Potential diagnostic application of the baculovirus-expressed recombinant truncated nucleocapsid protein of peste des petits ruminants virus in ELISA.

The study describes the expression of recombinant truncated nucleocapsid protein (NP) of peste des petits ruminants (PPR) virus in the baculovirus system (PPRV-rBNP) and its potential application as a diagnostic antigen in ELISA for diagnosis of PPR in sheep and goats. The PPRV N-terminal immunogenic region (1-266 aa) of the NP coding sequence was amplified and cloned into the pFastBac HT A vector. The PPRV-rBNP with a molecular weight of ∼30 kDa was expressed in an insect cell system using generated recombinant baculovirus through Bac-to-Bac® Baculovirus Expression System. The crude PPRV-rBNP or Ni-NTA affinity-purified NP was characterized by SDS-PAGE and immunoblot using standard PPRV-specific sera. The PPRV-rBNP reacted well with PPRV anti-N specific monoclonal and polyclonal antibodies and PPRV-specific antiserum, suggesting that the expressed PPRV-rBNP is in its native form. The crude PPRV-rBNP as a diagnostic antigen was evaluated either as a coating antigen or standard positive control antigen in the Avidin-Biotin ELISA using the known standard panel reagents. The results showed that the expressed PPRV-rBNP can be an alternative diagnostic antigen to E. coli expressed recombinant PPRV-NPN and the utility of PPRV-rBNP avoids the need to use live PPRV antigen in the diagnostic ELISA. Hence, this allows scope in the future for large-scale field application of the recombinant antigen-based assays for diagnosis/surveillance and monitoring of PPR at the eradication as well as post-eradication phases in endemic countries or PPR non-endemic countries.

Comparative diagnostic efficacy of Avidin-Biotin recombinant nucleoprotein competitive ELISA for serosurveillance and monitoring of peste des petits ruminants in sheep and goats.

In this study extensive evaluation of Avidin-Biotin recombinant nucleoprotein competitive ELISA (ABrC-ELISA) was carried out by mass screening of a large number of sera to make use of this assay for serosurveillance and seromonitoring of peste des petits ruminants (PPR) in sheep and goats to evaluate its diagnostic efficacy value and strengthen findings associated with the assay. The recombinant PPR virus (PPRV) nucleoprotein was over-expressed in E. coli, Ni-NTA affinity-purified, and characterized and used as coating diagnostic antigen in ABrC-ELISA, and evaluated using the field sera from animals. On evaluation of the diagnostic performance or efficacy of this assay using the pre-vaccinated and post-vaccinated sera of sheep and goats (n = 1437), the ABrC-ELISA showed a relative diagnostic sensitivity of 87.2% (95% CI: 84.1-90%) and diagnostic specificity of 92.0% (95% CI: 90-93.7%), against well-established existing indigenous H protein-specific PPR competitive ELISA kit with an accuracy of 90.1% (95% CI: 88.5-91.7%) and good or substantial agreement of Cohen's Kappa value of 0.79 ± 0.017 SE (95% CI: 0.76 to 0.82). These findings suggest that the ABrC-ELISA is a potential additional diagnostic tool of a rapid, sensitive, and specific assay for the detection of the PPRV nucleoprotein antibodies in sera of sheep and goats. This PPR Ab Chek kit can be used extensively under field conditions for serosurveillance, and seromonitoring of PPR in sheep and goats at the eradication /post-eradication phase in disease-controlled countries or PPR non-enzootic countries.

Assessment of post-vaccination immune response to peste des petits ruminants virus in small ruminants in the central and western regions of India.

The cross-sectional serosurvey for post-vaccination assessment of peste des petits ruminants (PPR) virus (PPRV) antibodies in sheep and goats was carried out in different states in the central and western regions of India after the implementation of vaccination under the PPR control programme. The serum samples (n = 4687) were collected from sheep (n = 1539) and goats (n = 3148) from August 2017 to March 2018 at various epidemiological units (n = 301) of the studied regions using a stratified random sampling method and PPR competitive ELISA kit was employed to detect PPRV antibodies. The results revealed 34, 21, 52, 74, 68, and 65% of prevalence of PPRV antibodies in small ruminants in Madhya Pradesh, Goa, Chhattisgarh, Maharashtra, Gujarat, and Rajasthan states, respectively, with a difference in seropositivity in sheep and goats across the states in sheep (p < 0.01) and goats (p < 0.01). Further, this serosurvey revealed that 60% of the epi-units (n = 185) had > 50% prevalence of post vaccination PPRV antibodies across states due to variations in vaccination rates and patterns. The vaccination coverage and the reported outbreaks varied between the states in the studied regions. Due to continuous vaccination under the control program, the reported PPR outbreaks have progressively declined in most of the studied states, and the PPR risk areas are confined to a few districts and sporadically, outbreaks are reported indicating the effectiveness of vaccination. These findings provide valuable information on potential PPRV episystems, and will assist with activities regarding intensive surveillance, vaccination, biosecurity, and modification of policy decisions towards designing and implementing control and eradication measures. Further, the present situation necessitates continuous mass vaccination and active surveillance programs to make these regions free from PPR in consonance with the PPR Global Control and Eradication Strategy under the PPR Global Eradication Program. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-022-00796-6.

Avidin-Biotin recombinant nucleoprotein competitive ELISA for the detection of peste des petits ruminants virus antibodies in sheep and goats.

The present study describes the development of a truncated recombinant peste des petits ruminants virus (PPRV) nucleoprotein (rPPRV-NPN) and its polyclonal antibodies-based immuno-diagnostic assay, Avidin-Biotin (AB) recombinant nucleoprotein competitive ELISA (ABrC-ELISA) for the detection of PPRV antibodies in the sheep and goats. The PPRV N-terminal immunogenic region (1-266 aa) of nucleoprotein (NPN) coding sequence was amplified and cloned into the pETite vector. The rPPRV-NPN with a molecular weight of ∼ 30 kDa was expressed in E. coli, purified, and characterized by SDS-PAGE and immunoblot using standard PPRV specific sera. The Ni-NTA affinity-purified rPPRV-NPN as coating antigen and its hyperimmune serum as competitive antibodies raised in guinea pigs were evaluated as diagnostic reagents in ABrC-ELISA using the known standard panel of sera. The threshold (cut-off) Percentage Inhibition (PI) value was determined as 45 (mean ± 3 SD) based on the reactivity of the known sheep and goats sera to PPRV antibodies [negative (n = 140) and positive (n = 98)] and the assay had a sensitivity of 97 % (95 % Confidence Interval (CI): 91.3-99.4 %) and specificity of 100 % (95 % CI: 97.4-100 %) with an excellent Area under curve (AUC) of 0.997 (95 % CI: 0.99-1.0). On evaluation of diagnostic performance of the assay using the sheep and goats sera (n = 391) from vaccinated, infected, and non-vaccinated animals, the ABrC-ELISA showed the relative diagnostic sensitivity of 95.88 % (95 % CI: 92.56-98.01 %) & 98.77 % (95 % CI: 96.43-99.74 %) and diagnostic specificity of 97.97 % (95 % CI: 94.19-99.58 %) & 90.54 % (95 % CI: 84.64-94.73 %) against indigenous PPR competitive ELISA kit & IDvet Screen® PPR Competition kit, respectively. The study showed that ABrC-ELISA is rapid, sensitive, and specific and can be a better alternative assay for the detection of the PPRV antibodies in the sera of small ruminants for serosurveillance / seromonitoring of PPR not only at the eradication and post-eradication phases in the disease-controlled endemic countries but also in the PPR non-endemic countries.

Evaluation of the diagnostic potential of recombinant leptospiral OMP A-like protein (Loa22) and transmembrane (OmpL37) protein in latex agglutination test for serodiagnosis of leptospirosis in animals.

Leptospirosis is a re-emerging zoonotic disease of animals and humans caused by pathogenic Leptospira, which has major public health concerns. The study is aimed to express the recombinant outer membrane protein (OMP) A-like protein (rLoa22) and transmembrane (rOmpL37) protein of Leptospira interrogans serovar Hardjo in the Escherichia coli and their evaluation as a diagnostic antigen in the latex agglutination test (LAT) to detect anti-leptospiral antibodies in the sera of animals. The Loa22 and OmpL37 genes lacking signal peptide coding sequences were individually amplified (522 and 963 bp), by polymerase chain reaction, and directionally cloned into a pETite N-His Kan vector for expression. The expressed purified proteins were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblot, which confirmed leptospiral specific reactive protein with a molecular weight of ~19 and 36 kDa, respectively. The sensitized latex beads coated with these OM proteins separately were evaluated in LAT using cattle sera of microscopic agglutination test (MAT) confirmed positive (n = 53) and negative (n = 52) cases of leptospirosis. The rLoa22 LAT and rOmpL37 LAT revealed the relative diagnostic sensitivity of 94·34 and 96·23%, diagnostic specificity of 92·31 and 96·15% and accuracy of 93·33 and 96·19%, with the excellent agreement of Cohen's kappa value of 0·87 and 0·92, respectively. After extensive evaluation, this rapid recombinant protein-based field diagnostic test can be applied as a screening test for the detection of anti-leptospiral antibodies in the sera of animals in the field conditions.

Peste Des Petits Ruminants in Atypical Hosts and Wildlife: Systematic Review and Meta-Analysis of the Prevalence between 2001 and 2021.

Peste des petits ruminants (PPR) or goat plague is considered a leading, highly contagious, and most lethal infectious viral disease of small ruminants affecting the worldwide livestock economy and international animal trade. Although sheep and goats are the primarily affected, the PPR Virus (PPRV) host range has expanded to other livestock (large ruminants) and wildlife animals over the last few decades, resulting in serious concern to the ongoing PPR global eradication program, which is primarily optimized, designed, and targeted towards accessible sheep and goat population. A systematic review and meta-analysis study was conducted to estimate the prevalence and spill-over infection of PPRV in large ruminants (bovine and camel) and wildlife. Published articles from 2001 to October 2021 on the "PPR" were searched in four electronic databases of PubMed, Scopus, Science direct, and Google Scholars. The articles were then selected using inclusion criteria (detection/prevalence of PPRV in bovine, camel, and wildlife population), exclusion criteria (only sheep or goats, lack of prevalence data, experimental trial, test evaluation, and reviews written in other languages or published before 2001), and the prevalence was estimated by random effect meta-analysis model. In the current study, all published articles belonged to Africa and Asia. The overall pooled prevalence of PPR estimates was 24% (95% CI: 15-33), with 30% in Asia (95% CI: 14-49) and 20% in Africa (95% CI: 11-30). The overall estimated pooled prevalence at an Africa-Asia level in bovine and camel was 13% (95% CI: 8-19), and in wildlife, it was 52% (95% CI: 30-74) with significant heterogeneity (I2 = 97%) in most pooled estimates with a high prevalence in atypical hosts and wildlife across Asia and Africa. Over the last two decades, the host range has increased drastically in the wildlife population, even for prevalent PPR in the unnatural hosts only for a short time, contributing to virus persistence in multi-host systems with an impact on PPR control and eradication program. This observation on the epidemiology of the PPRV in unnatural hosts demands appropriate intervention strategies, particularly at the livestock-wildlife interface.

Towards eradication of peste des petits ruminants: post-vaccination evaluation in sheep and goats in Southern Peninsular India.

The cross-sectional seroprevalence study of the peste des petits ruminants (PPR) in sheep and goats was carried out in the Southern Peninsular region of India to ascertain the prevalence of PPR virus (PPRV) antibodies at the epidemiological units (epi-units) level in the small ruminant population. The serum samples were collected from various epi-units (villages) in the different states and union territory (UT) in Southern Peninsular region using a stratified random sampling methodology from August 2017 to March 2018. A total of 6643 serum samples [sheep (n = 2785) and goats (n = 3858)] were collected from 360 epi-units and were screened by PPR competitive ELISA kit for the detection of PPRV antibodies. The results revealed that the seroprevalence of PPR in small ruminants in Telangana, Andhra Pradesh, Karnataka, Tamil Nadu, and Kerala states, and Puducherry UT was 87.0%, 66.4%, 64.3%, 47.8%, 11.4%, and 50.4%, respectively in the studied region. Further, the results of the chi-squared test revealed that the PPRV antibodies across different states and UT in the region were associated (sheep-χ2 = 218.8, p < 0.01; goats-χ2 = 827.1, p < 0.01), as all the states and UT adopted the PPR vaccination programme. The study also implies that the small ruminants in some of the epi-units (n = 102) had < 30% seroprevalence, which necessitates comprehensive intensive vaccination and active surveillance programmes to make this region as PPR free zone.

Expression of Recombinant Leptospiral Surface Lipoprotein-Lsa27 in E. coli and Its Evaluation for Serodiagnosis of Bovine Leptospirosis by Latex Agglutination Test.

The expressed recombinant leptospiral surface adhesion lipoprotein (Lsa27) of pathogenic Leptospira in E. coli was evaluated for the detection of Leptospira antibodies in cattle sera by latex agglutination test (LAT). The Lsa27 lacking signal peptide coding gene sequences from L. interrogans serovar Pomona was amplified (~ 660 bp) by PCR and the amplicon was cloned into pETiteN-HisKan vector. The expressed recombinant Lsa27 histidine-tagged fusion protein (rLsa27) was Ni-NTA affinity purified under denaturation followed by renaturation methods. The purified rLsa27 was characterized by SDS-PAGE and immunoblot, which confirmed the leptospiral protein with a MW of ~ 25 kDa. Further, the prepared sensitized latex beads coated with rLsa27 were evaluated as a diagnostic antigen for detection of pathogenic Leptospira antibodies by using known microscopic agglutination test (MAT) positive (n = 74) and negative (n = 62) sera for Leptospira antibodies in LAT, which revealed the relative diagnostic sensitivity of 91.89% and specificity of 87.10% against the gold standard serological test, MAT. Furthermore, on evaluation of developed rLsa27 LAT using serum samples from cattle associated with the history of abortions and reproductive disorder (n = 309), the relative sensitivity of 96.15%, and specificity of 89.11% were observed. Therefore, this rapid field test using the rLsa27 is first of its kind and it could be used as a screening test for the detection of Leptospira antibodies or it can be complemented by other diagnostics for the diagnosis /surveillance of bovine leptospirosis.

Seroprevalence of Peste des petits ruminants in small ruminants in the North Eastern Region of India.

A seroprevalence study of the peste des petits ruminants (PPR) in small ruminants was carried out in the different states (Assam, Manipur, Meghalaya, Mizoram, Nagaland, and Tripura) in the North Eastern Region (NER) of India using serum samples collected from April 2017 to March 2018. A total number of 4,163 sera [sheep (n = 508) and goats (n = 3,655)] collected from 345 epi­units/villages covering 176 municipalities in NER were screened by competitive ELISA kit for the detection of PPR virus antibodies. The results revealed that the seroprevalence of PPR in small ruminants in Assam, Manipur, Meghalaya, Mizoram, Nagaland, and Tripura was 34.3%, 10.3%, 4.7%, 15.7%, 14.7%, and 5.5%, respectively with an overall 14.5% prevalence.Association between the presence of antibodies and goats has been showed to be significant (p < 0.01) at the NER level level and within every single state. This manuscript highlights the need for continuous monitoring of this important disease as for the severe economic impact PPR may have in the affected countries.

Scorecard method for assessing the severity of peste des petits ruminants in sheep and goats.

A methodology to assess the clinical severity of peste des petits ruminants (PPR) in sheep and goats in the field condition was developed using a scorecard by considering five specific cardinal clinical signs (pyrexia, oculo-nasal discharge, oral lesions, respiratory signs, and diarrhoea) of disease. The scores were assigned for the signs based on the severity of the disease that ranged from 1 (low) to 4 (high). The assigned weightage for signs, morbidity, and mortality was 0.75, 0.05 and 0.2, respectively summing up to unity. The scoring and weightages and guidelines were devised by Delphi technique based on the field investigation, field veterinarian's assessment and specific inputs from PPR experts. The estimated Weighted Score Index (WSI) was considered to classify the severity into mild (WSI < 40) or moderate (WSI 41-60) or severe (WSI > 60) form. This scorecard will help preliminarily to the extent for the identification of the suspected flocks with a required case definition at the first instance, before making decisions on what merits further field investigation. This is first of its kind of methodology to assess the disease pattern in small ruminants and could be used as a disease severity assessment tool in different geographical areas in endemic settings.