Seroepidemiology of pertussis in the Japanese population

To ascertain just how widely Bordetella pertussis infection occurs in Japan, we assessed the immune level against pertussis in various age-groups in the Japanese population. Using sera mainly obtained from 6 to 75-year old subjects who visited the Tokyo Metropolitan Komagome Hospital, we determined the levels of bacterial agglutinins to Bordetella pertussis, of anti-filamentous hemagglutinin and of anti-pertussis toxin. The bacterial agglutinin titers in the older age-groups seemed to be slightly lower than those in the younger age-groups, but no obvious differences between the older and the younger age-groups were found in the levels of anti-filamentous hemagglutinin and anti-pertussis toxin. The older subjects were assumed to acquire these antibodies through clinical or subclinical natural pertussis infections because, prior to 1951, mass vaccination against pertussis was not practice in Japan. Our results reasonably indicate that Bordetella pertussis widely infests Japan, so that vaccination is inevitably warranted to prevent pertussis epidemics and to lower the pertussis case rates.     

Maternal immunity provides protection against pertussis in newborn piglets

Pertussis continues to be a significant cause of morbidity and mortality in infants and young children worldwide. Methods to control the disease are based on vaccination with either whole-cell or acellular vaccines or treatment with antibiotics. However, despite worldwide vaccination infants are still at the highest risk for the disease. Here we used our newly developed newborn-piglet model to investigate whether transfer of maternal immunity can protect newborn piglets against infection with Bordetella pertussis. Pregnant sows were vaccinated with heat-inactivated B. pertussis or treated with saline (controls). Newborn piglets were allowed to suckle colostrum and milk for 4 to 5 days before they were challenged with 5 x 10(9) CFU of bacteria intrapulmonarily. Elevated levels of B. pertussis-specific secretory immunoglobulin A (S-IgA) and IgG antibodies were found in the colostrum and serum of vaccinated sows but not in those of control sows. Subsequently, significant levels of specific IgG and S-IgA were detected in the serum and bronchoalveolar lavage fluid of piglets born to vaccinated sows. Following infection with 5 x 10(9) CFU of B. pertussis, clinical symptoms, pathological alterations, and bacterial shedding were significantly reduced in piglets that had received passively transferred immunity. Thus, our results demonstrate that maternal immunization might represent an alternative approach to provide protection against pertussis in young infants.     

Serum Immunoglobulin G Antibody Responses to Bordetella pertussis Lipooligosaccharide and B. parapertussis Lipopolysaccharide in Children with Pertussis and Parapertussis

Serum immunoglobulin G (IgG) antibodies against the lipooligosaccharide (LOS) of Bordetella pertussis and the lipopolysaccharide (LPS) of Bordetella parapertussis were measured by enzyme-linked immunosorbent assay in paired sera from 40 children with pertussis and 14 with parapertussis. Wide differences in the individual responses were noted. Both anti-LOS and -LPS IgG levels increased significantly in the children with pertussis, as did anti-LPS but not anti-LOS in those with parapertussis.     

Western blot analysis of antibody responses of young infants to pertussis infection

Western blot and agglutination techniques were used to analyze the antibody responses toBordetella pertussis in 27 infants less than six month of age with presumed pertussis infection. The antibody response to theBordetella pertussis adhesins filamentous hemagglutinin, pertactin and agglutinogens, and to theBordetella pertussis toxins pertussis toxin and adenylate cyclase-hemolysin were compared. Infection induced intense antibody responses to filamentous hemagglutinin, pertussis toxin and adenylate cyclase-hemolysin. Antibodies to agglutinogens were never detected, and antibodies to pertactin were rarely detected in infected infants' sera. Therefore, determination of anti-agglutinogens levels only is not suitable for the serological diagnosis of pertussis in young infants. Use of purified filamentous hemagglutinin, pertussis toxin and adenylate cyclase-hemolysin in Western blot analysis may improve the serodiagnosis ofBordetella infections. However, care must be exercised in distinguishing between the antibody response in young infants and maternally derived antibodies.     

Triplex real‐time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis

Xu Y, Xu Y, Hou Q, Yang R, Zhang S. Triplex real‐time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis. APMIS 2010; 118: 685–91. A triplex real‐time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis was developed. Three targets were used for amplification in a single tube: the insertion sequence IS481 and the pertussis toxin promoter region (ptxP) for B. pertussis, and the insertion sequence IS1001 for B. parapertussis. The performance of this PCR assay was evaluated in parallel in three single‐target real‐time PCR assays using DNA extracted from B. pertussis and B. parapertussis reference strains and nasopharyngeal swabs taken from 105 patients who had been coughing for more than 7 days. The minimum detection limit of the triplex PCR was one to five colony‐forming units (CFU) of B. pertussis and 1 CFU of B. parapertussis per reaction, and the coefficients of both intra‐ and inter‐assay variation were less than 7%. Results were available within 4 h. Of the 105 nasopharyngeal samples, seven were culture positive and 23 were PCR positive for B. pertussis. All culture‐positive samples were also PCR positive. Our single‐tube triplex real‐time PCR assay proved to be sensitive, specific and suitable for simultaneous detection and discrimination of B. pertussis and B. parapertussis.     

Hydrophobic adherence and phase variation in Bordetella pertussis

The hydrophobicity of Bordetella pertussis was assayed by measuring the ability of cells in suspension to adhere to a polystyrene surface. The quantity of adhered bacteria was measured by the binding of enzyme-conjugated anti B. pertussis antibodies. Hydrophobic adherence of non-pathogenic variant strains was about 20% of that exhibited by pathogenic strains. Hydrophobicity was a stable trait as it did not change with passaging or storage. Assays of a series of characterized stable variants suggested that the Filamentous Hemagglutinin (FHA) is the cell surface moiety responsible for hydrophobic adherence in B. pertussis.     

Protective role of immunoglobulin G antibodies to filamentous hemagglutinin and pertactin ofBordetella pertussis inBordetella parapertussis infection

An outbreak of parapertussis was studied prospectively in 38 first and second grade pupils of an elementary school. Eleven (29%) children were confirmed to be culture positive forBordetella parapertussis. Serum samples were collected from 31 children for assay of antibodies to filamentous hemagglutinin (FHA), pertactin (PRN), and pertussis toxin ofBordetella pertussis. At the first sampling, ten children were found to have a cough and 21 were asymptomatic. Of the latter, 12 remained asymptomatic and eight developed cough within 11 to 53 days (mean ± standard deviation, 31±12 days) after sampling. One child was identified as culture positive forBordetella pertussis and, thus, not included in the analysis ofBordetella parapertussis infection. The mean levels of IgC antibodies to FHA and PRN were significantly higher in the 12 asymptomatic children than in the eight children who later developed cough or in 20 healthy control children of the same age (for FHA, p=0.009 and < 0.001, respectively; for PRN, p=0.002 and 0.002, respectively). These preliminary data suggest thatBordetella parapertussis infection is more prevalent than documented, and that children with high levels of IgG antibodies to FHA and PRN can remain asymptomatic.     

Role of pertussis toxin in causing symptoms ofBordetella parapertussis infection

Whooping cough can be caused by eitherBordetella pertussis orBordetella parapertussis. Although the two species share an almost complete DNA identity,Bordetella parapertussis does not produce pertussis toxin, which is thought to be the main virulence factor ofBordetella pertussis. In order to elucidate the role of pertussis toxin in causing the typical symptoms of whooping cough, clinical information from 33 patients with culture-positiveBordetella parapertussis infection was collected and compared to that from 331 patients with infection caused byBordetella pertussis. Isolated strains ofBordetella parapertussis lacked pertussis toxin expression, as was demonstrated by negative tests for histamine sensitization. This was further substantiated in vivo by a significantly lower leukocyte count in the parapertussis group as compared to the pertussis group. Frequencies of typical symptoms of whooping cough, such as paroxysmal coughing, whooping and vomiting, were almost identical in the two groups. Nocturnal coughing and contact anamnesis were noted more often in theBordetella pertussis group. Children in the parapertussis group were significantly more often vaccinated with whole-cell pertussis vaccine than children infected withBordetella pertussis. The results indicate that pertussis toxin may not play a decisive role in causing the typical symptoms of whooping cough, such as paroxysmal coughing, whooping and vomiting.     

Virulence of a Bordetella pertussis strain expressing a mutant adenylyl cyclase with decreased calmodulin affinity

Bordetella pertussis, the pathogen responsible for whooping cough, produces a toxic calmodulin-sensitive adenylyl cyclase which enters animal cells and increases intracellular cAMP. A point mutant of B. pertussis with abolished adenylyl cyclase catalytic activity was over 1000-fold less pathogenic to newborn mice than wild-type bacteria, demonstrating the importance of the adenylyl cyclase for B. pertussis virulence (Gross et al. ). The B. pertussis adenylyl cyclase is highly sensitive to calmodulin with an apparent K_d for calmodulin of approximately 1 nM. The importance of this high-affinity calmodulin binding for virulence in vivo was examined by the creation of a B. pertussis point mutant (Trp-242 to Glu-242) with 200-fold lower calmodulin affinity than the native enzyme. This mutant B. pertussis strain retained its virulence in a newborn mouse model of pertussis, but the time course for establishment of a lethal infection in vivo was significantly delayed for the mutant strain. These data illustrate that high-affinity calmodulin binding is not obligatory for the activity of this toxin but is important for the rate for establishment of a lethal infection.     

Accumulation of avirulent Bordetella pertussis Bvg mutants in the course of experimental whooping cough in mice

The duration and dynamics of accumulation of insertional Bordetella pertussis Bvg^? mutants were studied in the lungs of laboratory mice after intranasal and intravenous challenge by virulent bacteria of the causative agent of whooping cough. The ability of the virulent B. pertussis bacteria to remain for a long time in the body of mice was established. Using real-time PCR, a hundred genome equivalents of DNA of B. pertussis were detected in lungs of mice 2 months after infection regardless of type of challenge. Using the bacterial test bacteria were identified during only 4 weeks after challenge. Avirulent Bordetella pertussis Bvg^? mutants were accumulated in the period of infection. The percentage of the avirulent bacteria in the B. pertussis population reached 50% in 7–9 weeks after challenge. The obtained results show that laboratory mice can be used to study the dynamics of accumulation of B. pertussis insertion mutants in vivo and confirm the hypothesis that there is accumulation of insertion avirulent Bordetella pertussis Bvg^? mutants during development of pertussis infection in human.     

Correlation of natural autoantibodies and cardiovascular disease‐related anti‐bacterial antibodies in pericardial fluid of cardiac surgery patients

Our previous studies showed that anti‐citrate synthase (anti‐CS) immunoglobulin (Ig)M natural autoantibodies are present in healthy individuals without previous antigen stimulation, but no studies have investigated their presence in the pericardial fluid (PF). Therefore, we detected the natural anti‐CS IgG/M autoantibody levels in plasma and PF of cardiac surgery patients and investigated their relationship with cardiovascular disease‐associated bacterial pathogens. PF and blood samples of 22 coronary artery bypass graft (CABG) and 10 aortic valve replacement (AVR) patients were tested for total Ig levels, natural autoantibodies and infection‐related antibodies using enzyme‐linked immunosorbent assay (ELISA) and Luminex methods. The B cell subsets were measured by flow cytometry. The total Ig subclass levels were four to eight times lower in PF than in plasma, but the natural anti‐CS IgM autoantibodies showed a relative increase in PF. The frequency of CD19⁺ B lymphocytes was significantly lower in PF than in blood (P = 0·01), with a significant relative increase of B1 cells (P = 0·005). Mycoplasma pneumoniae antibody‐positive patients had significantly higher anti‐CS IgM levels. In CABG patients we found a correlation between anti‐CS IgG levels and M. pneumoniae, Chlamydia pneumoniae and Borrelia burgdorferi antibody titres. Our results provide the first evidence that natural autoantibodies are present in the PF, and they show a significant correlation with certain anti‐bacterial antibody titres in a disease‐specific manner.     

Age-Related Differences in Patterns of Increased Bordetella pertussis Antibodies

During the period 2008 to 2010, we identified 11,386 serum samples with increased (positive) levels of antibodies recognizing Bordetella pertussis antigens. We sought to characterize the distribution of positive antibody result patterns in relation to patient age. IgG and IgA antibodies recognizing pertussis toxin (PT) and filamentous hemagglutinin (FHA) were quantified using a multianalyte immunodetection assay. Four mutually exclusive positive result patterns were observed: increased FHA antibodies only, increased PT IgA but not IgG, increased PT IgG but not IgA, and increased PT IgG and IgA. In patients <21 years old, the predominant pattern was increased PT IgG but not IgA, whereas in patients ≥21 years old, it was increased FHA antibodies only. The proportion of positive serum samples exhibiting increased PT IgA but not IgG was <20% in all age categories but showed a stepwise rise with age. The proportions of positive serum samples exhibiting increased PT IgG and IgA were similar (26 to 32%) in the age categories spanning 11 to 60 years of age but lower in the <11- and >60-year-old groups. In 3 of 5 age categories, a significant rise in the proportion of positive serum samples exhibiting increased FHA antibodies only occurred in 2010. Patterns of positive B. pertussis antibody results varied with age. The predominance of increased FHA antibodies only in patients >20 years old suggests that many adults thought to have B. pertussis infections actually have other infections that induce FHA-reactive antibodies. Similarly, the 2010 rise in the frequency of increased FHA antibodies only in some age groups suggests an increase in non-B. pertussis infections.     

Construction of genetically attenuated bacteria <Emphasis Type="Italic">Bordetella pertussis</Emphasis> devoid of dermonecrotic toxin activity that produces modified nontoxic form of pertussis toxin

Recombinant (attenuated) bacteria Bordetella pertussis that contains a knock-out mutation in dnt gene and produces a nontoxic derivative of pertussis toxin have been constructed. Immunological properties of mutant bacteria B. pertussis of KS strain have studied. It is demonstrated that recombinant bacteria B. pertussis of KS strain devoid of activity of dermonecrotic toxin protect the structure of mutant dnt genes upon cultivation on selective nutrient media and long-term exposure in organisms of laboratory animals. The intranasal immunization of mice with live B. pertussis bacteria of the attenuated KS strain protects animals from infection with virulent strain of pertussis pathogen, which can be comparable with an OSO-3 industrial standard sample.     

Seroprevalence of pertussis antitoxin (anti‐PT) in Sweden before and 10 years after the introduction of a universal childhood pertussis vaccination program

Hallander HO, Andersson M, Gustafsson L, Ljungman M, Netterlid E. Seroprevalence of pertussis antitoxin (anti‐PT) in Sweden before and 10 years after the introduction of a universal childhood pertussis vaccination program. APMIS 2009; 117: 912–22. The prevalence of IgG ELISA antibodies against pertussis toxin (anti‐PT) was studied in two Swedish seroepidemiological studies. One was performed in 1997 when the new pertussis vaccination program was 1 year old (n = 3420). In 2007, when Pa vaccines had been used countrywide for 10 years in the universal child vaccination program, this study was repeated to analyze the effect of vaccination on anti‐PT prevalence (n = 2379). Before the statistical analysis of seroprevalence, children vaccinated within the last 2 years before the serosurveys were excluded. The results indicate a reduced exposure to Bordetella pertussis in the population. The proportion of sera without measurable anti‐PT antibodies increased significantly, aggregated over all comparable age groups, from 3.8% in people sampled in 1997 to 16.3% in people sampled in 2007. For cord blood, 1% was without measurable anti‐PT antibodies in 1997 compared to a significantly higher level, 12%, in 2007. With anti‐PT concentrations of ≥50 and ≥100 EU/ml as cutoff points for ‘recent infection’ the proportion above the cutoff points for younger children was significantly higher in 1997 than in 2007 at both cutoff points. For all adults, 20 years of age and older, the difference in proportions above the lower cutoff point was close to statistically significant, comparing 1997 with 2007. This was not the case at 100 EU/ml. In the 1997 samples of children, there was a significant downward trend of ‘recent infections’ at both cutoff points for three sampled age groups between 5 and 15 years of age from 21% at 5.0–5.5 years of age to 7% at 14.7–15.7 years for the lowest cutoff. In the 2007 samples of children, on the contrary, there was a significant continuous upward trend of ‘recent infections’, at both cutoff points, for four sampled age groups between 4 and 18 years of age – from 4% at 4–5 years of age to 16% at 17–18 years at the lowest cutoff. The continuous increase, with age of children with high anti‐PT concentrations, supports the recent change in the general Swedish childhood vaccination program to include a pre‐school booster at 5–6 years and a school‐leaving booster at 14–16 years of age.     

Stability of Antibodies to Bordetella Antigens in German Adults

To estimate the rate of asymptomatic exposure to Bordetella pertussis antigens in the German adult population and to evaluate the stability of antibodies to these antigens, antibody levels against Bordetella antigens and their variability over time were measured in German adult blood donors. One hundred forty-six regular blood donors (41 females, 105 males) were tested repeatedly for antibodies of isotypes IgG and IgA to pertussis toxin, filamentous hemagglutinin (FHA) and pertactin over a period of 2–5 years. Overall, 86% and 56% had IgG or IgA antibodies to pertussis toxin, respectively, 100% and 92% had IgG or IgA antibodies to FHA, respectively, and 83% and 93% had IgG or IgA antibodies to pertactin, respectively. One significant titer increase of both IgG anti-FHA and IgG anti-pertactin, one of IgG anti-FHA, and two of IgA anti-FHA as well as one significant decrease of IgG anti-pertussis toxin were observed during an observation period of 480.5 person-years. Antibody concentrations in men and women were not different. The data show that the level of antibodies to pertussis toxin, FHA, and pertactin remains stable over several years. Furthermore, depending on the definition of serological evidence, the rate of significant increases or decreases suggesting unrecognized exposure to Bordetella antigens was estimated to be between <0.2 and 1.0 per 100 person-years in the population studied. Electronic Publication     

Anti-prothrombin antibodies are associated with adverse pregnancy outcome

Citation Marozio L, Curti A, Botta G, Canuto EM, Salton L, Tavella AM, Benedetto C. Anti‐prothrombin antibodies are associated with adverse pregnancy outcome. Am J Reprod Immunol 2011; 66: 404–409 Problem Women with antiphospholipid antibodies (aPL) such as lupus anticoagulant, anticardiolipin antibodies, and anti‐β₂ glycoprotein‐1 antibodies are at high risk of late pregnancy complications, such as severe pre‐eclampsia, placental insufficiency, and fetal loss. It has been observed that aPL consists of a heterogeneous group of antibodies targeting several phospholipid‐binding plasma proteins, including also anti‐prothrombin (anti‐PT), anti‐protein S (anti‐PS), and anti‐protein C (anti‐PC) antibodies. Their potential role in late pregnancy complications is not known. The aim of this work was to investigate the association between those autoantibodies and histories for adverse pregnancy outcome. Method of study Anti‐PT, anti‐PS, and anti‐PC antibodies were evaluated in 163 patients with previous severe pre‐eclampsia, fetal death, and/or placental abruption and in as many women with previous uneventful pregnancies, negative for aPL. Results The prevalence of anti‐PT antibodies was higher in cases than in controls (OR, 95% CI: 10.92, 4.52–26.38). The highest prevalence was observed in subjects with fetal death. Conclusion Anti‐PT antibodies appear to be associated with adverse pregnancy outcome, irrespectively of aPL.     

Maternal and neonatal anti‐cytomegalovirus IgG level and risk of postnatal cytomegalovirus transmission in preterm infants

Immunological mechanisms influencing the risk of mother‐to‐child cytomegalovirus (CMV) transmission in preterm infants have not been studied sufficiently. In this study, the correlation between maternal and neonatal serum anti‐CMV IgG levels and risk of postnatal CMV transmission in preterm infants was assessed. Anti‐CMV IgG levels of 79 CMV seropositive mothers and their 94 infants were determined in peripheral blood samples collected within 3 days after delivery. Postnatal CMV infection was detected in 39/94 (41%) infants by PCR on urine at term‐equivalent age (gestational age 40 weeks) after congenital infection was excluded. Maternal or infant anti‐CMV IgG levels were not significantly different between infants with and without postnatal CMV infection. The anti‐CMV IgG infant–mother ratio showed a significant positive correlation with gestational age (range 25–32 weeks, R² = 0.218, P < 0.001), reaching 1.0 at 32 weeks of gestation. Anti‐CMV IgG infant–mother ratio was significantly lower in infants with postnatal CMV infection (P = 0.015). In conclusion, the risk of postnatal CMV transmission is related to low gestational age and low anti‐CMV IgG infant–mother ratio. J. Med. Virol. 85:689–695, 2013. © 2013 Wiley Periodicals, Inc.     

The Bordetella pertussis Type III Secretion System Tip Complex Protein Bsp22 Is Not a Protective Antigen and Fails To Elicit Serum Antibody Responses during Infection of Humans and Mice

The type III secretion system (T3SS) of pathogenic bordetellae employs a self-associating tip complex protein Bsp22. This protein is immunogenic during infections by Bordetella bronchiseptica and could be used as a protective antigen to immunize mice against B. bronchiseptica challenge. Since low-passage clinical isolates of the human pathogen Bordetella pertussis produce a highly homologous Bsp22 protein (97% homology), we examined its vaccine and diagnostic potential. No Bsp22-specific antibodies were, however, detected in serum samples from 36 patients with clinically and serologically confirmed whooping cough disease (pertussis syndrome). Moreover, although the induction of Bsp22 secretion by the laboratory-adapted 18323 strain in the course of mice lung infection was observed, the B. pertussis 18323-infected mice did not mount any detectable serum antibody response against Bsp22. Furthermore, immunization with recombinant Bsp22 protein yielded induction of high Bsp22-specific serum antibody titers but did not protect mice against an intranasal challenge with B. pertussis 18323. Unlike for B. bronchiseptica, hence, the Bsp22 protein is nonimmunogenic, and/or the serum antibody response to it is suppressed, during B. pertussis infections of humans and mice.     

Seroprevalence of IgG antibodies against Bordetella pertussis in healthy individuals aged 4–24 years in Turkey

The distribution of IgG antibodies to Bordetella pertussis was investigated in serum samples from 550 subjects, aged 4–24 years, to determine the optimal age for booster immunisation. Levels of antibody to B. pertussis antigens were determined using an ELISA that measures a mixture of pertussis toxin, filamentous haemagglutinin and lipopolysaccharide. Geometric mean titres of anti-pertussis antibodies in subjects aged 4–6 years were significantly lower than those in other age groups, which reflects waning immunity following vaccination. High positive titres in older children and adolescents suggested acquired B. pertussis infection, and booster doses at the ages of 7 and 15 years are therefore suggested.