Significant Increase In IgG Antibodies Research Articles

Department of Microbiology, Collage of Veterinary Medicine, University of Diyal, Iraq

This study was done at the college of Veterinary Medicine, University of Diyala, from September 2021 to April 2022 to assess the seroprevalence and molecular detection of avian influenza virus (AIV) H5N8 in layer chickens. All samples came from 8 commercial flocks of layer chickens. Clinical sings included, severe respiratory infection with enlargement of the head, neck, and prominent edema of the face, followed by excessive lacrimation, sinusitis, severe rales, and cyanosis in all parts of unfeather skin predominantly the wattles comb, and the legs. In this regard, ELISA was used as a serological test and RT-PCR as a molecular detection technique. Accordingly, (364) serum samples from 8 probable AIV (H5N8) infected layer farms were collected and subjected to ELISA test using commercial kit that specific for IgG antibodies to AIV H5N8. The results showed a significant increase of IgG antibodies in such serum samples at days 70 and 200 of age. According to the instruction manual of ELISA kit manufacturer and to mean titers of layers of present study, all farms are infected with AIVH5N8 strain. However, at the age of 200 days, 48 postmortem tissue samples including trachea, lung and liver of affected birds were collected from clinically and sub-clinically infected flocks from all farms. These samples were processed, RNA extracted and submitted to RT-PCR using specific primers for H5N8 strain. The results showed that 32 out of the 48 tissue samples (66.6%) tested positive for H5N8.The resulted Amplicon (320bp) was commercially sequenced and analyzed. The sequencing of the local AIV H5N8 revealed a 99% sequence identity with the reference sequences. The detected strain was registered in GenBank data (NCBI) under acc.number ON247929.1. Phylogenetic tree for locally detected virus in comparison to data from of NCBI was created and showed that the investigated S1(ON247929.1, AIV (A/laying hens/Iraq/(H5N8) segment 4 hemagglutinin (HA) gene, partial cds, local sample is closely related to reference isolates from the NCBI acc. no. of MW961428.1, MW961444.1, MW961476.1, MW961436.,1 and MW961484.1. These strains of the Influenza H5 virus have been deposited from Nigeria.

6-C-Linked trehalose glycolipids signal through Mincle and exhibit potent adjuvant activity

Many studies have investigated the Mincle-mediated agonist activity of α,α′-trehalose-6,6́-glycolipids, however, none have considered how the position, or absence, of the ester moiety influences Mincle-mediated agonist activity. We prepared a variety of 6-C-linked α,α′-trehalose glycolipids containing inverted esters, ketone, carboxy or no carbonyl moieties. Modelling studies indicated that these derivatives bind to the CRD of Mincle in a manner similar to that of the prototypical Mincle agonist, trehalose dibehenate (TDB), with NFAT-GFP reporter cell assays confirming that all compounds, apart from derivatives with short alkyl chains, led to robust Mincle signalling. It was also observed that a carbonyl moiety was needed for good Mincle-mediated signalling. The ability of the compounds to induce mIL-1 β and mIL-6 production by bone marrow-derived macrophages (BMDMs) further demonstrated the agonist activity of the compounds, with the presence of a carbonyl moiety and longer lipid chains augmenting cytokine production. Notably, a C20 inverted ester led to levels of mIL-1β that were significantly greater than those induced by TDB. The same C20 inverted ester also led to a significant increase in hIL-1β and hIL-6 by human monocytes, and exhibited no toxicity, as demonstrated using BMDMs in an in vitro Sytox Green assay. The ability of the inverted ester to enhance antigen-mediated immune responses was then determined. In these studies, the inverted ester was found to augment the OVA-specific Th1/Th7 immune response in vitro, and exhibit adjuvanticity that was better than that of TDB in vivo, as evidenced by a significant increase in IgG antibodies for the inverted ester but not TDB when using OVA as a model antigen.

Serological and Molecular Detection of Local Infectious Bronchitis Virus in Vaccinated Broiler Chickens in Diyala Governorate, Iraq

The infectious bronchitis virus (IBV) is one of the most important Coronaviridae viruses, infecting the upper respiratory tract of chickens and leading to considerable losses in the poultry industry across the globe. Many outbreaks have recently occurred among IBV-vaccinated chicken farms in the Diyala Governorate of Iraq resulting in significant economic losses. As a result, the purpose of the present study was to investigate whether IBV can be a source of infection spread in IBV-vaccinated commercial broiler flock farms in Diyala Governorate. In this regard, ELISA was used as a serological test and RT-PCR as a molecular detection technique. Serum samples were collected from chickens suspected of IBV at 16 and 23 days of age. The results showed a significant increase of IgG antibodies in such serum samples at days 16 and 23 of age indicating the infections of the broilers with IBV. However, at the age of 2-3 weeks, the samples of kidney, liver, trachea, and lungs were collected from clinically and sub-clinically infected flocks, and also postmortem samples were sampled from all farms. Two sets of previously reported primers were created for this purpose in the S1-protein gene region. According to the findings of the present investigation, IBV was found in 83% of samples. Finally, despite immunization with IB4/91, IBV was prevalent in broiler chicken farms in the study area confirmed by serology and molecular biology tests. This finding indicates the possibility of genetic difference between the locally discovered IBV and the administered IBV vaccine. A study on the production of local vaccines can be useful in controlling IBV infection

Nonspecific Regulation of the Number of Immunocompetent Cells Under the Influence of DT Toxoid in Children With Glomerulonephritis.

BackgroundStudies aimed at identifying the mechanisms of the immunoregulatory effect of vaccination with diphtheria and tetanus toxoid on the parameters of adaptive immunity in children with kidney pathology are limited. The study aimed to study the effect of revaccination against diphtheria and tetanus on the proliferation and differentiation of immunocompetent cells, the formation of specific antibodies, and the course of the disease in children with glomerulonephritis (GN).MethodsThe study included 45 children with glomerulonephritis (GN) aged 5 to 15 years, in remission from 6 months up to 4 years. Of these, 25 children were revaccinated with DT toxoid (Diphtheria-Tetanus toxoid with reduced antigenic content) and 20 were in the control group (not vaccinated). The frequency of development of local and systemic reactions and the course of GN were assessed. The subpopulation structure of lymphocytes was studied in dynamics after 1-6-12 months by flow cytometry and IgG levels to diphtheria and tetanus were studied by ELISA.ResultsIn 92% of children with GN, the post-vaccination period was uneventful. 8% showed a rise in temperature up to 37.3°C, without the development of local reactions. During the year, none of the patients had an exacerbation of GN or a concomitant disease. After revaccination with DT toxoid, a significant increase in IgG antibodies against diphtheria and tetanus was revealed, which persisted after 12 months - 7.5 [5.1-10.8] IU/mL (p <0.001) and 7.2 [4.8-10.7] IU/mL (p <0.001), respectively. In the post-vaccination period, a multidirectional change in the concentration of T-lymphocytes was noted: with an initially increased level, their percentage after revaccination with DT toxoid decreases from 83 (81-86) % to 78 (76-80)% after a month (p = 0.04) and up to 75 (69-79)% after 12 months (p<0.001). In the control group, such a decrease was not observed. A similar picture was observed for T-helpers, cytotoxic T-lymphocytes, and in patients with an initially low percentage of cytotoxic T-lymphocytes, on the contrary, its increase was noted (p<0.001), which is comparable with the value of this parameter in the group of children with initially normal value (H = 0.54, p = 0.76). The same patterns were observed in the change in the content of B-cells: one month after revaccination, the relative level of B-cells in patients with an initially lowered value increased (p = 0.02) and remained for 12 months (p<0.001).ConclusionRevaccination with DT toxoid in children with GN not only does not cause undesirable changes in the system of immunocompetent cells but also has an immunomodulatory effect, which contributes to the favorable maintenance of the remission period of the disease.

Диагностика Helicobacter pylori методом иммуноферментного анализа у больных с калькулёзным холециститом

In recentyears, the interest in the significance of Helicobacter pylori in various pathological processes is growing. The most studied is the role of Helicobacter pylori in gastroduodenal pathology. Gallstone disease is one of the most common diseases of the digestive system. Inflammatory changes in the biliary tract aggravate the clinical course of gallstone disease both in the preoperative and in the postoperative period and cause strictures, deformations of the extra hepatic bile ducts in the long term. One of the most threatening complications of gallstone disease is acute cholecystitis. Determination of IgG to Helicobacter pylori in complex diagnosis is important in patients with calculous cholecystitis. We conducted a prospective study of patients (n = 105) aged 30 to 50 years, suffering from different variants of calculous cholecystitis, in the preoperative and postoperative period by enzyme immunoassay. Enzyme-linked immunosorbent assay is reliable, affordable, least invasive method for diagnosing H. pylori infection. In patients with calculous cholecystitis, we marked a significant increase in IgG antibodies to Helicobacter pylori before surgical intervention and a subsequent reduction 10 days after it. Diagnosis of Helicobacter pylori infection of the gallbladder increases therapeutic efficiency and helps to develop preventive measures for complications of gallstone disease.

Detection of Experimental Swine Trichinellosis Using Commercial Elisa Test.

The aim of the study carried out on ten young (10-week old) pigs of the native Polish Large White breed experimentally infected with a low dose of 300 invasive muscle larvae (ML) of Trichinella spiralis was intravital detection of trichinellosis using the E-S ELISA test, determination of a variation level of IgG antibodies against excretory-secretory (E-S) antigens of T. spiralis muscle larvae and finally, describing the intensity of T. spiralis larvae infection in selected muscles. The pig sera were collected at 7 and 9 days prior to the experimental infection with T. spiralis and at 9, 14, 20, 23, 25, 27, 30, 33, 37, 41, 46 days post-infection (d.p.i.). The anti-T. spiralis IgG antibodies were detected by a commercial E-S ELISA test (PrioCHECK Trichinella Ab). Average intensity of the T. spiralis infection in the examined muscles of pigs ranged from 1.52 up to 43.09 larvae/g. The studies revealed that the E-S antigen in the ELISA test did not show cross-reaction with the sera of pigs infected with Oesophagostomum spp. The ELISA assay did not recognize trichinellosis in pigs until 27 days after the T. spiralis infection. The anti-T. spiralis IgG antibodies were first detected on day 30 post-infection. A statistically significant increase of IgG antibodies against T. spiralis ML E-S antigens was first observed between days 27-30 (p<0.01) post-infection, and a further significant rise in the antibody level occurred between days 27 and 33 (p<0.01); 30 and 33 (p<0.01); 33 and 37 (p<0.05) following infection.

Immunogenicity and safety of pneumococcal conjugate polysaccharide and free polysaccharide vaccines alone or combined in HIV-infected adults in Brazil

BackgroundStreptococcus pneumoniae is a leading cause of hospitalization in HIV-infected adults therefore pneumococcal vaccine is recommended. The ideal antipneumococcal vaccine and effective vaccination regimen remain controversial and needs further evaluation. MethodsTo assess the efficacy of pneumococcal vaccines alone and combined, a randomized, blinded clinical trial was conducted in Brazil with 331 HIV-patients aged 18–60, with CD4-T cell count ≥200cells/mm3. Two interventions 60 days apart were done in three schedules: 23-valent pneumococcal polysaccharide vaccine (PPV23)/placebo; 7-valent pneumococcal conjugate vaccine (PCV7)/placebo; and PCV7 plus PPV23. Safety and reactogenicity were evaluated, and immunogenicity was assessed by an IgG enzyme-linked immunosorbent assay to S. pneumoniae serotypes 6B, 9V and 14, performed at baseline, 60 and 180 days after first intervention. Comparison of immunogenicity was based on geometric mean concentration (GMC), percentages of individuals with serotype-specific IgG≥0.35μg/mL and ≥1.0μg/mL and proportion of individuals with ≥4-fold increase in specific antibody concentrations for each serotype. ResultsDemographic and HIV conditions were similar, and both vaccines were well tolerated across vaccine groups. Significant increase in IgG-antibodies was observed to all serotypes evaluated. A greater proportion of PCV7 recipients reached and sustained IgG antibody concentrations at least four times as high as those at baseline, for serotypes 6B and 9V. A PPV23 dose after PCV7 did not enhance immunogenicity. ConclusionsIn this first trial conducted with HIV-infected immunologically stable adults in South America, both PPV23 and PCV7 were safe and immunogenic. Evidence suggesting PCV7 was more immunogenic than PPV23, as it elicited higher and persistent ≥4-fold increase of antibodies for 6B and 9V serotypes in a greater proportion of HIV-patients is noteworthy. Despite current recommendation of schedules combining PCV7 and PPV23, there is little evidence to support this practice and we did not observe benefits in this combination.

Oral and aerosol immunization with viable or inactivated Actinobacillus pleuropneumoniae bacteria: antibody response to capsular polysaccharides in bronchoalveolar lavage fluids (BALF) and sera of pigs.

To investigate the antibody response after local application of lung-pathogenic bacteria, pigs were immunized with viable or inactivated Actinobacillus pleuropneumoniae by the oral and aerogenous route. After 3 weeks class-specific immunoglobulins against purified A. pleuropneumoniae capsular polysaccharides (CP) were determined in serum and BALF by ELISA. A significant increase of IgA antibodies was found in BALF but not in sera of all immunized pigs. Oral immunization with viable A. pleuropneumoniae and aerosol immunization with either viable or inactivated bacteria resulted in a significant increase of IgG antibodies to the CP antigen in BALF, whereas only aerosol exposure to viable bacteria resulted in a significant increase in IgG antibodies in serum. A significant increase in anti-CP IgM in BALF was observed after aerosol exposure but not after oral immunization. IgM antibodies towards CP increased significantly by both routes of immunization with viable bacteria. The anti-CP activity of all three isotypes in sera and BALF was low in all groups compared with the positive controls, although inoculation of viable A. pleuropneumoniae led to higher levels of antibody concentration than inactivated bacteria. Our results indicate a traffic of primed lymphocytes from the gut into the bronchoalveolar airways and further support the hypothesis that polysaccharide-specific B cells may functionally mature at the mucosal surfaces.

Laboratory diagnosis of Mycoplasma pneumoniae respiratory tract infections in children

Mycoplasma pneumoniae is a common respiratory tractpathogen that causes up to 40% of cases of community-acquired pneumonia in children [1]. A specific diagnosis isessential because treatment of M. pneumoniae infection withβ-lactam antibiotics is ineffective. In routine laboratories,serology remains an important diagnostic tool [2, 3].However, it can only provide a retrospective diagnosis andpaired samples are required. Recently developed PCRtechniques show high levels of specificity and sensitivity forthe rapid detection of M. pneumoniae in clinical specimens[4, 5]; however, PCR alone is not always sufficient for adiagnosis [3]. In lieu of a gold-standard diagnostic method,we aimed to evaluate the laboratory methods currently usedto diagnose M. pneumoniae infection in order to find the onemost suitable for rapidly diagnosing the illness, especially inthe early phase of disease.During a 15-month period, a throat swab (viscose swab)andfirstserumspecimenweretakenonadmissionfromatotalof 75 children (42 males, 33 females; mean age 6.2 years;range 4 months–14 years) who were hospitalized fortreatment of a respiratory tract infection (RTI). According tothe guidelines of the British Thoracic Society, 53 of thesechildren had pneumonia, 16 pharyngitis and 6 tracheobron-chitis. A second serum specimen was obtained 9–24 dayslater.ThethroatspecimenswereexaminedforthepresenceofM. pneumoniae using culture in methylene blue–glucosediphasic medium (Oxoid, Basingstoke, UK), a sandwichenzyme-immunoassay for antigen detection (EIA-Ag Virion/Serion, Germany) and a PCR technique for DNA detection(extracted DNA was amplified by the primer pair P1-1 andP1-3 for the P1 adhesin gene [6]). Antibodies against M.pneumoniae were measured using the immunofluorescenceassay (IFA) for IgG and IgM (Vircell, Granada, Spain),enzyme-linked immunosorbent assay (ELISA) for IgG(Platelia BioRad, Marnes-la-Coquette, France) and IgA(Virotech, Russelsheim, Germany), and capture-ELISA forIgM (Platelia BioRad). Toconfirm thespecificityofIgMandIgAantibodydetection,theWesternblottechnique(Virotech)wasadditionallyperformed.Thismethodiscurrentlythemostspecific for detecting anti-M. pneumoniae antibodies [3]. ForIgM, a positive result requires ≥9 bands including P1protein; for IgA, >24 bands are required including P1protein. In the present study a current or recent infectionwas considered to have occurred definitely if at least two ofthe following criteria were met: (1) positive PCR and/orculture, (2) positive IgM antibodies (in the first and/or thesecond sample), (3) seroconversion or significant increase ofIgG antibodies (fourfold increase for IFA, twofold increasefor ELISA), or high IgG titers >40 AU/ml for ELISA.M. pneumoniae was detected by PCR in the throat-swabspecimens of 11 of 75 patients, by culture in three and bythe antigen detection test in only one. All culture-positivesamples were also positive by PCR. The complete resultsobtained using the various methods for the samples of eachof the 75 children studied are shown in Table 1.According to the diagnostic criteria, a definite diagnosisof current or recent M. pneumoniae infection was obtainedfor a total of 12 of the 75 pediatric patients (Table 1; patients1–12; ten with pneumonia and two with pharyngitis). Four

Immunogenicity of synthetic Rhodococcus equi virulence-associated protein peptides in neonate foals

Rhodococcus equi infection is considered the most common cause of pneumonia in foals less than 6 months of age. Immunization of foals and/or mares may become a procedure of choice for prevention. The present work documents the antibody response of neonate foals to R. equi virulence-associated protein (Vap) vaccine candidate peptides. A mixture of 4 R. equi (ATCC 33701) Vap peptides was selected based on their hydrophilicity and recognition by naturally acquired IgG antibodies from 13 adult horses and 33 neonate foals from France and Japan. They were combined with a water-based nanoparticular adjuvant to promote a protective immune response including both Th1 cytokine pattern and antibody response. A single intramuscular injection resulted in an IgG antibody response 30 days later, although inconsistently. In responding animals, no bias in IgG subclass distribution was observed, and antibody response was associated with enhanced serum opsonic activity. In conclusion, data indicate that synthetic Vap peptides combined with nanoparticular adjuvant were immunogenic and resulted in a significant increase in IgG antibodies against the corresponding virulent R. equi strain in a majority of foals.

Humoral response against heat shock proteins and other mycobacterial antigens after intravesical treatment with bacille Calmette-Guérin (BCG) in patients with superficial bladder cancer.

Few studies have analysed the antibody response during intravesical BCG immunotherapy for superficial bladder cancer. We have examined the evolution in serum antibody response against several heat shock proteins (hsp), including the recombinant mycobacterial hsp65 and the native protein P64 from BCG, GroEL from Escherichia coli (hsp60 family), recombinant mycobacterial hsp70 and the E. coli DnaK (hsp70 family), against purified protein derivative of tuberculin (PPD) and the AG85 complex of Mycobacterium bovis BCG, as well as against tetanus toxoid in 42 patients with a superficial bladder tumour, 28 treated with six intravesical BCG instillations and 14 patients used as controls. We also analysed the lymphoproliferative response of peripheral blood mononuclear cells against PPD in this population. Data of antibody responses at 6 weeks post BCG were available in all 28 patients, and at 4 month follow up in 17 patients. All patients who demonstrated a significant increase in IgG antibodies against PPD at 4 months follow up had a significant increase already at 6 weeks of follow up. In contrast, IgG antibodies against hsp increased significantly from 6 weeks to 4 months post-treatment. A significant increase in IgG antibodies against PPD, hsp65, P64, GroEL, and hsp70 at 4 months follow up was observed in 10/17, 8/17, 10/17, 4/17 and 8/17 patients. Native P64 protein elicited a higher antibody response than recombinant mycobacterial hsp65. No increase in antibody response was observed against Dnak from E. coli, against AG85 or tetanus toxoid after BCG therapy. An increase in IgG antibodies against P64 at 4 months follow up compared with pretreatment values was found to be a significant predictor of tumour recurrence (P<0.01). Further studies with a larger number of patients are needed to confirm the value of the antibody response against P64 as a clinical independent prognostic factor.

Analysis of the class-specific immune response to Yersinia enterocolitica virulence-associated antigens in oro-gastrically infected rabbits

The antibody response was analysed in rabbits oro-gastrically infected (i) with virulent (plasmid-carrying) Yersinia enterocolitica 0:3 (v-rabbits) and (ii) with the avirulent (plasmid-cured) derivative (av-rabbits). In an immunoblot assay with whole cell lysate proteins from the infecting virulent Yersinia strain, a significant IgG response was evident in convalescent sera of v-rabbits and of av-rabbits, demonstrating that all rabbits seroconverted. However, v-rabbits mounted a stronger immune response to the cell lysate proteins than av-rabbits and the immune response persisted for a longer time. The post-challenge sera also reacted with whole cell lysate proteins of Escherichia coli and Salmonella typhimurium, indicating cross-reactivity between the different members of Enterobacteriaceae. In contrast, the antibody response against plasmid-encoded released proteins (RPs) appeared specific for infection with virulent strains in that the sera of av-rabbits failed to recognize the plasmid-encoded proteins. Six days following challenge with the virulent Yersinia strain all animals mounted a serum IgM and IgA response to RPs, followed by IgG antibodies on day 9. While the IgM and IgA serum antibody response rapidly decreased (within five-seven and eight-ten months, respectively), IgG antibodies to RPs were still present one year after challenge. Fourteen months after the first infection both the av-rabbits and the v-rabbits were reinfected with the virulent Yersinia strain and the antibody response to RPs was monitored. The v-rabbits only responded with a significant increase of IgG antibodies, indicating that they were primed to the RPs whereas the av-rabbits produced IgM, IgA and IgG specific antibodies like those seen in the primary response of v-rabbits. This study indicates that the rabbit model is helpful and adequate to analyse the character and kinetics of the antibody response during Yersinia infection.

Toxoplasma antibody titers in renal transplant recipients. Pretransplant evaluation and posttransplant follow-up of 73 patients.

In 73 consecutive kidney transplant recipients, anti-Toxoplasma antibodies were determined before transplantation and during a 3-year follow-up after transplantation. In 9 patients, antibody titers increased significantly after transplantation. Antibody titers to various viral antigens determined in parallel remained unchanged, suggesting that the anti-Toxoplasma antibody increase was not due to polyclonal nonspecific stimulation. In 2 of the 24 pretransplant seronegative patients, acquired toxoplasmosis was diagnosed serologically after transplantation, with the observation of a strong IgM and IgG antibody response. The incidence of toxoplasmosis in this group of patients was not found to be significantly different from that in a normal population, suggesting that transmission of Toxoplasma from the transplanted kidney may not be a significant mode of contamination. Among the 49 patients who were seropositive before transplantation, reactivation of toxoplasmosis was suspected in 7 cases on the basis of a significant increase in IgG antibodies. Reactivation occurred more frequently in patients treated with azathioprine and antithymocyte globulin, and a direct relationship between administration of steroids and antibody increase was demonstrated in three patients. Although toxoplasmosis has occasionally been reported as a major infectious problem in kidney transplant recipients, our clinical and serological data show that the potential risk of developing Toxoplasma infection is low since none of the patients with either acquired or reactivated toxoplasmosis developed clinical disease.