Samples Of Animal Origin Research Articles

Detection of Hepatitis E virus in samples of animal origin collected in Hungary

Hepatitis E virus (HEV) is an enterically transmitted human pathogen. HEV infections are mainly associated with acute, self-limited, icteric hepatitis with an average mortality rate of 1%. Animal reservoirs are considered to play an important role in the maintenance of the virus and in the spread of HEV to humans. HEV-induced seroconversion was described in several species, however clinical hepatitis in animals has not been observed to date. HEV strains from animals are genetically closely related to human HEV isolates, which supports the opinions on the zoonotic transmission of the virus. In this expansive study the occurrence of HEV was investigated in Hungarian wild and domesticated animal samples. HEV RNA was detected by reverse transcription-polymerase chain reaction in liver samples of wild boars, roe deer, and deer. The investigations of domestic swine samples detected HEV in 39% of the investigated Hungarian pig farms. Simultaneous investigation revealed no definite difference between liver and faeces samples of domestic pigs in the frequency of HEV positivity. The highest (36%) incidence of HEV infection was found among the 11–16-week-old pigs. Samples from domestic cattle and rodents collected in pig farms, forests and meadows were tested negative for HEV RNA. Phylogenetic analysis of partial sequences amplified within the ORF1 and ORF2 regions of selected strains revealed that the detected viruses belong to three subgroups of the third genogroup of HEV, and are closely related to human and swine HEV strains detected in different countries. The investigations revealed widespread distribution of HEV in Hungarian wild ungulate and domesticated swine populations, with considerable genetic diversity among the strains.

Detection of methicillin-resistant Staphylococcus aureus ST398 in food samples of animal origin in Spain

Detection of methicillin-resistant Staphylococcus aureus ST398 in food samples of animal origin in Spain

Genetic diversity analysis of 10 STR's loci used for forensic identification in canine hair samples

Pets live with people and place biological samples everywhere, which may be useful in a forensic context linking suspects and victims, to an occurrence. The genetic diversity of canine microsatellite loci PEZ1, FHC2054, FHC2010, PEZ5, PEZ20, PEZ12, PEZ3, PEZ6, PEZ8 and FHC2079 was investigated in a population of 63 Portuguese dogs. Preliminary results show that the studied markers appeared to be polymorphic, thus contributing to increase the potential of forensic samples of animal origin. The number of alleles per locus, varied between five (FHC2010) to 20 (PEZ3) and allelic frequencies were estimated.

Bifidobacterial survival in surface water and implications for microbial source tracking

Bifidobacteria are common in the intestines of humans and other animals. Sorbitol-fermenting Bifidobacteria (SFB) are associated with human faecal pollution. The ratio of total Bifidobacteria (TB) to SFB has been proposed to be a means for determining the source (human or animal) of faecal pollution. In this study, SFB were detected in all sewage samples (n = 39) and in 42% of samples of animal origin (n = 24): pig (5/6 positive), poultry (4/8), horse (1/4), and cow (0/6). The TB:SFB ratio was significantly higher in animal samples than in sewage samples (p < 0.01), with the exception of stored poultry manure, which had ratios (1.4-11) similar to the sewage samples (1-17). The lowest ratio in the other animal samples was 40 (pig). The TB:SFB ratio in sewage remained stable over time in surface water, and it may be a useful screening tool to determine the source of faecal pollution in surface water. Bifidobacteria died off in a log linear manner at 4 and 22 degrees C, with rates of disappearance of 0.16 and 0.58 log(days-1), respectively. Faecal coliforms (FC) disappeared more slowly (0.09 and 0.43 log(days-1), respectively). The FC:SFB ratio proved to be an inadequate tool for determining the age of sewage pollution.

Detection of vanA and vanB2-containing enterococci from food samples in Spain, including Enterococcus faecium strains of CC17 and the new singleton ST425

Two-hundred-twenty-nine food samples of animal origin were tested to know the prevalence of vancomycin-resistant enterococci (VRE) after a decade of avoparcin ban as animal growth promoter in Spain. VRE with acquired mechanism of resistance were detected in 9 of these 229 samples (3.9%, obtained from chicken, veal and rabbit), and one VRE per food sample was further characterized. The vanA gene was identified in seven isolates (2 E. faecium, 3 E. durans, and 2 E. hirae), and the vanB2 gene in the remaining 2 isolates (identified as E. faecium). The two vanB2 isolates showed a phenotype of multiresistance that included, in addition to vancomycin, also ampicillin, erythromycin, tetracycline, streptomycin, kanamycin, ciprofloxacin, chloramphenicol and trimethoprim-sulfamethoxazole and contained, among others, erm(B), tet(M), ant(6), and aph(3′)-III genes. Most of vanA enterococci showed erythromycin and tetracycline resistance and contained the erm(B) and tet(M) genes. One vanA- and both vanB2-positive E. faecium isolates were classified by MLST analysis into the CC17 clonal complex (ST17 and ST78), and one additional vanA isolate was included in a new sequence type named ST425 (singleton). Co-transference by conjugation of erm(B) and vanA genes was demonstrated in one vanA-positive E. faecium isolate. The inclusion of vanB2 cluster into Tn 5382 structure was demonstrated in the two vanB2 isolates, as well as the linkage pbp5-Tn 5382, and beta-haemolysis and gelatinase production was identified in one of them. Food sample of animal origin could be a vehicle of transference of VRE of vanA and vanB2 type that could be transferred to humans.

Detection of multiple-antimicrobial resistance and characterization of the implicated genes in Escherichia coli isolates from foods of animal origin in Tunis.

Phenotypic and genotypic characterization of antimicrobial resistance was conducted for 98 Escherichia coli isolates recovered from 40 food samples of animal origin (poultry, sheep, beef, fish, and others) obtained in supermarkets and local butcheries in Tunis during 2004 and 2005. Susceptibility to 15 antimicrobial agents was tested by disk diffusion and agar dilution methods, the mechanisms of resistance were evaluated using PCR and sequencing methods, and the clonal relationship among isolates was evaluated using pulsed-field gel electrophoresis. High resistance was detected to tetracycline, sulphonamides, nalidixic acid, ampicillin, streptomycin, and trimethoprim-sulfamethoxazole (29 to 43% of isolates), but all isolates were susceptible to cefotaxime, ceftazidime, cefoxitin, azthreonam, and amikacin. One-third of the isolates had multiresistant phenotypes (resistance to at least five different families of antimicrobial agents). Different variants of blaTEM, tet, sul, dfrA, aadA, and aac(3) genes were detected in most of the strains resistant to ampicillin, tetracycline, sulphonamide, trimethoprim, streptomycin, and gentamicin, respectively. The presence of class 1 and class 2 integrons was studied in 15 sulphonamide-resistant unrelated E. coli strains, and 14 of these strains harbored class 1 integrons with five different arrangements of gene cassettes, and a class 2 integron with the dfrA 1 + sat + aadA 1 arrangement was found in one strain. This study revealed the high diversity of antimicrobial resistance genes, some of them included in integrons, in E. coli isolates of food origin.

Genotypic Characterization ofPseudomonas aeruginosaIsolated from Human and Animal Sources in Egypt

Two different techniques for the molecular typing of Pseudomonas aeruginosa were used to study the epidemiology of P. aeruginosa strains. Colonization with P. aeruginosa was studied by taking samples of human origin collected from urine, sputum samples of patients suffering from lung manifestations and patients exposed to third-degree burns. In addition, samples of animal origin were collected from mastitic milk and lung tissues of slaughtered calves and from the internal organs of diseased chickens. Typing of 18 isolates was performed by random amplified polymorphic DNA analysis and amplified fragment length polymorphism analysis. Computer-aided cluster analysis indicated that similar groups of related isolates were obtained by each method.

Genotyping of Acanthamoeba isolates from clinical and environmental specimens in Iran

In this study, 15 Acanthamoeba isolates from AK patients and 10 environmental samples (water, soil and animal-origin samples) were classified at the genotype level based on the sequence analysis of the Diagnostic Fragment 3 (DF3) of Acanthamoeba small subunit rRNA gene. The obtained results revealed that most of these Acanthamoeba strains belonged to genotype T4 both in clinical and environmental samples. The presence T11 genotype in clinical samples was also revealed after the genotyping analysis and to our knowledge this is the first report of T11 genotype in Iran. Moreover, the isolation of T4 genotype from cow faeces in this study highlights a possible transmission of Acanthamoeba through animal faeces in Iran. Overall, the widespread distribution of pathogenic Acanthamoeba T4 across the environmental sources and the increasing number of contact lens wearers in Iran, demands more awareness within the public and health professionals as this pathogen is emerging as a risk for human health in Iran and worldwide.

Chromatographic Residue Analysis of Sulfonamides in Foodstuffs of Animal Origin

The widespread use of antibiotics in veterinary medicine for prophylactic, therapeutic and growth promoting purposes may result in the presence of residues in foodstuff of animal origin. Sulfonamides, N‐derivatives of 4‐amino‐benzenesulfonamide, belong to a diverse class of synthetic antibiotics consisting of chemically related compounds. A number of them e.g., sulfamethazine, sulfadiazine, sulfaquinoxaline and sulfamethoxypyridazine, are widely used in animal husbandry. Their residues in food are of great concern because of their potential carcinogenic character and because they may contribute to the development of antibiotic resistance in humans. These facts have induced most of the regulatory activities on sulfonamides. Regulatory bodies in the European Union as well as in other countries have set different maximum residue limits in various tissues. These regulations have stimulated the development of fast, accurate and sensitive analytical methods for monitoring sulfonamide residues in food samples of animal origin. Gas chromatography methods tend to be time‐consuming due to the required previous cleanup and derivatization procedures, thus, HPLC methods with UV or MS detection have the leading position. These and other aspects of current analytical methodology, including sample preparation and cleanup, are reviewed and discussed. Emphasis is given to confirmatory methods since these comprise useful tools to regulatory agencies, and identifications based on these methods can be used in support of regulatory action.

Effective monitoring for ractopamine residues in samples of animal origin by SPR biosensor and mass spectrometry

Ractopamine (RCT) is a member of the β-2-agonist (β-agonist) family. It is licensed for use as an animal growth promoter in more than 20 countries worldwide, including the United States and Canada, but is either not licensed or prohibited by over 150 others, including those within the European Union. The issue of the use of RCT in livestock bound for human consumption has risen to prominence recently following the decision by The People's Republic of China to ban the import of pork from a number of processing plants after finding traces of RCT in shipments from the U.S.A. In order to monitor for the illegal use of such compounds within Europe, there is a requirement to have a robust and reliable testing scheme capable of the detection of low concentrations of RCT. In the present study an optical biosensor screening assay was developed. The developed assay was compared with a liquid chromatography/mass spectrometry/mass spectrometry (LC–MS/MS) confirmatory procedure. These methods were used to study the ability to detect RCT in pigs following treatment. Both testing procedures were capable of detecting low μg kg −1 concentrations of the drug in urine and liver. Liver was found to be a less suitable sample matrix, with RCT residue levels being undetectable after 5 days withdrawal of the drug. Urine samples however still contained detectable RCT residues several weeks after withdrawal. The correlation (as measured by r 2) between the biosensor and LC–MS/MS methods was 0.99 and 0.97 for urine and liver samples, respectively. It is concluded that testing regimes based on RCT analysis in liver are less likely to detect illegal administration of the drug than those based on urine analysis. Urine samples provide an excellent matrix for the detection of RCT residues for an extended period post withdrawal.

Pesticide and non-dioxin-like polychlorinated biphenyls (NDL-PCBs) residues in foodstuffs from Ismailia city, Egypt

Samples of vegetables and cereals from Egypt were screened for 113 pesticides, of which 68 were quantified, using gas chromatography-mass spectrometry (GC/MS) with limits of detection (LODs) ranging from 0.02 to 1.9 µg kg−1. In addition, the residues of 17 non-dioxin-like polychlorinated biphenyls (NDL-PCBs) were measured in samples of animal origin (meat, dairy products and seafood) using high-resolution gas chromatography (HRGC)/high-resolution mass spectroscopy (HRMS). None of the cereal samples and 72.7% of the vegetables contained any detectable levels of the pesticides. Detectable residues, not exceeding the current European Union maximum residue limits (MRLs) were found in 27.3% of vegetables. The estimated daily intake for detected pesticides was well below their corresponding acceptable daily intake (ADI), with exposure ranges between 0.002% of the ADI for profenofos and 0.2% of the ADI for lambda-cyhalothrin. The sum concentration of 17 PCBs congeners varied between 2.5 and 322 ng g−1 fat, corresponding to 1.7-216 ng g−1 fat for the seven indicator PCBs. The highest values were measured in seafood, the lowest in dairy products. Hexa-CB 153, 138 and hepta-CB180 were the congeners with the highest contribution. PCBs congener profiles found in most of the samples were consistent with the expected profile for Aroclor 1260 and 1262. PCBs’ contamination levels reported in this paper were many times lower than in developed countries, except for chicken samples. Also, the dietary intake of seven indicator PCBs due to the consumption of food of animal origin (4.84 ng kg−1 body weight day−1) from Ismailia city, Egypt, is several times lower than the intake in European Union countries.

ELISA and HPLC methods for atrazine and simazine determination in trophic chains samples

The aim of the research was to determine optimal conditions for atrazine determination in trophic chain samples by means of an antigen-coated tube enzyme-linked immunosorbent assay (ELISA). The ELISA method was used for analysis of a selection of samples and the results and method requirement compared with HPLC. The 2 h competitive ELISA showed a minimum detection limit of 0.05 ng mL −1 and a dynamic range 0.1–2 ng mL −1. Investigation of atrazine concentration in a selection of trophic chain samples indicated that the content of atrazine (μg kg −1) in soil samples was 3.2–85.4, vegetable roots 32.9–148.9, green parts of plants 67.7–136.4, cereals 42.4–91.5 and samples of animal origin 1.3–8.4. The correlation between results obtained by HPLC and ELISA methods was 0.97. In addition, simazine content was determined by the HPLC method in which the detection limits were 0.2 μg g −1 for atrazine and 0.3 μg g −1 for simazine. The content (μg kg −1) of simazine in soil samples was 13.5–15.5, in vegetables roots 29.5–93.7, in green parts of plants 34.6–72.6 and in cereals 158–189. The study demonstrates the utility and convenience of the simple, practical and cost-effective ELISA method in a non-immunoassay laboratory for the analysis of food and environmental samples. The method is ideal for the rapid screening of large numbers of samples in laboratories where access to HPLC facilities is limited or lacking. In addition the investigation demonstrates the presence of significant levels of atrazine and simazine in trophic chain samples collected from different areas of the region. As expected, the highest concentration of both herbicides was found in plants.

Optimisation of the determination of thiamin, 2-(1-hydroxyethyl)thiamin, and riboflavin in food samples by use of HPLC

The aim of this study was first to optimise and validate a method using an enzyme-mixture to liberate protein- and phosphate-bound thiamin and riboflavin in food by the use of ultrasonication and HPLC, and second to include the quantitation of the vitamin B 1 active compound 2-(1-hydroxyethyl)thiamin (HET). The enzyme-mixture consisted of α-amylase, proteinase, and phosphatase. The use of ultrasonication in the enzyme treatment enabled the results for vitamin B 1 and B 2 to be obtained in 1 day. In consequence of an incomplete release of phosphate-bound thiamin of some of the batches of enzymes used, thiamin was quantitated as the sum of thiaminmonophosphate and thiamin. The vitamin B 1 active compound, HET was detected and quantitated separately. The standard deviations for the method were 3.7%, 4.7%, and 13.3% for thiamin, riboflavin, and HET, respectively. The relative bioactivity of HET is similar to the bioactivity of thiamin. In the samples of animal origin the content of HET represented 7–24% of the content of thiamin, while in dried yeast the content of HET was 37% of the content of thiamin. Quantitation of vitamin B 1 in food by a post-column derivatisation is recommended to include separate quantitation of thiamin and HET.

Methicillin-resistant Staphylococcus aureus (MRSA) in foods of animal origin product in Italy

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a global health concern. The present study regarded 160 S. aureus strains that had been isolated from 1634 foodstuff samples of animal origin in a previous survey conducted in Italy during 2003–2005. The strains were characterized by detecting the mecA gene, the production of type A to D staphylococcal enterotoxins (SEs), and studying their resistance properties against several antibiotics; their ecological origin was determined by biotyping. Of the 160 analyzed S. aureus strains six (3.75%) were mecA positive and derived from six different samples; four isolates were from bovine milk and two from dairy products (pecorino cheese and mozzarella cheese). Two strains isolated from milk belonged to the non-host-specific biovar while the others to the ovine biovar. The strain isolated from mozzarella cheese belonged to the non-host-specific biovar and the strain isolated from pecorino cheese to the ovine biovar. All the MRSA strains isolated were enterotoxigenic; two strains synthesized SEA/SED two SED and one SEC. All the strains showed resistance to at least one of the antibiotics tested but none was resistant to glycopeptides.

PCR-based detection of cow's milk in goat and sheep cheeses marketed in the Czech Republic

A method based on the polymerase chain reaction (PCR) principle was validated for detecting cow&amp;rsquo;s milk in goat and sheep cheeses. DNA was isolated from the cheeses using the isolation kit Invisorb Spin Food I by Invitek Co., designed for the samples of animal origin. The PCR method applied utilizes the sequence of the mitochondrial gene coding cytochrome b which is specific for mammals. It uses the common forward primer and the reverse primer species-specific. After electrophoresis, cow DNA was characterised by the fragment of the size of 274 bp, goat DNA by the fragment of 157 bp, and sheep DNA by the fragment of 331 bp. The detection limit of the PCR method described (1%) was determined with model samples made from pure goat cheese with a defined addition of cheese made from cow&amp;rsquo;s milk. The method validated was applied in the analysis of 17 goat cheeses and 7 sheep cheeses obtained from retail trade. Products of Czech, Slovak, French, Dutch, and Italian origin were examined. The presence of undeclared cow&amp;rsquo;s milk was detected in three kinds of goat cheese and in one of sheep cheese. &amp;nbsp;

Application of the Collision Cell (CCT) in the ICP-MS Analytical System

Our laboratory has seen a sharp rise in the number of requests for the analysis of smaller and smaller concentrations of elements from foods, plants, soil, organic fertiliser, irrigation and ground water, sewage, sewage sludge, raw material of food, as well as human and animal origin samples. From the above elements the inductively coupled plasma mass spectrometer is capable of analysing the smallest concentration of elements. Our method of analysis is to use our ICP-MS instrument, together with a ICP-MS with Collision Cell Technology (CCT). The CCT method has better detection limit, with 1-3 magnitudes, compared to the normal ICP-MS analytical method. The CCT has better detection limits mainly for the following elements in the periodical system: analysis of arsenic, selenium, germanium, vanadium and chromium. Additionally a collision cell can be applied for the analysis of silicium, sulphur, zink, copper, iron, calcium, magnesium and potassium in smaller concentrations.

PCDDs, PCDFs and dioxin-like PCBs in food of animal origin (Slovakia)

The levels of 17 toxic polychlorinated dibenzo- p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and 12 non- ortho and mono- ortho polychlorinated biphenyls (PCBs) in 73 foodstuff samples of animal origin collected from shops and small farms in selected districts of Slovak Republic are presented in this paper. The concentrations expressed as WHO-TEQ in analysed samples ranged for PCDDs/PCDFs from 0.25 pg/g fat in pork to 75 pg/g fat in cod liver. The TEQ concentrations of non- ortho PCBs were between 0.007 and 181 pg/g fat and mono- ortho PCBs between 0.0083 and 66.5 pg/g fat. The mean concentrations in freshwater fish and imported species of marine fish were 0.089 pg TEQ/g fresh weight for PCDDs/PCDFs, 0.17 pg TEQ/g fresh weight for non- ortho PCBs and 0.034 pg TEQ/g fresh weight for mono- ortho PCBs. The mean total concentration of PCDDs/PCDFs and dioxin-like PCBs in samples of infant milk formula imported from EU countries was 0.98 pg TEQ/g fat.

Comparative evaluation of PCR assays for the robust molecular detection of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis ( MAP) can cause a very serious, often-fatal disease, namely paratuberculosis, in several animal species, especially ruminants. Recently, it has also been implicated in the pathogenesis of Infectious Bowel Disease of man. The aim of this study was to develop a molecular method for the routine detection and identification of MAP, from tissue samples of animal origin. The proposed assay would have to combine optimum performance and cost, with high reproducibility. To this goal, three laboratories in Greece and the Czech Republic undertook different parts of a study that involved evaluation of DNA extraction procedures, and PCR assays, for MAP detection. For DNA extraction we used one in-house, and one commercial method, and for the PCR we assessed a number of different assays, starting with the evaluation of primer specificity with an extended GenBank database search. Based on these results, we chose to assess a one-tube nested, 2 two-tube nested, and a single PCR assay, targeted to different genomic regions of the IS900 element. These four methods were applied on positive and negative control samples, consisted of pure bacterial cultures and formalin-fixed paraffin-embedded (FFPE) tissue samples collected from cattle with paratuberculosis and chickens with M. avium subsp. avium infection. Based on the criteria of reliability and cost, the procedure that performed better was the one-tube nested PCR assay combined with the in-house DNA extraction method. The agreement of the results obtained by the three collaborating laboratories indicates the reliability of the proposed assay even under different laboratory conditions.

Virulence and antimicrobial-resistance gene profiles determined by PCR-based procedures for Salmonella isolated from samples of animal origin

The virulence (V) and antimicrobial resistance (R) gene profiles of Salmonella isolates collected from samples of animal origin in a Spanish Region were determined by PCR-based procedures, and compared with V and R gene profiles of clinical strains from the same region. Some V-profiles were serotype-specific, i.e. S. Panama carried all the V-genes tested (invE/A, phoP/Q, stn, iroB, slyA, hin/H2, and agfA), whereas S. Enteritidis lacked hin/H2, and S. Hadar and all but one of the S. Typhimurium isolates, lacked agfA. Salmonella V-plasmids (spv ) were found in serotypes Enteritidis, non-motile [9,12:-:-], Typhimurium and its monophasic variant [4,5,12:i:-]. Twenty-six out of the 56 animal-origin isolates showed some drug resistance. In the nalidixic acid-R isolates, mutations in the Ser-83 and/or Gly 133 codons in the gyrA gene were identified. Seventeen R-genes grouped into different R-profiles were detected, [pse1–aadA2–sul1–tet(G)–floR]-profile being the most frequent. Most of the serogroup B isolates, collected from pork or beef products, displayed multidrug-R and carried class 1-sul1 integrons. The integrons contained the following gene cassettes: pse1, aadA2, dfr17–aadA5, dfr1–aadAla, oxa1–aadA1a and dfr12–aadA2. All of them, except the dfr17–aadA5 gene cassette configuration , were also present in integrons of clinical isolates. Integrons could be plasmid- and/or chromosome-located, and those with oxa1–aadA1a and dfr12–aadA2 gene cassettes were located in spv -plasmids, which also carried the transposon associated genes tpnA, tpnR and merA . Conversely, all S. Enteritidis, S. Tennessee, and non-motile isolates collected from poultry samples showed drug susceptibility to all antibiotics tested. Moreover, among S. Enteritidis clinical isolates both ampicillin-R (encoded by tem1 genes, plasmid- or chromosome-located) and quinolone-R (due to mutations involving gyrA gene) were frequent. The efficiency, rapidity, and flexibility of the PCR-protocols applied was high. All of them can be performed directly on aliquots of a bacterial culture, using sequence-specific primers, the same basic equipment, and the same basic PCR-program.

PCR-based procedures in detection and DNA-fingerprinting of Salmonella from samples of animal origin

The usefulness of selected PCR-protocols for the detection of Salmonella in 117 samples of animal origin (17 raw minced meat, 27 raw chicken meat, 8 raw sausages, and 25 egg samples, as well as 18 poultry faecal, and caecal swabs samples) and DNA-fingerprinting typing is shown. To establish an accurate PCR-procedure for Salmonella detection the following parameters were evaluated: two pre-PCR concentration procedures, centrifugation and immunomagnetic separation (IMS) using Dynabeads anti- Salmonella; the specificity and sensitivity of 10 sets of primers; and different conditions of the amplification reaction. In light of the results obtained from the use of PCR-based procedures alone or in combination with conventional methods, the following findings can be underlined: First, IMS is more efficient than centrifugation in the recovery of Salmonella. Second, the selected IMS/PCR-detection protocol is less time-consuming (45 h) than the IMS/culture procedure (90 h), and a good concordance between them was found when the Kappa coefficient was calculated (0·87). Third, PCR-ribotyping technique showed a very low discrimination power, being able to differentiate only three profiles. Fourth, RAPD technique using specific primers supports previous works in which it was proposed as a simple and useful tool for discriminating isolates between and within serotypes. Fifth, The efficiency, rapidity, and flexibility of the PCR-protocols applied were high, and they can be performed using two PCR-programs and the same basic equipment.