Serotype-specific Polymerase Chain Reaction Research Articles

Using a new serotype-specific Polymerase Chain Reaction (PCR) and sequencing to differentiate between field and vaccine-derived African Horse Sickness viruses submitted in 2016/2017

The outer capsid viral protein 2 (VP2) of African horse sickness virus, encoded by the most variable genome segment 2 (Seg-2), is the primary target for AHSV-specific neutralising antibodies and thus determines the virus serotype. Full length segment 2 sequences from more than 100 AHSVs isolated over the last 80 years were compared and single nucleotide polymorphisms (SNPs) identified between the reference strains and recent field viruses. Regions unique to each individual serotype were identified and primers designed to differentially amplify each of the nine serotypes. The sequences of resulting amplicons contained a significant amount of SNPs to discriminate between field viruses and reference strains or live attenuated viruses. The new serotype specific RT-PCR were subsequently used to determine the prevalence of different AHSV serotypes associated with samples submitted to the Agricultural Research Council - Onderstepoort Veterinary Research Institute during the 2016 / 2017 season. Subsequent sequencing of the PCR products were used to determine if the infections were caused by field or vaccine-derived strains. The serotypes of 70 AHSV positive diagnostic samples submitted to the ARC-OVR were determined. Serotypes 2 and 6 were the most prevalent, while Serotype 1 was the only serotype where sequences identical to the ALV or reference strains were detected in field samples. Based on this study, the incidence of vaccine-derived AHS infections submitted from southern Africa were low. This serotype-specific RT-PCR and sequencing assay could assist with the surveillance and control of equines movement nationally and internationally. It could also provide valuable scientific guidance on the policies and guidelines regulating vaccination and trade of equines in South Africa.

Population structure, epidemiology and antibiotic resistance patterns of Streptococcus pneumoniae serotype 5: prior to PCV-13 vaccine introduction in Eastern Gambia.

BackgroundStreptococcus pneumoniae serotype 5 is among the most common serotypes causing invasive pneumococcal disease (IPD) in The Gambia. We anticipate that introduction of the 13-valent pneumococcal conjugate vaccine (PCV-13) into routine vaccination in The Gambia will reduce serotype 5 IPD. However, the emergence of new clones that have altered their genetic repertoire through capsular switching or genetic recombination after vaccination with PCV-13 poses a threat to this public health effort. In order to monitor for potential genetic changes post-PCV-13 vaccination, we established the baseline population structure, epidemiology, and antibiotic resistance patterns of serotype 5 before the introduction of PCV-13.MethodsFifty-five invasive S. pneumoniae serotype 5 isolates were recovered from January 2009 to August 2011 in a population-based study in the Upper River Region of The Gambia. Serotyping was done by latex agglutination and confirmed by serotype-specific Polymerase Chain Reaction (PCR). Genotyping was undertaken using Multilocus Sequence Typing (MLST). Antimicrobial sensitivity was done using disc diffusion. Contingency table analyses were conducted using Pearson’s Chi2 and Fisher’s exact test. Clustering was performed using Bionumerics version 6.5.ResultsMLST resolved S. pneumoniae serotype 5 isolates into 3 sequence types (ST), namely ST 289(6/55), ST 3339(19/55) and ST 3404(30/55). ST 289 was identified as the major clonal complex. ST 3339, the prevalent genotype in 2009 [84.6 % (11/13)], was replaced by ST 3404 [70.4 % (19/27)] in 2010 as the dominant ST. Interestingly, ST 3404 showed lower resistance to tetracycline and oxacillin (P < 0.001), an empirical surrogate to penicillin in The Gambia.ConclusionsThere has been an emergence of ST 3404 in The Gambia prior to the introduction of PCV-13. Our findings provide important background data for future assessment of the impact of PCV-13 into routine immunization in developing countries, such as The Gambia.

Serotype specific polymerase chain reaction identifies a higher prevalence of streptococcus mutans serotype k and e in a random group of children with dental caries from the Southern region of India.

Background:The development of dental caries has been associated with the oral prevalence of Streptococcus mutans. Four serotypes of S. mutans have been reported, namely serotype c, e, f, and k that are classified based on the composition and linkages of cell wall polysaccharides, response to physiological reactions, sero-specificity and 16s rRNA homology. Although the oral prevalence of S. mutans serotype c in Indian subjects with or without caries is known, the prevalence of the other three serotypes, e, f, and k are not known. Hence in this study, we have investigated the occurrence of the e, f, and k serotypes in children with or without caries within the age group of 6-12 years.Materials and Methods:Genomic DNA isolated from whole saliva of caries active (CA) and caries free (CF) groups were first screened for the presence of S. mutans by strain specific polymerase chain reaction (PCR). Those samples that tested positive for the presence of S. mutans were further analyzed by serotype specific PCR to identify the prevalence of the serotypes.Results:Strain specific PCR indicated a higher prevalence of S. mutans in CA group (80%) relative to CF group (43%). Further analysis of the S. mutans positive samples in both groups indicated a higher prevalence of serotype k and e, followed by serotype f in CA group.Conclusion:The present data clearly establishes a novel S. mutans serotype prevalence hierarchy in children from this region, compared with those that have been reported elsewhere. Besides, the data are also clinically significant as the occurrence of serotype k has been associated with infective endocarditis.

A new variant of Actinobacillus pleuropneumoniae serotype 3 lacking the entire apxII operon

Actinobacillus pleuropneumoniae is an important respiratory pathogen causing pleuropneumonia in pig. The species is genetically characterized by the presence of 4 RTX (Repeats in the Structural ToXin) toxin genes: apxI, apxII, and apxIII genes are differentially present in various combinations among the different serotypes, thereby defining pathogenicity; the apxIV gene is present in all serotypes. Polymerase chain reaction (PCR)-based apx gene typing is done in many veterinary diagnostic laboratories, especially reference laboratories. The present report describes the isolation of atypical A. pleuropneumoniae from 4 independent cases from 2 countries. All isolates were beta-nicotinamide adenine dinucleotide (β-NAD) dependent and nonhemolytic but showed strong co-hemolysis with the sphingomyelinase of Staphylococcus aureus on sheep blood agar. Classical biochemical tests as well as Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and sequence-based analysis (16S ribosomal RNA [rRNA] and rpoB genes) identified them as A. pleuropneumoniae. Apx-toxin gene typing using 2 different PCR systems showed the presence of apxIV and only the apxIII operon (apxIIICABD). None of the apxI or apxII genes were present as confirmed by Southern blot analysis. The 16S rRNA and rpoB gene analyses as well as serotype-specific PCR indicate that the isolates are variants of serotype 3. Strains harboring only apxIV and the apxIII operon are possibly emerging types of A. pleuropneumoniae and should therefore be carefully monitored for epidemiological reasons.

Occurrence and characterisation of enterohaemorrhagic Escherichia coli isolates from diarrhoeic calves

The presence of major virulence factors of enterohaemorrhagic Escherichia coli (EHEC; stx1, stx2, eae, Ehly) were determined among isolates from 158 diarrhoeic calves by multiplex polymerase chain reaction (PCR). Strains positive for virulence factors were subjected to serotype specific PCR assays for O157:H7 and O111 antigens. Additionally, serogroups were determined by three monovalent antisera for O26, O111 and O157 somatic antigens and enterohaemolysin production were also shown phenotypically. Thirteen (8.2%) calves carried strains positive for one or more of the virulence factors tested, and eleven (6.9%) calves harboured the shiga toxin-producing strains (stx1 or stx2). stx1 was detected in eight (5%) and stx2 in three (1.9%) calves. eae and Ehly were observed in the same frequency (6.3%) and were detected in parallel. Of the 13 virulence-positive strains, the predominant genotype was (stx1/eae/Ehly) at 53.8%. None of the EHEC in this study belonged to O157:H7 or O111 serotypes, but four strains (30.7%) belonged to the O26 serogroup. The results show the possible role of stx1/eae in calf diarrhoea and the particular importance of O26 EHEC. Calves can also act as a reservoir for EHEC and in the transmission of the disease to humans.

Fièvre catarrhale ovine aux Pays-Bas : aperçu sur trois ans

Since August 2006, the Netherlands has been facing outbreaks of bluetongue (BT) caused by serotype 8 (BTV-8). In this first BT-season about 470 affected holdings were reported in the southern part of the country. It was believed that restrictions to animal movements slowed down the northwards spread of BTV. After a relatively mild winter, BT simultaneously resurfaced in July 2007 at many locations indicating that BTV-8 had survived well. Thousands of affected holdings across the country were reported during that year. After another mild winter, a vaccina­tion campaign for serotype 8 was launched in May 2008, with massive vaccination of sheep, goats and cattle. In 2008, less than 150 outbreaks were reported. The reported BTV-8 cases were in the north-eastern part of the country where the level of natural immunity and the willingness to vaccinate were rela­tively low. This third year with outbreaks was followed by a cold winter. In 2009, no BTV-positive animals were reported from mid-March on. Based on a questionnaire, the willingness of farmers to (re)vaccinate animals tended to decline in 2009, but for cattle farmers this was still at an acceptable level. It is questionable whether farmers will (re)vaccinate again in 2010. Optimism is growing with respect to control, and possibly eradi­cation of BTV-8, but re-emergence after a silent year is a serious possibility. One additional year of vaccination is thus probably required for eradication. In September 2008, because of the enhanced risk of BTV-1 intro­duction, polymerase chain reaction (PCR) positive results were confirmed for BTV-8\net2006 by sequencing the amplicons of the serogroup-specific PCR test and by serotype-specific PCR testing. BTV-6 was found in infected animals in three different herds, irrespective of vaccination. The sequence of genome seg­ment 10 of this virus is genetically close to BTV-2, whereas other segments are all close to those of the modified-live vaccine for serotype 6. This suggests that BTV-6\net2008 is a reassortant of serotypes 2 and 6. Extensive monitoring and additional cases revealed an unusual epidemiology. No other affected animals were detected, but BTV-6 positive animals on a few additional farms were found. The distance between affected holdings was 30-50 kilometres, and there was no epidemiological link between the affected farms. All BTV-6 positive animals were PCR-negative in March 2009, with no new reports of BTV-6 infection. In October 2008, a BTV-1 positive bull was officially reported one month after import. In the same herd, four additional PCR-positive animals were found. Three of these also originated from France and were positive for BTV-8. Detailed investiga­tions revealed a French variant of BTV-8 never found in the Netherlands. We concluded that these animals were infected in France and that BTV-8 appeared to evolve differently in dif­ferent regions in Europe. The fifth animal was positive for BTV-6 and was moved from the BTV-6 affected area. Since no other BTV-positive animals were found in the respective herds and in the one-kilometre radius zone, no spread of BTV-1, BTV-6 and BTV-8 seemed to have occurred in this area. Furthermore, export of Dutch pregnant heifers began again in December 2008, with 2000 to 3000 animals per month. Export is allowed on the basis of vaccination (against serotype 8) before pregnancy and negative PCR testing. Since, no animals were found PCR-positive by the serogroup-specific PCR test, we con­cluded that the Netherlands was free of any BTV circulation in 2009. The situation in 2010 will show whether the Netherlands can be considered as BTV-free again.

A serotype-specific polymerase chain reaction for identification of Pasteurella multocida serotype 1.

A serotype-specific polymerase chain reaction (PCR) assay was developed for detection and identification of Pasteurella multocida serotype 1, the causative agent of avian cholera in wild waterfowl. Arbitrarily primed PCR was used to detect DNA fragments that distinguish serotype 1 from the other 15 serotypes of P. multocida (with the exception of serotype 14). Oligonucleotide primers were constructed from these sequences, and a PCR assay was optimized and evaluated. PCR reactions consistently resulted in amplification products with reference strains 1 and 14 and all other serotype 1 strains tested, with cell numbers as low as 2.3 cells/ml. No amplification products were produced with other P. multocida serotypes or any other bacterial species tested. To compare the sensitivity and further test the specificity of this PCR assay with traditional culturing and serotyping techniques, tissue samples from 84 Pekin ducks inoculated with field strains of P. multocida and 54 wild lesser snow geese collected during an avian cholera outbreak were provided by other investigators working on avian cholera. PCR was as sensitive (58/64) as routine isolation (52/64) in detecting and identifying P. multocida serotype 1 from the livers of inoculated Pekins that became sick or died from avian cholera. No product was amplified from tissues of 20 other Pekin ducks that received serotypes other than type 1 (serotype 3, 12 x 3, or 10) or 12 control birds. Of the 54 snow geese necropsied and tested for P. multocida, our PCR detected and identified the bacteria from 44 compared with 45 by direct isolation. The serotype-specific PCR we developed was much faster and less labor intensive than traditional culturing and serotyping procedures and could result in diagnosis of serotype 1 pasteurellosis within 24 hr of specimen submission.

A multiplex PCR for Massachusetts and Arkansas serotypes of infectious bronchitis virus

Infectious bronchitis virus (IBV), the prototype of the coronavirus family, is an enveloped, single-stranded RNA virus with a genome size of approximately 27·6 kilobase. Infectious bronchitis virus causes an acute, highly contagious respiratory and urogenital disease of chickens which results in significant economic losses in commercial broilers, layers and breeders. A rapid, highly sensitive and specific method is needed in the differential diagnosis of infections of different serotypes. A multiplex polymerase chain reaction (PCR) method was developed and optimized to simultaneously detect Massachusetts (Mass) and Arkansas (Ark) serotypes of IBV. One common primer and two serotype specific primers were chosen from the S1 gene sequences of IBV and used in one PCR reaction. Under optimized PCR conditions, two serotype specific PCR products, 1026 bp for Mass and 896 bp for Ark, respectively, were amplified and detected by agarose gel electrophoreses. The specificity of the technique was verified by using 20 different strains and isolates of IBV, and other avian bacterial and viral pathogens. Using a serial 10-fold dilution of the artificial mixture of both Mass and Ark samples, the detection limit was found to be 5 pg RNA after 35 cycles of PCR. The multiplex PCR was able to detect and differentiate both serotypes in embryonated eggs that were co-infected with different EID50virus titers of Mass 41 and Ark 99. The multiplex PCR developed in this study will be valuable for rapid identification, differential diagnosis, and epidemiological studies of these two serotypes of IBV infections.