Molecular characterisation of IBDV isolates in Turkey revealed reassortant strains.

1. Infectious bursal disease (IBD) is an acute, highly contagious viral disease of chickens caused by a virus (IBDV) which has a bi-segmented, double-stranded RNA genome. It has five viral proteins in its structure; the VP1 gene is encoded in segment B and the other four are in segment A.2. In this study, bursae of Fabricius and spleen samples taken from chickens suspected of having clinical or subclinical IBD from a total of 50 chicken flocks located in different geographical regions of Turkey were examined.3. The RT-PCR analysis of the VP2 gene showed that 30 of the 50 samples (60%) tested positive. Eight positive isolates were chosen and RT-PCR was performed to amplify the VP1 gene.4. The study showed that reassortant field strains that cause clinical or subclinical disease are currently circulating in broiler flocks across Turkey.

Giant Cell Tumor of the Proximal Phalanx: Report of two Cases Treated by Two Different Methods and Review of the Literature.

The hand is an extremely rare site for giant cell tumor (GCT). There are only a few reported cases of GCT including the hand, and even fewer reporting involvement of phalanges. GCTs in small bones are typically more aggressive and have higher local recurrence and rate of metastasis in younger patients compared to long bone involvement, so the treatment is more clinically challenging in the hand. In this study, we present the management of giant cell tumors of the proximal phalanxin two patients treated with two different method; ray resection and arthrodesis using an iliac crest graft. Key words: giant cell tumor, phalanx, hand, recurrence.

Effects of oregano juice on eggshell microbial load, layer embryo development, hatching results, and growth at the first 2 weeks after hatch.

One of the factors affecting the hatching results and chick quality is the implementation of an appropriate disinfection program with effective disinfectants. This study was carried out to evaluate the effectiveness of oregano juice as a disinfection of hatching layer eggs as an alternative to formaldehyde. In the study, 1800 eggs collected from 48-week old Akbay white layer breeders were used. Eggs were divided into 3 equal disinfection groups (fumigation with formaldehyde, 50% oregano juice, and 100% oregano juice). In the study, the characteristics of the eggshell, microbial load, embryo development, egg weight loss, hatching results, chick weight and quality, and performance in the first 2 weeks were determined. No differences were observed among the disinfection groups in egg shell characteristics, microbial load in the shell, hatchability of fertile and set eggs, embryonic deaths, and body weight, body weight gain, and feed conversion ratio in female chicks. Egg weight loss was higher (P < 0.001) in the 50% oregano juice group (13.75%) compared to the formaldehyde fumigation group (12.11%). Chick quality score was found to be lower in the formaldehyde fumigation group (96.44) compared to the other two oregano juice groups (P < 0.001). The chicks hatched from the eggs in the formaldehyde fumigation group consumed more feed (119 g/week) in the first 2 weeks (P < 0.001). The use of 50% oregano juice in the disinfection of hatching eggs did not have any negative effects compared to the fumigation process with formaldehyde. Therefore, it is concluded that disinfection with 50% oregano juice was effective in reducing the microbial load in the egg shell as much as formaldehyde fumigation, and even it was superior in chick quality score.

Effects of cage type on performance, welfare, and microbiological properties of laying hens during the molting period and the second production cycle.

As most of the unenriched cages will soon switch to enriched cages, it is important to characterize all the effects in the laying hens for sustainable production. Laying hens can be used in several production periods by applying molting. The aim of this study was to determine the cage type (unenriched and enriched) on performance, welfare, and microbiological properties of laying hens during the molting period and the second production cycle. Overall, 840 brown laying hybrids were used in the experiment. Laying hens were reared on two different cage types (unenriched cage (UEC) and enriched cage (EC)) in the same poultry house. When the hybrids were 75 weeks old, they were subjected to force molting with whole grain barley. Performance, welfare, microbiological, and serological data of laying hens were obtained from 73 to 107 weeks of age. Egg production, egg weight, feed conversion ratio, breaking strength, albumen and yolk index, Haugh unit, feather condition, and breaking force of femur and metatarsus were better in the post-molting period. However, keel bone deformities and Newcastle disease virus antibody titers are the worst in the post-molting period. Stiffness of femur and metatarsus was increased with period. These results indicate that necessary precautions should be taken against the problems that may occur in the direction of bone and health. During the molting period, hens kept in EC had lower egg production but they returned to egg production at a high rate. EC type had a positive effect on egg production, feed conversion ratio, feather and foot condition, and breaking force of metatarsus.

Effect of Lonicera japonica extract on Mycoplasma gallisepticum in naturally infected broiler flocks.

1. In this study, the effect of chlorogenic acid extract from Lonicera japonica Thunb. on Mycoplasma gallisepticum infections and the performance of broiler flocks was investigated. 2. A total of 360 Ross-308 broiler chicks taken from M. gallisepticum seropositive flocks were divided equally into three groups designated as control (nothing administered), antibiotic (Tylosin tartrate given for the first 3 d and d 20-22) and test group (chlorogenic acid extract given twice a day on d 16 and 22). 3. Broiler performance analysis, serological tests (slide agglutination), molecular identification (polymerase chain reaction) and histopathological examination were performed to detect M. gallisepticum. 4. The results show that chlorogenic acid not only increases live body weight but is also an alternative treatment option in M. gallisepticum-infected broiler flocks.

The Radioprotective Effects of Caffeic Acid Phenethyl Ester and Thymoquinone on Oxidative and Nitrosative Stress in Liver Tissue of Rats Exposed to Total Head Irradiation.

OBJECTIVE: The aim of this study is to investigate the effects of addition of caffeic acid phenethyl ester (CAPE) and thymoquinone (TQ) on oxidative and nitrosative stress in the liver tissue of irradiated rats. METHODS: Forty Sprague-Dawley rats were divided into five groups to test the radioprotective effectiveness of TQ and CAPE administered by intraperitoneal injection. Appropriate control groups were also studied. RESULTS: Liver antioxidant capacity, as measured by levels of total superoxide scavenger activity (TSSA), non-enzymatic superoxide scavenger activity (NSSA) and glutathione-S-transferase (GST) activity except superoxide dismutase (SOD) activity, were statistically lower in the irradiation (IR) group compared to all other groups. Total superoxide scavenger activity and NSSA were statistically higher in the IR plus TQ and IR plus CAPE groups compared to all other groups. In contrast, glutathione peroxidase (GSH-Px) activity was significantly found to increase in the IR plus CAPE group compared to control groups. The xanthine oxidase (XO), nitric oxide synthase (NOS) activities, nitric oxide (NO●) and malondialdehyde (MDA) levels in the IR group were statistically higher than in the other groups. Moreover, XO activity in the IR plus TQ group was statistically lower than all other groups including the IR plus CAPE group. In addition, NO● level was found to increase in all groups when compared to the normal control group. CONCLUSIONS: Thymoquinone and CAPE decrease oxidative and nitrosative stress markers and have antioxidant effects, which also increase antioxidant capacity in the liver tissue of irradiated rats.

Detection of mycoplasma species in turkeys by culture and polymerase chain reaction.

The purpose of the present study was to investigate the presence of pathogenic mycoplasma species in the turkey population of Turkey. Tracheal samples randomly collected from a total of 624 apparently healthy meat-type turkeys at a commercial abattoir located in the north of the country were examined by culture and genus- and species-specific polymerase chain reaction (PCR) assays for mycoplasma. In the direct plating onto solid specific media, mycoplasma growth was observed from 1.4% (9/624) of the samples, which were confirmed to belong to the Mycoplasma genus by genus-specific PCR. Mycoplasma iowae (MI) and M. meleagridis (MM) were identified by the species-specific PCR from eight and one of the samples, respectively. However, genus-specific PCR amplification was obtained from 2.6% (16/624) of the samples which produced turbidity in the liquid media. Interestingly, these positive samples were different from those obtained from solid agar and mycoplasma growth was not observed when the broth samples were inoculated onto solid media. In the species-specific PCR analysis of the broth samples, MI, MM and M. gallisepticum were identified from twelve, two and two samples, respectively. The inconsistency between the results obtained from liquid and solid media raises questions about the efficiency of isolation procedures for mycoplasma and this warrants further investigation.

Anesthesia-related complications in living liver donors: the experience from one center and the reporting of one death.

Living donor liver transplantation has become an alternative therapy for patients with end-stage liver disease. Donors are healthy individuals and donor safety is the primary concern. The objective of this study was to evaluate the anesthetic complications and outcomes for our donor cases; we report one death. The charts of the patients who underwent donor hepatectomy from February 1997 to June 2007 were retrospectively reviewed. Right hepatectomy (resection of segments 5-8) was done in 101 donors, left lobectomy (resection of segments 2-3) in 11 donors, and left hepatectomy (resection of segments 2-4) in one donor. Minor anesthetic complications were shoulder pain, pruritus and urinary retention related to epidural morphine, and major morbidity included central venous catheter-induced thrombosis of the brachial and subclavian vein, neuropraxia, foot drop and prolonged postdural puncture headache. One of 113 donors died from pulmonary embolism on the 11th postoperative day. This procedure has some major risks related to anesthesia and surgery. Although careful attention will lower complication rate, we have to keep in mind that the risks of donor surgery will not be completely eliminated.

Polymerase chain reaction detection of Salmonella spp. in fecal samples of pet birds.

The aims of this study were 1) to determine the prevalence of Salmonella in clinically ill birds in aviaries in Ankara, Turkey, and 2) to compare conventional culture and polymerase chain reaction (PCR) for detection of Salmonella in feces from clinically ill pet birds. In the study, 185 fecal samples (feces and/or swabs) collected from the pet birds kept in the seven different aviaries in the city of Ankara were investigated for the existence of Salmonella spp. by bacterial isolation and PCR. The conventional isolation and identification methods were performed for Salmonella isolation from fecal cultures. Suspected colonies were confirmed with the Salmonella polyvalent O antiserum and serogrouped with Salmonella group-specific antiserum. PCR was performed after the fecal swabs were incubated for 18 hr in 10 ml of tetrathionate broth. Three (1.63%) out of 185 fecal samples were found to harbor Salmonella spp. by conventional identification tests and were found to belong to serogroup B. Five (2.7%) swab samples were found to harbor Salmonella DNA by PCR tests. As a conclusion, PCR following incubation of clinical samples in pre-enrichment broth seemed to be a fast and practicable method for Salmonella spp. diagnosis when compared to protracted labor-intensive conventional culture techniques.

Restriction fragment length polymorphism typing of infectious bursal disease virus field strains in Turkey.

Infectious bursal disease (IBD), also known as Gumboro disease, is a highly contagious, immunosuppressive disease of immature chickens. It is caused by IBD virus (IBDV) and is responsible for major economic losses in the poultry industry worldwide. In this study, 280 bursa samples from 56 commercially reared chicken flocks in Turkey with clinical symptoms of IBD were examined for IBDVs using the reverse transcription (RT)-polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) assay. The assay was conducted on a 743-bp fragment of the VP2 gene with the restriction enzymes BstNI, MboI, and SspI. The results indicate the existence of field isolates with new molecular patterns different from those previously published that may well be unique and specific to geographical regions.

Prophylactic use of low-dose, on-demand, intramuscular hepatitis B immunoglobulin and lamivudine after liver transplantation.

The combination of hepatitis B immunoglobulin (HBIG) and antivirals (nucleos[t]ide analogs) has extended the applicability of orthotopic liver transplantation (OLT) for patients with hepatitis B virus (HBV)-related liver disease. However, HBIG administrations have an extremely high cost. Herein, we evaluated our results with low-dose, on-demand, intramuscular HBIG plus lamivudine (LAM) prophylaxis after OLT. The HBV DNA status in 40 patients at the time of OLT determined the treatment: group A (n = 22), HBV DNA (-), no antiviral pretreatment; group B (n = 11), HBV DNA (-), after LAM; group C (n = 3), HBV DNA (+) after LAM (LAM resistance/Adefovir [ADV] unavailable); group D (n = 2), HBV DNA (+), no antiviral pretreatment; and group E (n = 2), HBV DNA (-) after LAM + ADV (LAM resistance/ADV available). Five patients died within 12 months after OLT unrelated to HBV infection. The remaining 35 patients were followed for a median duration of 16 months (range, 6-93 months). Only two recipients from group C, who were transplanted despite LAM resistance + no ADV pretreatment, revealed recurrent HBV infections at 14 and 16 months posttransplantation; they were then treated successfully with ADV as it became available. The third group C recipient had undetectable HBV DNA at 18 months after OLT. The mean cumulative doses of HBIG administered within the first, second, and third years were 34,014, 5258, and 5090 IU, respectively. In conclusion, low-dose, on-demand, intramuscular HBIG plus (LAM +/- ADV) prophylaxis is a safe, efficient, and cost-effective regimen to prevent recurrent HBV infection following OLT. OLT despite untreated LAM resistance may require sustained higher serum HBsAb levels after surgery.

Efficacy of right anterior sector drainage reconstruction in right-lobe live-donor transplantation.

BACKGROUND: Without including the middle hepatic vein (MHV) into right-lobe grafts, venous drainage of the anterior segment (AS) has always been a concern. The efficacy and the necessity of additional venous reconstruction in the AS for graft viability and function are still controversial. METHODS: Since February 2002, 57 right-lobe grafts were implanted into adult recipients. The mean graft to ideal recipient weight ratio (GRWR) was 1.3% (minimum: 0.8%, maximum 2.1%). All minor venous tributaries to the MHV less than 5 mm in diameter were ligated. The management of MHV tributaries was categorized into three groups: (A) no major tributaries (n = 33); (B) major tributaries ligated (n = 11); and (C) major tributaries reconstructed (n = 13). Reconstructions were established using vascular grafts (PTF, n = 8; cadaveric, n = 2) or joining with right hepatic vein orifice (n = 6). The groups were homogenous in age, gender, MELD (17 +/- 6, 16 +/- 4, 18 +/- 7), graft weight (817 +/- 181, 838 +/- 152, and 855 +/- 159 g), and graft/ideal liver weight ratio (0.54 +/- 0.12, 0.57 +/- 0.13, and 0.57 +/- 0.1, respectively). We investigated the volume of ascites, serum bilirubin, albumin, and liver enzyme levels and INR on postoperative day 4 (POD4) and POD21. RESULTS: Two patients died of MRSA sepsis on POD18 and POD23 (MELD: 24 and 28) after initial graft function. Their graft weights were 800 g and 980 g, and their volumes were 47% (group B) and 62% (group A) of the ideal liver mass of the recipients, respectively. One PTF and another cadaveric vascular graft were found occluded 4 and 7 days after implantation without any negative consequence. At 6-month follow-up, the remaining 55 patients were alive with primary liver grafts. Vascular reconstructions were patent except for 2 early occlusions. Among the 3 groups, no significant difference was found on POD4. Three weeks after transplantation, the mean AST level in the major ligation group (46.7 +/- 8.14) was significantly higher than in the minor ligatation group (29.6 +/- 8.6) but not in the major reconstruction group (33.7 +/- 3.7; P = .03 and P = .29). The mean albumin level was highest after minor ligation (3.2 +/- 0.18), which was significantly better than the major reconstruction (2.7 +/- 0.1) but not the major ligation (3.1 +/- 0.14) cohorts (P = .02 and P = .13). CONCLUSIONS: In this study of a limited number of cases, right-lobe liver grafts with GRWR of > or =1.1% displayed optimal graft function without additional venous reconstruction to the AS.

Effect of magnesium sulfate on renal ischemia-reperfusion injury in streptozotocin-induced diabetic rats.

OBJECTIVE: Ischemia/reperfusion (I/R) injury is a major cause of acute organ dysfunction and I/R related acute renal failure is a common clinical problem. Diabetes mellitus is defined as a risk factor for the development of acute renal injury as diabetic nephropathy compromises the renal tolerance to ischemia. The aim of this study was to investigate the protective effect of magnesium sulfate in a diabetic rat renal I/R injury model. MATERIALS AND METHODS: Diabetes mellitus was induced using streptozotocin. Thirty-five rats were divided into five groups: Group I: Nondiabetic sham group; Group II: Diabetic sham group; Group III: Diabetic I/R group; Group IV: Diabetic I/R + prophylactic (preischemic) MgSO4; and Group V: Diabetic I/R + therapeutic (following reperfusion) MgSO4 group. MgSO4 was administered 200 mg/kg intraperitoneally. Renal I/R (45 min ischemia + 4 h reperfusion) was induced in both kidneys. Histomorphological, immunohistochemical (caspase-3 and iNOS) and biochemical (BUN, Creatinine) methods were performed to assess the blood and tissue samples. RESULTS: Histomorphological injury scores and immunostaining intensities (for both caspase-3 and iNOS) were significantly lower in the MgSO4 administered groups (prophylactic and therapeutic) than in the Diabetic IR group. There were no significant differences in biochemical parameters (BUN, Cr) between the MgSO4 administered groups and the Diabetic IR group. CONCLUSIONS: In the present study, it was demonstrated by histomorphological and immunohistochemical methods that magnesium sulfate administration before ischemia or following reperfusion significantly reduced renal I/R injury in a diabetic rat model.

Comparison of the RIFLE, AKIN, and KDIGO Diagnostic Classifications for Acute Renal Injury in Patients Undergoing Liver Transplantation.

BACKGROUND: The aim of this study was to determine the incidence and peri-operative risk factors for acute renal injury (ARI) and their relationship with mortality rate through the use of 3 different ARI diagnostic classifications in patients after liver transplantation (LT). METHODS: We retrospectively investigated the medical records of adult LT recipients. Post-operative ARI was determined with the Risk, Injury, Failure, Loss, and End-Stage Renal Failure (RIFLE), Acute Kidney Injury Network (AKIN), and Kidney Disease: Improving Global Outcomes (KDIGO) classifications. RESULTS: We analyzed 440 adult patients. The post-operative incidence of ARI was 8.0% by the RIFLE classification, 14.31% by the AKIN classification, and 14.54% by the KDIGO classification. Significant risk factors for ARI were pre-operative albumin (odds ratio [OR], 0.776; 95% confidence interval [CI], 0.473-1.273 for AKIN; and OR, 0.724; 95% CI, 0.444-1.182 for KDIGO) and hemoglobin levels (OR, 2.830; 95% CI, 1.157-6.9261 by RIFLE), intra-operative red blood cell transfusion (OR, 1.072; 95% CI, 1.004-1.143 by AKIN; and OR, 1.077; 95% CI, 1.011-1.147 by KDIGO), and blood loss (OR, 1.00; 95% CI, 0.999-1.000 by RIFLE). The early mortality rate was 7.9% in our series. CONCLUSIONS: The RIFLE, AKIN, and KDIGO ARI classifications classify the severity of renal dysfunction in patients who have undergone LT. Direct associations were found between higher mortality rates and severity of renal disease.

Effect of analgesia administration timing on early post-operative period characteristics: a randomized, double-blind, controlled study.

This study investigated the effect of time of analgesia administration in 64 patients undergoing total abdominal hysterectomy. Patients received standard general anaesthesia and were divided randomly into two equal groups. At the time of fascia closure, patients in the intra-operative (Iop) group received 0.5 mg/kg pethidine intravenously. On arrival in the post-anaesthesia care unit, the same dose of pethidine was given to patients in the post-operative (Pop) group. All patients then used a patient-controlled analgesia pump to administer pethidine analgesia as required. Times to extubation, response to verbal stimulation and orientation, post-operative pain scores and analgesic consumption were recorded. Times to extubation and response to verbal stimulation were significantly longer in the Iop group. Pain scores, analgesic consumption and additional analgesic requirements were significantly higher in the Pop group in the first 2 h post-operatively. In conclusion, intra-operative administration of pethidine provided better pain management than post-operative administration.

Acute Renal Injury Evaluation After Liver Transplantation: With RIFLE Criteria.

BACKGROUND: The aim of this study was to identify acute renal injury (ARI) through the use of RIFLE (risk, injury, failure, loss, end-stage kidney disease) criteria and to investigate perioperative risk factors for ARI in liver transplantation (LT). METHODS: We reviewed medical records of adult LT patients retrospectively. Postoperative ARI was staged with RIFLE criteria by the 1st and 7th days of the surgery. RESULTS: We analyzed 440 adult LT patients, categorized as risk (R), injury (I), or failure (F) according to the RIFLE criteria. In this study, in the first postoperative day, incidence of ARI was 7.95%; all of them were R-class, and, on the 7th day, the incidence of ARI was 7.27%, as R-class 6.59% and I-class 0.68%. Significant risk factors were detected within the first postoperative day including pre-operative hemoglobin levels <9 g/dL (P = .019), intra-operative transfusion of red blood cells (RBCs) (P = .049) and fresh-frozen plasma (FFP) (P = .049), blood loss (P = .011), and post-reperfusion syndrome (P = .023). Multivariate analysis revealed risk factors for ARI as RBCs (odds ratio [OR], 1.049; P = .247) and FFP (OR, 1.017; P = .627) transfusion and blood loss (OR, 1.000; P = .021) (blood loss OR: 0.9996952300184; 95% confidence interval: 0.9994356774026 to 0.999548500399). The only significant risk factor for the 7th postoperative day was the Model for End-Stage Liver Disease (MELD) score (>20) (P = .002). CONCLUSIONS: This study showed that RBC and FFP transfusion, perioperative blood loss, and MELD score >20 are risk factors for LT-related ARI. Also normalization of hemoglobin levels with non-blood products in patients with preoperative low hemoglobin levels can diminish the need for RBC and that can prevent ARI.

Reversible expression of flagella in Campylobacter spp.

The in vitro phase variation of flagella and the transition rates between flagellate and aflagellate phenotypes in Campylobacter species including C. jejuni, C. coli, C. lari (thermophilic campylobacters), C. fetus subsp. fetus, C. fetus subsp. venerealis and C. hyointestinalis were investigated. The change from the flagellate to aflagellate phenotype was detected in all of the 12 Campylobacter strains studied. When measured in a motility medium, flagellate to aflagellate transition in thermophilic campylobacters, C. fetus and C. hyointestinalis strains occurred at a rate of 1.8 x 10(-3) to 7.5 x 10(-3), 3.0 x 10(-4) to 7.8 x 10(-4) and 1.8 x 10(-5) to 7.7 x 10(-6) per cell per generation, respectively. Transition from aflagellate to flagellate phenotype occurred at a rate of 5.8 x 10(-6) to 9.3 x 10(-6) per cell per generation in thermophilic campylobacters and 1.0 x 10(-6) to 1.5 x 10(-6) in C. fetus strains. No reversion from aflagellate to flagellate phenotype could be detected in C. hyointestinalis strains. It was concluded that the ability to reversibly express flagella was inherent in the wild-type strains and the transition rates for both directions were consistent for each strain.

Motile aeromonads in the feces and carcasses of broiler chickens in turkey.

Motile Aeromonas spp. were surveyed in chicken feces, carcasses, scalding water, and chilling water during slaughtering in a commercial processing plant in Turkey. Fecal and carcass samples of 351 chickens from 15 different flocks were examined by selective isolation techniques. All of the 15 flocks were positive for motile aeromonads. Motile aeromonads were detected in 14.8%, 90.5%, and 100% of fecal, carcass, and chilling water samples, respectively, while scalding water samples were negative for these organisms. Aeromonas hydrophila was the predominant species in both feces (51.9%) and carcass (66.9%) samples, followed by A. caviae and A. sobria. These results suggested that during the slaughtering process the spread of motile aeromonads from the intestinal contents to carcasses via processing water caused a heavy contamination of chicken carcasses.

Antimicrobial susceptibility of Pasteurella haemolytica and Pasteurella multocida isolated from pneumonic ovine lungs.

The in vitro susceptibilities of Pasteurella haemolytica biotypes A and T and P multocida from pneumonic ovine lungs to penicillin, ampicillin, oxytetracycline, chloramphenicol, erythromycin, kanamycin, neomycin, gentamicin, streptomycin and lincomycin were determined by the disk diffusion method. All the isolates were sensitive to chloramphenicol and resistant to lincomycin. The isolates of P haemolytica biotype A were consistently more sensitive to penicillin, ampicillin, oxytetracycline, erythromycin and streptomycin than those of biotype T.

Serotypes and electrophoretic protein profiles of Pasteurella haemolytica isolated from pneumonic ovine lungs.

Serotypes and SDS-PAGE protein profiles of P. haemolytica isolated from pneumonic ovine lungs were investigated. Of 268 P. haemolytica isolates, 232 (86.6%) were serotypable. A total of 12 serotypes were recognized in 20 different geographic origins of central Turkey. The most common serotype was A2, followed by A7, A1 and T4. Serotypes A13, A14, A16 and T15 could not be detected. In SDS-PAGE, marked differences between major bands of biotype A and T strains were found. In numerical analysis of protein profiles, biotype A and T strains were separated at 58% similarity level. Biotype A isolates produced a cluster at 80% similarity level, and biotype T isolates at 92% similarity level. No single cut off level was able to discriminate between each serotype studied and isolates could not be clustered on the basis of their geographic origins.