Molecular survey of feline immunodeficiency virus (FIV) infection in Namibian cats.

Feline Immunodeficiency Virus (FIV) is one of the most important infectious diseases of cats, with potential implications in wildlife conservation. Unfortunately, FIV screening and surveillance in domestic cats remains limited in several African countries, including Namibia. In this study, 279 blood samples from domestic cats in Namibia were analyzed for FIV diagnosis by PCR. The cats represented various regions and were cared for by people largely from rural areas with limited financial means. Only 1.43 % of the samples tested positive, unexpectedly low given their outdoor lifestyles. The infected cats, primarily adult and unsterilized, showed no typical FIV symptoms, suggesting subclinical infections. Genetic analysis of the detected strains indicated a unique FIV strain cluster in Namibia, although with a certain within-country variability, in the absence of consistent geographical clustering. The present study represents the first detection and genetic characterization of FIV in the Namibian domestic cat population. Although the infection frequency was low, also in the rural free-roaming population, the features of the enrolled population could have biased the estimation, suggesting the need for more extensive surveys involving diseased and older cats as well. Additionally, because of the long-lasting subclinical nature of the infection, frequent monitoring activities should be performed that allow prompt isolation of infected animals and the implementation of appropriate control measures if necessary.

Molecular epidemiology of Canine circovirus in domestic dogs and wildlife in Namibia, Africa.

Canine circovirus (CanineCV) is a DNA virus affecting domestic dogs and other wild carnivore species. Despite the potential implications for dogs' health and wildlife conservation, data on CanineCV presence, epidemiology and genetic features from Africa is still poor. In the present study, biological specimens collected between 2020 and 2022 from a total of 32 jackals and 575 domestic dogs were tested for the presence of CanineCV DNA to evaluate its frequency. Furthermore, sequencing was conducted on positive samples to characterize the strains and compare them with publicly available sequences through phylogenetic analysis. A high CanineCV prevalence was observed both in jackals (43.75%; 95 CI: 28.17% - 60.67%) and domestic dogs (27.13%; 95 CI: 23.66% - 30.91%). All aside from one Namibian strain formed an independent clade, suggestive of extremely rare introduction events, followed by local persistence, circulation, and evolution. Remarkably, different recombination events were observed involving strains from both jackals and domestic dogs, which testify to the likely strain exchange between these populations. Distinctive amino acid residues were also observed in jackals. The limitations of the considered host populations however prevent a definitive conclusion on host adaptation, biological, and clinical features. Further studies should be performed to expand our current knowledge of the CanineCV disease scenario in Namibia, other African regions, and associated host species in Africa.

Low Seroprevalence of WNV in Namibian Dogs Suggests a Limited Effectiveness as Sentinels for Infection Monitoring.

West Nile virus (WNV) is an important zoonotic Flavivirus responsible for mild fever to severe neurological disease in humans and horses. Despite the occurrence of major previous outbreaks in Namibia and the likelihood of the current endemicity of the virus, only limited investigations and monitoring activities of WNV have been performed in the country. The use of animal sentinels is a valuable approach toward investigating the infection presence in an area and to predict the potential occurrence of human outbreaks. Serological investigations in dogs hold several advantages, considering their infection susceptibility, the ease of sample handling, and the evaluation of risk factors of pet owners that share the same habit with their pets. To evaluate the usefulness of such a sero-epidemiological investigation in Namibia, a broad serosurvey was performed in 2022 that included 426 archived domestic dog samples from eight Namibian regions. Although the ELISA prevalence, indicative of Flavivirus infection, was relatively high (16.43%; 95 CI: 13.10-20.39%), the virus neutralization test confirmed only a minority of cases, highlighting a prevalence of 2.82% (95 CI: 1.47-4.90%), significantly lower than in Namibian donkeys and reports from other countries. Variables that could explain the recorded differences remain to be explored, including animal exposure, variable vector presence, distribution, and feeding preferences. The study results suggest the limited usefulness of dogs as sentinels for WNV monitoring in Namibia.

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.

SARS-CoV-2 in Namibian Dogs.

The pandemic of coronavirus disease 19 (COVID-19) has focused the attention of researchers, and especially public opinion, on the role of the human-animal-environment interface in disease emergence. At the beginning of the COVID-19 pandemic, media reports regarding the role of pets in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused significant concern and social anxiety. Although nowadays proven negligible in developed countries, essentially no studies have been performed in low-income African areas where companion animals are often raised differently from high income countries, and the contact patterns occurring in these scenarios could affect the epidemiological scenario. An extensive molecular biology survey was performed from March 2022 to September 2022 on Namibian dogs residing in urban and rural areas, showing a low but not negligible SARS-CoV-2 prevalence (1%; 95CI: 0.33-2.32%) of 5 out of 500. In only one instance (i.e., a 4-year-old female Labrador) was there a clear association that could be established between the infections of the owner and animal. In all other cases, no evidence of human infection could be obtained and no episodes of COVID-19 were reported by the owners. Although no consistent evidence of pet-to-pet transmission was proven in the present study, a cautionary principle suggests intensive and dedicated investigation into companion animal populations, especially when animal contact is frequent and a particularly susceptible population is present.

Molecular epidemiology of canine parvovirus in Namibia: Introduction pathways and local persistence.

Canine parvovirus is a member of the Carnivore protoparvovirus 1 species that, after a relatively recent origin, has reached a worldwide distribution. Like other ssDNA viruses, it is featured by a remarkable evolutionary rate and thus genetic variability. CPV-2 is responsible for a severe systemic infection affecting especially domestic dogs. However, other carnivores, including wild species, are susceptible and thus represents a menace to wildlife conservation too. Despite the relevance of the topic, molecular epidemiology data are scarce and outdated in certain areas of the world, like Africa and, in particular, Namibia. The present study investigates the occurrence and genetic features of CPV in Namibian domestic dogs and jackals. The VP2 of detected strains was characterized and analyzed to assess the viral circulation and link among host species, Namibian districts and foreign countries. With the only exception of one New-CPV-2a, all the detected strains belonged to the CPV-2c antigenic variant and were closely related to strains of Asian origin. Nevertheless, a dedicated phylogeographic analysis revealed that the introduction was more likely mediated by other African countries, highlighting the challenge of controlling illegal animal imports across land borders. Similarly, the absence of any geographical clustering within Namibia testify a substantially unconstrained viral circulation among districts. The absence/incomplete vaccination status reported by the animal owners could have significantly contributed to the infection's success after its introduction. Finally, infection of a wild jackal was also proven. Although the limited wild animals' sample size prevents any definitive conclusion, the identity of the sequences from the jackal and the ones originating from the domestic dogs suggests a potential inter-species transmission. The epidemiological and clinical implications in wild specie remain obscure.

Phylodynamic and phylogeographic reconstruction of beak and feather disease virus epidemiology and its implications for the international exotic bird trade.

Beak and feather disease virus (BFDV) infects domestic and wild psittacine species and is able to cause progressive beak, claw and feather malformation and necrosis. In addition to having an impact on the health and welfare of domesticated birds, BFDV represents a significant threat to wild endangered species. Understanding the epidemiology, dynamics, viral migration rate, interaction between wild and domestic animals and the effect of implemented control strategies is fundamental in controlling the spread of the disease. With this in mind, a phylodynamic and phylogeographic analysis has been performed on a database of more than 400 replication-associated protein (Rep) gene (ORF1) sequences downloaded from Genbank including some recently generated sequences from fifteen samples collected in Namibia. The results allowed us to reconstruct the variation of viral population size and demonstrated the effect of enforced international bans on these dynamics. A good correlation was found between viral migration rate and the intensity of animal trade between regions over time. A dominant flux of viral strains was observed from wild to domestic populations, highlighting the directionality of viral transmission and the risk associated with the capturing and trade of wild birds. Nevertheless, the flow of viruses from domestic to wild species was not negligible and should be considered as a threat to biodiversity. Therefore, considering the strong relationship demonstrated in this study between animal trade and BFDV viral fluxes more effort should be made to prevent contact opportunities between wild and domestic populations from different countries in order to control disease spread.

Molecular characterization of avipoxviruses circulating in Windhoek district, Namibia 2021.

Samples from eleven birds (chicken, dove and peacock) with symptoms of fowlpox, caused by the avipoxvirus (APV), were collected in seven different areas of the Windhoek district, Namibia between April and October 2021. A fragment of the 4b core protein and the DNA polymerase gene of APV were amplified by PCR from the DNA of the samples and sequenced. Phylogenetic analysis revealed that the viruses present in the chickens all belonged to clade A1 while the viruses in the doves and peacock were from subclade A3.1. This is the first report of subclade A3.1 avipoxvirus in peacock. In addition, all of the samples obtained from chickens were shown by PCR to be positive for the integration of reticuloendotheliosis virus while those from the doves and peacocks were negative. This study is the first characterization of avipoxvirus in Namibia and provides additional information on the presence of avipoxvirus in southern Africa.

A Comparison Between Manual Count, Flow Cytometry and Quantitative Real-Time Polymerase Chain Reaction as a Means of Determining Babesia rossi Parasitaemia in Naturally Infected Dogs.

PURPOSE: Light microscopic manual count is the current gold standard for parasite quantification. The ability to determine parasite density in whole blood is crucial to understanding disease pathogenesis and finding a suitable automated method of Babesia rossi parasite quantification would facilitate higher throughput and provide results that are more objective. This study investigated both peripheral capillary and central venous whole blood to estimate the correlations between light microscopy, flow cytometry and quantitative real-time polymerase chain reaction (qPCR). METHODS: Peripheral capillary and central venous blood were sampled from 40 naturally B. rossi-infected dogs and 10 healthy control dogs. Samples were analysed by reverse line blot hybridization assay to confirm a mono-B. rossi infection. Capillary blood parasite density was detected using light microscopic manual counting and venous blood parasitaemia detected by manual counts, flow cytometry and qPCR. RESULTS: A significant correlation was found between the venous manual counts and flow cytometry (rs = 0.465; P < 0.001), as well as qPCR (rs = - 0.500; P < 0.001). A significant correlation was also observed between the capillary manual counts compared to venous manual counts (rs = 0.793; P < 0.001), flow cytometry (rs = 0.399; P = 0.004), and qPCR (rs = - 0.526; P < 0.001). CONCLUSIONS: The study results suggest that qPCR is of value as an alternative to the gold standard manual count for detecting B. rossi parasitaemia in canine whole blood and that flow cytometry may be useful with further refinement of issues such as background fluorescence and the influence of reticulocytes.

Development and validation of a multiplex, real-time PCR assay for Babesia rossi and Babesia vogeli.

Canine babesiosis is caused by tick-transmitted intraerythrocytic protozoan parasites occurring worldwide. In southern Africa, babesiosis is caused by Babesia rossi and B. vogeli and is one of the most common and important infectious diseases affecting dogs. There is no reliable, rapid and sensitive method for the detection of these parasites, especially when parasitaemia is low. The aim of this study was to develop a sensitive and specific multiplex TaqMan® MGB PCR assay for the diagnosis of canine babesiosis infections occurring in southern Africa, and to discriminate between Babesia rossi and B. vogeli. The fitness of purpose of the assay was to confirm diagnosis of suspect or clinical cases, and estimate prevalence of infection for research purposes. A total of 648 published sequences were used to design the assay. A set of group-specific canine Babesia spp. primers were designed to amplify a 117 nucleotide region of the 18S rRNA gene of all canine Babesia spp. Species-specific TaqMan® MGB probes were developed for B. rossi, B. vogeli, B. canis and B. gibsoni, but analytical validation was only performed for B. rossi and B. vogeli as a multiplex assay. The assay had a broad dynamic range and amplified B. rossi and B. vogeli efficiently (98.6% and 94.7% respectively). The assay was sensitive, with a 95% LOD of 10-2.67% parasitized erythrocytes (PE) for B. rossi and 10-2.03% PE for B. vogeli, and specific, with no cross reaction between B. rossi and B. vogeli and no detection of other haemoparasites that infect dogs, such as Ehrlichia canis and Anaplasma platys. Consistent repeatability within and between PCR runs was shown. This assay will be able to accurately and rapidly confirm babesiosis in canines and allow for treatment to be administered in the early stages of the disease, speeding up the recovery time in affected dogs.