Influence of Haemolysis on the Mineral Profile of Cattle Serum

Haemolysis of serum samples is the leading cause of preanalytical errors in clinical laboratories. Little is known about the potential alterations in the concentrations of mineral elements in haemolyzed serum and the phenomenon has not been specifically studied in bovine serum samples. We investigate how haemolysis affects the mineral content of bovine samples. We used ICP-MS to measure the concentrations of 12 mineral elements (Ca, Co, Cr, Cu, Fe, Mg, Mn, Mo, Ni, P, Se and Zn) in bovine whole blood, serum and gradually haemolyzed samples and observed significant differences between the different types of samples, particularly in the Fe and Zn concentrations. However, in practice, the high interindividual variability makes it difficult to establish whether a given value corresponds to normal or haemolyzed samples. In response to this problem, we propose to consider that a result is significantly biased when the haemolysis threshold (the degree of haemolysis above which the concentration of an element in serum is significantly altered) of a given element is surpassed. The haemolysis threshold values for the different elements considered were found as follows: 0.015 g Hb L−1 for Fe, 2 g for Zn, 4 g for Cr and 8 g for Ca, Se and Mo.

Correlation Between Serology Test Result of Leptospira sp. With The Representation of Histopathological Lesions on The Cattle Kidneys

Leptospirosis is an eminent diseases among human and animal health. As a zoonosis disease, the occurrence of leptospirosis is not clearly understood in animal. Furthermore, the lesion caused by Leptospira sp. is not well demonstrated. This study aimed to analyze the correlation between the result of serological test using microscopic agglutination test (MAT) and the representation of histopathological lesion in kidney from the cattle. This study used 28 samples consist of cattle serum and kidney organs. The serum was tested using MAT and kidney was tested using histopathology. The data was reported semi quantitatively and tested using Spearman test. The result showed that there is no correlation between the result of serological test to the representation of histopathological lesion from the kidney of cattle. It is supported by the coefficient correlation (0,05) and probability value p=0,78 (p≥0,05). In conclusion, the result of Leptospira sp. serological test either seropositive or seronegative uncorrelated to the representation of histopathological lesion from the cattle kidney.

Comparative evaluation of the immunodominant proteins of Brucella abortus for the diagnosis of cattle brucellosis

Background and Aim: The present serodiagnosis of brucellosis in livestock is based on the whole cell or smooth lipopolysaccharide of the Brucella organism in which specificity is hampered by the cross-reactivity, especially with the antibodies against Yersinia enterocolitica O:9 organism. The problem can be addressed by screening for better immunodominant antigens. Hence, the present study was undertaken to screen protein antigens of Brucella abortus for their diagnostic potential in cattle brucellosis. Materials and Methods: Protein antigens of B. abortus (n=10) non-reactive to antibodies against Y. enterocolitica O:9 were selected, expressed in Escherichia coli, assessed the reactivity of expressed recombinant proteins by Western blot, standardized indirect-enzyme-linked immunosorbent assay (ELISA) for detecting Brucella antibodies in cattle serum, and comparative evaluation was done. Results: All the selected protein antigens were expressed and in the Western blot with Brucella antibodies positive cattle serum, six recombinant (Brucella protein 26 [BP26], Cu-Zn Superoxide dismutase [SodC], B. abortus I-1885, Serine protease, Bacterioferritin, and Brucella Lumazine Synthase [BLS]) proteins showed reaction whereas none of the proteins showed reactivity with Brucella negative cattle serum. ELISA has been done using known Brucella positive and negative cattle sera samples (n=113 each) in which the performance of recombinant proteins in diagnosing brucellosis was in the order of BP26 > BLS > SodC followed by rest of the proteins. BP26 based ELISA was found to be better with area under the curve as 0.953, and diagnostic sensitivity, diagnostic specificity, and Youden's index of 90.27%, 95.58%, and 0.8584, respectively, with the excellent agreement (k=0.85). Conclusion: BP26 could be a potential diagnostic antigen among the immunodominant proteins of B. abortus in ruling out Y. enterocolitica O:9 infection while diagnosing brucellosis in cattle herds.

The detection of antibody to Bacillus anthracis recombinant PA in vaccinated animal serum

Anthrax is a worldwide zoonosis in animals and human. In Mongolia, the confirmed case of anthrax outbreak is reported every year over the past decade. The prevention and control measure of animal anthrax is vaccination using spore of attenuated Sterne strain, but horse does not get vaccinated in Mongolia. In this study, we constructed the recombinant plasmid for over expression of anthrax protective antigen (PA)/GST fusion protein in pGEX-6P-1 vector and purified the recombinant PA (r-PA) using glutathione Sepharose column under native and denaturing conditions. Since both forms of r-PA were recognized by specific antibody against PA, ELISA system to detect antibody titer in vaccinated bovine serum was constructed. Total of 890 vaccinated cattle serum were collected from 178 cattle at 0, 3, 5, 8 and 12 months’ post vaccination. As negative control, 200 cattle serum from Umnugovi aimag were selected which does not have anthrax foci. All serum was tested by rPA indirect ELISA and, antibody to PA were detected in vaccinated cattle serum but were not detected in negative serum. Therefore, rPA should be used in as monitoring of the anthrax vaccination.

Performance of the Dot-blot test method for detecting antibodies to Sarcocystis spp. in cattle

ABSTRACT: Serological techniques can detect antibodies against Sarcocystis spp., Neospora caninum and Toxoplasma gondii antigens in single or mixed infections. Immunofluorescent antibody tests (IFAT) is considered the gold standard technique for Sarcocystosis diagnostic in cattle serum and a positive IFAT result reflects Sarcocystis spp. infection. Therefore, the aims of the present study were to compare IFAT and Dot-blot for sarcocystosis diagnostic in experimentally infected mice and to investigate serological cross-reactions with N. caninum and T. gondii in these methods. Mice (Mus musculus) were inoculated intraperitoneally with bradizoites of Sarcocystis spp. or tachyzoites of N. caninum or T. gondii. Serum samples were obtained and analyzed by IFAT and Dot-blot for the three protozoa. Serum from N. caninum and T. gondii experimentally infected mice were tested by IFAT and reacted only to N. caninum or T. gondii antigens, respectively. Specific antibodies against Sarcocystis spp. were present in all animals experimentally infected with this protozoan, with IFAT titers from 10 to 800. Serum samples from mice experimentally infected with Sarcocystis spp., N. caninum and T. gondii and tested by Dot-blot demonstrated no cross reaction between protozoa. A Dot-blot using Sarcocystis spp. antigen appears to be a good alternative to IFAT in the serological diagnosis of Sarcocystosis.

The validation of a commercial enzyme-linked immunosorbent assay and the effect of freeze-thaw cycles of serum on the stability of cortisol and testosterone concentrations in Aceh cattle

Background: To obtain accurate measurements of cortisol (C) and testosterone (T) in Aceh cattle, commercial enzyme-linked immunosorbent assay (ELISA) kits need to be carefully validated. Moreover, repeated freeze-thaw cycles during the storage of the samples may affect the stability of the hormones in the serum. Here, the reliability of C and T concentration measurements in the serum of Aceh cattle, was tested using commercial C and T ELISA kits designed to measure human C and T concentrations. Further, the effect of repeated freeze-thaw cycles on the stability of C and T concentrations in the serum was evaluated. Methods: Commercial C (Cat. no. EIA-1887) and T (Cat. no. EIA-1559) ELISA kits from DRG Instruments GmbH were validated through an analytical validation test (i.e., parallelism, accuracy, and precision) and a biological validation test (for C: effect of transportation on the C secretion; for T: the concentrations of T between bulls and cows). To test the effects of freeze-thaw cycles, cattle serum was subjected to the following treatments: (i) remained frozen at -20OC (control group); (ii) exposed to freeze-thaw cycles for two, four, six, and eight times (test groups). Results: Parallelism, accuracy, and precision tests showed that both C and T ELISA kits adequately measured C and T in the serum of Aceh cattle. Concentrations of C post-transportation were significantly higher than pre-transportation (p<0.01). Concentrations of T in bulls were significantly higher than in cows (p<0.01). After four to eight freeze-thaw cycles, C concentrations were significantly lower compared to the control group (all p < 0.05). In contrast, T concentrations remained stable (all p>0.05). Conclusions: Commercial C (EIA-1887) and T (EIA-1559) ELISA kits are reliable assays for measuring serum C and T, respectively, in Aceh cattle. Repeated freeze-thaw cycles significantly affected the stability of serum C, but did not for T.

The validation of a commercial enzyme-linked immunosorbent assay and the effect of freeze-thaw cycles of serum on the stability of cortisol and testosterone concentrations in Aceh cattle

Background: To obtain accurate measurements of cortisol (C) and testosterone (T) in Aceh cattle, commercial enzyme-linked immunosorbent assay (ELISA) kits need to be carefully validated. Moreover, repeated freeze-thaw cycles during the storage of the samples may affect the stability of the hormones in the serum. Here, the reliability of C and T concentration measurements in the serum of Aceh cattle, was tested using commercial C and T ELISA kits designed to measure human C and T concentrations. Further, the effect of repeated freeze-thaw cycles on the stability of C and T concentrations in the serum was evaluated. Methods: Commercial C (Cat. no. EIA-1887) and T (Cat. no. EIA-1559) ELISA kits from DRG Instruments GmbH were validated through an analytical validation test (i.e., parallelism, accuracy, and precision) and a biological validation test (for C: effect of transportation on the C excretion; for T: the concentrations of T between bulls and cows). To test the effects of freeze-thaw cycles, cattle serum was subjected to the following treatments: (i) remained frozen at -20OC (control group); (ii) exposed to freeze-thaw cycles for two, four, six, and eight times (test groups). Results: Parallelism, accuracy, and precision tests showed that both C and T ELISA kits adequately measured C and T in the serum of Aceh cattle. Concentrations of C post-transportation were significantly higher than pre-transportation (p<0.05). Concentrations of T in bulls were significantly higher than in cows (p<0.05). After four to eight freeze-thaw cycles, C concentrations were significantly lower compared to the control group (all p < 0.05). In contrast, T concentrations remained stable (all p>0.05). Conclusions: Commercial C (EIA-1887) and T (EIA-1559) ELISA kits are reliable assays for measuring serum C and T, respectively, in Aceh cattle. Repeated freeze-thaw cycles significantly affected the stability of serum C, but did not for T.

A seroepidemiological study of bovine cysticercosis in Bali and Nusa Tenggara, Indonesia

Background and Aim: Taenia saginata hazardously affects human and animal health. The distribution of this disease is found almost all over the world. The study aimed to obtain epidemiological information concerning prevalence and the distribution of bovine cysticercosis in Bali and Nusa Tenggara, Indonesia. Materials and Methods: A total of 267 community-owned Bali cattle serum samples from the provinces of Bali, West Nusa Tenggara, and East Nusa Tenggara were examined. The study was conducted by examining the serum of Bali cattle using enzyme-linked immunosorbent assay technique. Risk factors related to cysticercosis that analyzed were sex, breeding type, age, physical condition, source of drinking water, pen condition, and latrine availability. Results: Seven of 91 Bali cattle sera from all regencies/cities in Bali showed a positive result. Those positive sera were originated from Buleleng (1), Gianyar (2), Denpasar (2), and Klungkung (2). Meanwhile, four of 92 Bali cattle sera from West Nusa Tenggara and seven of 84 from East Nusa Tenggara occurred antibodies against T. saginata. We identified that two risk factors that influence the incidence of T. saginata infection in Bali cattle in Bali were the sex and the cattle breeding type. Conclusion: Through this research can be made a map of bovine cysticercosis in Bali cattle in Bali and Nusa Tenggara region. By mapping the disease, it is recommended that the animal health officers should be more accurate when conducting postmortem examination, especially on cattle from a positive region.

Comparative evaluation of two commercial ELISA kits for detection of antibodies against Akabane virus in cattle serum

Background Akabane disease (AD), a barrier to international trade for endemic areas with far economic impact on the countries, is caused by Akabane virus (AKAV). Commercial enzyme-linked immunosorbent assay (ELISA) is a commonly used diagnostic technique for AKAV infection, including the IDEXX and IDVET ELISA kits. However, the comparative evaluation of the IDEXX and IDVET ELISA kits has not been published. The object of this study was to evaluate the test performance of the two commercial ELISA kits in detecting serum anti-AKAV antibodies in cattle. Results With virus neutralization test (VNT) as the “relative gold standard”, the diagnostic sensitivity (DSe) was 80.39% (123/153) and 93.46% (143/153) for the IDEXX and IDVET ELISA kit, when suspect samples were included. The diagnostic specificity (DSp) for the IDEXX and IDVET ELISA kit was 93.48% (502/537) and 82.31% (442/537), respectively. Conclusion Both of the tested ELISA kits could be applied to detect antibodies against AKAV in cattle serum. The IDVET ELISA kit had a higher DSe. The IDEXX ELISA kit possessed the higher DSp. These results have important implications if the kits are used to screen herds or individual cattle in surveillance programs, or at border crossings for import-export inspection and quarantine.