DNA-SEQUENCE-BASED DIAGNOSIS OF MASTITIS FROM A MILK SAMPLE

Abstract: A rapid method for diagnosing mastitis is described. The method is based on DNA sequence identification comprising the steps of determining the presence of the following DNA sequences in a milk sample: a DNA sequence specific for somatic cells for indicating inflammation; a DNA sequence specific for a mastitis pathogen for indicating infection; and a DNA sequence specific for an antibiotic-resistance-encoding gene of a pathogen for assisting a proper drug therapy. Test kits and genus- or species-specific oligonucleotides and their use in said method are also described.

Description: DNA-seq ence-based diagnosis of mastitis from a milk sample Technical Field of the Invention The invention relates to a method for diagnosing mastitis by measuring the presence of specific DNA sequences in a milk sample. The invention further relates to a test kit and specific oligonucleotides for use in the method. Description of Related Art Mastitis can be defined as an inflammation of the mammary gland. Mastitis is a disease the object of which can be a female of any mammalian species including man. From the economical point of view mastitis is the most important disease of dairy heifers and cows. In most cases mastitis is a result of colonization of the mammary gland by pathogenic bacteria (infection mastitis) . In addition, physical injuries or local mechanical or chemical stresses in udder organs are able to trigger a local inflammation cascade without the involvement of any primary bacterial infection. Accordingly, these cases are also considered as mastitis, more precisely sterile mastitis. The inflammatory state is associated with varying degrees of pathological damage to the mammary epithelium, resulting in subclinical or clinical mastitis. Clinical mastitis is often an acute inflammation, but inflammatory reactions in subclinical mastitis are often so mild that this state could easily remain unrecognized and without medical care. Most cases of subclinical mastitis represent a chronic subclinical form. Many recent surveys on the prevalence of dairy cow mastitis in western countries indicate that 30-50% of dairy cows suffer from mastitis, in most cases subclinical mastitis. Consequently, bovine mastitis causes vast economical losses to dairy farmers worldwide through reduced milk production, medical expenses and slaughter of chronic cases. A mastitis diagnosis should be based on both an infection study and a study on the state of inflammation. The methods for detecting mastitis are classified as direct and indirect ones. Bacteriological isolation and exa in- ation is a test, which directly shows whether the milk studied is infected or not. This bacteriological approach relies on the assumption that the mammary gland is normally aseptic and the appearance of bacteria indicates infection mastitis. Because inflammation is a complex and multifac- toral process, there are various tests, including both direct and indirect ones, for measuring inflammation within the mammary gland. Mastitis is known to cause many biochem¬ ical and biophysical changes in the composition of milk. These changes arise as a consequence of microbial invasion or non-microbial factors followed by counter-reactions of the body to these stimuli. The changes include active mobilisation of leucocytes (representing somatic cells together with epithelium cells) from the blood into the milk gland, passive diffusion of blood and alveoli epi- thelium cell components like proteins and ions, caused by local cell damage and tissue injury, following an alterna¬ tion in the permeability of microcirculatory vessels. The most commonly used marker for inflammation is somatic cell counting (SCC) , which can be performed directly with a particle counter or a light microscope or indirectly with tests estimating the amount of SCC by parameters like milk viscosity after detergent treatment in the California Mastitis Test (CMT) , the most common cowside screening test for inflammation. In most cases mastitis is caused by infection of certain Staphylococcus or Streptococcus bacteria. As regards bovine mastitis among staphylococcal species S . aureus and certain coagulase negative species (CNS) , especially S . hyicus , S . chromogenes (also classified as S . hyicus ssp. chromogenes ) , S . εimulans , S . epidermidis , and S . xylosus are the most common ones. Among streptococci the important bovine mastitis pathogens include Str . agalac- tiae , Str. dysgalactiae, Str. uberis and Str. bovis species. Only a few percent of infection mastitis is caused by some other bacteria like Eεcherichia coli and Actinomy- ceε or by eukaryotic microbes, yeasts and molds. S . aureus is the most common pathogen causing clinical mastitis. CNS have been considered as minor pathogens because they are inhabitants of the skin and mucous membranes. CNS are, however, important, because they are commonly involved in subclinical mastitis and also in clinical mastitis among heifers. Presently streptococcal mastitis pathogens are still important especially in cases of chronic mastitis and mastitis primarily caused by teat canal trauma. The contribution of different streptococcal and staphylococcal species on bovine mastitis seems to depend on the habits and practise of both animal husbandry and veterinarian therapy. Especially the transition from hand milking to milking machines and the wide use of broad- spectrum antibiotics have had strong influence on the species spectrum of mastitis pathogens. Streptococcal species, especially Str . agalactiae, were the most frequent mastitis pathogens until the use of milking machines spread in the 1950's and 1960's, when they were replaced mainly by S . aureus . In the 1970's the importance of CNS as bovine mastitis pathogens was still marginal. During the last twenty years the CNS have increased their proportions with a simultaneous decrease in the proportion of S . aureus as mastitis pathogens. This change coincides with the wide- spread use of broad specturm antibiotics like penicillin-G that is still the most important antibiotic drug in mastitis therapy. One serious consequence of the use of antibiotics has been the emergence of antibiotic-resistant strains. Presently this also holds true for bovine mastitis patho- gens. Genes encoding antibiotic resistance are able to recoiubine by transposons and to transfer further between bacterial species by conjugation, transformation or transduction. Especially the CNS have been found to receive and further transfer these kinds of antibiotic resistance genetic determinants more easily than S. aureus (Owens & Watts, J. Dairy Sci. 71 (1988) 1934-1939; Muhammad et al . , Amer. J. Vet. Res. 54 (1993) 1432-1440) . Consequently, differences in susceptibilities between S . aureus and CNS to various antibiotics widely used in bovine mastitis therapy may have caused a selective advantage to CNS over S. aureus and increased the proportion of CNS as mastitis pathogens. About one third of S. aureus strains isolated from bovine mastitis have proved to be resistant to penicillin- G. This proportion is somewhat smaller among CNS species (Myllys, J. Dairy Res. 62 (1995) 51-60) . The level of peni¬ cillin resistance encoded by β-lactamase gene blaZ is dependent on the levels of transcriptional and transla- tional expression of this gene. The tests used for penicil¬ lin resistance screening are not always sensitive enough to detect strains expressing the blaZ gene weakly. This is problematic, because this kind of false negative test result may lead to false penicillin therapy and to positive selection of blaZ gene among mastitis pathogens. Presently, good laboratory practice of mastitis diagnosis includes both an inflammation study and a bac¬ teriological study of the milk sample. As a rapid and simple test for measuring the degree of inflammation, the CMT is sensitive enough in detecting clinical mastitis, but not always subclinical mastitis. Even the SCC as a direct test is not sensitive enough for the diagnosis of subclini¬ cal mastitis due to great physiological and individual variations in the somatic cell level of milk. Repeated testing and interquarter comparison of SCC levels between the four quarters of the udder shall improve the probabil¬ ity of detecting the positive subclinical cases, for it is statistically rare that all four quarters of the udder are inflammed simultaneously and to the same degree. A bacteri- ological study should include both an isolation trial of the mastitis pathogen and furthermore, if a pathogen is detected, antimicrobial susceptibility testing for a proper antibiotic treatment. As a test, the bacteriological culti¬ vation of the milk sample is slow, because bacterial col- onies can be analyzed at the earliest after one day of incubation, but typically after two days. It is possible to perform antimicrobial susceptibility testing only after the isolation of the bacterium. If the traditional Bauer-Kirby disk diffusion method (Bauer et al . , Am. J. Clin. Pathol. 45 (1966) 493-496) is used, it will take about one day. Presently certain biochemical and enzymatic tests are more rapid giving results in a few hours. This kind of bacteri¬ ological study of a milk sample still takes altogether at least one day, but typically two days, before all the necessary data required for a correct mastitis diagnosis and therapy by a veterinarian is available. A delay of even one day in the start of the care of mastitis will cause considerable economical losses to the farmers. Consequent¬ ly, a more rapid mastitis diagnosis is highly desirable among both veterinarians and farmers. Summary of the Invention Accordingly it is an object of the present invention to shorten the time needed for a proper mastitis diagnosis, i.e. a diagnosis indicating the presence of inflammation, infection and drug resistance. Another object of the inven¬ tion is to enable identification of a possible infection agent. These and other objects are accomplished by an alternative approach to performing a mastitis diagnosis which is wholly based on measuring the presence of specific DNA sequences in a milk sample. The present invention therefore provides a method for diagnosing mastitis comprising the steps of determining the presence of the following DNA sequences in a milk sample: a DNA sequence specific for somatic cells for indicating inflammation; a DNA sequence specific for a mastitis pathogen for indicating infection; and a DNA sequence specific for an antibiotic-resistance-encoding gene of a pathogen for assisting a proper drug therapy. Another object of the present invention is to provide a test kit and oligonucleotides useful in the DNA- sequence-based method for diagnosing mastitis. The present invention thus provides a test kit for diagnosing mastitis from a milk sample comprising at least the following oligonucleotides: an oligonucleotide which specifically hybridizes with a DNA sequence specific for somatic cells; an oligonucleotide which specifically hybridizes with a DNA sequence specific for a mastitis pathogen; and an oligonuc¬ leotide which specifically hybridizes with a DNA sequence specific for an antibiotic-resistance-encoding gene. The present invention further provides oligonucleotides for use in a method for diagnosing mastitis from a milk sample, said oligonucleotide specifically hybridizing with a masti¬ tis-pathogen-specific DNA sequence substantially from the 16S-23S rRNA spacer region of the pathogen. Brief Description of the Drawings Figure 1 is a flow chart illustrating the principle of the invention. Figure 2 presents the 16S-23S rRNA intergenic spacer sequences of the ten most important bovine mastitis bac- terial species. (GenBank accession numbers are given in brackets, selected species-specific subsequences are in bold print and flanking 16S and 23S rRNA gene sequences are in brackets.) Detailed Description of the Invention The present invention makes it possible to perform a proper mastitis diagnosis in only a few hours or even in a shorter time. Despite this short time the invention makes it possible to identify a pathogen even at the species level and to test for antibiotic resistance. The principle of the invention is illustrated in the flow chart of Figure 1. DNA is isolated from a milk sample from a female mammal¬ ian suspected to suffer from mastitis. The mammalian can be of any species, including man. Preferably it is a dairy heifer or a cow. For inflammation study the presence of a host-specific target sequence is determined, and for infec¬ tion study the presence of a target sequence specific for a mastitis pathogen and a target sequence specific for antibiotic resistance are determined. This kind of molecu¬ lar genetic tests do not require any bacterial cultivation and isolation, but can be performed directly on the iso¬ lated DNA. Preferably the host-specific target sequence is from the conservative region of a bovine 18S rRNA gene and preferably the pathogen-specific target sequence is sub¬ stantially from the 16S-23S rRNA spacer region of a Streptococcus or Staphylococcus mastitis bacterium, it is especially from any of SEQ ID NO l-ll or from the comple¬ mentary strands thereof. The target sequence specific for an antibiotic-resistance-encoding gene is preferably from the blaZ gene encoding resistance to β-lactam antibiotics. The present invention is very useful for detecting bacterial mastitis pathogens especially from the genus Streptococcus or Staphylococcus , in which case it is prac- tical to first identify a genus-specific sequence and thereafter, if desired, the appropriate species-specific sequence. Preferred embodiments of the invention are also set forth in the dependent claims. The presence of the target sequences can be deter- mined in any conventional way e.g. by hybridization or preferably by polymerase chain reaction (PCR) as previously described e.g. in Ehrmann et al . , FEMS Microbiol. Lett. 117 (1994) 143-150 and Barry et al. , PCR Methods and Applica¬ tions 1 (1991) 51-56, respectively. For hybridization purposes, the isolated DNA is first denaturated and then reacted with e.g. a labeled complementary probe under conditions enabling hybridizaton, whereafter the amount of hybrid-bound probe is measured. In PCR, a primer pair is used which recognizes complementary strands of the DNA segment to be enzymatically amplified. The amplified DNA segment (usually about 0.2 - 2 kb) can then be detected e.g. by gel electrophoresis. Oligonucleotides of about 15 to 25 nucleotides are preferably used as hybridizing probes or primer pairs for PCR. "Oligonucleotide" as used herein means a relatively short preferably synthetic nucleotide molecule usually com¬ prising less than 100 nucleotides. Oligonucleotides "specifically hybridizing" means oligonucleotides which are complementary to the target sequence or which are sufficiently complementary to hybrid¬ ize with said target sequence but not with interfering sequences. "Substantially" as used in connection with a DNA sequence substantially from the 16S-23S rRNA spacer region means that said sequence is mainly derived from the spacer region, although it may comprise further sequences e.g. from the flanking 16S rRNA and/or 23S rRNA genes as shown (in brackets) in Figure 2. "Target sequence" means the sequence to which the oligonucleotide probe or primer hybridizes. Identification of a mastitis pathogen is required for infection study. More than 99% of the mastitis patho¬ gens are bacteria. A successful strategy in nucleic-acid- sequence-based identification of bacterial species has shown to be the application of phylogenetic differences among sequences of ribosomal RNA operons (rrn ) present in every prokaryotic chromosome (Barry et al . , BioTechnology 8 (1990) 233-236) . The 5S, 16S and 23S RNA genes of the rrn operons contain both conservative and variable regions. These conservative regions allow the design of universal oligonucleotide primers with which the amplification of rrn DNA segments from any bacterial DNA is possible by PCR. Nucleotide sequences of these amplified DNA segments can be determined and species-specific oligonucleotides can be designed based on the observed differences in sequences among various bacterial species. So far most species- specific oligonucleotides have been derived from variable regions of 16S and 23S rRNA genes. However, there are situations where very little variation is observed within said genes, in which case species-specific sequences derived from the spacer (intergenic) region between the 16S and 23S rRNA genes offer a considerable alternative. In fact, the intergenic regions have been found to present higher sequence variability than the variable regions of rRNA genes (Barry et al . , PCR Methods and Applications 1 (1991) 51-56; EP-A1-0452596) . In order to find diagnostically useful species- or genus-specific nucleotide sequences for the identification of mastitis bacteria, nucleotide sequence data on rrn operons is required. However, it turned out that this kind of published sequence data on relevant mastitis bacteria is very limited (until Nov '95 only sequences of 16S-23S rRNA gene of S . aureus (Gϋrtler & Barrie, Microbiology 141 (1995) 1255-1265) and Str. agalactiae (GenBank Ace. n:o L31412) have been described in various sequence data banks available) . Consequently, the 16S-23S rRNA gene spacer sequences of the ten most important Staphylococcus and Streptococcus species causing bovine mastitis were deter¬ mined and are presented in Figure 2 (SEQ ID NO 1-11) . The nucleotide sequences of the conservative and consequently general oligonucleotides used as primer pairs for the PCR amplifications of these 16S-23S rRNA gene segments are presented in Table I SEQ ID NO 36/37 and 38/39. The 16S-23S rRNA spacer sequences from the ten mastitis bacteria species were further analyzed in order to design species- and genus-specific oligonucleotides for each case. This approach turned out to be successful and the species-specific oligonucleotide primer pairs for each of the ten mastitis bacteria species considered in this study could be designed as shown in Table II (SEQ ID NO 12- 31) . These sequences are derived from SEQ ID NO 1-11 or their complementary strands, i.e. they are either subse¬ quences or inverted complementary subsequences of SEQ ID NO 1-11. The regions they derive from are in bold print in Figure 2. Furthermore, it was possible to design Strepto¬ coccus and Staphylococcus genus-specific primer pairs and they are also included in Table II (SEQ ID NO 32/33 and 34/35) . Species and genus specificities of each of the twelve primer pairs of Table II were tested by PCR and the results are summarized in Table III. As shown with each primer pair, a PCR product was obtained species- and genus- specifically as designed and expected without any false positive or negative PCR amplifications. For the infection study of mastitis, it is desirable to link mastitis pathogen identification with antimicrobial resistance screening. In the present invention, the simul¬ taneous analysis of bacterial species and the screening of (a) desired resistance gene(s) from the same DNA sample is possible by PCR with specific primer pairs. In practise, the simultaneous screening of one or a few genetic resis¬ tance determinants is reasonable. The most widely used antimicrobial drug for mastitis is penicillin and its derivatives like ampicillin, which are members of the β- lactam antibiotic group. Accordingly the screening of the gene blaZ encoding β-lactamase is one of the priority choices in mastitis diagnosis. As demonstrated in Table IV, the general primer pair BLAZ I/BLAZ II (SEQ ID NO 42/43, see Table I) for blaZ gene is a proper choice to screen penicillin-resistant strains among mastitis bacterial strains tested. In principle screening for any genotypic resistance determinant can be performed from the DNA sample if sequence data required for oligonucleotides as a probe or primers is available. Bacteriological study of a masti¬ tis milk sample by PCR with specific primer pairs can be performed directly and correctly after the DNA has been isolated from the milk sample as demonstrated in Table V. For bacterial DNA isolation, a relatively simple and rapid procedure has been described in Example 6. The level of SCC in a milk sample can be estimated for inflammation study on the basis of the amount of eukaryotic DNA in the sample (FI patent 90788) . This strategy requires a method which distinguishes eukaryotic DNA from prokaryotic and viral DNAs. This requires the use of techniques by which eukaryotic DNA can be identified based on the unique genetic information that is present in eukaryotic DNA, but absent in prokaryotic or viral DNAs. For example histone genes, ribosomal RNA (rRNA) genes and their most conservative regions represent this kind of ubiquitous and conservative eukaryotic DNA markers. Two oligonucleotide sequences of this kind, Plb/P2b (SEQ ID NO 40/41) , derived from 18S rRNA gene sequences (FI patent 90788) are shown in Table I. These two oligonucleotides specifically hybridize with the conservative regions of eukaryotic 18S rRNA genes and accordingly can be used as probes for these genes or alternatively as a primer pair for the amplification of these gene regions by polymerase chain reaction (PCR) . The amplified eukaryotic 18S rRNA gene fragment can be sequenced. There are sequence differ¬ ences between the 18S rRNA genes of different eukaryotic species like human (Homo sapiens ) , bovine (Bos taurus) and so on (FI patent 90788) . If required, these sequence differences can be applied to the design of eukaryote species-specific oligonucleotides. In practise, this is required only occasionally for inflammation study. The general oligonucleotides Plb/P2b (SEQ ID NO 40/41) described in Table I are specific enough to estimate SCC levels in mastitis milk samples as demonstrated in Table VI (example 7) . Only in those rare cases (frequency of less than 1%) where a eukaryotic microbe that is a yeast or a mold causes mastitis, specific oligonucleotides derived from eukaryotic microbe 18S rRNA gene sequences could be applicable. In summary, for the purpose of a mastitis diagnosis both the infection study and the inflammation study can be performed by PCR with specific primer pairs simultaneously after DNA from the mastitis milk sample has been isolated using relatively simple and rapid procedures as shown in examples 6 and 7. The time required for isolating DNA from a milk sample for PCR reactions was less than 130 in. The DNA amplification step took about 110 min with a conven¬ tional PCR device (example 2) , but only 20 min with an ATC- type PCR device (example 3) . An analysis of PCR products with agarose gel electrophoresis (with 150 V voltage in 1.5% agarose in lx TBE buffer) in the presence of ethidiu bromide could be performed in 15 min. It is also possible to use alternative systems to measure (semi)quantitatively the amounts of ds-DNA after PCR reactions e.g. fluoromet- rically or immunologically using ds-DNA specific stain or antibodies, respectively. Altogether, the time required for a DNA-sequence-based mastitis diagnosis was less than about 4 h or 3 h when a conventional or ATC-type PCR device, respectively, was operated for PCR. Because of the bacteri¬ ological cultivation of a milk sample, a conventional mas¬ titis diagnosis requires at least 24 hours and typically double the time. Accordingly, the DNA-sequence-based masti- tis diagnosis described here will drastically increase the speed of mastitis diagnosis to at least eight-fold without any loss of necessary information required for a correct diagnosis. It should be understood that the detailed descrip¬ tion above and the following specific examples, while indi¬ cating preferred embodiments of the invention, are given by way of illustration only, since various changes and modi¬ fications within the spirit and scope of the invention will become apparent to those skilled in the art and are intended to be included within the scope of the claims. Example 1 Extraction of bacterial DNA for sequence determina¬ tion Each mastitis bacterial strain included in this study was grown overnight in a proper culture medium (Tryptic Soy Broth) . Bacterial cells were collected by centrifugation (14,000 g/ 5 min) at 4°C and washed twice with 10 mM Tris-HCl-buffer, pH 7.0. The washed bacterial cells were suspensed in 10 mM Tris-HCl-buffer, pH 7.0, containing 12% (w/v) PEG 6000 and 10 g/ l lysozyme (Sigma Chemical Co, St. Louis, U.S.A.) , and the suspension was incubated for 30 min at 37°C. The cells were collected by centrifugation (10,000 g/ 15 min) at 4 C C, lysis buffer (10 mM EDTA-20mM Tris-HCl-buffer, pH 7.0, containing 3% (w/v) SDS) was added to the cell pellet, and the cell suspension was incubated for 30 min at room temperature. The clarified cell solution was extracted twice with a phenol-chloroform- isoamylalcohol (25:24:1) mixture, after which the bacterial DNA was precipitated from the water phase with 200 mM NaCl and 50% (v/v) isopropanol. The DNA was collected by centri- centrifugation and washed with 70% EtOH, dried and resus- pended in proper volume of TE-buffer (lOmM Tris-HCl, 1 mM EDTA, pH 8.0) . Example 2 Amplification and sequencing of 16S-23S rRNA inter¬ genic regions The 16S-23S spacer regions were amplified from the bacterial DNA by PCR (Saiki et al. , Science 239 (1988) 487- 491) . The primer sequences used for amplifying the DNA segment represented conservative regions at the 3'-end of the 16S rRNA-gene and at the 5'- end of the 23S rRNA-gene. The oligonucleotide sequences of the primer pairs 16-1D/23- ID (SEQ ID NO 36/37) and 16-1A/23-1B (SEQ ID NO 38/39) used for staphylococcal and streptococcal species, respectively, are shown in Table I. The PCR reactions were carried out in DNA Thermal Cycler 480 (Perkin Elmer, Norwalk, U.S.A.) using DynaZyme DNA Polymerase kit (Finnzymes, Espoo, Finland) . A typical PCR reaction mixture contained sterile distilled water, reaction buffer (10 mM Tris-HCl, pH 8.8, 1.5 mM MgCl 2 , 50 mM KCl, 0.1% Triton), 200 μM dNTP, 0.6 μM of each primer, 5 ng of bacterial DNA and 1.2U (0.6 μl) DynaZyme DNA polymerase solution / 100 μl of reaction mixture. The reactions were overlayed with mineral oil to prevent evaporation of the mixture. The parameters of the PCR reaction cycle were: 30 sec at 95°C (denaturation) , 30 sec at 55°C (annealing) and 30 sec at 72°C (extention) . The cycle number was 30. Before the first cycle the sample tubes were incubated for 2 min at 95°C. The PCR amplification was finished with 10 min extention at 72°C followed by a cooling step down to 4°C. The total time required for the PCR amplifications of the DNA samples was about 110 minutes. The PCR reaction products (expected size about 500-700 bp) were analyzed with agarose gel electrophoresis. The excess primers and nucleotides were removed from the products with QIAquick PCR-purification kit (QIAGEN, Hilden, Germany) . The 16S-23S rRNA intergenic spacers were sequenced directly from the PCR products by cycle sequencing using a Circumvent Thermal Cycle Dideoxy DNA-sequencing kit (New England Biolabs. Inc., Beverly, U.S.A.) . In sequencing reactions 3 μl (50-100 ng) of the obtained DNA solution was used and the reactions were carried out in the PCR thermal cycler. The parameters of the sequencing reaction cycle were: 40 sec at 95°C, 30 sec at 55°C and 2 min at 72°C. The cycle number was 15. The sequencing samples were analyzed with polyacrylamide gel electrophoresis. In the cases where the PCR product consisted of primary and secondary products, the primary product was excised from the agarose gel and purified with QIAquick gel extraction kit (QIAGEN, Hilden, Germany) . This DNA was used as a template in a second round of PCR, after which the PCR products were purified as above. PCR products of some samples (S. aureus , S . simulans, S . epidermidis , S . hyicus and S . xylosus) were cloned using a TA Cloning Kit (Invit- rogen, San Diego, U.S.A.) and sequenced using a Sequenase Version 2.0 DNA-sequencing kit (United States Biochemical, Cleveland, U.S.A.) because a direct sequencing method failed to give enough readable sequence. Example 3 Design of species- and genus-specific oligonucleo- tides for identification of mastitis bacterial species The species- and genus-specific sequence regions were analyzed from the 16S and 23S rRNA intergenic nucleo¬ tide sequences of each of the ten mastitis bacterial species studied, and they are presented in Figure 2 (SEQ ID NO l-ll) . In the case of S . simulans, two sequence variants were determined. Based on the sequence analysis, oligonuc¬ leotides suitable for use as species- and genus-specific primers were designed from both ends of the spacer (near to or including a few nucleotides from the 16S rRNA and 23S rRNA genes) and they are presented in Table II (SEQ ID NO 12-35) . The oligonucleotides were synthesized by the oligo¬ nucleotide synthesis service unit at the Department of Biochemistry, University of Oulu, Oulu, Finland by coromer- cially available equipment according to the manufacturer's instructions. PCR reactions were carried out as described in example 2, but instead of general primers, one of the species- or genus-specific primer pairs described in Table II was used. In addition, the MgCl 2 -concentration was 3.0 mM for primer pairs Dy I & Dy II, Ep I & Ep II and Str I & Str II, and .0 mM for primer pairs Ag I & Ag II and Bo I & Bo II. The PCR products (expected size about 250-350 bp) were analyzed with agarose gel electrophoresis. Table III summarizes reactions with each primer pair and each bacterial DNA tested. Example 4 Rapid PCR with specific primer pairs PCR reactions with specific primer pairs presented in Table II were also carried out in a RapidCycler thermal cycler (Idaho Technology, Idaho Falls, U.S.A.) . With this type of cycler (ATC, Air Thermal Cycler) it is possible to significantly reduce the time required for DNA amplifica¬ tions. PCR reactions contained a reaction buffer (67 mM Tris-HCl, pH 8.8, 16 mM (NH 4 ) 2 S0 4 ; 0.01 % Tween-20) , 3 mM MgCl 2 (except for 4 mM for the primer pair Hy I/Hy II and 5 mM for BlaZ I/BlaZ II) , 200 μM dNTP, 0.25 μg/μl of BSA, sterile water, 5 ng of bacterial DNA and 0.6 μM of each primer. The reaction volume was 10 μl and glass capillaries were used instead of microcentrifuge tubes. The parameters of the rapid PCR cycle were: J sec at 94°C, 0 sec at 55°C and 15 sec at 72°C, the cycle number being 30. The program was preceded by a denaturation step for 15 sec at 94°C. PCR products were analyzed with agarose gel electrophoresis as in example 2. The PCR products obtained with the rapid PCR were similar to the PCR products obtained with the conventional PCR. In summary, with the rapid PCR the amplifications were completed in 20 min, which was about six times faster than the completion of the amplifications with the conventional PCR (about 110 min) , as shown in examples 2 and 3. Example 5 Determination of bacterial resistance to β-lactam antibiotics The presence of β-lactamase gene (blaZ) in bacterial DNA was determined by PCR amplification with specific primers, BLAZI and BLATZII (SEQ ID NO 42/43). The primers applied for amplifying the blaZ gene region were based on a comparison of available GenBank blaZ gene sequences from Bacillus cereus and Staphylococcus aureus (for sequences and references see Table I) . The PCR reactions and the analysis of the PCR products (expected size about 260 bp) were carried out as described in example 2. For comparison, the antibiotic resistance was tested also by analyzing the growth of bacteria in Bacto tryptic soy broth (Difco) medium containing 25 μg/ml of ampicillin (Boehringer Mann¬ heim) . Bacteria were incubated overnight at 37°C with constant agitation. The results from these experiments are summarized in Table IV. As shown, the results from the DNA- sequence-based detection of blaZ gene by PCR are in fully agreement with the results from the conventional cultiva¬ tion tests. The resistance was previously determined only for S . simulans ATCC 11631, which is a penicillin-resistant genotype (penR, for ref. see ATCC Catalogue of Bacteria and Bacteriophages, 18th edition, 1992) . It was used as a positive control. Example 6 Infection study from mastitis milk samples by PCR Twenty μl of a milk sample in a 1.5 ml Eppendorf tube was mixed with 200 μl of phosphate-buffered saline (PBS) , and further centrifuged for 5 min at 12,000 g. The supernatant was remowed by pipetting. The remaining visible pellet containing cells was suspended into STE-sucrose buffer (100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 20% sucrose) containing mutanolysin 500 units/ml (Sigma) . The suspension was incubated at 37°C for 60 min, and further centrifuged for 5 min at 12,000 g. The supernatant was discarded and the remaining pellet was suspended into 20 μl of lysis buffer (50 mM KCl, 10 mM Tris-HCl pH 8.0, 1% Tween 20, 1 mg/ml proteinase K) . The suspension was incubated for 60 min at 60°C. Finally, the suspension was boiled in a waterbath for 10 min. PCR reactions (15 μl in volume) were carried out as described in example 2 using bacterial genus- or species-specific or eubacterial primer pairs, but instead of purified DNA, 1 μl of lysed cell suspension obtained above and cycle number 40 instead of 30 were used. The PCR products were analyzed as described in example 2 (expected size about 250-350 bp when a species- or genus- specific primer pair was used or about 500-700 bp when a eubacterial primer pair was used) . For comparison, conven¬ tional bacteriological study was done for the same mastitis milk samples. The test strategy and the results are shown in Table V demonstrating that the results from the bacteri¬ ological study of mastitis by PCR with specific primer pairs were fully in agreement with the results from the conventional bacteriological study. In fact, in two cases the results obtained from the DNA-sequence-based study were more precise than those obtained from the bacteriological diagnosis. Example 7 Inflammation study from mastitis milk For estimation of the level of somatic cells in a milk sample, PCR with the primer pair derived from 18S rRNA gene sequences (Table I) was done. One μl of milk was added to 200 μl of 40 mM NaOH in a 1.5 ml Eppendorf tube in order to reach a proper dilution. The diluted sample was boiled for 10 min in a waterbath to lyse the somatic cells. The PCR reaction (15 μl in volume) was carried out as described in example 2, using the primer pair Plb/P2b (SEQ ID NO 40/41) , but instead of purified DNA, 1 μl of lysed cell mixture obtained above was used. The PCR products (expected size about 170 bp) were analyzed .with agarose gel electrop¬ horesis and the amount of DNA in the PCR products was quantitated by a densitometer. For comparison, the same milk samples were also analyzed by two conventional SCC tests, that is the CMT (California Mastitis Test) and Fossomatic cell counting. These tests were performed by a clinical laboratory. The results from these comparative studies have been summarized in Table VI. These results demonstrate that the DNA-based SCC determination is as informative as the conventional SCC tests. Table I Sequences of oligonucleotides used as general primer pairs for molecular genetic mastitis diagnosis Target gene Ofigonudeotide SEQ 10 NO Sequence Directtαn Refera Eukaryotic 18S rRNA gene P1 b Eukaryotic 1 ΘS rRNA gene P2b Eubacterial 16S rRNA gene I β-I D Eubacteriel 23S rRNA gene 23-10 Stap ylococcal 1 S rRNA ganβ I β-I D Staphylococcal 23S rRNA gβna 23-1 D Streptococcal 16S rRNA gβna 16-1A Straptococcal 23S rRNA gen* 23-1B Penicillin resistance gene blaZ BlaZ l Penicillin resistance gene blaZ BlaZ II a ) Fl-patent 90788 b) alignments of streptococcal and staphylococcal 16S and 23S rRNA gene sequences available in GenBank eg. X59028, X59030, X58317, 222809, X59032, X68417, X68425, S60799 c) GenBank accession numbers M15195, M15526, Z04121, X16471, X52734 Table II S t rep t ococcus and Staphylococcus species- and genus-specific oligonucleotide sequences substantially from 16S-23S rRNA intergenic spacerregions suitable as primers and probes for molecular genetic mastitis diagnosis Oβgo- Length U'-TI tπt) I16S-23S) Ispacer) 12 GGAAACCTGCCATTTGCG 13 TAACTTAACCTTATTAACCTAG 14 GGAAGCACGTTTGGGTATT 15 AACCTTATTTTGGTTCTGTTG 16 TGGAACACGTTAGGGTCG 17 CTTTTACTAGTΔTATCTTAACTΔ 18 TAAGGAACACGTTGGTTAAG 19 TCCΔGTCCTTAGACCTTCT 20 TCTTCAGAAGATGCGGAATA 21 TAAGTCAAACGTTAACATACG 22 ACGGAATATCGCTTTTAAGC 23 CGTTTACATTCGGCTTTCG 24 TCTACGAAGATGAGGGATA 25 TTTCCΔCCATATTTTGΔATTGT 26 TACGGAATATCGCCTTAGG 27 AAAACATCTGTCATCCGAAG 28 CTTTCTAAGGATATATTCGG 29 ATTGTGAGTAATCGTTTGCC 30 TCTTTAGAAGΔTGΔCAGAGG 31 TGACTTTTAACΔCGACGAAG 32 TGTTTAGTTTTGAGΔGGTCTTG 33 CGTGGAATTTGΔTATAGΔTATTC 34 GGAATAACGTGACATATTGTA 35 TTCACTCGGTTTTGCTTGG (STRA = Streptococcus agalactiae , STRB = Str . bov±s , STRD = Str . dysgalact±a , STRU = Str . uberls, STAA = SεapΛyiococcus aureus, STAC = S. cΛrojnogene., STAE = S. epidermidis , STAH = S. hyicus , STAS = S. simulans , STAX = S. xyl osus , STR = Streptococcus sp. , STA = Staphylococcus sp. Table III Species and genus specificities of the 12 primer pairs described in Table II as determined separately by PCR amplification of bacterial DNA from each of the ten mastitis Streptococcus or Staphylococcus species Bacterial spacm- PCR amplification with primer pair' STR STA STRA STRB STRD STRU STAA STAC STAE STAH STAS STAX Str. agalβctiαe Str. bo vis Str. dysgalactiae Str. ubaris S. aureus S. chromogenes S. epidermidis S. hyicus S. simulans S. xylosus • Primer pairs: STRA = Ag I & Ag II, STRB = Bo I & Bo II, STRD = Dy I & Dy II, STRU = Ub I & Ub II, STAA = Au I & Au II, STAC = Chr I & Chr II, STAE = Ep I & Ep II, STAH = Hy I & Hy II, STAS = Si I Si Si II, STAX = Xy I & Xy II, STR = Str I £ Str II and STA = Sta I & Sta II ■+ = PCR product obtained - = no PCR product Table IV Resistance of certain mastitis bacterial strains to β-lactam antibiotics as determined by cultivation test and by PCR Bacterial strain Observed sensitivity Presence of blaZ gene to ampicillin (25 μg/ml) determined by PCR in growth medium Str . agalactiae ATCC 27956 S Str. bovis ATCC 27960 S Str. dysgalactiae ATCC 27957 S Str . uberis ATCC 27958 S S . aureus ATCC 25923 S S . chromogenes ATCC 43764 S S . epidermidis ATCC 12228 R S. hyicus KNS 264/92 S S. simulans ATCC 11631 (penR) R S. xylosus ATCC 12162 S S = sensitive (no growth), R = resistant (growth), - = no PCR product with BLAZI and BLAZII primers, + = PCR product obtained with BLAZI and BLAZII primers (table I) Table V Bacteriological study from mastitis milk samples as determined by conventional bacteriological cultivation and examination procedures and by PCR with specific primer pairs Sample Microbiological Primer pair U3βd for PCR b) No. study" EB C) STA STAA STAC STAE STAH STAS STAX STR STRA STRB STRD STRU a) standard bacteriological study performed for mastitis diagnosis by a clinical laboratory b) for nucleotide sequences for primer pairs see Table I and Table II c) EB = eubacterial primer pair, 16-1D/23-1D (see Table I) d) CNS = coagulase negative staphylococci Table VI Inflammation study from mastitis milk samples as determined by CMT, Fossomatic cell counting and by PCR with the specific primer pair Plb/P2b described in Table I Milk sample No. CMT value Fossomatic cell PCR product counting (cells/ml) relative intensity* 1 1 18 000 0.045 2 2 189 000 0.180 3 3 479 000 0.249 4 4 2 530 000 0.355 5 5 8 468 000 0.420 CMT = California mastitis test (CMT 1 = < 150 000 cells/ml, CMT 2 = 150 000 - 300 000 cells/ml, CMT 3 = 300 000 - 800 000 cells/ml, CMT 4 = 800 000 - 5 million cells/ml, CMT 5 = > 5 million cells/ml) * the amount of the PCR product was measured as a relative intensity of the band in the agarose gel by a densitometer SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Alatossava, Tapani Forsman, Paivi Tilsala-Timisjarvi, Anu (ii) TITLE OF INVENTION: DNA-sequence-based diagnosis of mastitis from a milk sample (iii) NUMBER OF SEQUENCES: 43 (iv) CORRESPONDENCE ADRESS: (A) ADRESSEE: Oulutech Ltd. (B) STREET: Teknologiantie 1 (C) CITY: Oulu (E) COUNTRY: Finland (F) POSTAL CODE (ZIP): 90570 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 291 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Streptococcus agalactiae (B) STRAIN: ATCC 27956 ( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 1 : CTAAGGATAA GGAAACCTGC CATTTGCGTC TTGTTTAGTT TTGAGAGGTC TTGTGGGGCC 60 TTAGCTCAGC TGGGAGAGCG CCTGCTTTGC ACGCAGGAGG TCAGCGGTTC GATCCCGCTA 120 GGCTCCATTG AATCGAAAGG TTCAAATTGT TCATTGAAAA TTGAATATCT ATATCAAATT 180 CCACGATCTA GAAATAGATT GTAGAAAGTA ACAAGAAAAT AAACCGAAAA CGCTGTGAAT 240 ATTTAATGAG TTTTCTAGTT TTAAAGAAAC TAGGTTAATA AGGTTAAGTT A 291 ( 2 ) INFORMATION FOR SEQ ID NO : 2 : ( i ) SEQUENCE CHARACTERISTICS : ( A) LENGTH : 278 base pairs ( B ) TYPE : nucleic acid ( C ) STRANDEDNESS : double ( D ) TOPOLOGY : linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Streptococcus bovis (B) STRAIN: ATCC 27960 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CTAAGGATAA ACGGAAGCAC GTTTGGGTAT TGTTTAGTTT TGAGAGGTCT TGTGGGGCCT 60 TAGCTCAGCT GGGAGAGCGC CTGCTTTGCA CGCAGGAGGT CAGCGGTTCG ATCCCGCTAG 120 GCTCCATTGA ATCGAAAGAT TCAAAGATTG TCCATTGAAA ATTGAATATC TATATCAAAT 180 TCCACGATTC AAGAAATTGA ATTGTAGATA GTAACAAGAA ATAAACCGAA AGCGCTGTGA 240 TTTAATGAGT TTAAGGTCAA CAGAACCAAA ATAAGGTT 278 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 287 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Streptococcus dysgalactiae (Lancefield's group C) (B) STRAIN: ATCC 27957 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CTAAGGAAAT GGAACACGTT AGGGTCGTCT TATTTAGTTT TGAGAGGTCT TGTGGGGCCT 60 TAGCTCAGCT GGGAGAGCGC CTGCTTTGCA CGCAGGAGGT CAGCGGTTCG ATCCCGCTAG 120 GCTCCATTAG GATAGAGATA TCCTAAAAAC TGTCCATTGA AAATTGAATA TCTATATCAA 180 ATTCCACGAT CAAGAAATTG ATTGTACGAA TAGTAACAAG AAAATAAACC GAAAACGCTG 240 TGAATAATCA AGAGTTTTTC TAGTTAAGAT ATACTAGTAA AAGATAA 287 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 342 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Streptococcus uberis (B) STRAIN: ATCC 27958 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: CTAAGGATAA GGAACACGTT GGTTAAGTCT TATTTAGTTT TGAGAGGTCT TGCAAGACGC 60 AGAGACAAAC TGTGGGGCCT TAGCTCAGCT GGGAGAGCGC CTGCTTTGCA CGCAGGAGGT 120 CAGCGGTTCG ATCCCGCTAG GCTCCATAGG ATACAGTTCA ACTGAACTTA ATAGAAGTGA 180 AGTTTCATTG TATCTTAGTA TAGTCCATTG AAAATTGAAT ATCTATATCA AATTCCACGA 240 TCATGAAAAT GATTGTAGAA AAGTAACAAG AAATAAACCG AAAAAAAACG ATAAACGCGA 300 ACATATTAAA AAAAATCAAG AAGGTCTAAG GACTGGAAAT AA 342 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 460 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Staphylococcus aureus subsp. aureus (B) STRAIN: ATCC 25923 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: CTAAGGATAT ATTCGGAACA TCTTCTTCAG AAGATGCGGA ATAACGTGAC ATATTGTATT 60 CAGTTTTGAA TGTTTATTTA ACATTCAAAA AATGGGCCTA TAGCTCAGCT GGTTAGAGCG 120 CACGCCTGAT AAGCGTGAGG TCGGTGGTTC GAGTCCACTT AGGCCCACCA TTATTTGTAC 180 ATTGAAAACT AGATAAGTAA GTAAAATATA GATTTTACCA AGCAAAACCG AGTGAATAAA 240 GAGTTTTAAA TAAGCTTGAA TTCATAAGAA ATAATCGCTA GTGTTCGAAA GAACACTCAC 300 AAGATTAATA ACGTGTTTAA ATCTTTTTAT AAAATAAAAC GTTTAGCAGA CAATGAGTTA 360 AATTATTTTA AAGCAGAGTT TACTTATGTA AATGAGTATT TAAAATAATG AAAACGAAGC 420 CGTATGTTAA CGTTTGACTT ATAAAAATGG TGGAAACATA 460 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 281 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Staphylococcus chromogenes (B) STRAIN: ATCC 43764 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CTAAGGATAA TATACGGAAT ATCGCTTTTA AGCGATAAGG AATAACGGAG ACATATTGTA 60 TTCAGTTTTG AATGCTCATT TTCGAGGCAT TCAACATTGT ACATTGAAAA CTAGATAAGT 120 AAGTATAGAT TTTACCAAGC AAAACCGAGT GACAAGCGAA AAGCTTGAAA CAAAAATTAT 180 CGCTAGTCGT CGACAGACSA CTCACAATAA TTAATAACTG GTGGATGTTG GTTATTGTTT 240 AATTCGAAAG CCGAATGTAA ACGATTGCCA AAACATCAAA A 281 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 264 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Staphylococcus epidermidis (B) STRAIN: ATCC 12228 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CTAAGGATAT ATTCGGAACA TCTTCTACGA AGATGAGGGA TAACGTGACA TATTGTATTC 60 AGTTTTGAAT GTTTATTAAC ATTCTTTGTA CATTGAAAAC TAGATAAGTA AGTAAGATTT 120 TACCAAGCAA AACCGAGTGA ATAGAGTTTT AAATAAGCTT GAATTCATAA ATAATCGCCT 180 AGTGTTCGAA AGAACACTCA CAAGATTAAT AACTAGTTTT AGCTATTTAT TTTTAATAAC 240 AATTCAAAAT ATGGTGGAAA CATA 264 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 375 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Staphylococcus hyicus (B) STRAIN: KNS 264/92 (isolated from a clinical sample) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CTAAGGATAA TATACGGAAT ATCGCCTTAG GCATACGGAA TAACGAAGAC ATATTGTATT 60 CAGTTTTGAA TGCTCATTTT GAGGATTCAA CATTGTACAT TGAAAACTAG ATAAGTAAGT 120 ATAGATTTTA CCAAGCAAAA CCGAGTGACA AGCGAAAAGC TTGAAACAAA AAATTATCGC 180 TAGTCGTCGA CAGCGACTCA CAATAATTAA TAACTGGTGG ATGTTGGTTA ATGTTTACTT 240 CGGATGACAG ATGTTTTGAA AACGTTTGTC AGTCTATGAA TCGCAAACAA GAGCGAAGGC 300 CGTTACTTCC GTAAGCAACT GAGTGATTTG TGCCGCGATG AAGCCGAATG CAAACGATTG 360 CCAAAACATC ATAAA 3 7 5 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 339 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Staphylococcus simulans (B) STRAIN: ATCC 11631 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CTTTCTAAGG ATATATTCGG AACAGTTTCG CAGGAAACTG AAACGGAATA ACGTGACATA 60 TTGTATTCAG TTTTGAATGT TTATTTGAAA CATTCAACGT GAGATGGGCC TATAGCTCAG 120 CTGGTTAGAG CGCACGCCTG ATAAGCGTGA GGTCGGTGGT TCGAGTCCAC TTAGGCCCAC 180 CATTTTGATT TTTTGTACAT TGAAAACTAG ATAAGTAAGT AAAAAATAGA TTTTACCAAG 240 CAAAACCGAG TGAATTAGAG TTTTAAAAGC TTTATTCATT TAAATGAATC GCTAGTAATC 300 AATTGCCGAC GGCAAACGAT TACTCACAAT ATTAATAAC 339 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 248 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Staphylococcus simulans (B) STRAIN: ATCC 11631 ( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 10 : CTTTCTAAGG ATATATTCGG AACAGTTTCG CAGGAAAYTG AAACNGGAAT AACGTGACAT 60 ATTGTATTCA GTTTTGAATG TTTATTTGAA ACATTCAAAG ATTGTACATT GAAAACTAGA 120 TAAGTAAGTA AAAAATAGAT TTTACCAAGC AAAACCGAGT GAATTAGAGT TTTAAAAGCT 180 TTATTCATTT AAATGAATCG CTAGTAATCA ATTGCCGACG GCAAACGATT ACTCACAATA 240 TTAATAAC 248 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 285 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Staphylococcus xylosus (B) STRAIN: ATCC 12162 ( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 11 : CTAAGGATAT ATTCGGAACA TCTTCTTTAG AAGATGACAG AGGAATAACA TTGACATATT 60 GTATTCAGTT TTGAATGCTC ATTGGAGTAT TCAGTGCATA ATTTGTACAT TGAAAACTAG 120 ATAAGTAAGT AAAATATATA GATTTTACCA AGAAAAACCG AGTGAATTAG AGTTTTAAAT 180 AAGCTTGAAT TCAAAAAGAA ATAATCGCTA GTGTTCGAAA GAACACTCAC AGATTAATAA 240 CATTTTGGGT TTTTAACCGA CTTCGTCGTG TTAAAAGTCA AAAAA 285 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Streptococcus agalactiae (B) STRAIN: ATCC 27956 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GGAAACCTGC CATTTGCG 18 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Streptococcus agalactiae (B) STRAIN: ATCC 27956 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: TAACTTAACC TTATTAACCT AG 22 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Streptococcus bovis (B) STRAIN: ATCC 27960 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GGAAGCACGT TTGGGTATT 19 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Streptococcus bovis (B) STRAIN: ATCC 27960 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: AACCTTATTT TGGTTCTGTT G 21 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Streptococcus dysgalactiae (Lancefield's Group C) (B) STRAIN: ATCC 27957 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: TGGAACACGT TAGGGTCG 18 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Streptococcus dysgalalactiae (Lancefield' s Group C) (B) STRAIN: ATCC 27957 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: CTTTTACTAG TATATCTTAA CTA 23 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Streptococcus uberis (B) STRAIN: ATCC 27958 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: TAAGGAACAC GTTGGTTAAG 20 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Streptococcus uberis (B) STRAIN: ATCC 27958 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: TCCAGTCCTT AGACCTTCT 19 (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Staphylococcus aureus subsp. aureus (B) STRAIN: ATCC 25923 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: TCTTCAGAAG ATGCGGAATA 20 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Staphylococcus aureus subsp. aureus (B) STRAIN: ATCC 25923 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: TAAGTCAAAC GTTAACATAC G 21 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Staphylococcus chromogenes (B) STRAIN: ATCC 43764 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: ACGGAATATC GCTTTTAAGC 20 (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Staphylococcus chromogenes (B) STRAIN: ATCC 43764 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: CGTTTACATT CGGCTTTCG 19 (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Staphylococcus epidermidis (B) STRAIN: ATCC 12228 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: TCTACGAAGA TGAGGGATA 19 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Staphylococcus epidermidis (B) STRAIN: ATCC 12228 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: TTTCCACCAT ATTTTGAATT GT 22 (2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Staphylococcus hyicus (B) STRAIN: KNS 264/92 (isolated from a clinical sample) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: TACGGAATAT CGCCTTAGG 19 (2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Staphylococcus hyicus (B) STRAIN: KNS 264/92 (isolated from a clinical sample) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: AAAACATCTG TCATCCGAAG 20 (2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Staphylococcus simulans (B) STRAIN: ATCC 11631 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: CTTTCTAAGG ATATATTCGG 20 (2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Staphylococcus simulans (B) STRAIN: ATCC 11631 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: ATTGTGAGTA ATCGTTTGCC 20 (2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Staphylococcus xylosus (B) STRAIN: ATCC 12162 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: TCTTTAGAAG ATGACAGAGG 20 (2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Staphylococcus xylosus (B) STRAIN: ATCC 12162 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: TGACTTTTAA CACGACGAAG 20 (2) INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Streptococcus sp. (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: TGTTTAGTTT TGAGAGGTCT TG 22 (2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Streptococcus sp. (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: CGTGGAATTT GATATAGATA TTC 23 (2) INFORMATION FOR SEQ ID NO: 34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Staphylococcus sp. (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: GGAATAACGT GACATATTGT A 21 (2) INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Staphylococcus sp. (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: TTCACTCGGT TTTGCTTGG 19 (2) INFORMATION FOR SEQ ID NO: 36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: GGTGAATACG TTCCCGGG 18 (2) INFORMATION FOR SEQ ID NO: 37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: CTTACAGCTC CCCAAAGCAT 20 (2) INFORMATION FOR SEQ ID NO: 38: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: GTCGGAATCG CTAGTAATCG 20 (2) INFORMATION FOR SEQ ID NO: 39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: GGGTTCCCCC ATTCGGA 17 (2) INFORMATION FOR SEQ ID NO: 40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: AGGAATTCCC AGTAAGTGC 19 (2) INFORMATION FOR SEQ ID NO: 41: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: AGATAGTCAA GTTCGACCG 19 (2) INFORMATION FOR SEQ ID NO: 42: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: GCTCATATTG GTGTTTATGC 20 (2) INFORMATION FOR SEQ ID NO: 43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: ATCACTATAT GTCATTGAAG C 21

Claims: Claims 1. A method for diagnosing mastitis comprising the steps of determining the presence of the following DNA sequences in a milk sample: a DNA sequence specific for somatic cells for indicating inflammation; a DNA sequence specific for a mastitis pathogen for indicating infection; and a DNA sequence specific for an antibiotic-resis¬ tance-encoding gene of a pathogen for assisting a proper drug therapy. . A method according to claim 1 wherein the presence of the DNA sequences is determined using oligonuc- leotides specifically hybridizing with said DNA sequences either as primer pairs for amplification by PCR or as hybridization probes. 3. A method according to claim 2 wherein the presence of said DNA sequences is determined by PCR using oligonucleotides comprising about 15 to 25 nucleotides as primer pairs. . A method according to claim 1 wherein said DNA sequence specific for somatic cells is from the conserva¬ tive region of a bovine 18S rRNA gene. 5. A method according to claim 1 wherein said pathogen-specific DNA sequence is substantially from the 16S-23S rRNA spacer region of a Streptococcus or Staphylo¬ coccus mastitis bacterium. 6. A method according to claim 5 wherein said pathogen-specific DNA sequence is from any of SEQ ID NO 1- 11 or from the complementary strands thereof. 7. A method according to claim 1 wherein said DNA sequence specific for an antibiotic-resistance-encoding gene is from the blaZ gene encoding resistance to β-lactam antibiotics. 8. A test kit for use in a method for diagnosing mastitis from a milk sample comprising at least the following oligonucleotides: an oligonucleotide which specifically hybridizes with a DNA sequence specific for somatic cells; an oligonucleotide which specifically hybridizes with a DNA sequence specific for a mastitis pathogen; and an oligonucleotide which specifically hybridizes with a DNA sequence specific for an antibiotic-resistance- encoding gene. 9. A test kit according to claim 8 comprising oligonucleotides which specifically hybridize with masti¬ tis-pathogen-specific DNA sequences substantially from the 16S-23S rRNA spacer region of Streptococcus or Staphylococ- cus bacteria. 10. A test kit according to claim 8 comprising at least the following primers for PCR amplification: an oligonucleotide primer pair which specifically hybridizes with DNA sequences specific for somatic cells; an oligonucleotide primer pair which specifically hybridizes with DNA sequences specific for a mastitis pathogen; and an oligonucleotide primer pair which specifically hybridizes with DNA sequences specific for an antibiotic- resistance-encoding gene. 11. A test kit according to claim 10 comprising primer pairs which specifically hybridize with DNA sequences specific for the conservative regions of a eucaryotic 18S rRNA gene; mastitis-pathogen-specific DNA sequences substan¬ tially from the 16S-23S rRNA spacer region of the pathogen; and DNA sequences specific for the blaZ gene. 12. An oligonucleotide for use in a method for diagnosing mastitis from a milk sample, said oligonucleo¬ tide specifically hybridizing with a mastitis-pathogen- specific DNA sequence substantially from the 16S-23S rRNA spacer region of the pathogen. 13. An oligonucleotide according to claim 12 specifically hybridizing with a subsequence of any of SEQ ID NO 1 to 11 or with a subsequence of the complementary strands thereof. 14. An oligonucleotide according to claim 13 selected from SEQ ID NO 12-35.

Inventors: ALATOSSAVA JOUKO TAPANI, FORSMAN PAEIVI TUULIKKI, TILSALA-TIMISJAERVI ANU KYLLIK, ALATOSSAVA, JOUKO, TAPANI, FORSMAN, PAEIVI, TUULIKKI, TILSALA-TIMISJAERVI, ANU, KYLLIKKI
Applicants: OULUTECH LTD, OULUTECH LTD.
Application year: 1997
Main classification: C12Q1
IPC codes: C07K14, C12Q1