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Abstract Peste des petits ruminants (PPRV), a highly contagious viral disease, causes significant economic losses concerning sheep and goats. Recently, PPR viruses (PPRVs), have adopted new hosts and lineage IV of PPRVs represents genetic diversity within the same lineage. 350 samples, including blood, swabs, and tissues from sheep/goats, were collected during the 2020–2021 disease outbreaks in Pakistan. These samples were analysed through RT-PCR and three isolates of PPRV with accession numbers, MW600920, MW600921, and MW600922, were submitted to GenBank, based on the partial N-gene sequencing. This analysis provides a better understanding of genetic characterizations and a targeted RT-PCR approach for rapid PPRV diagnosis. An IELISA test was developed using the semi-purified antigen MW600922 isolate grown in Vero cells. The PPRV isolates currently present high divergence with the Turkish strain; conversely, similarities equivalent to 99.73% were observed for isolates collected from Pakistan. The developed indirect ELISA (IELISA) test demonstrated antibody detection rates at dilutions of 1:200 for antibodies (serum) and 1:32 for antigens. In comparison to cELISA, high specificity (85.23%) and sensitivity (90.60%) rates were observed. In contrast to the virus neutralization test (VNT), IELISA was observed to be 100% specific and 82.14% sensitive in its results. Based on these results, serological surveys conducted for PPR antibodies using IELISA can be a more effective strategy on a larger scale. Furthermore, our results demonstrate a significant breakthrough in the research in terms of cost-effectiveness and storage efficiency, and the developed IELISA test is highly recommended for use in developing countries.

Simple Summary: Hemorrhagic septicemia (HS) is an acute, fatal, and septicemic bacterial disease of cattle and buffaloes caused by Pasteurella multocida (P. multocida). The present study aimed to carry out HS surveillance through multi-stage random sampling constituting 692 cattle and buffalo serum samples sourced from two Indian states, four districts, eight clusters, 56 epiunits, and 306 households (HHs) by indirect enzyme-linked immunosorbent assay (iELISA). Significantly higher (p < 0.0001) iELISA positives were observed in Gujarat compared to Karnataka state. Of the 306 households visited, 9 of 27 epiunits (33.33%) in Karnataka and 24 out of 29 epiunits in Gujarat were iELISA positive (82.75%) and an association was found to be significant (p = 0.0002). However, a non-significant association to species, age, or lactation was recorded, although indigenous cattle breeds had a higher HS sero-prevalence compared to crossbreeds. This study highlights important baseline data on HS sero-prevalence in two major milk-producing states of India at different strata. Hemorrhagic septicemia (HS) is a highly contagious and fatal disease of cattle and buffaloes caused by P. multocida. Both conventional and molecular methods are applied in parallel for rapid diagnosis of HS outbreaks and the periodical surveillance strategy to identify risk areas for HS is ignored. The current cross-sectional study aimed to estimate sero-prevalence and associated risk factors for HS in cattle and buffaloes in non-vaccinated regions of two Indian states. HS surveillance was carried out through the multi-stage random sampling technique at different strata. The study employed a questionnaire incorporating host factors (species, breed, sex, age, and lactation) and demographic parameters (state, district, block/cluster and village/epiunits, and household). First, two Indian states known for high milk production were selected followed by two districts within each state, subsequently four clusters within each district, finally 5–10 epiunits within clusters and 5–8 households within clusters were randomly selected to collect cattle and buffalo samples. The chi-square/p values and maps were prepared to represent disease prevalence and to correlate disease risk factors at different strata. A total of 692 cattle and buffalo serum samples were sourced from two states of the country (Karnataka-285 and Gujarat-407). In the first strata, antibodies to P. multocida were high in Gujarat (14.49%, CI: 11.22–18.30) compared to Karnataka (3.85%, CI: 1.94–6.80) with significant (p < 0.0001) association between the states. In the second strata, one of the four districts investigated revealed the highest sero-prevalence (18.61%, CI: 13.81–24.24) with statistical significance (p = 0.01) between the districts. Among clusters, one out of eight clusters showed the highest sero-prevalence (23.02%, CI: 16.59–30.54) with statistical significance (p = 0.03) between the clusters in the third strata. At epiunit level (fourth strata), 9 out of 27 epiunits (33.33%) visited in Karnataka and 24 out of 29 epiunits sampled in Gujarat were sero-positive (82.75%) in iELISA. At the household level, out of 306 HH visited, 40 HH had at least one positive animal (13.07%) and the p value between HH in the two states was highly significant (p = 0.0002). Chi-square analysis did not find any association of HS sero-prevalence to species, age, and lactation. However, significantly higher (p < 0.05) sero-prevalence was recorded in indigenous cattle breeds (16.56%) compared to crossbreeds (6.59%). Various immunoprophylactics and antibiotic therapies are effective against HS, but inappropriate disease reporting and failure to implement adequate vaccination control measures are the gaps identified. The present study highlights the current scenario of HS sero-prevalence in two of the high milk-producing states of India, which will be useful for stakeholders for undertaking the implementation of surveillance and control strategies for the regions. [ABSTRACT FROM AUTHOR]

Peste des petits ruminants (PPRV), a highly contagious viral disease, causes significant economic losses concerning sheep and goats. Recently, PPR viruses (PPRVs), have adopted new hosts and lineage IV of PPRVs represents genetic diversity within the same lineage. 350 samples, including blood, swabs, and tissues from sheep/goats, were collected during the 2020–2021 disease outbreaks in Pakistan. These samples were analysed through RT-PCR and three isolates of PPRV with accession numbers, MW600920, MW600921, and MW600922, were submitted to GenBank, based on the partial N-gene sequencing. This analysis provides a better understanding of genetic characterizations and a targeted RT-PCR approach for rapid PPRV diagnosis. An IELISA test was developed using the semi-purified antigen MW600922 isolate grown in Vero cells. The PPRV isolates currently present high divergence with the Turkish strain; conversely, similarities equivalent to 99.73% were observed for isolates collected from Pakistan. The developed indirect ELISA (IELISA) test demonstrated antibody detection rates at dilutions of 1:200 for antibodies (serum) and 1:32 for antigens. In comparison to cELISA, high specificity (85.23%) and sensitivity (90.60%) rates were observed. In contrast to the virus neutralization test (VNT), IELISA was observed to be 100% specific and 82.14% sensitive in its results. Based on these results, serological surveys conducted for PPR antibodies using IELISA can be a more effective strategy on a larger scale. Furthermore, our results demonstrate a significant breakthrough in the research in terms of cost-effectiveness and storage efficiency, and the developed IELISA test is highly recommended for use in developing countries. Summary: Peste des petits ruminants (PPRV) is a transboundary, highly contagious, and economically significant viral disease affecting small ruminants and wildlife. PPRV, a disease that only targets animals, is the focus of the Global Eradication Programme (PPRV GEP), which aims to eradicate the disease by 2030. Following the completion of the first phase of the GEP (2017–2021), Pakistan has initiated the second phase: PPRV presence and the implementation of a control strategy. Rapid and accurate laboratory diagnosis is vital to the disease's effective control and eradication. In the present study, we have improved diagnosis by reverse transcriptase polymerase chain reaction (RT-PCR), which not only can detect low viral concentrations but also contributes to the genetic analysis of lineage-IV viruses. However, the development of cost-effective indirect ELISA (iELISA) may allow for the analysis of serum samples obtained from larger populations of small ruminants. [ABSTRACT FROM AUTHOR]

Abstract The aim of this study was to detect trypomastigote forms of Trypanosoma vivax, in blood smears, DNA of T. vivax and anti-T. vivax antibodies in samples from buffalos reared in the lowlands of Maranhão, Brazil. Blood samples were collected from 116 buffalos and 25 ectoparasite specimens. Blood smears were produced to diagnose forms compatible with Trypanosoma spp.; the indirect enzyme-linked immunosorbent assay (iELISA) and lateral-flow immunochromatography (Imunotest®) serological tests were used; and the polymerase chain reaction (PCR) was used to make molecular diagnoses. No forms compatible with Trypanosoma spp. were observed in blood smears. Among the 116 serum samples analyzed, 79.31% and 76.72% were positive in the ELISA and rapid tests, respectively. One sample was positive in the molecular test. Twenty-five lice of the species Haematopinus tuberculatus were collected. When subjected to PCR for detection of DNA of T. vivax, all of them were negative. The louse specimens were negative for T. vivax. There were no statistically significant differences (p < 0.05) in the presence of T. vivax in this region, in relation to the animals’ age and sex. It can be concluded that these protozoa are circulating in the buffalo herd of the lowlands of Maranhão displaying crypitc parasitemias.

Introduction: Lumpy skin disease (LSD) is a highly contagious vector-borne viral disease of cattle. LSD has emerged in Bangladesh in 2019, causing significant economic losses due to its high morbidity and mortality. This research was designed to isolate, identify, and assess the immunogenicity of LSD virus (LSDV) using nodular tissue samples obtained from affected cattle during the 2019-20 outbreak across nine districts of Bangladesh. Methods: To determine the presence of LSDV in nodular tissues, we initially used iiPCR and PCR, followed by histopathological examination. 151 were positive via iiPCR and PCR among the 180 collected samples. The PCR positive 151 samples were then inoculated into 10-day-old embryonated chicken eggs via the CAM route to isolate LSDV, confirmed through PCR. Subsequently, partial sequencing and phylogenetic analysis of the P32 gene were performed to determine the origin of the circulating LSDV strain. The immunogenicity of selected LSDV strains was assessed through an ELISA test. Results: The PCR results revealed a distinct positive band at 192 bp in both the nodular tissue samples and the LSDV isolated from chicken embryo inoculations. Microscopic analysis of the nodular lesions revealed thickening of the epidermis, ballooning degeneration of keratinocytes, and proliferation of follicular epithelia. Additionally, mononuclear infiltration was observed at the demarcation line between infected and healthy tissue, with necrosis of muscular tissues beneath the epidermis. The LSDV isolate from Bangladesh exhibited a close genetic relationship with LSDV strains isolated from neighboring and other regional countries including India, Myanmar, and Mongolia. This observation strongly suggests the possibility of a transboundary spread of the LSD outbreak in Bangladesh during 2019-2020. The results of the immunogenicity test showed that the serum antibody titer remained at a protective level for up to 18 months following secondary immunization with inactivated LSDV antigen. This finding suggests that the inactivated LSDV antigen could be a potential vaccine candidate to protect cattle in Bangladesh against LSDV. Conclusion: In conclusion, our research successfully isolated, identified, and characterized LSDV in cattle nodular tissues from the 2019-20 outbreak in Bangladesh. Furthermore, it provided insights into the probable origin of the circulating strain and investigated a potential vaccine candidate to protect cattle in the region from LSDV. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

IntroductionLumpy skin disease (LSD) is a highly contagious vector-borne viral disease of cattle. LSD has emerged in Bangladesh in 2019, causing significant economic losses due to its high morbidity and mortality. This research was designed to isolate, identify, and assess the immunogenicity of LSD virus (LSDV) using nodular tissue samples obtained from affected cattle during the 2019–20 outbreak across nine districts of Bangladesh.MethodsTo determine the presence of LSDV in nodular tissues, we initially used iiPCR and PCR, followed by histopathological examination. 151 were positive via iiPCR and PCR among the 180 collected samples. The PCR positive 151 samples were then inoculated into 10-day-old embryonated chicken eggs via the CAM route to isolate LSDV, confirmed through PCR. Subsequently, partial sequencing and phylogenetic analysis of the P32 gene were performed to determine the origin of the circulating LSDV strain. The immunogenicity of selected LSDV strains was assessed through an ELISA test.ResultsThe PCR results revealed a distinct positive band at 192 bp in both the nodular tissue samples and the LSDV isolated from chicken embryo inoculations. Microscopic analysis of the nodular lesions revealed thickening of the epidermis, ballooning degeneration of keratinocytes, and proliferation of follicular epithelia. Additionally, mononuclear infiltration was observed at the demarcation line between infected and healthy tissue, with necrosis of muscular tissues beneath the epidermis. The LSDV isolate from Bangladesh exhibited a close genetic relationship with LSDV strains isolated from neighboring and other regional countries including India, Myanmar, and Mongolia. This observation strongly suggests the possibility of a transboundary spread of the LSD outbreak in Bangladesh during 2019–2020. The results of the immunogenicity test showed that the serum antibody titer remained at a protective level for up to 18 months following secondary immunization with inactivated LSDV antigen. This finding suggests that the inactivated LSDV antigen could be a potential vaccine candidate to protect cattle in Bangladesh against LSDV.ConclusionIn conclusion, our research successfully isolated, identified, and characterized LSDV in cattle nodular tissues from the 2019–20 outbreak in Bangladesh. Furthermore, it provided insights into the probable origin of the circulating strain and investigated a potential vaccine candidate to protect cattle in the region from LSDV.

Hemorrhagic septicemia (HS) is a highly contagious and fatal disease of cattle and buffaloes caused by P. multocida. Both conventional and molecular methods are applied in parallel for rapid diagnosis of HS outbreaks and the periodical surveillance strategy to identify risk areas for HS is ignored. The current cross-sectional study aimed to estimate sero-prevalence and associated risk factors for HS in cattle and buffaloes in non-vaccinated regions of two Indian states. HS surveillance was carried out through the multi-stage random sampling technique at different strata. The study employed a questionnaire incorporating host factors (species, breed, sex, age, and lactation) and demographic parameters (state, district, block/cluster and village/epiunits, and household). First, two Indian states known for high milk production were selected followed by two districts within each state, subsequently four clusters within each district, finally 5–10 epiunits within clusters and 5–8 households within clusters were randomly selected to collect cattle and buffalo samples. The chi-square/p values and maps were prepared to represent disease prevalence and to correlate disease risk factors at different strata. A total of 692 cattle and buffalo serum samples were sourced from two states of the country (Karnataka-285 and Gujarat-407). In the first strata, antibodies to P. multocida were high in Gujarat (14.49%, CI: 11.22–18.30) compared to Karnataka (3.85%, CI: 1.94–6.80) with significant (p < 0.0001) association between the states. In the second strata, one of the four districts investigated revealed the highest sero-prevalence (18.61%, CI: 13.81–24.24) with statistical significance (p = 0.01) between the districts. Among clusters, one out of eight clusters showed the highest sero-prevalence (23.02%, CI: 16.59–30.54) with statistical significance (p = 0.03) between the clusters in the third strata. At epiunit level (fourth strata), 9 out of 27 epiunits (33.33%) visited in Karnataka and 24 out of 29 epiunits sampled in Gujarat were sero-positive (82.75%) in iELISA. At the household level, out of 306 HH visited, 40 HH had at least one positive animal (13.07%) and the p value between HH in the two states was highly significant (p = 0.0002). Chi-square analysis did not find any association of HS sero-prevalence to species, age, and lactation. However, significantly higher (p < 0.05) sero-prevalence was recorded in indigenous cattle breeds (16.56%) compared to crossbreeds (6.59%). Various immunoprophylactics and antibiotic therapies are effective against HS, but inappropriate disease reporting and failure to implement adequate vaccination control measures are the gaps identified. The present study highlights the current scenario of HS sero-prevalence in two of the high milk-producing states of India, which will be useful for stakeholders for undertaking the implementation of surveillance and control strategies for the regions.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In our scholarly endeavor at Oxford University, we meticulously investigated refined methodologies for detecting anti-brucella antibodies within the serum of ovine species. This study rigorously evaluates two predominant diagnostic approaches: the Rose Bengal Plate Test (RBPT) and the indirect Enzyme-Linked Immunosorbent Assay (iELISA). The RBPT, recognized for its expeditious nature, is a slide-based agglutination test employing antigens from Brucella melitensis biovar 1, specifically the Weybridge strain number 99. In contrast, iELISA represents an indirect detection paradigm, utilizing specific antigens to accurately identify antibodies within serum samples. Both diagnostic procedures commence with the standardization of serum samples and reagents to ambient temperature. This is followed by intricate protocols encompassing antigenantibody interactions and meticulous observation of outcomes. The RBPT is characterized by its immediate visual agglutination, typically manifesting within minutes. Conversely, the iELISA approach discerns antibodies through a sequence of enzyme-mediated reactions, culminating in observable color variations. This comprehensive study encompassed the analysis of 259 serum samples extracted from female sheep, stratified across three distinct age brackets. Within this cohort, 37 out of 259 samples (14.29%) yielded positive results through RBPT, whereas a significantly larger proportion, 222 out of 259 (85.71%), were determined to be negative. In the realm of ELISA, 67 out of 259 samples (25.87%) were positively identified, leaving 192 out of 259 (74.13%) in the negative spectrum. These samples were meticulously collected from eight disparate fields in the Thi-Qar Province, located in the southern region of Iraq. The collection period spanned from November 2022 to June 2023, encompassing animals that exhibited clinical signs of the disease or had histories of abortion. Our findings lead to a compelling conclusion: the ELISA technique demonstrates superior sensitivity and accuracy in detecting specific antibodies within physiological fluids when juxtaposed with the RBPT method. This revelation holds profound implications for the field of veterinary diagnostics, particularly in the context of brucellosis in sheep. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

Meixue Yao,1,2,&ast; Xiaohan Guo,1,2,&ast; Xiling Wu,1,2 Qiongqiong Bai,1,2 Mingjun Sun,3 Dehui Yin1,2 1Center for Medical Statistics and Data Analysis, School of Public Health, Xuzhou Medical University, Xuzhou, People’s Republic of China; 2Key Laboratory of Human Genetics and Environmental Medicine, School of Public Health, Xuzhou Medical University, Xuzhou, People’s Republic of China; 3Laboratory of Zoonoses, China Animal Health and Epidemiology Center, Qingdao, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Dehui Yin, Key Laboratory of Human Genetics and Environmental Medicine, School of Public Health, Xuzhou Medical University, No. 129 Tongshan Road, Xuzhou, 221004, People’s Republic of China, Email yindh16@xzhmu.edu.cnBackground: Brucellosis is a zoonotic disease that causes substantial public health problems and endangers the development of animal husbandry in endemic areas. Early diagnosis of infected animals and humans is a crucial step in reducing the incidence of brucellosis. In this study, we designed different combinations of Brucella major outer membrane proteins (omps) including omp10, omp16, omp19, omp25, omp31 and BP26 as antigens and evaluated their efficiency in serodiagnosis for brucellosis. The efficiency assay was conducted using the method of indirect enzyme-linked immunosorbent assay (iELISA) together with a collection of brucellosis-positive sera and healthy sera from multiple species (161 from human, 120 from goat and 144 from cattle). The diagnostic effectiveness of each omp combination was analyzed by receiver operating characteristic (ROC) curve with the software GraphPad Prism version 6.05.Results: The omp25/omp31/BP26 combination showed the best efficiency in diagnosis for human brucellosis. The area under the ROC curve (AUC) was 0.995 and, compared with the serum tube agglutination test (SAT) and the Rose Bengal plate agglutination test (RBPT), the positive and negative diagnostic accuracies of iELISA were 94.59% (105/111) and 100.0% (50/50), respectively. Evaluation of the 120 goat and 144 cattle serum samples showed that the best combination for diagnosing both omp31/BP26, the AUC was 0.9262 in goat and 0.9344 in cattle, and compared with those of SAT and RBPT, the positive and negative diagnostic accuracies in goat were 72.73% (48/66) and 100.0% (54/54), respectively. The positive and negative diagnostic accuracies in cattle were 79.79% (75/94) and 100.0% (50/50), respectively. Cross-reaction assays showed that omp25/omp31/BP26 and omp31/BP26 do not cross with other common pathogens.Conclusion: The results indicated that combinations of omps, as protein antigens, can be used to diagnose brucellosis with high accuracy in human, goat and cattle.Keywords: brucellosis, diagnosis, outer membrane proteins, iELISA

Paratuberculosis (pTB) also known as Johne’s disease (JD), is a chronic infectious disease of animals caused by Mycobacterium avium subsp. paratuberculosis (MAP). It is also a serious public health concern as MAP is responsible for Crohn’s disease in human beings. This infectious disease is so far unexplored in animals in Khyber Pakhtunkhwa (KP) province of Pakistan. Therefore, this study was proposed to investigate the presence of MAP by indirect ELISA (iELISA), associated histopathological lesions and PCR in sheep (Ovis aries) and goats (Capra aegagru hircus) in district Peshawar. Serum and fecal samples were collected at random both from commercial farms and abattoirs. Additionally, tissue samples (intestine and mesenteric lymph node (MLN)) were collected at random from sheep and goats at abattoirs of district Peshawar. Analyses of serum samples by iELISA revealed the presence of antibodies against MAP in 9% sheep and 5% goats. Ziehl Nielsen (ZN) staining exhibited acid-fast bacilli (AFB) in 31% sheep and 23% goat’s fecal impression smears. Gross pathology in intestinal samples (thickness, corrugation) was observed in 30% sheep and 22% goats, while MLN exhibited gross lesions (hemorrhages, edematous swelling) in 35% sheep and 27% goats. Histopathological lesions were observed in intestine and MLN in 26% and 19% sheep, and 17% and 14% goats, respectively. Additionally, PCR revealed the presence of MAP in tissue samples of 5% sheep and 3% goat. This study concluded that MAP is present in the small ruminants of district Peshawar. The infection was confirmed by PCR and iELISA. The presence of MAP could be a serious threat to livestock and public health in the region. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A large number of Southern Song dynasty costumes were unearthed from Huangsheng tomb in Fuzhou city, Fujian province. The unlined clothing was among the funeral objects, and made of the most common leno fabric, which was light and transparent, and the clothing was in extremely poor condition. To protect the unlined clothing, scientific evaluation and protection were carried out. Three‐dimensional microscopy was used to analyse the weave structure and X‐ray fluorescence spectroscopy (XRF) and indirect enzyme‐linked immunosorbent assay (iELISA) were used to analyse scientifically the printed gold and colour paint components of the unlined clothing. The results showed that the structure of the unlined clothing was a two‐warp twisted leno, the printed gold contained Au, Cu, Fe and other elements, the coloured paint contained Pb and Hg elements, and the binding material component of the coloured paint was type I collagen. Based on the results of the analysis, the silk fibroin consolidation method was used to improve resistance to the stress and strain caused by the needle–thread repair method, and the needle–thread repair method was then used to restore the original clothing shape. This study provides a new approach for the protection of fragile silk fabrics in the future. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

Parainfluenza virus 5 (PIV5), a member of paramyxoviruses, causes respiratory and neurological infection in several animal species. Whereas information on PIV5 infection in digestive system is very scarce. Here, we successfully isolated one PIV5 strain from diarrheic piglets. After four times plaque purification and ultracentrifugation, the paramyxovirus‐like particles were observed by electron microscopy. The genome‐wide phylogenetic analysis showed that the isolated strain was closely related to the PIV5 strain from a lesser panda and pigs in China. Therefore, we characterized this isolated PIV5 and found that this virus could haemagglutinate red blood cells from both guinea pigs and chickens. Further, we observed that this PIV5 could infect cell lines from various host species including pig, human, monkey, bovine, dog, cat, rabbit, hamster and mouse, which was confirmed with the immunofluorescent assay. To evaluate the distribution of PIV5 in the field, we developed an indirect ELISA (iELISA) for the first time to detect the specific antibodies based on recombinant nucleocapsid protein. A total of 530 porcine serum samples were tested and the PIV5‐positive rate was 75.7%. To our knowledge, this is the first report describing the full characterization of PIV5 strain isolated from a diarrheic piglet. The ability of this PIV5 strain to infect a wide range of mammalian cell types indicates that PIV5 can transmit across different species, providing a remarkable insight into potential zoonosis. The virus strain and iELISA developed in this study can be used to investigate the pathogenesis, epidemiology, and zoonotic potential of PIV5. [ABSTRACT FROM AUTHOR]

This study aimed to detect Brucella antibodies in the sera of dairy cows and to identify Brucella species in the milk of seropositive cows. A total of 100 sera and 100 milk samples were collected from two 50-cows groups (group 1 with and group 2 without a history of reproductive problems and/or decreased milk production). Rose Bengal plate test and indirect ELISA were used to explore Brucella antibodies in the serum samples and thereafter milk samples of seropositive cows were undergone PCR analysis using Brucella genus specific primers and 3 pairs of species specific primers for identification of B. abortus, B. melitensis and B. suis. The RBPT showed 22 cows were carriers for the Brucella antibodies, 18 in group 1 and 4 in group 2 whereas the iELISA showed only 10 cows out of these 22 cows were positive, 9 in group 1 and only 1 cow in group 2. The PCR assay, which was performed on milk samples of the RBPT positive cows, revealed 18 samples were positive for the Brucella genus and the Brucella abortus species and were negative for Brucella melitensis and Brucella suis species. As a conclusion, the results of this study showed that brucellosis has been encountered in cows with or without a history of reproductive problems, and the RBPT followed by PCR assay for milk samples of the seropositive cows could provide more specific detection than performing either test alone and could be more useful for rapid screening of brucellosis in dairy cows.

Strangles is a prevalent infectious and contagious disease among Equidae that is caused by Streptococcus equi subspecies equi. This study was conducted on 184 horses that were studied in terms of carrying S. equi and its frequency among the horse population kept in equestrian clubs around Tabriz. The serum samples were collected from different equestrian clubs and were assayed using an iELISA (Indirect Enzyme-Linked Immunosorbent Assay) test for anti-SeM antibody in Streptococcus equi. The relative frequency of carriers was 53.3% in stallions and 24.5% in mares (p0.05). The results of Chi-squared test showed that the most positive results were observed in Arpadresi area of Tabriz city (p

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.

Objective Real-time reverse transcription-polymerase chain reaction is the gold standard for the diagnosis of COVID-19, but it is necessary to utilize other tests to determine the burden of the disease and the spread of the outbreak such as IgG-, IgM-, and IgA-based antibody detection using enzyme-linked immunosorbent assay (ELISA). Materials and Methods We developed an indirect ELISA assay to quantitatively measure the amount of COVID-19 IgG, IgM, and IgA antibodies present in patient serum, dried blood, and plasma. Results The population cutoff values for positivity were determined by receiver operating characteristic curves to be 1.23 U/mL, 23.09 U/mL, and 6.36 U/mL for IgG, IgM, and IgA, respectively. After albumin subtraction, the specificity remained >98% and the sensitivity was 95.72%, 83.47%, and 82.60%, respectively, for IgG, IgM, and IgA antibodies to the combined spike subunit 1 receptor binding domain and N proteins in serum. Plasma and dried blood spot specimens were also validated on this assay. Conclusion This assay may be used for determining the seroprevalence of SARS-CoV-2 in a population exposed to the virus or in vaccinated individuals. [ABSTRACT FROM AUTHOR]

Brucellosis in swine is a contagious disease with greater zoonotic potential caused by Brucella suis. The study describes PAN India swine brucellosis sero-prevalence in 5431 stratified random serum samples collected during 2018–2019 from 26 out of 29 states and two out of seven union territories. The serum samples were tested for anti-Brucella antibodies by indirect ELISA and overall, 4.33% apparent prevalence (AP) was recorded. The AP is ≥ 10% in five states among 26 states, P ≥ 50% in four districts out of 117 districts screened and cent percent prevalence in two epi units out of 264 sampled. Significantly high seropositivity (p < 0.05) in male (6.08%) than female pigs (3.46%) and in ≥ 24-month-old pigs indicated older and male pigs as potential carriers of the disease. The study recorded endemicity of the swine brucellosis in few regions of India requiring periodical surveillance for control of the disease. Brucella testing of boars before breeding and awareness among farmers and veterinarians will aid in reduction of disease burden in the absence of vaccination policy. [ABSTRACT FROM AUTHOR]

ABSTRACT: Enzootic bovine leukosis (EBL) is an infectious disease caused by bovine leukemia virus (BLV) that affects cattle worldwide. Agar gel immunodiffusion (AGID) was the reference test for EBL diagnosis for many years, but enzyme-linked immunosorbent assay (ELISA) showed higher sensitivity, was faster to perform, and resulted in an objective reading. However, the importation of ELISA kits is lengthy and expensive, and currently, no AGID kits are available in Brazil. The aim of this work was to standardize an indirect ELISA (iELISA) for EBL diagnosis using BLV antigens produced in Tadarida brasiliensis lung (Tb1Lu) cells, which are Bovine viral diarrhea virus (BVDV) free, unlike fetal lamb kidney (FLK) cells, currently used for this purpose. Following standardization, iELISA results were compared with those obtained by AGID and the commercial Chekit Leucose-Serum ELISA. Compared to AGID, iELISA had 94,44% sensitivity, 75.68% specificity, 79.10% positive predictive value (PPV) and 93.30% negative predictive value (NPV), with 84% concordance and a Kappa index of 0.699. Compared to the Chekit Leucose-Serum ELISA, iELISA showed 92.60% sensitivity, 87.09% specificity, 90.27% PPV and 90,00% NPV, with 90.27% concordance and a Kappa index of 0.801. Taking into account the high agreement with the traditional tests and the absence of non-specific reactions with BVDV, the developed assay could be used as diagnostic method to control EBL in Brazil.

Vaccination is the practical solution for the prevention of rabies in dogs. Assessment of the immunogenicity of vaccination includes estimation of specific rabies virus neutralizing antibodies (VNA) in the target species. We undertook a study to estimate the levels of VNA in free-roaming dogs with a history of rabies vaccination in Bengaluru city, India. We compared the rapid fluorescent focus inhibition test (RFFIT) and an in-house quantitative indirect ELISA (iELISA). The study area comprised the jurisdiction of Bruhat Bengaluru Mahanagara Palike (BBMP), the Bengaluru civic body. The BBMP, along with several non-government organizations (NGO), were conducting a trap- neuter- vaccinate- release program for the prevention of dog rabies. Serum samples were collected from 250 free-roaming dogs from representative regions of BBMP, of which 125 had a VNA titre of 0.5 IU or more by the RFFIT. Furthermore, 126 dogs showed percent positivity values (PP values) more than the cut off PP value of 57.1 by the iELISA, accounting for 50.4% of satisfactory post-vaccinal serum conversion. The sensitivity and specificity of the iELISA was 94.4% and 95.2%, respectively. Based on these data, a quantitative iELISA may be a complementary tool for sero-monitoring immune responses of free-ranging animals after rabies vaccination. [ABSTRACT FROM AUTHOR]

Small ruminant (sheep and goats) brucellosis caused by Brucella melitensis remains widely recognized as a major zoonosis causing profound economic, animal production, and public health consequences. Even though definite diagnosis depends on the isolation of the bacterial agent from clinical and post-mortem specimens, a presumptive diagnostic based on the assessment of specific serological response is used in routine diagnostics for the purpose of disease control or animal trade. The Rose Bengal test, the complement fixation test, the enzyme-linked immunosorbent assay, or the fluorescence polarization assay are considered by the World Animal Health Organization--OIE as a suitable serological test for diagnosing B. melitensis infection on a herd and individual animal level. The aim of this study was to assess agreement among results of the Rose Bengal, complement fixation test, and indirect enzyme-linked immunosorbent assay using small ruminant sera samples collected through brucellosis surveillance program in Bosnia and Herzegovina. A subset of these samples from non-vaccinated animals (2250) was reused and tested on each test. Agreement among test results was assessed pair vise using Kappa statistical analysis with correspondent 95% CI. Additionally, Landis-Koch scale was used for the classification of observed agreement based on established Kappa. The highest agreement was found between the complement fixation test and the Rose Bengal test (0.643), while the lowest was between the enzyme-linked immunosorbent assay and the rose Bengal test (0.533). Choice of serologic tests and testing protocols used in brucellosis surveillance programs depends on the program aim, alongside specific epidemiological, animal production, economic, and cultural circumstances. [ABSTRACT FROM AUTHOR]

Abstract Background The tick-borne intra-erythrocytic apicomplexan Babesia caballi is one of the etiological agents of equine babesiosis, an economically important disease of equids in most tropical and subtropical areas of the world. Discovering candidate antigens for improved diagnostic tools and vaccines remains needed for controlling equine babesiosis. This study describes the B. caballi sbp4 (Bcsbp4) gene and protein (BcSBP4) and analyzes its antigenicity in infected equids. Methods BLAST searches of an uncurated B. caballi assembly genome using the B. bovis SBP4 as a query were carried out, followed by PCR amplification and sequencing of a newly identified BcSBP4. Characterization of this novel gene and protein was performed by bioinformatics analysis, western blots, immunofluorescence (IFA) and an in vitro neutralization test using anti SBP4 peptide antibodies. Antigenicity of recombinant BcSBP4 (rBcSBP4) was tested with sera from field animals (n = 18) using an indirect ELISA (iELISA). Results Babesia caballi genome searches using B. bovis SBP4 as a query allowed identification of a novel gene termed Bcsbp4. The Bcsbp4 gene encodes for a protein of 30.58 kDa, which is fully conserved among B. caballi isolates from USA and Egypt. Bioinformatics analysis indicates that BcSBP4 contains a signal peptide and lacks additional transmembrane domains. Expression of BcSBP4 in blood stages of B. caballi was confirmed by western blot and IFA using antibodies against synthetic peptides representing putative B-cell epitopes of BcSBP4 predicted by in silico analysis. In vitro neutralization tests using anti-BcSBP4 peptide antibodies showed a marginal, but statistically significant inhibitory effect on the infectivity of B. caballi merozoites in horse red blood cells. Sera from eight B. caballi-infected equids, but none out of ten negative equid control sera, gave a positive signal in an rBcSBP4 based iELISA. Conclusions The Bcsbp4 gene is expressed in B. caballi blood stages. The BcSBP4 protein is a potential candidate for developing a novel serological test that could detect B. caballi infection in equids in tropical and subtropical countries worldwide.

Abstract Background Antibiotic treatment of horses with strangles is reported to impair the development of immunity to subsequent exposure to Streptococcus equi ssp equi (S. equi). However, apart from a single clinical report, evidence‐based studies for this hypothesis are lacking. Hypothesis/Objective To determine whether penicillin treatment during clinical strangles influences the development or persistence of seropositivity to S. equi‐specific antibodies. Animals A natural outbreak of strangles with 100% morbidity in 41 unvaccinated mature Icelandic horses. Methods A prospective longitudinal study of acute clinical strangles from onset through full recovery approximately 10 months after the index case. Horses were monitored clinically 6 times for S. equi, as well as serologically for antibodies to antigens A and C of S. equi using an enhanced indirect ELISA. Seven horses received penicillin within 11 days of onset of fever (Group 1), 5 between 16 and 22 days after onset of fever (Group 2), and the remainder (Group 3, n = 29) received no antibiotics during clinical disease. The proportions of seropositive horses in each group were compared using an extension of Fisher's exact test with P

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.

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.

Brucellosis caused by facultative intracellular bacteria, Brucella, remains a global threat to both animal and human health. In this study we aimed to identify potential risk factors of bovine brucellosis and to assess the knowledge, attitudes, and practices (KAPs) of livestock keepers in Hisar, India. A standardized questionnaire was used to collate information regarding potential risk factors of bovine brucellosis and livestock owners’ KAPs. A total of 127 livestock keepers were involved. Serum samples from their animals (n = 635) were tested for the presence of antibodies against Brucella by Rose Bengal Plate Test (RBPT) and indirect enzyme-linked immunosorbent assay (iELISA). Out of these, 78 (61.4%) of the herds had at least one seropositive animal, and 302 (47.6%) of the cattle were seropositive. Univariate and multivariate analysis revealed significant associations between intensive farm type (OR = 4.6; 95% CI, 1.6–16.7; P = 0.009), hygienic disposal of aborted fetuses (OR = 0.3; 95% CI, 0.08–0.9; P = 0.04) and herd seropositivity for brucellosis. The majority, 96 (75.6%) of the respondents, were males aged 18–50, and 82 (64.6%) owned a small-backyard farm. Only 51 (40.2%) of the participants knew about brucellosis; out of them, 54.9% (28/51) could not identify clinical signs of brucellosis. Six (11.8%) participants indicated abortion as the most noticeable clinical sign, and 45.1% indicated that consumption of raw milk is associated with high risk of contracting brucellosis. A large proportion of respondents confirmed that milk from their animals was regularly consumed (86.6%) and sold (59.8%) to other people. These results suggest that bovine brucellosis is endemic in Haryana, where Brucella-contaminated milk is likely being regularly sold. Brucellosis control efforts in Haryana should include education programs to raise awareness of the disease and means to control it in cattle and to prevent zoonotic transmission. [ABSTRACT FROM AUTHOR]

Brucellosis in small ruminants caused mainly due to Brucella melitensis is an important zoonotic disease characterized by abortion, retained placenta, infertility, orchitis, epididymitis and rarely arthritis. Small ruminants are the main source of economy for the rural and marginally poor farmers and brucellosis is resulting in huge economic losses due to abortions and infertility and causing public health concern among the small ruminant keepers. Bovine brucellosis control programme has been implemented in India and small ruminants are left out of the programme mainly due to paucity of brucellosis status. The present cross‐sectional study based on stratified random sampling was undertaken during 2017–18 to provide the nationwide brucellosis sero‐prevalence in small ruminants. A total of 24,056 small ruminant serum samples (sheep samples = 8,103 [male—2,440 and female—5,663] and goat samples = 15,953 [male—4,331 and female—11,622]) sourced from 27 out of 29 states and two out of seven union territories (UTs), 350 districts of total 640 districts (54.68% of the Indian districts) and from 1,462 villages out of 6,40,867 villages (43.83% of the Indian villages). The serum samples were tested by indirect ELISA and overall brucellosis apparent and true prevalence of 7.45 (95% CI: 7.13–7.79) and 3.79 (95% CI: 3.44–4.17) was recorded. Significantly higher brucellosis sero‐prevalence (p <.0001) was observed in sheep (11.55%) than goats (5.37%). Similarly, brucellosis seropositivity was highly significant in females compared to males in both sheep and goats. Countrywide, greater than 5% brucellosis sero‐prevalence in sheep and goats was recorded in 14 and 10 states, respectively, indicating endemicity of the disease. The study provided the latest update on nationwide spatial sero‐prevalence of small ruminant brucellosis which will aid government to strengthen regular surveillance and vaccination to reduce the disease burden and public health problems in the country. [ABSTRACT FROM AUTHOR]

Brucellosis is a well-known contagious and zoonotic infection of different animals which influences the health status of animal and economy of underdeveloped/ developing countries. Therefore, this study was conducted to estimate the seroprevalence and risk factors of Brucella infection in dairy cattle through reliable diagnostic techniques. A total of 300 cattle was included for blood sampling kept at peri-urban areas of Sargodha, Sahiwal and Chiniot districts of Punjab. All the serum samples were tested through RBPT antigen, i-ELISA and RT-PCR. Results showed that 12.66% animals were seropositive for Brucella infection and all seropositive samples were positive for Brucella genus specific RT-PCR. Abortion history, gender, repeat breeding, lactating animals and pregnancy status were key factors associated with seropositivity of animals. Results on logistic regression analysis indicated significant association of different potential risk factors such as history of abortion, repeat breeding and lactating stage with brucellosis in animals. From the results of this study, it is suggested that there is a need of continuous screening of dairy animals to control brucellosis and to minimize the risk of human brucellosis. [ABSTRACT FROM AUTHOR]

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.

Brucellosis is among the most important zoonotic infectious diseases worldwide affecting both humans and domestic animals. The present study aimed to determine and compare the seroprevalence of brucellosis among rural and periurban dairy cattle farms of four Iranian provinces from 2017 to 2019. We applied different serological tests, including RBT, SAT, and iELISA to evaluate the brucellosis prevalence among 2808 dairy cattle. Species-specific multiplex PCR and biotyping tests were also used to further identify the implicated Brucella species. Serological screening using RBT, SAT, and iELISA led to 157 (5.6%), 112 (3.9%), and 139 (4.9%) positive results among tested cattle, respectively. Brucella abortus biovars 1 (2 cases) and biovars 3 (42 cases) were identified by biotyping experiments and multiplex PCR in all 44 tested lymph node samples. Further, Cohen’s kappa statistical analysis revealed that the best degree of agreement was seen between RBT and iELISA (99.4%), followed by SAT/iELISA (98.5%) and finally RBT/SAT (98.4%). Our results also showed a significantly lower seroprevalence of brucellosis in periurban dairy cattle when compared to rural dairy cattle population (p value= 0.01). These results reflect the need for better vaccine coverage using RB51 combined with an appropriate test-and-slaughter program in the rural dairy cattle population. [ABSTRACT FROM AUTHOR]

Trichinella is a zoonotic nematode traditionally detected worldwide in both domestic and wild animals. In South America, along with the occurrence of this parasite in domestic pigs and wild boars, there are reports of infection in wild carnivores. Brazil is considered free of the domestic cycle of Trichinella, but there is unpublished serological evidence of infection in wild boars, which changed the Brazilian status in OIE regarding the disease after an official communication. We investigated Trichinella spp. infection in wild boars and wild carnivores in the Southeastern region of Brazil. A total of 136 samples were tested, 121 from wild boars and 15 from wild carnivores. Artificial enzymatic digestion (AED) tests were performed on muscle samples from 37 wild boars and 15 wild carnivores, and 115 serum samples from wild boars were tested by iELISA. Seven serum samples from wild boars tested positive (7/115 = 6.1%, 95% CI 3.0–12.0), but no larvae were found in the AED. There was no significant difference between sex, age, and location of the samples. The serological results suggest that a wild cycle of Trichinella spp. may occur in Brazil, but further analyses should be performed to confirm the presence of the parasite.

Vaccination is the practical solution for the prevention of rabies in dogs. Assessment of the immunogenicity of vaccination includes estimation of specific rabies virus neutralizing antibodies (VNA) in the target species. We undertook a study to estimate the levels of VNA in free-roaming dogs with a history of rabies vaccination in Bengaluru city, India. We compared the rapid fluorescent focus inhibition test (RFFIT) and an in-house quantitative indirect ELISA (iELISA). The study area comprised the jurisdiction of Bruhat Bengaluru Mahanagara Palike (BBMP), the Bengaluru civic body. The BBMP, along with several non-government organizations (NGO), were conducting a trap- neuter- vaccinate- release program for the prevention of dog rabies. Serum samples were collected from 250 free-roaming dogs from representative regions of BBMP, of which 125 had a VNA titre of 0.5 IU or more by the RFFIT. Furthermore, 126 dogs showed percent positivity values (PP values) more than the cut off PP value of 57.1 by the iELISA, accounting for 50.4% of satisfactory post-vaccinal serum conversion. The sensitivity and specificity of the iELISA was 94.4% and 95.2%, respectively. Based on these data, a quantitative iELISA may be a complementary tool for sero-monitoring immune responses of free-ranging animals after rabies vaccination.

Bovine brucellosis is an important zoonotic disease caused by Brucella abortus. The distribution of bovine brucellosis in West Bengal has not been reported so far. Here, a longitudinal epidemiological study was conducted from April 2002 to March 2016 to determine the distribution, pattern and trend of bovine brucellosis in different organized and unorganized farms of West Bengal, India. Bovine serum samples were examined for identification of brucellosis by Rose Bengal Plate Test and indirect ELISA. Results envisaged that the prevalence of brucellosis is 11.12 times higher in organized farm (average 6.6%) compared to rural areas (average 0.6%) with overall prevalence was 3.9%. The highest and lowest level of sero-positivity was observed in 2015 and 2011 with 9.8% and 0.5% respectively. In reference to the year 2002, trend of brucellosis was decreasing up to 2013 in a fluctuating manner (odd ratio ranging from 1.7 to 2.3); however, it again increased in 2014 and 2015 with final decrease in 2016. In organized farms, highest (12.6%) and lowest (0.6%) level of prevalence was recorded in 2015 and 2011 respectively. In rural areas, the highest and lowest level of prevalence was observed in 2010 and 2008 with 1.8% and 0.15% respectively. Chi square statistics revealed that location of cattle (χ2 =713.8, df=1, p

Abstract Background Human chromogranin A (CgA) is a ~ 49 kDa secreted protein mainly from neuroendocrine cells and endocrine cells. The CgA values in the diagnosis of tumor, and in the potential role in prognostic and predictive tumor as a biomarker. Results The synthesized gene of CgA coding area was cloned and expressed as fusion protein CgA-His in procaryotic system. Then the purified CgA-His protein was mixed with QuickAntibody-Mouse5W adjuvant, and injected into mice. The CgA-His protein was also used as coating antigen to determine the antiserum titer. By screening, a stable cell line named 4E5, which can generate anti-CgA monoclonal antibody (mAb), was obtained. The isotype of 4E5 mAb was IgG2b, and the chromosome number was 102 ± 4. Anti-CgA mAb was purified from ascites fluid, and the affinity constant reached 9.23 × 109 L/mol. Furthermore, the specificity of the mAb was determined with ELISA, western blot and immunohistochemistry. Results indicated that the mAb 4E5 was able to detect chromogranin A specifically and sensitively. Conclusions A sensitive and reliable method was successfully developed for rapid production of anti-CgA mAb for immunohistochemistry diagnosis in this study, and the current study also provides conclusive guidelines for preparation of mAbs and implements in immunohistochemistry diagnosis.

Background: Difficulty in detection of silent carriers of Streptococcus equi is a key reason for its continued spread to immunologically naïve groups of horses. Objective: To determine whether clinical examination, markers of inflammation, or serology differentiate silent carriers of S. equi in recovered comingled horses. Animals Ninety‐eight warmblood yearlings and 72 unaffected mares on a large breeding farm (outbreak A), 38 mature Icelandic horses at a riding stable (outbreak B), and 27 mixed breed horses at a boarding stable (outbreak C). Methods: Prospective observational study 6 months to 2 years after strangles outbreaks. Carriers were defined as any animal positive on culture or qPCR to S. equi from nasopharyngeal lavage or guttural pouch endoscopy and lavage. Most horses had complete physical exams and 1 group included evaluation of white blood cell counts and serum amyloid A. Sera from all horses was tested for antibodies to antigens A and C of S. equi using an enhanced indirect ELISA. Descriptive statistics were calculated. Data were compared using paired t tests, Wilcoxon ranked test, chi square, or the Fishers exact test. Significance was set at P <.05. Results: Apart from weanlings at 6 months in outbreak A, there was no significant association between any clinical markers or serology with carrier state (P =.06‐1). Moreover, 3/12 culture positive carriers were seronegative to S. equi. Conclusions and Clinical Importance: Silent carriers of S. equi do not differ clinically or on markers of inflammation to their noncarrier herd‐mates. Moreover, serology alone will not distinguish carriers in comingled horses. [ABSTRACT FROM AUTHOR]

As per the report from the OIE in 2005, infectious laryngotracheitis (ILT) has not been yet reported in Ethiopia. Hence, considering the evident clinical signs on-field associated with the disease, it felt that there is a need to identify the disease and to protect the chicken population. The study was, therefore, aimed at identifying the seroprevalence of ILT virus from the samples collected from chickens in backyard system, so as to notify its prevalence and setup recommendations for further research in the future. Consequently, cross-sectional study was conducted in eleven purposefully selected peasant associations (PA) of Ada'a district from January to May 2019 to determine ILT in backyard chickens. A total number of 426 sera sample of backyard chickens were randomly collected from 11 PA and each sera was exposed to an indirect enzyme-linked immunosorbent assay (iELISA), at the National Animal Health Diagnostic and Investigation Center, Ethiopia. Out of 426 samples, 233 (54.7%) samples were found positive for ILT virus-specific antibody. The highest prevalence was recorded in Wajitu (83.3%), whereas the least was in Giche (40.7%) PA. There was a statistically significant difference (p < 0.05) among seroprevalence and study PA. The result of this study revealed that a high prevalence of ILT virus is circulating among backyard chickens in the selected PA of Ada'a district, which could significantly affect the poultry sector. Thus, further studies on the circulating strains and the epidemiology of the disease should be carried using a molecular diagnostic test. [ABSTRACT FROM AUTHOR]