Molecular characterization of recombinant LSDV isolates from 2022 outbreak in Indonesia through phylogenetic networks and whole-genome SNP-based analysis.

Lumpy skin disease (LSD) is a transboundary viral disease of cattle and water buffaloes caused by the LSD virus, leading to high morbidity, low mortality, and a significant economic impact. Initially endemic to Africa only, LSD has spread to the Middle East, Europe, and Asia in the past decade. The most effective control strategy for LSD is the vaccination of cattle with live-attenuated LSDV vaccines. Consequently, the emergence of two groups of LSDV strains in Asian countries, one closely related to the ancient Kenyan LSDV isolates and the second made of recombinant viruses with a backbone of Neethling-vaccine and field isolates, emphasized the need for constant molecular surveillance. This current study investigated the first outbreak of LSD in Indonesia in 2022. Molecular characterization of the isolate circulating in the country based on selected LSDV-marker genes: RPO30, GPCR, EEV glycoprotein gene, and B22R, as well as whole genome analysis using several analytical tools, indicated the Indonesia LSDV isolate as a recombinant of LSDV_Neethling_vaccine_LW_1959 and LSDV_NI-2490. The analysis clustered the Indonesia_LSDV with the previously reported LSDV recombinants circulating in East and Southeast Asia, but different from the recombinant viruses in Russia and the field isolates in South-Asian countries. Additionally, this study has demonstrated alternative accurate ways of LSDV whole genome analysis and clustering of isolates, including the recombinants, instead of whole-genome phylogenetic tree analysis. These data will strengthen our understanding of the pathogens' origin, the extent of their spread, and determination of suitable control measures required.

SARS-CoV-2 Infection in Beaver Farm, Mongolia, 2021.

We report an outbreak of COVID-19 in a beaver farm in Mongolia in 2021. Genomic characterization revealed a unique combination of mutations in the SARS-CoV-2 of the infected beavers. Based on these findings, increased surveillance of farmed beavers should be encouraged.

High detection frequency and genetic diversity of porcine circovirus 3 (PCV-3) in Namibian backyard farms and warthogs.

Since its first identification in 2015, porcine circovirus 3 (PCV-3) has been reported worldwide with a high frequency and in the presence of several clinical conditions, although its impact on pig health and productivity is still debated. Data on the presence of PCV-3 in Africa are, however, limited. A previous study performed on commercial pigs in Namibia failed to identify the pathogen. In the present study, the viral circulation in backyard farms, characterised by lower biosecurity measures and frequent animal exchange between farms, was assessed. The susceptibility of warthogs to PCV-3 infection and their potential epidemiological role were also evaluated. Tonsils from 77 pigs from backyard piggeries and 55 warthogs were collected in different regions of Namibia and tested by PCR. Positive samples were sequenced and compared to PCV-3 strains circulating globally. Forty-two out of 77 pigs (54.54 %) and 12 out of 55 warthogs (21.82 %) tested positive, demonstrating the presence of PCV-3 in the country and suggesting that the high biosecurity measures implemented in the commercial farms that previously tested negative for PCV-3 probably prevented viral introduction. The partial ORF2 gene was successfully sequenced in samples from 27 pigs and 6 warthogs. Genetically, the identified strains were part of 3 distinct groups which included both backyard pigs and warthogs from different regions of Namibia. There is also evidence for the occurrence of multiple introduction events most likely from Asian countries, either directly into Namibia or through other African countries. Considering the strict Namibian regulations on live animal importation, understanding the source of viral introduction is challenging, although semen importation or the habit of feeding backyard pigs with human food waste might have played a role. Pig exchanges between farms for breeding purposes or wildlife movements could also have been involved in PCV-3 dispersal within Namibia. Despite the significant advances in the field, further studies should be undertaken to properly understand PCV-3 epidemiology in Namibia and its impact on pig productivity and wildlife health.

Harnessing Attenuation-Related Mutations of Viral Genomes: Development of a Serological Assay to Differentiate between Capripoxvirus-Infected and -Vaccinated Animals.

Sheeppox, goatpox, and lumpy skin disease caused by the sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV), respectively, are diseases that affect millions of ruminants and many low-income households in endemic countries, leading to great economic losses for the ruminant industry. The three viruses are members of the Capripoxvirus genus of the Poxviridae family. Live attenuated vaccines remain the only efficient means for controlling capripox diseases. However, serological tools have not been available to differentiate infected from vaccinated animals (DIVA), though crucial for proper disease surveillance, control, and eradication efforts. We analysed the sequences of variola virus B22R homologue gene for SPPV, GTPV, and LSDV and observed significant differences between field and vaccine strains in all three capripoxvirus species, resulting in the truncation and absence of the B22R protein in major vaccines within each of the viral species. We selected and expressed a protein fragment present in wildtype viruses but absent in selected vaccine strains of all three species, taking advantage of these alterations in the B22R gene. An indirect ELISA (iELISA) developed using this protein fragment was evaluated on well-characterized sera from vaccinated, naturally and experimentally infected, and negative cattle and sheep. The developed wildtype-specific capripox DIVA iELISA showed >99% sensitivity and specificity for serum collected from animals infected with the wildtype virus. To the best of our knowledge, this is the first wildtype-specific, DIVA-capable iELISA for poxvirus diseases exploiting changes in nucleotide sequence alterations in vaccine strains.

Comparison of the Whole-Genome Sequence of the African Swine Fever Virus from a Mongolian Wild Boar with Genotype II Viruses from Asia and Europe.

African swine fever (ASF) is a highly contagious and severe viral hemorrhagic disease in domestic and wild pigs. ASF seriously affects the global swine industry as the mortality rate can reach 100% with highly virulent strains. In 2007, ASF was introduced into the Caucasus and spread to Russia and later into other European and Asian countries. This study reported the first whole-genome sequence (WGS) of the ASF virus (ASFV) that was detected in a Mongolian wild boar. This sequence was then compared to other WGS samples from Asia and Europe. Results show that the ASFV Genotype II from Mongolia is similar to the Asian Genotype II WGS. However, there were three nucleotide differences found between the Asian and European genome sequences, two of which were non-synonymous. It was also observed that the European Genotype II ASFV WGS was more diverse than that of the Asian counterparts. The study demonstrates that the ASFV Genotype II variants found in wild boars and domestic pigs are highly similar, suggesting these animals might have had direct or indirect contact, potentially through outdoor animal breeding. In conclusion, this study provides a WGS and mutation spectrum of the ASFV Genotype II WGS in Asia and Europe and thus provides important insights into the origin and spread of ASFV in Mongolia.

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.

Phylogenetic analysis of Newcastle disease virus detected in Eritrea between 2017 and 2021.

Thirty-five samples collected from chickens in 13 commercial farms in Eritrea between 2017 and 2021 following reports of disease were screened for Newcastle disease virus. Seventeen samples (50%) were shown to be positive by RT-PCR. An initial analysis of partial fusion (F) gene sequences of 10 representative samples indicated that the viruses belonged to subgenotype VII.1.1. Subsequently, full F gene sequence analysis of four of these representative samples confirmed the genotype of the viruses but also revealed that they were not identical to each other suggesting different origins of the VII.1.1 subgenotype viruses circulating in Eritrea. These data have implications for the control of Newcastle disease within the poultry population in Eritrea.

A novel multiplex qPCR­HRM assay for the simultaneous detection of four abortive zoonotic agents in cattle, sheep, and goats.

Abortifacient pathogens induce substantial economic losses in the livestock industry worldwide, and many of these pathogens are zoonotic, impacting human health. As Brucella spp., Coxiella burnetii, Leptospira spp., and Listeria monocytogenes cause abortion, rapid differential molecular diagnostic tests are needed to facilitate early and accurate detection of abortion to establish effective control measures. However, the available molecular methods are laborious, time-consuming, or costly. Therefore, we developed and validated a novel multiplex real-time polymerase chain reaction (qPCR) method based on high-resolution melting (HRM) curve analysis to simultaneously detect and differentiate four zoonotic abortifacient agents in cattle, goats, and sheep. Our HRM assay generated four well-separated melting peaks allowing the differentiation between the four zoonotic abortifacients. Out of 216 DNA samples tested, Brucella spp. was detected in 45 samples, Coxiella burnetii in 57 samples, Leptospira spp. in 12 samples, and Listeria monocytogenes in 19 samples, co-infection with Brucella spp. and Coxiella burnetii in 41 samples, and 42 samples were negative. This assay demonstrated good analytical sensitivity, specificity, and reproducibility. This is a valuable rapid, cost-saving, and reliable diagnostic tool for detecting individual and co-infections for zoonotic abortifacient agents in ruminants.

Aerosol vaccination of chicken pullets with irradiated avian pathogenic Escherichia coli induces a local immunostimulatory effect.

The present study investigated the expression of cytokines and cellular changes in chickens following vaccination with irradiated avian pathogenic Escherichia coli (APEC) and/or challenge. Four groups of 11-week-old pullets, each consisting of 16 birds were kept separately in isolators before they were sham inoculated (N), challenged only (C), vaccinated (V) or vaccinated and challenged (V+C). Vaccination was performed using irradiated APEC applied via aerosol. For challenge, the homologous strain was administered intratracheally. Birds were sacrificed on 3, 7, 14 and 21 days post challenge (dpc) to examine lesions, organ to body weight ratios and bacterial colonization. Lung and spleen were sampled for investigating gene expression of cytokines mediating inflammation by RT-qPCR and changes in the phenotype of subsets of mononuclear cells by flow cytometry. After re-stimulation of immune cells by co-cultivation with the pathogen, APEC-specific IFN-γ producing cells were determined. Challenged only birds showed more severe pathological and histopathological lesions, a higher probability of bacterial re-isolation and higher organ to body weight ratios compared to vaccinated and challenged birds. In the lung, an upregulation of IL-1ß and IL-6 following vaccination and/or challenge at 3 dpc was observed, whereas in the spleen IL-1ß was elevated. Changes were observed in macrophages and TCR-γδ+ cells within 7 dpc in spleen and lung of challenged birds. Furthermore, an increase of CD4+ cells in spleen and a rise of Bu-1+ cells in lung were present in vaccinated and challenged birds at 3 dpc. APEC re-stimulated lung and spleen mononuclear cells from only challenged pullets showed a significant increase of IFN-γ+CD8α+ and IFN-γ+TCR-γδ+ cells. Vaccinated and challenged chickens responded with a significant increase of IFN-γ+CD8α+ T cells in the lung and IFN-γ+TCR-γδ+ cells in the spleen. Re-stimulation of lung mononuclear cells from vaccinated birds resulted in a significant increase of both IFN-γ+CD8α+ and IFN-γ+TCR-γδ+ cells. In conclusion, vaccination with irradiated APEC caused enhanced pro-inflammatory response as well as the production of APEC-specific IFN-γ-producing γδ and CD8α T cells, which underlines the immunostimulatory effect of the vaccine in the lung. Hence, our study provides insights into the underlying immune mechanisms that account for the defense against APEC.

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and play a unique role within the immune system by linking the innate and adaptive immune responses. These cells can phagocytize and then present captured antigens to effector immune cells, triggering a diverse range of immune responses. This paper demonstrates a standardized method for the in vitro generation of bovine monocyte-derived dendritic cells (MoDCs) isolated from cattle peripheral blood mononuclear cells (PBMCs) and their application in evaluating vaccine immunogenicity. Magnetic-based cell sorting was used to isolate CD14+ monocytes from PBMCs, and the supplementation of complete culture medium with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of CD14+ monocytes into naive MoDCs. The generation of immature MoDCs was confirmed by detecting the expression of major histocompatibility complex II (MHC II), CD86, and CD40 cell surface markers. A commercially available rabies vaccine was used to pulse the immature MoDCs, which were subsequently co-cultured with naive lymphocytes. The flow cytometry analysis of the antigen-pulsed MoDCs and lymphocyte co-culture revealed the stimulation of T lymphocyte proliferation through the expression of Ki-67, CD25, CD4, and CD8 markers. The analysis of the mRNA expression of IFN-γ and Ki-67, using quantitative PCR, showed that the MoDCs could induce the antigen-specific priming of lymphocytes in this in vitro co-culture system. Furthermore, IFN-γ secretion assessed using ELISA showed a significantly higher titer (**p < 0.01) in the rabies vaccine-pulsed MoDC-lymphocyte co-culture than in the non-antigen-pulsed MoDC-lymphocyte co-culture. These results show the validity of this in vitro MoDC assay to measure vaccine immunogenicity, meaning this assay can be used to identify potential vaccine candidates for cattle before proceeding with in vivo trials, as well as in vaccine immunogenicity assessments of commercial vaccines.

Molecular characterization of porcine circovirus-2 and -3 in pigs in Tanzania.

Like in many other African countries, pig production is increasing in Tanzania. To support farmers and stakeholders, it is important to increase our understanding of porcine pathogens present in the country. Currently, little is known about the circulation of porcine circovirus-2 (PCV-2) and porcine circovirus-3 (PCV-3). For this reason, samples from 124 pigs collected throughout the country between 2018 to 2022 were screened by PCR for the presence of PCV-2 and PCV-3. Sequencing and phylogenetic analysis of positive amplicons identified two PCV-2 genotypes (a and d). Limited genetic heterogenicity was observed among the PCV-3. This study provides important data on pathogens present in pigs in Tanzania and should be of use veterinary authorities involved in porcine disease management.

Poxvirus Infections in Dairy Farms and Transhumance Cattle Herds in Nigeria.

Lumpy Skin disease (LSD) is an economically important disease in cattle caused by the LSD virus (LSDV) of the genus Capripoxvirus, while pseudocowpox (PCP) is a widely distributed zoonotic cattle disease caused by the PCP virus (PCPV) of the genus Parapoxvirus. Though both viral pox infections are reportedly present in Nigeria, similarities in their clinical presentation and limited access to laboratories often lead to misdiagnosis in the field. This study investigated suspected LSD outbreaks in organized and transhumance cattle herds in Nigeria in 2020. A total of 42 scab/skin biopsy samples were collected from 16 outbreaks of suspected LSD in five northern States of Nigeria. The samples were analyzed using a high-resolution multiplex melting (HRM) assay to differentiate poxviruses belonging to Orthopoxvirus, Capripoxvirus, and Parapoxvirus genera. LSDV was characterized using four gene segments, namely the RNA polymerase 30 kDa subunit (RPO30), G-protein-coupled receptor (GPCR), the extracellular enveloped virus (EEV) glycoprotein and CaPV homolog of the variola virus B22R. Likewise, the partial B2L gene of PCPV was also analyzed. Nineteen samples (45.2%) were positive according to the HRM assay for LSDV, and five (11.9%) were co-infected with LSDV and PCPV. The multiple sequence alignments of the GPCR, EEV, and B22R showed 100% similarity among the Nigerian LSDV samples, unlike the RPO30 phylogeny, which showed two clusters. Some of the Nigerian LSDVs clustered within LSDV SG II were with commonly circulating LSDV field isolates in Africa, the Middle East, and Europe, while the remaining Nigerian LSDVs produced a unique sub-group. The B2L sequences of Nigerian PCPVs were 100% identical and clustered within the PCPV group containing cattle/Reindeer isolates, close to PCPVs from Zambia and Botswana. The results show the diversity of Nigerian LSDV strains. This paper also reports the first documented co-infection of LSDV and PCPV in Nigeria.

The Spread of Peste Des Petits Ruminants Virus Lineage IV in West Africa.

Monitoring the transboundary spread of peste des petits ruminants (PPR) virus is an essential part of the global efforts towards the eradication of PPR by 2030. There is growing evidence that Lineage IV is becoming the predominant viral lineage, replacing Lineage I and II in West Africa. As part of a regional investigation, samples collected in Burkina Faso, Côte d'Ivoire, Guinea and Ghana were screened for the presence of PPRV. A segment of the nucleoprotein gene from positive samples was sequenced, and phylogenetic analysis revealed the co-circulation of Lineage II and IV in Burkina Faso, Côte d'Ivoire and Guinea, and the identification of Lineage IV in Ghana. These data will be of importance to local and regional authorities involved in the management of PPRV spread.

Avian influenza H5N1 in a great white pelican (Pelecanus onocrotalus), Mauritania 2022.

In February 2022, mortalities among great white pelicans (Pelecanus onocrotalus) were reported in the Parc National de Diawling, southwestern Mauritania. Samples were collected and processed, indicating the presence of high pathogenicity avian influenza subtype H5N1. A nearly complete genome was generated for one sample, revealing a high similarity [> 99.5% (H5) nucleotide sequence identity] with Clade 2.3.4.4b H5N1 identified in Europe in 2022.

Comparative assessment of lyophilized and wet reagents for the molecular detection of H5N1 high pathogenic avian influenza virus and H9N2 low pathogenic avian influenza virus.

Global surveillance for Avian Influenza Virus (AIV) in birds is essential for assessing public and animal health risks and real-time polymerase chain reaction (RT-qPCR) is among the official methods recommended by the World Organisation for Animal Health (WOAH) to confirm the presence of the virus in laboratory specimens. Yet, in low-resource setting laboratories, the detection of AIV can be hampered by the need to maintain a cold chain for wet reagents. In such cases, alternatives should be ready to maximize surveillance capacities and mining of AIV. Therefore, we compared two lyophilized RT-qPCR reagents (1st - 5 × CAPITAL™ 1-Step qRT-PCR Probe Reagent, lyophilized kit, and 2nd - Qscript lyo 1-step-kit) to the WOAH recommended protocol by Nagy et al., 2020 using QuantiTect Probe RT-PCR-kit as wet reagent. The comparative study panel comprised 102 RNA samples from two AIV subtypes, i.e. H5 and H9 subtypes. Despite that the wet reagent exhibited the lowest limit of detection (LOD) compared to the two lyophilized reagents, the inter-assay agreement was substantial between the 1st lyophilized reagent and the comparator with 95.1% of shared positive results. Cohen's-kappa was fair between the 2nd lyophilized reagent and the comparator with 75.5% of shared positive results. Agreement using the statistical test Bland-Altman was good for samples with Cq-values < 25 for all reagents, revealing discrepancies when the viral load is low. This trend was especially evident while using the 2nd lyophilized reagent. Similar trends were obtained using the same lyophilized reagents but following the protocol by Heine et al., 2015 with AgPath-ID™ One-Step RT-PCR as a comparator, showing that Cq-values increase using lyophilized reagents but correlate strongly with the wet reagent. Further, inter-assay agreement between reagents improved when the protocol from Heine et al., 2015 was applied, suggesting a higher resilience to chemistry changes allowing easier reagents interchangeability.

Highly pathogenic avian influenza H5N1 virus outbreak among Cape cormorants (Phalacrocorax capensis) in Namibia, 2022.

In January 2022, significant mortality was observed among Cape cormorants (Phalacrocorax capensis) on the west coast of Namibia. Samples collected were shown to be positive for H5N1 avian influenza by multiplex RT-qPCR. Full genome analysis and phylogenetic analysis identified the viruses as belonging to clade 2.3.4.4b and that it clustered with similar viruses identified in Lesotho and Botswana in 2021. This is the first genomic characterization of H5N1 viruses in Namibia and has important implications for poultry disease management and wildlife conservation in the region.

Aerosol delivered irradiated Escherichia coli confers serotype-independent protection and prevents colibacillosis in young chickens.

Escherichia coli causes colibacillosis in chickens, which has severe economic and public health consequences. For the first time, we investigated the efficacy of gamma-irradiated E. coli to prevent colibacillosis in chickens considering different strains and application routes. Electron microscopy, alamarBlue assay and matrix assisted laser desorption/ionization time-of- flight mass spectrometry showed that the cellular structure, metabolic activity and protein profiles of irradiated and non-treated E. coli PA14/17480/5-ovary (serotype O1:K1) were similar. Subsequently, three animal trials were performed using the irradiated E. coli and clinical signs, pathological lesions and bacterial colonization in systemic organs were assessed. In the first animal trial, the irradiated E. coli PA14/17480/5-ovary administered at 7 and 21 days of age via aerosol and oculonasal routes, respectively, prevented the occurrence of lesions and systemic bacterial spread after homologous challenge, as efficient as live infection or formalin-killed cells. In the second trial, a single aerosol application of the same irradiated strain in one-day old chickens was efficacious against challenges with a homologous or a heterologous strain (undefined serotype). The aerosol application elicited better protection as compared to oculonasal route. Finally, in the third trial, efficacy against E. coli PA15/19103-3 (serotype O78:K80) was shown. Additionally, previous results of homologous protection were reconfirmed. The irradiated PA15/19103-3 strain, which also showed lower metabolic activity, was less preferred even for the homologous protection, underlining the importance of the vaccine strain. In all the trials, the irradiated E. coli did not provoke antibody response indicating the importance of innate or cell mediated immunity for protection. In conclusion, this proof-of-concept study showed that the non-adjuvanted single aerosol application of irradiated "killed but metabolically active" E. coli provided promising results to prevent colibacillosis in chickens at an early stage of life. The findings open new avenues for vaccine production with E. coli in chickens using irradiation technology.

A Phylogeographic Analysis of Porcine Parvovirus 1 in Africa.

Porcine parvovirus 1 (PPV1) is recognized as a major cause of reproductive failure in pigs, leading to several clinical outcomes globally known as SMEDI. Despite being known since the late 1960s its circulation is still of relevance to swine producers. Additionally, the emergence of variants such as the virulent 27a strain, for which lower protection induced by vaccines has been demonstrated, is of increasing concern. Even though constant monitoring of PPV1 using molecular epidemiological approaches is of pivotal importance, viral sequence data are scarce especially in low-income countries. To fill this gap, a collection of 71 partial VP2 sequences originating from eight African countries (Burkina Faso, Côte d'Ivoire, Kenya, Mozambique, Namibia, Nigeria, Senegal, and Tanzania) during the period 2011-2021 were analyzed within the context of global PPV1 variability. The observed pattern largely reflected what has been observed in high-income regions, i.e., 27a-like strains were more frequently detected than less virulent NADL-8-like strains. A phylogeographic analysis supported this observation, highlighting that the African scenario has been largely shaped by multiple PPV1 importation events from other continents, especially Europe and Asia. The existence of such an international movement coupled with the circulation of potential vaccine-escape variants requires the careful evaluation of the control strategies to prevent new strain introduction and persistence.

Investigation and sequence analysis of psittacine beak and feather disease virus and avian polyomavirus from companion birds in Windhoek, Namibia.

The commercial farming and trading of parrots and ornamental birds as companion animals are important economic activities in many countries. Some of the bird species farmed/traded are captured from the wild or are closely related to wild birds and therefore represent a risk of pathogen exchange/introduction. Beak and feather disease virus (BFDV) and avian poliomavirus (APV) are among the viruses with the biggest impact on companion bird populations and have been detected in different hosts worldwide. Despite their relevance for both domesticated and wild birds, our knowledge of BFDV and APV epidemiology remains limited in several African countries. In the present study, 143 cloacal swabs were collected from companion birds in Windhoek, Namibia, and tested by polymerase chain reaction for BFDV and APV. Of the samples tested, 35/143 (24.48%) tested positive for BFDV; 11/143 (7.69%) were positive for APV; and 6/143 (4.2%) tested positive for both pathogens. Positive amplicons, consisting of segments of the ORF1 and VP1 genes, were sequenced and compared with sequences from viruses identified in other countries. Four Namibian-only clades of BFDV were identified, loosely related to foreign strains, which suggest the occurrence of multiple introduction events in the past, potentially from South Africa, followed by local, independent evolution. In contrast, the Namibian APV sequences were identical to each other and form a single clade. In both instances, no correlation was observed between the sampling host and the viral phylogeny, suggesting the absence of host-specific adaptation and a remarkable, unconstrained viral circulation within Namibian borders. Therefore, while regulations and control measures developed against foreign strain introduction have proven to be effective over time, the spread of BFDV and APV within Namibia's borders appears undeterred. Additional resources should be dedicated to limit strain circulation in commercial farming facilities, markets and small-scale traders.

Evidence indicating transmission of porcine parvovirus 1 between warthogs and domestic pigs in Namibia.

Thirty swine samples collected from different regions of Namibia between 2019 and 2020 were screened for the presence of porcine parvovirus 1 (PPV1) by PCR. Eleven samples (37%) were positive. Phylogenetic analysis of a partial sequence of the structural protein gene (VP2) identified two distinct clusters, one which contained sequences that were highly similar to PPV1 previously identified in warthogs in Namibia. These results indicate possible PPV1 transmission between warthogs and domestic pigs and highlight the importance of wildlife as sources of pathogens.