°C until use.The present study was carried out during the DNA amplification: PCR were carried out in 50 µl period between April, 2008 and, March 2010 in reaction volumes containing 5 µl template DNA, the department of Food Hygiene, Faculty of 1 mM MgCl , 1 mM of dNTP, 5 µl of x 10 PCR Veterinary Medicine Assiut University. 2 buffer (Quiagen), 1.25 unit of Taq DNA polymerase a) Collection of samples: A total of 240 random (Ampli Taq Gold Quiagen) and 20 pM of each raw milk samples were collected from cows, primer.The sequences of the primers are given in buffaloes, sheep and goats in Assiut and Qena Table -1.cities, Egypt (30 samples each).Each sample was Unfortunately, reference strain of Mycoplasma mixed and tested for heat treatment using Storch could not be obtained by Molecular Biology test (Lampert, 1975).
Research and Genetic Engineering Center, Assiut b) Isolation of Mycoplasma species: Enrichment University, Egypt. of Mycoplasma species was adopted using The PCR cycles consisted of pre-heating at Mycoplasma enrichment broth and then incubated 94°C for 9 min, denaturation at 94°C for 30 sec. at 37°C for 3 days.Isolation was done using min, annealing at 60°C for 1 min and extension at 72°C for 1 min.the amplifications were performed the multiplication of pathogenic microorganisms.for 35 cycles in a model T professional basic 070-The highest incidence (30.00%) of Mycoplasma 701 thermocycler, in the Molecular Biology species was recorded from the examined milk Research and Genetic Engineering Center, Assiut samples collected from Qena City.Nearly similar University, Egypt, with a final extension step at results were recorded by El-Shabiny and Abo-el 72°C for 7 min.The PCR products were Makarrem, (1994).Zaitoun and Eissa, (1994) visualized using a 2.5% agarose gel containing found that 17.65% of the examined buffaloe's milk 0.5 µg of ethidium bromide/ml in relation to DNA with clinical mastitis were culturally infected by mass ladder standard (1000-bp DNA ladder Mycoplasma.Infection with Mycoplasma in the Quiagen).
dairy animals is attributed to several causes illustrated by Edmonson and Bramely, (2004) and

Results and Discussion
Polanivel et al., (2008).However, unhygieneic Many Mycoplasma species are pathogenic sanitary measures during milking and /or defects to animals, human and plants and they are, of pre-miliking and post-milking may play an therefore, of great concern in human and astonishing role.veterinary medicine (Maniloff et al., 1992 and From the results recorded in Table 3 it is Ross, 1993).Mycoplasma are often host different clear that the most prevalent isolated species was species, and ruminants especially cattle, harbour M. bovirhinis which was isolated from 6.67% of a number of different species.
cow's milk samples collected from Assiut City.Results illustrated in Table-2 revealed that Concerning M. bovis, similar results was Mycoplasma species was isolated from 6.67, obtained by McDonald et al., (2009) as M. bovis was not detected in any tank milk samples by 3.33, 3.33 and 6.67% of the examined cow milk, PCR or culture, where 7 of 66 (10.61%) and 6 of buffalo milk, sheep milk and goat milk samples 51 (11.76%) cows were infected with the collected from Assiut City, while, 3.33, 6.67, 10.00 and 10.00% of the same samples that organism (Gonzalez et al., 1992).Data recorded collected from Qena City proved to harbor this in Table 4 showed that M. arginini, was the most prevalent species in the examined milk samples bacterium, respectively.Presence of Mycoplasma collected from Qena City.M. arginini, M. in milk is not surprising in view of the fact that bovirhinis, M. species group 7 and M. dispar they are widely distributed in nature and could be isolated in a variable percentage from the contaminate milk.The problem was complicated examined cows, buffaloes, sheep and goats milk by the absence of cool system that may enhance  (Poveda et al., 2002).The small size and correspondingly lower concentrations of cellular disease in both sheep and goat it occurs primarily constituents of mycoplasmas results in smaller in Mediterranean countries but is also reported from many other areas of the world (Jones, 1987).signals that are more difficult to resolve from the The existence of Mycoplasma in goat milk clarify background noise than those obtained from that the intra mammary pathogen in goats are mammalian cells or even from larger microassociated with poor hygienic condition in organisms.Additionally, the genetic variation housing and in the milking parlors (Contreras et between strains isolated from local samples and al., 2002).Previous results published by Elthe others all over the world affect the results, so, Shabiny and Abo-el Makarrem, (1994) referred we suggested making a genetic sequencing for to M. bovirhinis as an unconventional mastitis the isolated microorganisms and creating specific pathogen responsible for sever clinical mastitis in primers for the locally isolated strains.dairy buffaloes at Beni-Suef, Egypt.However, There is no treatment of mycoplasmal infection, Hirose et al., (2001) concluded that M. bovirhinis its control relies on identification of infected animal was a secondary respiratory infection rather than by culture of composite or quarter milk samples mastitis pathogen.from all milking and dry animals in the herd Using PCR for detection of M. bovirhinis, (Bushnell, 1984).Great care should be used when non of the 5 isolated strains, gave a positive purchasing the animals.All action should be used results as indicated in Figure -1.Although PCR upon the understanding of the high contagious nature, based methods offer a substantial advantage in slow recovery rates and the in-effectiveness of that they reduce the average diagnostic time.treatment of Mycoplasma infection is likely to increase Positive results from PCR assays enable a full as a consequence of a perceived bioterrorism threat.investigation to take place, however negative

Table - 2: Incidence of Mycoplasma species in the examined milk samples Table -3: Incidence of different Mycoplasma species recovered from the examined milk samples collected from Assiut City
Detection of Mycoplasma species in raw milk of lactating animals of Assiut and Qena city in Egypt samples collected from Qena City.smallest self-replicating microorganisms known The important Mycoplasma causes natural