Journal of Dairy Science
Volume 89, Issue 10 , Pages 3886-3890, October 2006

Conventional Identification of Streptococcus uberis Isolated from Bovine Mastitis in Argentinean Dairy Herds

  • L. Odierno

      Affiliations

    • Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, 5800 Río Cuarto, Córdoba, República Argentina
    • Corresponding Author InformationCorresponding author.
    • ,
  • L. Calvinho

      Affiliations

    • Estación Experimental Agropecuaria Rafaela, Instituto Nacional de Tecnología Agropecuaria, 2300 Rafaela, Santa Fe, República Argentina
    • ,
  • P. Traverssa

      Affiliations

    • Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, 5800 Río Cuarto, Córdoba, República Argentina
    • ,
  • M. Lasagno

      Affiliations

    • Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, 5800 Río Cuarto, Córdoba, República Argentina
    • ,
  • C. Bogni

      Affiliations

    • Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, 5800 Río Cuarto, Córdoba, República Argentina
    • ,
  • E. Reinoso

      Affiliations

    • Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, 5800 Río Cuarto, Córdoba, República Argentina

Received 27 December 2005; accepted 26 May 2006.

Article Outline

Abstract 

The objective of this study was to evaluate a conventional scheme for identifying Streptococcus uberis strains isolated from bovine mastitis. Seventy-five gram-positive, catalase-negative cocci were collected from cows with mastitis from 19 dairy herds located in the east-central region of Argentina. Five American Type Culture Collection strains and bovine isolates were identified by the API 20 Strep system and by restriction fragment length polymorphism analysis of 16S rDNA. A conventional scheme based on 11 biochemical tests was selected for identification of Strep. uberis strains: the Christie–Atkins–Munch-Petersen reaction; hydrolysis of Arg, esculin, and sodium hippurate; growth in inulin, mannitol, raffinose, salicin, and sorbitol; and growth at 45°C and in 6.5% NaCl. Reference strains and 25 bovine isolates were classified accurately to the species level by the conventional scheme in a blind assay. Each reference strain and each bovine isolate were identified as belonging to the same species following these 3 methods. The remaining 50 isolates identified as Strep. uberis by the API 20 Strep system and 16S rDNA RFLP were assayed by the conventional scheme. This scheme correctly identified 47 (94%) of 50 isolates as Strep. uberis by comparing their biochemical profile with that of the reference strain. Three (6%) of the 50 isolates were classified as Strep. uberis by the API 20 Strep system and by 16S rDNA RFLP and were identified as Enterococcus faecalis by the conventional scheme. Thirty percent of the Strep. uberis strains showed biochemical profiles identical to the Strep. uberis American Type Culture Collection 27958 strain. Seventy percent of the Strep. uberis strains demonstrated variability compared with the reference strain, resulting in 19 different biochemical profiles. The conventional scheme proposed in this study resulted in a relatively low number of misidentifications and could biochemically identify not only typical, but also atypical Strep. uberis strains. This conventional scheme can be considered an adequate method for identifying Strep. uberis strains isolated from bovine mastitis because of its affordable cost in developing countries, and it may contribute to determining the frequency of isolation of Strep. uberis strains in Argentinean dairy herds.

Key words: Streptococcus uberis, bovine mastitis, conventional identification

 

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Introduction 

Bovine mastitis is the most costly disease among dairy cows. Estimates of losses caused by mastitis range from US$35 to $295 per cow per year (DeGraves and Fetrow, 1993). In Argentina milk production losses have been estimated at US$221million a year (Asociación Argentina de lucha contra la mastitis, 1983).

Streptococcus uberis is known worldwide as an environmental pathogen responsible for a high proportion of cases of clinical and subclinical mastitis in lactating cows and is also the predominant organism isolated from mammary glands during the nonlactating period (Bradley, 2002; Khan et al., 2003). Accurate and cost-effective methods of identifying mastitis pathogens are important for the diagnosis, surveillance, and control of this economically important disease among dairy cows (McDonald, 1984; Leigh, 1999).

Gram-positive, catalase-negative, esculin-hydrolyzing cocci isolated from cases of clinical and subclinical mastitis are commonly categorized as Strep. uberis in routine diagnostic laboratories. The heavy workload of mastitis diagnostic services does not allow more accurate identification. Upon closer examination, however, many of these bacteria may in fact be very different from this major streptococcal pathogen. The differential scheme for mastitis streptococci and enterococci of the US National Mastitis Council (National Mastitis Council, 1990) lists Strep. uberis, Streptococcus bovis, Streptococcus equinus, Enterococcus faecalis, Enterococcus faecium, and Enterococcus saccharolyticus as esculin-hydrolyzing species and Streptococcus dysgalactiae and Streptococcus equi as esculin-variable species. More recently, 2 new esculin-hydrolyzing streptococcal species, Streptococcus parauberis (Williams and Collins, 1990) and Streptococcus plurianimalium (Devriese et al., 1999), were isolated from bovine mastitis. However, according to different authors (King, 1981; Calvinho et al., 1991; Khan et al., 2003), Strep. uberis, Strep. dysgalactiae, Strep. bovis, Strep. equinus, and E. faecalis represent the esculin-positive or esculin-variable cocci most frequently isolated from mastitic cows. Farrow and Collins (1984) examined a collection of strains, most of which were nonhuman isolates, and reported that the phenotypically described Strep. bovis and Strep. equinus type strains belonged to a single DNA group and corresponded to the same species.

Identification of Strep. uberis is currently based on observation of the cultural and morphological characteristics, determination using biochemical tests, and enzyme activity (Leigh, 1999; Khan et al., 2003). On the other hand, several commercial microbial identification systems have also been used to differentiate Strep. uberis from the other streptococci and enterococci isolated from bovine mastitis (Watts, 1989; Freney et al., 1992), and more recently, molecular tools such as PCR-based protocols have been proposed to provide an accurate identification of Strep. uberis isolates (Hassan et al., 2001; Schlegel et al., 2003; Kawata et al., 2004). Among these, RFLP analysis of 16S rDNA was proposed as a general method for bacterial identification and typing (Jayarao et al., 1992). Perhaps the greatest disadvantage of identification techniques based on commercial rapid systems and molecular tools is that these methods can be expensive for most laboratories that wish to offer analyses at an affordable cost. The objective of this study was to evaluate a conventional scheme based on 11 biochemical tests for identification of Strep. uberis strains collected from the mammary glands of cows with mastitis from 19 dairy herds located in the east-central region of Argentina.

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Materials and Methods 

Bacteria 

Five American Type Culture Collection (ATCC) strains, sent by M. Gottschalk and B. Jayarao, including Streptococcus agalactiae ATCC 27956, Strep. dysgalactiae ATCC 27957, Strep. uberis ATCC 27958, Strep. equinus ATCC 27960, and E. faecalis ATCC 19433, were used in this study. In addition, over a period of 12 mo, 75 bacterial cultures were collected and presumptively identified as streptococci or enterococci by colony appearance, gram stain reaction, and catalase test (Hogan et al., 1999). The collected isolates were obtained from mammary glands of cows with mastitis from 19 dairy herds located in the east-central region of Argentina. Four isolates per dairy herd were stored at −20°C in Todd–Hewitt broth (Sigma-Aldrich Co., St. Louis, MO) with 20% glycerol. All ATCC strains and bovine isolates were subcultured from storage media onto 5% sheep blood agar plates and were identified by the API 20 Strep system (bioMérieux, Inc., Durham, NC; Poutrel and Ryniewicz, 1984) and by 16S rDNA RFLP, as previously described (Jayarao et al., 1992).

Biochemical Tests 

Eleven biochemical tests were selected for identification of Strep. uberis strains: the Christie–Atkins–Munch-Petersen (CAMP) reaction (Hogan et al., 1999); esculin hydrolysis (Hogan et al., 1999); sodium hippurate hydrolysis (Baron et al., 1994a); Arg hydrolysis (McDonald and McDonald, 1976); growth in inulin, mannitol, raffinose, salicin, and sorbitol (McDonald and McDonald, 1976); and growth at 45°C and in 6.5% NaCl (Baron et al., 1994b). The Todd–Hewitt broth was used as a basal medium for temperature- and salt-tolerance tests. Five reference strains and 25 bovine isolates previously identified by the API 20 Strep system and by 16S rDNA RFLP, were classified to the species level by biochemical tests in a blind assay. Five representative colonies, obtained by streaking 5μL of Todd–Hewitt broth-glycerol onto 5% sheep blood agar plates, were picked from each plate and used in each biochemical test. The remaining 50 isolates of bovine origin, identified by the API 20 Strep system and by 16S rDNA RFLP, were subjected to the biochemical tests, and the results were compared with biochemical profiles obtained from reference strains. Each isolate was identified to the species level when 8 of the 11 biochemical tests were identical to one of the reference strains and at least 2 were different from each of the other reference strains.

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Results 

Reference strains were identified to the species level by the API 20 Strep system and by 16S rDNA RFLP. Biochemical tests of reference strains were determined by using the conventional scheme proposed for Strep. uberis identification. Reference strains were classified accurately to the species level in a blind assay by comparing their biochemical profiles with previously reported results (McDonald and McDonald, 1976; Garvie and Bramley, 1979; Watts, 1988; Lämmler, 1991; Devriese et al., 1999; Facklam, 2002; National Mastitis Council, 2002; Fortin et al., 2003; Khan et al., 2003). Table 1 shows the identification of the reference strains by the conventional scheme obtained after each biochemical test was repeated 3 times for each isolate.

Table 1. Identification of reference strains by the conventional scheme1
Hydrolysis of Growth in
Reference strainsCAMP reactionARGESCHIPGrowth in 6.5% NaClGrowth at 45°CINUMANRAFSALSOR
Streptococcus agalactiae ATCC 27956++−+−−−−−+−
Streptococcus dysgalactiae ATCC 27957−+−−−−−−−−−
Streptococcus uberis ATCC 27958−+++−−++−++
Streptococcus equinus ATCC 27960−−++−−−−++−
Enterococcus faecalis ATCC 19433−+++++−+−++

1CAMP=Christie–Atkins–Munch-Petersen; ARG=arginine; ESC=esculin; HIP=sodium hippurate; INU=inulin; MAN=mannitol; RAF=raffinose; SAL=salicin; SOR=sorbitol.

Twenty-five gram-positive, catalase-negative cocci isolated from the mammary glands of cows with mastitis were identified by the API 20 Strep system and by 16S rDNA RFLP as Strep. agalactiae (n=5), Strep. dysgalactiae (n=5), Strep. uberis (n=5), Strep. equinus (n=5), and E. faecalis (n=5). In the blind experiment, these strains were successfully classified to the species level by the conventional scheme. Reference strains and the 25 bovine isolates were identified as belonging to the same species by the 3 methods.

The remaining 50 isolates identified as Strep. uberis by the API 20 Strep system and 16S rDNA RFLP were assayed by the conventional scheme. This scheme, based on 11 biochemical tests, correctly identified 47 (94%) of the 50 isolates as Strep. uberis by comparing their biochemical profiles with that of the reference strain. All Strep. uberis strains were negative for growth in 6.5% NaCl. Most of them (81 to 98%) were positive not only for growth in mannitol, salicin, and sorbitol, but also for hydrolysis of Arg, esculin, and sodium hippurate, and they were negative for the CAMP reaction and for growth at 45°C and in raffinose. However, only 64% of the Strep. uberis strains were positive for growth in inulin (Table 2).

Table 2. Biochemical tests of 47 Streptococcus uberis strains isolated from bovine mastitis
Biochemical testStrep. uberis ATCC 27958Percentage1
CAMP2 reaction−81
Arg3+83
Esculin3+89
Sodium hippurate3+96
6.5% NaCl4−100
45°C5−98
Inulin4+64
Mannitol4+98
Raffinose4−94
Salicin4+94
Sorbitol4+96

1Percentage of strains showing reactions in biochemical tests identical to those obtained for the reference strain Strep. uberis ATCC 27958.

2CAMP=Christie–Atkins–Munch-Petersen.

3Hydrolysis of.

4Growth in.

5Growth at.

Three (6%) of the 50 isolates were classified as Strep. uberis by the API 20 Strep system and by 16S rDNA RFLP but were identified as E. faecalis by the conventional scheme.

Fourteen (30%) of the 47 Strep. uberis strains showed biochemical profiles identical to the Strep. uberis ATCC 27958 strain. These strains were positive for growth in inulin, mannitol, and sorbitol, and for hydrolysis of Arg, esculin, and sodium hippurate, whereas they were negative for the CAMP reaction and for growth at 45°C and in 6.5% NaCl and raffinose. The remaining 33 (70%) Strep. uberis strains showed some variability with respect to the Strep. uberis reference strain, resulting in 19 different biochemical profiles, designated A to S (Table 3).

Table 3. Atypical biochemical profiles of 33 Streptococcus uberis strains isolated from bovine mastitis
ProfileBiochemical testAtypical biochemical profilesNo. (%)of Strep. uberis strains1
ASorbitol2−1/33 (3)
BSodium hippurate3−2/33 (6)
CMannitol2−1/33 (3)
DInulin2−5/33 (15)
EEsculin3−4/33 (12)
FCAMP reaction+2/33 (6)
GGrowth at 45°C+1/33 (3)
HArg3−2/33 (6)
IRaffinose2+1/33 (3)
JSalicin2−1/33 (3)
KCAMP reaction+5/33 (15)
Inulin2−
LCAMP reaction+1/33 (3)
Raffinose2+
MArg3−1/33 (3)
Inulin2−
NInulin2−1/33 (3)
Sorbitol2−
OEsculin3−1/33 (3)
Inulin2−
Sorbitol2−
PCAMP reaction+1/33 (3)
Arg3−
Inulin2−
QEsculin3−1/33 (3)
Inulin2−
Salicin2−
REsculin3−1/33 (3)
Inulin2−
Raffinose2+
SArg3−1/33 (3)
Esculin3−
Inulin2−

1Number and percentage of Strep. uberis strains with atypical biochemical profiles showing only the tests that differ from those obtained for the reference strain Strep. uberis ATCC 27958 [CAMP (Christie–Atkins–Munch-Petersen) reaction −, hydrolysis of Arg +, hydrolysis of esculin +, hydrolysis of sodium hippurate +, growth in 6.5% NaCl −, growth at 45°C −, growth in inulin +, growth in mannitol +, growth in raffinose −, growth in salicin +, growth in sorbitol +].

2Growth in.

3Hydrolysis of.

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Discussion 

Streptococcal mastitis causes tremendous economic loss in bovine milk production, and Strep. uberis has become one of the most important environmental etiological agents responsible for cases of clinical and subclinical mastitis in lactating and nonlactating cows.

In this study, we proposed an identification scheme for Strep. uberis based on 11 biochemical tests. We were able to determine the agreement between the conventional scheme proposed and 2 other methods, the API 20 Strep system and 16S rDNA RFLP. This scheme correctly identified 47 (94%) of 50 isolates as Strep. uberis by comparing their biochemical profiles with that of a reference strain. The results showed that 3 of the 50 bovine isolates identified as E. faecalis by the conventional scheme were in fact Strep. uberis by the API 20 Strep system and 16S rDNA RFLP. The most common mistake was to identify Strep. uberis strains as Enterococcus spp. (Watts, 1989; Fortin et al., 2003). The conventional scheme proposed in this study resulted in a relatively low number of misidentifications.

In this study, all the strains classified by the conventional scheme as Strep. uberis were negative for growth in 6.5% NaCl, which agrees with the results of McDonald and McDonald (1976), Watts (1988), and the National Mastitis Council (2002). However, Fortin et al. (2003) reported that nearly 32% of the Strep. uberis strains in that study were positive for growth in 6.5% NaCl.

Nine of the 47 (19%) bovine isolates characterized as Strep. uberis were CAMP reaction-positive, although Strep. uberis reference strain ATCC 27958 was not. These findings were similar to those of McDonald and McDonald (1976) and Lämmler (1991), but differed from those reported by Watts (1988), the National Mastitis Council (2002), and Khan et al. (2003).

Growth in inulin, which has been recommended as a routine test for identification of Strep. uberis (Hogan et al., 1999; Fortin et al., 2003), was observed in only 64% of the isolates classified as Strep. uberis in this study. This result agrees with the work of Khan et al. (2003), who reported that 92 (69%) of 132 Strep. uberis strains were positive for growth in inulin. In contrast, Watts (1988) reported that 100% (n=33) of Strep. uberis strains were negative for growth in inulin, whereas Lämmler (1991) and Devriese et al. (1999) found that 100 and 98%, respectively, of Strep. uberis strains were positive for growth in inulin.

In addition, 83% of the isolates characterized as Strep. uberis were positive for hydrolysis of esculin, which agrees with the results of Lämmler (1991), who reported that 83% (n=36) of Strep. uberis strains were positive for esculin hydrolysis. Other authors (Garvie and Bramley, 1979; Khan et al., 2003), however, have reported that all Strep. uberis strains evaluated were positive for hydrolysis of esculin.

The CAMP reaction, hydrolysis of esculin, and growth in inulin have been recognized as variable characteristics for Strep. uberis (Lämmler, 1991; Fortin et al., 2003; Khan et al., 2003) and can therefore be considered dubious for identification of Strep. uberis strains. However, Fortin et al. (2003) proposed a flow chart for the identification of catalase-negative, non-β-hemolytic, gram-positive cocci isolated from milk samples. The authors suggested that the CAMP reaction, the leucine amino-peptidase test, hydrolysis of esculin and of sodium hippurate, and growth in inulin and in raffinose should be routinely done.

The results obtained in this study showed that the conventional scheme proposed could biochemically identify not only typical but also atypical Strep. uberis isolates. This conventional scheme may be considered an adequate method to identify Strep. uberis strains isolated from bovine mastitis because of its affordable cost in developing countries, and it may contribute to determining the frequency of isolation of the Strep. uberis strains in Argentinean dairy herds, especially in herd-monitoring when regular sampling from all animals is performed.

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Acknowledgments 

We acknowledge J. Chaves and José A. Giraudo for providing some of the milk samples. Thanks are due to M. Gottschalk and B. Jayarao for kindly providing the bacterial reference strains. This research was supported by grants from Agencia Córdoba Ciencia de la Provincia de Córdoba and Secretaría de Ciencia y Técnica de la Universidad Nacional de Río Cuarto, Argentina. E. Reinoso holds a postdoctoral fellowship with the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET).

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Supplementary data 

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References 

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PII: S0022-0302(06)72431-6

doi:10.3168/jds.S0022-0302(06)72431-6

Journal of Dairy Science
Volume 89, Issue 10 , Pages 3886-3890, October 2006

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