Development and validation of an epitope-blocking ELISA using an anti-haemagglutinin monoclonal antibody for specific detection of antibodies in sheep and goat sera directed against peste des petits ruminants virus.

Peste des petits ruminants (PPR) is a contagious and economically important disease affecting production of small ruminants (i.e., sheep and goats). Taking into consideration the lessons learnt from the Global Rinderpest Eradication Programme (GREP), PPR is now targeted by the international veterinary community as the next animal disease to be eradicated. To support the African continental programme for the control of PPR, the Pan African Veterinary Vaccine Centre of the African Union (AU-PANVAC) is developing diagnostics tools. Here, we describe the development of a blocking enzyme-linked immunosorbent assay (bELISA) that allows testing of a large number of samples for specific detection of antibodies directed against PPR virus in sheep and goat sera. The PPR bELISA uses an anti-haemagglutinin (H) monoclonal antibody (MAb) as a competitor antibody, and tests results are interpreted using the percentage of inhibition (PI) of MAb binding generated by the serum sample. PI values below or equal to 18% (PI ≤ 18%) are negative, PI values greater than or equal to 25% (PI ≥ 25%) are positive, and PI values greater than 18% and below 25% are doubtful. The diagnostic specificity (DSp) and diagnostic sensitivity (DSe) were found to be 100% and 93.74%, respectively. The H-based PPR-bELISA showed good correlation with the virus neutralization test (VNT), the gold standard test, with a kappa value of 0.947. The H-based PPR-bELISA is more specific than the commercial kit ID Screen® PPR Competition (N-based PPR-cELISA) from IDvet (France), but the commercial kit is slightly more sensitive than the H-based PPR-bELISA. The validation process also indicated good repeatability and reproducibility of the H-based PPR-bELISA, making this new test a suitable tool for the surveillance and sero-monitoring of the vaccination campaign.

Isolation, molecular characterization and sero-prevalence study of foot-and-mouth disease virus circulating in central Ethiopia.

BACKGROUND: Ethiopian livestock production and productivity is still very low due to widespread of diseases. Among the diseases, foot-and-mouth disease (FMD) is an extremely contagious and acute viral disease that causes significant economic problems in the country. A cross sectional study design was conducted from September 2015 to May 2016 to isolate and characterize FMD virus from outbreak cases; determine the sero-prevalence of antibodies against FMD virus (FMDV), and assess potential risk factors associated with sero-prevalence of the disease in selected areas of central Ethiopia. A multistage sampling technique was employed to select the study animals. Isolated viruses were characterized by antigen ELISA (IZLER, Brescia, Italy) and by genetic analysis of the sequence of the viral protein 1 (VP1). Sero-prevalence was determined using an ELISA for antibodies against non-structural proteins of FMDV based on the 3ABC proteins (ID Screen® FMD NSP Competition, ID-VET, Grabels, France). Risk factors for sero-prevalence of antibodies against FMD virus was investigated using logistic regression analysis. RESULT: From outbreak investigation, 28.8% (n = 378) cattle showed signs and lesions suggestive of FMD and 34 samples were subjected to virus isolation. Twenty eight of these cultures exhibited cytopathic effect (CPE) and were serotyped as O, A and SAT 2 FMD viruses. One A and two SAT 2 isolates named A-ETH-19-2015, SAT 2-ETH-18-2015 and SAT 2-ETH-20-2015 were further characterized by phylogenetic analysis. The overall sero-prevalence of antibodies against non-structural proteins of FMDV was 24.2% (n = 574). Cattle herds with crossbreed cattle, with older cattle (> 2 years), and kept together with small ruminants had higher sero-prevalences of antibodies against FMDV (p < 0.05). CONCLUSION: This study showed that FMD was present in the study areas. Among the associated risk factors, breed, age and herd composition were significantly associated with presence of antibodies against FMD virus. Three different serotypes (A, O and SAT 2) were responsible for the outbreaks of the disease. Genetic analysis indicated that the isolated viruses clustered differently from previous outbreaks. Thus, further molecular analyses coupled with protection potential of the existing vaccines against the isolates should be performed.

Seroprevalence and associated risk factors of equine herpesvirus type-1/-4 in selected districts of Northwest Amhara, Ethiopia.

This study aimed to estimate the prevalence, determine the distribution, and identify the epidemiological risk factors of EHV-1/-4 infections in selected districts of Northwest Amhara Region. 460 serum samples were collected from equines using multistage cluster sampling technique, and a competitive Enzyme-linked immunosorbent assay (cELISA) was performed. Various risk factors for the occurrence of EHV-1/-4 were considered. Statistical analysis was performed using R version 4.3.1. 65.9% (303) equids were tested positive for antibodies against EHV-1/-4. Based on district, the highest prevalence was recorded in Wogera (86.1%), while the lowest was in Debark (47.4%). There was a significant difference (p <0.05; 95% CI: 1.1067993-3.682843) in the prevalence of EHV-1/-4 among species and donkeys are 2.019 times more likely to get an EHV infection than horses. The prevalence of EHV-1/-4 was highest in equids with the age of 3-8 years and lowest in < 3 years, and the difference was statistically significant (p <0.05; 95% CI: 1.9812042-6.771820). Statistically significant variation (p <0.05; 95% CI: 1.1173822-2.684013) was also observed between sex of equids in which females had 1.73 times higher chance to get EHV infection than males. Higher prevalence was found in lactating equids (81.6%), followed by pregnant equids (74.6%), and dry equids (66.4%). Generally, this study indicated a high and wide distribution of EHV-1/-4 infection in the study area, which needs due attention. Devising strategies to prevent and minimize the spread and occurrence of the infection is crucial.

Sequencing and Analysis of Lumpy Skin Disease Virus Whole Genomes Reveals a New Viral Subgroup in West and Central Africa.

Lumpy skin disease virus (LSDV) is a member of the capripoxvirus (CPPV) genus of the Poxviridae family. LSDV is a rapidly emerging, high-consequence pathogen of cattle, recently spreading from Africa and the Middle East into Europe and Asia. We have sequenced the whole genome of historical LSDV isolates from the Pirbright Institute virus archive, and field isolates from recent disease outbreaks in Sri Lanka, Mongolia, Nigeria and Ethiopia. These genome sequences were compared to published genomes and classified into different subgroups. Two subgroups contained vaccine or vaccine-like samples ("Neethling-like" clade 1.1 and "Kenya-like" subgroup, clade 1.2.2). One subgroup was associated with outbreaks of LSD in the Middle East/Europe (clade 1.2.1) and a previously unreported subgroup originated from cases of LSD in west and central Africa (clade 1.2.3). Isolates were also identified that contained a mix of genes from both wildtype and vaccine samples (vaccine-like recombinants, grouped in clade 2). Whole genome sequencing and analysis of LSDV strains isolated from different regions of Africa, Europe and Asia have provided new knowledge of the drivers of LSDV emergence, and will inform future disease control strategies.

Application of the Nagoya Protocol to veterinary pathogens: concerns for the control of foot-and-mouth disease.

The Nagoya Protocol is an international agreement adopted in 2010 (and entered into force in 2014) which governs access to genetic resources and the fair and equitable sharing of benefits from their utilisation. The agreement aims to prevent misappropriation of genetic resources and, through benefit sharing, create incentives for the conservation and sustainable use of biological diversity. While the equitable sharing of the benefits arising from the utilisation of genetic resources is a widely accepted concept, the way in which the provisions of the Nagoya Protocol are currently being implemented through national access and benefit-sharing legislation places significant logistical challenges on the control of transboundary livestock diseases such as foot-and-mouth disease (FMD). Delays to access FMD virus isolates from the field disrupt the production of new FMD vaccines and other tailored tools for research, surveillance and outbreak control. These concerns were raised within the FMD Reference Laboratory Network and were explored at a recent multistakeholder meeting hosted by the European Commission for the Control of FMD. The aim of this paper is to promote wider awareness of the Nagoya Protocol, and to highlight its impacts on the regular exchange and utilisation of biological materials collected from clinical cases which underpin FMD research activities, and work to develop new epidemiologically relevant vaccines and other diagnostic tools to control the disease.

Porcine circovirus-2 in Africa: Identification of continent-specific clusters and evidence of independent viral introductions from Europe, North America and Asia.

Porcine circovirus-2 (PCV-2) is associated with several disease syndromes in domestic pigs that have a significant impact on global pig production and health. Currently, little is known about the status of PCV-2 in Africa. In this study, a total of 408 archived DNA samples collected from pigs in Burkina Faso, Cameroon, Cape Verde, Ethiopia, the Democratic Republic of the Congo, Mozambique, Nigeria, Senegal, Tanzania and Zambia between 2000 and 2018 were screened by PCR for the presence of PCV-2. Positive amplicons of the gene encoding the viral capsid protein (ORF2) were sequenced to determine the genotypes circulating in each country. Four of the nine currently known genotypes of PCV-2 were identified (i.e. PCV-2a, PCV-2b, PCV-2d and PCV-2 g) with more than one genotype being identified in Burkina Faso, Ethiopia, Nigeria, Mozambique, Senegal and Zambia. Additionally, a phylogeographic analysis which included 38 additional ORF2 gene sequences of PCV-2s previously identified in Mozambique, Namibia and South Africa from 2014 to 2016 and 2019 to 2020 and available in public databases, demonstrated the existence of several African-specific clusters and estimated the approximate time of introduction of PCV-2s into Africa from other continents. This is the first in-depth study of PCV-2 in Africa and it has important implications for pig production at both the small-holder and commercial farm level on the continent.

Seroepidemiology of Peste des Petits ruminants in sheep and goats in the selected district of Horu Guduru Zone, Western Ethiopia.

Peste des Petits Ruminants (PPR) is an acute, highly contagious, economically important transboundary viral disease of small ruminants. The objectives of this cross-sectional study were to estimate the seroprevalence and associated risk factors of PPR in small ruminants in selected districts of Horo Guduru Zone, Western Ethiopia. A total of 806 blood samples, corresponding to 124 flocks comprised of 387 sheep and 419 goats, were collected using multi-stage sampling. The sera were tested using competitive Enzyme-Linked Immunosorbent Assay (c-ELISA). A pre-tested questionnaire was used to collect data on potential risk factors for PPRV infection. Pearson's Chi-Square and logistic regression analyses were used to see the association of the seroprevalence with potential risk factors. The flock-level overall seroprevalence of PPR was 27.42% (95% confidence interval [CI]: 19.79-36.15%). An overall animal level seroprevalence of 5.71% (95% CI: 4.21-7.54%) was recorded with 6.98% (95% CI: 4.65-10.00%) seroprevalence in sheep and 4.53% (95% CI: 2.75-6.99%) in goats. There was a significant association between the seroprevalence and risk factors that were sharing common grazing land (P = 0.032), the introduction of newly purchased animals into the flock (P = 0.011), and uncontrolled animal movements in goats (P = 0.018). Sharing common grazing land with other flocks in goats (P = 0.026) and midland altitude in sheep (P = 0.029) were the other predictors of PPR. The seropositivity in unvaccinated small ruminants suggests the circulation of the virus. Therefore, awareness creation, vaccination, strengthening the disease surveillance system and large-scale epidemiological and molecular studies are suggested.

Cryptosporidium and Giardia infections in dairy calves in southern Ethiopia.

Giardia and Cryptosporidium are the most common enteric protozoan parasites causing diarrhea in humans and animals worldwide. This study was conducted with the objectives of estimating prevalence and identifying risk factors for Cryptosporidium and Giardia infections in dairy calves in selected districts of southern Ethiopia. Fecal samples (n = 330) were collected from calves in 92 farms. The monoclonal antibody-based commercial direct immunofluorescent kit was used to test the samples for Cryptosporidium oocysts and Giardia cysts. A questionnaire survey was also administered to collect data on potential risk factors of infections. The results showed a farm-level prevalence of 69.6% (95% confidence interval [CI]: 59.1-78.7%) for Cryptosporidium and 38.04% (95% CI: 28.1-48.8%) for Giardia. Likewise, an overall animal level prevalence of 13.0% (95% CI: 9.6-17.2%) for Cryptosporidium and 9.7% (95% CI: 6.7-13.4%) for Giardia was found. At the farm level, multivariate logistic regression model showed that calves in smallholder farms were 5.3 times more likely to shed Cryptosporidium oocysts than calves in commercial farms (p=0.019). However, in case of Giardia, calves in commercial farms were 5.5 times more likely to shed cysts than calves in smallholder farms (p=0.037). Calves with diarrhea were nearly three times more likely to be positive for Cryptosporidium oocysts than those with normal feces (p=0.027). At the animal level, larger farms and younger calves were associated with Giardia cysts shedding, while larger herd size and lose fecal consistency were associated with Cryptosporidium oocysts shedding. Giardia and Cryptosporidium infection are endemic in the studied dairy farms. Therefore, detailed molecular epidemiological studies are essential to identify the role of domestic animals in the transmission of infections to humans and vice versa, and to determine the best options for prevention and control of cryptosporidiosis and giardiasis.

Molecular characterization of foot-and-mouth disease viruses circulating in Ethiopia between 2008 and 2019.

One of the constraints to controlling foot-and-mouth disease (FMD) in East Africa is the incomplete knowledge of the specific FMD virus (FMDV) strains circulating and the way in which these viruses move across countries in the region. This retrospective study focuses on Ethiopia, which has one of the largest FMD-susceptible livestock populations in Africa. Analyses of FMDV positive samples collected between 2008 and 2019 demonstrate that serotypes O (n = 175), A (n = 51) and SAT 2 (n = 33) were present in the country. Phylogenetic analysis of the VP1 sequences for these viruses showed that there were at least seven different FMD viral clades circulating during this period: O/EA-3, O/EA-4, A/AFRICA/G-I, A/AFRICA/G-IV, A/AFRICA/G-VII, SAT2/VII and SAT2/XIII. Although these results only represent a snapshot and might not reflect all FMDV lineages that were present, they highlight the importance of serotype O, as well as the complexity and co-existence of FMDV serotypes in Ethiopia and surrounding countries. These sequence data also support the idea that there are two FMDV ecosystems existing in East Africa. Data from retrospective studies, such as these presented here, will be beneficial for vaccine selection and vaccination campaigns to control FMDV within Ethiopia.

Efficacy of albendazole against nematode parasites isolated from a goat farm in Ethiopia: relationship between dose and efficacy in goats.

A suspected case of albendazole resistance in a goat farm of Hawassa University was examined using faecal egg count reduction test (FECRT), controlled anthelmintic efficacy test and egg hatch assay (EHA) to verify the development of resistance and/or the need for higher doses of the drug in goats than in sheep. The experiment was conducted in 12 sheep (2 groups: treatment versus control) and 24 goats (4 groups: 3 treatments versus control, n = 6; per group) following artificial infection with infective larvae of Haemonchus contortus and Oesophagostomum columbianum. The first group of sheep and goats were treated orally with albendazole at the dose rate of 3.8 mg/kg body weight (i.e. manufacturer's recommended dose for sheep) while the second group of sheep and the fourth group of goats were left untreated. The second and the third group of goats were treated with albendazole at 5.7 and 7.6 mg/kg respectively. The FECRT showed an efficacy of albendazole in goats to be 65.5, 81.4 and 84.1% at the dose rate of 3.8, 5.7 and 7.6 mg/kg body weight respectively while in sheep it was 62% at the dose rate of 3.8 mg/kg. Increasing the dose to 1.5 the sheep recommended dose induced minor improvement of efficacy in goats; however the efficacy was almost the same at 1.5 and twice the dose recommended for sheep. Worm counts at day 15 post-treatment revealed that H. contortus has developed resistance to albendazole. EHA results also supported these findings. On the other hand, O. columbianum was 100% susceptible at all dose levels tested.

Evaluation of the efficacy of anthelmintics sold on Ethiopian markets against Haemonchus contortus in experimentally infected Sheep.

A total of 54 lambs, aged between 6-8 months were experimentally infected with Haemonchus contortus to evaluate the efficacy of different anthelmintic brands sold on Ethiopian markets using the faecal egg count reduction test (FECRT) and controlled anthelmintic efficacy trial. Accordingly four different albendazole (Alzole, Analgon-300, Albenjung_s and Ahshialben-300), two tetramisole (Tetsole and Ashitetra 600) and two tetramisole-oxyclozanide (Tetraclozan sheep and Tetraclozash 900) brands were evaluated at the dosage rates recommended by the manufacturers. Animals were allocated into nine groups of six animals each, and balanced for faecal egg counts (FEC), based on their pre-treatment FEC and treatments were randomized among the groups. One group was kept untreated as a control. Faecal egg count was conducted on day 30 post-infection (day of treatment) and on the 10th day post-treatment. Evaluation of anthelmintics based on FECRT revealed high efficacy (99.55-100% reduction in FEC) for all anthelmintic brands tested against H. contortus. The worm count reduction test using controlled anthelmintic efficacy trial also supported the above finding with 99-100% efficacy of the tested anthelmintics. Therefore, the suspicion on the anthelmintic products as being substandard in quality is not credible, at least, for the brands investigated in this study and it might rather be attributed to under dosing. The need for a good extension system for livestock producers with regard to good anthelmintic usage practices, in light of the inevitable development of anthelminitic resistance, is emphasized. Regular surveillance and laboratory quality evaluation of the anthelmintic products in Ethiopia is indicated.

Molecular epidemiological update of Peste des Petits Ruminants virus (PPRV) in Ethiopia.

Peste des Petits ruminants (PPR) is a devastating disease of small ruminants with high morbidity and mortality rates among susceptible animals. The disease is endemic in much of Africa, the Middle East and Asia and constitutes one of the major hurdles to the improvement of small-ruminant production in these countries. The causal agent of PPR, the Small Ruminant Morbillivirus (SRMV), previously known as PPR virus (PPRV) belongs to the genus Morbillivirus within the family Paramyxoviridae. SRMV can be categorized into four genetically distinct lineages (I to IV). Suspicion of PPR was first reported in Ethiopia in 1977 and since then genetic characterization of circulating viruses has identified lineages III and IV in the country. This study was undertaken to provide an update on the molecular epidemiology of PPR in Ethiopia by analysing animal tissue samples collected between 2011 and 2017. PPR positive samples were identified in four regions of the country. Sequence and phylogenetic analysis of fourteen RT-PCR positive amplicons revealed that all of the SRMV in the samples from 2010 to 2017 belong to sub-clade II of clade I of lineage IV. No lineage III viruses were identified.

Evaluation of the safety, immunogenicity and efficacy of three capripoxvirus vaccine strains against lumpy skin disease virus.

The safety, immunogenicity and efficacy of three commercially available vaccines against lumpy skin disease (LSD) in cattle have been evaluated using a combination of vaccine challenge experiments and the monitoring of immune responses in vaccinated animals in the field. The three vaccines evaluated in the study included two locally produced (Ethiopian) vaccines (lumpy skin disease virus (LSDV) Neethling and Kenyan sheep and goat pox (KSGP) O-180 strain vaccines) and a Gorgan goat pox (GTP) vaccine manufactured by Jordan Bio-Industries Centre (JOVAC). The latter vaccine was evaluated for the first time in cattle against LSDV. The Ethiopian Neethling and KSGPO-180 vaccines failed to provide protection in cattle against LSDV, whereas the Gorgan GTP vaccine protected all the vaccinated calves from clinical signs of LSD. There was no significant difference in protective efficacy detected between two dosage levels (P=0.2, P=0.25, and P=0.1 for KSGP, Neethling and Gorgan vaccines, respectively). Additionally, the Gorgan GTP vaccinated cattle showed stronger levels of cellular immune responses measured using Delayed-Type Hypersensitivity (DTH) reactions at the vaccination site indicating higher levels of immunogenicity produced by the GTPV vaccine in cattle, as opposed to the other two vaccines. This study indicated, for the first time, that the Gorgan GTP vaccine can effectively protect cattle against LSDV and that the Neethling and KSGP O-180 vaccine were not protective. The results emphasise the need for molecular characterization of the Neethling and KSGP O-180 vaccine seed viruses used for vaccine production in Ethiopia. In addition, the potency and efficacy testing process of the Ethiopian LSD Neethling and KSGP O-180 vaccines should be re-evaluated.