Comparison of serologic tests for detection of Brucella infections in cattle and water buffalo (Bubalus bubalis)

OBJECTIVE: To estimate sensitivity and specificity of 4 commonly used brucellosis screening tests in cattle and domestic water buffalo of Trinidad, and to compare test parameter estimates between cattle and water buffalo. ANIMALS: 391 cattle and 381 water buffalo. PROCEDURE: 4 Brucella-infected herds (2 cattle and 2 water buffalo) and 4 herds (2 of each species) considered to be brucellosis-free were selected. A minimum of 100 animals, or all animals > 1 year of age, were tested from each herd. Serum samples were evaluated for Brucella-specific antibodies by use of standard plate agglutination test (SPAT), card test (CT), buffered plate agglutination test (BPAT), and standard tube agglutination test (STAT). A Bayesian approach was used to estimate sensitivity and specificity of diagnostic tests without the use of a gold standard, assuming conditional independence of tests. RESULTS: Sensitivity and specificity estimates in cattle, respectively, were SPAT, 66.7 and 98.9; CT, 72.7 and 99.6; BPAT, 88.1 and 98.1; and STAT, 80.2 and 99.3. Corresponding test estimates in water buffalo, respectively, were SPAT, 51.4 and 99.3; CT, 90.4 and 99.4; BPAT, 96.3 and 90.7; and STAT, 75.0 and 98.8. Sensitivity of the CT and specificity of the BPAT were different between cattle and water buffalo with at least 95% probability. CONCLUSIONS AND CLINICAL RELEVANCE: Brucellosis serologic test performance varied by species tested, but BPAT had the highest sensitivity for screening cattle and water buffalo. Sensitivity and specificity of more than 2 screening tests can be estimated simultaneously without a gold standard by use of Bayesian techniques.     

Evaluation of a fluorescence polarization assay for the detection of serum antibodies to Brucella abortus in water buffalo (Bubalus bubalis)

The fluorescence polarization assay (FPA) was evaluated for the serological diagnosis of brucellosis in water buffalo (Bubalus bubalis) in southern Italy. This assay uses O-polysaccharide prepared from Brucella abortus lipopolysaccharide conjugated with fluorescein isothiocyanate as a tracer. It has many methodological advantages over older, more established tests and can be performed in a fraction of the time. Sera from 890 buffalos from the Campania Region - 526 positive sera and 364 negative sera according to the complement fixation test (CFT) - were evaluated in this study. All samples were tested with the Rose Bengal test (RBT), CFT, and FPA in parallel and in blind fashion. Sensitivities (Sn) were 84.5% and 92.6%, and specificities (Sp) were 93.1% and 91.2% for RBT and FPA, respectively, relative to CFT. Finally, receiver operating characteristic (ROC) analysis suggested a cut-off value of 117 millipolarization (mP) units. On the whole, these results suggested that FPA might replace RBT in the diagnosis of buffalo brucellosis for its better performance relative to CFT, its adjustable cut-off useful in different epidemiological situations, its reliability, ease of performance, and for its potential application in field and high-throughput laboratories.     

Evaluation of indirect TaSP enzyme-linked immunosorbent assay for diagnosis of tropical theileriosis in cattle (Bos indicus) and water buffaloes (Bubalus bubalis) in Egypt

The aim of the present study was to evaluate the validity of Theileria annulata surface protein (TaSP)-ELISA, in comparison with traditional microscopic test, for the diagnosis of T. annulata infection among Egyptian baladi cattle (Bos taurus) and water buffaloes (Bubalus bubalis). Molecular confirmation of infection using T. annulata merozoite surface (Tams-1) target amplification by PCR was used as a gold standard. A total of 76 clinically suspected animals including 64 baladi cattle and 12 water buffaloes were investigated in the current study by the three methods. Based on the PCR-confirmed results, the evaluation study revealed higher sensitivity of TaSP-ELISA (72.9% and 75%) as compared to microscopic examination (58.3% and 50%) among cattle and buffaloes, respectively. On the other hand, the specificity of TaSP-ELISA in diagnosis of T. annulata infection was higher (87.5%) in baladi cattle as compared to water buffaloes (37.5%). In conclusion, TaSP-ELISA was shown to be suitable for the diagnosis of T. annulata infection in cattle under field conditions.     

Evaluation of brucellosis RB51 vaccine for domestic water buffalo (Bubalus bubalis) in Trinidad

Thirty-two young domestic water buffalo (Bubalus bubalis) were obtained from a brucellosis-free farm to determine effectiveness of RB51 vaccination for prevention of Brucella infection under natural-exposure conditions in Trinidad. Study animals (20 males and 12 females 5–20 months old) were assigned to vaccination or control groups, using a block randomization design ensuring equal sex distributions between groups. The vaccination group received commercially available RB51 at the recommended calfhood dose of (1.0–3.4)×10¹⁰ colony-forming units (CFU) and controls received 2 ml sterile saline. Vaccination did not result in positive serologic results as measured by four traditional agglutination tests: standard tube agglutination test (STAT), standard plate agglutination test (SPAT), buffered plate agglutination test (BPAT), and card agglutination. Study animals were maintained in a brucellosis-positive herd in southern Trinidad with an estimated 56% prevalence to allow for natural exposure to B. abortus, which was evaluated using STAT, SPAT, BPAT, and card tests. Animals were sampled seven times over 2 years and were classified as positive if they had persistent agglutination titers or had Brucella isolated from specimens collected at completion of the study. Five of the original 32 study animals were lost to follow-up during the field trial. Six of the 14 (43%) vaccinated animals completing the study were classified as positive for Brucella infection—as were two of the 13 (15%) control animals (P=0.21). Isolates from four vaccinates and one control were confirmed as B. abortus biovar 1.     

Cross comparison of seminal plasma proteins from cattle and buffalo (Bubalus bubalis)

The objective of this study was to evaluate seminal plasma proteins from cattle and buffalo (Bubalus bubalis), to identify differences between related species. Sixteen buffaloes and 16 cattle between 30 and 60 months of age were used. Semen collection was performed by electroejaculation, followed by macroscopic and microscopic subjective analyses. After analysis, the samples were centrifuged at 800 g for 10 min, and the supernatant (seminal plasma) was recentrifuged at 10,000 g for 30 min at 4°C. The total protein concentration was determined by the Bradford method, and the proteins were digested in solution for mass spectrometry (nLC-MS/MS). Multivariate statistical analysis was used to evaluate the proteomics results by non-hierarchical clustering the considering exponentially modified protein abundance index (emPAI). Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used for clustering. Proteomics identified 78 proteins, and multivariate analysis showed 4 that were over-expressed in buffaloes (cystatin C, prosaposin, peptide YY and keratin type II cytoskeletal 5) and 9 in cattle (spermadhesin-1, seminal plasma protein PDC-109, ribonuclease 4, metalloproteinase inhibitor 2, acrosin inhibitor 1, seminal ribonuclease, C-type natriuretic peptide, angiogenin-1 and osteopontin). Among the proteins identified in seminal plasma, the C-type natriuretic peptide and metalloproteinase inhibitors were described for the first time in buffaloes. Some protease inhibitors were found over-expressed in buffaloes, and important proteins in seminal plasma of cattle were not identified or were found at lower expression levels in buffaloes, which can contribute to reproductive performance in this species.     

Genetic relationship and diversity analysis of Indian water buffalo (Bubalus bubalis)

The water buffalo (Bubalus bubalis) is an important dairy animal on the Indian subcontinent and in Southeast Asian countries. The diversity and differentiation among 12 populations or breeds of buffalo were studied. Data were generated and analyzed from 527 animals belonging to 10 recognized breeds and 2 additional populations of Indian buffalo by using 22 microsatellite loci. Relationships among buffalo breeds and populations were estimated based on genetic distances. The Bayesian analysis grouped 12 populations into 8 distinctive clusters. Geographically close breeds clustered together, except for the Jaffarabadi and Murrah, which were not in geographic contiguity. The Mantel test revealed nonsignificant correlations between genetic and geographic distances. This supports the hypothesis that buffaloes have been domesticated at different places for specific purposes. The phylogenetic relationship based on microsatellite loci supported the breed classification based on body size. The Toda breed, which is considered to be endangered, had genotypes similar to those of the surrounding buffalo populations.     

Comparative evaluation of enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of dourine

The detection of antibodies against Trypanosoma equiperdum in 689 equid sera was compared by enzyme-linked immunosorbent assay (ELISA), the complement fixation test (CFT) and an indirect immunofluorescent test (IIF). CFT was the least sensitive technique, susceptible to anti-complementary factors and the most technically demanding. IIF was more sensitive, but was only suitable for testing limited numbers of samples. In this study, ELISA was the most sensitive test, the least labour intensive and lends itself to a considerable degree of automation. It is suggested that ELISA would be relatively easy to standardise between laboratories and an ELISA protocol could be adopted as the internationally approved test for equine health certification.     

An enzyme-linked immunosorbent assay for detection of Brucella abortus in cattle using monoclonal antibodies

One group of 24 cattle was vaccinated with the usual calfhood dose of B. abortus strain 19 and a further 27 cattle were similarly vaccinated but as adults. Twenty-four cattle (12 from each group) and a control group of 12 cattle were subsequently challenged with B. abortus strain 544. Two monoclonal antibodies (MA (A) and MA (B) ) conjugated to horseradish peroxidase were used independently in a competitive enzyme-linked immunosorbent assay (ELISA) to test the serums. After vaccination with B. abortus strain 19, the performance of the monoclonal antibodies was in general agreement with the CFT as fewer calfhood vaccinates were positive 12 weeks after vaccination to the ELISA with MA (A) and MA (B) than adult vaccinates. After challenge, MA (A) and MA (B) ELISA tests detected the infected cattle earlier than the CFT, but more positive reactions occurred in the cattle that proved uninfected at slaughter.     

Comparison of carcass and meat characteristics of Brahman grade cattle (Bos indicus) and crossbred water buffalo (Bubalus bubalis)

This study was conducted to determine and compare the live traits, carcass yield and the physical, chemical, processing and sensory properties of meat from 20 Brahman grade cattle (crossbred cattle) and crossbred water buffalo (Philippine carabao × Murrah, Bulgarian or Indian) with an average age and weight of 29 months and 434 kg, respectively. The animals were fed a similar diet for 180 days before slaughter. A survey was performed to establish consumer meat preference between the two species. Live weights before slaughter of the two species were found to be different (P < 0.05) with the crossbred water buffalo showing higher bodyweight. However, the crossbred water buffalo had a lower (P < 0.05) dressing yield based on hot carcass weight and chilled carcass weight. The lower dressing yield of crossbred water buffalo can be attributed to its higher (P < 0.05) weights of edible and non‐edible slaughter by‐products. The chilled carcass yield of the forequarters and hindquarters from crossbred cattle and crossbred water buffalo were comparable when expressed as percent of live weight and chilled carcass weight. The estimated lean yield was higher in crossbred cattle than crossbred water buffalo. Crude protein, ash, fat, cholesterol, myofibrillar, sarcoplasmic and insoluble protein contents of the beef and carabao meat were all similar. Water holding capacity, pH, muscle fiber diameter, tenderness, firmness and marbling score in carabeef were all comparable to the beef. Redness was found to be higher (P < 0.05) in carabeef than the beef as measured with a color meter. The consumer meat preference survey showed that 55.9% selected the beef while 44.1% preferred carabeef. The color and amount of fat on the outside of the beef sample were primary in buyer selection.     

Label‐free proteome of water buffalo (Bubalus bubalis) seminal plasma

The study aimed to describe the Bubalus bubalis seminal plasma proteome using a label‐free shotgun UDMS^E approach. A total of 859 nonredundant proteins were identified across five biological replicates with stringent identification. Proteins specifically related to sperm maturation and protection, capacitation, fertilization and metabolic activity were detected in the buffalo seminal fluid. In conclusion, we provide a comprehensive proteomic profile of buffalo seminal plasma, which establishes a foundation for further studies designed to understand regulation of sperm function and discovery of novel biomarkers for fertility. MS data are available in the ProteomeXchange with identifier PXD003728.     

Bioinformatics analysis of candidate genes for milk production traits in water buffalo (Bubalus bubalis)

Tropical Animal Health and Production - Water buffalo (Bubalus bubalis) is an important livestock species in developing countries due to its contribution to meat, milk production, and a certain...     

Prevalence,biology, and distribution pattern of Sarcocystis infection in water buffalo (Bubalus bubalis) in Iran

This study was conducted to determine the prevalence, distribution pattern, and the Sarcocystis species involved in slaughtered water buffaloes (Bubalus bubalis) in the Khuzestan, Iran by macroscopic and histological examination. The esophagus, heart, diaphragm, tongue, masseter, and thigh muscles were investigated. Esophagus and thigh muscles of only 3 of the 100 examined water buffaloes (3%) were infected with macroscopic Sarcocystis, whereas at microscopic level Sarcocystis were found in 83 of the 100 examined animals (83%). The highest prevalence rate of microscopic cysts was found in masseter muscle (57.1%) and then followed by tongue, diaphragm, esophagus, heart, and finally, thigh muscles (30.0%). There was no significant difference between males (83.6%) and females (82.0%) or between two investigated age groups (≤2 years old, 78% versus >2 years old, 88%). Based on the size of the sarcocysts, thickness of the walls and location of the cysts, Sarcocystis buffalonis as a macroscopic form and Sarcocystis levinei and Sarcocystis dubeyi as the microscopic forms were diagnosed in the examined muscles of the buffaloes of this area. Sarcocystis fusiformis was not seen in the examined organs of these buffaloes. The high prevalence rate of microscopic Sarcocystis in this region indicates that dogs have a more significant role than cats in transmission of these protozoa.     

Comparison of a commercial enzyme-linked immunosorbent assay with hemagglutination inhibition assay for serodiagnosis of swine influenza virus (H1N1) infection.

A commercial indirect swine influenza virus (SIV) H1N1 enzyme-linked immunosorbent assay (ELISA) was compared with the hemagglutination inhibition (HI) assay by testing 72 samples from experimentally infected pigs and 780 field samples of undefined SIV status. The HI assay was performed using SIV isolates A/Swine/IA/73 for H1N1 and A/Swine/IA/8548-1/98 for H3N2. The ELISA used an SIV isolated in 1988. The results showed that HI and ELISA detected an antibody in 11 and 6, respectively, of 72 serum samples collected from pigs experimentally infected with a 1992 SIV isolate (A/Swine/IA/40776/92). The presence of antibodies in these experimental samples was confirmed by HI tests in which all 72 samples were positive against the homologous virus, a more recent H1N1 SIV isolate (A/Swine/NVSL/01) supplied by National Veterinary Services Laboratories, Ames, Iowa, and a 1999 H1N1 isolate currently used in a commercial vaccine. On testing 780 field samples, an overall agreement of 85.5% was generated between the HI and ELISA. This study demonstrated that the ELISA is a useful serodiagnostic screening test at herd level for detecting swine antibodies against SIV. However, a new SIV isolate representing current SIV strains circulating in the field is needed to replace the older isolates used in the HI and ELISA to increase the test accuracy for serodiagnosis of SIV.     

Ear-tag retention and identification methods for extensively managed water buffalo (Bubalus bubalis) in Trinidad

Thirty-two young domestic water buffalo were studied to evaluate ear-tag retention during an epidemiologic field trial. Plastic ear-tags were placed in both ears before the start of the trial, which was implemented in an extensively managed domestic water buffalo herd of approximately 1000 animals in Trinidad from 1999–2001. The presence or absence of ear-tags was recorded at the times of animal handling. The rate of ear-tag loss was modeled using a parametric survival analysis assuming an exponential rate of loss. A gamma distribution was used to estimate the amount of time that each animal would be positively identified based only on the presence or absence of one or more ear-tags. The overall median ear-tag retention was 272 days. The estimated rate of ear-tag loss was 0.0024 ear-tags lost per day. The use of ear-tags alone might not be sufficient for long-term identification of extensively managed animal populations.     

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 6?C10?months were used to evaluate serological responses to Brucella abortus strain RB51 (RB51) vaccination in a dose?Cresponse 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?CIII 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).