ISOLATION, CLONING AND PHYLOGENETIC ANALYSIS OF pld AND cp40 , VIRULENCE FACTORS OF A MEXICAN ISOLATE OF

Background. Caseous lymphadenitis is a worldwide distribute disease that affects the sheep and goat industry. Corynebacterium pseudotuberculosis ovis is a facultative intracellular Gram-positive bacterium, considered the etiologic agent of the disease. Complete genome sequences of Mexican isolates have been obtained and different strain has been previously characterized. The study of virulence factors allows establishing potential candidates for the development of vaccines and diagnostic tools. Objective. To identify the complete pld and cp40 genes sequence from a Mexican isolate of Corynebacterium pseudotuberculosis ovis , principal virulence factors. Methodology . Corynebacterium pseudotube rculosis isolate 2J-L was obtained from an abscess of a sheep of the State of Jalisco. The complete pld and cp40 genes were amplified by PCR, cloned into a replicative vector and sequenced by Sanger automatic sequencing. Gene sequences conservancy was established, analyzing homology across previously reported genes of Mexican strains MEX1, MEX2, MEX9, MEX25 and MEX29. Results . Sequences of pld and cp40 genes from isolate 2J-L presented high percentages of similarity (99%) in comparison with the sequences of other isolates of Corynebacterium pseudotuberculosis , reported in the GenBank database. The analysis of nucleotide sequence homology and phylogenetic tree based on pld and cp40 directed the observation that 2J-L is related to Mexican strain MEX29 and MEX25. Phylogenetic results agreed on the idea that strains biovar ovis and biovar equi are groupings on different clades. Finally, results indicate that Mexican strains are more similar among strains isolated from the same host type, without geography distance influence. Implications. The analysis pointed out that both genes conserve their sequences in comparison with Mexican and international strains, which encourages the research continuity for vaccine and diagnostic tools development using proteins PLD and CP40 as antigen targets. Conclusions. The complete pld and cp40 genes from Mexican isolate 2J-L were amplified, cloned and analyzed; important virulence factors from Corynebacterium pseudotuberculosis .


INTRODUCTION
Corynebacterium pseudotuberculosis biovar ovis is the causal agent of Caseous Lymphadenitis (CLA), a dangerous contagious chronic disease that affects small ruminants, being the reason for great economic losses for the livestock industry (Bernard, 2012). CLA conduce to a deterioration in the organic condition of infected animals, reduction in milk and meat production (Schreuder et al., 1990;Collett et al., 1994), skin and carcasses condemnation and reproductive disorders Faeza et al., 2019). C. pseudotuberculosis is considered an emergent health problem (Moussa et al., 2016), characterized by lesions associated with encapsulated abscesses that prevent the action of antibiotics and indiscriminate use of them have generated multiresistant strains (Gallardo et al., 2019). C. pseudotuberculosis is a Gram-positive, facultative intracellular bacteria, belongs to the order Actynomicetales and family Corynebacteriaceae (Bernard, 2012), representing an important pathogen responsible for a large variety of diseases among humans (Trost et al., 2010), ovine and caprine (Parise et al., 2018), equine (Muñoz et al., 2016), bovine (Viana et al., 2017), dromedary (Tejedor et al., 2000), alpaca (Sprake and Gold, 2012) and llama . The conventional protocols conducted in farms for establishing the prevention and control are still inefficient (Windsor et al., 2014;Punjataewakupt et al., 2019). Despite the ongoing success of classical vaccines for controlling different bacterial diseases, commercial vaccines for CLA are still facing the difficulty of not developing safety and effective protection. Glanvac™ series (Zoetis, Australia), Caseous D -T™ and Case -Bac™ (Colorado Serum Company, USA), Biodectin™ (Zoetis, España), LinfoVac (Laboratory Vencofarma, Brasil) are vaccines used in farms immunization schedules, been available in almost all producer countries, but remains the prevalence of the disease (Paton, 1995;Paton et al., 2003;Bastos et al., 2012). For that reason, studies continue to identify the correct combination of molecules that active and increase the immune system, that could be used for generating a vaccine with a protective immunogenic response. The genes pld and cp40 have been identified as the main virulence factors of C. pseudotuberculosis. The potential of PLD (exotoxin) (Correa et al., 2018) and CP40 (endoglycosidase) (Shadnezhad et al., 2016) have been evaluated in separated experimental assays, and they are capable of induced increases IgG and cellular immune mediators like INF-ɣ and IL-12. Both proteins are secreted antigens detected in culture supernatant from C. pseudotuberculosis, in addition, their expression has been identified during in vivo and in vitro assays (Rebouças et al., 2011;Correa et al., 2018).
Mexico has active production of small ruminants, been demonstrated by various studies the presence of CLA in different regions of the country. Six Mexican strains with different biovar, isolated from horse, goat and sheep, were complete sequenced and studied in silico. Besides a putative drug target was predicted and in silico analysis compared 46 strains showed two genes clusters (Restriction Modification system, exclusively in biovar ovis and CRISPR-Cas cluster, which is only present in biovar equi strains) able to establish the identification between different biovar (Parise et al., 2018). Another study carried out the identification of 57 isolates of C. pseudotuberculosis by bacteriological tests and the amplification of 16S rRNA, rpoB and pld genes, as well as, genes involved in virulence and pathogenicity (Guerrero et al., 2018). The virulence factor pld and cp40 continue to be an objective of study due to their potential for diagnostic and vaccine development. The aim of the present study was to analyze virulence factors, pld and cp40 from a Mexican isolate of Corynebacterium pseudotuberculosis ovis compared with those reported in Mexican strains from different regions.

Bacterial strain and growth conditions
Corynebacterium pseudotuberculosis isolate 4-2.2LJ (C43) of biovar ovis previously identify was renamed in this work as 2J-L and used for genetic material extraction and genes amplification (Guerrero et al., 2018). This isolate was obtained from a naturally occurring case of Caseous lymphadenitis in a sheep of Jalisco State, Mexico. Corynebacterium pseudotuberculosis 2J-L was previously identified by biochemical miniaturized API Coryne test (Biomeriux 77 Lab., REF, 20900. France) and Multiplex PCR. In addition, the DNA of Corynebacterium pseudotuberculosis ATCC® 43926™ was used as a positive control for the PCR amplification of pld and cp40. Bacteria was growing in 5% sheep blood agar and incubated at 37°C for 24-48 hours in microaerophilic conditions. Culture morphology was evaluated, and Gram staining was used to confirm the purity of the colonies. A single colony was cultured in 5mL of brain heart infusion (BHI) medium (Oxoid, Hampshire, England) with microaerophilic conditions at 37 °C for 24 hours, for using the culture in DNA extraction. Escherichia coli strain DH5α (Invitrogen, USA) was used as a host for transformation with pGEM-T Easy vector containing pld and cp40 genes. Transformed E. coli strains were cultivated in Luria broth (10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl per liter) containing 100μg of ampicillin per mL.

Genomic
DNA extraction from C. pseudotuberculosis 2J-L and ATCC 43926 Bacterial DNA was extracted from C. pseudotuberculosis 2J-L and ATCC 43926 strain using commercial Fast ID Genomic DNA Extraction Kit (Genetic ID NA, Inc. v. 8.3,USA). Accordantly with the manufacture instruction in a labeled 2mL vial was added 200mg of bacteria pellet homogenized with 1,000µL Genomic Lyse buffer premixed with Proteinase K (20mg/mL), vortex thoroughly until a homogeneous slurry was obtained. Samples were incubated at 65 °C for 30 min and centrifuged at 12,000 g for 5 min in a microcentrifuge (Corning, USA). Later 500µL of supernatant was transferred into a new labeled 2mL vial, and mixed with equal amount of Genomic Bind buffer, centrifuged at 12,000 g for 5 min. The supernatant was put through the DNA Binding Column by centrifugal force at 12,000 g for 3 min. One step of wash was performed with 800µL of Genomic Wash buffer and three steps with 800µL of 75% ethanol using centrifugation 12,000 g for 3 min. For DNA collection 100μL of elution solution was added into the column, spin at 12,000 rpm for 1 min, been DNA collected in a new 1.5 mL vial and stored at -20 0 C.

Identification of Corynebacterium pseudotuberculosis isolate 2J-L by PCR
Molecular identification of C. pseudotuberculosis was based on Multiplex PCR amplification of a partial sequence of 16s rRNA, rpoB, and pld genes (Table 1) according to the protocol previously published (Pacheco et al., 2007). Multiplex PCR was performer in a final reaction volume of 25μL containing 12.5 μL 2X Quiagen Master Mix, 1μL of each primer (100 pmol/μL) and 2μL of DNA with a concentration approximately of 30 ng/ μL. Reactions were carried out in a thermal cycler (Techne TC 512, USA) under the following conditions: initial denaturation at 95 °C for 3 min; 40 cycles of 95 °C for 1 min, 58 °C for 40 s and 68 °C for 1 min 30 s; final extension at 68 °C for 7 min. Amplified products were resolved by electrophoresis in 1.0% (w/v) agarose, stained with ethidium bromide (0.5 mg/mL) and visualized in a transilluminator (Mini Bis Pro, DNR Bio-imagen system, USA).

Amplification of complete pld and cp40 genes
To develop a primer pair specific for a complete pld and cp40 genes amplification, software's Primer 3 Plus (https://primer3plus.com/) and Primer Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) were used for oligonucleotide design based on pld (L16587.1) and cp40 (JF299259) sequence from GenBank. Restriction sites were included in primers for NcoI (5' end) and XhoI (3' end) recognition, to facilitate future cloning steps ( Table 2). All PCR reactions were performed in the same thermal cycler (Techne TC 512, USA) in a total reaction volume of 25μl, containing 12.5 μL 2X Quiagen Master Mix, 1μL of each primer (100 pmol/μL) and 2μL of DNA (30 ng/ μL). Amplification programs began with a denaturation step of 94°C for 5 min and ended with a final extension of 72°C for 7 min. In addition, 30 cycles for pld gen amplification were performed, each one involving denaturation at 94°C for 1 min, annealing at 59°C for 1 min, and synthesis at 72°C for 2 min. For cp40 gene amplification 35 cycles were performed under the following conditions: denaturation steps at 94°C for 1 min, annealing at 66°C for 1 min and synthesis at 72 °C for 1 min 30 sec. The amplified products were detected by ethidium bromide (0.5 mg/mL) staining after electrophoresis in 1.0% agarose gels, visualized in a transilluminator (Mini Bis Pro, DNR Bio-imagen system, USA. Cloning strategy and sequencing PCR products were purified using a Wizard® SV Gel and PCR Clean-Up System kit (Promega, USA) and cloned directly into pGEM-T Easy, which is supplied cut with T overhangs. According to the manufacturer's instructions plasmid and the purified PCR products were ligated with T4 DNA ligase (Promega, USA) reaction and ligation mixture was transformed into the competent cell, Escherichia coli DH5α (Invitrogen, USA) for plasmid propagation. Recombinants plasmid were identified by restriction enzymatic digestion with NotI/ NcoI / XhoI. DNA sequencing was performed by Sanger methods, on CINVESTA Center (Mexico).

Analysis of sequence data
Molecular evolutionary genetics analysis software (MEGA v10.0) was used to correct the sequences and manually placed at the same position for generating the phylogenetic trees based on pld and cp40, in comparison with sequences of C. pseudotuberculosis strains deposited in the database GenBank (Table 3).
In addition, the analysis of the similarity and identity of the genes was performed using BLASTn. Finally, phylogenetic analyses of the 2J-L isolate were inferred using the Maximum Likelihood method and JTT matrix-based model. Percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was shown next to the branches. Initial trees for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with a superior log-likelihood value.

Identification of
Corynebacterium pseudotuberculosis isolate 2J-L, pld and cp40 genes amplification by PCR.
The identity of isolate 2J-L was confirmed as Corynebacterium pseudotuberculosis by Multiplex PCR (Figure 1). The complete pld and cp40 genes were amplified from Corynebacterium pseudotuberculosis Mexican isolate 2J-L by PCR (Figure 2).
Resultant PCR fragments were ligated into the pGEM-T Easy vector and recombinant plasmids were identified by enzymatic digestion (Figure 3).

Nucleotide sequence analysis of pld and cp40 from isolate 2J-L
Sequencing recombinant plasmids allowed the analysis of the sequence of pld and cp40 genes from Corynebacterium pseudotuberculosis isolate 2J-L. Nucleotide sequence alignments allowed the identification of pld with 99.91% of similarities in comparison to sequences of Mexican strains MEX1(CP017711.1), MEX2(CP046644.1), MEX9 (CP014543.1), MEX25(CP013697.1) and MEX29 (CP016826.1) with a change in position 606 with a Cytosine instead of Thymine (C / T). In addition, the cp40 gene resulted identical with a 100% of similarity to sequence of the strain MEX29, and 99.91% in comparison with MEX1, MEX2, MEX9 with a variation in position 60 (C / T) and a variation in position 739 (G / C) respect to MEX25 (Figure 4).
Sequences obtained in this study have been deposited in the GenBank database under the accession number pld 2JL: OL347711 and cp40 2jL: OL347712.

Table 3. Strains used in phylogenetic analyses were obtained from the database GenBank.
Direct Sub: Direct submission on the NCBI page.

Phylogenetic analysis of Corynebacterium pseudotuberculosis isolate 2J-L
Phylogenetic relations of the 2J-L isolate were established based on pld and cp40 sequences in comparison to other strains of C. pseudotuberculosis, with the construction of two separate phylogenetic trees ( Figure 5). Comparative phylogenetic analysis grouped isolate 2J-L in the same clusters with biovar ovis strain supported by high bootstrap values, 92% for cp40 and 99% for pld sequence. Both phylogenetic analyses revealed that isolate 2J-L is considerably closer to MEX29 and MEX 25 Mexican strains, included in a subgroup with 4681 and 29156 strains.

DISCUSSION
Mexico is one of the main consuming and producing countries of sheep and goat meat in America (Morales, 2020). Earlier studies have identified and sequenced the complete genome of Corynebacterium pseudotuberculosis strains from both biovar ovis and   Phylogenetic dendrograms were constructed based on the pld (A) and cp40 (B) genes of Corynebacterium pseudotuberculosis 2J-L in comparison to other related sequences. The evolutionary history was inferred by using the Maximum Likelihood method and JTT matrix-based model. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Initial trees for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with a superior log-likelihood value. Evolutionary analyses were conducted in MEGA X.

A B
equi, obtained from outbreaks in the different States of Mexico (Parise et al., 2018;Muñoz et al., 2016). Molecular identification, biochemical characterization and pathogenicity factors such as fag A, fag B, fag C, fag D and hsp60 have been analyzed of Corynebacterium pseudotuberculosis isolates from Jalisco State (Guerrero et al., 2018). One of the samples derived from that study, sample 4-2.2LJ (C43) from sheep origin, was used in this work and renamed as isolate 2J-L, for pld and cp40 identification for future use as potential candidates for vaccine and diagnostic development. Multiplex PCR products confirmed 2J-L previous identification as C. pseudotuberculosis, based on the amplification of partial rpoB, 16S rRNA and pld. Multiplex PCR has been validated by different authors, due to the combination of that variety of genes that allow Corynebacterium pseudotuberculosis differentiation concerning the closest species Corynebacterium diphtheria and Corynebacterium ulceran . New primers design, PCR schedules and cloning steps used in this work were effective to obtain the complete genes and their insertion into pGEM-T Easy plasmid. These results constitute an advantage and the starting point for obtaining the proteins by recombinant technology in future assays.
Analysis of nucleotide sequence of pld and cp40 genes from isolate 2J-L allowed establishing differences and similarities respect other Mexican strains previously reported. Sequences of pld and cp40 genes from isolate 2J-L presented high percentages of similarity in comparison with the sequences of other isolates of Corynebacterium pseudotuberculosis, reported in the GenBank database. The highest percentages of identity were presented concerning Mexican strain MEX25 and MEX29, as well as with the strains 4681 from an Australian sheep and the isolate 29156 from a bovine of Israel (Sousa, 2019a). The results show that these genes are highly conserved due to the role of proteins PLD and CP40, as virulence and pathogenicity factors of Corynebacterium pseudotuberculosis (Hodgson et al., 1990;Shadnezhad et al., 2016).
Phylogenetic results are in concordance with those obtained by other authors where strains biovar ovis and biovar equi are grouped on different clades (Soares et al., 2013;Oliveira et al., 2016). Isolate 2J-L was grouped to Mexican strains biovar ovis, with preference to the type of host. Results obtained according to the analyzes of the complete genome of Mexican strains MEX1, MEX9, MEX25, MEX29, showed that strains were grouped predominantly depending on the type of host, and not due to geographical proximity. But like in other studies, also isolate 2J-L was found in the same clade with strains biovar ovis from different types of hosts, related in the same clade with strain 29156, isolated from a bovine of Israel (Sousa, 2019a). Isolate 2J-L was obtained from a sheep belonging to a farm with ovine and caprine coexisting together, where this farm could have different sources of biovar ovis strains. Biovar ovis strains contain a high degree of clonality, with more sequences conserved and the capacity to infect different hosts. While biovar ovis strains formed mix groups with a representation of different types of hosts, strains belonging to biovar equi are under a constant mutational process, presenting established strain differences that make their phylogenetic tree distribution more specific and separate by group, even by type of host (Soares et al., 2013).
Previous studies revealed that MEX1 strain (NZ_CP017711.1) was isolated from the abscess of a goat in the Tlaxcala region, MEX2 (CP046644.1) from a goat of Puebla and MEX29 strain (NZ_CP016826.1) from a sheep of the Rio Frio de Juarez, all regions with 40-50 Km approximately between isolates. In addition, strain MEX25 (NZ_CP013697.1) was isolated from a sheep and MEX9 (NZ_CP014543.1) from a goat, both isolated from the Guanajuato region, with 450 km concerning previous isolates. Isolate 2J-L was obtained from Jalisco, Guadalajara with 276 Km respect Guanajuato and 666 Km of Tlaxcala. Despite the difference in the geographic location of isolates MEX25, MEX29, and 2J-L, all derived from sheep, there were grouped in the same clade. The same behavior was observed for MEX1, MEX2 and MEX9, all derived from goats, grouped closer phylogenetically (Parise et al., 2018). The gene sequence analysis (pld and cp40) indicates that Mexican strains are more similar among strains isolated from the same type of host, corroborated in previous reports with the complete genome of Mexican strains (Parise et al., 2018).

CONCLUSIONS
Caseous Lymphadenitis remains an important disease with a tremendous negative impact on the economy of small ruminate producers in Mexico and all over the world. We provide gene sequence of pld and cp40 from a Corynebacterium pseudotuberculosis Mexican isolate, focusing on their conserved sequence and homology compared to genes from Mexican strains previously reported. Both genes from isolate 2J-L present a conserved sequence concerning genes on Mexican strains and might be relevant for vaccine and diagnostic development to improve controls protocols for CLA in Mexico.