Serotyping Methods Research Articles

Establishment and application of PCR method in serotyping of Streptococcus pneumonia

To establish a method based on PCR for serotyping of Streptococcus pneumonia isolates. PCR serotyping method was applied for investigating the serotypes of S. pneumonia strains. 12 pairs of primers targeting different serotypes of S. pneumonia were designed and synthesized. After optimizing the PCR amplification reaction, sensitivity and specificity of each pair was performed. We applied the PCR methods for testing the serotypes of the isolated S. pneumonia strains. Each pair of primers showed satisfied PCR sensitivity and specificity. Of all 119 S. pneumonia strains tested by PCR serotyping method, 113 isolates were identified (3, 5, 6A/B, 9A/V, 14, 18, 19A, 19F, 23F) with 6 isolates were unable to be serotyped. We developed a simple, reliable and economic method for S. pneumonia serotyping which could be used for testing the serotypes of S. pneumonia that had been prevailed among general population.

Two five-plex PCRs methods for identification of common Salmonella spp. serotypes

In Tunisia, Salmonella is the most common bacterial agent responsible for childhood diarrhoea. Currently, isolation of the bacterium by microbiological and biochemical methods and confirmation of the serotype by serological method remain as the "gold standard". This study aimed to differentiate among the most common serotypes of Salmonella spp. via two rapid five-plex PCRs assay (MPCR) to evaluate the molecular serotyping method compared with the gold standard serotyping technique. The two five-plex PCRs assays were designed for the simultaneous detection of six genetic loci from Salmonella enterica serovar Typhimurium and four from S. enterica serovar Typhi. Sixty-one Tunisian strains (46 collected from patients and 15 from food) were isolated during the period 2002–2007. The STM and STY primers were able to discriminate all tested Salmonella serotypes that represent the most common clinical and food strains of S. enterica subsp. enterica in our laboratory. All strains belonged to 19 different serotypes: 15 serotypes gave unique amplification patterns compared each other and the other 4 serotypes were grouped into two pairs that gave the same molecular profile. We resolved this problem through the addition of a monoplex PCR. Salmonella typhimurium ATCC 14028 consistently produced the same molecular profile as S. typhimurium laboratory isolates. Interestingly, seven strains of Anatum serovar produced two different PCR profiles with these primers: five strains had the same amplification pattern STM 2,4,5 and STY 2; however, two strains had another molecular profile STM 2,3,5 and STY 2; so the reproducibility of this method was reduced to 93%. The MPCR system is a rapid, specific, and cost-effective molecular method that gas been proved to have efficient discrimination in serotyping of the most common isolates of S. enterica subsp. enterica.

Pneumococcal serotypes causing pediatric meningitis in Turkey: application of a new technology in the investigation of cases negative by conventional culture

The surveillance of serotypes causing invasive pneumococcal disease (IPD) provides further insight into the pathogenesis of pneumococcal disease and is important in order to track vaccine impact. Although the Quellung reaction has been accepted as the standard method for serotyping, prior antibiotic use causes a gap in studies based on bacterial culture. A total of 31 cerebrospinal fluid (CSF) samples found to be positive for Streptococcus pneumoniae by polymerase chain reaction (PCR) targeting the ply gene during an active surveillance were tested in a Bio-Plex multiplex antigen detection assay capable of detecting 14 serotypes/groups (1, 3, 4, 5, 6A, 6B, 7F/A, 8, 9V, 14, 18, 19A, 19F, and 23F). Twenty-seven CSF samples could be serotyped. The most common serotypes were serotypes 5 (n = 7), 19F (n = 5), 1 (n = 3), and 23F (n = 3). Theoretical coverage rates by the heptavalent (PCV7), 10-valent (PCV10), and 13-valent (PCV13) pneumococcal conjugate vaccines for bacterial meningitis were 48.1, 85.2, and 92.3%, respectively, for all age groups and 71.4, 85.7, and 100.0%, respectively, for those under 2 years of age. We propose that antigen detection assay used in conjunction with a PCR assay can be effectively applied in CSF samples to detect the pneumococcal serotypes, especially when the patient may have already been treated and, therefore, the cultures would be negative.

Characterization of Campylobacter jejuni and Campylobacter coli strains isolated in the region of Nis, Serbia

Campylobacter jejuni and Campylobacter coli represent one of the main causes of bacterial diarrhoea in humans. Although the disease is usually mild and self-limiting, severe chronic sequelae may occur, such as reactive arthritis, Guillain-Barré and Miller Fisher syndromes. Serotyping is used as an epidemiological marker, while post-infective polyneuropathies are associated with several O serotypes. Strains of C. jejuni and C. coli were serotyped based on heat stable (HS) and heat labile (HL) antigens, as well as biotypes to determine strain diversity. Campylobacter spp. was isolated using selective blood media with antibiotics. Differentiation to the species level was done by a combination of biotyping tests and by a PCR-based RFLP test. The isolates were characterised by Penner and Lior serotyping methods. The serotypes showed diversity without predominant serotypes. 24 HS serotypes were detected among 29 C. jejuni strains, and seven serotypes among nine C. coli strains. HL serotyping method successfully typed 62.5% of strains. Among 16 C.jejuni strains 14 serotypes were detected, and three among four C. coli strains. A C.jejuni strain associated with a patient with Guillain-Barré syndrome was typed as biotype II, O:19. The biotyping and serotyping results have indicated that C. jejuni and C. coli strains in the region of Nis, Serbia are diverse and could be probably of unrelated sources of origin or reservoirs. The strain associated with the Guillain-Barré syndrome patient was serotype O:19, one of the most common in this post-infective complication.

Evaluation of a multiplex PCR assay as an alternative method for Listeria monocytogenes serotyping

Listeria monocytogenes serotyping is commonly used as the first level of characterisation in the epidemiological surveillance of food and clinical isolates and is therefore widely accepted. The aim of this study was to define a scheme for multiplex molecular serotyping of L. monocytogenes based on a previously described PCR assay and then to evaluate and compare this new procedure with conventional serotyping by agglutination. The study included 1204 Listeria strains collected from food products in France, from March 2005 to October 2006. Two multiplex PCR assays were designed to cluster L. monocytogenes strains into five molecular serogroups: IIa, IIb, IIc, IVa, IVb in agreement with the most commonly encountered serotypes. Amplification of the prfA gene was added to the multiplex PCR to check for L. monocytogenes species; forty-eight (4%) of the isolates tested belonged to the genus Listeria but were not L. monocytogenes. Using this first multiplex PCR, the concordance between conventional and molecular methods was 90.6%, 97.8%, 100% and 100%, for 1/2a, 1/2c, 1/2b and 4b serotypes respectively. False results were observed for some atypical 1/2a, 3a and 1/2c strains. Therefore, this lack of specificity was resolved by using an additional PCR assay based on amplification of the flaA gene, a specific target of 1/2a and 3a strains. When applying the second PCR assay to IIa and IIc molecular serogroup strains, total agreement was obtained between molecular and conventional serotyping methods with a lower level of discrimination for the molecular one. This study proposes to define a strategy for molecular serotyping using both PCR assays: a multiplex and the flaA PCR in order to assign the atypical 1/2a, 3a and 1/2c strains. Moreover, prs gene detection was added for Listeria genus recognition as a positive control in association with flaA detection. Indeed, this molecular serotyping scheme could be considered as a useful and rapid method for first-level characterisation of the most frequently encountered L. monocytogenes serotypes.

A rapid screen of broth enrichments for Salmonella enterica serovars enteritidis, Hadar, Heidelberg, and Typhimurium by Using an allelotyping multiplex PCR that targets O- and H-antigen alleles.

Salmonella continues to cause significant foodborne outbreaks, best illustrated with recent outbreaks associated with peanut butter, raw tomatoes, and serrano peppers. To ascertain the likely source of the outbreak, bacterial typing is essential to this process. While PCR has become an important detection tool for pathogens in foods, PCR can also identify strain differences by targeting gene(s) or sequences exhibiting polymorphisms or variability in its distribution within the bacterial population. Over 2,500 Salmonella enterica serovars identified based on antigenic differences in lipopolysaccharide and flagellin have been identified to date. We developed an allelotyping PCR scheme that identifies the O and H alleles associated with S. enterica serovars Enteritidis, Hadar, Heidelberg, Typhimurium, and others, with the same antigen alleles but in different O- and H-allele combinations (e.g., S. enterica Kentucky), and validated it as a screen to identify samples contaminated with these Salmonella serovars. We correctly identified poultry samples containing S. enterica serovars Enteritidis, Kentucky, and Typhimurium from our multiplex screen of primary enrichments of environmental drag swabs. PCR agreed well (kappa values = 0.81 to 1.0) with conventional serotyping methods used to type salmonellae isolated from primary enrichment. Coupled with Salmonella-specific PCR, such as invA, this allelotyping PCR could prove useful in the identification of Salmonella and specific S. enterica serovars present in foods or the environment and could decrease the time and cost associated with conventional serotyping methods.

Genetic Diversity of the Pneumococcal Capsule: Implications for Molecular-Based Serotyping

Streptococcus pneumoniae remains an important pathogen despite licensure of a seven-valent pneumococcal protein conjugate vaccine. As a result, serotyping strains remains of paramount importance to both assess the effectiveness of current vaccines and closely monitor for the emergence of nonvaccine strains. Given the limitations of the quellung reaction, both molecular and immunology-based serotyping methods have been pursued. Currently, the most promising assay combines an immunologic assay with multiplex PCR of serotype-specific genes. The key limitation with a molecular-based assay is the plasticity of the pneumococcus, as capsular transformation or point mutations could easily result in serotype misclassification. Based on the currently available techniques, a comprehensive immunology-based assay appears to be the most promising alternative to the quellung reaction. In the future, assays that utilize high-throughput sequencing technology and/or matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) could lead to a novel pneumococcal serotyping method.

Escherichia coli O123 O antigen genes and polysaccharide structure are conserved in some Salmonella enterica serogroups

The serotyping of O and H antigens is an important first step in the characterization of Salmonella enterica. However, serotyping has become increasingly technically demanding and expensive to perform. We have therefore sequenced additional S. enterica O antigen gene clusters to provide information for the development of DNA-based serotyping methods. Three S. enterica isolates had O antigen gene clusters with homology to the Escherichia coli O123 O antigen region. O antigen clusters from two serogroup O58 S. enterica strains had approximately 85 % identity with the E. coli O123 O antigen region over their entire length, suggesting that these Salmonella and E. coli O antigen regions evolved from a common ancestor. The O antigen cluster of a Salmonella serogroup O41 isolate had a lower level of identity with E. coli O123 over only part of its O antigen DNA cluster sequence, suggesting a different and more complex evolution of this gene cluster than those in the O58 strains. A large part of the Salmonella O41 O antigen DNA cluster had very close identity with the O antigen cluster of an O62 strain. This region of DNA homology included the wzx and wzy genes. Therefore, molecular serotyping tests using only the O41 or O62 wzx and wzy genes would not differentiate between the two serogroups. The E. coli O123 O-antigenic polysaccharide and its repeating unit were characterized, and the chemical structure for E. coli O123 was entirely consistent with the O antigen gene cluster sequences of E. coli O123 and the Salmonella O58 isolates. An understanding of both the genetic and structural composition of Salmonella and E. coli O antigens is necessary for the development of novel molecular methods for serotyping these organisms.

Identification of 20 Common Human Enterovirus Serotypes by Use of a Reverse Transcription-PCR-Based Reverse Line Blot Hybridization Assay

The more than 100 human enterovirus (HEV) serotypes can also be classified into four species, HEV-A to -D, based on phylogenetic analysis of multiple gene regions. Current molecular typing methods depend largely on reverse transcription-PCR (RT-PCR) amplification and nucleotide sequencing of the entire or 3' half of the VP1 gene. An RT-PCR-based reverse line blot (RLB) hybridization assay was developed as a rapid and efficient approach to characterize common HEVs. Twenty HEV serotypes accounted for 87.1% of all HEVs isolated at an Australian reference virology laboratory from 1979 to 2007. VP1 sequences of all known HEV prototype strains were aligned to design one sense primer and three antisense primers for RT-PCR. After sequencing of the complete VP1 genes of 37 previously serotyped examples of the commonest 20 serotypes and alignment of these VP1 sequences with GenBank sequences, four serotype-specific probes for each serotype were designed for RLB. The RT-PCR-RLB assay was then applied to 132 HEV isolates, made up of the previously sequenced 37 isolates and another 95 serotyped clinical isolates. The RT-PCR-RLB genotypes corresponded with the serotypes for 131/132 isolates; the one exception was confirmed by VP1 sequencing, and the genotype was confirmed by repeat conventional serotyping. Genotyping by RT-PCR-RLB complements traditional serotyping methods and VP1 sequencing and has the advantages of convenience, speed, and accuracy. RT-PCR-RLB allows detection of specific enteroviral serotypes or genotypes associated with HEV outbreaks and significant disease.

“Dodgy 6As”: Differentiating Pneumococcal Serotype 6C from 6A by Use of the Quellung Reaction

The Quellung reaction has long been the standard method for pneumococcal serotyping ([7][1]); however, the new serotype 6C is reportedly indistinguishable from serotype 6A using this method ([8][2]). We report an anomalous Quellung reaction associated with serotype 6C, which may aid in diagnostic

A 10-year retrospective study on listeriosis in Latvia, 1998-2007

This study was undertaken to describe the trends in human listeriosis in Latvia over the 10-year period from 1998 to 2007 as a means of characterizing the epidemiological and clinical features of this disease. A total of 90 listeriosis cases were identified for the period from 1998 to 2007. During the 10-year study period the overall incidence of listeriosis was 0.4 per 100 000 of the population. Overall, 77% of all described clinical cases were characterized by meningoencephalitis and/or sepsis. The overall mortality, including abortions and perinatal infant deaths, was 6.7% (6/90). A marked clustering of listeriosis cases was observed from September to December 2000 possibly indicating one large outbreak. The lack of serotyping and molecular typing methods for subtyping of Listeria (L.) monocytogenes isolates in the present laboratory-based surveillance system is one of the main reasons why there were no officially documented listeriosis outbreaks in Latvia up to the present date. Therefore, serotyping and molecular typing should be introduced for all clinical isolates of L. monocytogenes to identify any listeriosis outbreaks as early as possible. Such measures will allow the application of appropriate actions at the food industry level, thus either preventing or significantly reducing the real burden of listeriosis in Latvia.

Differential Effects of Pneumococcal Vaccines against Serotypes 6A and 6C

Because classic pneumococcal serotyping methods cannot distinguish between serotypes 6A and 6C, the effects of pneumococcal vaccines against serotype 6C are unknown. Pneumococcal vaccines contain serotype 6B but not serotypes 6A and 6C. We used a phagocytic killing assay to estimate the immunogenicity of the 7-valent conjugate vaccine (PCV7) in children and the 23-valent polysaccharide vaccine (PPV23) in adults against serotypes 6A and 6C. We evaluated trends in invasive pneumococcal disease (IPD) caused by serotypes 6A and 6C in the United States, using active surveillance. Serum specimens from PCV7-immunized children had median opsonization indices of 150 and < 20 for serotypes 6A and 6C, respectively. Similarly, only 52% of adults (25 of 48) vaccinated with PPV23 showed opsonic indices of > 20 against serotype 6C. During 1999--2006, the incidence of serotype 6A IPD decreased by 91% (from 4.9 to 0.46 cases per 100,000 persons; P < .05) among individuals aged < 5 years and by 58% (from 0.86 to 0.36 cases per 100,000 persons; P < .05) among those aged > or = 5 years. Although the incidence of 6C IPD showed no consistent trend (range, 0-0.6 cases per 100,000 persons) among individuals aged < 5 years, it increased from 0.25 to 0.62 cases per 100,000 persons (P < .05) among those aged > or = 5 years. PCV7 introduction has led to reductions in serotype 6A IPD but not serotype 6C IPD in the United States.

Assay for 5′ Noncoding Region Analysis of All Human Rhinovirus Prototype Strains

Increasing recognition of the association of rhinovirus with severe lower respiratory tract illnesses has clarified the need to understand the relationship between specific serotypes of rhinovirus and their clinical consequences. To accomplish this, a specific and sensitive assay to detect and serotype rhinovirus directly from clinical specimens is needed. Traditional methods of serotyping using culture and serum neutralization are time-consuming, limited to certain reference laboratories, and complicated by the existence of over 100 serotypes of human rhinoviruses (HRVs). Accordingly, we have developed a sequence-based assay that targets a 390-bp fragment accounting for approximately two-thirds of the 5' noncoding region (NCR). Our goal was to develop an assay permitting amplification of target sequences directly from clinical specimens and distinction among all 101 prototype strains of rhinoviruses. We determined the sequences of all 101 prototype strains of HRV in this region to enable differentiation of virus genotypes in both viral isolates and clinical specimens. We evaluated this assay in a total of 101 clinical viral isolates and 24 clinical specimens and compared our findings to genotyping results using a different region of the HRV genome (the VP4-VP2 region). Five specimens associated with severe respiratory disease in children did not correlate with any known serotype of rhinovirus and were found to belong to a novel genogroup of rhinovirus, genogroup C. Isolates were also found that corresponded to the genogroup A2 variant identified in New York and Australia and two other novel group A clusters (GAC1 and GAC2).

Serotype 6C is associated with penicillin-susceptible meningeal infections in human immunodeficiency virus (HIV)-infected adults among invasive pneumococcal isolates previously identified as serotype 6A in South Africa

A newly described pneumococcal serotype (6C) is indistinguishable from serotype 6A when using the conventional Quellung serotyping method. Serotype 6A isolates were screened by polymerase chain reaction (PCR) for the wciN region of the capsular locus. This study detected serotype 6C among invasive pneumococcal disease (IPD) isolates from national laboratory-based surveillance (2005–2006) in South Africa. No serotype 6C isolates were identified among 23 serotype 6A cases from children enrolled in a 9-valent pneumococcal conjugate vaccine trial (1998–2005). Of 8167 IPD cases reported nationally, viable isolates were available for serotyping in 87% of cases ( n = 7080). Quellung serotyping identified 608 serotype 6A isolates, of which 606 were further tested for serotype 6C. PCR confirmed serotype 6C in 5% (30/606) of the isolates tested. Serotype 6C isolates were: less likely than 6A to cause disease in children compared with adults (6/30 (20%) vs. 311/550 (57%); P < 0.001); more likely to cause laboratory-confirmed meningitis (15/30 (50%) vs. 167/578 (29%); P = 0.01); and more likely to demonstrate susceptibility to penicillin (non-susceptibility 0/30 vs. 129/578 (22%); P = 0.004). No association with gender, human immunodeficiency virus (HIV) co-infection or case fatality rate was observed. Although serotype 6C prevalence was low, its epidemiology may differ from the other serogroup 6 pneumococci. Our data from the vaccine efficacy trial suggest that cross-protection of the conjugate vaccine is against true serotype 6A strains.

Development of rapid coagulase serotyping method by PCR and microplate hybridization

We developed a novel PCR-based method for coagulase serotyping. Coagulase gene amplicons biotinylated by PCR were identified by microplate hybridization (MPH) using serotype-specific probes. The conventional serotyping method, which is strongly dependent on coagulase activity, may sometimes give a mistaken determination of the serotype, especially in cases where there is high coagulase activity. In contrast, the results of PCR-MPH are not affected by coagulase activity. Furthermore, once the isolated colonies are obtained, it only takes 3 h to perform PCR-MPH, and the interpretation of the results is entirely objective. We compared PCR-MPH with the conventional method for 90 strains of coagulase-producing Staphylococcus aureus. The same results were found for both the PCR-MPH and conventional methods, and thus, our results indicate that PCR-MPH is a rapid, objective, and reliable method for coagulase serotyping.

Use of Phenotypic and Molecular Serotype Identification Methods To Characterize Previously Nonserotypeable Group B Streptococci

Among 1,762 isolates of Streptococcus agalactiae (group B streptococcus [GBS]), 207 (12%) initially nonserotypeable isolates were tested by improved conventional serotyping methods (Lancefield antigen extraction with 0.1 and 0.2 N HCl, latex agglutination assays, and use of antisera against all known serotypes [Ia, Ib, and II to IX]) and a molecular serotype identification system (multiplex PCR-based reverse line blot [mPCR/RLB] assays targeting serotype-specific sites in the region spanning cpsH to cpsM). Serotypes were assigned to 71 (34%) of the 207 isolates by using antisera and to 204 (98.5%) of them by mPCR/RLB. Sequencing of a portion of the cpsE-cpsF-cpsG region of 141 persistently nonserotypeable isolates and 1 with discrepant conventional and molecular serotyping results was attempted. Major mutations were identified in 34 isolates (24%), including 11 (8%) from which no amplicons were obtained and 23 (16%) with sequence variation compared with published sequences; of the latter, 21 (15%) were associated with amino acid changes. By contrast, mutations were identified in only 12 (2.3%) of 516 serotypeable isolates for which this region has been sequenced previously. In summary, an improved serotyping scheme allowed serotype identification of more than one-third of the previously nonserotypeable GBS isolates. Molecular serotypes were assigned to almost all of the isolates by mPCR/RLB. Significant mutations (with no amplicons or with associated amino acid changes) were found in the cpsE-cpsF-cspG region of a higher proportion of nonserotypeable than of serotypeable isolates (32/141 versus 8/516; P < 0.001), but further investigation is needed to determine the genetic basis for most nonserotypeable GBS isolates.

Molecular typing of colonizing Streptococcus agalactiae strains by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) in a Chennai based hospital

Streptococcus agalactiae is reported to be an asymptomatic vaginal colonizer in Indian women, although it is considered one of the major causes of neonatal infections in many European countries. DNA based molecular typing methods are more reliable than the conventional serotyping method for identification and typing of this pathogen. In the present study, we have evaluated genetic diversity among colonizing S. agalactiae strains (n=86) by using a PCR-based genotyping method i.e. Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR). With ERIC-PCR fingerprinting at 60% similarity level in a dendrogram generated by UPGMA cluster analysis, 10 different ERIC groups were identified, which were subdivided into 62 distinct genotypes at ≥ 95% similarity level. Based on these findings, we demonstrate that ERIC-PCR is a simple, rapid, and inexpensive tool with sufficient discriminatory power and is applicable for characterization and genotyping of a large number of clinical isolates of S. agalactiae at molecular level.

Development of a multiplex PCR test for identification of Actinobacillus pleuropneumoniae serovars 1, 7, and 12

A PCR assay for simultaneous species identification and separation of Actinobacillus pleuropneumoniae serovars 1, 7 and 12 was developed. Primers specific for genes involved in biosynthesis of the capsular polysaccharides ( cps genes) of serovars 1, 7, and 12 were combined with a species-specific PCR test based on the omlA gene. The PCR test was evaluated with the serovar reference strains of A. pleuropneumoniae as well as 183 Danish field isolates. For all typable strains, a complete correspondence was found between results obtained with the multiplex PCR test and results from the traditional serotyping methods. Among eight serologically cross-reacting strains designated K1:O7, seven isolates produced amplicons of similar sizes as serovar 1 and one isolate produced amplicons of similar sizes as serovar 7. The species specificity of the assay was evaluated using a collection of 126 strains representing 25 different species within the family Pasteurellaceae including 45 field strains of the phylogenetically affiliated species Actinobacillus lignieresii. All these isolates tested negative for the cps genes by the multiplex PCR test except for 6 isolates of A. lignieresii. Five of these isolates produced an amplicon identical to the cps gene of serovar 7, whereas one isolate produced an amplicon identical to the cps gene of serovar 1. In addition, four isolates of Actinobacillus genomospecies 1 tested positive for the omlA gene but negative for the cps genes. The test represents a convenient and specific method for serotyping A. pleuropneumoniae in diagnostic laboratories.

Isolation and Identification of Ornithobacterium rhinotracheale in Broiler Breeder Flocks of Guilan Province, North of Iran

The aims of the present study were to isolate and serotype, determine the Seroprevalence, Drug susceptibility and diagnosis of infection by Polymerase Chain Reaction (PCR). In this study 460 serum samples and 220 tracheal swabs, 90 ovaries and oviduct swabs, 90 misshapen egg shells swabs were collected from 22 broiler breeder flocks of 5 companies. Serological results showed that all of the 22 flocks (100%) were positive for ORT infection. Ornithobacterium rhinotracheale (ORT) antibodies were detected in 289 (62/83%) out of the 460 serum samples. ORT was detected from tracheal swabs of seven flocks (31/81% or 3/18% out of 220 tracheal swabs). There was significant correlation between flock different ages and ORT titers (p<0.05), but correlation of flock ages and ORT isolates was not significantly different (p>0.05). Seven flocks infected with ORT were detected positive in PCR but bacteria were Isolated from only five culture. No ovaries and oviducts, misshapen egg shell swabs yielded ORT. A 784 bp fragment of the 16S rRNA gene was amplified using ORT specific primers in the PCR. All the isolates were identified as serotype A by Rapid Agglutination Test. Drug sensitivity test using standard disk diffusion technique was performed with 27 antibiotics. Antibiotic susceptibility for Quinolons family was seen more than the others and Cephalosporins family except to Cephalexin. The isolates were 80-100% susceptible to Tetracycline family and the most antibiotic resistant were seen for Aminopenicillins, Polypeptides, Sulfanamides and 80-100% resistant to Aminoglycoside family. Eighty percent of the isolates were resistant to Licomycin and 60% were moderate sensitive to Lincomycin. This study is the first report of prevalence of ORT, bacterial isolation, biochemical characteristics, serotyping and molecular method (PCR) in broiler breeder flocks in Guilan province of Iran.

Serotyping and Population Genetics of Invasive Haemophilus influenzae

One of the objectives of a recent study by Satola et al. ([2][1]) was to use PCR capsule gene typing to evaluate the accuracy of the slide agglutination method for serotyping (SAST) Haemophilus influenzae . Of the 360 invasive H. influenzae isolates tested, 64 (or 17.5%) were found to be