ORIGINAL ARTICLE

http://dx.doi.org/10.1016/j.ram.2015.06.007

Genotyping and study of the pauA and sua genes of Streptococcus uberis isolates from bovine mastitis

Genotipificación y estudio de los genes pauA y sua de aislamientos de Streptococcus uberis de mastitis bovina

 

Melina S. Perrig^a,b, María B. Ambroggio^a, Fernanda R. Buzzola^c, Iván S. Marcipar^a,b, Luis F. Calvinho^d,e, Carolina M. Veaute^a, María Sol Barbagelata^a,*

a. Laboratorio de Tecnología Inmunológica, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Santa Fe, Argentina
b. Consejo Nacional de Investigaciones Científicas y Técnicas, CONICET, Argentina
c. Instituto de Investigaciones en Microbiología y Parasitología Médica, Universidad de Buenos Aires-Consejo Nacional de Investigaciones Científicas y Técnicas (IMPaM, UBA-CONICET), Facultad de Medicina, Buenos Aires, Argentina
d. Estación Experimental Agropecuaria Rafaela, Instituto Nacional de Tecnología Agropecuaria (INTA), Rafaela, Santa Fe, Argentina
e. Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral, Santa Fe, Argentina

 

*Corresponding author.
E-mail address: msolbar@yahoo.com.ar (M.S. Barbagelata).

Received 22 December 2014; accepted 22 June 2015
Available online 23 October 2015

© 2015 Asociación Argentina de Microbiología. Published by Elsevier España, S.L.U. This is an open access article under the CCBY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

 

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Abstract

This study aimed to determine the clonal relationship among 137 Streptococcus uberis isolates from bovine milk with subclinical or clinical mastitis in Argentina and to assess the prevalence and conservation of pauA and sua genes. This information is critical for the rational design of a vaccine for the prevention of bovine mastitis caused by S. uberis. The isolates were typed by random amplified polymorphic DNA (RAPD) analysis and by pulsed-field gel electrophoresis (PFGE). The 137 isolates exhibited 61 different PFGE types and 25 distinct RAPD profiles. Simpson's diversity index was calculated both for PFGE (0.983) and for RAPD (0.941), showing a high discriminatory power in both techniques. The analysis of the relationship between pairs of isolates showed 92.6 % concordance between both techniques indicating that any given pair of isolates distinguished by one method tended to be distinguished by the other. The prevalence of the sua and pauA genes was 97.8 % (134/137) and 94.9 % (130/137), respectively. Nucleotide and amino acid sequences of the sua and pauA genes from 20 S. uberis selected isolates, based on their PFGE and RAPD types and geographical origin, showed an identity between 95 % and 100 % with respect to all reference sequences registered in GenBank. These results demonstrate that, in spite of S. uberis clonal diversity, the sua and pauA genes are prevalent and highly conserved, showing their importance to be included in future vaccine studies to prevent S. uberis bovine mastitis.

Keywords: Bovine mastitis; Molecular typing; Streptococcus uberis; PauA; SUAM.

Resumen

Este estudio pretendió determinar la relación clonal entre 137 aislamientos de S. uberis obtenidos de leche de bovinos con mastitis clínica o subclínica en la Argentina, como así también la prevalencia y la conservación de los genes sua y PauA entre dichos aislamientos. Esta información es crítica para el diseño racional de una vacuna que prevenga la mastitis bovina por S. uberis. Los aislamientos se tipificaron molecularmente por amplificación al azar del ADN polimórfico (RAPD) y mediante electroforesis de campos pulsados (PFGE). Los 137 aislamientos mostraron 61 pulsotipos mediante PFGE y 25 tipos de RAPD diferentes. Los índices de Simpson calculados fueron 0,983 por PFGE y 0,941 por RAPD; esto evidencia el elevado poder discriminatorio de ambas técnicas. El análisis de la relación entre pares de aislamientos mostró un 92,6 % de concordancia entre ambas técnicas, lo que indica que cualquier par de aislamientos que fue distinguido por un método tendió a ser distinguido por el otro. La prevalencia de los genes sua y puaA fue del 97,8 % (134/137) y 94,9 % (130/137), respectivamente. Las secuencias de nucleótidos y de aminoácidos codificados por los genes sua y pauA de los 20 aislamientos de S. uberis seleccionados sobre la base de su tipo de PFGE y RAPD y origen geográfico tuvieron un porcentaje de identidad de entre 95 % y 100 % con respecto a todas las secuencias de referencia registradas en GenBank. Estos resultados demuestran que, a pesar de la diversidad clonal de S. uberis, los genes sua y pauA son prevalentes y están altamente conservados y deberían ser incluidos en futuros estudios de vacunas para prevenir mastitis bovina causada por S. uberis.

Palabras clave: Mastitis bovina; Tipificación molecular; Streptococcus uberis; PauA; SUAM.

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Introduction

Bovine mastitis is one of the most costly diseases of dairy cattle as a consequence of antibiotic treatment expenses, decreased milk production and quality, and increased animal replacement rate^6,30. In Argentina, milk yield losses in cows suffering from mastitis were reported to be of about $4.3/cow/days⁴¹. Bovine intramammary infections (IMI) are caused by both contagious and environmental bacteria. Streptococcus uberis is one of the most prevalent environmental pathogens associated with subclinical and clinical IMI both in lactating and non-lactating cows^7,27. In addition, this pathogen can persist in the mammary gland causing chronic IMI⁴⁷. Traditional control procedures based on milking time hygiene and antibiotic therapy are considered adequate to reduce incidence of most contagious pathogens, but are often insufficient for the control of IMI caused by S. uberis²⁰. Consequently, the interest has focused on the development of immunoprophylactic strategies. However, a significant obstacle in the design of an effective vaccine is the high level of genetic variability of different isolates of S. uberis, frequently involving virulence factor genes^18,31. In this context, epidemiological studies of regional isolates are extremely useful to detect the frequency and distribution of bacterial types associated with IMI and to identify target molecules for the development of immunogens and therapeutic agents. Methods based on DNA analysis including restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), pulsed field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) have been successfully applied to fulfill this need. PFGE is the most discriminatory method for typing bacteria²², whereas RAPD is a more straightforward method given its relatively lower costs, execution time demanded, less expensive equipment requirements and sensitivity. Both methods have been widely used to study the genetic variability of many bacterial species, including important human pathogens^22,37.

Several S. uberis virulence factors have been described^16,20,23,24. Among them, plasminogen activator A (PauA)³⁵ and S. uberis adhesion molecule (SUAM)² have shown potential as protective immunogens. The former is a protease capable of activating plasmin, which in turn, degrades proteins producing small peptides and free amino acids used by bacteria as a nitrogen source. This factor has been related to early mammary gland colonization²¹ while SUAM both to adherence and internalization, through its binding to lactoferrin²⁸, and to bacterial persistence in bovine mammary epithelial cells in vitro².

The wide genetic diversity observed in S. uberis indicates that virulence is not associated with any specific molecular type⁴³. Therefore, immunologic prevention strategies against this organism should be directed to a multitude of different factors included in field isolates from individual herds. Nevertheless, the difficulty associated with S. uberis genetic variability can be currently overcome through bioinformatic tools that allow to analyze DNA sequence encoding genes from virulence factors which are found in a large number of isolates. Thus, epidemiological studies on gene distribution together with their sequence analysis will contribute to identify potential antigens as vaccine components.

The main objectives of this work were to determine the clonal relationship between S. uberis isolates from milk of cows suffering from subclinical or clinical mastitis in Argentina and to assess the prevalence and conservation of the pauA and sua genes.

Materials and methods

S. uberis collection and DNA preparation

A total of 137 S. uberis strains were isolated from milk of 122 cows suffering from clinical and subclinical mastitis between November 2010 and July 2012. Milk samples were obtained from 35 dairy herds located in the main dairy regions of Argentina (Fig. 1). Clinical mastitis was characterized by evidence of inflammation (abnormal milk, heat, swelling or pain of affected quarters) while subclinical mastitis by lack of local or systemic signs of mammary inflammation and somatic cell counts ≥200,000 cells/ml.

Figure 1. The total number (n = 137) of S. uberis isolates recovered from bovines suffering from subclinical or clinical mastitis were from 35 dairy farms located in the main dairy region of Argentina (black and gray globes). Gray globes indicate geographical origin of isolates selected for the sua and pauA sequencing.

S. uberis isolates were initially identified by standard conventional biochemical tests such as esculin hydrolysis, hippurate hydrolysis, growth on 6.5 % NaCl, growth on bile-esculin-agar and the CAMP test²⁵. Restriction fragment length polymorphism (RFLP) of 16S rDNA¹⁴ was performed for molecular confirmation. S. uberis ATCC 27958 was used as reference strain.

Genomic DNA was extracted using a modification of the method described by Hill and Leigh¹³. The detailed protocol is available at pubmlst.org/suberis/info/protocol.shtml.

Molecular typing of S. uberis strains

Molecular typing was carried out by PFGE and RAPD-PCR. Pulsed-field electrophoresis was performed in accordance with a previously described method⁵. Briefly, electrophoresis was performed with the CHEF-DR III SYSTEM (Bio-Rad Laboratories) using 1 % Pulsed Field Certified™ (BioRad Laboratories) in 0.5× Tris-borate-EDTA (TBE) buffer at 11.3 °C for 23 h. The electrophoresis conditions were as follows: initial switch time value of 5 s, final switch time of 35 s at a gradient of 6 V/cm. After electrophoresis, the gel was stained with ethidium bromide solution (0.5 mg/l) and then destained with deionized water. Then the DNA bands were visualized with a UV transilluminator and a digital image of PFGE patterns was obtained using Molecular Imager® Gel Doc™ XR+ System (BioRad, Laboratories Inc., Richmond, CA, USA).

RAPD-PCR was performed as previously described⁴⁵ using primer OPE-4. The amplified products were separated by gel electrophoresis in 1.5 % agarose gel, stained with ethidium bromide. Banding pattern similarities were analyzed using the Dice correlation coefficient. Values for tolerance and optimization were set at 1.5 % and 1 %, respectively. The cluster analysis was based on UPGMA, Unweighted Pair Group Method with Arithmetic averages, and data were analyzed with the TREECON software for Windows³⁹. Isolates indicating more than 3 DNA fragment differences and a similarity of < 80 % following the dendrogram analysis were classified as different PFGE or RAPD types, whereas fragment variations and a similarity of > 80 % following the dendrogram analysis were defined as PFGE or RAPD subtypes. Isolates showing identical patterns (100 % pattern similarity index) or a similarity index of > 80 % were interpreted as belonging to the same PFGE or RAPD type¹. The typing experiments were repeated at least twice.

Calculation of concordance, Simpson's index of diversity and Wallace's coefficients

Simpson's index of diversity was calculated to measure the discriminatory power of the typing systems. This index indicates the probability that 2 strains randomly sampled from a population will belong to 2 different types⁴. Wallace's coefficients were used to explore the correlation between results produced by PFGE and RAPD. These were calculated using EpiCompare version 1.0 (http://www3.ridom.de/epicompare/). Group concordance was evaluated by cross-classification of all possible pairs of isolates as previously described³⁴. All possible pairs of isolates were cross-classified according to matched or mismatched types. The resulting 2 × 2 table was evaluated by the chi-square test and the percentage of concordant cells was calculated using the GraphPad Prism 5 (Version 5, USA) software.

PCRs and DNA sequencing

DNA amplification for the sua gene was performed using oligonucleotide primers derived from published sequences²³. Amplification of the pauA gene was performed using the following primers, which were designed using DNAstar Primer Select software (DNAstar, Madison, WI), 5'-TTTTTAATATTAATGCTTTTG-3' and 5'-AGAAAAATTTAATGGATAC-3'. The reaction mixture was made with 150 ng/µl template DNA, 0.8 µM oligonucleotide primers, 0.2 µM of each of the four dNTPs, 1.25 U Taq polymerase and 3 mM MgCl₂, in a final volume of 50 µl. The expected amplicon size was 874 bp. PCR conditions were 5 min at 95 °C, 30 cycles of [1 min at 94 °C, 1 min at 49 °C and 1 min at 72 °C], 5 min at 72 °C.

The PCR products from 20 isolates of S. uberis were selected for sequencing. The following criteria were taken into account for isolate selection: isolates had to belong to different geographical regions and/or had to be epidemiologically unrelated (Table 1; Figs. 1 and 2). DNA was purified using the Wizard®SV Gel and PCR Clean-Up System (Promega) according to the manufacturers' protocol and eluted in 30 µl of sterile MilliQ water. Positive PCR products were sequenced in both directions using the same specific PCR primers for the pauA gene while for the sua gene the following oligonucleotides were added: sua2: 5'-GAA TTC ACA CAA TCT GAC GAG GT-3'; sua3: 5'-GAA TTC GAA GTT GGG GCA TAC-3' and sua4: 5'-GAA TTC CCA AGT GCT CCG GTC T-3' to the PCR primers. DNA samples (30-50 ng/µl) were sequenced by ABI3130XL sequencer analyzer (Applied Biosystems) from the sequencing service of the Biotechnology Institute of INTA Castelar, Argentina.

Table 1. Characteristics of S. uberis isolates according to their PFGE type.

Figure 2. Dendrogram of pulsed-field gel electrophoresis (PFGE) (A) and random amplified polymorphic DNA (RAPD) (B) profiles of 137 Streptococcus uberis subclinical mastitis isolates collected from 35 dairy herds. Isolate code and PFGE type (A) and RAPD type (B) of each strain are also represented in the dendrogram. The dendrogram was produced by using Dice coefficients and an unweighted pair group method using arithmetic averages (UPGMA).

Molecular and data analysis

Forward and reverse sequences were aligned using Sequencher-DNA Sequencig Software Demo 5.1 (Gene Codes Corporation) and the consensus sequence files converted to FASTA format. Sequences were analyzed by searching the GenBank database of the National Center for Biotechnology Information via the Basic Local Alignment Search Tool (BLAST) network service. Then, the sequences were compared between them and against all existing allelic types from the database using the Codon Code Aligner software (Codon Code Corporation, Dedham, MA). The pauA sequences were also compared through the Food Microbe Tracker database at www.pathogentracker.net⁴⁸.

Results

Typability and discrimination of PFGE typing

The molecular epidemiological analysis of S. uberis isolates from milk identified by RFLP was performed on 137 isolates from 35 dairy farms. Digested chromosomal DNA generated 1-23 fragments ranging from 48.5 to 436.5 kb in size, which could be resolved by PFGE (Fig. 2a). Epidemiologically related groups were identified based on a minimum 80 % similarity. The analysis of the 137 isolates revealed 61 types of PFGE patterns, 29 of which were unique isolates, and 32 types, containing 2-8 isolates per type. Isolate distribution in groups was: 19 types with 2 isolates (PF 04, 9, 11, 12, 15, 16, 18, 21, 23, 25, 27, 37, 43, 45, 46, 47, 55, 56 and 57), 5 types with 3 isolates (PF 13, 19, 32, 33 and 40), 5 types with 4 isolates (PF 20, 31, 34, 35 and 39), 2 types with 6 isolates (PF14 and PF29) and 1 type with 8 isolates (PF1) (Table 1). Genotype grouping appeared to be neither related to the mastitis type (clinical or subclinical) nor to the farm origin. Epidemiologically related groups were observed among isolates from different farms. Similarities ranging between 80.1 % and 100 % were established in isolates retrieved from the same farm in only 11 types (PF 1, 4, 14, 19, 20, 29, 31, 32, 34, 35 and 47), suggesting a common source or horizontal transmission of the pathogen. Fifteen out of the 137 studied isolates gave a poor PFGE quality pattern; therefore, they could not be included in the analysis. These strains were classified as non-typeable, resulting in 90.5 % typability for this technique according to the Epicompare software analysis.

Typability and discrimination of RAPD-PCR typing

Products obtained after RAPD-PCR amplification were 0.5-8.0 kb in size (Fig. 2b). All samples exhibited at least one of these products: 1632 bp, 1800 bp, and 2430 bp. Differences among isolates were given by products ranging between 1800 and 1632 bp in size. The analysis was carried out in 137 samples using the same criteria for PFGE which allowed the identification of 25 clonal patterns. The patterns were assigned to 5 types with only one isolate and 20 types, containing 2-17 isolates. The composition of the groups was as follows: 4 types with 2 isolates (RD-7, 12, 20 and 22), 3 types with 3 isolates (RD-5, 8 and 15), 1 type with 4 isolates (RD-19), 5 types with 5 isolates (RD-4, 11, 16, 18 and 23), 1 type with 7 isolates (RD-14), 2 types with 8 isolates (RD-1 and 6), 1 with 9 isolates (RD-3), 1 with 11 isolates (RD-9), 1 with 13 isolates (RD-13), and 1 with 17 isolates (RD-10). As for PFGE, no association was found either between mastitis type or farm of origin. Thirteen isolates could not be assigned to any group given the poor quality observed in their electrophoretic pattern, and therefore they were classified as non-typeable resulting in 89 % typability for this technique according to the Epicompare software analysis.

Comparison of the typing methods

To compare the discriminatory power of PFGE and RAPD, we calculated the Simpson's diversity index for both methods (Table 2). PFGE and RAPD yielded different Simpson index values of 0.983 (95 % CI 0.978-0.989) and 0.941 (95 % CI 0.927-0.955), respectively. To compare the congruence between type assignments using PFGE and RAPD, we calculated the Wallace coefficients. Wallace coefficients for PFGE and RAPD indicated a weak bidirectional correspondence (0.042-0.172) between types generated by both methods. However, cross-classification of the isolates, based on matched or mismatched schemes by PFGE and RAPD, showed that the 2 typing systems were 92.6 % concordant (Table 3).

Table 2. Simpson's diversity indices of the genotyping methods for all typed isolates.

Table 3. Cross-classification of all possible pairs of isolates based on matched or mismatched PFGE and RAPD types.

Detection and evaluation of the sua and pauA genes

PauA, encoded by the pauA gene, and SUAM, encoded by the sua gene, have been described as two of S. uberis major virulence factors and as potential vaccine immunogens against this pathogen. We studied the distribution of these genes among a population of S. uberis from our country and analyzed their sequences in 20 selected isolates (Table 1; Fig. 1). Table 1 and Fig. 1 show S. uberis isolates selected according to geographical differences and/or epidemiological dissimilarity. The encoding genes for SUAM and PauA were present in the majority of S. uberis isolates from different Argentinean dairy areas. The pauA gene was detected in 94.9 % (130/137) whereas the sua gene in 97.8 % (134/137).

Nucleotide sequences of the sua gene from the 20 S. uberis selected isolates showed 99 % identity with respect to the unique reference sequence from the GenBank (DQ232760.1)². In concordance with the identity of nucleotide sequences, the amino acid sequences from mastitis isolates showed between 97 % and 99 % identity with respect to the reference sequence (GenBank ABB52003.1)². Amino acids encoded by codons with single mutations in the sua gene are shown in Table 4. About 5-13 amino acid changes were detected in the 20 isolates. It is worth mentioning that the changes in encoded amino acids were repeated in the different isolates and suggesting conserved mutations (Table 4).

Table 4. Amino acids encoded by codons with single mutations in the S. uberis adhesion molecule coding gene sua of S. uberis.

 

The difference in amino acid sequence from the majority are bolded.

Nucleotide sequences of the pauA gene from the 20 selected S. uberis mastitis isolates showed between 95 % and 100 % identity with respect to all the 55 sequences registered in GenBank and to 30 sequences registered in the Food Microbe Tracker database. Similarly, the amino acid sequences showed 96-100 % identity with respect to the reference sequences. Amino acids encoded by codons with single mutations in pauA are shown in Table 5. About 1-6 mutations that resulted in amino acid change were detected in 11 isolates. Sequence data of the sua and pauA genes were registered in GenBank under accession numbers KT006548-KT006587.

Table 5. Amino acids encoded by codons with single mutations in the plasminogen activator coding gene pauA of S. uberis.

Discussion

S. uberis is a well-known pathogen causing bovine IMI worldwide. However, there is scant epidemiological information on S. uberis isolated from bovine milk in Argentina¹⁹. In the present study, PFGE and RAPD were used for the molecular characterization of S. uberis isolated from cows belonging to herds located in the main dairy areas of Argentina. Based of this information a reliable representative set of isolates was selected for the study of the pauA and sua genes.

As shown in previous studies^1,32,36,43, PFGE has proven to be a highly discriminatory method. In the present study, PFGE was able to resolve many isolates that were indistinguishable by RAPD-PCR. Some isolates from different herds that were defined as belonging to the same type (100 % similarities) were associated with different types of mastitis (both clinical and subclinical), in agreement with previous studies^15,29. The PFGE patterns for S. uberis showed great variation; among the 137 isolates (collected from 122 cows on 35 farms), 61 distinct PFGE profiles were observed. This high level of heterogeneity is in accordance with classical epidemiological studies from Argentina and other countries^1,19,32,42. However, we found eleven cases of one type isolated from different animals in the same dairy herd. This finding suggests that cows were infected by the same organism, either from a common source or by spread from one quarter or cow to another. Poor milking hygiene and faulty milking machine functioning could contribute to S. uberis transmission among cows⁴⁷. The total epidemiological data reported in this work allows to consider S. uberis as an environmental opportunistic pathogen, with a limited number of dominant types and a great variety of strains.

RAPD typing relies on non-stringent reaction conditions for the amplification of arbitrary target sites²⁶. Low stringent reaction conditions are associated with difficulty to achieve high pattern repeatability¹⁰. Target DNA concentration conditions and the thermal cycling program were standardized in our fingerprinting method to yield constant and reproducible results. The analysis of the 137 isolates distinguished 25 distinct RAPD profiles. Conserved band pattern obtained with RAPD-PCR disagreed with those previously reported by groups from New Zealand and United States^11,45. These discrepancies indicate that the method does not allow to compare results from different geographical origins. However, RAPD allows to perform epidemiological studies in less time at lower costs⁴⁵.

The comparative analysis of RAPD and PFGE results indicated that isolates belonging to the same type according to RAPD exhibited different PFGE patterns. The discriminatory power and congruence between both methods were compared using the Simpson's index of diversity and Wallace's coefficients. Our results indicated that both typing methods had a high discriminatory power between epidemiologically non-related isolates as indicated by the Simpson's index of diversity. Although the Simpson's diversity index of the FPGE method was higher than that of the RAPD method, the difference was modest. The congruence between types defined by PFGE and RAPD yielded low values, as reflected by the Wallace coefficients (0.042 and 0.172), indicating a weak bidirectional agreement among types generated by both methods. PFGE is a technique of choice for short-term or local epidemiological studies, since even minor genetic changes can lead to a three-fragment difference in the PFGE banding pattern¹. Moreover, several studies have shown that PFGE has greater discriminatory power than MLST^32,38,40. Gillespie and Oliver¹² compared RAPD and PFGE for differentiation of S. uberis isolates, concluding that PFGE had higher discriminatory power than RAPD on the basis of the number of groups that could be differentiated by each method. Similar results were obtained in the present study. However, considering the high concordance between both methods (96.2 %), we can conclude that any given pair of isolates distinguished by one method tended to be distinguished by the other. To our knowledge, this is the first study reporting a statistical analysis for comparison of these two techniques applied for S. uberis molecular epidemiology.

A successful vaccine should confer broad protection against a multitude of strains. This approach requires a detailed knowledge of the epidemiology and pathogenesis of the organism. Despite the severe economic impact caused by this infection, the virulence factors associated with S. uberis pathogenesis are not well understood². Plasminogen activators such as PauA have been proposed as important intermediaries to obtain nutrients for optimal growth of the organism⁴⁴. In addition, the successful establishment of IMI depends on adherence, internalization, and intracellular persistence²⁸. The SUAM was found to play a central role in S. uberis adherence to bovine mammary epithelial cells⁸, contributing to infection persistence. These two virulence factors have been previously studied as potential immunogens^3,21. However, it is unknown whether genetic variability could have modified the effectiveness they demonstrated in preclinical trials. In the present study, 94.9 % of the strains studied carried the pauA gene. The prevalence of the pauA gene was previously reported as ranging from 61.5 % in Argentina to 100 % in India^17,33,36. To get further insight, we studied the nucleotide and amino acid sequences from 20 selected S. uberis mastitis isolates. We observed an identity greater than 95 % with respect to all the 55 sequences registered in GenBank and to the 30 sequences registered in the Food Microbe Tracker database. Furthermore, amino acid sequences showed a low number of mutations. Mutations at positions 43, 67, 217, 343, 371, 631, 718 and 724 of the consensus sequences coincided with those described by Zadoks et al⁴⁷. In fact, the mutations observed in other positions found in this work were not described until now. Therefore, pauA is present in most isolates and interestingly, it is a highly conserved gene. Correspondingly, the presence of the sua gene was detected in 97.8 % of the 137 isolates. Previous works reported a prevalence of the sua gene of 83.3 %³³ in Argentina, 84.6 % in India³⁶ and 100 % in New Zealand, United States and England^23,46. In addition, the nucleotide and amino acid sequences from the 20 selected S. uberis mastitis isolates had an identity greater than 97 % with respect to the reference sequence (ABB52003.1). The study of amino acid sequences showed several repeated mutations in different isolates. Yuan et al. described similar results in sua gene conservation among isolates from different countries⁴⁶. The relevance of the present findings, compared with previous studies, relies on the higher number (n = 137), the strict criteria used for the selection and broad distribution of the isolates analyzed. All together these results demonstrate the high prevalence and conservation of the sua and pauA genes in Argentinean S. uberis isolates, which was also reflected in their amino acid sequence analysis. To our knowledge, this is the first study that compared these two virulence factors in sequences from field isolates versus reported GenBank sequences from different countries.

It has been suggested that the genetic clonal diversity of S. uberis is an obstacle for the development of an effective vaccine since a broadly-reactive immunogen with field strains has not been obtained so far^9,21. However, previous works and the present study demonstrated that S. uberis isolates belonging to different PFGE profiles showed conserved gene sequences³². In addition, it has to be taken in account that the minimal variation observed in gene carriage highlights a potential problem with respect to the development of subunit vaccines against S. uberis, since vaccines based on a single antigen may not provide protection against all strains of the pathogen even using conserved genes. Thus, we found that those isolates that did not carry the sua gene harbor the pauA gene and vice versa (Table 1), and only in two isolates (1.45 %) none of these genes were detected. All together, our results showed that the pauA and sua genes are conserved across different geographical areas and are present in most S. uberis isolates despite the high genetic variability observed, which provides further support for the use of these virulence factors as potential vaccine components against S. uberis.

Ethical disclosures

Protection of human and animal subjects

The authors declare that the procedures followed were in accordance with the regulations of the relevant clinical research ethics committee and with those of the Code of Ethics of the World Medical Association (Declaration of Helsinki).

Confidentiality of data

The authors declare that no patient data appear in this article.

Right to privacy and informed consent

The authors declare that no patient data appear in this article.

Funding

This work was supported by grants from FONCyT (M.S.B., PICT Bicentenario 2010-1309), Q5 (F.R.B., PICT Bicentenario 2010-0733), (L.F.C. and I.S.M., PICT 1175) and from Fundación Banco de la Provincia de Santa Fe (M.S.B.).

Conflict of interest

The authors declare that they have no conflicts of interest.

Acknowledgements

We thank M.V. Liliana Tirante (Lactodiagnóstico Sur, Olivos, Buenos Aires) for providing the isolates and Dr. Andrea Puebla (Instituto de Biotecnología, CICVyA Castelar, INTA) for sequencing service. M.S.P. is a fellow from CONICET, I.S.M. and F.R.B. are independent and adjunct members of the research career of CONICET.

References

1. Abureema S, Smooker P, Malmo J, Deighton M. Molecular epidemiology of recurrent clinical mastitis due to Streptococcus uberis: evidence of both an environmental source and recurringinfection with the same strain. J Dairy Sci. 2014;97:285-90.         [ Links ]

2. Almeida RA, Luther DA, Park HM, Oliver SP. Identification, isolation, and partial characterization of a novel Streptococcus uberis adhesion molecule (SUAM). Vet Microbiol.2006;115:183-91.         [ Links ]

3. Almeida RA, Luther DA, Patel D, Oliver SP. Predicted antigenic regions of Streptococcus uberis adhesion molecule (SUAM) are involved in adherence to and internalization into mammaryepithelial cells. Vet Microbiol. 2010;148:323-8.         [ Links ]

4. Carrico JA, Silva-Costa C, Melo-Cristino J, Pinto FR, de Lencastre H, Almeida JS, Ramirez M. Illustration of a common framework for relating multiple typing methods by application to macrolide-resistant Streptococcus pyogenes. J Clin Microbiol.2006;44:2524-32.         [ Links ]

5. Chung M, de Lencastre H, Matthews P, Tomasz A, Adamsson I, Aires de Sousa M, Camou T, Cocuzza C, Corso A, Couto I, Dominguez A, Gniadkowski M, Goering R, Gomes A, Kikuchi K, Marchese A, Mato R, Melter O, Oliveira D, Palacio R, Sa-Leao R, Santos Sanches I, Song JH, Tassios PT, Villari P. Molecular typing of methicillin-resistant Staphylococcus aureus by pulsed-field gel electrophoresis: comparison of results obtained in a multilaboratory effort using identical protocols and MRSA strains. Microb Drug Resist. 2000;6:189-98.         [ Links ]

6. De Vliegher S, Fox LK, Piepers S, McDougall S, Barkema HW. Invited review: Mastitis in dairy heifers: nature of the disease, potential impact, prevention, and control. J Dairy Sci. 2014;95:1025-40.         [ Links ]

7. Douglas VL, Fenwick SG, Pfeiffer DU, Williamson NB, Holmes CW. Genomic typing of Streptococcus uberis isolates from cases of mastitis, in New Zealand dairy cows, using pulsed-field gel electrophoresis. Vet Microbiol. 2000;75:27-41.         [ Links ]

8. Fang W, Almeida RA, Oliver SP. Effects of lactoferrin and milk on adherence of Streptococcus uberis to bovine mammary epithelial cells. Am J Vet Res. 2000;61:275-9.         [ Links ]

9. Finch JM, Winter A, Walton AW, Leigh JA. Further studies on the efficacy of a live vaccine against mastitis caused by Streptococcus uberis. Vaccine. 1997;15:1138-43.         [ Links ]

10. Gallego FJ, Martinez I. Method to improve reliability of random-amplified polymorphic DNA markers. Biotechniques. 1997;23:663-4.         [ Links ]

11. Gillespie BE, Jayarao BM, Pankey JW, Oliver SP. Subtyping of Streptococcus dysgalactiae and Streptococcus uberis isolated from bovine mammary secretions by DNA fingerprinting. Zentralbl Veterinarmed B. 1998;45:585-93.         [ Links ]

12. Gillespie BE, Oliver SP. Comparison of an automated ribotyping system. Pulsed-field gel electrophoresis and randomly amplified polymorphic DNA fingerprinting for differentiation of Streptococcus uberis strains. Biotechnology. 2004;3:165-72.         [ Links ]

13. Hill AW, Leigh JA. DNA fingerprinting of Streptococcus uberis: a useful tool for epidemiology of bovine mastitis. Epidemiol Infect. 1989;103:165-71.         [ Links ]

14. Jayarao BM, Dore JJ Jr, Oliver SP. Restriction fragment length polymorphism analysis of 16S ribosomal DNA of Streptococcus and Enterococcus species of bovine origin. J Clin Microbiol. 1992;30:2235-40.         [ Links ]

15. Jayarao BM, Schilling EE, Oliver SP. Genomic deoxyribonucleic acid restriction fragment length polymorphism of Streptococcus uberis: evidence of clonal diversity. J Dairy Sci. 1993;76:468-74.         [ Links ]

16. Jiang M, Babiuk LA, Potter AA. Cloning, sequencing and expression of the CAMP factor gene of Streptococcus uberis. Microb Pathog. 1996;20:297-307.         [ Links ]

17. Khan IU, Hassan AA, Abdulmawjood A, Lammler C, Wolter W, Zschock M. Identification and epidemiological characterization of Streptococcus uberis isolated from bovine mastitis using conventional and molecular methods. J Vet Sci. 2003;4:213-24.         [ Links ]

18. Lang P, Lefebure T, Wang W, Zadoks RN, Schukken Y, Stanhope MJ. Gene content differences across strains of Streptococcus uberis identified using oligonucleotide microarray comparative genomic hybridization. Infect Genet Evol. 2009;9:179-88.         [ Links ]

19. Lasagno MC, Reinoso EB, Dieser SA, Calvinho LF, Buzzola F, Vissio C, Bogni CI, Odierno LM. Phenotypic and genotypic characterization of Streptococcus uberis isolated from bovine subclinical mastitis in Argentinean dairy farms. Rev Argent Microbiol. 2011;43:212-7.         [ Links ]

20. Leigh JA. Streptococcus uberis: a permanent barrier to the control of bovine mastitis? Vet J. 1999;157:225-38.         [ Links ]

21. Leigh JA, Finch JM, Field TR, Real NC, Winter A, Walton AW, Hodgkinson SM. Vaccination with the plasminogen activator from Streptococcus uberis induces an inhibitory response and protects against experimental infection in the dairy cow. Vaccine. 1999;17:851-7.         [ Links ]

22. Li W, Raoult D, Fournier PE. Bacterial strain typing in the genomic era. FEMS Microbiol Rev. 2009;33:892-916.         [ Links ]

23. Luther DA, Almeida RA, Oliver SP. Elucidation of the DNA sequence of Streptococcus uberis adhesion molecule gene (sua) and detection of sua in strains of Streptococcus uberis isolated from geographically diverse locations. Vet Microbiol. 2008;128:304-12.         [ Links ]

24. Matthews KR, Almeida RA, Oliver SP. Bovine mammary epithelial cell invasion by Streptococcus uberis. Infect Immun. 1994;62:5641-6.         [ Links ]

25. Odierno L, Calvinho L, Traverssa P, Lasagno M, Bogni C, Reinoso E. Conventional identification of Streptococcus uberis isolated from bovine mastitis in Argentinean dairy herds. J Dairy Sci. 2006;89:3886-90.         [ Links ]

26. Olive DM, Bean P. Principles and applications of methods for DNA-based typing of microbial organisms. J Clin Microbiol. 1999;37:1661-9.         [ Links ]

27. Oliver SP. Frequency of isolation of environmental mastitiscausing pathogens and incidence of new intramammary infection during the nonlactating period. Am J Vet Res. 1988;49:1789-93.         [ Links ]

28. Patel D, Almeida RA, Dunlap JR, Oliver SP. Bovine lactoferrin serves as a molecular bridge for internalization of Streptococcus uberis into bovine mammary epithelial cells. Vet Microbiol. 2009;137:297-301.         [ Links ]

29. Phuektes P, Mansell PD, Dyson RS, Hooper ND, Dick JS, Browning GF. Molecular epidemiology of Streptococcus uberis isolates from dairy cows with mastitis. J Clin Microbiol. 2001;39:1460-6.         [ Links ]

30. Piepers S, De Vliegher S, de Kruif A, Opsomer G, Barkema HW. Impact of intramammary infections in dairy heifers on future udder health, milk production, and culling. Vet Microbiol. 2009;134:113-20.         [ Links ]

31. Pullinger GD, Lopez-Benavides M, Coffey TJ, Williamson JH, Cursons RT, Summers E, Lacy-Hulbert J, Maiden MC, Leigh JA. Application of Streptococcus uberis multilocus sequence typing: analysis of the population structure detected among environmental and bovine isolates from New Zealand and the United Kingdom. Appl Environ Microbiol. 2006;72:1429-36.         [ Links ]

32. Rato MG, Bexiga R, Nunes SF, Cavaco LM, Vilela CL, Santos- Sanches I. Molecular epidemiology and population structure of bovine Streptococcus uberis. J Dairy Sci. 2008;91:4542-51.         [ Links ]

33. Reinoso EB, Lasagno MC, Dieser SA, Odierno LM. Distribution of virulence-associated genes in Streptococcus uberis isolated from bovine mastitis. FEMS Microbiol Lett. 2011;318: 183-8.         [ Links ]

34. Robinson DA, Hollingshead SK, Musser JM, Parkinson AJ, Briles DE, Crain MJ. The IS1167 insertion sequence is a phylogenetically informative marker among isolates of serotype 6B Streptococcus pneumoniae. J Mol Evol. 1998;47:222-9.         [ Links ]

35. Rosey EL, Lincoln RA, Ward PN, Yancey RJ, Leigh JA. PauA: a novel plasminogen activator from Streptococcus uberis. FEMS Microbiol Lett. 1999;178:27-33.         [ Links ]

36. Shome BR, Bhuvana M, Mitra SD, Krithiga N, Shome R, Velu D, Banerjee A, Barbuddhe SB, Prabhudas K, Rahman H. Molecular characterization of Streptococcus agalactiae and Streptococcus uberis isolates from bovine milk. Trop Anim Health Prod. 2012;44:1981-92.         [ Links ]

37. Soyer Y, Alcaine SD, Schoonmaker-Bopp DJ, Root TP, Warnick LD, McDonough PL, Dumas NB, Grohn YT, Wiedmann M. Pulsedfield gel electrophoresis diversity of human and bovine clinical Salmonella isolates. Foodborne Pathog Dis. 2010;7:707-17.         [ Links ]

38. Van Belkum A, Tassios PT, Dijkshoorn L, Haeggman S, Cookson B, Fry NK, Fussing V, Green J, Feil E, Gerner-Smidt P, Brisse S, Struelens M. Guidelines for the validation and application of typing methods for use in bacterial epidemiology. Clin Microbiol Infect. 2007;13 Suppl. 3:1-46.         [ Links ]

39. Van de Peer Y, De Wachter R. TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Appl Biosci. 1994;10:569-70.         [ Links ]

40. Vimont S, Mnif B, Fevre C, Brisse S. Comparison of PFGE and multilocus sequence typing for analysis of Klebsiella pneumoniae isolates. J Med Microbiol. 2008;57:1308-10.         [ Links ]

41. Vissio C, Agüero DA, Raspanti CG, Odierno LM, Larriestra AJ. Productive and economic daily losses due to mastitis and its control expenditures in dairy farms in Córdoba, Argentina. Arch Med Vet. 2015;47.         [ Links ]

42. Wang L, Chen W, Zhang L, Zhu Y. Genetic diversity of Streptococcus uberis isolates from dairy cows with subclinical mastitis in Southern Xinjiang Province, China. J Gen Appl Microbiol. 2013;59:287-93.         [ Links ]

43. Wang SM, Deighton MA, Capstick JA, Gerraty N. Epidemiological typing of bovine streptococci by pulsed-field gel electrophoresis. Epidemiol Infect. 1999;123:317-24.         [ Links ]

44. Ward PN, Leigh JA. Genetic analysis of Streptococcus uberis plasminogen activators. Indian J Med Res. 2004;119:136-40.         [ Links ]

45. Wieliczko RJ, Williamson JH, Cursons RT, Lacy-Hulbert SJ, Woolford MW. Molecular typing of Streptococcus uberis strains isolated from cases of bovine mastitis. J Dairy Sci. 2002;85:2149-54.         [ Links ]

46. Yuan Y, Dego OK, Chen X, Abadin E, Chan S, Jory L, Kovacevic S, Almeida RA, Oliver SP. Short communication: Conservation of Streptococcus uberis adhesion molecule and the sua gene in strains of Streptococcus uberis isolated from geographically diverse areas. J Dairy Sci. 2014;97: 7668-73.         [ Links ]

47. Zadoks RN, Gillespie BE, Barkema HW, Sampimon OC, Oliver SP, Schukken YH. Clinical, epidemiological and molecular characteristics of Streptococcus uberis infections in dairy herds. Epidemiol Infect. 2003;130:335-49.         [ Links ]

48. Zadoks RN, Schukken YH, Wiedmann M. Multilocus sequence typing of Streptococcus uberis provides sensitive and epidemiologically relevant subtype information and reveals positive selection in the virulence gene pauA. J Clin Microbiol.2005;43:2407-17.         [ Links ]