World Organisation For Animal Health Research Articles

Epidemiological Analyses of the First Incursion of the Epizootic Hemorrhagic Disease Virus Serotype 8 in Tunisia, 2021-2022.

Epizootic hemorrhagic disease (EHD) is a non-contagious arthropod-transmitted viral disease and a World Organization for Animal Health (WOAH)-listed disease of domestic and wild ruminants since 2008. EHDV is transmitted among susceptible animals by a few species of midges of genus Culicoides. During the fall of 2021, a large outbreak caused by the epizootic hemorrhagic disease virus (EHDV), identified as serotype 8, was reported in Tunisian dairy and beef farms with Bluetongue virus (BTV)-like clinical signs. The disease was detected later in the south of Italy, in Spain, in Portugal and, more recently, in France, where it caused severe infections in cattle. This was the first evidence of EHDV-8 circulation outside Australia since 1982. In this study, we analyzed the epidemiological situation of the 2021-2022 EHDV outbreaks reported in Tunisia, providing a detailed description of the spatiotemporal evolution of the disease. We attempted to identify the eco-climatic factors associated with infected areas using generalized linear models (GLMs). Our results demonstrated that environmental factors mostly associated with the presence of C. imicola, such as digital elevation model (DEM), slope, normalized difference vegetation index (NDVI), and night-time land surface temperature (NLST)) were by far the most explanatory variables for EHD repartition cases in Tunisia that may have consequences in neighboring countries, both in Africa and Europe through the spread of infected vectors. The risk maps elaborated could be useful for disease control and prevention strategies.

Comparative analysis of tuberculin and defined antigen skin tests for detection of bovine tuberculosis in buffaloes (Bubalus bubalis) in Haryana state, India.

Bovine tuberculosis (bTB) is a chronic disease that results from infection with any member of the Mycobacterium tuberculosis complex. Infected animals are typically diagnosed with tuberculin-based intradermal skin tests according to World Organization of Animal Health which are presently in use. However, tuberculin is not suitable for use in BCG-vaccinated animals due to a high rate of false-positive reactions. Peptide-based defined skin test (DST) antigens have been identified using antigens (ESAT-6, CFP-10 and Rv3615c) which are absent from BCG, but their performance in buffaloes remains unknown. To assess the comparative performance of DST with the tuberculin-based single intradermal test (SIT) and the single intradermal comparative cervical test (SICCT), we screened 543 female buffaloes from 49 organized dairy farms in two districts of Haryana state in India. We found that 37 (7%), 4 (1%) and 18 (3%) buffaloes were reactors with the SIT, SICCT and DST tests, respectively. Of the 37 SIT reactors, four were positive with SICCT and 12 were positive with the DST. The results show that none of the animals tested positive with all three tests, and 6 DST positive animals were SIT negative. Together, a total of 43 animals were reactors with SIT, DST, or both, and the two assays showed moderate agreement (Cohen's Kappa 0.41; 95% Confidence Interval (CI): 0.23, 0.59). In contrast, only slight agreement (Cohen's Kappa 0.18; 95% CI: 0.02, 0.34) was observed between SIT and SICCT. Using a Bayesian latent class model, we estimated test specificities of 96.5% (95% CI, 92-99%), 99.7% (95% CI: 98-100%) and 99.0% (95% CI: 97-100%) for SIT, SICCT and DST, respectively, but considerably lower sensitivities of 58% (95% CI: 35-87%), 9% (95% CI: 3-21%), and 34% (95% CI: 18-55%) albeit with broad and overlapping credible intervals. Taken together, our investigation suggests that DST has a test specificity comparable with SICCT, and sensitivity intermediate between SIT and SICCT for the identification of buffaloes suspected of tuberculosis. Our study highlights an urgent need for future well-powered trials with detailed necropsy, with immunological and microbiological profiling of reactor and non-reactor animals to better define the underlying factors for the large observed discrepancies in assay performance, particularly between SIT and SICCT.

Post-Vaccination Sero-Monitoring of Peste des Petits Ruminants in Sheep and Goats in Karnataka: Progress towards PPR Eradication in India.

Peste des petits ruminants (PPR) presents economic challenges in enzootic countries impacting small ruminant productivity. The state of Karnataka, India, implemented a mass vaccination campaign in alignment with the PPR-Global Eradication Programme (GEP) and the National Strategic Plan for PPR eradication. This study was conducted from January to March 2023 to assess seroconversion in post-vaccinated goats and sheep at the epidemiological unit (epi-unit) level, aligning with the World Organisation for Animal Health (WOAH) and the Food and Agriculture Organization (FAO) guidelines in the PPR Global Control and Eradication Strategy (GCES). Before vaccination, 3466 random serum samples were collected from small ruminants of three age groups (6-12 months, 1-2 years, and >2 years) across 116 epi-units, spanning 82 taluks in 28 districts. Post-vaccination sero-monitoring included 1102 serum samples collected from small ruminants of the 6-12-month age group only, across 111 epi-units covering 64 taluks in 23 districts. The PPRV antibody status was determined using an indigenous hemagglutinin (H) protein monoclonal antibody-based competitive ELISA kit. Pre-vaccination, the PPR seropositivity rates were 55%, 62%, and 66% in the age groups of 6-12 months, 1-2 years, and >2 years, respectively, with a 61% PPRV antibody prevalence across all the age groups. Notably, 41% of the epi-units exhibited antibody prevalence rates of ≥70%, indicating a substantial population immunity, possibly attributed to the previous vaccination program in the state since 2011. In contrast, only 17% of the epi-units had below 30% seroprevalence rates, emphasizing the need for intensified vaccination. Statistical analysis of the data revealed significant correlations (p < 0.05) between the presence of PPRV antibodies and host factors such as species, breed, and sex. Post-vaccination seroprevalence in the 6-12 months age group was found to be 73.4%, indicating the use of an efficacious vaccine. On the evaluation of vaccination immunity in the 6-12 months age group, it was revealed that over 69% of the epi-units achieved a response surpassing ≥70%, indicating a significant improvement from 42% of the epi-units in pre-vaccination. For active PPR eradication, a mass vaccination campaign (>95% coverage) targeting small ruminant populations aged >4 months is advocated, aiming to achieve the desired herd immunity of >80%. This study offers crucial insights into PPR baseline seroprevalence/immunity status and vaccine efficacy, guiding national strategies towards a PPR-free India and further supporting the global eradication initiative.

Evaluation of the diagnostic assays detecting red sea bream iridovirus infection at different severity levels

Red sea bream iridovirus (RSIV) is a highly contagious viral infection that affects various fish species and poses a significant threat to the global aquaculture industry. Thus, accurate and timely diagnosis is paramount for sustainable management of fish health. This study rigorously evaluated the diagnostic efficacy of various polymerase chain reaction (PCR) assays, focusing on those recommended by the World Organization for Animal Health (WOAH) and the assays newly proposed by WOAH's Aquatic Animals Health Standards Commission. Specifically, this study assessed conventional PCR, nested PCR, modified 1-F/1-R, and real-time PCR assays using a 95% limit of detection (LoD95%), as well as diagnostic sensitivity (DSe) and specificity (DSp) tests across different RSIV severity grades (G0−G4). In previous studies, the LoD95% for the 1-F/1-R and 4-F/4-R conventional assays were 225.81 and 328.7 copies/reaction, respectively. The modified 1-F/1-R exhibited a lower LoD95% of 51.32 copies/reaction. Notably, the nested PCR had an LoD95% of 11.23 copies/reaction, and the real-time PCR assay had an LoD95% of 12.02 copies/reaction. The DSe varied across RSIV severity grades, especially in the lower G0−G2 grades. The nested PCR and modified 1-F/1-R assays displayed the highest DSe, making them particularly useful for early-stage screening and detection of asymptomatic carriers. In addition, the PCR assays did not cross-react with any other aquatic pathogens except RSIV. Our findings significantly advanced the diagnostic capabilities of RSIVD by suggesting that nested PCR and modified 1-F/1-R assays are particularly promising for early detection. We propose their inclusion in future WOAH guidelines for a more comprehensive diagnostic framework.

Comparative evaluation of assay performance for SARS-CoV-2 detection in animal oral samples, lung homogenates, and phosphate-buffered saline using the TaqPath COVID-19 Combo kit.

A One Health approach has been key to monitoring the COVID-19 pandemic, as human and veterinary medical professionals jointly met the demands for an extraordinary testing effort for SARS-CoV-2. Veterinary diagnostic laboratories continue to monitor SARS-CoV-2 infection in animals, furthering the understanding of zoonotic transmission dynamics between humans and animals. A RT-PCR assay is a primary animal screening tool established within validation and verification guidelines provided by the American Association of Veterinary Laboratory Diagnosticians (AAVLD), World Organisation for Animal Health (WOAH), and the U.S. Food and Drug Administration (FDA). However, differences in sample matrices, RNA extraction methods, instrument platforms, gene targets, and cutoff values may affect test outcomes. Therefore, targeted validation for a new sample matrix used in any PCR assay is critical. We evaluated a COVID-19 assay for the detection of SARS-CoV-2 in feline and canine lung homogenates and oral swab samples. We used the commercial Applied Biosystems MagMAX Viral/Pathogen II (MVP II) nucleic acid isolation kit and TaqPath COVID-19 Combo kit, which are validated for a variety of human samples, including nasopharyngeal and oropharyngeal swab samples. Our masked test showed a high detection rate and no false-positive or false-negative results, supporting sample extension to include feline oral swab samples. Our study is a prime example of One Health, illustrating how a COVID-19 assay designed for human testing can be adapted and used to detect SARS-CoV-2 in oral swab samples from cats and likely dogs, but not lung homogenates.

Determining patterns of tissue tropism for IHHNV, GAV and YHV-7 infection in giant black tiger shrimp (Penaeus monodon) using real-time RT-qPCR

Disease management in shrimp aquaculture is heavily dependent on accurate and rapid pathogen detection and diagnosis. Understanding the presence and distribution of pathogens within various tissues of the shrimp host is critical to ensure detection efforts are targeted for optimal sensitivity and reliability. Modern advancements in molecular technologies, including nucleic acid extraction and RT-qPCR, have yielded significant improvements in pathogen detection capabilities. Despite these advancements and their widespread adoption within both research and industry applications, evidence to inform their optimised use, such as revisions to tissue-specific viral detection to support selection of appropriate target tissues, has not been established. To address these gaps, this study aimed to establish contemporary evidence of viral tissue tropism for three major shrimp pathogens. TaqMan qPCR analysis was used to determine tissue-specific loading of viral pathogens in naturally infected Penaeus monodon pleopod, gill, hepatopancreas, lymphoid organ, abdominal muscle tissue, hindgut and ventral nerve cord. The viral targets analysed included Penaeus stylirostris penstyldensovirus 1 (PstDV1), formerly named infectious hypodermal haematopoietic necrosis virus (IHHNV) and, two genotypes of the yellow head virus complex, including genotype 2 known as gill associated virus (YHV-2/ GAV), and genotype 7 (YHV-7). QPCR analyses demonstrated the highest level of genetic IHHNV detection from gill tissue, followed sequentially by hindgut, pleopod, hepatopancreas, lymphoid organ, ventral nerve cord and abdominal muscle. For the yellow head viruses (GAV and YHV-7), no significant differences in genetic viral detection were demonstrated, although a non-significant advantage was observed for lymphoid organ and hindgut tissue. Beyond establishing contemporary evidence of viral distribution to improve understanding of host-viral interactions, these findings offer supporting evidence for revisions to appropriate target tissue recommendations, within the World Organisation for Animal Health (WOAH, founded as OIE) Manual of Diagnostic Tests for Aquatic Animals, for optimised modern shrimp pathogen detection.

The One Health Concept.

The concept of One Health has been developed as the appreciation that human health is intricately connected to those of other animals and the environment that they inhabit. In recent years, the COVID-19 pandemic and noticeable effects of climate change have encouraged national and international cooperation to apply One Health strategies to address key issues of health and welfare. The United Nations (UN) Sustainable Development Goals have established targets for health and wellbeing, clean water and sanitation, climate action, as well as sustainability in marine and terrestrial ecosystems. The One Health Quadripartite comprises the World Health Organization (WHO), the World Organization for Animal Health (WOAH-formerly OIE), the United Nations Food and Agriculture Organization (FAO) and the United Nations Environment Programme (UNEP). There are six areas of focus which are Laboratory services, Control of zoonotic diseases, Neglected tropical diseases, Antimicrobial resistance, Food safety and Environmental health. This article discusses the concept of One Health by considering examples of infectious diseases and environmental issues under each of those six headings. Biomedical Scientists, Clinical Scientists and their colleagues working in diagnostic and research laboratories have a key role to play in applying the One Health approach to key areas of healthcare in the 21st Century.

Assessing Reassortment between Bluetongue Virus Serotypes 10 and 17 at Different Coinfection Ratios in Culicoides sonorenesis.

Bluetongue virus (BTV) is a segmented, double-stranded RNA orbivirus listed by the World Organization for Animal Health and transmitted by Culicoides biting midges. Segmented viruses can reassort, which facilitates rapid and important genotypic changes. Our study evaluated reassortment in Culicoides sonorensis midges coinfected with different ratios of BTV-10 and BTV-17. Midges were fed blood containing BTV-10, BTV-17, or a combination of both serotypes at 90:10, 75:25, 50:50, 25:75, or 10:90 ratios. Midges were collected every other day and tested for infection using pan BTV and cox1 (housekeeping gene) qRT-PCR. A curve was fit to the ∆Ct values (pan BTV Ct-cox1 Ct) for each experimental group. On day 10, the midges were processed for BTV plaque isolation. Genotypes of the plaques were determined by next-generation sequencing. Pairwise comparison of ∆Ct curves demonstrated no differences in viral RNA levels between coinfected treatment groups. Plaque genotyping indicated that most plaques fully aligned with one of the parental strains; however, reassortants were detected, and in the 75:25 pool, most plaques were reassortant. Reassortant prevalence may be maximized upon the occurrence of reassortant genotypes that can outcompete the parental genotypes. BTV reassortment and resulting biological consequences are important elements to understanding orbivirus emergence and evolution.

Mycoplasma agalactiae Vaccines: Current Status, Hurdles, and Opportunities Due to Advances in Pathogenicity Studies.

Contagious agalactia (CA) is a serious multietiological disease whose classic etiological agent is Mycoplasma agalactiae and which causes high morbidity and mortality rates in infected herds. CA is classified as a notifiable disease by the World Organization for Animal Health due to its significant worldwide economic impact on livestock, primarily involving goat and sheep farms. The emergence of atypical symptoms and strains of M. agalactiae in wildlife ungulates reestablishes its highly plastic genome and is also of great epidemiological significance. Antimicrobial therapy is the main form of control, although several factors, such as intrinsic antibiotic resistance and the selection of resistant strains, must be considered. Available vaccines are few and mostly inefficient. The virulence and pathogenicity mechanisms of M. agalactiae mainly rely on surface molecules that have direct contact with the host. Because of this, they are essential for the development of vaccines. This review highlights the currently available vaccines and their limitations and the development of new vaccine possibilities, especially considering the challenge of antigenic variation and dynamic genome in this microorganism.

Animal welfare as the basis of One Health: A UN convention on animal welfare, health, and protection poses a realistic solution to improved animal welfare and human health

Abstract Animal welfare is closely linked to animal health and human welfare and health, as the World Organisation for Animal Health (WOAH) emphasizes in its Global Animal Welfare Strategy of 2017. As a result of recent pandemics, the Food and Agriculture Organization of the United Nations (FAO), the UN Environment Programme (UNEP), the World Health Organization (WHO), and the WOAH have created the One Health High Level Expert Panel (OHHLEP), committed to animal health and human health in the One Health Joint Plan of Action (2022–2026) and pledged to “[work] together for the Health of Humans, Animals, Plants and the Environment.” Despite these important acknowledgments and efforts from leading world organizations, including the One Health quadripartite, the latter has so far been reluctant to actively pursue the adoption of a legally binding commitment to improve the protection and welfare of animals globally. Animal welfare remains without relevant provisions. In the following, it is argued this must be integrated into the OHHLEP in addition to the creation of an “Animal Think and Do Tank” in the form of a technical working group, which would finalize a UN Convention on the issue, as proposed, e.g., in the drafted UN Convention on Animal Health and Protection (UNCAHP) 2018. The “Animal Think and Do Tank,” within its One Health context, must manifest a transdisciplinary approach, since – unlike globally binding legislation – policies, agreements, and declarations of various world organizations do not suffice to bring about a fundamental change in animal welfare. Acknowledging that animal protection is a complex and multifaceted issue involving ethical, cultural, social, religious, political, economic, and scientific dimensions (GAL Association, 2018 , Preamble), these aspects must be represented within the decision making process regarding the Convention’s content. Besides veterinarians, experts of further disciplines related to One Health must be consulted – such as lawyers and legal academics specializing in animal, international, and UN law, competent representatives of the animal welfare and animal rights movements, and ethicists and economists. Measures must be taken to improve transparency and overall confidence in the dialogue between the actors, including competent and not specifically scientific representatives of the animal protection and animal rights movement as elements of civil society to advocate for the fundamental interests of animals. One Health impact statement This article proposes that a UN Convention on Animal Health and Protection would further the enforceability of a One Health approach to human, environmental, and animal health. Science has proven that these elements are intertwined, but they remain disparate at a legislative level in many jurisdictions. It is suggested that placing animal welfare provisions on a UN-Convention footing by utilizing the positioning of the WOAH and UNEP is the most effective way of rendering One Health policy a global reality. Cooperation between professionals is imperative: scientists, policy makers, and lawyers must work together.

Anti-rabies humoral immune response in cats after concurrent vs separate vaccination against rabies and feline leukaemia virus using canarypox-vectored vaccines.

Some expert groups recommend that cats should be vaccinated with non-adjuvanted feline leukaemia virus (FeLV) and rabies vector vaccines, which, in the European Union, are currently not licensed for concurrent use and have to be administered at least 14 days apart (different from the USA) and thus at separate visits, which is associated with more stress for cats and owners. The aim of this study was to assess the anti-rabies antibody response in cats after vaccination against rabies and FeLV at concurrent vs separate (4 weeks apart) visits using two canarypox-vectored vaccines (Purevax Rabies and Purevax FeLV; Boehringer Ingelheim) and to evaluate the occurrence of vaccine-associated adverse events (VAAEs). Healthy FeLV antigen-negative client-owned kittens (n = 106) were prospectively included in this randomised study. All kittens received primary vaccinations against rabies (week 0) and FeLV (weeks 4 and 8). After 1 year, the study group (n = 52) received booster vaccinations against rabies and FeLV concurrently at the same visit (weeks 50-52). The control group (n = 54) received booster vaccinations against rabies (weeks 50-52) and FeLV (weeks 54-56) separately. Anti-rabies virus antibodies (anti-RAV Ab) were determined by fluorescent antibody virus neutralisation assay at weeks 4, 50-52 and 54-56, and compared between both groups using a Mann-Whitney U-test. Four weeks after the first rabies vaccination, 87/106 (82.1%) kittens had a titre ⩾0.5 IU/ml and 19/106 (17.9%) had a titre <0.5 IU/ml. Four weeks after the 1-year rabies booster, all cats had adequate anti-RAV Ab according to the World Organisation for Animal Health (⩾0.5 IU/ml), and the titres of the study group (median = 14.30 IU/ml) and the control group (median = 21.39 IU/ml) did not differ significantly (P = 0.141). VAAEs were observed in 7/106 (6.6%) cats. Concurrent administration of Purevax FeLV and Purevax Rabies vector vaccines at the 1-year booster does not interfere with the development of anti-RAV Ab or cause more adverse effects and thus represents a better option than separate vaccination visits for cats and owners.

Mycoplasma mycoides subspecies capri, an uncommon mastitis and respiratory pathogen isolated in a German flock of goats

Mycoplasma mycoides ssp. capri (Mmc) is one of the etiological microorganisms of contagious agalactia, which is among the diseases causing the highest economical losses in small ruminants. We report a disease outbreak in a German flock that led to significant suffering of goats characterized by mastitis, arthritis, pleuropneumonia and sudden deaths. Mmc was persistently isolated from many animals both from milk, and from a number of different swab and tissue samples. A number of closely related Mycoplasma spp. have to be taken into consideration to rule out important animal epizootics listed by European Animal Health Law and the World Organisation for Animal Health (WOAH). Some goats developed cross-reacting antibodies against Mycoplasma mycoides ssp. mycoides. Although Mmc is believed to be an uncommon microorganism in Germany, this study highlights that veterinarians should consider this pathogen in their work during herd health monitoring in Central Europe. Although eradication was not fully achieved, autogenous vaccination significantly seemed to improve animal health and welfare.

The Live Attenuated Vaccine Strain "ARRIAH" Completely Protects Goats from a Virulent Lineage IV Field Strain of Peste Des Petits Ruminants Virus.

Peste des petits ruminants (PPR) is a transboundary viral disease that affects small ruminants, such as goats and sheep, in Africa, the Middle East, and Asia, causing substantial damage to livelihoods and disrupting livestock trade. Although Russia is PPR virus (PPRV)-free, controlling PPRV in neighboring countries is the top national priority. Recent PPR outbreaks in Mongolia and other countries in the Middle East caused by a lineage IV virus represent a risk of transboundary emergence in neighboring countries, including China, Kazakhstan, and Russia. In the present study, we assessed the potency and safety of the ARRIAH live attenuated PPRV vaccine (lineage II) in Zaannen and Nubian goat breeds by challenging them with a virulent lineage IV Mongolia/2021 isolate. For comparison, two commercial vaccines of Nigeria75/1 strain were used. The ARRIAH-vaccinated animals showed an increase in body temperature of 1-1.5 °C above the physiological norm, similar to the animals vaccinated with Nigeria75/1 vaccines. In all vaccinated groups, the average rectal temperature never exceeded 39.4-39.7 °C throughout the infection period, and no clinical signs of the disease were observed, demonstrating vaccine efficacy and safety in the current experimental setting. However, the control group (mock vaccinated) challenged with Mongolia/2021 PPRV exhibited moderate-to-severe clinical signs. Overall, the findings of the present study demonstrate that the ARRIAH vaccine strain has a promising protective phenotype compared with Nigeria75/1 vaccines, suggesting its potential as an effective alternative for curbing and controlling PPR in affected countries. Although the ARRIAH vaccine against PPR is not currently endorsed by the World Organization for Animal Health due to its incomplete safety and potency profile, this study is the first step to provide experimentally validated data on the ARRIAH vaccine.

Development of a highly sensitive TaqMan method based on multi-probe strategy: its application in ASFV detection.

The establishment of high sensitive detection method for various pathogenic microorganisms remains constantly concerned. In the present study, multi-probe strategy was first systematically investigated followed by establishing a highly sensitive TaqMan real-time fluorescent quantitative PCR (qPCR) method for detecting African swine fever virus (ASFV). Briefly, four probes based on the B646L gene of ASFV were designed and the effects of different combinations of the probes in a single TaqMan qPCR assay on the detection sensitivity were investigated. As less as 0.5-5 copies/μl of the ASFV gene was detected by the established TaqMan qPCR assay. Furthermore, plasmid harboring the B646L in water samples could be concentrated 1000 times by ultrafiltration to enable a highly sensitive detection of trace viral nucleic acids. Moreover, no cross-reactivity was observed with other common clinical swine viruses such as PCV2, PCV3, PCV4, PEDV, PDCoV, CSFV, PRRSV, and PRV. When detecting 173 clinical porcine serum samples, the coincidence rate between the developed method and WOAH (World Organization of Animal Health) recommended method was 100%. This study might provide an integrated strategy to achieve higher detection sensitivity of trace pathogenic microorganisms and applicably sensitive TaqMan-based qPCR assays.

A toolbox for manipulating the genome of the major goat pathogen, Mycoplasma capricolum subsp. capripneumoniae.

Mycoplasma capricolum subspecies capripneumoniae (Mccp) is the causative agent of contagious caprine pleuropneumonia (CCPP), a devastating disease listed by the World Organisation for Animal Health (WOAH) as a notifiable disease and threatening goat production in Africa and Asia. Although a few commercial inactivated vaccines are available, they do not comply with WOAH standards and there are serious doubts regarding their efficacy. One of the limiting factors to comprehend the molecular pathogenesis of CCPP and develop improved vaccines has been the lack of tools for Mccp genome engineering. In this work, key synthetic biology techniques recently developed for closely related mycoplasmas were adapted to Mccp. CReasPy-Cloning was used to simultaneously clone and engineer the Mccp genome in yeast, prior to whole-genome transplantation into M. capricolum subsp. capricolum recipient cells. This approach was used to knock out an S41 serine protease gene recently identified as a potential virulence factor, leading to the generation of the first site-specific Mccp mutants. The Cre-lox recombination system was then applied to remove all DNA sequences added during genome engineering. Finally, the resulting unmarked S41 serine protease mutants were validated by whole-genome sequencing and their non-caseinolytic phenotype was confirmed by casein digestion assay on milk agar. The synthetic biology tools that have been successfully implemented in Mccp allow the addition and removal of genes and other genetic features for the construction of seamless targeted mutants at ease, which will pave the way for both the identification of key pathogenicity determinants of Mccp and the rational design of novel, improved vaccines for the control of CCPP.

TaqMan real-time RT‒PCR assay with an internal amplification control for rapid detection of Bluetongue virus

Bluetongue (BT) is an arbovirus disease caused by Bluetongue virus (BTV), which has spread all over the world. In this study, a qRT-PCR assay with an internal amplification control was established for preventing false-negative results in Bluetongue virus (BTV) detection. The primers and probes for BTV NS3 were based on the qRT-PCR method for the detection of BTV in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (2022) of the World Organization for Animal Health (WOAH). The GAPDH gene was selected as the internal amplification control, and the primers and probes for the GAPDH gene were designed based on the analysis of conserved sequences in the genome sequences of cattle, sheep and goats. Optimization of the reaction conditions, specificity, sensitivity and repeatability tests were conducted, and a qRT-PCR method was established for the simultaneous detection of BTV and the reference gene GAPDH. The results showed that the optimal primers and probe concentrations of BTV-NS3 were 0.30 μmol/L and 0.25 μmol/L, and were 0.15 μmol/L and 0.20 μmol/L for the reference gene GAPDH. The established method has no cross-reaction with other viruses, which showed good specificity. The minimum detection of BTV- and GAPDH-positive plasmid standard copies was 6.685 copies/μL and 209 copies/μL, respectively. The intra-assay and interassay CVs of the Ct values measured by BTV were less than 1.2%, and the intra-assay and interassay CVs of the Ct values measured by GAPDH were less than 0.7%, which suggested a high degree of repeatability and stability for this assay. The established BTV qRT-PCR method was used to detect BTV in 88 sheep serum samples stored in the laboratory, revealing 4 BTV-positive samples with a positive rate of 4.55%, which was consistent with the result obtained by nested PCR for detecting BTV recommended by WOAH. The qRT-PCR method established in this study for simultaneously detecting BTV and the internal reference gene GAPDH has high sensitivity, strong specificity and high repeatability. The addition of an internal reference gene can effectively control the detection accuracy and enable false-negative results to be avoided. It can provide a reference for the standardization of qRT-PCR methods for Bluetongue disease.

Progress in vaccines against peste des petits ruminants virus

Peste des petits ruminants (PPR) is a highly contagious and economically important viral disease of both domestic (goats and sheep) and wild small ruminants. Due to the devastating effect of this disease on livestock and livelihoods, the Food and Agriculture Organization of the United Nations (FAO) and the World Organization for Animal Health (WOAH) endorsed the Global Strategy for the Control and Eradication of PPR (PPR GCES) and launched the PPR Global Eradication Programme (PPR GEP) to eradicate PPRV by 2030. In order to achieve this goal, a potent, safe and efficacious live-attenuated PPR vaccine with long-lasting immunity is available for immunoprophylaxis. However, the live-attenuated PPR vaccines are thermolabile and require maintenance of an effective cold chain to deliver to the field. In addition, infected animals cannot be differentiated from vaccinated ones (DIVA). To overcome these limitations, some new generation PPR vaccines have been developed: poxvirus vaccine, positive and negative marker vaccine through reverse genetic approach, chimeric vaccine, anti-idiotypic vaccine, subunit vaccine, virus-like particles vaccine, edible vaccine and combined vaccines. Novel recombinant PPR DIVA vaccines were evaluated in goats for safety and efficacy, and all vaccinated animals were clinically protected against an intranasal PPRV challenge. Furthermore, newly developed ELISAs were capable of differentiating between infected and vaccinated animals. Therefore, these DIVA vaccines and the associated tests can facilitate the serological monitoring process and speed up global PPR eradication through vaccination.

Expression of recombinant Florida clade 2 hemagglutinin in baculovirus expression system: A step for subunit vaccine development against H3N8 equine influenza virus.

Equine influenza (EI) is a transmissible viral respiratory sickness of the Equidae family. Two viruses, H7N7 and H3N8 caused EI; however, H7N7 has not been detected for decades. H3N8 has circulated and bifurcated into Eurasian and American lineages. The latter subsequently diversified into Kentucky, South America, and Florida sub-lineages. Florida clade 1 (FC1) and Florida clade 2 (FC2) strains are the only circulating EI viruses (EIVs) in the meantime. Immunization is considered the major means for the prevention and control of EI infection. Using disparate technologies and platforms, several vaccines have been developed and commercialized. According to the recommendations of the World Organization for Animal Health (WOAH), all commercial vaccines shall comprise representatives of both FC1 and FC2 strains. Unfortunately, most of the commercially available vaccines were not updated to incorporate a representative of FC2 strains. The purpose of this research was to develop a new EI vaccine candidate that incorporates the hemagglutinin (HA) antigen from the currently circulating FC2. In this study, we report the expression of the full-length recombinant HA gene of FC2 in the baculovirus expression system. The HA recombinant protein has been proven to maintain its biological characteristics by hemadsorption (HAD) and hemagglutination tests. Moreover, using a reference-specific serum, the specificity of the HA has been confirmed through the implementation of immunoperoxidase and western immunoblotting assays. In conclusion, we report the expression of specific biologically active recombinant HA of FC2, which would act as a foundation for the generation of an updated EI subunit or virus vector vaccine candidates.

Prevalence and zoonotic risk factors of Mycobacterium bovis tuberculosis in cattle at the cattle-wildlife-human interface in South and East Cameroon.

Bovine tuberculosis (bTB) is a contagious and notifiable disease, which is prevalent in cattle populations of many countries and in several wildlife species worldwide. However, the role of wildlife in the transmission and/or maintenance of bTB at the human-wild animal-animal interface and the epidemiology of zoonotic disease are poorly understood in Cameroon, where many wildlife species exist. This study aimed to estimate the prevalence and zoonotic risk factors of bTB at the cattle-wildlife-human interface in the South and East regions of Cameroon. We conducted a descriptive cross-sectional study from May to October 2022 in the southern region (Vallée du Ntem and Dja et Lobo) and eastern region (Haut Nyong and Lom et Djérem) of Cameroon to determine risk factors for bTB in Zebu Bororo, Goudali, Ndama, and Simmental cattle breeds. A comparative intradermal tuberculin testing (CIDT) was performed on 160 cattle randomly selected from herds using the threshold recommended by the World Organization for Animal Health. An interviewee-administered questionnaire was used to gather epidemiological data on sociodemographics, interaction between cattle and wildlife, and awareness of zoonotic tuberculosis (TB) from 90 cattle professionals. The prevalence of bTB at the herd level and associated risk factors were estimated using multiple logistic regression models. Based on the comparative intradermal tuberculin test (CIDT), the estimated prevalence of bTB in 160 cattle (Zebu Bororo, Goudali, Ndama, and Simmental) in South and East Cameroon was 6.8% (4.35%-9.41%) and 1.8% (0%-3.6%) for threshold values 3 mm and 4 mm, respectively. The prevalence obtained by simple intradermal tuberculin test (IDT) was 0.6% (0%-1.2%) for a threshold value 4 mm. Univariate analysis revealed three risk factors associated with bTB with significant odds ratios (OR; p = 0.05): herd size (OR = 4.88; 95% confidence interval [CI]: 1.24-32.56); cattle aged>10 years (OR = 0.17; 95% CI: 0.05-0.53); and victims of bTB organ seizure (OR = 0.015; 95% CI: 0.002-0.067). Multivariate analysis showed that being a cattle herder and contact between wildlife and livestock due to forage was significantly associated with bTB exposure (adjusted OR = 0.02; p = 0.001). Bovine TB is prevalent in cattle of the South and East Cameroon. Comparative IDT of cattle reared in the epidemiological and environmental context of the study areas yielded better results at a threshold of 3 mm than at a threshold of 4 mm recommended by the World Health Organization. Factors associated with exposure to/appearance of bTB were high herd size, cattle aged >10 years old, seizures of tuberculous organs, shepherding as a profession, and contact between cattle and wildlife can be due to lack of forage.

Characterisation of common hypothetical surface peptides between protozoan parasites (Perkinsus olseni) originating from different geographical locations.

Perkinsus olseni and P. marinus are classified as notifiable pathogens by the World Organisation for Animal Health and are known to cause perkinsosis in a variety of molluscs globally. Mass mortalities due to these parasites in farms and in the wild have been a recurrent issue. Diagnosis for these protozoans is currently done using Ray's fluid thioglycollate medium method followed by optical microscopy or molecular assays. Both require a high level of skill and are time-consuming. An immunoassay method would make the diagnosis of perkinsosis quicker and cheaper. The present study used mass spectrometry-based proteomics to investigate common hypothetical surface peptides between different geographical isolates of P. olseni, which could be used to develop immunoassays in the future. Two peptides were identified: POLS_08089, which is a 42.7 kDa peptide corresponding to the 60S ribosomal subunit protein L4; and POLS_15916, which is a conserved hypothetical protein of 55.6 kDa. The identification of peptides may allow the development of immunoassays through a more targeted approach.