Neospora caninum IgG avidity tests: an interlaboratory comparison

Avidity tests can be used to discriminate between cattle that are acutely and chronically infected with the intracellular parasite Neospora caninum. The aim of this study was to compare the IgG avidity ELISA tests being used in four European laboratories. A coded panel of 200 bovine sera from well documented naturally and experimentally N. caninum infected animals were analysed at the participating laboratories by their respective assay systems and laboratory protocols. Comparing the numeric test results, the concordance correlation coefficients were between 0.479 and 0.776. The laboratories categorize the avidity results into the classes "low" and "high" which are considered indicative of recent and chronic infection, respectively. Three laboratories also use an "intermediate" class. When the categorized data were analysed by Kappa statistics there was moderate to substantial agreements between the laboratories. There was an overall better agreement for dichotomized results than when an intermediate class was also used. Taken together, this first ring test for N. caninum IgG avidity assays showed a moderate agreement between the assays used by the different laboratories to estimate the IgG avidity. Our experience suggests that avidity tests are sometimes less robust than conventional ELISAs. Therefore, it is essential that they are carefully standardised and their performance continuously evaluated.

Anti-Leishmania infantum IgG Antibody Avidity in Visceral Leishmaniasis

IgG avidity tests are used to discriminate acute from chronic infections. There are few reports on the IgG avidity profile of patients with visceral leishmaniasis (VL). This study investigated the anti-Leishmania IgG avidity in patients with classic VL (n = 10), patients showing clinical cure after treatment (n = 18), and asymptomatic subjects with at least one positive Leishmania test (n = 20). All subjects were from areas in Brazil where VL is endemic. Serum samples were collected from each subject on two different occasions. IgG avidity was evaluated by Western blotting. The proportion of high-avidity antibodies was higher in all samples from patients with classic VL. In contrast, low-avidity antibodies predominated in subjects with a history of VL, including 13 cases (72.2%) in the first assessment and 14 (77.8%) in the second. Fifteen (75%) of the asymptomatic subjects presented a predominance of low-avidity antibodies in the first assessment, and the frequency of high-avidity antibodies increased over time in seven subjects (35%) of this group. Antibodies against the 14- and/or 16-kDa antigen fraction were detected in the first assessment in all patients with classic VL, in 10 (55.5%) treated patients, and in 10 (50%) asymptomatic subjects. These were high-avidity antibodies in most cases. In the asymptomatic group, an increase in IgG avidity against the 14- and/or 16-kDa antigen fraction was observed in three cases (15%). The results indicate distinct responses in infected and asymptomatic subjects, probably associated with the length of time after infection. In this respect, IgG avidity tests represent a new approach to better characterize asymptomatic VL.

Toxoplasmose aguda em gestantes de Araraquara-SP: avaliação da aplicabilidade do teste de avidez de IgG anti-toxoplasma

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Human herpesvirus 6 in multiple sclerosis and encephalitis

Human herpesvirus 6 (HHV-6) was identified from patients with HIV and lymphoproliferative diseases in 1986. It is a β-herpesvirus and is divided into two subgroups, variants A and B. HHV-6 variant B is the cause of exanthema subitum, while variant A has not yet definitely proven to cause any disease. HHV-6, especially variant A, is a highly neurotropic virus and has been associated with many diseases of the central nervous system (CNS) such as encephalitis and multiple sclerosis (MS). The present studies were aimed to elucidate the role of HHV-6 and its two variants in neurological infections. Special attention was given to study the possible role of HHV-6 in the pathogenesis of MS. We studied the expression of HHV-6 antigens using immunohistochemistry in brain autopsy samples from patients with MS and controls. HHV-6 antigen was identified in 70% of MS specimens whereas 30% of control specimens expressed HHV-6 antigen. Serum and cerebrospinal fluid (CSF) samples were collected from patients with MS and patients with other neurological diseases (OND) from patients visiting Helsinki University Central Hospital Neurological Outpatient Clinic during the years 2003 and 2004. In addition, we studied 53 children with suspected encephalitis. We developed an immunofluorescence IgG-avidity assay for the detection of primary HHV-6A and HHV-6B infection. For HHV-6B antibodies, no differences were observed between patients with MS and OND. For HHV-6A both seroprevalence and mean titers were significantly higher in MS compared to OND. HHV-6A low-avidity IgG antibodies, suggestive of primary infection, were found in serum of two, three and one patient with definite MS, possible MS and OND, respectively. From pediatric patients with suspected encephalitis, six serum samples (11.3%) contained low-avidity antibodies, indicating a temporal association between HHV-6A infection and onset of encephalitis. Three out of 26 patients with CDMS and four out of 19 patients with CPMS had HHV-6 antibodies in their CSF compared to none of the patients with OND (p=0.06 and p=0.01, respectively). Two patients with CDMS and three patients with CPMS appeared to have specific intrathecal synthesis of HHV-6A antibodies. In addition, oligoclonal bands (OCB) were observed in the CSF of five out of nine MS patients tested, and in two the OCBs reacted specifically with HHV-6 antigen, which is a novel finding. These results indicate HHV-6 specific antibody production in the CNS and suggest that there is a subset of MS patients with an active or chronic HHV-6A infection in the CNS that might be involved in the pathogenesis of MS. Our studies suggest that HHV-6 is an important causative or associated virus in some neurological infections, such as encephalitis and it might contribute to the development of MS, at least in some cases. In conclusion, HHV-6 is a neurotropic virus that should be taken into consideration when studying acute and chronic CNS diseases of unknown origin.

Ocorrência da infecção por Toxoplasma gondii e avaliação da imunização em caprinos do sertão do cabugi, Rio Grande do Norte

Toxoplasmosis is one zoonosis caused by Toxoplasma gondii protozoan. Goats, amongst the production animals, are one of the species most susceptible to this parasite, being one them main involved agents in ovine and goat abortions, determining great economic losses and implications for public health, since the presence it parasite in the products of goat origin, consist in one of the main sources of infection for the man. In this study 244 blood samples in 8 farms situated in 4 cities from the Sertão do Cabugi region, Rio Grande do Norte State, northeast of Brazil and, tested by ELISA assay. The results had shown a prevalence of 47.13% for anti- T. gondii antibodies and a significant association between positivity and variable evaluated as age, locality and property. The IgG avidity assay evaluated in 115 positive samples was carried to discriminate acute and chronic infection. Twelve samples (10.4%) had presented antibodies of low avidity while 103 (89.6%) presented high avidity antibodies; indicating that most of the animals was precocious exposure to the parasite. Significant difference was verified only for the variable sex. We also evaluate the capacity of recombinant adenoviruses codifying SAG1, SAG2, SAG3 and CMV in inducing activation of specific immune response in goat. These 109 animals received 109 pfu of the AdSAG1, AdSAG2, AdSAG3, AdCMV or PBS in vaccine protocol with 3 immunizations. Serum samples of the each animal, before and after mmunization, had been submitted to the ELISA. The results demonstrate that the immunizations had induced the production of IgG antibodies specific against T. gondii proteins

Distribution of antibodies against Neospora caninum, BVDV and BHV-1 among cows in brazilian dairy herds with reproductive disorders.

Neospora caninum, bovine viral diarrhea virus (BVDV) and bovine herpes virus 1 (BHV-1) are worldwide spread pathogens associated with reproductive problems in cattle. The present work aimed to observe the infection pattern of these three pathogens in two dairy herds with distinct reproductive managements from Triângulo Mineiro, Minas Gerais State, Brazil. The herds were not vaccinated against either N. caninum, BVDV or BHV-1. Blood samples were collected and analyzed for presence of specific antibodies, and N. caninum IgG avidity was measured in N. caninum positive samples. In herd 1, 34 out of 174 sampled cows (20%) had antibodies to N. caninum and the seropositivity of BVDV and BHV-1 were 62% and 86%, respectively. Of 69 sampled cows in herd 2. 7 (10%) had antibodies to N. caninum, and 49% and 39% were seropositive to BVDV and BHV-1, respectively. The IgG avidity profiles indicated that N. caninum had been present in both herds for some years and that herd 1 had an ongoing horizontal spread of the parasite. The results indicate that the studied reproductive pathogens were present in the herds and partly may have contributed to their reproductive problems.

Seroepidemiological analysis of toxoplasmosis in college students

Background Toxoplasmosis is a zoonosis caused by an obligate intracellular parasite, Toxoplasma gondii, which affects warm-blooded animals including humans. Its prevalence rates usually vary in different regions of the planet. Methods In this study, an analysis of the seroprevalence of toxoplasmosis among Brazilian students was proposed by means of IgG specific antibodies detection. The presence of anti-Toxoplasma gondiiantibodies by indirect fluorescent antibody test (IFAT) was also evaluated in order to compare it with enzyme-linked immunosorbent assay (ELISA) and to assess the use of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and o-phenylenediamine dihydrochloride chromogens. Results The IFAT method showed a seroprevalence of 22.3%. These results were similar to those obtained by ELISA (24.1%). The seroprevalence was directly estimated from the IgG avidity, which showed that in a sample of 112 students, three of them had acute infection, an incidence of 1.6% in the studied population. Conclusion In this study, the use of different chromogenic substrates in immunoenzymatic ELISA assays did not display different sensitivity in the detection of T. gondii-reagent serum. The extrapolation of results to this population must be carefully considered, since the investigation was conducted on a reduced sample. However, it allows us to emphasize the importance of careful and well prepared studies to identify risk factors for toxoplasmosis, to adopt preventive measures and to offer guidance to at-risk populations about the disease.

Human bocavirus infection diagnosed serologically among children admitted to hospital with community-acquired pneumonia in a tropical region

Human bocavirus (HBoV) is a human virus associated with respiratory disease in children. Limited information is available on acute infection with HBoV among children admitted to hospital with community-acquired pneumonia in tropical regions and the current diagnosis is inadequate. The aims were to diagnose and describe acute HBoV infections among children hospitalized for community-acquired pneumonia. In Salvador, Brazil, 277 children with community-acquired pneumonia were prospectively enrolled. Paired serum samples were tested by IgG, IgM, and IgG-avidity enzyme immunoassays (EIAs) using recombinant HBoV VP2. HBoV DNA was detected in nasopharyngeal aspirates and serum by a quantitative polymerase-chain reaction (PCR). HBoV DNA was detected in nasopharyngeal aspirates of 62/268 (23%) children and 156/273 (57%) were seropositive. Acute primary HBoV infection was reliably diagnosed (bearing at least two acute markers: Positive IgM, a fourfold increase/conversion of IgG, low IgG avidity or viremia) in 21 (8%) of 273 patients, 90% of 20 had HBoV DNA in nasopharyngeal aspirates, 83% with a high DNA load. The median age of infection with HBoV was 16 months, range 5-36.Community-acquired pneumonia was confirmed radiographically in 85% of 20 patients with acute HBoV infection diagnosed serologically. HBoV DNA was found in nasopharyngeal aspirates of 42/246(17%) children without an acute primary HBoV infection and available nasopharyngeal aspirate. Four children with HBoV secondary immune responses were detected, lacking both IgM and viremia. HBoV infection was diagnosed accurately in children aged 5-36 months with community-acquired pneumonia confirmed radiographically. PCR of nasopharyngeal aspirates is not a reliable marker of acute HBoV infection. J. Med. Virol. 84:253-258, 2012. (C) 2011 Wiley Periodicals, Inc.

CASE-CONTROL STUDY OF AN OUTBREAK OF ACUTE TOXOPLASMOSIS IN AN INDUSTRIAL PLANT IN THE STATE OF SÃO PAULO, BRAZIL

Foodborne diseases represent operational risks in industrial restaurants. We described an outbreak of nine clustered cases of acute illness resembling acute toxoplasmosis in an industrial plant with 2300 employees. These patients and another 36 similar asymptomatic employees were diagnosed with anti-T. gondii IgG titer and avidity by ELISA. We excluded 14 patients based on high IgG avidity and chronic toxoplasmosis: 13 from controls and one from acute disease other than T. gondii infection. We also identified another three asymptomatic employees with T.gondii acute infection and also anti-T. gondii IgM positive as remaining acute cases. Case control study was conducted by interview in 11 acute infections and 20 negative controls. The ingestion of green vegetables, but not meat or water, was observed to be associated with the incidence of acute disease. These data reinforce the importance of sanitation control in industrial restaurants and also demonstrate the need for improvement in quality control regarding vegetables at risk for T. gondii oocyst contamination. We emphasized the accurate diagnosis of indexed cases and the detection of asymptomatic infections to determine the extent of the toxoplasmosis outbreak.

Case-control study of an outbreak of acute toxoplasmosis in an industrial plant in the state of São Paulo, Brazil

Foodborne diseases represent operational risks in industrial restaurants. We described an outbreak of nine clustered cases of acute illness resembling acute toxoplasmosis in an industrial plant with 2300 employees. These patients and another 36 similar asymptomatic employees were diagnosed with anti-T. gondii IgG titer and avidity by ELISA. We excluded 14 patients based on high IgG avidity and chronic toxoplasmosis: 13 from controls and one from acute disease other than T. gondii infection. We also identified another three asymptomatic employees with T.gondii acute infection and also anti-T. gondii IgM positive as remaining acute cases. Case control study was conducted by interview in 11 acute infections and 20 negative controls. The ingestion of green vegetables, but not meat or water, was observed to be associated with the incidence of acute disease. These data reinforce the importance of sanitation control in industrial restaurants and also demonstrate the need for improvement in quality control regarding vegetables at risk for T. gondii oocyst contamination. We emphasized the accurate diagnosis of indexed cases and the detection of asymptomatic infections to determine the extent of the toxoplasmosis outbreak.

Ocorrência da infecção por Toxoplasma gondii e avaliação da imunização em caprinos do sertão do cabugi, Rio Grande do Norte

Toxoplasmosis is one zoonosis caused by Toxoplasma gondii protozoan. Goats, amongst the production animals, are one of the species most susceptible to this parasite, being one them main involved agents in ovine and goat abortions, determining great economic losses and implications for public health, since the presence it parasite in the products of goat origin, consist in one of the main sources of infection for the man. In this study 244 blood samples in 8 farms situated in 4 cities from the Sertão do Cabugi region, Rio Grande do Norte State, northeast of Brazil and, tested by ELISA assay. The results had shown a prevalence of 47.13% for anti- T. gondii antibodies and a significant association between positivity and variable evaluated as age, locality and property. The IgG avidity assay evaluated in 115 positive samples was carried to discriminate acute and chronic infection. Twelve samples (10.4%) had presented antibodies of low avidity while 103 (89.6%) presented high avidity antibodies; indicating that most of the animals was precocious exposure to the parasite. Significant difference was verified only for the variable sex. We also evaluate the capacity of recombinant adenoviruses codifying SAG1, SAG2, SAG3 and CMV in inducing activation of specific immune response in goat. These 109 animals received 109 pfu of the AdSAG1, AdSAG2, AdSAG3, AdCMV or PBS in vaccine protocol with 3 immunizations. Serum samples of the each animal, before and after mmunization, had been submitted to the ELISA. The results demonstrate that the immunizations had induced the production of IgG antibodies specific against T. gondii proteins

A serological follow-up of toxocariasis patients after chemotherapy based on the detection of IgG, IgA, and IgE antibodies by enzyme-linked immunosorbent assay.

A serological follow-up study was carried out on 27 children (1–12 years old) with visceral and/or ocular toxocariasis, after treatment with thiabendazole. A total of 159 serum samples were collected in a period ranging from 22–116 months. Enzyme-linked immunosorbent assays (IgG, IgA, and IgE ELISA) were standardized, using excretory–secretory antigens obtained from the second-stage larvae of a Toxocara canis culture. The sensitivity found for the IgG, IgA, and IgE ELISA, as determined in visceral toxocariasis patients, was 100%, 47.8%, and 78.3%, respectively. Approximately 84% of the patients presented single or multiple parasitosis, as diagnosed by stool examination, yet such variables did not appear to affect the anti-Toxocara immune response. Titers of specific IgE antibody showed a significant decrease during the first year after treatment, followed by a decrease in the IgA titers in the second year, and in the IgG titers from the fourth year onwards. Sera from all patients presented high avidity IgG antibodies, indicating that they were in the chronic phase of the disease. Moreover, 1 year after treatment, the level of leukocytes, eosinophils, and anti-A isohemagglutinin in patients decreased significantly. The present data suggest that IgE antibodies plus eosinophil counts are helpful parameters for patient followup after chemotherapy.

Passive transfer of IgG anti-GM1 antibodies impairs peripheral nerve repair.

Anti-GM1 antibodies are present in some patients with autoimmune neurological disorders. These antibodies are most frequently associated with acute immune neuropathy called Guillain-Barré syndrome (GBS). Some clinical studies associate the presence of these antibodies with poor recovery in GBS. The patients with incomplete recovery have failure of nerve repair, particularly axon regeneration. Our previous work indicates that monoclonal antibodies can inhibit axon regeneration by engaging cell surface gangliosides (Lehmann et al., 2007). We asked whether passive transfer of human anti-GM1 antibodies from patients with GBS modulate axon regeneration in an animal model. Human anti-GM1 antibodies were compared with other GM1 ligands, cholera toxin B subunit and a monoclonal anti-GM1 antibody. Our results show that patient derived anti-GM1 antibodies and cholera toxin beta subunit impair axon regeneration/repair after PNS injury in mice. Comparative studies indicated that the antibody/ligand-mediated inhibition of axon regeneration is dependent on antibody/ligand characteristics such as affinity-avidity and fine specificity. These data indicate that circulating immune effectors such as human autoantibodies, which are exogenous to the nervous system, can modulate axon regeneration/nerve repair in autoimmune neurological disorders such as GBS.

Thromboxane A2 acts as tonic immunoregulator by preferential disruption of low-avidity CD4+ T cell-dendritic cell interactions

Interactions between dendritic cells (DCs) and T cells control the decision between activation and tolerance induction. Thromboxane A2 (TXA2) and its receptor TP have been suggested to regulate adaptive immune responses through control of T cell-DC interactions. Here, we show that this control is achieved by selectively reducing expansion of low-avidity CD4(+) T cells. During inflammation, weak tetramer-binding TP-deficient CD4(+) T cells were preferentially expanded compared with TP-proficient CD4(+) T cells. Using intravital imaging of cellular interactions in reactive peripheral lymph nodes (PLNs), we found that TXA2 led to disruption of low- but not high-avidity interactions between DCs and CD4(+) T cells. Lack of TP correlated with higher expression of activation markers on stimulated CD4(+) T cells and with augmented accumulation of follicular helper T cells (TFH), which correlated with increased low-avidity IgG responses. In sum, our data suggest that tonic suppression of weak CD4(+) T cell-DC interactions by TXA2-TP signaling improves the overall quality of adaptive immune responses.

DNA immunization: Induction of higher avidity antibody and effect of route on T cell cytotoxicity

Immunizations of mice with plasmid DNAs encoding ovalbumin (OVA), human Ig, and hen egg lysozyme were compared with doses of soluble protein (without adjuvant) that induced similar IgG responses. The route of immunization influenced the magnitude of the antibody (Ab) response in that intradermal (i.d.) injection elicited higher IgG Ab levels than i.m. injection in both DNA- and protein-immunized mice. Although total IgG levels were similar to soluble protein controls, the avidity of the anti-OVA Abs generated by DNA immunization were 100- and 1,000-fold higher via the i.m. or i.d. route, respectively. However, despite the generation of high-avidity Ab in DNA-immunized mice, germinal centers could not be detected in either DNA- or protein-immunized mice. Examination of the IgG subclass response showed that IgG2a was induced by i.m. DNA immunization, coinciding with elevated interferon γ production, whereas a dominant and elevated IgG1 response, coinciding with detectable interleukin 4 production, was generated after i.d. immunization with DNA or soluble OVA and hen egg lysozyme but not human Ig protein. As expected, cytotoxic T cell (CTL) responses could be detected only after DNA immunization. I.d. immunization produced the strongest CTL responses early (2 weeks) but was similar to i.m. later. Therefore, DNA immunization can differ from protein immunization by its ability to induce rapid CTL responses and higher avidity Ab, both of which are advantageous for vaccination.