Comparison of coccidioidal complement fixation and quantitative immunodiffusion serology at a reference laboratory.

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.

Comparative evaluation of RBPT, I-ELISA, and CFT for the diagnosis of brucellosis and PCR detection of Brucella species from Ethiopian sheep, goats, and cattle sera.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Serological screening for Coxiella burnetii in the context of early pregnancy loss in dairy cows.

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.

Seroprevalence and risk factors for brucellosis in cattle in selected districts of Jimma zone, Ethiopia.

A cross-sectional study was carried out in Jimma town and Chora Botor district of Jimma zone from February 2014 to May 2014 to determine seroprevalence and risk factors of brucellosis in cattle. A total of 348 blood samples (174 each from zebu and crossbreed) were collected. The sera were separated and screened by Rose Bengal plate test (RBPT), and positive sera were retested by complement fixation test (CFT) for confirmation. The overall seroprevalence of bovine brucellosis was 1.4 and 0.3 % as tested by RBPT and CFT, respectively. The seroprevalence of bovine brucellosis in indigenous and crossbreed cattle was 1.1 and 0.6 % and 1.7 and 0 % using RBPT and CFT, respectively. Retained fetal membrane was the only risk factor found to be significantly associated with seropositivity of brucellosis in this study (p = 0.019). The overall seroprevalence of brucellosis was very low. However, due to the zoonotic and economic importance of the disease, prevention and control measures are required to stop further spread of the disease. To effectively implement this, the One Health (OH) is the most constructive approach we recommend.

Retrospective analysis of the prevalence of Brucella antibodies in sheep in the Karas Region of Namibia.

In this retrospective study, complete Brucella serology data from the annual national brucellosis testing program and disease investigation for the years 2008-2010 was collated and analyzed to estimate the prevalence of brucellosis in sheep in the Karas Region of Namibia. A total of 22,994 serological results from 762 flocks screened using the Rose Bengal test (RBT) and confirmed using the complement fixation test (CFT) were analyzed. An overall prevalence of 0.14% was recorded over the 3 years. Yearly prevalence was 0.19% (2008), 0.05% (2009), and 0.18% (2010). At district level, brucellosis prevalence was estimated to be between 0 and 0.49%. On positive farms (n = 32), prevalence was between 2.25 and 30%. True prevalence was zero at district level and in all the three study years. We concluded that the prevalence of Brucella antibodies in sheep was low taking into account that some farmers may have vaccinated against the disease. The low prevalence confirms the effectiveness of existing brucellosis control measures implemented by the official veterinary services.

Brucellosis in nomadic pastoralists and their goats in two provinces of the eastern Algerian high plateaus.

A 31-months study was conducted to elucidate the prevalence of brucellosis in nomadic pastoralists and their goats in two provinces of the eastern Algerian high plateaus. Five hundred eight human and 4955 animal sera were screened with the Rose Bengal plate test and the complement fixation test for confirmation. Uterine fluids from aborting goats were subjected to microbiological analyses to determine the biovars responsible for abortions. The overall seroprevalence was 0.98% among animals and 15.84% among herds. A significant correlation was recorded between occurrence of brucellosis and herd size (r = 0.4046, P < 0.0001) as well as age (χ(2) = 5.809, P = 0.0159) and sex of animals (χ(2) = 20.09, P < 0.0001); 89.65% of human cases were related to positive herds and the infection rate was higher in men (7.6%) than in women (6%) and children (0.92%). Brucella melitensis biovar 3 was the only aetiology of brucellosis-associated abortion in goats of the studied region.

Effect of incubation temperature on the diagnostic sensitivity of the glanders complement fixation test.

The complement fixation test (CFT) is the only serological test prescribed by the World Organisation for Animal Health (OIE) for the diagnosis of glanders in international trading of equids. However, false-positive reactions have caused financial losses to the animal owners in the past, and false-negative tests have resulted in the introduction of glanders into healthy equine populations in previously glanders-free areas. Both warm (incubation at 37°C for 1 h) and cold (overnight incubation at 4°C) procedures are recommended by the OIE for serodiagnosis of glanders. In a comparison of the sensitivity and specificity of the two techniques, using the United States Department of Agriculture antigen, warm CFT was found to be significantly less sensitive (56.8%; p < 0.0005) than the cold CFT (83.6%). Cold CFT thus increases the detection rate of glanders but a lower diagnostic specificity has to be accepted. The immunoblot was used as the gold standard.

Seroprevalence investigation of bovine brucellosis in Macenta and Yomou, Guinea.

Brucellosis is one of the world's major zoonotic diseases associated with reproductive disorders and a potential infection of human. Brucellosis leads to serious economic losses due to late-term abortion, stillbirth, weak calves, and sterility. In Guinea, the data on brucellosis was only detected as far back as 10 years ago. The purpose of this study was to estimate the prevalence of bovine brucellosis in the provinces of Macenta and Yomou of Guinea. A structured questionnaire was used in the clinical study, and 345 cattle were clinically examined. Three hundred serum samples were initially subjected to the Rose Bengal test (RBT); the positive results of which were confirmed by the complement fixation test (CFT). The investigation indicated that farmers had little information on brucellosis. Hygroma, abortion, sterility, and placental retention were the observed symptoms. Of the 29 RBT-positive samples, 26 were confirmed by CFT. The prevalence of brucellosis in Macenta and Yomou was 12 and 5.33 %, respectively. In both provinces, the prevalence mean was 8.67 %. This study highlighted the immediate necessity to carry out a strengthened surveillance of human and animal brucellosis to obtain as many data as possible in order to establish strategies for prevention and management of brucellosis in Guinea.

Development and validation of a chemiluminescent western blot assay for glanders (Burkholderia mallei) serodetection.

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.

Sero-prevalence and risk factors study of brucellosis in small ruminants in Southern Zone of Tigray Region, Northern Ethiopia.

This study reports a prevalence and risk factor survey of brucellosis in small ruminants in Southern Zone of Tigray Region, Northern Ethiopia between October 2011 and April 2012 to determine the sero-prevalence of small-ruminant brucellosis and to identify associated risk factors for the occurrence of disease in small ruminants under extensive production system. Multistage random sampling was followed to select locations, flocks, and individual animals. Laboratory analysis of serum samples provided sero-prevalence estimates for flocks and geographic location. Information on risk factors at the individual and flock level was obtained by examination of individual animal and a questionnaire interview to flock owners. The overall individual animal-level sero-prevalence of brucellosis in small ruminants was 3.5 % and flock level sero-prevalence was 28.3 %, and the within-flock sero-prevalence was ranged from 0 % to 22.2 % based on the Complement Fixation Test. Multivariable logistic regression showed that the major risk factors for flock level sero-positivity were flock size and abortion history. This study showed that small-ruminant brucellosis is prevalent in the study area. Larger flock size and history of previous abortion in the flock were major risk factors identified for sero-positivity of small-ruminant brucellosis.

Development and validation of a method for purification of mallein for the diagnosis of glanders in equines.

BACKGROUND: The allergic test of mallein is one of the most frequently used tests, together with the Complement Fixation Test (CFT), for the diagnosis of glanders in endemic areas. Mallein, a purified protein derivative (PPD), is produced similarly to PPD tuberculin and the end product is a primarily proteic antigen, which is only poorly purified. The immuno-allergic activity of mallein is believed to be due to a high molecular weight group of proteins present in the antigen. To improve the quality of the antigen, in terms of sensitivity and specificity, a new method of mallein production was developed, in which purification was accomplished by ultrafiltration in a Tangential Flow Filtration system (TFF). RESULTS: The TFF methodology efficiently separated the high and low molecular weight protein groups of mallein. The five TFF-purified malleins, produced from Burkholderia mallei strains isolated from clinical cases of glanders in Brazil, proved to be more potent than standard mallein in the induction of an allergic reaction in sensitized animals. Regarding specificity, two of the purified malleins were equivalent to the standard and three were less specific. CONCLUSION: Some of the TFF-purified malleins showed considerable potential to be used as an auxiliary test in the diagnosis of glanders.

Serological profile of buffalo (Bubalus bubalis) female calves vaccinated with standard Brucella abortus strain 19 vaccine using rose bengal, 2-mercaptoethanol and complement fixation tests.

The serological profiles of 21 female buffaloes vaccinated between 3 and 8 months of age using Brucella abortus strain 19 (S19) were evaluated by rose bengal (RBT), 2-mercaptoethanol (2ME) and complement fixation (CFT) tests. The serum strains were collected in day zero, 15, 30, 45, 60th days and subsequently to each 30 months, until 720th day after vaccination. No animal showed reaction in day zero. In 15th day above 95% of animals revealed reaction in all tests. All the animals presented absence of reactions in CFT, RBT and 2ME tests at 270, 300 and 360 days after vaccination, respectively. Our finding highlighted early response in CFT compared than other conventional agglutination tests. None of animals presented oscillation of titers or reactions in any test after 360 day of study, which enables the use of these tests after this period without interference of antibodies from S19 vaccine origin between 3 and 8 months in buffalo heifers.

Preliminary comparison of different immune and production components in local and imported Saanen goats reared under a sub-tropical environment.

Three objectives were included in this research work. The first objective compared different immune components in healthy mature males, mature females, and female kids of local and imported Saanen goats, reared under a sub-tropical environment. The significantly differing immune components were the blood monocyte percent, blood CD8 count, and the total white blood cell count. The second objective compared the performance of Saanen versus local does. The means of the milk yield and prolificacy of the imported Saanen does were significantly higher than those of the local does (p<0.05). The third objective compared the immune responses (hemagglutination-HA titers) and complement fixation (CF) titers in mature does of the two breeds to chicken red blood cells (c-RBC). The HA titers showed a significant seroconversion only in imported Saanen (p<0.05) but not in local does; however, the CF titers increased significantly at 4 weeks following priming with c-RBC in local (p<0.05) but not in the imported Saanen does. The impact of the differences in blood immune components and responses to antigens in the compared goats on protection potential against prevalent diseases in the sub-tropical zone of the eastern Mediterranean countries is discussed.

Performance evaluation of two serological tests for contagious bovine pleuropneumonia (CBPP) detection in an enzootic area using a Bayesian framework.

A Bayesian approach, allowing for conditional dependence between two tests was used to estimate without gold standard the sensitivities of complement fixation test (CFT) and competitive enzyme-linked immunosorbent assay test (cELISA) and the serological prevalence of CBPP in a cattle population of the Central Delta of the Niger River in Mali, where CBPP is enzootic and the true prevalence and animals serological state were unknown. A significant difference (P = 0.99) was observed between the sensitivities of the two tests, estimated at 73.7% (95% probability interval [PI], 63.4-82.7) for cELISA and 42.3% (95% PI, 33.3-53.7) for CFT. Individual-level serological prevalence in the study population was estimated at 14.1% (95% PI, 10.8-16.9). Our results indicate that in enzootic areas, cELISA performs better in terms of sensitivity than CFT. However, negative conditional sensitivity dependence between the two tests was detected, implying that to achieve maximum sensitivity, the two tests should be applied in parallel.

Serological and bacteriological responses of water buffalo (Bubalus bubalis) vaccinated with two doses of Brucella abortus strain RB51 vaccine.

Thirty-two water buffalo (Bubalus bubalis) calves aged 6­10 months were used to evaluate serological responses to Brucella abortus strain RB51 (RB51) vaccination in a dose-response study and to compare the use of two selective media for the isolation of RB51. The animals were randomly divided into three treatment groups. Groups I-III received the recommended vaccine dose (RD) twice 4 weeks apart, RD twice 18 weeks apart and saline once, respectively. Lymph nodes were excised from the three groups and subjected to bacteriological examination to determine the frequency of detection of RB51. Pre- and post-vaccination blood samples were collected and tested for B. abortus antibodies using the buffered plate agglutination test (BPAT), complement fixation test (CFT), and dot-blot assay. Sera taken at all post-inoculation weeks (PIW) were negative for field strain B. abortus using the BPAT. Antibody responses to RB51 were demonstrated in all vaccinates but not in controls by CFT and dot-blot assay from 1 PIW up to 16 weeks following booster vaccination. The agreement for both assays was 80.7% and there was a linear interdependence with a Pearson's correlation coefficient value of 0.578. The frequency of isolation of RB51 from the two selective media used was not significantly different (P > 0.05).

Brucellosis due to Brucella suis in a swine herd associated with a human clinical case in the State of São Paulo, Brazil.

Brucella suis has been recognized as the major etiological agent of human brucellosis in areas free from Brucella melitensis infection. However, with changes in swine management, the occurrence of swine brucellosis has decreased as has the human incidence of B. suis infection. A swine brucellosis outbreak within a herd from Jaboticabal (São Paulo, Brazil) was detected in July 2006. The herd comprised approximately 300 sows and 1,500 finishing animals. Many sows within this herd experienced abortions, while others exhibited vaginal discharge; three sows suffered posterior paralysis. Among 271 sows, 254 (93.7%) tested positive for brucellosis by complement fixation, and among 62 randomly bled finishing animals, 17 (27.4%) also tested positive. The B. suis biovar 1 was cultured from 14 aborted fetuses and six sows. Brucella was identified using routine methods. Fourteen farm workers were tested using agglutination tests, with three workers showing evidence of Brucella antibody titers. A 39-year-old woman, who worked with maternal pigs and had direct contact with aborted fetuses, presented an agglutinating titer of 480 IU/mL and displayed clinical signs of infection. Our findings suggest that despite a reduction of swine brucellosis throughout Brazil, B. suis infection still occurs, thereby posing a zoonotic risk.

The value of fluorescence polarisation assay in verification of problematic sera from pigs for brucellosis.

The aim of the study was an evaluation of fluorescence polarisation assay (FPA) as a potential tool improving specificity of serological diagnosis of brucellosis in pigs. The evaluation was done by comparing the results of FPA with the results of rose bengal test (RBT), serum agglutination test (SAT), complement fixation test (CFT) and ELISA when false positive sera were tested. One hundred ninety porcine samples, reacting positively in at least one classical serological assay were used. We observed that among 198 sera, 104 were also positive in FPA. The studies confirmed that porcine FPA adds little as far as specificity in comparison to other methods is concerned.

A minor role of CD4+ T lymphocytes in the control of a primary infection of cattle with Mycoplasma mycoides subsp. mycoides.

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides, is an important livestock disease in Africa. The current control measures rely on a vaccine with limited efficacy and occasional severe side effects. Knowledge of the protective arms of immunity involved in this disease will be beneficial for the development of an improved vaccine. In previous studies on cattle infected with M. mycoides subsp. mycoides, a correlation was detected between the levels of mycoplasma-specific IFN-γ-secreting CD4+ T lymphocytes and reduced clinical signs. However, no cause and effect has been established, and the role of such cells and of protective responses acquired during a primary infection is not known.We investigated the role of CD4+ T lymphocytes in CBPP by comparing disease patterns and post mortem findings between CD4+ T cell depleted and non-depleted cattle. The depletion was carried out using several injections of BoCD4 specific murine monoclonal antibody on day 6 after experimental endotracheal infection with the strain Afadé. All cattle were monitored clinically daily and sacrificed 28-30 days post-infection. Statistically significant but small differences were observed in the mortality rate between the depleted and non-depleted animals. However, no differences in clinical parameters (fever, signs of respiratory distress) and pathological lesions were observed, despite elimination of CD4+ T cells for more than a week. The slightly higher mortality in the depleted group suggests a minor role of CD4+ T cells in control of CBPP.

Serological testing of cattle experimentally infected with Mycoplasma mycoides subsp. mycoides Small Colony using four different tests reveals a variety of seroconversion patterns.

BACKGROUND: To study the specific antibody response to infection with Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC), the agent of Contagious Bovine Pleuropneumonia (CBPP), we examined three panels of sera collected during three experimental infection trials in African cattle. The methods used included an in-house complement fixation test (CFT), a commercially available CFT, a competitive antibody ELISA (cELISA) and the immunoblotting test (IBT). In addition, lung tissue samples were examined by culture. RESULTS: A total of 89% (51/59) of all experimentally infected animals tested positive on at least one of the serological tests throughout the trial. The specific antibody titres to the MmmSC infection became positive first by CFT (6 to 9 days post infection [dpi]), followed by IBT (9 to 13 dpi) and cELISA (13 to 16 dpi). Individual animals were found to display remarkably distinct seroconversion patterns, which allowed their classification into i) early high responders, ii) late high responders, and iii) low responders. In accordance with other studies, none of the present serological tests was capable of detecting all CBPP infected animals. CONCLUSION: Comparison of the assays' performance in terms of sensitivity and specificity raises serious questions as to their reliability for identification of infected individuals in the field. In view of these limitations, a combination of CFT and cELISA can markedly improve CBPP diagnosis at single-animal level.

The first International Standard anti-Brucella melitensis Serum.

The World Organisation for Animal Health (OIE) requested an International Standard anti-Brucella melitensis Serum (ISaBmS) to standardise diagnostic tests and reagents for sheep and goats. The agreed criteria were the highest dilution (in negative serum) of the standard which must give a positive result and the lowest dilution (in negative serum) which must simultaneously give a negative result. The two dilutions for each assay were, respectively: indirect enzyme-linked immunosorbent assay (iELISA) 1/64 and 1/750, competitive ELISA (cELISA) 1/8 and 1/300, fluorescent polarisation assay (FPA) 1/16 and 1/200, Rose Bengal test (RBT) 1/16 and 1/200. The OIE International Standard Serum (OIEISS) will remain the primary standard for the RBT; the ISaBmS is an additional standard. It was impossible to set criteria for the complement fixation test, therefore the OIEISS will remain the primary standard. The ISaBmS can be used to standardise iELISA, cELISA and FPA to diagnose sheep and goat brucellosis. This standard should facilitate harmonisation of tests used for brucellosis surveillance and international trade in these species.