Variation of pneumococcal Pilus-1 expression results in vaccine escape during Experimental Otitis Media [EOM].

UNLABELLED: The pneumococcal Pilus-1 enhances attachment to epithelial cells in the respiratory tract and subsequent invasion. Pilus-1 expression is bi-stable and positively regulated by the RlrA transcriptional regulator. To delineate the role of pilus-1 in Experimental Otitis Media (EOM), we evaluated colonization and disease due to a Streptococcus pneumoniae (SP) wild type strain (Taiwan19F-14 wt) and its otherwise isogenic pilus-1 and pilus-2 deficient mutant (Taiwan19F-14 ΔPI-1/PI-2-) as well as potential for a chimeric protein (RrgB321) vaccine candidate for prevention of middle ear (ME) disease. METHODS: Chinchillas were challenged intranasally with either Taiwan19F-14 wt or Taiwan19F-14PI-1/PI-2 deficient mutant. ME status was assessed and direct cultures performed. New cohorts of animals were immunized with RrgB321 or alum. Intranasal challenge with Taiwan19F-14 wt [erythromycin susceptible E(S)] was performed. Subsequently, a second cohort of animals was immunized and challenged with either Taiwan19F-14 wt or a Pilus-1 over-expressing mutant [Taiwan19F-14+pMU1328_Pc-rlrA mutant; E resistant (R)] strain. Pilus-1 expression was analyzed in SP isolated from nasopharynx (NP) and ME fluids by flow cytometry. RESULTS: Culture positive EOM developed following challenge with either wild type SP (Taiwan19F-14) or its pilus-1 deficient mutant. Culture positive EOM developed following challenge with wild type in both RrgB321 immunized and control animals. Pilus-1 expression in ME fluids was significantly higher in controls compared to immunized chinchillas. In second cohort of immunized and control animals challenged with the over-expressing Pilus-1 mutant, delayed development of EOM in the immunized animals was observed. Pneumococci recovered from ME fluid of immunized animals were no longer E(R) signifying the loss of the pMU1328_Pc-rlrA plasmid. CONCLUSION: Pneumococcal pilus-1 was not essential for EOM. Regulation of Pilus-1 expression in ME fluids in the presence of anti RrgB321 antibody was essential for survival of S. pneumoniae. Pneumococci have evolved mechanisms of regulation of non-essential surface proteins to evade host defenses.

Use of the Chinchilla model to evaluate the vaccinogenic potential of the Moraxella catarrhalis filamentous hemagglutinin-like proteins MhaB1 and MhaB2.

Moraxella catarrhalis causes significant health problems, including 15-20% of otitis media cases in children and ~10% of respiratory infections in adults with chronic obstructive pulmonary disease. The lack of an efficacious vaccine, the rapid emergence of antibiotic resistance in clinical isolates, and high carriage rates reported in children are cause for concern. In addition, the effectiveness of conjugate vaccines at reducing the incidence of otitis media caused by Streptococcus pneumoniae and nontypeable Haemophilus influenzae suggest that M. catarrhalis infections may become even more prevalent. Hence, M. catarrhalis is an important and emerging cause of infectious disease for which the development of a vaccine is highly desirable. Studying the pathogenesis of M. catarrhalis and the testing of vaccine candidates have both been hindered by the lack of an animal model that mimics human colonization and infection. To address this, we intranasally infected chinchilla with M. catarrhalis to investigate colonization and examine the efficacy of a protein-based vaccine. The data reveal that infected chinchillas produce antibodies against antigens known to be major targets of the immune response in humans, thus establishing immune parallels between chinchillas and humans during M. catarrhalis infection. Our data also demonstrate that a mutant lacking expression of the adherence proteins MhaB1 and MhaB2 is impaired in its ability to colonize the chinchilla nasopharynx, and that immunization with a polypeptide shared by MhaB1 and MhaB2 elicits antibodies interfering with colonization. These findings underscore the importance of adherence proteins in colonization and emphasize the relevance of the chinchilla model to study M. catarrhalis-host interactions.

Kinetic analysis and evaluation of the mechanisms involved in the resolution of experimental nontypeable Haemophilus influenzae-induced otitis media after transcutaneous immunization.

Transcutaneous immunization (TCI) is a simple and needle-free method with which to induce protective immune responses. Using a chinchilla model of nontypeable Haemophilus influenzae (NTHI)-induced otitis media (OM), we examined the efficacy afforded by TCI with a novel chimeric immunogen called 'chimV4' which targets two critical adhesins expressed by NTHI, outer membrane protein P5 and the majority subunit of NTHI Type IV pilus, PilA. Experimental OM was first established in cohorts of animals, and then TCI performed via a therapeutic immunization regime by rubbing vaccine formulations on hydrated pinnae. The kinetics of resolution of established experimental disease was evaluated by clinically-relevant assessments of OM, bacterial culture of planktonic and adherent NTHI within the middle ear and gross examination of the relative amount of NTHI mucosal biofilms within the middle ear space. Within seven days after primary TCI, a significant reduction in the signs of OM, significantly fewer NTHI adherent to the middle ear mucosa and significant resolution of mucosal biofilms was detected in animals that received chimV4+ the adjuvant LT(R192G-L211A), compared to animals administered LT(R192G-L211A) alone or saline by TCI (p<0.05) with eradication of NTHI within an additional seven days. The mechanism for rapid disease resolution involved efflux of activated dermal dendritic cells from the pinnae after TCI, secretion of factors chemotactic for CD4(+) T-cells, induction of polyfunctional IFNγ- and IL-17-producing CD4(+) T-cells and secretion of host defense peptide within the middle ear. These data support TCI as a therapeutic intervention against experimental NTHI-induced OM and begin to elucidate the host response to immunization by this noninvasive regimen.

Doenças de chinchilas (Chinchilla lanigera) / Diseases of chinchilla (Chinchilla lanigera)

As doenças de chinchilas foram estudadas através da avaliação de laudos de necropsia entre janeiro de 1997 e dezembro de 2011. Em 202 chinchilas necropsiadas, 189 (93,5%) tiveram o diagnóstico determinado, e 13 (6,5%) tiveram diagnóstico inconclusivo, por ausência de lesões ou autólise acentuada. Dentre as 202 chinchilas computadas, 162 eram fêmeas (80%), 37 eram machos (18%), e em quatro chinchilas (2%) o sexo não foi anotado. As chinchilas tinham entre um dia a 12 anos de idade. As doenças foram agrupadas nas seguintes categorias: doenças inflamatórias, doenças causadas por intoxicações, doenças causadas por agentes físicos, doenças metabólicas, doenças parasitárias, doenças degenerativas, distúrbios circulatórios, neoplasmas, distúrbios do desenvolvimento e "outros distúrbios". As doenças inflamatórias foram as mais prevalentes (52 casos [25,7%]) e foram representadas por casos de gastrite (10 casos), listeriose (5 casos), septicemia (5 casos), broncopneumonia bacteriana (4 casos), enterite necrosante (4 casos), piometra (4 casos), diarreia com isolamento de Proteus sp. (3 casos), abscessos subcutâneos e em linfonodos (2 casos), endometrite (2 casos), otite (2 casos), pielonefrite (2 casos), abscesso do ligamento redondo do fígado (1 caso), pneumonia fibrinosa (1 caso), pneumonia intersticial (1 caso), hepatite e colecistite com isolamento de Salmonella sp. (1 caso), histiocitose pulmonar (1 caso), miosite linfo-histiocítica (1 caso) e um caso de dermatofitose (Trichopyton metagrophytes). O segundo grupo de doenças mais prevalentes foram as intoxicações (22,3%), representado por 45 casos de intoxicação por salinomicina. As doenças causadas por agentes físicos (21 casos [10,4%]) incluíam casos de traumas causados por outros animais (8 casos), automutilação após injeção intramuscular (8 casos), prolapso de reto (3 casos) e parto distócico (2 casos). A categoria de doenças metabólicas foi representada por 16 casos (7,9%) de lipidose hepática. As doenças parasitárias (8 casos [4%]) consistiram em infestação por pulga (4 casos), piolho (3 casos) e giardíase (1 caso). Doenças degenerativas (4 casos [2,5%]) incluíam insuficiência renal crônica (2 casos), necrose aleatória de hepatócitos (1 caso) e necrose muscular de origem desconhecida (1 caso). Os distúrbios circulatórios incluíram dois casos (0,99%) de insuficiência cardíaca congestiva. Neoplasmas foram representados por dois casos (0,99%) de adenocarcinoma gástrico. Um caso de atresia ani, associado a ausência do trato reprodutor, intestino grosso e rins policíticos representou a categoria de distúrbios do desenvolvimento (0,5%). Algumas doenças não se enquadraram nas categorias acima e foram enquadradas em "outros distúrbios" (38 casos [18,8%]). Nesta categoria, doenças dentárias foi o distúrbio mais comum, diagnosticado em 9% (18 de 202) de todas as chinchilas examinadas. Seguido por casos de hipertermia (14 casos), dois casos de anemia, dois casos de metaplasia de células adiposas do córtex da adrenal, e dois casos de mucometra.(AU)

Diseases of chinchilla were studied through the review of necropsy reports of 202 postmortem examinations carried out in this species from January 1997 to December 2011. One hundred and eighty nine of these necropsies (93.5%) had a conclusive diagnosis and in 13 (6.5%) a conclusive diagnosis was not reached due either absence of lesions or advanced autolysis. One hundred and sixty two (80%) of the necropsied chinchilla were females and 37 (18%) were males and the sex was not recorded in four cases (2%). Ages of necropsied chinchillas varied from one day to 12-years-old. The encountered diseases were grouped in the following categories: inflammatory diseases, diseases caused by intoxications, diseases caused by physical agents, metabolic diseases, parasitism, degenerative diseases, circulatory disturbances, developmental disorders and "other disorders". Inflammatory diseases were the most prevalent (52 cases [25.7%]) and included gastritis (10 cases), necrotizing enteritis (6 cases), listeriosis (5 cases), septicemia (5 cases), bacterial bronchopneumonia (4 cases), pyometra (4 cases), diarrhea associated with Proteus sp. (3 cases), subcutaneous and lymph node abscesses (2 cases), endometritis (2 cases), otitis (2 cases), pyelonephritis (2 cases), abscesses in hepatic the round ligament (1 cases), fibrinous pneumonia (1 case), interstitial pneumonia (1 case), hepatitis and cholecystitis associated with Salmonella sp. (1 case), pulmonary histiocytosis (1 case), and dermatophytosis by Trichopyton metagrophytes (1 case). The second most prevalent group of diseases was that caused by intoxications (22,3% of the cases) including 45 cases of intoxication by salinomycin. Diseases caused by physical agents (21 cases [10.4%]) included trauma cases caused by other animals (8 cases), self mutilation secondary to intramuscular injection (8 cases), rectal prolapsed (3 cases) and dystocia (2 cases). Metabolic diseases were represented by 16 cases (7.9%) of hepatic lipidosis. Parasitic diseases were represented by 8 cases (4%) flea (4 cases) lice (3 cases) infestations and one case o giardiasis. Degenerative diseases (4 cases or 2,5%) included two cases of chronic renal failure, one case of scattered hepatocellular necrosis and one case of muscle necrosis of unknown origin. Circulatory disturbances included two cases (0.99%) of congestive heart failure. Neoplasms were represented by two cases (0.99%) of gastric adenocarcinoma. Developmental disorders included one (0,5%) case of atresia ani associated with polycystic kidneys and absence of reproductive tract and large intestine. Thirty eight cases (18.8%) did not fit in any of the above categories and were placed as "other disorders". In this category dental disease was the most commonly (8 cases or 9%) diagnosed disorder, followed by 14 cases (6,9%) of hyperthermia, tow cases of anemia, two cases of fat metaplasia of adrenal cortex and two cases of mucometra.(AU)

Infecção sistêmica por Yersinia enterocolitica em chinchilas (Chinchilla laniger) / Systemic infection by Yersinia enterocolitica in chinchillas (Chinchilla laniger)

Yersinia enterocolitica é uma bactéria Gram-negativa que causa infecções em diversas espécies de mamíferos. O agente, geralmente, provoca infecções restritas ao intestino e linfonodos mesentéricos, porém a infecção pode se tornar sistêmica ocasionando lesões em outros órgãos como fígado e baço. Neste trabalho descrevem-se dois surtos de infecções sistêmicas causadas pela Yersinia enterocolitica em criatórios comerciais de chinchilas no Rio Grande do Sul (Brasil). Os proprietários relatavam que os animais acometidos apresentavam apatia, anorexia e morte. Foram encaminhados 13 animais para a realização de necropsia. No exame post mortem dos animais observou-se esplenomegalia, hepatomegalia e áreas multifocais esbranquiçadas no fígado, baço, pulmões, rins e intestino. No exame microscópico visualizou-se infiltrado inflamatório de neutrófilos e macrófagos, necrose, deposição de fibrina e ocasionalmente pode ser observado coco-bacilos no centro das áreas de necrose. No cultivo bacteriológico obteve-se o crescimento de Yersinia enterocolitica nos animais provenientes dos dois criatórios. O agente foi isolado de amostras no fígado, baço, intestino e pulmões dos animais necropsiados, além do cultivo de fezes de animais de uma das propriedades acometidas. A yersiniose, portanto, é uma patologia que deve ser investigada em casos de mortalidade de chinchilas.

Yersinia enterocolitica is a Gram-negative bacterium, which causes infections in several mammalian species. It is often recognized as an agent causing intestinal and mesenteric lymph nodes lesions. However, Yersinia enterocolitica infection may also become systemic, with lesions in others organs such as liver and spleen. This paper describes outbreaks of systemic infection due to Yersinia enterocolitica in two commercial chinchilla breeders in Rio Grande do Sul (Brazil). Owners reported that affected animals showed apathy, anorexia prior to death. Macroscopic examination performed in 13 animals revealed splenomegaly, hepatomegaly and multifocal whitish pinpoint foci in liver, spleen, lung, kidney and intestine. Microscopically, the affected tissues had infiltration of neutrophils and macrophages, as well as fibrin and necrosis with central areas containing cocobacilli bacteria. Yersinia enterocolitica was isolated from liver, spleen, lung and intestine samples from animals of both breeders, and from feces of chinchillas of one of the breeders. Therefore, yersiniosis is a disease to be investigated in cases of mortality of chinchillas.

Chinchilla laniger can be used as an experimental model for Taenia solium taeniasis.

Chinchilla laniger has been reported as an experimental definitive host for Taenia solium; however no information about its suitability and yield of gravid tapeworm proglottids containing viable and infective eggs has been published. In total 55 outbred female chinchillas were infected with 4 cysticerci each; hosts were immunodeppressed with 6 or 8 mg of methyl-prednisolone acetate every 14 days starting the day of infection and their discomfort was followed. Kinetics of coproantigen ELISA or expelled proglottids was used to define the infection status. Efficiency of tapeworm establishment was 21% and of parasite gravidity was 8%; chinchillas showed some degree of suffering along the infection. Viability of eggs obtained from gravid proglottids was tested comparing methods previously published, our results showed 62% viability with propidium iodide, 54% with trypan blue, 34% with neutral red, 30% by oncosphere activation and 7% with bromide 3-(4,5-dimetil-tiazol-2-il)-2,5-difenil-tetrazolio (MTT) reduction; no statistical differences were obtained between most techniques, except activation. Four piglets were infected with 50,000 eggs each, necropsy was performed 3 months later and, after counting the number of cysticerci recovered, the percentage of infection was similar to data obtained with T. solium eggs recovered from humans. Our results demonstrate that the experimental model of T. solium taeniasis in C. laniger is a good alternative for providing eggs and adult tapeworms to be used in different types of experiments; optimization of the model probably depends on the use of inbred hosts and on the reduction of infected animals' suffering.

Immunization with recombinant Streptococcus pneumoniae neuraminidase NanA protects chinchillas against nasopharyngeal colonization.

Immunization with recombinant S. pneumoniae neuraminidase NanA (rNanA) resulted in a significant reduction in pneumococcal colonization in the chinchilla model. The bacteria were eliminated from the nasopharynx 1 week earlier than that from the control cohort. Our data suggest that rNanA affords protection against pneumococcal nasopharyngeal colonization.

Immunization with native or recombinant Streptococcus pneumoniae neuraminidase affords protection in the chinchilla otitis media model.

Streptococcus pneumoniae neuraminidase has been implicated as a virulence factor in the pathogenesis of pneumococcal otitis media. In this study, native neuraminidase was partially purified from cultures of S. pneumoniae by serial chromatography with DEAE-Sepharose and Sephacryl S-200. Recombinant neuraminidase, a 3,038-bp fragment of the neuraminidase A (nanA) gene, was cloned into the pET-28b vector and then expressed at high levels in Escherichia coli. Chinchillas were immunized subcutaneously with either the gel-purified native or recombinant neuraminidase, and all responded with elevated titers of antineuraminidase antibody in serum. Immunization with neuraminidase resulted in a significant reduction in nasopharyngeal colonization as well as in the incidence of otitis media with effusion. These data demonstrate for the first time that neuraminidase affords protection against S. pneumoniae nasopharyngeal colonization and experimental otitis media.

[Sensitization to chinchillas on exposure in households] . / Sensibilisierung gegenüber Chinchilla bei Exposition im Haushalt.

The contact to chinchilla in households may lead to sensibilization and allergic rhinitis in children and adults. In nasal provocation tests on 6 sensibilized patients suffering on perennial rhinitis and/or asthma one child and three adults showed positive reactions with chinchilla hair extract. Allergen avoidance is recommended.

Properties of recombinant HtrA: an otitis media vaccine candidate antigen from non-typeable Haemophilus influenzae.

Non-encapsulated or non-typable Haemophilus influenzae (NTHi) is a major cause of middle ear infections in young children. HtrA has been identified as a vaccine candidate antigen from NTHi; therefore physicochemical characterization of this antigen is important for vaccine development. Recombinant NTHi HtrA has been expressed in E. coli and shown to have serine protease activity. Several mutant, recombinant HtrA proteins were expressed and purified to obtain suitable vaccine antigens lacking protease activity. Two mutants with alterations at the putative active site His91 and Ser197, designated H91A and S197A were examined by circular dichroic spectropolarimetry (CD) to evaluate secondary structure. The S197A mutant had a more random secondary structure compared to wild-type rHtrA or H91A. It is likely that improper folding of S197A accounts for its lack of immunoprotective properties in a chinchilla model of otitis media.

Production of sequence specific polyclonal antibodies to human parathyroid hormone 1-37 by immunization with multiple antigenic peptides.

To generate site-specific antibodies to the N-terminal bioactive fragment of the parathyroid hormone hPTH 1-37, multiple antigenic peptide systems (MAP) for immunization were used. Two 10 residue fragments and a 14 residue fragment derived from knowledge of the secondary structure of hPTH 1-37 were selected to be synthesized as MAPs. Each peptide (hPTH 1-10, hPTH 9-18, and hPTH 24-37) was synthesized directly onto a branching heptalysine core matrix by automated solid phase synthesis. The hPTH 1-10 and the hPTH 24-37 MAP were highly immunogenic in rabbits. Ten polyclonal antisera obtained from rabbits were characterized by epitope mapping. Antigenic determinants were found as follows: 1) Sera K1-K3 raised to MAP 1-10 showed a predominant binding sequence at hPTH 1-5. 2) Sera K4-K6 raised to MAP 8-18 preferentially bound to residues 9-14. 3) Immunizing with hPTH 24-37 MAP led to antisera characterized as follows: serum K7 recognized residues 24-37, the sequence used for immunization, sera K8, K9 and K10 bound to residues 24-37 and 26-34. In summary, the favoured regions as deduced from the secondary structure of hPTH 1-37 were covered by the produced antibodies.

Isotype-specific rabbit antibodies against chinchilla immunoglobulins G, M, and A.

Chinchillas have become a preferred animal model for studying otitis media, and are also useful in studying insulin release, gastrin physiology, intestinal infection, and hepatocellular pathophysiology. Immunopathologic studies in the model, however, have been limited by absence of specific antibody reagents against chinchilla immunoglobulins. We describe a method for preparing isotype-specific rabbit antibodies against the heavy-chain components of chinchilla immunoglobulins G, M, and A. Chromatographic techniques were used to isolate chinchilla immunoglobulins from serum and breast milk; heavy-chain fractions were isolated and used as antigens to produce isotype-specific antibodies in New Zealand White rabbits. Enzyme-linked immunosorbent assay of these antisera disclosed anti-light chain cross-reactivity, which was removed by affinity chromatography. The isolation and affinity purification techniques were highly reproducible. The availability of these reagents should greatly enhance the utility of the chinchilla in modeling human disease.

The relationship between antigen levels and middle ear inflammation in antigen-induced otitis media in the chinchilla.

The relationship between antigen levels in middle ear effusions (MEE) and the degree of middle ear inflammation was studied in an antigen-induced otitis media model, using chinchillas sensitized with human serum albumin (HSA). The degree of middle ear inflammation was evaluated by both tympanometric analysis, and cytological and biochemical analyses of the MEE. Middle ear inflammation develops after HSA challenge with a remarkable decrease in HSA levels in the MEE. This inflammation persists even when HSA levels are no longer detectable in the MEE. These findings show that local challenges with an antigen induce a certain degree of middle ear inflammation, which continues even after complete elimination of the antigen from the middle ear through an immunological defense mechanism.

Biochemical and cytological studies of immune-complex-induced otitis media in the chinchilla.

The pathogenesis of immune complex (IC)-induced otitis media in the chinchilla was studied through cytological and biochemical analyses of middle ear fluid (MEF) recovered after instillation of premade IC. The number to total leukocytes was 3.03 +/- 2.13 X 10(6)/cm3, and mainly involved neutrophils (72.3%) and macrophages (22.7%). The mean value of total protein, alpha 1-antitrypsin (alpha 1-AT) and alpha 2-macroglobulin (alpha 2-M) was 27.1 mg/ml, 189.5 and 75.2 mg/dl. The number of leukocytes had a significant correlation with the levels of total protein, alpha 1-AT and alpha 2-M (P less than 0.01). The inflammatory reaction induced by premade IC is characterized by an increased vascular leakage and an infiltration of leukocytes into the locus. The percentage of macrophages in the total leukocytes was larger in IC-induced otitis media than that in antigen-induced otitis media. These findings suggest that cellular events in the early stage of IC-induced otitis media may be different from antigen-induced otitis media.

Humoral immune response in chinchillas to the capsular polysaccharides of Streptococcus pneumoniae.

Vaccines made from the capsular polysaccharides of Streptococcus pneumoniae have been shown to reduce the incidence of pneumococcal disease in certain populations and have recently been evaluated for their ability to elicit protection against experimental pneumococcal otitis media in a chinchilla model. In this study, chinchillas were vaccinated with a dodecavalent preparation of pneumococcal capsular polysaccharides (PCP) to obtain more information on the immunogenicity of these polysaccharide antigens. All 12 PCP types elicited an antibody response, but the optimum PCP dose and the kinetics of the antibody response varied among types. Immunological paralysis was demonstrated with an immunogenic dose of PCP after primary immunization with a large PCP dose (25 micrograms or more). Pertussis vaccine acted as neither an immunoadjuvant nor an immunosuppressant in the serum antibody response to type 7F PCP in chinchillas.

Immunologic study on the inner ear. Immunoglobulins in perilymph.

The immunoglobulin composition of perilymph (PL) was measured using electroimmunodiffusion, and transfer of serum antibodies to PL was studied using a passive hemagglutination test in chinchillas and guinea pigs. The mean values of IgG an albumin in PL were two to four times greater than those in CSF. In guinea pigs, IgA was found in 93% of the PL and 32% of the CSF samples, but in chinchillas only trace amounts of IgA were found in 50% of the PL and 15% of the CSF samples. No IgM was detected in PL or CSF of either species. This study suggests that a greater portion of the immunoglobulins in PL probably is derived from perilymphatic blood vessels as a filtrate and that the perilymphatic inner ear immune system is independent from that of the CSF.