Abstracts Page 1 - LSDV, ASF, CSF

Molecular and Serodetection of Lumpy Skin Disease Virus Circulating in Cattle and Arthropod Vectors in West and Central Africa

Anise N. Happi1*, John Fadele1,2, Oluwatobi Adedokun1, Ayotunde E. Sijuwola1, Olusola A. Ogunsanya1, Femi M. Saibu1, Akeemat O. Ayinla1, Faburay Bonto3,4, Corrie Brown5, Christian T. Happi1,2

1 African Centre of Excellence for Genomics of Infectious Diseases, Redeemer’s University, Ede, Osun State, Nigeria

2 Department of Biological Sciences, Redeemer’s University, Ede, Osun State, Nigeria

3U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Foreign Animal Disease Diagnostic Laboratory, National Bio and Agro-Defense Facility USDA, Manhattan, KS 66502, USA

4U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Foreign Animal Disease Diagnostic Laboratory, Plum Island Animal Disease Center, Greenport, NY 11957, USA

5LifeStock International, Athens, GA, USA / *Corresponding author – happia@run.edu.ng 

Background. Lumpy Skin Disease virus (LSDV), is the causative agent of Lumpy Skin Disease, a transboundary disease of large ruminants known to cause economic losses and food insecurity in developing countries. Preventive efforts face challenges due to the emergence of recombinant vaccine strains, and resultant vaccine failures. However, being a transboundary disease, the Capri pox virus strain circulating is poorly characterized. 

Method. This study used a cross-sectional design to investigate the prevalence of the LSD virus and characterised the strains circulating in cattle populations in three neighbouring countries in West and Central Africa. A total of 159 oral/nasal swabs, 129 skin scabs, and 10 nodule aspirates were collected from cattle with clinical signs suggestive of LSD across farms, cattle markets, and abattoirs in Cameroon, Benin Republic, and Nigeria. Additionally, fly and tick samples were collected and pooled from the sampling locations and sampled animals, respectively. Serological tests using ELISA were performed on 172 cattle plasma samples, while swabs, skin scabs, and nodule aspirates were analysed using real-time PCR. All LSD PCR-positive samples were sequenced using the shotgun NGS on the Illumina platform. 

Results. Overall PCR positivity (6.8%) for LSD was recorded from swab samples (3.8%), skin scabs (9.3%), and nodule aspirates (10%) of the 298 samples tested, while 1.2% of plasma samples were LSDV seropositive. Five animals were PCR-positive for both skin scab and swab samples. In Cameroon, Benin Republic and Nigeria, 4.45%, 1.67% and 2.44% of cattle tested positive for LSDV, respectively. Additionally, a pool of flesh flies and ticks tested positive for LSDV, all from Cameroon. Furthermore, two full and four partial LSDV genomes were assembled.

Conclusion. Our study confirms the presence of LSDV in sampled cattle across Cameroon, Benin Republic, and Nigeria. It also suggests flesh flies (Sarcophaga spp.) as potential mechanical vectors of LSDV. The ongoing genomic characterization of LSDV will reveal the LSDV strains circulating in West and Central Africa, which will inform the development of countermeasures. 

Risk Factor Evaluation, Molecular and Genomic Surveillance of African Swine Fever in Southern Nigeria

Anise N. Happi1*, Alhaji S. Olono1,2, John Fadele1,2, Olusola A. Ogunsanya1, Ayotunde E. Sijuwola1, Femi M. Saibu1, Oluwatobi Adedokun1, Akeemat O. Ayinla1, Faburay Bonto3,4, Corrie Brown5, Christian T. Happi1,2

1African Centre of Excellence for Genomics of Infectious Diseases, Redeemer’s University, Ede, Osun State, Nigeria

2Department of Biological Sciences, Redeemer’s University, Ede, Osun State, Nigeria

3U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Foreign Animal Disease Diagnostic Laboratory, National Bio and Agro-Defense Facility USDA, Manhattan, KS 66502, USA

4U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Foreign Animal Disease Diagnostic Laboratory, Plum Island Animal Disease Center, Greenport, NY 11957, USA

5LifeStock International, Athens, GA, USA / *Corresponding author – happia@run.edu.ng

Background. African Swine Fever (ASF) is a highly contagious viral disease that continues to threaten pig farming in Nigeria, causing severe economic losses. Despite its enzootic status, annual outbreaks persist, raising concerns about viral evolution, reservoir hosts, and biosecurity practices. This study investigates the epidemiology and genomic characteristics of ASF in Nigeria to understand the factors contributing to its recurrence. 

Method. A cross-sectional study was conducted in 40 pig farms across Nigeria, including both outbreak and non-outbreak periods. Whole blood samples from pigs and tick samples from pig environments were collected and analyzed using polymerase chain reaction (PCR) and whole-genome sequencing. Farm assessments and structured questionnaires were used to evaluate risk factors. Bioinformatic and Statistical analyses were performed to determine viral genotype and evaluate epidemiological drivers.

Results. All ASF-positive samples belonged to genotype II, confirming its dominance in Nigeria. No seasonal variation in ASF positivity was observed, suggesting that human-mediated factors, rather than climatic conditions, drive transmission. Ticks were not identified as reservoirs, reinforcing the absence of a sylvatic cycle in West Africa. Poor biosecurity measures, including inadequate quarantine, frequent pig-wildlife interactions, and low outbreak reporting rates due to economic concerns, were major risk factors.

Conclusion. ASF persistence in Nigeria is mainly driven by human practices and biosecurity deficiencies. Strengthening surveillance, incentivizing outbreak reporting, and implementing stricter biosecurity protocols are crucial for controlling ASF in smallholder pig farms.

Development of long-read targeted whole genome sequencing for African and classical swine fever viruses.

Chester D. McDowell1, Taeyong Kwon1, Patricia Assato1, Emily Mantlo1, Jessie D. Trujillo1, Natasha Gaudreault1, Jayme A. Souza-Neto1, Leonardo C. Caserta2, Roman M. Pogranichniy1, Diego G. Diel2, Juergen A. Richt1*

1 Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA

2 Department of Population Medicine and Diagnostic Sciences, Cornell University College of Veterinary Medicine, Ithaca, NY, USA

*Corresponding author – jricht@vet.k-state.edu

Background: African swine fever virus (ASFV) and classical swine fever virus (CSFV) are high consequence swine pathogens. ASFV is a large DNA virus with genome sizes of 170-190 kilobases (kB). CSFV is an RNA virus with a genome size of ~12 kB. Due to the reduced cost and availability of sequencing, it is vital to have whole genome sequencing (WGS) protocols in place for the rapid complete genetic characterization of ASFV and CSFV for outbreak and surveillance situations.

Methods: Primer panels spanning the genomes of ASFV or CSFV were developed to generate ~10kB (ASFV) or ~6kB (CSFV) amplicons, enabling whole genome amplification in 5 PCR reactions (ASFV) or 2 RT-PCR reactions (CSFV). The protocols were tested on viral DNA or RNA extracted from clinical samples collected from pigs experimentally infected with ASFV or CSFV, as well as viral RNA from virus stocks strains representing the 3 known CSFV genotypes. Amplicons were sequenced on the Oxford Nanopore MinION platform. 

Results: These targeted WGS protocols resulted in average coverages greater than 1,000X for ASFV, 99% of the genome covered, and greater than 10,000X for CSFV, 97% to 99% of the genome covered. The ASFV targeted WGS protocol has been optimized for genotype II ASFVs. The CSFV targeted WGS protocol has potential universal application for the detection of all currently known CSFV genotypes. 

Conclusions: These targeted WGS protocols will be important tools for the complete genomic characterization of these high consequence swine viruses in outbreak and surveillance situations globally                                         

Comparison of Library Preparation Methods for the Sequencing of African Swine Fever Virus using Illumina Platform

Amy L. Berninger1*, Jim L. Pierce1, Lizhe Xu2, Roger W. Barrette2, Steven M. Lakin3, Patrick T. Ababio4, Theophilius Odoom4, Bonto Faburay3, Vivian K. O’Donnell2

1 Oak Ridge Institute for Science and Education (ORISE), Oak Ridge, TN, USA 

2 Foreign Animal Disease Diagnostic Laboratory, National Veterinary Services Laboratories, Animal and Plant Health Inspection Service, U.S. Department of Agriculture, Plum Island Animal Disease Center, Plum Island, New York, USA

3 National Bio- and Agro-defense Facility (NBAF), Animal and Plant Health Inspection Service, U.S. Department of Agriculture, Manhattan, KS USA

4 Accra Veterinary Laboratory of Veterinary Services Directorate, Ghana

*Corresponding author – Amy.Berninger@usda.gov

Background: The goal of this project was to evaluate the performance of three different DNA library preparation kits (Illumina DNA Prep, Nextera XT DNA Library Prep Kit, and the ExpressPlex Library Prep Kit) to sequence the whole genome of African Swine Fever Virus (ASFV).

Methods: For evaluation, the following samples were used: 4 blood samples from swine experimentally infected with ASFV, provided by the Foreign Animal Disease Diagnosticians School and 17 swine tissue samples collected in outbreak regions of Ghana. Two sequencing approaches were tested: whole genome sequencing and amplicon sequencing. Amplicon generation of ASFV was achieved by following the ASFV Tiled PCR amplification protocol, using LongAmp Taq DNA polymerase (NEB) as a modification. Libraries were prepared using Illumina DNA Prep Kit (Illumina), Nextera XT (Illumina), and ExpressPlex™ Library Prep Kit (SeqWell). Samples were multiplexed and loaded on the MiSeq System. Bioinformatic analysis was performed using our own ASFV fast pipeline.

Results: The Illumina DNA Prep kit produced the highest depth of coverage and percent genome sequenced out of all three kits, with Nextera XT and SeqWell having a similar performance to each other. When ASF tiled amplicon sequencing was performed, an increase in depth of coverage and percent genome sequenced was observed, as compared to the WGS for all three kits. 

Conclusion: Our results indicate when sequencing the whole ASF genome, libraries prepared with the Illumina DNA Prep Kit generated higher resolution of the genomes with greater depth of coverage and percent genome sequenced, offering the best opportunity to genotype the samples. 

Assessment of the reversion to virulence and protective efficacy in pigs receiving the live attenuated classical swine fever recombinant vaccine candidate FlagT4G.

Elizabeth Ramirez-Medina1*, Lauro Velazquez-Salinas1, Alyssa Valladares1, Ayushi Rai1,2, Leeanna Burton1*, Leandro Sastre1, Ediane Silva1, Llilianne Ganges3,4, and Manuel V. Borca1*

1 Plum Island Animal Disease Center, ARS, USDA, Greenport, NY 11955, USA2 Oak Ridge Institute for Science and Education (ORISE), Oak Ridge, TN 37830, USA*3 IRTA, Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Bellaterra, 08193 Barcelona, Spain4 WOAH Reference Laboatory for Classical Swine Fever, IRTA-CReSA, 08193 Barcelona, Spain*Corresponding authors e-mail: 

Leeanna Burton – Leeanna.Burton@usda.gov

Elizabeth Ramirez-Medina – Elizabeth.Ramirez@usda.gov

Manuel V. Borca – Manuel.Borca@usda.gov

Abstract: Control of classical swine fever virus (CSFV) in endemic countries relies on vaccination using live attenuated vaccines (LAV). Most of these LAV do not allow for the differentiation of vaccinated animals from infected animals (DIVA) based in their serological re-sponse. FlagT4G vaccine is a novel candidate that confers robust protective immunity early after vaccination and shows DIVA capabilities. This report presents the characterization of FlagT4G virus in terms of the stability of its genomic and attenuated phenotype assessed by a reversion to virulence protocol, as well as its protective efficacy by determining the minimal protective dose. Results presented here demonstrate that after five consecutive passages in groups of 5-week-old susceptible domestic pigs, FlagT4G virus remains genetically stable, and its attenuated phenotype remained unaltered. In terms of efficacy, FlagT4G virus induced solid protection against the intranasal challenge with 105 tissue culture infectious dose (TCID50) of virulent field isolate Brescia virus even with a vaccine dose as low as 102 TCID50. Results presented here indicate that FlagT4G vaccine may be a useful tool for CSFV control.

Surveillance and Management Strategies for African Swine Fever (ASF) in Central Luzon, Philippines

Virginia M. Venturina1,2,*, Romeo S. Gundran2, Ronalie B. Rafael2, Roderick T. Salvador2, Marvin Bryan S. Salinas3, Errol Jay Y. Balagan3, Phebe M. Valdez4, Ma-Jian R. Dela Cruz 1, Lianne Kathleen P. Salazar 1, Lohreihlieh P. Parayao1, Corrie C. Brown5and Bonto Faburay6,7

1 Center for Transboundary Animal Diseases (CenTrAD), Central Luzon State University, Science City of Muñoz, Nueva Ecija, 3120, Philippines

2 Department of Veterinary Paraclinical Sciences, College of Veterinary Science and Medicine, Central Luzon State University, Science City of Muñoz, Nueva Ecija, 3120, Philippines

3 Department of Basic Veterinary Sciences, College of Veterinary Science and Medicine, Central Luzon State University, Science City of Muñoz, Nueva Ecija, 3120, Philippines

4 Department of Veterinary Clinical Sciences, College of Veterinary Science and Medicine, Central Luzon State University, Science City of Muñoz, Nueva Ecija, 3120, Philippines

5 LifeStock International, Athens, GA, USA

6 U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Foreign Animal Disease Diagnostic Laboratory, National Bio and Agro-Defense Facility USDA, Manhattan, KS 66502, USA

7 U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Foreign Animal Disease Diagnostic Laboratory, Plum Island Animal Disease Center, Greenport, NY 11957, USA

*Corresponding author – vmventurinadvm@clsu.edu.ph

Background. African swine fever (ASF) has emerged as a major concern in the agricultural sector of the Philippines. This viral infection targets primarily the domestic pig populations, resulting in extensive outbreaks that have profoundly curtailed the livelihood in the region of the Philippines that used to dominate the national swine population.

Methods. Whole blood samples were collected from 277 pigs in 7 provinces of Central Luzon, Philippines. Serology and PCR was done on the samples. Farm assessments and structured questionnaires were used to evaluate risk factors. Bioinformatic and Statistical analyses were performed to determine viral genotype and evaluate epidemiological drivers.

Results. The overall prevalence of ASFV in Central Luzon was 26.7% with the highest prevalence found in Bataan (80.5%) and Nueva Ecija (55.0%). African swine fever virus (ASFV) p72 gene was detected in the blood by real-time PCR. Insufficient veterinary care, deficiencies in biosecurity measures such as the lack of perimeter fencing and disinfection protocols on certain farms was identified as critical risk factors contributing to the infection of ASFV.

Conclusion. These findings reveal the extent of ASFV infection in pigs in a major pig-producing region, suggesting the need to address the biosecurity gaps and other risk factors associated with its spread.

Virginia M. Venturina1,2,*, Romeo S. Gundran2, Ronalie B. Rafael2, Roderick T. Salvador2, Marvin Bryan S. Salinas3, Errol Jay Y. Balagan3, Phebe M. Valdez4, Ma-Jian R. Dela Cruz 1, Lianne Kathleen P. Salazar 1, Lohreihlieh P. Parayao1, Corrie C. Brown5and Bonto Faburay6,7

1 Center for Transboundary Animal Diseases (CenTrAD), Central Luzon State University, Science City of Muñoz, Nueva Ecija, 3120, Philippines

2 Department of Veterinary Paraclinical Sciences, College of Veterinary Science and Medicine, Central Luzon State University, Science City of Muñoz, Nueva Ecija, 3120, Philippines

3 Department of Basic Veterinary Sciences, College of Veterinary Science and Medicine, Central Luzon State University, Science City of Muñoz, Nueva Ecija, 3120, Philippines

4 Department of Veterinary Clinical Sciences, College of Veterinary Science and Medicine, Central Luzon State University, Science City of Muñoz, Nueva Ecija, 3120, Philippines

5 LifeStock International, Athens, GA, USA

6 U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Foreign Animal Disease Diagnostic Laboratory, National Bio and Agro-Defense Facility USDA, Manhattan, KS 66502, USA

7 U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Foreign Animal Disease Diagnostic Laboratory, Plum Island Animal Disease Center, Greenport, NY 11957, USA

*Corresponding author – vmventurinadvm@clsu.edu.ph

Background. African swine fever (ASF) has emerged as a major concern in the agricultural sector of the Philippines. This viral infection targets primarily the domestic pig populations, resulting in extensive outbreaks that have profoundly curtailed the livelihood in the region of the Philippines that used to dominate the national swine population.

Methods. Whole blood samples were collected from 277 pigs in 7 provinces of Central Luzon, Philippines. Serology and PCR was done on the samples. Farm assessments and structured questionnaires were used to evaluate risk factors. Bioinformatic and Statistical analyses were performed to determine viral genotype and evaluate epidemiological drivers.

Results. The overall prevalence of ASFV in Central Luzon was 26.7% with the highest prevalence found in Bataan (80.5%) and Nueva Ecija (55.0%). African swine fever virus (ASFV) p72 gene was detected in the blood by real-time PCR. Insufficient veterinary care, deficiencies in biosecurity measures such as the lack of perimeter fencing and disinfection protocols on certain farms was identified as critical risk factors contributing to the infection of ASFV.

Conclusion. These findings reveal the extent of ASFV infection in pigs in a major pig-producing region, suggesting the need to address the biosecurity gaps and other risk factors associated with its spread.