Glanders, caused by Burkholderia mallei, is a zoonotic disease of equids. Serologic testing for glanders is required by disease-free countries before international movement of equids. The World Organisation for Animal Health Terrestrial Manual recommends the complement fixation test (CFT) for clearance of individual animals for movement, but the CFT is prone to false-positive results. A colorimetric western blot (WB) assay was developed and validated to resolve false-positive CFT results; however, that assay is relatively time-consuming, and the interpretation is subjective. We present here a procedurally similar chemiluminescent WB assay that performs comparably to the validated colorimetric WB assay and offers noticeable benefits of decreased time-to-result and greater ease of interpretation.

The incidence of coccidioidomycosis continues to increase. The diagnosis frequently relies on non-invasive diagnostic testing with immunodiffusion and complement fixation (CF) testing the current gold standard. A direct comparison of quantitative immunodiffusion and CF for IgG antibodies has not been previously reported. In a comparison of 368 samples, there was close concordance observed (360/368 = 97.8%) (P-value < .001). These tests can be considerably interchangeable in the reference laboratory setting.
There are several diagnostic methodologies available in coccidioidomycosis. Direct comparisons of these methods are limited. Prior studies have not compared quantitative immunodiffusion to complement fixation testing. Our results show these tests are highly concordant.

Brucellosis is an economically devastating animal disease and has public health concern. Serological methods such as Rose Bengal Plate Test (RBPT), Complement Fixation Test (CFT), and Indirect-Enzyme-Linked Immunosorbent Assay (I-ELISA) have been used to detect brucellosis. However, there is limited comparative evaluation studies and lack of molecular confirmation of the causative agents in the study areas. The study was aimed to compare RBPT, I-ELISA, CFT, and confirmation using Polymerase Chain Reaction (PCR). A total of 2317 sera samples were collected from brucellosis-affected areas of Ethiopia with no vaccination history. All sera were subjected to comparative serological assays. Post-cross tabulation, sensitivity, and specificity were determined using Receiver Operating Characteristics (ROC) curve analysis software. PCR was performed on 54 seropositive samples using genus- and species-specific primers.
Among the 2317 sera tested for comparative serological assays, 189 (8.16%) were positive for RBPT, 191 (8.24%) for I-ELISA, and 48 (2.07%) for CFT. Sensitivity to RBPT was 100% (95%) in shoats and 74% (95%) in cattle. Specificity on RBPT was 98.69% (95%), 99.28% (95%), 100% (95%) in sheep, goats, and cattle, respectively. CFT sensitivity was 4 (95%) in sheep, 9.65 (95%) goats, and 72 (95%) cattle. Specificity on CFT was 100% (95%) for sheep, goats, and cattle. A 223bp Brucella genus-specific and 156bp B. abortus species-specific detected. However, B. melitensis not detected.
In this study, I-ELISA was the most sensitive and specific test. RBPT detected all Brucellosis-infected sheep and goats; nevertheless, it showed false positive in sheep and goats and false negative in cattle. The presence of B. abortus in small and large ruminants was confirmed by PCR. This is the first report of B. abortus detection in small ruminant in Ethiopia. B.abortus detected in non-preferred hosts. The findings suggest further study on molecular epidemiology of Brucella species.

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.

Burkholderia mallei is the etiologic agent of glanders. It is difficult to diagnose this zoonotic disease in its early stages. Some methods such as the complement fixation test (CFT) cause some problems for veterinary authorities and financial losses to animal owners due to false-positive results. The mallein test requires appropriate laboratory equipment and skilled personnel. To quickly and accurately diagnose the disease, especially in areas where animals cannot be kept, new methods (such as the Western blot test [WBT]) should be used to identify the disease. This study designed and optimized the Western blot (immunoblot) test using sera from 84 glanderous equids, and the sensitivity and specificity of ELISA and CFT were compared with the WBT. ELISA tests are based on B. mallei antigens whereas a purified lipopolysaccharide-containing B. mallei antigen is used in the WBT. The sensitivity and specificity of the tests were estimated using the cut-off values recommended by the test developers. The WBT and ELISA were significantly more specific than the CFT. The ELISA based on B. mallei antigens was significantly less sensitive than the CFT. Given their comparable sensitivities and specificities, the CFT (95.7%, 98.5%), the WBT (95%, 100%) and the ELISA (85%, 100%) should be further developed. The CFT is still the prescribed technique for serological investigation of equids for trade purposes to certify individual animals without glanders. Therefore, more efforts should be made to further improve and optimize the WBT and ELISA tests.

Primary cold agglutinin disease (CAD) is a monoclonal antibody (M-protein) and complement-mediated chronic hemolytic disease process. Antibody glycosylation can play a role in both antibody half-life and complement fixation. Recently, M-protein light chain (LC) glycosylation has been shown to be associated with AL amyloidosis. We hypothesized that M-protein LC glycosylation is also associated with cold agglutinin (CA) titers and CA-mediated hemolysis.
A cross-sectional study of patients undergoing CA titer evaluation underwent mass spectrometric analysis for M-proteins and M-protein LC glycosylation. A subset of serum samples also underwent evaluation for the ability to trigger cold hemolysis in vitro. M-protein and M-protein LC glycosylation rates were compared across CA titer groups, clinical diagnosis, direct antiglobulin testing (DAT) results, and cold in vitro hemolysis rates.
Both M-protein and M-protein LC glycosylation rates significantly differed across CA titer groups with the highest rates in those with elevated CA titers. M-protein LC glycosylation occurred almost exclusively on IgM kappa M-proteins and was significantly associated with positive DAT results and a clinical diagnosis of CAD. Cold in vitro hemolysis was demonstrated in two patients who both had a CA titer of more than 512 but there was no significant association with CA titer group or M-protein LC glycosylation status.
M-protein LC glycosylation is significantly associated with higher CA titer levels. Given the role that antibody glycosylation can play in antibody half-life and complement fixation, further studies are needed to clarify the effects of LC glycosylation within the context of CAD.

Q fever is one of the commonest infectious diseases worldwide. A Coxiella burnetii prevalence of 97.6% has been found by ELISA and PCR tests of the bulk tank milk in dairy cattle farms of Hungary. The herd- and individual-level seroprevalence rates of C. burnetii in the examined dairy cows and farms have dramatically increased over the past ten years. Three high-producing industrial dairy farms were studied which had previously been found ELISA and PCR positive for C. burnetii by bulk tank milk testing. Coxiella burnetii was detected in 52% of the 321 cows tested by ELISA. Pregnancy loss was detected in 18% of the cows between days 29-35 and days 60-70 of gestation. The study found a higher seropositivity rate (80.5%) in the cows that had lost their pregnancy and a seropositivity of 94.4% in the first-bred cows that had lost their pregnancy at an early stage. The ELISA-positive pregnant and aborted cows were further investigated by the complement fixation test (CFT). In dairy herds an average of 66.6% individual seropositivity was detected by the CFT (Phase II) in previously ELISA-positive animals that had lost their pregnancy and 64.5% in the pregnant animals. A higher (Phase I) seropositivity rate (50.0%) was found in the cows with pregnancy loss than in the pregnant animals (38.5%). The high prevalence of C. burnetii in dairy farms is a major risk factor related to pregnancy loss.

In South Africa, brucellosis testing and record-keeping are done by several laboratories, thus it is difficult to access any organized data to assess the status of the disease. This study evaluated the seropositivity for brucellosis using Rose Bengal test and complement fixation test in suspect cattle, sheep, goats and pigs sera submitted to Bacterial Serology Laboratory, Agricultural Research Council-Onderstepoort Veterinary Research (ARC-OVR) from nine provinces in the country during the period 2007-2015. This retrospective data analysis was conducted to estimate the occurrence of brucellosis in the country from the submitted samples, identify variables that affected seropositivity for brucellosis, investigate existing gaps in data recording and make recommendations on important variables to facilitate better data capture and inferences on brucellosis. Nine years of data were collated and analysed to detect association (seropositivity over time regarding animal species and location). Of the 764,276 animals tested, the distribution of samples was 90.50% (691,539/764,276), 5.19% (39,672/764,276), 3.92% (29,967/764,276) and 0.41% (3,098/764,276) for cattle, sheep, goats and pigs, respectively. The seropositivity for brucellosis by animal species was 6.31% (43,666/691,539, 95% CI: 6.26-6.37), 2.09% (828/39,672, 95% CI: 1.95-2.23), 0.63% (189/29,967, 95% CI: 0.55-0.73) and 0.13% (4/3,098, 95% CI: 0.05-0.33) in cattle, sheep, goats and pigs respectively. The data available did not capture information on the age, sex, breed and other host risk factors that would have been related to seropositivity for brucellosis. The data provide an understanding of the disease occurrence and confirm that brucellosis is enzootic in South Africa. Improved and standardized data collection can be used to pro-actively drive, monitor, change or formulate policies to mitigate the challenges brought about by brucellosis in the livestock sector in South Africa.

Coccidioidomycosis is most frequently diagnosed serologically, and the quantitative test for complement-fixing antibodies is considered prognostically useful. Because complement-fixing antibody testing is complex, labor-intensive, and poorly standardized, an enzyme-linked immunoassay (ELISA) alternative would be attractive. In this report, we restrict the complement-fixing, antibody-binding domain to a 200-amino-acid recombinant peptide of the known antigen. Over-lapping truncations of this peptide do not bind complement-fixing antibodies, suggesting that the responsible epitope(s) are conformational. Further, anchoring the antigenic peptide to the ELISA plate by means of a C-terminal biotin-mimic peptide tag instead of allowing the peptide to randomly adhere to the plastic plate improves sensitivity of antibody detection by 1-2 logs in different sera. The newly developed ELISA shows a significant quantitative correlation with complement-fixing antibody titers. This ELISA shows potential as the basis for a new quantitative assay for coccidioidal antibodies.

Equine trypanosomosis comprises different parasitic diseases caused by protozoa of the subgenus Trypanozoon: Trypanosoma equiperdum (causative agent of dourine), Trypanosoma brucei (nagana) and Trypanosoma evansi (surra). Due to the absence of a vaccine and the lack of efficacy of the few available drugs, these diseases represent a major health and economic problem for international equine trade. Development of affordable, sensitive and specific diagnostic tests is therefore crucial to ensure the control of these diseases. Recently, it has been shown that a small RNA derived from the 7SL gene (7SL-sRNA) is produced in high concentrations in sera of cattle infected with Trypanosoma congolense, Trypanosoma vivax and Trypanosoma brucei. Our objective was to determine whether 7SL-sRNA could serve as a marker of active infection in equids experimentally infected with Trypanosoma equiperdum by analysing the sensitivity, specificity and stability of the 7SL-sRNA. Using a two-step RT-qPCR, we were able to detect the presence of 7SL-sRNA between 2 and 7 days post-infection, whereas seroconversion was detected by complement fixation test between 5 and 14 days post-infection. There was a rapid loss of 7SL-sRNA signal from the blood of infected animals one day post-trypanocide treatment. The 7SL-sRNA RT-qPCR allowed an early detection of a treatment failure revealed by glucocorticoid-induced immunosuppression. In addition, the 7SL-sRNA remains detectable in positive sera after 7 days of storage at either 4°C, room temperature or 30°C, suggesting that there is no need to refrigerate serum samples before analysis. Our findings demonstrate continual detection of 7SL-sRNA over an extended period of experimental infection, with signals detected more than six weeks after inoculation. The detection of a strong and consistent 7SL-sRNA signal even during subpatent parasitemia and the early detection of treatment failure highlight the very promising nature of this new diagnostic method.