Exposure to Brucella Species, Coxiella burnetii, and Trichinella Species in Recently Imported Camels from Sudan to Egypt: Possible Threats to Animal and Human Health.

Brucellosis and coxiellosis/Q fever are bacterial infections caused by Brucella species and Coxiella burnetii, respectively; camels are highly susceptible to both pathogens. Trichinellosis is a parasitic infection caused by various Trichinella nematode species. Reportedly, camels are susceptible to experimental infection with Trichinella spp., but information on this potential host species is scarce. All three infections are of zoonotic nature and thus of great public health concern. The current study aimed to determine antibodies against the three pathogens in recently imported camels (n = 491) from Sudan at the two main ports for the entrance of camels into southern Egypt using commercial indirect ELISAs. Samples were collected in two sampling periods. The seropositivity rates of Brucella spp., C. burnetii, and Trichinella spp. were 3.5%, 4.3%, and 2.4%, respectively. Mixed seropositivity was found in 1% for Brucella spp. and C. burnetii. Marked differences were found between the two study sites and the two sampling periods for Brucella. A higher rate of seropositivity was recorded in the Red Sea/older samples that were collected between 2015 and 2016 (4.3%, 17/391; odds ratio = 9.4; p < 0.030) than in those collected in Aswan/recent samples that were collected between 2018 and 2021 (0/100). Concerning C. burnetii, samples collected during November and December 2015 had a significantly higher positivity rate than the other samples (13%, 13/100; OD = 4.8; p < 0.016). The same effect was observed for antibodies to Trichinella spp., with samples collected during November and December 2015 showing a higher positivity rate than the other samples (7%, 7/100; OD = 10.9; p < 0.001). This study provides valuable information on the seroprevalence of Brucella spp. and additional novel information on C. burnetii and Trichinella spp. in recently imported camels kept in quarantine before delivery to other Egyptian regions. This knowledge can be utilized to reduce health hazards and financial burdens attributable to brucellosis, Q fever, and trichinellosis in animals and humans in Egypt.

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.

Harnessing Attenuation-Related Mutations of Viral Genomes: Development of a Serological Assay to Differentiate between Capripoxvirus-Infected and -Vaccinated Animals.

Sheeppox, goatpox, and lumpy skin disease caused by the sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV), respectively, are diseases that affect millions of ruminants and many low-income households in endemic countries, leading to great economic losses for the ruminant industry. The three viruses are members of the Capripoxvirus genus of the Poxviridae family. Live attenuated vaccines remain the only efficient means for controlling capripox diseases. However, serological tools have not been available to differentiate infected from vaccinated animals (DIVA), though crucial for proper disease surveillance, control, and eradication efforts. We analysed the sequences of variola virus B22R homologue gene for SPPV, GTPV, and LSDV and observed significant differences between field and vaccine strains in all three capripoxvirus species, resulting in the truncation and absence of the B22R protein in major vaccines within each of the viral species. We selected and expressed a protein fragment present in wildtype viruses but absent in selected vaccine strains of all three species, taking advantage of these alterations in the B22R gene. An indirect ELISA (iELISA) developed using this protein fragment was evaluated on well-characterized sera from vaccinated, naturally and experimentally infected, and negative cattle and sheep. The developed wildtype-specific capripox DIVA iELISA showed >99% sensitivity and specificity for serum collected from animals infected with the wildtype virus. To the best of our knowledge, this is the first wildtype-specific, DIVA-capable iELISA for poxvirus diseases exploiting changes in nucleotide sequence alterations in vaccine strains.

A Highly Sensitive Indirect Enzyme-Linked Immunosorbent Assay (ELISA) Based on a Monoclonal Antibody Specific to Thermal Stable-Soluble Protein in Pork Fat for the Rapid Detection of Pork Fat Adulterated in Heat-Processed Beef Meatballs.

Processed foods containing pork fat tissue to improve flavor and gain economic benefit may cause severe issues for Muslims, Jews, and vegetarians. This study aimed to develop an indirect enzyme-linked immunosorbent assay (iELISA) based on a monoclonal antibody specific to thermal stable-soluble protein in pork fat tissue and apply it to detect pork fat tissue in heat-processed (autoclave, steam, roast, and fry) beef meatballs. To develop a sensitive iELISA, the optimal sample pre-cooking time, coating conditions, primary and secondary dilution time, and various buffer systems were tested. The change in the iELISA sensitivity with different 96-well microtiter microplates was confirmed. The detection limit of iELISA performed with an appropriate microplate was 0.015% (w/w) pork fat in raw and heat-treated beef. No cross-reactions to other meats or fats were shown. These results mean that the iELISA can be used as an analytical method to detect trace amounts of pork fat mixed in beef.

Evaluation of an iELISA for detection and quantification of rabies antibodies in domestic dog sera.

Many rabies endemic-countries have recognized rabies as a public health problem that can be eliminated. As a result, some countries have started implementing small-scale vaccination programs with the aim of scaling them up. Post-vaccination serological monitoring is crucial to assess the efficacy of these programs. The recommended serological tests, the rapid fluorescent focus inhibition test, and the fluorescent antibody virus neutralization (FAVN) are accurate; however, the procedures require considerable expertise and must be carried out in high containment facilities, which are often not available in rabies endemic countries. Given these constraints, enzyme linked immunosorbent assays (ELISAs) have been considered as alternative methods to neutralization tests. This is the first study to evaluate, under field conditions, the performance of the commercial rabies indirect-ELISA (iELISA), the PlateliaTM Rabies II kit ad usum Veterinarium kit, using sera from domestic dogs. Serum samples were collected from two groups of community dogs in northern Tanzania: i) dogs with no history of vaccination against rabies (n = 100) and ii) dogs vaccinated with the Nobivac Canine Rabies® vaccine (n = 101) four weeks previously. When compared to the gold standard FAVN test, the iELISA was found to be 99% specific and 98% sensitive and there was a significant correlation between the two tests (p < 0.001, r = 0.92). Given these findings, we conclude that the PlateliaTM Rabies II kit ad usum Veterinarium can be considered a valuable tool for the rapid assessment of vaccination status of animals in vaccination programs.

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.

Seroepidemiology Study of Porcine Epidemic Diarrhea Virus in Mexico by Indirect Enzyme-Linked Immunosorbent Assay Based on a Recombinant Fragment of N-Terminus Domain Spike Protein.

Porcine epidemic diarrhea (PED) is an intestinal disease caused by the porcine epidemic diarrhea virus (PEDV) and affects Mexico's swine industry. Despite the disease initially being described in Mexico in 2013, there has been no research into the virus's seroepidemiology carried out in Mexico. Thus, the goal of this study was to develop an indirect ELISA (iELISA) based on a recombinant N-terminal domain truncated spike (S) protein (rNTD-S) of PEDV to evaluate serum obtained from different pig-producing states in Mexico. A total of 1054 sera were collected from pig farms, slaughterhouses, and backyard production in the states of Aguascalientes, Guanajuato, Hidalgo, Jalisco, Morelos, Queretaro, Sinaloa, and Veracruz between 2019 and 2021. The rNTD-S protein was expressed in E. coli BL21 (DE3) cells. Negative and positive serum samples used in the iELISA were previously tested by Western blot. According to our findings, 61.66% of the serum samples (650/1054) were positive, with Jalisco having the highest percentage of positive samples, at a rate of 21.44% (226/1054). This is the first seroepidemiology study of PEDV carried out in Mexico, revealing that the virus is still circulating since the initial outbreak; furthermore, it provides an overview of PEDV's spread and high level of persistence across the country's key swine-producing states.

Development of the first PCR for detection of Psoroptes ovis var. cuniculi infection and its comparison to microscopic examination and serological assay in rabbits.

Psoroptes mites are the common ecto-parasites of wild and domestic animals worldwide, which causes considerable economic losses in livestock industry. Microscopy is deemed to be the 'gold standard' for the diagnosis of Psoroptes mite infection but it has poor sensitivity for low mite infections and/or sub-clinical infections. To overcome these shortcomings, we screened four genes to develop a sensitive and specific PCR for the detection of Psoroptes mite infection in rabbits, and confirmed its practicability in detecting early infection and monitoring treatment outcome with traditional microscopy and serological tests. Results showed that PCR assay targeting ITS2 (ITS2-PCR) had a high specificity and sensitivity (detection limit: 40.3 pg/µL DNA) for detecting P. ovis DNA. In rabbits artificially infected with P. ovis, all three diagnostic tests showed the same detection rate from 14 days post infection (dpi) to 42 days dpi. However, these diagnostic tests behave differently at 7 dpi and after treatment: at 7 dpi, the detection rate of ITS2-PCR was higher than rPsoSP3-based iELISA and traditional microscopy (ITS2-PCR: 88.9%, rPsoSP3-iELISA: 77.7%, microscopy: 33.3%); at 7 days post treatment (dpt), positivity rates of ITS2-PCR and microscopy rapidly decreased to 0.00% and 11.1%, whereas rPsoSP3-iELISA remained 100% positive rate. Furthermore, the comprehensive comparisons of diagnostic performance and features of three diagnostic tests at 7 dpi were performed. Compared to ITS2-PCR or rPsoSP3-iELISA, microscopy had the lowest sensitivity, and the agreement between these assays was low (κ < 0.3). Field study showed that ITS2-PCR showed a higher detection rate than microscopy (19.4% and 11.1%, respectively). Our results suggested that the ITS2-PCR developed in this study provided a new laboratory tool for diagnosis of P. ovis var. cuniculi infection, and it had advantages over microscopic examination in detection low-level mite infections and serological assay in monitoring treatment outcome.

Serological and Bacteriological Surveillance of Glanders Among Horses in Central Region of Iran.

Glanders is the oldest and very contagious disease among horses caused by Burkholderia mallei. The disease occurs as a chronic form in horses. Hence, because of the prolonged shedding, numerous horses can potentially get infected by one horse with glanders. Glanders is endemic in Iran and this causes occasional occurrence in horse population of the country. The aim of this study was to determine the incidence of B.mallei infection in horses in two central provinces of Iran. A total of 517 serum samples were collected from stable horses in Tehran and Alborz provinces. Among the studied horses, seven presented fever, anorexia, dyspnea, subcutaneous abscesses, nasal and cutaneous discharges, emaciation, and lymphadenopathy. Nasal and ocular discharges and subcutaneous abscesses were sampled for bacterial culture and PCR. The sera were examined by means of complement fixation test (CFT) and indirect enzyme-linked immunosorbent assay (iELISA). Seropositive cases were further examined by Mallein test. The results derived from the present study indicated that only 1.35% of the studied horses were positive in CFT, iELISA and Mallein test, of which only in 42.85% B.mallei was successfully cultured on blood agar and glycerinated nutrient media and confirmed by PCR. Periodic serological tests along with quarantine can benefit reduction of the occurrence of the disease in horses in Iran.

A Candidate Antigen of the Recombinant Membrane Protein Derived from the Porcine Deltacoronavirus Synthetic Gene to Detect Seropositive Pigs.

Porcine deltacoronavirus (PDCoV) is an emergent swine coronavirus which infects cells from the small intestine and induces watery diarrhea, vomiting and dehydration, causing mortality in piglets (>40%). The aim of this study was to evaluate the antigenicity and immunogenicity of the recombinant membrane protein (M) of PDCoV (rM-PDCoV), which was developed from a synthetic gene obtained after an in silico analysis with a group of 138 GenBank sequences. A 3D model and phylogenetic analysis confirmed the highly conserved M protein structure. Therefore, the synthetic gene was successfully cloned in a pETSUMO vector and transformed in E. coli BL21 (DE3). The rM-PDCoV was confirmed by SDS-PAGE and Western blot with ~37.7 kDa. The rM-PDCoV immunogenicity was evaluated in immunized (BLAB/c) mice and iELISA. The data showed increased antibodies from 7 days until 28 days (p < 0.001). The rM-PDCoV antigenicity was analyzed using pig sera samples from three states located in "El Bajío" Mexico and positive sera were determined. Our results show that PDCoV has continued circulating on pig farms in Mexico since the first report in 2019; therefore, the impact of PDCoV on the swine industry could be higher than reported in other studies.

Development of an Indirect Enzyme-Linked Immunosorbent Assay Based on the Yeast-Expressed CO-26K-Equivalent Epitope-Containing Antigen for Detection of Serum Antibodies against Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea (PED) is a severe contagious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), which leads to high mortality in piglets. In this study, by analyzing a total of 53 full-length spike genes and COE domain regions of PEDVs, the conserved COE fragment of the spike protein from the dominant strain SC1402 was chosen as the target protein and expressed successfully in Pichia pastoris (P. pastoris). Furthermore, an indirect enzyme-linked immunosorbent assay (iELISA) based on the recombinant COE protein was developed for the detection of anti-PEDV antibodies in pig sera. The results showed that under the optimized conditions, the cut-off value of COE-based indirect ELISA (COE-iELISA) was determined to be 0.12. Taking the serum neutralization test as standard, the relative sensitivity of the COE-iELISA was 94.4% and specificity 92.6%. Meanwhile, no cross-reactivity to other porcine pathogens was noted with this assay. The intra-assay and inter-assay coefficients of variation were less than 7%. Moreover, 164 vaccinated serum samples test showed that overall agreement between COE-iELISA and the actual diagnosis result was up to 99.4%. More importantly, the developed iELISA exhibited a 95.08% agreement rate with the commercial ELISA kit (Kappa value = 0.88), which suggested that the expressed COE protein was an effective antigen in serologic tests and the established COE-iELISA is reliable for monitoring PEDV infection in pigs or vaccine effectiveness.

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.

Study on antigenic protein Omp2b in combination with Omp31 and BP26 for serological detection of human brucellosis.

BACKGROUND: Brucellosis is a very common zoonosis in certain localized areas worldwide, with a high prevalence in most developing countries. The detection of brucellosis still faces many challenges such as the need for more sensitive and specific diagnostic antigens. METHODS: To evaluate the efficacy of Brucella outer membrane proteins (Omps) Omp2b in combination with omp31 and BP26 as diagnostic antigens for the serological detection of human brucellosis, these proteins were prepared by a prokaryotic expression system. Human brucellosis-positive and-negative sera were collected, and the detection effects of the diagnostic antigens were evaluated using an established indirect ELISA (iELISA) method. Receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC), true positives, true negatives, false positives, false negatives, accuracy, positive predictive value, negative predictive value, analytical specificity, and sensitivity were obtained to evaluate the effectiveness of Omp2b and antigen combinations. RESULTS: The iELISA results showed that the AUC of the antigenic proteins was 0.9100, 0.9387, 0.9343, and 0.9448, respectively, and that the combination of Omp31 and BP26 improved the accuracy and was superior to that of Omp2b alone. Analysis at the determined cut-off values showed that the analytical sensitivity of the assay was 0.8739 (95% CI:0.7974-0.9293) and the analytical specificity was 0.8539 (95% CI:0.7632-0.9199) when using Omp2b alone and 0.8649 when using the combination of Omp2b + BP26 (95% CI:0.7869-0.9223) with an analytical specificity of 0.9213 (95% CI:0.8446-0.9678) and 0.8468 (95% CI:0.7662-0.9082) and an analytical sensitivity of 0.9101 (95% CI:0.8305-0.9604). When Omp2b + Omp31 + BP26 was combined, the analytical sensitivity and specificity were 0.8559 (95% CI:0.7765-0.9153) and 0.9326 (95% CI:0.8590-0.9749), respectively. Protein antigens, including antigen combinations, did not cross-react with Yersinia enterocolitica O9 and E. coli O157: H7, indicating that their specificity was better than that of lipopolysaccharide (LPS). CONCLUSIONS: Compared with individual Omp2b, antigen combinations improved the effectiveness in detecting brucellosis, but were still not as effective as LPS antigen. Omp2b, combined with Omp31 and BP26 as diagnostic antigens, can be used to detect human brucellosis.

Diagnostic Accuracy of an Indirect Enzyme Linked Immunosorbent Assay (iELISA) for Screening of Babesia bovis in Cattle from West Africa.

The epidemiology of corresponding tick-borne diseases has changed as a result of the recent introduction of Rhipicephalus (Boophilus) microplus to West Africa. The current study aimed to assess the diagnostic performance of an indirect ELISA for the detection of Babesia bovis infection in cattle. In a cross-section study, using a Bayesian Latent Class Model and iELISA diagnostic test for cattle babesiosis due to Babesia bovis, accuracy has been assessed with RT-PCR as an imperfect reference test. A total of 766 cattle were tested. The optimal diagnostic performances were obtained with 5% percentage of positivity. Sensitivity and specificity were, respectively, 0.94 [Cr. I.: 0.85−0.99] and 0.89 [Cr. I.: 0.87−0.92]. Additional diagnostic characteristics revealed that the Positive Predictive Value (PPV) and Negative Predictive Value (NPV) were 96.6% [Cr. I.: 92.7−100%] and 82.2% [Cr. I.: 72−93%]. Overall, this test well discriminates an infected status from an uninfected status considering the area under the ROC curve (AUC) which was 0.78 [Cr. I: 0.72−0.85] and a Diagnostic Odds Ratio (DOR) of 127.8 [Cr. I.: 10.43−1562.27]. The AUC was significantly higher than 0.5 (p < 10−5). In consequence, this serologic assay could be suitable in moderate to high prevalence assessments.

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.

Establishment of indirect ELISA method for Salmonella antibody detection from ducks based on PagN protein.

BACKGROUND: Salmonella as an important food-borne zoonotic bacterial pathogen, infection in ducks is a recessive infection, however, it can also cause high mortality and threat to food safety. Preventing and controlling the infection and transmission of Salmonella in ducks critically require rapid and sensitive detection method. Full-length Salmonella-specific protein PagN was induced and expressed in E.coil BL21 and was purified as an antigen to establish an indirect enzyme-linked immunosorbent assays (iELSA) detection kit. RESULTS: The recombinant PagN protein has a molecular weight of 43 kDa containing a His-tag, was recognized by an anti-Salmonella positive serum by Western blot assay. The optimal concentration of PagN as a coating antigen in the iELISA was 1 µg/mL, and the optimal dilution of enzyme-labeled secondary antibody was 1:4000 (0.025 µg/mL). The cutoff OD450 value was established at 0.268. The iELISA kit showed high selectivity since no cross-reaction with E. coli, Staphylococcus aureus and Streptococcus was observed. iELISA method and Dot-blot test were performed on 100 clinical sera samples collected from duck farms, and the actual coincidence rate was 89% (89/100). 613 duck serum samples from 3 different farms were tested using established method and commercial ELISA kit. The concordance between the two methods was 94.1%. CONCLUSION: Anti-PagN based iELISA can serve as a useful tool for diagnosis of Salmonella infection.

Development and Optimization of Indirect ELISAs for the Detection of Anti-Capripoxvirus Antibodies in Cattle, Sheep, and Goat Sera.

Sheeppox (SPP), goatpox (GTP), and lumpy skin disease (LSD) are economically significant pox diseases of ruminants, caused by sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV), respectively. SPPV and GTPV can infect both sheep and goats, while LSDV mainly affects cattle. The recent emergence of LSD in Asia and Europe and the repeated incursions of SPP in Greece, Bulgaria, and Russia highlight how these diseases can spread outside their endemic regions, stressing the urgent need to develop high-throughput serological surveillance tools. We expressed and tested two recombinant truncated proteins, the capripoxvirus homologs of the vaccinia virus C-type lectin-like protein A34 and the EEV glycoprotein A36, as antigens for an indirect ELISA (iELISA) to detect anti-capripoxvirus antibodies. Since A34 outperformed A36 by showing no cross-reactivity to anti-parapoxvirus antibodies, we optimized an A34 iELISA using two different working conditions, one for LSD in cattle and one for SPP/GTP in sheep and goats. Both displayed sound sensitivities and specificities: 98.81% and 98.72%, respectively, for the LSD iELISA, and 97.68% and 95.35%, respectively, for the SPP/GTP iELISA, and did not cross-react with anti-parapoxvirus antibodies of cattle, sheep, and goats. These assays could facilitate the implementation of capripox control programs through serosurveillance and the screening of animals for trade.

Development of CD163 receptor-based enzyme-linked immunosorbent assay for diagnosis of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) is an important economical disease in the global swine industry. The accurate detection of the PRRS virus (PRRSV) antigen is essential for the disease control and prevention programme. In this study, an indirect enzyme-linked immunosorbent test (PRRSVCD163-iELISA) was developed for the detection of the PRRSV antigen in samples of post-mortem swine tissue using the recombinant pig CD163 receptor protein as the capture ligand. The test was found to be specific for PRRSV, with no cross-reactions with other prevalent pig viral pathogens. The assay was validated by testing 217 post-mortem porcine tissue samples and the results were found to be satisfactory with a relative accuracy of 88.88%. Our assay is also quite precise, with intra- and inter-assay CVs of 6% and 10%, respectively. These findings imply that the PRRSVCD163-iELISA developed is capable of detecting the PRRSV antigen in swine post-mortem tissue samples. This research showed that porcine CD163, the PRRSV cellular receptor, can be exploited to build a diagnostic technique for the detection of PRRSV antigen. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03376-z.