Sequence Analysis of Three Genes of Mycoplasma bovis Isolates from Egyptian Cattle and Buffaloes

The present study concerned with phylogentic analysis of three genes (gapA, p 40 pseudogene and uvrC) related to adhesion and virulence of Mycoplasma bovis (M. bovis) in cows and buffaloes. In this study, 625 milk samples were collected from clinical and subclinical mastitis in cows and buffaloes in two Egyptian Governorates Fayoum (south of Cairo Governorate) and Dakahlia (south of Cairo Governorate) for the detection of M. bovis infection by isolation and PCR. Mycoplasma infection was higher in cows (31.6%) than buffaloes (14.3%) suffered from clinical mastitis at Fayoum. The incidence of clinical mastitis in cows (20%) is higher than that in buffaloes (10%) at Dakahlia. Concerning subclinical cases, the incidence was higher at Dakahlia (30.19% and 38.3%) than Fayoum (20.6% and 12%) in cows and buffaloes respectively. Phylogentic analysis of nucleotides and amino acids of gapA gene showed 100% identity of the two isolates. The study proved that p40 gene is present in bovine as pseudogene and its protein did not expressed. The amino acids of uvrC gene of our field isolates showed 50 a.a substitutions. In conclusion M. bovis isolate isolated in the current study had identical gapA gene in both cow and buffalo in DNA and amino acid sequences (aa), whereas uvrC gene showed 50 aa substitutions in which could affect the antigenicity and p40 gene showed no protein expression and present in bovine as pseudogene. Original Research Article Eissa et al.; BMRJ, 14(3): 1-10, 2016; Article no.BMRJ.25014 2


INTRODUCTION
Mycoplasmas cause a variety of different diseases in ruminants and can affect the udder, respiratory and genital tracts, joints and conjunctiva. In Europe, the most commonly encountered pathogenic mycoplasma in cattle is M. bovis, which is often associated with mastitis in adult dairy herds, but can also cause pneumonia, polyarthritis and synovitis in native beef herds [1], as well as pneumonia in calves where the disease is endemic [2] and [3]. It is capable of rapid spread as witnessed in Ireland [4,5]. In Egypt, [6] concerned with comparative molecular study of M. bovis isolated from cows and buffaloes suffered from mastitis. PCR plus sequencing of variable surface protein A (VspA) gene were used for identification and characterization of M. bovis isolates. Phylogenetic and sequence analysis showed that, the isolated Egyptian strains were grouped in two groups.
Adherence to host cells is a prerequisite for colonization and infection [7]. Several proteins such as P26 and Vsps (variable surface proteins) are involved in cytadherence of M. bovis [8] and [9]. Recent results suggest that other proteins could also be implicated in the first step of infection [10]. One protein that has received attention as a potential vaccine target in many species is the conserved glyceraldehyde-3phosphate dehydrogenase (GAPDH) protein. In addition to its role in glucose metabolism, GAPDH had been shown to bind cellular matrixes, [11,12] and it has been postulated to be a virulence factor [13]. These properties of GAPDH suggest that this protein could be used as a protective antigen. Pseudogenes are abundant in most organisms, but their function is still unclear, and they are thought to be simple molecular fossils [14]. The uvrC gene is species specific and well conserved within each M. bovis and M. agalactiae, it is sufficiently different between the two species in order to facilitate a good resolution of these two mycoplasmas which are difficult to be identified by other genetic methods such as using their gene sequences. On the other hand, uvrC seems to be the uvrC sufficiently conserved within each species in spite of high genetic and antigenic heterogeneity which is found amongst M. bovis and M. agalactiae strains. Therefore, it is an ideal target gene for PCR-based identification of M. bovis and M. agalactiae [15].
The aim of the present work is sequence analysis of two virulent strains of M. bovis isolated from cattle and buffalo using three genes.

Sampling
Milk samples were collected from clinical and subclinical cases (detected by California mastitis test), 164 buffaloes and 461 dairy cows in two Egyptian Governorates Fayoum and Dakahlia which were examined for the detection of M. bovis. The animals were examined during period from January 2012 to March 2014 both clinical and subclinical cases.

Isolation and Identification of Mycoplasma bovis
Milk samples were cultured in PPLO broth media, Difco TM PPLO broth USA [16] and maintained at 37°C for 3-7 days. Biochemical characterization of the isolated purified strains was carried out [17].

DNA extraction
The DNA extraction from the biochemically identified isolates was carried out using QIA Amp ® DNA Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions.

PCR amplification
Three oligonucleotide primers specific gene for M. bovis were used. Table (1)

Primer Sequences were Tabulated in
The primer was prepared by Macrogen Company, South Korea.
The PCR reaction mixture (total volume 50 µl) was 25 µl Master Mix (2x), Fermentas company Cat.no. K1080, USA, 3 µl target DNA, 1 µl of each primer (10 pmol) and mixture was completed to 50 µl by RNAse, DNAse free sterile dis. water. PCR was performed on a Bio-Rad thermal Cycler (S1000™ Thermal cycler, USA). Conditions for PCR were as follows: 1 cycle of 2 min at 94°C, 35 cycles of 30 sec at 94°C, 30 sec annealing at the appropriate temperature for the primer combination used (56°C gapA and p40 genes, 52°C uvrC gene) 2 min extension at 72°C and a final cycle of 5 min at 72°C. Aliquots of the samples were loaded on a 1.5% agarose gel, the bands resolved by electrophoresis and the gel stained with ethidium bromide and photographed.

Sequencing and Sequence Analysis
The amplified fragments were purified using Gene Jet PCR purification kit; Fermentas (Cat no. KO701). Five published Mycoplasma bovis in GenBank uvrC, GapA and p40 genes and two M. agalactiae were selected as Reference sequences. Sequencing was performed by Macrogen Company (South Korea) and identification of homologies between nucleotide and amino acid sequences of the M. bovis were compared with other strains published on GenBank using BLAST 2.0 and PSI-BLAST search programs, (National Center for Biotechnology Information NCBI" http://www.ncbi.nlm.nih.gov/), respectively The obtained nucleotide sequences comparisons and their multiple alignments with reference M. bovis as well as the deduction of amino acid sequences were done using the BioEdit sequence alignment editor [19], CLUSTALX software for multiple sequence alignment [20], ClustalW software for multiple sequence alignment [21], ClustalV [22] and MegAlign (DNASTAR, Lasergene®, Version 7.1.0, USA) [23]. The phylogenetic trees were constructed using MegAlign for tree reconstruction of sequences by Neighbor-joining method based on ClustalW. Bootstrapping values were calculated using a random seeding value of 111 [21]. ClustalV was used when end gaps were faced. Sequence divergence and identity percent were calculated by MegAlign The structural character of uvrC, GapA and p 40 protein sequence was identified by Protean (DNASTAR, Lasergene®, Version 7.1.0. USA).

RESULTS
The incidence of mycoplasma infection was higher (31.6%) in cows suffered from clinical mastitis at Fayoum Governorates than that at Dakahlia (20%), while the incidence in subclinical cases was higher at Dakahlia (30.19%) than that at Fayoum (20.6%).
Although there were difference in the incidence of infection in the two Governorate, but there are no significance differences (P<0.05).

Polymerase Chain Reaction Results
PCR technique was used for the detection of Mycoplasma bovis isolated from cows and buffaloes. The examined isolates were amplified PCR fragment size 1007 bp of gapA gene, 797 bp of p 40 pseudogene and 1626 bp of uvrC gene.

Sequence Submission to GenBank
The following Acc.
The analysis of uvrC gene amino acids of our field isolate of cattle (Egy-10-FA-14) was (100%) similar when compared with the reference strain (PG45) and the field strains on GenBank (Figs. 9,10), while the field isolate of buffalo (Egy-11-DK-14) was 89.5% similar with the our field isolate of cattle and the reference strain PG45 with 50 amino acid substitution (89-355 a.a) ( Table 4).

DISCUSSION
The present study started with investigation of cow and buffalo dairy herds in Fayoum & Dakahlia Governorates for the detection of mycoplasma infection. Cows and buffaloes suffered from clinical mastitis at Fayoum showed higher incidence (31.6% and 14.3%) than that at Dakahlia (20% and 10%). The incidence of infection in subclinical cases in buffaloes and cows was higher (38.3% and 30.19%) at Dakahlia than that of Fayoum (12% and 20.6%). These results disagreed with [24], who mentioned that the prevalence of Mycoplasm infection in clinical mastitis cows ranged from (28.6% to 73%) and buffaloes (60%-100%) in other Governorates of Egypt.
Three genes (gapA, p 40 pseudogene and uvrC) were studied, gapA nucleotide sequence showed 100% identity between our M. bovis field isolates from cows and buffaloes. Similarity with the reference strain (PG45) was 98.2%.
GapA gene and its protein considered as virulence factor of M. bovis with its ability to elicit the immue response of cattle due to the infection. [18]. They also mentioned that the gapA protein can be used for a vaccine to prevent the disease caused by M. bovis.
Concerning analysis of p 40 pseudogene nucleotide sequence of our field isolates showed (97.5%) identity when compared with each other, when compared with (PG45) reference strain on GenBank showed similarity (99.8% and 97.3% respectively). These results agreed with Thomas et al. [10].
Constitutive genes which are expressed in all living organisms have basic functions such as replication, transcription and translation, are good candidates for genetic differentiation of species, The uvrC gene of M. bovis is a suitable conserved target for diagnosis of M. bovis using PCR [10] uvrC amino acid sequence analysis of the two isolates from cows and buffaloes showed differences due to 50 a.a. substitutions, these changes could affect the antigenicity of isolates.

CONCLUSION
In the current study M. bovis isolate had identical gapA gene in both cow and buffalo in DNA and amino acid sequences (aa), whereas uvrC gene showed 50 aa substitutions in which could affect the antigenicity and p40 gene showed no protein expression and present in bovine as pseudogene.