Complement Fixation Test Titres Research Articles

Detectable immunological response in Brucella abortus s 19 vaccinated herds of cattle in vilachchiya veterinary range, north central province, Sri Lanka

Brucellosis a zoonotic disease caused by Gram-negative, intracellular, coccobacillus. Brucellosis, an economically important disease in livestock with zoonotic implications has been reported worldwide. It is transmitted via contaminated animals and their products. Clinical signs of animal brucellosis are abortions, reduced fertility, retention of fetal membranes, endometritis, and substantial decline in milk production over an animal’s lifespan. Orchitis, epididymitis and chronic hygroma had been reported in bulls. The current study focused on to studying the detectable immunological responses to Brucella S 19 live attenuated vaccine using serological tests in cattle of dry zone farms. A questionnaire-based survey was carried out in selected farms with and without a previous history of abortion respectively. Animals that were in 1st to 5th lactation in the 18 farms were selected for screening by Rose Bengal Plate Test (RBPT) and positives were confirmed by Complement Fixation Test (CFT). Three farms each with and without history of abortion were selected for Brucella S19 vaccination from the RBPT and CFT negative farms. Sixty non pregnant heifers over 6 months were selected, and 1ml each of S19 vaccine was given subcutaneously to 30 animals while 30 animals were kept as controls. The serum samples were collected and screened for Brucellosis by RBPT and CFT at 2, 4, 6, 8 and 10 weeks of post vaccination. The second week after the vaccination, 71.42% of the animals showed over 1:4 CFT levels while 28.57% of them did not show any antibody titres for Brucellosis in the Brucella endemic farms. In Brucella non-endemic farms, 68.75% of animals showed over 1:4 CFT titre at 2 weeks after the vaccination. However, 31.25% of animals did not show any CFT titre. No persistent and detectable antibody responses were found 10 weeks post vaccination in heifers of endemic and non-endemic herds. The titers of CFT indicate IgG or IgM humeral response, cell mediated immune response of live attenuated vaccine has not been monitored in the study. Thus, seropositivity after the 10 weeks post vaccination may indicate an infection of Brucellosis since no antibodies from vaccination were found. Further investigation with highly sensitive tests such as ELISA is recommended to have a better insight into this aspect in the future.

Correlation of toxoplasmosis with small‑for‑gestational‑age newborns.

Toxoplasma gondii infection in pregnant women could lead to significant changes during the pregnancy, affect the outcomes of pregnancy and the timing of labour. Small‑for‑gestational‑age (SGA) newborns are defined by birthweight below the 10th percentile for gestational age. We tested an association between latent toxoplasmosis in pregnant women and deliveries of SGA babies. For testing, we included 1,647 women who gave birth to a singleton baby at ≥ 37 weeks of gestation. The complement-fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) tests for IgG and IgM were used. The latent form of toxoplasmosis was defined as a CFT titre of 1:8 or higher, together with index positivity IgG ELISA > 1.1 and negative IgM. There were 406 (24.7 %) women positive, and 1,241 (75.3 %) women negative for latent toxoplasmosis. Of all deliveries. 190 were SGA‑positive and 1,457 were SGA‑negative. Our study found a statistically significant association between latent toxoplasmosis and SGA foetuses born at term. The Pearson chi-square model was statistically significant (χ2(1) = 7.365, p = .007). The odds ratio was 1.567. Pregnant women with latent toxoplasmosis giving birth at ≥ 37 weeks of gestation have a 1.567 times higher risk of delivering an SGA baby (Tab. 2, Fig. 1, Ref. 30).

Toxoplasmosis impact on prematurity and low birth weight.

BackgroundToxoplasma gondii, one of the most common parasites, causes toxoplasmosis, one of the most frequent zoonotic diseases worldwide. T. gondii infects about one-third of the world’s population. T. gondii infection is generally considered a major risk for spontaneous abortion, prematurity and low birth weight in the animal sphere. Less commonly, a toxoplasma serological profile is correlated with the particular data of delivery. Acute T. gondii infection during pregnancy often leads to spontaneous abortion and/or a severe injury of the eyes, brain, and other structures of the foetus. Latent T. gondii infection of pregnant women could lead to less obvious but important changes during pregnancy, including the end product of pregnancy and the timing of labour. This study aimed to contribute to the current knowledge by comparing serological T. gondii profiles of pregnant women with prematurity and low birth weights of newborns.Material and methodsA retrospective study design was adopted. The study participants included a cohort of 1733 pregnant women who consecutively gave birth to their children and underwent regular antenatal biochemical screening between the 14th and 16th weeks of pregnancy. Prematurity was defined as the liveborn preterm delivery in gestational age of pregnancy <37 weeks. Low birth weight was defined as weight at birth of ≤2499 grams. The complement-fixation test (CFT) provided serological profiles for toxoplasmosis that expresses the overall levels of toxoplasma immunoglobulins of all classes. Enzyme-linked immunosorbent assay (ELISA) tests for IgG and IgM were used simultaneously. IgM positivity helped to differentiate acute from the latent stage of toxoplasmosis. Birth data, especially the week of delivery and fetal weight, were evaluated accordingly.ResultsOf the 1733 pregnant women, 25% were diagnosed as latent toxoplasma positive, and 75% as toxoplasma negative. There were 87 premature deliveries versus 1646 timely births. We observed 88 low birth weights and 1645 normal fetal weights. We found a statistically significant association between latent toxoplasmosis and prematurity, χ2(1) = 5.471, p = .019 and between latent toxoplasmosis and low birth weight of newborns, χ2(1) = 7.663, p = .006. There was a 1.707 times higher risk of prematurity for toxoplasma-positive women, while the risk for low birth weight was 1.861 times higher. The strength of both tests of association was mild. We tested the correlation between the levels of CFT titres and week of delivery and weight of newborns. No association was found between the level of latent toxoplasmosis and the week of delivery and fetal weight.ConclusionLatent toxoplasmosis was associated with premature birth rate and lower birth weight of newborns. The odds of premature delivery was 1.7 and low birth weight 1.9 times higher in women with latent toxoplasmosis compared to toxoplasma negative women. Even though the strength of the association in our large sample is relatively mild, the combination of latent toxoplasmosis with other adverse factors could cause serious harm. Whole CFT and specific IgG levels of latent toxoplasmosis are not linked to the severity of prematurity or low birth weight in newborns.

Acute phase biomarkers, oxidants, antioxidants, and trace minerals of mobile sheep flocks naturally infected with brucellosis

This study assayed the acute phase responses of sheep seropositive to Brucella. Sera collected from ewes (n=160) were subjected to serological tests of Brucella, Rose Bengal plate agglutination test (RBPAT), buffer acidified plate agglutination test (BAPAT), and complement fixation test (CFT). Results revealed that CFT was the most predictive test of brucellosis followed by BAPAT then RBPAT. The moderate predictive blood biochemical parameters were zinc and ascorbic acid. Ewes with low CFT titre (chronic) had low fibrinogen, copper, NO, and GPx. Seropositive animals had high blood concentrations of ascorbic acid and zinc.

Detection of brucellosis and leptospirosis in feral pigs in New South Wales

To determine the presence and estimate the prevalence of Brucella suis, Leptospira interrogans serovar Pomona (hereafter L. pomona) and Leptospira borgpetersenii serovar Hardjo (hereafter L. hardjo) in feral pigs culled in New South Wales (NSW), Australia. During 2012 and 2013, 239 serum samples were collected from feral pigs killed as pests or game in NSW. All sera were subjected to the rose-bengal test for B. suis, with positives subjected to the complement fixation test (CFT). Attempts were made to detect B. suis by culture and PCR on CFT-positive samples. All sera were tested separately for the presence of L. pomona and L. hardjo antibodies using the microscopic agglutination test. Of 238 samples tested, 7 were positive (4 with CFT titres ≥ 32) for B. suis antibodies (3% seroprevalence). However, B. suis was not cultured or detected by PCR. Of 239 sera tested for L. pomona antibodies, 126 samples were positive (53%) and 9 (4%) were positive for L. hardjo. The findings are the first tangible evidence that feral pigs in northern NSW harbour B. suis, providing a plausible explanation for recent human and canine cases of brucellosis related to pig hunting. The increased seroprevalence of L. pomona occurred in years preceded by flooding and rodent plagues, suggesting a potential for zoonotic infection much greater than previously realised. Advice to the community should focus on encouraging the adoption of improved hygiene practices during pig hunting and consideration of vaccinating livestock against leptospirosis.

Seroconversion and semen shedding in rams experimentally infected with Brucella ovis

AIM: To determine the time taken for rams to develop antibodies to Brucella ovis in serum, shed B. ovis in semen and develop lesions of epididymitis following infection with B. ovis.METHODS: Fifteen 19-month-old rams were artificially infected with B. ovis by inoculation of infected semen onto the nasal and rectal mucus membranes (Day 0). Serum was collected from each ram at 2 to 8-day intervals and tested at commercial laboratories using a complement fixation test (CFT) and an ELISA. Cut-off values for the CFT were 0–4/4 negative; 1/8–3/8 suspicious and 4/8–4/128 positive, and for the ELISA were <10% negative; ≥10 to <50% suspicious and ≥50% positive. Selected serum samples were also tested using a gel diffusion test (GDT). At 7 to 8-day intervals semen was collected for bacterial culture and the scrotal contents were palpated to identify lesions of epididymitis. The study was terminated after 56 days.RESULTS: On Day 28 B. ovis was isolated from the semen of one ram and by Day 49 it was isolated from the semen of 10 rams. All 10 rams had suspicious or positive ELISA or CFT titres by Day 36 and 56, respectively. The GDT results were all negative on Day 36 and in general did not become positive in individual rams until 7–28 days after semen shedding commenced. Epididymitis was detected in one ram on Day 36; by Day 56 eight rams had epididymitis detectable by scrotal palpation.CONCLUSIONS: The B. ovis ELISA test identified infected rams at an earlier stage than the CFT; this was at 19–36 days after exposure. Rams can begin shedding B. ovis in semen as early as 28 days after exposure and lesions of epididymitis develop as early as 36 days after exposure.CLINICAL RELEVANCE: During a test and slaughter campaign for the control of B. ovis, the most appropriate serological re-testing interval is likely to be around 28 days (4 weeks) using the ELISA with or without the CFT, although caution is required in interpretation of “suspicious” ELISA results. Following a B. ovis breakdown, two negative CFT or ELISA tests 60 days apart are recommended to confirm freedom from infection, supporting current guidelines.

Investigating Coxiella burnetii infection in a breeding cattery at the centre of a Q fever outbreak.

The potential role of cats in transmitting Coxiella burnetii to humans was highlighted in a Q fever outbreak, linked to a caesarean section in a breeding queen, in an Australian small animal veterinary hospital. The objectives of this study were to evaluate the C burnetii seroreactivity of the breeding queen and other cats residing at the same breeding cattery (n = 27) and to evaluate C burnetii infection of the breeding queen by molecular and histological methods. Three assays [complement fixation test (CFT), indirect immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA)] were used for serological evaluation. Additionally, uterine and ovarian samples collected from the breeding queen 11 weeks post-parturition were assessed by routine and specialised histological methods and polymerase chain reaction. The breeding queen showed strong seropositivity using CFT (titre 1/32), IFA (titre phase I 1/8192 and phase II 1/8192) and ELISA; however, the reproductive tract showed no evidence of pathology or C burnetii infection. A number of cattery-confined cats were identified as seropositive to phase II and/or phase I C burnetii. Serological detection of C burnetii in a breeding cattery linked to a Q fever outbreak indicates likely infection by this bacterium in Australian feline populations, re-confirming the relevance of this zoonosis.

An evaluation of Irish cattle herds with inconclusive serological evidence of bovine brucellosis.

Since 1998, there has been a steady decline in herd restrictions and de-populations in Ireland due to bovine brucellosis. There is concern that the interpretation of laboratory results may become increasingly problematic, as brucellosis prevalence falls in Ireland. Therefore, the purpose of the current study was to evaluate the infection status of Irish herds and animals with inconclusive serological evidence of bovine brucellosis. During 12 months from September 1, 2004, laboratory and observational epidemiological data were collected from all Irish herds where animal testing identified at least one animal with a complement fixation test (CFT) reading greater than zero and/or a positive result to the indirect enzyme-linked immunosorbent assay (iELISA). Due to the observational nature of the study, we have robust estimates of the relative, but not the absolute, performance of the CFT, iELISA and brucellin skin test (BST). Herds were divided into three categories (Group A, B or C) on the basis of test results at initial assessment. A total of 639 herds were enrolled into the study, and observed for at least two years following enrolment. A rising CFT titre, with a CFT reading of 111 International CFT Units (IU) or greater at the subsequent blood test, was generally associated with herds where other evidence of infection was also available. Knowledge of the CFT reading at the initial and a subsequent blood test proved useful in distinguishing false-positive and true-positive brucellosis results. There was poor correlation between the CFT and iELISA results, and between the CFT and BST results. As a result of this study, national policy has been modified to include re-sampling of all animals with CFT readings of 20 IU or greater. This project has also led to a reduction in the number of herds restricted, as well as restriction duration. It has also contributed to a reduction in the number of herds listed for contiguous tests, and therefore the potential for contiguity testing of false positive results.

Infections and vaccinations preceding childhood Guillain-Barré syndrome: a prospective study

We performed a prospective, multicentre study in children with Guillain-Barré syndrome (GBS), diagnosed according to international criteria, to investigate the frequency and aetiology of antecedent diseases. All infections and vaccinations occurring within a 6-week period prior to the onset of GBS were documented. Stool cultures, standardised serological investigations and PCR analyses for 24 different infective agents were performed. Serological findings were regarded as significant if specific immunoglobulin (Ig)M or IgA antibodies were detected, if the IgM enzyme immunoassay or immunfluorescence assay findings were confirmed by immunoblot, if complement fixation test titres rose fourfold or if geometric titres were more than threefold higher than in uninfected control persons. Ninety-five children with GBS were included in the study over a 40-month period. Preceding events were reported in 82%. Microbiological studies carried out on 84 patients resulted in a probable diagnosis in 46 (55%). Coxsackieviruses (15%), Chlamydia pneumoniae (8%), cytomegalovirus (7%) and Mycoplasma pneumoniae (7%) were the most frequently involved agents. Serological evidence of a Campylobacter jejuni infection was found in six patients (7%). Eight children had been vaccinated during the 6 weeks preceding the onset of GBS; in six of these children concomitant infectious diseases were reported, and in one child the time between vaccination and GBS was extremely short. We conclude that, in contrast to adults, Campylobacter spp. does not seem to play a major role in childhood GBS in German-speaking countries. The aetiology of antecedent diseases is distributed over a wide spectrum of paediatric infectious diseases. Most of the children who had been vaccinated showed concomitant infectious diseases, thus obscuring the causative role for GBS.

Pathological and immunological characteristics of ewes experimentally infected with Mycoplasma mycoides subsp. mycoides SC strains isolated from cattle and sheep

The pathological features of experimental infection with Mycoplasma mycoides subsp. mycoides small colony ( Mmm SC) isolated from cattle (CBPP strain) and sheep (mastitis strain) were investigated in 16 out of 36 ewes using an intratracheal–endobronchial route of inoculation. Animals kept in-contact with infected animals, as well as those of a control group, were also studied. The immune response to two strains was also assessed. Complement fixation test (CFT) and immunoblotting (IBT) used for contagious bovine pleuropneumonia (CBPP) diagnosis, were performed on serum obtained from weekly blood samples. Only one ewe inoculated twice with the bovine isolate showed hyperplasia of the germinal centres in the lymph nodes and mucosa-associated lymphoid tissue in the lung. This ewe also exhibited a CFT titre of 1:160 simultaneously showing specific antibodies to 110, 98, 95, 62 and 48 kDa immunogenic bands. Interstitial pneumonia and thickening of pulmonary septa were observed in 10 inoculated and 6 in-contact ewes. Statistical analysis of the incidence of interstitial pneumonia indicated that the lesion was related to mycoplasma infection. The strongest immune response occurred at day 77 post-exposure to immunogenic antigens with molecular weights of 110, 98, 95, 62, 60 and 48 kDa in ewes from both inoculated groups. Taking into consideration the inoculum dose rate, ewes receiving the bovine isolate produced an earlier and more pronounced immune response than those receiving the ovine isolate. A mild response was seen in three ewes in-contact first and then inoculated with the ovine isolate. In spite of a relatively weak serologic reaction detected, the results suggested the possibility of contact transmission among animals.

The production of EAE-free lambs from infected damsusing multiple ovulation and embryo transfer

This investigation aimed to ascertain whether embryo transfer was a feasible method of breaking the disease cycle caused by Chlamydia psittaci (ovis). Ten naive ewe lambs were inoculated orally with the T76 and G188 isolates of C. psittaci (ovis) in late pregnancy. Five animals which sero-converted to the complement fixation test (CFT) were used as donors for a multiple ovulation and embryo transfer programme. Three ewes excreted chlamydiae at parturition 1 year after inoculation, with one animal exhibiting a CFT titre indicative of clinical disease. Twelve embryos collected from these three donors and transferred to seven disease-free recipients survived and were not infected, nor were their recipient dams. It therefore appears possible to infect ewe lambs during the final stages of pregnancy with the disease manifesting itself during the following breeding season. Embryos collected from infected animals do not appear to transmit the chlamydia causing enzootic abortion.

Serological crossreactivity between Brucella abortus and Yersinia enterocolitica 0:9 III. Specificity of the in vitro antigen-specific gamma interferon test for bovine brucellosis diagnosis in experimentally Yersinia enterocolitica 0:9-infected cattle

The course of immunological reaction in 10 Yersinia enterocolitica 0:9 experimentally-infected heifers was followed using the conventional brucellosis tests complement fixation test (CFT), serum agglutination test (SAT) and brucella card test (BCT), and a recently developed Brucella antigen-specific gamma interferon (IFN-γ) test. Initially, the animals were exposed orally to 10 10 colony-forming units (CFU) of Y. enterocolitica 0:9. Four weeks later, they were inoculated intravenously with 10 8 CFU of Y. enterocolitica 0:9 cells. After oral inoculation, the response in the conventional brucellosis tests was minimal. Only after intravenous inoculation were CFT and SAT titres and BCT reactions comparable to natural, false positive brucellosis reactors. After oral exposure the Brucellergen-stimulated release of IFN-γ peaked at values above the cut-off stimulation index of 2.5 in 80% of the heifers. After intravenous inoculation, stimulation indices above 2.5 were present in only 10% of the animals. Two B. abortus infected control cattle showed stimulation indices of 3.1 and 3.4, and a negative control animal exhibited a stimulation index of 1.0. These findings show, in contrast to a previous study, that the Brucellergen-specific IFN-γ assay cannot be used as a specific and discriminatory test for B. abortus infections.

Measles virus serology in Crohn's disease.

BACKGROUND: There is evidence that measles virus infection in early life may predispose to Crohn's disease. AIMS: To examine using serological methods a potential association between measles virus infection in...

Rangiferine brucellosis on Baffin Island.

The standard tube agglutination test (STAT) and the complement fixation test (CFT) were used to assess the seroprevalence of antibodies to Brucella spp. in caribou (Rangifer tarandus) from three populations on Baffin Island, Canada. During late winter from 1983 to 1986, sera from 17 of 40 North Baffin (43%), 11 of 33 Northeast Baffin (33%) and 12 of 82 South Baffin (15%) adult caribou had antibodies in the STAT at 1:50 or the CFT at 1:5. Seroprevalence increased as caribou matured with one (4%) of 25 calves, four (13%) of 31 yearlings, and 40 (26%) of 155 adult caribou being positive. However, seroprevalence did not differ with sex in any age class. Positive antibody titers were higher in adult females sampled in May, 3 to 4 wk before parturition, than in adult females sampled in late March and April. The strength of positive titers did not differ with the time of sampling among adult males. Pathologic signs of brucellosis were found in three (13%) of 23 caribou that were assumed to have active infections (caribou with CFT titers > 1:160). Brucella suis biovar 4 was isolated from 24 (60%) of 40 caribou from which lesions were submitted. Between 1986 and 1990, the annual incidence of reported human (Homo sapiens) cases averaged 3.4 (34:100,000) on Baffin Island.

Prevention of congenital toxoplasmosis in Szeged, Hungary.

Toxoplasma gondii infection of the fetus can only be discovered or prevented by the appropriate serological screening and subsequent treatment of the mother and her offspring. In Hungary, there is no obligatory toxoplasma screening for pregnant women and both the reporting and follow-up of congenital toxoplasmosis cases is limited. In 1987 we started a systematic study in the Szeged region of Hungary, in which all pregnant women were screened and appropriate treatment given to all mothers and their offspring where congenital toxoplasmosis was suspected. All pregnant women were routinely screened within the first 16 weeks of gestation for toxoplasma antibodies by complement fixation test (CFT). Seronegative cases were retested for possible seroconversion every second month. Patients with CFT titres > or = 1:256 were retested for anti-P30 immunoglobulin A (IgA), IgM and IgG antibodies by ELISA and/or SDS-PAGE-Western immunoblot in order to distinguish the acute and chronic phases of the infection. Up to the end of 1994, the sera of 17,735 gravidae were screened. Ten women were found to have seroconverted during pregnancy and 78 had high initial antibody levels accompanied by anti-P30 IgA antibodies at the very first screening. These two groups together were considered as definitely (10) or possibly (78) infected with Toxoplasma during pregnancy and were treated with Spiramycin. All of their offspring were also treated for one month and followed-up by systematic serological and clinical screening for 2 years. No congenital toxoplasmosis was found in any of the offspring. Antenatal, early diagnosis and treatment of toxoplasmosis in mothers, together with treatment and follow-up of their offspring, may considerably reduce the incidence of the disease in the offspring.

Hopkins syndrome associated with Mycoplasma infection

A 3-year-old boy who presented with flaccid weakness of the right lower limb I week after treatment for an acute attack of asthma is described. Sensation was normal. Electromyography suggested anterior horn cell damage and persisting weakness. These features suggested a diagnosis of Hopkins syndrome. Complement fixation test titer and agglutination test titer to Mycooplasma pneumoniae were significantly increased, suggesting a previously unreported association with this syndrome. We review possible infectious agents detected in previous reports.

Intestinal Chlamydia in finishing pigs.

Gut and blood samples from 119 finishing pigs derived from 11 farms were collected during routine slaughter at an abattoir. Sections of formalin-fixed, paraffin-embedded tissues were labeled immunohistochemically using genus-specific, mouse monoclonal antibody against chlamydial lipopolysaccharide; goat polyclonal antiserum against the major outer membrane protein of Chlamydia trachomatis; and mouse monoclonal antibody against the ovine abortion subtype of C. psittaci. Gut samples from 33 of 111 (29.7%) individual pigs stained positive with the genus-specific monoclonal antibody, and of these 30 of 32 (93.7%) also reacted with the C. trachomatis-specific antiserum. Labeled inclusions were restricted to mature enterocytes of the large intestine in 33 of 111 cases. Infection of small intestinal enterocytes was noted in only one of 82 ileal samples. The blood samples were tested for antichlamydial antibodies by enzyme-linked immunosorbent assay (ELISA) and complement fixation test (CFT). With ELISA, 95 of the 115 sera tested (82.6%) yielded positive antichlamydial reactions. With CFT, 34 of the 119 sera tested (28.6%) were unequivocally positive (> or = 1:10, 100% binding), and 10 (7.6%) yielded doubtful positive reactions (1:10, 50-75% binding). Positive ELISA and CFT titers showed poor agreement (kappa = 0.112), whereas the agreement between positive findings by immunohistochemical labeling and CFT was fair (kappa = 0.205).

A complement-fixation, enzyme-linked immunosorbent assay as an aid in the serological investigation of enzootic abortion in ewes

An enzyme-linked immunosorbent assay which detects complement fixed in an antigen/antibody complex ( CFELISA) was used to examine sera previously examined by the complement fixation test ( CFT) in an investigation of enzootic abortion in ewes ( EAE). The CFELISA was easy to perform and did not suffer from the many drawbacks of the CFT. Accepting a CFT titre of ≥32 to indicate infection, the CFELISA had a sensitivity of 98 per cent and a specificity of 100 per cent; the predictive value of a positive result was 100 per cent and that of a negative result was 98 per cent.

Evaluation of serological tests for diagnosis of Brucella melitensis infection of goats.

Five serological assays were evaluated for the diagnosis of brucellosis in goats: the rose bengal test (RBT), complement fixation test (CFT), radial immunodiffusion (RID) with Brucella and Yersinia enterocolitica O:9 polysaccharides, counterimmunoelectrophoresis (CIEP) with cytosol, and enzyme-linked immunosorbent assay (ELISA) with polyclonal and protein G conjugates and smooth lipopolysaccharide (S-LPS), native hapten polysaccharide (NH), or cytosol antigens. For optimal sensitivity, RBT had to be used with sera-antigen at a 3:1 dilution. In the RID test, Brucella melitensis biotype 1 NH could not be replaced by Brucella abortus biotype 1 or Y. enterocolitica 0:9 polysaccharides. In the ELISA, S-LPS and NH gave similar results and the protein G conjugate increased the specificity. With the sera from 55 B. melitensis culture-positive goats, the sensitivity was 100% for RBT, CFT (titer > or = 4), and ELISA with S-LPS or NH; 94% for RID; and 93% for CIEP. All tests were negative (100% specific) when testing the sera from 127 brucella-free goats. Larger discrepancies among the results of the serological tests were obtained with sera from goats of areas where brucellosis is endemic. When the sera of 20 young goats vaccinated subcutaneously (10(9) CFU of B. melitensis Rev 1) and bled 6 months later were examined, the specificities were as follows: NH ELISA, 60%; CFT and S-LPS ELISA, 75%; RBT, 80%; CIEP, 90%; and RID, 94%. With the sera from 10 young goats vaccinated conjunctivally (10(9) CFU of B. melitensis Rev 1) all tests were 100% specific 4 months after vaccination. The proportion of goats giving a positive reaction after vaccination decreased faster in RID than in other tests.

Evaluation of a DNA-based probe for the detection of cattle experimentally infected with Babesia bigemina.

A digoxigenin-labeled probe was used for hybridization to various preparations of Babesia bigemina-infected erythrocyte extracts. Dot blot hybridization and immunological detection of DNA hybrids revealed that the probe was specific for B. bigemina DNA because it did not hybridize to the DNA of B. bovis, a closely related species. Studies of sensitivity showed that the probe would bind to as little as 1 ng of B. bigemina DNA, but not to 1 microgram of the B. bovis DNA. The probe reacted with equal intensity against seven B. bigemina isolates from different geographic areas. The lowest percentage of B. bigemina-infected erythrocytes detected was 0.001%, a level of parasitemia not usually detected with the light microscope. Six intact, mixed-breed steers, approximately 3 years old, were inoculated with a blood stabilate containing B. bigemina-infected erythrocytes. Blood samples collected from day -1 to day 86 postinoculation (PI) and prepared for DNA extraction were analyzed in a dot blot hybridization assay using a nonradioactive DNA probe. Hybridization reaction (HR) signals were compared to results obtained by light microscopy (LM) examination of Giemsa-stained blood smears and to antibody titers of serum samples assayed with the complement fixation test (CFT) and indirect fluorescent antibody test (IFAT). Four of six inoculated steers became infected with B. bigemina as assessed by LM. The parasitemias were low (less than 0.01-0.05) at day 10 PI. Only three steers were serologically positive by CFT (titer 1:40-1:160) and IFAT (1:1280). All four infected steers had positive HR signals in the dot blot assay. The HR signals were observed from day 10 to day 77 PI and were usually correlated with the presence of parasites in blood as observed by LM. The HR signals varied in intensity for different blood samples from the experimental animals and with day of blood sample collection. Although the signal intensity did not correlate with the parasitemia level estimated by LM, the nucleic acid hybridization assay was more sensitive than LM, CFT, or IFAT for the detection of B. bigemina-infected cattle.