A study on the epidemiology of brucellosis in bovine population of peri-urban and rural areas of district Multan, southern Punjab, Pakistan.

BACKGROUND: Brucellosis is a zoonotic disease caused by a bacterial pathogen belonging to the genus Brucella. It is one of the most frequent bacterial zoonoses globally but unfortunately, it is still considered as a neglected disease in the developing world. Keeping in view, this study was conducted to determine the prevalence and risk determinants of brucellosis in large ruminants of peri-urban and rural areas of district Multan-Pakistan. For this purpose, blood samples (n = 490) were collected from the cattle (n = 245) and buffalo (n = 245) population of the study area and subjected to preliminary screening of brucellosis using local and imported RBPT reagents. All the samples were further analyzed using commercially available multi-specie indirect ELISA kit followed by their confirmation by PCR using genus and species-specific primers. Data obtained from lab analysis and questionnaires were subjected to statistical analysis for Pearson Chi-square, Odds Ratio and Confidence intervals (95%). RESULTS: The results showed that the maximum seropositivity was recorded with local RBPT reagent (VRI, Pakistan; 12.45%; 95%CI = 9.72-15.65%) followed by RBPT-IDEXX (12.24%; 95%CI = 9.52-15.45%) and RBPT-ID.vet (11.84%; 95%CI = 9.18-14.95%) however statistical difference was non-significant (P = 0.956). The ELISA results showed an overall seroprevalence rate of 11.22% (95%CI = 8.59-14.33%) with comparatively higher rate in cattle (12.65%; 95%CI = 8.82-17.44%) as compared to buffaloes (9.80%; 95%CI = 6.49-14.15%). The PCR analysis confirmed the presence of genus Brucella in all seropositive samples whereas frequency of B. abortus and B. melitensis in seropositive samples was 80% and 20%, respectively. The co-existence of both species was also observed in 5.45% samples. The statistical analysis showed a significant association of bovine brucellosis with herd size, breed, reproductive disorders, mode of insemination, educational status and farmers' awareness about brucellosis (P < 0.05). Conversely, locality, age, weight, gender, pregnancy status, parity and puberty status had no associations with brucellosis (P > 0.05). CONCLUSION: In conclusion, brucellosis is prevalent in large ruminants of district Multan, Pakistan. It is suggested to devise and implement stringent policies for the effective control and prevention of brucellosis in the region. Further, the current situation also warrants the need to strengthen interdisciplinary coordination among veterinarians and physicians in one health perspective to ensure and strengthen the human and animal health care systems in the region.

Seroprevalence of Toxoplasma gondii in water buffaloes and cats in Guangxi, China.

Toxoplasma gondii is a pathogen that poses a serious threat to human health and causes significant economic losses to the global livestock industry. The prevalence of toxoplasmosis infection has been reported to be high in humans and animals around the world, but the occurrence of the disease has not yet been reported in water buffaloes in Guangxi Zhuang Autonomous Region, southern China. To understand the overall seroprevalence of T. gondii infection in Guangxi, a total of 1041 water buffalo and 114 cat serum samples were examined using an indirect enzyme-linked immunosorbent assay (I-ELISA). Of the 1041 water buffaloes analyzed, an overall seroprevalence of 52.9% (551/1041) was obtained, with year, season, and city location being significant factors affecting the rate of T. gondii infection in water buffaloes (P < 0.001). The results also revealed a high seroprevalence of 57% (65/114) in cats. Given that buffalo milk and meat products are vital food sources, these findings suggest that toxoplasmosis in water buffaloes may be a public health threat. This study provides the first T. gondii seroprevalence data in Guangxi, which could contribute to the prevention and control of toxoplasmosis in the region.

Prevalence and risk factor investigation for exposure to Brucella species in surrogate stray cattle population reared in cow shelters in Punjab, India.

Bovine brucellosis is endemic in cattle in India, however not much is known on the prevalence of this disease in stray cattle populations of the country. This study aimed to estimate the prevalence and identify risk factors associated with brucellosis in the stray cattle populations reared in cow shelters (gaushalas) of Punjab, India. Blood samples were collected from 587 cattle reared in 23 cow shelters in 23 districts (one per district) of the Punjab and were tested using Rose Bengal plate test (RBPT), standard tube agglutination test (STAT) and Indirect Enzyme Linked Immunosorbent Assay (i-ELISA). Information on the sex and breed of the animal, total cattle population and presence of a separate shed for parturition were collected. An animal was considered exposed to Brucella infection based on a positive RBPT or STAT test and a positive i-ELISA test. Explanatory variables for the animal level disease status outcome variable were sex and breed of the animal and at the shelter level were shelter cattle population size and presence of a separate shed for parturition. Univariable binomial exact logistic regression analysis was conducted to assess the association of each explanatory variable with the binary outcome variable. Sixty-two animals were seropositive on RBPT, with an apparent seroprevalence of 10.56% (95% confidence interval [CI]: 8.33%, 13.31%) and the estimated true seroprevalence of 11.48% (95% CI: 8.9%, 14.64%). Sixty three animals were seropositive using STAT [apparent seroprevalence of 10.73% (95% CI: 8.48%, 13.50%) and the estimated true seroprevalence of 10.69% (95% CI: 8.27%, 13.67%)], and 68 using i-ELISA [an apparent seroprevalence of 11.58% (95% CI: 9.24%, 14.43%) and the estimated true seroprevalence of 13.28% (95% CI: 10.50%, 16.66%)]. Cross bred cattle had a lower risk of being test positive (odds ratio 0.16, p = 0.04) as compared to indigenous cattle. Due to a ban on cow slaughter in the country, roaming stray cattle infected with brucellosis present a permanent risk of introduction of disease to the dairy farms and other vulnerable populations.

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.

Evaluation of serological diagnostic tests for bovine brucellosis in dairy cattle herds in an endemic area: a multicenter study.

Brucellosis is known as one of the most common zoonotic diseases worldwide affecting both livestock and humans. It causes abortions, reduces milk production, and infertility in infected animals. The disease is routinely diagnosed through three serological techniques, such as rose bengal plate test (RBPT), standard agglutination test (SAT), and indirect enzyme-linked immunosorbent assay (I-ELISA). The aim of this study was to identify and compare the brucellosis seroprevalence among dairy cattle farms through these different serological tests. From 2112 sampled dairy cattle in different parts of Iran, RBPT, SAT, and I-ELISA led to 296 (14.02%), 215 (10.18%), and 297 (14.06%) positive results, respectively. Brucella abortus biovar 3 (62 cases) was identified as the most common cause of brucellosis in tested animals. Our results showed that the specificity and sensitivity of I-ELISA were higher than those obtained by RBPT and SAT. In this study, the overall agreement of RBPT and SAT with I-ELISA reached 95.21% and 94.12% in dairy cattle farms, respectively. Furthermore, Cohen's kappa statistical analysis revealed that the best degree of agreement was seen between RBPT and I-ELISA (0.80), followed by RBPT and SAT (0.78) and finally SAT and I-ELISA (0.72), thereby indicating a strong agreement between RBPT and I-ELISA methods and good agreement between SAT and I-ELISA methods. The McNemar analysis also showed that a significant difference exists between positive and negative results determined by SAT and I-ELISA methods (p < 0.0001). However, the positive and negative results determined by I-ELISA and RBPT did not show a significant difference (p = 0.9207). Therefore, I-ELISA was a more specific and sensitive serological test when compared to RBPT and SAT and could remarkably decrease non-specific reaction by improving the serological screening specificity for an accurate brucellosis diagnosis in endemic areas.

Neospora caninum seroprevalence in water buffaloes in Guangxi, China.

Neospora caninum is an important obligate intracellular apicomplexan parasite that causes spontaneous abortions in cattle and leads to huge economic losses to the farming industry. Although a high prevalence of N. caninum infection has been reported in Asia, data on the prevalence of water buffaloes in China remain unclear. To understand the seroprevalence of N. caninum infection in water buffaloes and its definitive host dogs in China, a total of 987 water buffalo sera from Guangxi Province were tested using an indirect enzyme-linked immunosorbent assay. We obtained an overall seroprevalence of 50.9% (502/987) for water buffalo samples. And the positive rate was higher in border cities (56.8%, 425/748) than in central cities (32.3%, 77/239). We further tested 240 serum samples from dogs in Guangxi and found an overall prevalence of 57.9% (139/240). The high prevalence of N. caninum infection in both dogs and water buffaloes was first reported in southern China, and these data will surely contribute to the prevention and control of the disease.

Bayesian Evaluation of Three Serological Tests for Diagnosis of Brucella infections in Dromedary Camels Using Latent Class Models.

Brucellosis is a zoonotic disease with significant economic and public health impacts. The disease has been found in ruminants, including camels, but clinical diagnosis of camel brucellosis is difficult due to the lack of clinical signs. Thus, this study aimed to estimate the sensitivity (Se) and specificity (Sp) of the Buffered Plate Antigen Test (BPAT), Rose Bengal Test (RBT), and indirect ELISA (i-ELISA) for the diagnosis of Brucella infection in dromedary camels imported from Sudan to Egypt. The secondary objective of the study was to calculate the animal-level true prevalence of Brucella infection in imported camels. A cross-sectional study was carried out on 921 apparently healthy camels randomly selected from those imported from Sudan and kept in the quarantine stations in the Shalateen area of the Red Sea Governorate, Egypt, between June 2018 and January 2019. Serum samples were collected and analyzed using BPAT, RBT, and i-ELISA. The posterior estimates [medians and 95% Bayesian probability intervals (95% BPI)] for Se and Sp of the three serological tests were obtained using Bayesian latent class models (BLCMs). The BLCM was fitted with the assumption that the BPAT and RBT tests were conditionally dependent on the true brucellosis status of camels. All tests had comparable and high Se (>86%) and Sp (>98%). The animal-level true prevalence of Brucella infection in imported camels was 8.6% (95% BPI: 6.8 - 10.7). Based on these findings, the three assays could be used for the initial screening of Brucella infection in camels. However, the BPAT and RBT are more suitable for use in camel brucellosis control and eradication program in Egypt because of their low unit cost and fast turnaround time compared to the i-ELISA. In addition, BPAT and RBT could be performed in the field where in-vivo tests are rarely used due to logistic and management constraints.

Self-Assembly of Porcine Parvovirus Virus-like Particles and Their Application in Serological Assay.

Porcine parvovirus (PPV) is widely prevalent in pig farms. PPV is closely related to porcine respiratory disease complex (PRDC) and porcine circovirus disease (PCVD), which seriously threatens the healthy development of the pig industry. Although commercial antibody detection kits are available, they are expensive and unsuitable for large-scale clinical practice. Here, a soluble VP2 protein of PPV is efficiently expressed in the E. coli expression system. The VP2 protein can be self-assembled into virus-like particles (VLPs) in vitro. After multiple steps of chromatography purification, PPV-VLPs with a purity of about 95% were obtained. An indirect, enzyme-linked immunosorbent assay (I-ELISA), comparable to a commercial PPV kit, was developed based on the purified PPV-VLPs and was used to detect 487 clinical pig serum samples. The results showed that the I-ELISA is a simple, cost-effective, and efficient method for the diagnosis of clinical pig serum and plasma samples. In summary, high-purity, tag-free PPV-VLPs were prepared, and the established VLP-based I-ELISA is of great significance for the sero-monitoring of antibodies against PPV.

Development of an indirect ELISA method based on the VP4 protein for detection antibody against duck hepatitis A virus type 1.

In Picornavirus, the VP4 gene is located inside the viral capsid, but the antibodies it produces have neutralizing activity. At the same time, we know that the VP4 gene is relatively conserved among the four structural proteins, which makes the usage VP4 protein-based antigen potentially meaningful. Therefore, we used purified duck hepatitis A virus type 1 (DHVA-1) recombinant VP4 protein as the coating antigen to establish an indirect method. The optimal antigen, serum and enzyme-labeled antibody dilutions were 1:400 (3.375 µg/mL), 1:80 and 1:1600, respectively. The optimal blocking buffer was 1% skimmed milk powder. The cutoff value was determined to be 0.203, and the analytical sensitivity was 1:1600. The established ELISA method has good specificity, repeatability and sensitivity. It has a high coincidence rate of 70.8 % with the DHVA-1 as the coating antigen. So, it can be used for the detection of DHAV-1 serum antibodies.

Validation of New ELISA Technique for Detection of Aflatoxin B1 Contamination in Food Products versus HPLC and VICAM.

Toxin-contaminated foods and beverages are a major source of illness, may cause death, and have a significant negative economic impact worldwide. Aflatoxin B1 (AFB1) is a potent toxin that may induce cancer after chronic low-level exposure. This study developed a quantitative recombinant AflR gene antiserum ELISA technique for aflatoxin B1 detection in contaminated food products. Aflatoxin B1 residuals from 36 food samples were analyzed with HPLC and VICAM. DNA was extracted from aflatoxin-contaminated samples and the AflR gene amplified using PCR. PCR products were purified and ligated into the pGEM-T vector. Recombinant plasmids were sequenced and transformed into competent E. coli (BL21). Molecular size and B-cell epitope prediction for the recombinant protein were assessed. The purified protein was used to induce the production of IgG antibodies in rabbits. Serum IgG was purified and labeled with alkaline phosphatase. Finally, indirect-ELISA was used to test the effectiveness of polyclonal antibodies for detection of aflatoxin B1 in food samples.

Development and evaluation of an indirect ELISA based on recombinant structural protein VP2 to detect antibodies against duck hepatitis A virus.

An indirect enzyme-linked immunosorbent assay (I-ELISA) based on the VP2 protein of duck hepatitis A virus type 3 (DHAV-3) was established in this study. The optimal dilutions of antigen, serum and goat anti-duck IgG conjugate were 1:1600 (2.23 µg/mL), 1:160 and 1:2000, respectively. The optimal blocking buffer was 1% skim milk. The cut-off value for the method was 0.25, and the analytical sensitivity of the method was 1:5120. The results of specific evaluation showed that except for DHAV-1, DHAV-3 antisera did not cross-react with any other common duck-sensitive pathogens, indicating that this method can be used to detect DHAV-3 and DHAV-1 antibodies. The coefficients of variation (CVs) were lower than 10 %. The coincidence rate between the VP2-DHAV-3-ELISA and the neutralization test was 93.3 %. In summary, the I-ELISA method based on VP2 protein has high sensitivity, specificity, and coincidence rate compared with the neutralization test and has advantages in serum monitoring. The I-ELISA method based on VP2 protein provides a simple and rapid method for the detection of anti-DHAV antibodies and the epidemiological monitoring of DHAV.

Evaluation of newly developed 'six recombinant secretary proteins based 'cocktail ELISA' and 'whole cell lysate' based 'indigenous ELISA' and tissue microscopy' with 'Gold standard' histo-pathology for the diagnosis of Johne's disease in slaughtered goats and buffaloes.

Three screening tests {(newly developed, six recombinant secretory proteins based 'cocktail ELISA', in-house robust 'indigenous ELISA' based on semi-purified protoplasmic antigens and tissue microscopy were evaluated with 'Gold standard', histo-pathology for the diagnosis of Johne's disease in goats and buffaloes. Serum and tissues {mesenteric lymph nodes and intestines) were driven from farmer's goats (n = 77) and buffaloes (n = 40) slaughtered for harvesting meat and farm goats (n = 77), died and necropsied. Twenty seven (35%) goats and 23 (57.5%) buffaloes were positive in all the four tests. Of 134 tissues screened by histo-pathology, 79.8% MLN and 76.8%, intestines, were positive for MAP infection. In tissue microscopy, 55.2 and 52.3%, goats and buffaloes were positive, respectively. Of 117 sera screened by i_ELISA, 58.4 and 70.0%, goats and buffaloes were positive, respectively. Whereas, c_ELISA detected 55.8 and 62.5%, goats and buffaloes, positives, respectively. Twelve tissues (70.5%) of goats necropsied were positive, both in tissue microscopy and histo-pathology. Most significant gross findings were serous atrophy of the fat and mild to moderate, diffuse thickening of terminal ileum, especially at ileo-caecal junction with or without transverse / longitudinal corrugations. In histo-pathology grade III and IV lesions were significantly low as compared to grade I and II. Of the four tests used for screening 268 samples, histo-pathology was most sensitive (78.3%), followed by i_ELISA (62.3%), c_ELISA (58.9%) and tissue microscopy (58.9%). Between two ELISA tests, c_ELISA using six recombinants secretory proteins, had higher specificity as compared to i_ELISA.

Development and evaluation of an indirect ELISA based on recombinant nonstructural protein 3A to detect antibodies to duck hepatitis A virus type 1.

To develop an indirect enzyme-linked immunosorbent assay(I-ELISA) method based on 3A protein of duck hepatitis A virus type 1(DHAV-1) for detection of DHAV-1 antibody, the recombinant protein 3A of DHAV-1 was expressed in E.coli and detected by Western blotting with DHAV-1 infected duck serum. A 3A-ELISA method using the expressed 3A protein as coating antigen for the detection of antibodies to DHAV-1 was developed. The optimal antigen, serum and enzyme-labeled antibody dilutions were 1:200(6.185 µg/ml), 1:20 and 1:2000, respectively. The optimal blocking buffer was 5% BSA. The cutoff value was determined to be 0.274, and the analytical sensitivity was 1:1280. There was no cross reaction between DHAV-1 infected duck serum and other common pathogenic duck serum, indicating that I-ELISA could be used to detect DHAV-1 infected duck serum. The coefficients of variation(CVs) were lower than 10%. The concordance between the I-ELISA based on the 3A subunit of DHAV-1 and that based on the whole DHAV-1 particle was 92.7%. Taken together, the 3A-ELISA method is a highly sensitive and specific test that could be used for screening for DHAV-1 infection and monitoring DHAV-1 antibody.

Indirect ELISA using a multi-epitope recombinant protein to detect antibodies against foot-and-mouth disease virus serotype O in pigs.

Foot-and-mouth disease (FMD) is a devastating animal disease. A previously developed multi-epitope protein B4 vaccine of the FMD virus (FMDV) serotype O provides safety advantages over inactivated vaccines and could be used to prevent and control FMD in pigs. Commercial enzyme-linked immunosorbent assay (ELISA) kits for assessing vaccine efficacy are available for the inactivated vaccines but not for the multi-epitope protein vaccine. In this study, multi-epitope protein B4 was expressed in Escherichia coli, and an indirect ELISA (I-ELISA) was developed to detect antibodies against FMDV serotype O in pigs. The specificity and sensitivity were 96.7% and 95.9%, respectively. B4-vaccinated pigs yielded B4 I-ELISA serum values that were positively correlated with clinical protection against challenge with FMDV serotype O. The I-ELISA's ability to detect antibodies from animals vaccinated with the inactivated vaccine was also evaluated, and the B4 I-ELISA values were significantly positively correlated with liquid-phase blocking ELISA (LPBE) titers (r = 0.6708, p < 0.0001); thus, the I-ELISA was also suitable for detection of antibodies from swine vaccinated with the inactivated vaccine.

Recombinant Fiber-2 Protein-Based Indirect ELISA for Antibody Detection of Fowl Adenovirus Serotype 4.

Clinical cases of hepatitis-hydropericardium syndrome (HHS) from fowl adenovirus serotype 4 (FAdV-4) have increased in China since 2013. Therefore, the development of a new serologic method for HHS detection is now urgent. Here, the FAdV-4 strain JSJ13 was used to construct a plasmid for prokaryotic expression of the JSJ13 fiber-2 protein. The protein, purified by affinity chromatography, was refolded by gradient dialysis. After coating a 96-well plate with the purified fiber-2 protein (1.5 µg/ml), standard serum and secondary antibodies (1:200 and 1:6000 dilutions, respectively) were used to develop an indirect ELISA (I-ELISA). Nine field-collected serum samples and JSJ13-positive serum were tested by I-ELISA and the results corresponded with those of the serum neutralization test. The I-ELISA was used to test 450 clinical serum samples from different parts of China. Chickens from nonvaccinated flocks had low antibody titers and low virus positivity rates. In contrast, FAdV-4 vaccinated chickens were strongly positive, and the positivity rates of all the flocks exceeded 73.3%. The newly developed I-ELISA with the recombinant JSJ13 fiber-2 protein as the antigen detected antibodies to FAdV-4 accurately and sensitively.

Evaluation of three serological tests for diagnosis of bovine brucellosis in smallholder farms in Pakistan by estimating sensitivity and specificity using Bayesian latent class analysis.

The present study aimed to estimate the herd-level sensitivity (Se) and specificity (Sp) of three commonly used serological tests in naturally-infected cattle and buffalo in smallholder farms in Pakistan. Between February and June 2015, a cross-sectional study was carried out in five districts of Punjab (Kasur, Okara, Pakpattan, Jhelum, and Bhakkar) and two districts of Sindh (Badin and Thatta). Serum samples from mixed farms of cattle (n=441) and buffalo (n=621) were collected and tested using the Rose Bengal test (RBT), indirect ELISA (I-ELISA) and competitive ELISA (C-ELISA). A Bayesian latent class analysis (LCA) approach was used to estimate the Se and Sp of these three serological tests and the true herd-level prevalence in each district. The model was fitted under the assumption of conditional independence between three tests and also conditional dependence by including covariances between the two ELISAs. In addition, the model was fitted using three different shapes of beta distributions to incorporate prior information in the model. The test with the highest Se was the C-ELISA, with a range from 76.3% (95% PCI (Posterior Credibility Interval), 62.6-88.2%) to 81.4% (95% PCI, 68.2-92.8%). The RBT was found to have the highest Sp (99.1-99.4%) of the tests. The highest estimated herd-level prevalence, 45% (95% PCI, 32-59%), was found in Jhelum district and the lowest in Thatta district, 1.1% (95% PCI 0.04-6.0%). The results of this study identified some discrepancy between the published literature on the level of Se of these tests, especially for RBT. It appears that RBT has lower Se and higher Sp when used in the field conditions of the present study. Consequently, it is recommended that none of the evaluated tests should be performed in isolation for the diagnosis of bovine brucellosis in the field conditions of Pakistan, but the use of tests in combination, with RBT and C-ELISA used in parallel returning optimal Se and Sp, is warranted.

Validation of an inhibition enzyme-linked immunosorbent assay system for the diagnosis of Toxoplasma gondii infection in buffaloes (Bubalus bubalis). / Validación de un sistema inmunoenzimático de inhibición de un anticuerpo para el diagnóstico de infección por Toxoplasma gondii en búfalos (Bubalus bubalis).

Toxoplasmosis is one of the most widespread zoonoses in the world, due to the existence of a wide variety of Toxoplasma gondii hosts, which include several domestic animal species. In Cuba, there is sustained production of the Bubalus species, which is highly adaptable and disease resistant, although it has been identified as a reservoir for a range of aetiological agents. Several countries have reported buffaloes as the intermediate host of T. gondii, noting the need to carry out epidemiological studies and confirm the possible presence of this parasitic infection in the Bubalus species. The current study was conducted to validate an inhibition enzyme-linked immunosorbent assay (i/ELISA) system for the diagnosis of T. gondii infection in buffaloes (Bubalus bubalis). This involved evaluating its performance in relation to that of a latex agglutination test. With buffalo sera, the i/ELISA assay showed a sensitivity of 100%, a specificity of 99.5%,and a concordance of 0.99 (considered very good) with respect to the reference diagnostic method. The conclusion is that i/ELISA performs extremely well as a serological test for the diagnosis of T. gondii in buffaloes.

La toxoplasmose est l'une des zoonoses les plus répandues dans le monde, ce qui s'explique par le très large spectre d'hôtes de Toxoplasma gondii, dont plusieurs espèces d'animaux domestiques. À Cuba, les buffles font l'objet d'un élevage durable et présentent de bonnes aptitudes d'adaptation et de résistance aux maladies, bien que cette espèce joue un rôle avéré de réservoir pour un certain nombre d'agents pathogènes. Des rapports émanant de plusieurs pays ont fait état du rôle joué par les buffles en tant qu'hôtes intermédiaires de T. gondii, d'où la nécessité d'effectuer des études épidémiologiques afin de confirmer la présence éventuelle de cette parasitose chez cette espèce. Les auteurs présentent des résultats d'une étude de validation d'une méthode immuno-enzymatique d'inhibition des anticorps pour le diagnostic de T. gondii chez le buffle (Bubalusbubalis). Pour ce faire, les performances de cette méthode ont été évaluées par rapport à celles du test d'agglutination au latex. La comparaison à partir de sérums de buffles a montré que la sensibilité de l'épreuve immuno-enzymatique était de 100 % et sa spécificité de 99,5%, avec une concordance par rapport à la méthode de référence de 0,99, ce qui est considéré un très bon résultat. Cette étude démontre l'aptitude de l'ELISA d'inhibition pour le diagnostic sérologique de T. gondii chez le buffle et conclut à son excellente performance diagnostique.

La toxoplasmosis es una de las zoonosis más difundidas en el mundo debido a que existe una amplia variedad de hospedadores de Toxoplasma gondii, entre los que se encuentran varias de las especies de animales domésticos. En Cuba, la especie bufalina se produce de manera sostenida con buena adaptabilidad y resistencia a las enfermedades, aunque se ha identificado como reservorio de diversos agentes etiológicos. En varios países se ha informado que los búfalos son hospedadores intermediarios de T. gondii y se ha indicado la necesidad derealizar estudios epidemiológicos y de comprobar la posible presencia de dicha parasitosis en esta especie. Este trabajo se realizó para validar un sistema inmunoenzimático de inhibición de un anticuerpo (ELISA/i) para el diagnósticode infección por T. gondii en búfalos (Bubalus bubalis). Para ello, se evaluó su rendimiento respecto a una prueba de aglutinación por látex. Frente a sueros de búfalo, el sistema inmunoenzimático demostró tener una sensibilidad del 100%, una especificidad del 99,5% y una concordancia de 0,99, considerada muy buena, respecto al método de diagnóstico de referencia. Se concluye que el ELISA/i permite el diagnóstico serológico de T. gondii en búfalos con un excelente rendimiento diagnóstico.

Comparative efficacy of serological diagnostic methods and evaluation of polymerase chain reaction for diagnosis of bovine brucellosis.

The aim of present study was to compare the diagnostic performance of the different Brucella abortus antigen based serological and molecular tests such as Rose Bengal Plate Test (RBPT), indirect enzyme linked immunosorbent assay (I-ELISA) and polymerase chain reaction (PCR). Out of 89 samples, 44 were positive by I-ELISA, 18 by RBPT and 21 by PCR. Substantial agreement was observed between PCR and I-ELISA (κ=0.48). A slight degree of agreement was observed between RBPT and I-ELISA and PCR (κ=0.18) and RBPT and PCR (κ=0.11). Indirect enzyme linked immunosorbent assay detected more samples as positive among these tests. In conclusion, I-ELISA can be routinely used for an accurate and efficient diagnosis of Brucella infection, because the chances of non-detection of an infected animal in I-ELISA are minimal. However, PCR could be used as a supplement and complement test along with I-ELISA for identification and differentiation of bovine brucellosis.

Comparison between in-house indirect ELISA and Dot-ELISA for the diagnosis of Fasciola gigantica in cattle.

This survey was done to investigate the efficacy of the in-house indirect ELISA (iELISA) and Dot-ELISA methods Prepared from excretion-secretory (ES Ag) and Crude (Cr Ag) antigens of Fasciola for sero-diagnosis of Fasciola gigantica in cattle. The liver specimens of slaughtered cattle were collected and their liver examined macroscopically and microscopically for infestation to fasciolosis. Sera from two groups of cattle, one infected with fasciolosis (n = 60) and the other non-infected with fasciolosis (n = 60), were used in the iELISA and Dot-ELISA test; grouping based on histopathology results. Except specificity, other parameters such as, sensitivity, accuracy, positive and negative predictive values of both Dot-ELISA and iELISA done with ES Ag were better than those of tests performed with Cr Ag. Interestingly, the reliability of two methods was very good similar to one another.

Expression, purification and immunochemical characterization of recombinant OMP28 protein of Brucella species.

Brucellosis is the lion's share of infectious disease of animals and it has a particular socio-economic importance for the Republic of Kazakhstan. Sixty percent of epizootic outbreaks of brucellosis identified in the Commonwealth of Independent States (CIS) originated from Kazakhstan in recent years. Definitive diagnosis of brucellosis remains a difficult task. Precisely for this reason, we evaluated a purified recombinant out membrane protein 28 (rOMP28) of Brucella species (Brucella spp.) produced in Escherichia coli (E. coli) as a diagnostic antigen in an Indirect ELISA (I-ELISA) for bovine brucellosis. The gene encoding OMP28 was synthesized using a two-round PCR procedure. In order to produce the rOMP28, the de novo synthesized DNA was cloned into the expression vector pET-22b(+). Then, the rOMP28 was expressed in E. coli system and characterized in the present study. We further estimated the diagnostic potential of purified rOMP28 of Brucella spp. for screening bovine sera. To determine if rOMP28 has a valuable benefit for use in the serodiagnosis of bovine brucellosis, rOMP28-based I-ELISA was performed. Brucella spp. positive (n=62) and Brucella spp. negative (n=28) samples from tube agglutination test (TAT) were positive (n=59) and negative (n=27) by I-ELISA, respectively. These findings show that the rOMP28 of Brucella spp. could be a good candidate for improving serological diagnostic methods for bovine brucellosis.