[Preparation of colloidal gold test strips for the detection of antibodies to peste des petits ruminants based on monoclonal antibodies to N protein].

A simple, fast, and visual method for detecting antibodies against peste des petits ruminants virus (PPRV) using colloidal gold strips was developed. In this study, the pET-32a-N was transformed into Escherichia coli Rosetta (DE3) for expression. Hybridoma cell lines were generated by fusing SP2/0 myeloma cells with splenocytes from immunized mice with the expressed and purified N protein of PPRV. The PPRV N protein was labeled with colloidal gold particles as the gold-labeled antigen. The N protein served as the gold standard antigen and as the test (T) line-coated antigen, while the monoclonal antibody served as the quality control (C) line-coated antibody to assemble the colloidal gold immunochromatographic test strips for detecting antibodies against the N protein of PPRV. Hybridoma cell line designated as 1F1 was able to stably secrete the monoclonal antibody against the N protein of PPRV. The titer of 1F1 monoclonal antibody in ascites was 1:128 000 determined by indirect enzyme-linked immunosorbent assays (ELISA), and the immunoglobulin subtype of the monoclonal antibody was IgG1, with kappa chain. The obtained monoclonal antibody was able to specifically recognize the N protein of PPRV, as shown by Western blotting and indirect immunofluorescent assay (IFA). The developed colloidal gold test strip method was able to detect PPRV antibodies specifically, and there was no difference between different batches of the test strips. Testing of a total of 122 clinical sera showed that the compliance rate of the test strip with ELISA test was 97.6%.The test strip assay developed in this study has good specificity, reproducibility, and sensitivity, and it can be used for the rapid detection of PPRV antibodies.

The evaluation of five serological assays in determining seroconversion to peste des petits ruminants virus in typical and atypical hosts.

Peste des petits ruminants (PPR) is an infectious viral disease, primarily of small ruminants such as sheep and goats, but is also known to infect a wide range of wild and domestic Artiodactyls including African buffalo, gazelle, saiga and camels. The livestock-wildlife interface, where free-ranging animals can interact with captive flocks, is the subject of scrutiny as its role in the maintenance and spread of PPR virus (PPRV) is poorly understood. As seroconversion to PPRV indicates previous infection and/or vaccination, the availability of validated serological tools for use in both typical (sheep and goat) and atypical species is essential to support future disease surveillance and control strategies. The virus neutralisation test (VNT) and enzyme-linked immunosorbent assay (ELISA) have been validated using sera from typical host species. Still, the performance of these assays in detecting antibodies from atypical species remains unclear. We examined a large panel of sera (n = 793) from a range of species from multiple countries (sourced 2015-2022) using three tests: VNT, ID VET N-ELISA and AU-PANVAC H-ELISA. A sub-panel (n = 30) was also distributed to two laboratories and tested using the luciferase immunoprecipitation system (LIPS) and a pseudotyped virus neutralisation assay (PVNA). We demonstrate a 75.0-88.0% agreement of positive results for detecting PPRV antibodies in sera from typical species between the VNT and commercial ELISAs, however this decreased to 44.4-62.3% in sera from atypical species, with an inter-species variation. The LIPS and PVNA strongly correlate with the VNT and ELISAs for typical species but vary when testing sera from atypical species.

Investigation of the Clinical and Diagnostic Aspects of Peste des Petits Ruminants (PPR) in Sheep from the Southern Region of Iraq.

In the southern region of Iraq, Peste des petits ruminants (PPR) has been identified and diagnosed. The study was done on (300) local sheep breeds of varying ages and sexes exhibiting PPR symptoms, while (25), healthy sheep breeds served as the control group. Additionally, the diagnosis of PPRV was confirmed by PCR. Infected sheep exhibit a variety of clinical symptoms. However, DNA sequencing was used to detect genetic links and genetic variation, and the results revealed a closed genetic relationship with the NCBI BLAST PPRV India isolate (GU014574.1) at total genetic variation (0.02-0.01%). Results indicate a large rise in PCV and ESR in conjunction with leukocytopenia and lymphocytopenia, a significant difference in clotting factor indices, and a significant increase in ALT, AST, and CK. In addition, there was a substantial variation in acute phase response. Postmortem examinations revealed various erosive lesions on the upper and lower gums, severe hemorrhagic enteritis, particularly of the small intestine, and obvious congestion of the lungs. Histopathological changes revealed an obvious flattening of the intestinal mucosa as well as an enlargement of the villi. In addition to a granuloma in the sub-mucosa, chronic inflammatory cells, primarily lymphocytes, were seen invading the mucosa. It has been determined that the sickness was circulating in the southern region of Iraq and severely afflicted sheep, which might result in significant economic losses owing to the detrimental effects of the virus that causes the disease on the various bodily parts.

Prokaryotic expression of the V protein of the peste des petits ruminants virus and development of an indirect ELISA.

In this study, we recombinantly expressed the V protein of the peste des petits ruminants virus (PPRV) and evaluated its diagnostic value for PPRV infection using an indirect ELISA (i-ELISA). The optimal concentration of the coated antigen of V protein was 15 ng/well at a serum dilution of 1:400, and the optimal positive threshold value was 0.233. A cross-reactivity assay showed that the V protein-based i-ELISA was specific to PPRV with consistent reproducibility and showed a specificity of 82.6% and a sensitivity of 100% with a virus neutralization test. Using the recombinant V protein as an antigen in ELISA is useful for seroepidemiological studies of PPRV infections.

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.

Identification and characterization of phage display-selected peptides having affinity to Peste des petits ruminants virus.

Phage display is a well-established technique used for selecting novel ligands having affinity to a plethora of targets including proteins, viruses, whole bacterial and mammalian cells as well as lipid targets. In the present study, phage display technology was used to identify peptides having affinity to PPRV. The binding capacity of these peptides was characterized through various formats of ELISA using phage clones, linear and multiple antigenic peptides. The whole PPRV was used as an immobilized target in a surface biopanning process using a 12-mer phage display random peptide library. After five rounds of biopanning, forty colonies were picked and amplified followed by DNA isolation and amplification for sequencing. Sequencing suggested 12 different clones expressing different peptide sequence Phage-ELISA was performed using all 12 phage clones. Results indicated that four phage clones i.e., P4, P8, P9 and P12 had a specific binding activity to PPR virus. Linear peptides displayed by all 12 clones were synthesized using solid phase peptide synthesis and subjected to virus capture ELISA. No significant binding of the linear peptides with PPRV was evident which may be due to loss of conformation of linear peptide after coating. When the four selected phage clones displayed peptide sequences were synthesized in Multiple antigenic peptide (MAP) format and used in virus capture ELISA, the results indicated significant binding of PPRV to the MAPs. It may be due to increased avidity and/or better projection of binding residues in 4-armed MAPs as compared to linear peptides. MAP-peptides were also conjugated on gold nanoparticles (AuNPs). Visual colour change from wine red to purple was observed on addition of PPRV in MAP-conjugated AuNPs solution. This colour change may be attributable to the networking of PPRV with MAP -conjugated AuNPs resulting in aggregation of AuNPs. All these results supported the hypothesis that the phage display selected peptides were capable of binding to the PPRV. The potential of these peptides to develop novel diagnostic or therapeutic agents remains to be investigated.

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.

Development of real-time RT-PCR systems for detection and quantitation of bovine enteric viral pathogens.

The enteric viruses in animals are responsible for severe and devastating losses to the livestock owners with a profound negative impact on animal, health, welfare, and productivity. These viruses are usually transmitted via the feco-oral route and primarily infect the digestive tract of the humans, bovines and different mammals as well as birds. Some of the important enteric viruses in ruminants are: Rotavirus A (RVA), Peste des petits virus (PPRV), Norovirus (NV), Bovine corona virus (BoCV) and Bluetongue virus (BTV). In the present study, sensitive, specific and reliable TaqMan probe-based RT-qPCRs were developed and standardized for the rapid detection and quantification of enteric viruses from fecal samples. The assays result in efficient amplification of the RVA, BTV and BoCV RNA with a limit of detection (LoD) of 5, 5 and 4 copies, respectively, which is 1000 times more sensitive than the traditional gel-based RT-PCR. The reproducibility of each assay was satisfactory, thus allowing for a sensitive and accurate measurement of the viral RNA load in clinical samples. In conclusion, real time PCR developed for these viruses are highly specific and sensitive technique for the detection of diarrheic viral pathogens of cattle and buffalo.

Evaluation of diagnostic performance of H-based blocking ELISA for specific detection of peste des petits ruminants in domestic sheep, goats, cattle and camels.

INTRODUCTION: Peste des petits ruminants virus (PPRV) causes a highly devastating disease of sheep and goats, peste des petits ruminants (PPR), which is targeted for global control and eradication by 2030. The serological diagnostic tool kits for accurate diagnosis of PPR have inherent strengths and weaknesses that require parallel validation and optimization across animal species. Thus, the objective of this study was to evaluate diagnostic performance of haemagglutinin based PPR blocking ELISA (HPPR- b-ELISA), that was developed by Africa Union Pan African Veterinary Vaccine Center for specific detection of anti- PPRV antibodies. METHODS: In preliminarily investigation, diagnostic performance of the HPPR-b-ELISA®, commercial PPR competition ELISA (c-ELISA) and virus neutralization test (VNT) were compared for the detection of anti-PPRV antibodies in goats, sheep, cattle and camels. RESULTS: The sensitivity and specificity of HPPR- b-ELISA® were 79.55 and 99.74%, respectively, compared to c-ELISA. The HPPR- b-ELISA® was in perfect agreement (κ = 0.86) with the c-ELISA in all sera collected from goats, sheep and cattle. There was almost perfect agreement between the species of goats (κ = 0.82) and sheep (κ = 0.98), while the agreement was substantial in cattle (κ = 0.78) and no agreement was observed in camels (κ = 0.00). Similarly, the sensitivity and specificity of the HPPR b-ELISA were 80 and 96.36%, respectively compared to VNT with almost perfect agreement in goats (κ = 0.83) and sheep (κ = 0.89), moderate in cattle (κ = 0.50) and none in camels (κ = 0.00). CONCLUSION: Our study revealed that HPPR- b-ELISA is a suitable and valid method that can alternatively be used for screening and monitoring of PPR in sheep, goats and cattle except for camels.

Epidemiology, clinical features, and molecular detection of orf virus in Haryana (India) and its adjoining areas.

Orf is an acute, highly contagious, and economically important viral disease of small ruminants. In this study, six orf suspected outbreaks among goats and sheep were investigated from Haryana state and adjoining areas of Rajasthan state during the year 2021. The disease was diagnosed on the basis of clinical signs and molecular identification. The causative agent of the disease, orf virus (ORFV), was confirmed using polymerase chain reaction (PCR) targeting immunodominant envelope antigen (B2L) gene and confirmed by sequencing. The morbidity in goats ranged from 8.75 to 100%, whereas in sheep, it ranged from 0 to 8%. The higher mortality was observed among flocks with mixed infections of orf and peste des petits (PPR) or orf and haemonchosis as compared to other outbreaks. The phylogenetic analysis of sequenced PCR products clustered the current study strains in the same clad with Indian as well as strains from other countries with nucleotide identity more than 99%, signifying a close genetic relationship. The study highlighted the circulation of strains of a single cluster among sheep and goats in Haryana and adjoining areas. Prompt diagnosis of the disease is highly important for facilitating the implementation of control measures to minimize the losses suffered by small and marginal farmers in this region. Further detailed studies are required to delineate the molecular details of ORFV for better understanding the dynamics and molecular epidemiology of strains circulating in the country and for designing the effective vaccines against the disease which are currently lacking in the country.

Environmental sampling for the detection of foot-and-mouth disease virus and peste des petits ruminants virus in a live goat market, Nepal.

Livestock markets are considered vital parts of the agricultural economy, particularly in developing countries where livestock keeping contributes to both food security and economic stability. Animals from diverse sources are moved to markets, they mix while they are there and are subsequently redistributed over wide geographic areas. Consequently, markets provide an opportunity for targeted surveillance for circulating pathogens. This study investigated the use of environmental sampling at a live goat market in Nepal for the detection of foot-and-mouth disease virus (FMDV) and peste des petits ruminants virus (PPRV), both of which are endemic. Five visits to the market were carried out between November 2016 and April 2018, with FMDV RNA detected on four visits and PPRV RNA detected on all five visits. Overall, 4.1% of samples (nine out of 217) were positive for FMDV RNA and 60.8% (132 out of 217) were positive for PPRV RNA, though the proportion of positive samples varied amongst visits. These results demonstrate that non-invasive, environmental sampling methods have the potential to be used to detect circulation of high priority livestock diseases at a live animal market and, hence, to contribute to their surveillance and control.

Nucleotide amplification and sequencing of the GC-rich region between matrix and fusion protein genes of peste des petits ruminants virus.

Peste des petits ruminants virus (PPRV) causes a highly devastating disease of sheep and goats, that threatens the conservation of small wild ruminants. The development of PPRV vaccines, diagnostics and therapeutics, greatly depends on in-depth genomic data. Yet, high guanine-cytosine (GC) content between matrix (M) and fusion (F) genes of PPRV poses difficulty for both primer design and nucleotide amplification. In turn, this has led into absence or low nucleotide sequence coverage in this region. This poses a risk of missing important part of the genome that could help to infer viral evolution. Here, an overlapping long-read primer-based amplification strategy was developed to amplify the GC-rich fragments between M-F gene junction using nexus gradient polymerase chain reaction (PCR). The resulting amplicons were sequenced by dideoxynucleotide cycle sequencing and compared with other PPRV nucleotide sequences available at GenBank. Our findings indicate clear PCR amplification products with expected size of the GC-rich fragments on agarose gel electrophoresis. The sequencing results of these fragments indicate 99.5 % nucleotide identity with PPRV strain KY628761. An extremely difficult PCR target of 67.4 % GC contents was successfully amplified and sequenced using this long-read primer approach. The long-read primer set may be used in tiling multiplex PCR for complete genome sequencing of PPRV.

Molecular Detection of Peste des Petits Ruminants Virus (PPRV) in Goats and in Sheep in Ibadan, Oyo State, Nigeria.

Peste des petits ruminants (PPR) is a vaccine-preventable transboundary animal disease of goats and sheep majorly, and is regarded as a major constraint to small ruminant production especially in developing countries like Nigeria. Despite different strategies that have been employed to control PPR in Nigeria, cases of the disease are still reported in PPR-vaccinated and unvaccinated small ruminant farms. In this study, molecular detection of field PPR virus (PPRV) strains was carried out to determine the presence of PPRV. A total of 135 samples (45 oculo-nasal swabs and 90 tissue samples) were purposively collected between August and October 2020 from goats and sheep at the Akinyele live small ruminant market and at Akinyele and Amosun abattoirs in Ibadan, Oyo State, Nigeria. Using reverse transcriptasepolymerase chain reaction with primers targeting the partial N-gene of PPRV, 10 out of the 135 (7.4%) field samples yielded positive results. The results of this study reveal that PPRV currently circulates in Ibadan. These findings underscore the need for continuous PPR surveillance, more extensive characterization of circulating PPRV strains and the importance of consistent use of quality vaccines in the country to achieve more effective preventive and control strategies against the disease.

Sialic Acid Activated Gold Nanoparticles as Rapid Affordable Reagent for Peste Des Petits Ruminants (PPR) Virus Detection.

Animal health issues are important for farming community in agriculture. Small ruminant populations such as goats and sheep often get affected with contagious diseases. Peste des petits ruminants caused by a virus which need to be detected quickly to isolate affected animals and stop the spread of disease. The H protein of Peste des petits ruminants virus has sialic acid specific receptor, therefore sialic acid reduce and stabilized gold-nanoparticles were synthesize by a simple one pot method and without chemically modifying the sialic acid. The gold nanoparticles showed targetspecific aggregation with viral particles via hemagglutinin-sialic acid binding. The PPR virus was readily detected at the dilution of 10-6 by sialic acid-AuNPs. While comparing with the standard monoclonal antibody based test used for the detection of Peste des petits ruminants virus, sialic acid-AuNPs gave detection faster in less than 2 minute.

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.

In vitro and in vivo analyses of co-infections with peste des petits ruminants and capripox vaccine strains.

BACKGROUND: Peste des petits ruminants (PPR) and goat pox (GTP) are two devastating animal epidemic diseases that affect small ruminants. Vaccination is one of the most important measures to prevent and control these two severe infectious diseases. METHODS: In this study, we vaccinated sheep with PPR and POX vaccines to compare the changes in the antibody levels between animals vaccinated with PPRV and POX vaccines alone and those co-infected with both vaccines simultaneously. The cell infection model was used to explore the interference mechanism between the vaccines in vitro. The antibody levels were detected with the commercial ELISA kit. The Real-time Quantitative PCR fluorescent quantitative PCR method was employed to detect the viral load changes and cytokines expression after the infection. RESULTS: The concurrent immunization of GTP and PPR vaccine enhanced the PPR vaccine's immune effect but inhibited the immune effect of the GTP vaccine. After the infection, GTP and PPR vaccine strains caused cytopathic effect; co-infection with GTP and PPR vaccine strains inhibited the replication of PPR vaccine strains; co-infection with GTP and PPR vaccine strains enhanced the replication of GTP vaccine strains. Moreover, virus mixed infection enhanced the mRNA expressions of TNF-α, IL-1ß, IL-6, IL-10, IFN-α, and IFN-ß by 2-170 times. GTP vaccine strains infection alone can enhanced the mRNA expression of IL-1ß, TNF-α, IL-6, IL-10, while the expression of IFN-α mRNA is inhibited. PPR vaccine strains alone can enhanced the mRNA expression of IFN-α, IFN-ß, TNF-α, and has little effect the mRNA expression of IL-1ß, IL-6 and IL-10. The results showed that GTP and PPR vaccine used simultaneously in sheep enhanced the PPR vaccine's immune effect but inhibited the immune effect of the GTP vaccine in vivo. Furthermore, an infection of GTP and PPR vaccine strains caused significant cell lesions in vitro; co-infection with GTP + PPR vaccine strains inhibited the replication of PPR vaccine strains, while the co-infection of GTP followed by PPR infection enhanced the replication of GTP vaccine strains. Moreover, virus infection enhanced the expressions of TNF-α, IL-1ß, IL-6, IL-10, IFN-α, and IFN-ß. CONCLUSIONS: Peste des petits ruminants and capripox vaccine strains interfere with each other in vivo and vitro.

Avidin-Biotin recombinant antigen capture ELISA for the detection of peste des petits ruminants virus in the clinical specimens of sheep and goats.

This study describes the development of Avidin-Biotin recombinant Antigen Capture ELISA (ABrAC ELISA) for the detection of the peste des petits ruminants virus (PPRV) antigens in the clinical specimens of sheep and goats. The assay uses the truncated recombinant PPRV N-terminal immunogenic region of nucleoprotein (rPPRV-NPN) as a reference positive antigen and its polyclonal antibodies as capture/detective antibodies and the rabbit PPRV polyclonal antibodies as coating antibodies. The cut-off value was determined as double times the mean reactivity of blank control based on the reactivity of the PPR confirmed negative and positive control panel samples. On assessing the specificity with the related differential diagnosis of the disease-causing viruses and bacteria, the assay showed specific detective reactivity to PPRV. Further, on evaluation using clinical specimens (n-274) of sheep and goats, the assay showed that the relative diagnostic sensitivity of 86.49 % (95 % confidence interval (CI): 71.23-95.46 %) and diagnostic specificity of 96.20 % (95 % CI: 92.91-98.25 %) against PPRV nucleoprotein-specific monoclonal antibody-based sandwich-ELISA (PPR s-ELISA) kit, with an accuracy of 94.89 % (95 % CI: 91.58-97.18 %) and Cohen's Kappa value of 0.791 + 0.055 SE (95 % CI: 0.68-0.90) with substantial agreements. The ABrAC-ELISA is an alternative method of an immunoassay for the rapid, sensitive, and specific detection of the PPRV antigens m the clinical specimens of sheep and goats for surveillance or diagnosis of PPR. This study also shows that the rPPRV-NPN and its specific polyclonal antibodies could be the sustainable source of safe diagnostic reagents without the need to handle the infectious virus during the eradication and post-eradication phases in endemic countries like India or PPR non-endemic countries.

"FastCheckFLI PPR-like"-A Molecular Tool for the Fast Genome Detection of PPRV and Differential Diagnostic Pathogens.

To assist the global eradication of peste des petits ruminants virus (PPRV), a molecular test for the rapid and reliable detection of PPRV was developed which additionally enables the detection of pathogens relevant for differential diagnostics. For this purpose, the necessary time frame of a magnetic bead-based nucleic acid extraction protocol was markedly shortened to 7 min and 13 s. The optimized extraction was run on a BioSprint 15 platform. Furthermore, a high-speed multi-well RT-qPCR for the genome detection of PPRV and additional important pathogens such as Foot-and-mouth disease virus, Parapoxvirus ovis, Goatpox virus, and Mycoplasmacapricolumsubsp.capripneumoniae was established and combined with suitable internal control assays. The here-described qPCR is based on a lyophilized master mix and takes only around 30 to 40 min. Several qPCR cyclers were evaluated regarding their suitability for fast-cycling approaches and for their diagnostic performance in a high-speed RT-qPCR. The final evaluation was conducted on the BioRad CFX96 and also on a portable Liberty16 qPCR cycler. The new molecular test designated as "FastCheckFLI PPR-like", which is based on rapid nucleic acid extraction and high-speed RT-qPCR, delivered reliable results in less than one hour, allowing its use also in a pen-side scenario.

Comparative evaluation of different antigen detection methods for the detection of peste des petits ruminants virus.

Peste des petits ruminants (PPR) is a fatal disease of small ruminants which has spread rapidly to previously PPR-free countries in recent decades, causing enormous economic losses in the affected regions. Here, two newly emerged PPR virus (PPRV) isolates from India and from the Middle East were tested in an animal trial to analyse their pathogenesis, and to evaluate serological and molecular detection methods. Animals infected with the two different PPRV isolates showed marked differences in clinical manifestation and scoring. The PPRV isolate from India was less virulent than the virus from the Middle East. Commercially available rapid detection methods for PPRV antigen (two Lateral Flow Devices (LFDs) and one antigen ELISA) were evaluated in comparison with a nucleic acid detection method. For this purpose, ocular and nasal swabs were used. Due to the easy non-invasive sampling, faecal samples were also analysed. For all rapid antigen detection methods, a high specificity of 100% was observed independent of the sample matrix and dilution buffers used. Both antigen ELISA and LFD tests showed highest sensitivities for nasal swabs. Here, the detection rate of the antigen ELISA, the LFD-PESTE-TEST and the LFD-ID Rapid-Test was 78%, 75% and 78%, respectively. Ocular swabs were less suitable for antigen detection of PPRV. These results reflect the increased viral load in nasal swabs of PPRV infected goats compared to ocular swabs. The faecal samples were the least suitable for antigen detection. In conclusion, nasal swab samples are the first choice for the antigen and genome detection of PPRV. Nevertheless, based on the excellent diagnostic specificity of the rapid tests, positive results generated with other sample matrices are solid. In contrast, negative test results can be caused on the reduced analytical sensitivity of the rapid antigen tests and must be treated with caution.

Characterisation of Peste Des Petits Ruminants Disease in Pastoralist Flocks in Ngorongoro District of Northern Tanzania and Bluetongue Virus Co-Infection.

Peste des petits ruminants (PPR) disease was first confirmed in Tanzania in 2008 in sheep and goats in Ngorongoro District, northern Tanzania, and is now endemic in this area. This study aimed to characterise PPR disease in pastoralist small ruminant flocks in Ngorongoro District. During June 2015, 33 PPR-like disease reports were investigated in different parts of the district, using semi-structured interviews, clinical examinations, PPR virus rapid detection test (PPRV-RDT), and laboratory analysis. Ten flocks were confirmed as PPRV infected by PPRV-RDT and/or real-time reverse transcription-polymerase chain reaction (RT-qPCR), and two flocks were co-infected with bluetongue virus (BTV), confirmed by RT-qPCR. Phylogenetic analysis of six partial N gene sequences showed that the PPR viruses clustered with recent lineage III Tanzanian viruses, and grouped with Ugandan, Kenyan and Democratic Republic of Congo isolates. No PPR-like disease was reported in wildlife. There was considerable variation in clinical syndromes between flocks: some showed a full range of PPR signs, while others were predominantly respiratory, diarrhoea, or oro-nasal syndromes, which were associated with different local disease names (olodua-a term for rinderpest, olkipiei-lung disease, oloirobi-fever, enkorotik-diarrhoea). BTV co-infection was associated with severe oro-nasal lesions. This clinical variability makes the field diagnosis of PPR challenging, highlighting the importance of access to pen-side antigen tests and multiplex assays to support improved surveillance and targeting of control activities for PPR eradication.