Development of Immunochromatographic Assay for the Rapid Detection of Mycoplasma capricolum subsp. capripneumoniae Antibodies

Mycoplasma capricolum subsp. capripneumoniae (Mccp) is the cause of contagious caprine pleuropneumonia (CCPP), which is a highly significant respiratory disease in goats leading to significant economic losses in Africa and Asia. Currently available procedures for the diagnosis of CCPP have some limitations in sensitivity, specificity, operation time, requirement of sophisticated equipment or skilled personnel, and cost. In this study, we developed a rapid, sensitive, and specific colloidal gold-based immunochromatographic assay (GICA) strip for the efficient on-site detection of antibodies against Mccp in the serum within 10 min. For the preparation of this colloidal GICA strip, recombinant P20 protein, the membrane protein of Mccp, was expressed by Escherichia coli prokaryotic expression system after purification was used as the binding antigen in the test. The rabbit anti-goat immunoglobulin G labeled with the colloidal gold was used as the detection probe, whereas the goat anti-rabbit immunoglobulin G was coated on the nitrocellulose membrane as the control line. The concentration of the coating antibody was optimized, and the effectiveness of this colloidal GICA strip was evaluated. Our results proved that the detection limit of the test strip was up to 1:64 dilutions for the Mccp antibody-positive serum samples with no cross-reactivity with other pathogens commonly infecting small ruminants,including goat pox virus, peste des petits ruminants virus, foot-and-mouth disease virus type A, or other mycoplasmas. Moreover, the colloidal GICA strip was more sensitive and specific than the indirect hemagglutination assay for the detection of Mccp antibodies. The 106 clinical serum samples were detected by the colloidal GICA strip compared with the complement fixation test, demonstrating an 87.74% concordance with the complement fixation test. This novel colloidal GICA strip would be an effective tool for the cost-effective and rapid diagnosis of CCPP in the field.

PREVALENCE OF TOXOPLASMOSIS AMONG SHEEP AND GOATS IN BAGHDAD AREA

The complement fixation test (CFT) and the direct fluorescent antibody test were used for detecting anti Toxoplasma gondii antibodies in sera obtained from 143 sheep and 44 goats. Complement fixing antibodies were detected in 38 (26.2%) for sheep sera and 24 (54.5%) of goat sera tested by CFT. On the other hand, 26(18.2%) of sheep sera were positive by the FAT. The combined use of CFT and FAT allows the differentiation between an acute or latent T.gondii infection.

Antibody Targets and Properties for Complement-Fixation Against the Circumsporozoite Protein in Malaria Immunity

The Plasmodium falciparum circumsporozoite protein (CSP) forms the basis of leading subunit malaria vaccine candidates. However, the mechanisms and specific targets of immunity are poorly defined. Recent findings suggest that antibody-mediated complement-fixation and activation play an important role in immunity. Here, we investigated the regions of CSP targeted by functional complement-fixing antibodies and the antibody properties associated with this activity. We quantified IgG, IgM, and functional complement-fixing antibody responses to different regions of CSP among Kenyan adults naturally exposed to malaria (n=102) and using a series of rabbit vaccination studies. Individuals who acquired functional complement-fixing antibodies had higher IgG, IgM and IgG1 and IgG3 to CSP. Acquired complement-fixing antibodies targeted the N-terminal, central-repeat, and C-terminal regions of CSP, and positive responders had greater antibody breadth compared to those who were negative for complement-fixing antibodies (p<0.05). Using rabbit vaccinations as a model, we confirmed that IgG specific to the central-repeat and non-repeat regions of CSP could effectively fix complement. However, vaccination with near full length CSP in rabbits poorly induced antibodies to the N-terminal region compared to naturally-acquired immunity in humans. Poor induction of N-terminal antibodies was also observed in a vaccination study performed in mice. IgG and IgM to all three regions of CSP play a role in mediating complement-fixation, which has important implications for malaria vaccine development.

263. Antibody Spill-Over vs. True Coccidioidal Meningitis in a Patient with HIV/AIDS and Disseminated Coccidioidomycosis

Background The diagnosis of coccidioidal meningitis merits life-long antifungal therapy given high rates of disease recurrence. Accurate diagnosis is important. Antibody spill-over into cerebrospinal fluid (CSF) can happen when serum titers are high. We present a case of antibody spill-over vs. true fluconazole-resistant coccidioidal meningitis. Methods A 49-year-old man presented with 6 months of intermittent fever, myalgias, decreased appetite, vomiting, diarrhea, unsteadiness and 60-pound weight loss. He was recently diagnosed with HIV and a prior lymph node biopsy had grown Coccidioides immitis (C. i) for which he was given fluconazole 100 mg twice daily. Figure 1. Timeline of Coccidioides immitis lab results in relation to treatment regimen. CF: Complement fixation, EIA: Enzyme immunoassay for Coccidioides antigen, CSF: Cerebrospinal fluid, c/mL: copies/mL, R: resistant, S: susceptible. Results Vitals revealed a temperature of 102°F. He was cachectic and a 0.5 cm right supraclavicular lymph node was palpable. No meningeal signs were appreciated. CD4 count was 50/µL (18%), HIV-1 viral load 2,969,945 copies/mL. Computed tomography (CT) of the abdomen/pelvis suggested lung and spleen involvement. Serum C. i enzyme immunoassay (EIA) was 1.38 ng/mL, immunodiffusion (ID) was positive and complement fixation (CF) titer was 1:256. C. i was isolated from expectorated sputum. CSF cell count was normal, but ID was positive and CF titer was 1:2 however, lab reported concern for spill-over due to high serum IgG titers. He left against medical advice with fluconazole 400 mg daily. He was hospitalized a month later for failure to thrive. MRI head revealed enlarged lateral and third ventricles with increased periventricular hyperintensity concerning for coccidioidal meningitis. Repeat serum studies were stable. CSF revealed CF 1:4 and C. i antigen by EIA 1.31ng/mL, distinguishing between spill-over and meningitis. Susceptibility results showed resistance to fluconazole and amphotericin B with minimum inhibitory concentrations (MICs) of 50 and 4 respectively, posaconazole susceptibility (MIC < 1) and itraconazole borderline (MIC 3.7). Despite amphotericin B resistance, it was used for bridge to posaconazole. ART was initiated after concern for immune reconstitution had resolved. Conclusion This case highlights the difficulty in making an accurate diagnosis of coccidioidal meningitis. It also describes a fluconazole-resistant C. i isolate in the setting of prolonged low-dose fluconazole therapy. Disclosures All Authors: No reported disclosures

692. Coccidioides sp. Infective Endocarditis: A Review of the Literature

Background Despite the endemic nature of Coccidioides sp. to the American Southwest, the incidence Coccidioides sp. infective endocarditis (CIE) is rare. Following successful treatment of a patient with CIE at our institution, we reviewed the literature to identify trends in disease presentation, patient characteristics, and outcomes. Methods We reviewed all cases of CIE reported since 1938. Details including patient demographics, underlying immunodeficiency, time to diagnosis, treatment, and outcome were collected for analysis of diagnostic challenges and survival. Results Including ours, we identified 11 published cases of CIE. The majority (7) occurred in men. 5 patients were of either African American or Hispanic descent. Of the 10 patients with reported ages, the median age was 35.5 years (range 3 weeks – 61 years). 5 patients had a previous diagnosis of coccidioidomycosis and only 3 had an immunocompromising condition. These comprised pregnancy, heart transplant, and juvenile inflammatory arthritis. Three cases had multi-valvular involvement, but the majority affected the mitral (5) and the aortic (4) valves. Only 2 of the 11 cases involved a prosthetic valve. Of the 8 cases with reported blood cultures, only 2 were positive. Ten of the 11 cases had extra-cardiac disease. Complement fixation (CF) titers were heterogenous with a median of 1:32 and a range of 1:1 to 1:2048. There was no obvious correlation between a patient’s CF titer and their survival. Average time to diagnosis was 3.5 months (range 2.5 – 36 months). Diagnosis was made post-mortem in 4 of the 11 cases. 6 patients (54%) did not survive. Notably, 2 of the fatal cases preceded the discovery of amphotericin B (1969) and 4 occurred prior to the discovery of fluconazole (1990). Of the five patients that survived, four required surgical intervention in addition to azole therapy. Conclusion CIE is a diagnostic and therapeutic challenge. The diagnosis itself is rare, culture incubation times are long, and the symptoms are often non-specific thus delaying definitive therapy. The introduction of azole therapy appears to have had significant impact on rates of survival. Despite this, successful management of CIE still requires concurrent surgical intervention with aggressive, indefinite anti-fungal therapy. Disclosures All Authors: No reported disclosures

Detection of Antibodies Brucella abortus in dairy cattle in Puspo District, Pasuruan using Rose Bengal Test and Complement Fixation Test

Brucellosis was an infectious disease caused by the genus brucella. Brucellosis in dairy cattle was caused by Brucella abortus that impaction an abortion. The aim of this study was to determine the presence of antibodies of the Brucella abortus in adult female dairy cattle in the Puspo district Pasuruan using Rose Bengal Test (RBT) and Complement Fixation Test (CFT). A descriptive analysis method with quantitative approach was used in this study. Meanwhile, interviews were conducted to get supporting information from the farmer. Used 100 samples. The results of the study showed 2% samples considered positive brucellosis tested by RBT. To confidence false-positive result, RBT test was followed by CFT. After tested by CFT, in this study there were no brucella antibodies in adult female dairy cattle in Puspo district, Pasuruan.

Serological diagnosis of gonorrhea

Alf. Cohn (Derm. Ztschr. Bd. 55, H. 2) gives detailed instructions for the preparation of a specific antigen, patient serum, and also describes in detail the technique of the Bordet-Gengou reaction itself. The author received as a result of his observations with acute gonorrhea in men 40% pos. responses, for chronic 60% and for complicated 80-100%. The results are especially demonstrative in gonorrhoid arthritis (100%). It is important to use the reaction in doubtful cases, when microscopic and bacteriological examinations do not give the necessary answer for making a diagnosis. If people who do not have gonorrhea are vaccinated, the reaction may be positive. If a positive reaction persists for months or years, you need to look for a focus with gonococci. According to the author, the complement fixation reaction cannot replace the previously tested diagnostic methods, but it complements them. Together with bacteriological and clinical research methods, the reaction sometimes helps to understand the diagnosis of difficult cases.

Seroprevalensi Brucellosis pada Sapi Potong di Papua Barat

Brucellosis merupakan penyakit bakterial yang disebabkan oleh Brucella abortus, yang menga-kibatkan kerugian ekonomi akibat terjadinya keguguran pada sapi betina yang bunting pada triwulan ketiga kebuntingan. Surveilans brucellosis dalam mendukung pengembangan peternakan sapi di Propinsi Papua Barat telah dilakukan oleh Balai Besar Veteriner Maros. Tujuan pelaksanaan surveilans ini adalah untuk mengetahui seroprevalensi brucellosis pada sapi potong di Papua Barat. Surveilans dilakukan di 4 kabupaten/kota, 14 kecamatan dan 33 desa. Sebanyak 684 sampel serum sapi telah diambil guna pengujian terhadap brucellosis. Sampling dilakukan pada lokasi kabupaten, kecamatan dan desa terpilih yang padat ternak. Metode pengujian dilakukan secara seri, dengan melakukan screening test menggunakan Rose Bengal Test, yang bila hasilnya positif diuji lebih lanjut dengan Complement Fixation Test. Hasil pengujian menunjukkan adanya reaktor brucellosis pada 5 ekor dari 684 ekor sapi yang diambil dan diuji serumnya. Seroprevalensi brucellosis pada sapi sebesar 0,73% di desa Remu Utara, kecamatan Sorong, kota Sorong sebanyak 1 ekor serta di desa KaliMerah, kecamatan Masni, kabupaten Manokwari. Untuk mencegah penularan dan penyebaran brucellosis pada sapi maka sapi reaktor brucellosis segera dipotong dengan pengawasan dari petugas.

Kondisi Brucellosis setelah Vaksinasi di Kecamatan Majauleng Kabupaten Wajo Sulawesi Selatan

Vaksinasi brucellosis telah dilakukan secara massal di Kabupaten Majauleng, Kabupaten Wajo, Sulawesi Selatan tahun 2013 dan 2014 menggunakan vaksin brucellosis RB51. Vaksinasi dimaksudkan untuk mengendalikan dan menekan angka prevalensi yang tinggi di Kabupaten Wajo yang mencapai angka 30%. Surveilans telah dilakukan oleh Balai Besar Veteriner Maros untuk melihat dan mengevaluasi prevalensi brucellosis yang terjadi beberapa tahun kemudian untuk melihat penurunan prevalensi brucellosis pasca vaksinasi. Pada tahun 2016 telah dilakukan pengujian sampel serum yang berasal dari Kabupaten Wajo. Sebanyak 118 serum telah diuji dengan Rose Bengal Test (RBT) yang diteruskan dengan Complement Fixation Test (CFT). Hasilnya menunjukkan bahwa brucellosis masih ditemukan di Kabupaten Wajo dengan angka prevalensi sebesar 25,42%. Prevalensi terbesar ditemukan di Kecamatan Takalalla yang mencapai 73,68%, sedangkan di Kecamatan Majauleng sebesar 3.13%. Sampling lanjutan dilakukan pada tahun 2020 di kecamatan terpadat populasi sapinya yaitu Kecamatan Majauleng. Prevalensi brucellosis pada tahun 2020 di dua desa di Kecamatan Majauleng meningkat menjadi sebesar 6 %.

Reduced risk of placental parasitemia associated with complement fixation on Plasmodium falciparum by antibodies among pregnant women

Background The pathogenesis of malaria in pregnancy (MiP) involves accumulation of P. falciparum-infected red blood cells (pRBCs) in the placenta, contributing to poor pregnancy outcomes. Parasite accumulation is primarily mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). Magnitude of IgG to pRBCs has been associated with reduced risk of MiP in some studies, but associations have been inconsistent. Further, antibody effector mechanisms are poorly understood, and the role of antibody complement interactions is unknown. Methods Studying a longitudinal cohort of pregnant women (n=302) from a malaria-endemic province in Papua New Guinea (PNG), we measured the ability of antibodies to fix and activate complement using placental binding pRBCs and PfEMP1 recombinant domains. We determined antibody-mediated complement inhibition of pRBC binding to the placental receptor, chondroitin sulfate A (CSA), and associations with protection against placental parasitemia. Results Some women acquired antibodies that effectively promoted complement fixation on placental-binding pRBCs. Complement fixation correlated with IgG1 and IgG3 antibodies, which dominated the response. There was, however, limited evidence for membrane attack complex activity or pRBC lysis or killing. Importantly, a higher magnitude of complement fixing antibodies was prospectively associated with reduced odds of placental infection at delivery. Using genetically modified P. falciparum and recombinant PfEMP1 domains, we found that complement-fixing antibodies primarily targeted a specific variant of PfEMP1 (known as VAR2CSA). Furthermore, complement enhanced the ability of antibodies to inhibit pRBC binding to CSA, which was primarily mediated by complement C1q protein. Conclusions These findings provide new insights into mechanisms mediating immunity to MiP and reveal potential new strategies for developing malaria vaccines that harness antibody-complement interactions.