Ubiquitin accumulation induced by the finger and palm sub-domains of NS5 modulates the replication of West Nile virus.

West Nile virus (WNV) causes encephalitis in human and animals. WNV is phylogenetically classified into at least five distinct genetic lineages with different pathogenicity. The pathogenesis of West Nile encephalitis is affected by ubiquitin accumulation in infected cells, but the mechanism is unknown. In this study, the association between ubiquitin accumulation and WNV pathogenicity was investigated. Ubiquitin accumulation was detected in cells infected with NY99 strain belonging to lineage-1, but not FCG and Zmq16 strains belonging to lineage-2. Substitution of the Finger and Palm sub-domains of NS5 from lineage-1 to -2 decreased ubiquitin accumulation and viral replication. Furthermore, the survival rate was increased, and viral replication and ubiquitin accumulation in the brain were attenuated, in mice inoculated with the substituted WNV compared with lineage-1 WNV. Therefore, the intracellular ubiquitin accumulation induced by the Finger and Palm sub-domains of NS5 is linked to the differences in pathogenicity among WNV lineages.

Expanding diversity of bunyaviruses identified in mosquitoes.

Mosquitoes interact with various organisms in the environment, and female mosquitoes in particular serve as vectors that directly transmit a number of microorganisms to humans and animals by blood-sucking. Comprehensive analysis of mosquito-borne viruses has led to the understanding of the existence of diverse viral species and to the identification of zoonotic arboviruses responsible for significant outbreaks and epidemics. In the present study on mosquito-borne bunyaviruses we employed a broad-spectrum RT-PCR approach and identified eighteen different additional species in the Phenuiviridae family and also a number of related but unclassified bunyaviruses in mosquitoes collected in Zambia. The entire RNA genome segments of the newly identified viruses were further analyzed by RNA sequencing with a ribonuclease R (RNase R) treatment to reduce host-derived RNAs and enrich viral RNAs, taking advantage of the dsRNA panhandle structure of the bunyavirus genome. All three or four genome segments were identified in eight bunyavirus species. Furthermore, L segments of three different novel viruses related to the Leishbunyaviridae were found in mosquitoes together with genes from the suspected host, the Crithidia parasite. In summary, our virus detection approach using a combination of broad-spectrum RT-PCR and RNA sequencing analysis with a simple virus enrichment method allowed the discovery of novel bunyaviruses. The diversity of bunyaviruses is still expanding and studies on this will allow a better understanding of the ecology of hematophagous mosquitoes.

Surveillance, Isolation, and Genetic Characterization of Bat Herpesviruses in Zambia.

Bats are of significant interest as reservoirs for various zoonotic viruses with high diversity. During the past two decades, many herpesviruses have been identified in various bats worldwide by genetic approaches, whereas there have been few reports on the isolation of infectious herpesviruses. Herein, we report the prevalence of herpesvirus infection of bats captured in Zambia and genetic characterization of novel gammaherpesviruses isolated from striped leaf-nosed bats (Macronycteris vittatus). By our PCR screening, herpesvirus DNA polymerase (DPOL) genes were detected in 29.2% (7/24) of Egyptian fruit bats (Rousettus aegyptiacus), 78.1% (82/105) of Macronycteris vittatus, and one Sundevall's roundleaf bat (Hipposideros caffer) in Zambia. Phylogenetic analyses of the detected partial DPOL genes revealed that the Zambian bat herpesviruses were divided into seven betaherpesvirus groups and five gammaherpesvirus groups. Two infectious strains of a novel gammaherpesvirus, tentatively named Macronycteris gammaherpesvirus 1 (MaGHV1), were successfully isolated from Macronycteris vittatus bats, and their complete genomes were sequenced. The genome of MaGHV1 encoded 79 open reading frames, and phylogenic analyses of the DNA polymerase and glycoprotein B demonstrated that MaGHV1 formed an independent lineage sharing a common origin with other bat-derived gammaherpesviruses. Our findings provide new information regarding the genetic diversity of herpesviruses maintained in African bats.

Prevalence and risk factors of bovine tuberculosis in slaughtered cattle, Malawi.

Bovine tuberculosis (bTB) is an infectious disease with significant socioeconomic, animal, and public health impacts. However, the prevalence of bTB remains largely unclear in Malawi due to a paucity of information. Additionally, the existence of multiple risk factors is postulated to enhance bTB transmission in animals. A cross-sectional survey to estimate the prevalence of bTB, animal characteristics and identify associated risk factors was conducted from slaughtered cattle at three major regional abattoirs (southern, central and northern regions) in Malawi. Out of a total of 1547 cattle examined, 154 (9.95%) had bTB-like lesions in various visceral organs and lymph nodes; one sample per animal was collected, processed, and cultured in the in the BACTEC Mycobacterial growth indicator tube (MGIT) 960 system. From the 154 cattle that showed tuberculous like lesions, only 112 were positive on MGIT and 87 were confirmed to have M. bovis based on multiplex PCR. Cattle from the southern region (odds ratio (OR) = 1.96, 95% CI: 1.03-3.85) and central region (OR = 2.00, 95% CI: 1.16-3.56) were more likely presented with bTB-like lesions at slaughter than from the northern region. The risk of having bTB-like lesions was higher in females (OR = 1.51, CI: 1.00-2.29), older cattle (OR = 2.17, CI: 1.34-3.37), and crossbreeds (OR = 1.67, 95% CI: 1.12-2.47) than in males, younger animals, and Malawi Zebu breed, respectively. The high prevalence of bTB is of critical concern and necessitates active surveillance and strengthening of the current control strategies under a One Health (OH) approach at the animal-human interface.

Isolation and Characterization of Distinct Rotavirus A in Bat and Rodent Hosts.

Rotavirus A (RVA) causes diarrheal disease in humans and various animals. Recent studies have identified bat and rodent RVAs with evidence of zoonotic transmission and genome reassortment. However, the virological properties of bat and rodent RVAs with currently identified genotypes still need to be better clarified. Here, we performed virus isolation-based screening for RVA in animal specimens and isolated RVAs (representative strains: 16-06 and MpR12) from Egyptian fruit bat and Natal multimammate mouse collected in Zambia. Whole-genome sequencing and phylogenetic analysis revealed that the genotypes of bat RVA 16-06 were identical to that of RVA BATp39 strain from the Kenyan fruit bat, which has not yet been characterized. Moreover, all segments of rodent RVA MpR12 were highly divergent and assigned to novel genotypes, but RVA MpR12 was phylogenetically closer to bat RVAs than to other rodent RVAs, indicating a unique evolutionary history. We further investigated the virological properties of the isolated RVAs. In brief, we found that 16-06 entered cells by binding to sialic acids on the cell surface, while MpR12 entered in a sialic acid-independent manner. Experimental inoculation of suckling mice with 16-06 and MpR12 revealed that these RVAs are causative agents of diarrhea. Moreover, 16-06 and MpR12 demonstrated an ability to infect and replicate in a 3D-reconstructed primary human intestinal epithelium with comparable efficiency to the human RVA. Taken together, our results detail the unique genetic and virological features of bat and rodent RVAs and demonstrate the need for further investigation of their zoonotic potential. IMPORTANCE Recent advances in nucleotide sequence detection methods have enabled the detection of RVA genomes from various animals. These studies have discovered multiple divergent RVAs and have resulted in proposals for the genetic classification of novel genotypes. However, most of these RVAs have been identified via dsRNA viral genomes and not from infectious viruses, and their virological properties, such as cell/host tropisms, transmissibility, and pathogenicity, are unclear and remain to be clarified. Here, we successfully isolated RVAs with novel genome constellations from three bats and one rodent in Zambia. In addition to whole-genome sequencing, the isolated RVAs were characterized by glycan-binding affinity, pathogenicity in mice, and infectivity to the human gut using a 3D culture of primary intestinal epithelium. Our study reveals the first virological properties of bat and rodent RVAs with high genetic diversity and unique evolutional history and provides basic knowledge to begin estimating the potential of zoonotic transmission.

Prevalence and diversity of Staphylococcus aureus in the Zambian dairy value chain: A public health concern.

Staphylococcus aureus is an important opportunistic pathogen of both humans and animals. It can cause several diseases, including mastitis, as well as food poisoning by production of heat-stable enterotoxins in food. The aim of this study was to determine the prevalence of S. aureus and the diversity of strains circulating in the Zambian dairy value chain, which have not been studied in detail before. Three provinces were covered by the study (Lusaka, Southern, and Western) and almost 2000 samples along the dairy value chain, covering both the informal and formal market sectors, were taken at two time points (dry and wet season), with a special focus on raw milk. Nearly 300 presumptive S. aureus isolates were confirmed by MALDI-TOF MS and real-time PCR. Raw milk from traditional and smallholder farms was widely contaminated with S. aureus; prevalence was 33-46% depending on the study province. Raw milk from milk collection centres, informal traders, traditional market sellers, and processors were also frequently contaminated with S. aureus. In addition, S. aureus was detected in several milk bucket swabs and nasal and hand swabs of milkers. From industrially processed (heat-treated) milk and dairy products, no S. aureus was isolated. Methicillin-resistant S. aureus (MRSA) were not detected, but around 10% of the S. aureus isolates carried lukS-PV, a marker gene for the virulence factor Pantone-Valentine leucocidin (PVL), which has been associated with severe diseases in human. Molecular typing identified a total of 44 spa types including 13 novel types: t18396, t18397, t18398, t18399, t18400, t18402, t18416, t20459, t20460, t20461, t20462, t20463, and t20464. Furthermore, 12 novel multi-locus sequence types were identified: ST7012, ST7100, ST7101, ST7177, ST7291, ST7304, ST7305, ST7344, ST7596, ST7597, ST7598, and ST7599, of which ST7012, ST7177, and ST7596 fall into the bovine-associated clonal linage CC97. The spa types t084, t267, t355, and the novel type t20464 were common in all three study provinces. The predominant spa type varied depending on the province. Whole genome sequencing (WGS) and core genome multi-locus sequence typing (cgMLST) indicates transmission of strains along the Zambian dairy chain with possible persistence in the chain over time. cgMLST also revealed a very close relatedness between some isolates from milkers and from raw milk or milk buckets. The high prevalence and wide spa type diversity of S. aureus, as well as possible direct or indirect transmission of (potentially highly virulent) S. aureus to humans along the Zambian dairy value chain, are of public health concern, particularly as milk and milk products are often consumed raw by the Zambian population.

Serological and molecular epidemiological study on swine influenza in Zambia.

Influenza A viruses (IAVs) cause highly contagious respiratory diseases in humans and animals. In 2009, a swine-origin pandemic H1N1 IAV, designated A(H1N1)pdm09 virus, spread worldwide, and has since frequently been introduced into pig populations. Since novel reassortant IAVs with pandemic potential may emerge in pigs, surveillance for IAV in pigs is therefore necessary not only for the pig industry but also for public health. However, epidemiological information on IAV infection of pigs in Africa remains sparse. In this study, we collected 246 serum and 605 nasal swab samples from pigs in Zambia during the years 2011-2018. Serological analyses revealed that 49% and 32% of the sera collected in 2011 were positive for hemagglutination-inhibition (HI) and neutralizing antibodies against A(H1N1)pdm09 virus, respectively, whereas less than 5.3% of sera collected during the following period (2012-2018) were positive in both serological tests. The positive rate and the neutralization titres to A(H1N1)pdm09 virus were higher than those to classical swine H1N1 and H1N2 IAVs. On the other hand, the positive rate for swine H3N2 IAV was very low in the pig population in Zambia in 2011-2018 (5.3% and 0% in HI and neutralization tests, respectively). From nasal swab samples, we isolated one H3N2 and eight H1N1 IAV strains with an isolation rate of 1.5%. Phylogenetic analyses of all eight gene segments revealed that the isolated IAVs were closely related to human IAV strains belonging to A(H1N1)pdm09 and seasonal H3N2 lineages. Our findings indicate that reverse zoonotic transmission from humans to pigs occurred during the study period in Zambia and highlight the need for continued surveillance to monitor the status of IAVs circulating in swine populations in Africa.

First report of Mycobacterium bovis in wild chacma baboons (Papio ursinus) at the human-wildlife interface area in Zambia.

Mycobacterium bovis (M. bovis) causes tuberculosis in mammals and is a major public health threat worldwide. While M. bovis has been reported in humans, domestic and wild ruminants at the human-wildlife-livestock interface area in Zambia, there is paucity of information on the role of primates as reservoir hosts. We screened seven wild chacma baboons (Papio ursinus) for tuberculosis at the human-wildlife interface area in Lochinvar National Park in the Kafue Flats, Zambia. Following necropsy, lung tissue and associated lymph nodes with tuberculous-like lesions collected from four adult male baboons were prepared for Mycobacterium culture. The isolates were initially typed using the Mycobacterium tuberculosis complex-discrimination multiplex PCR assay and further characterized by spoligotyping and 26-loci MIRU-VNTR. Mycobacteria were isolated from all four animals and identified as M. bovis by PCR. On Spoligotyping, all isolates belonged to SB 0120 spoligotype, which is similar to what was previously reported in humans, cattle and Kafue lechwe antelopes in Kafue Flats ecosystem. Furthermore, on MIRU-VNTR typing, the baboon isolates clustered with cattle and Kafue lechwe isolates from the same catchment area. This finding intimates probable cross-species transmission of M. bovis in the Kafue Flats ecosystem. Due to the close interaction of baboons and humans at interface areas in Zambia, our results have potential implications for public health. Equally, this finding raises concerns for conservation.

Brucella Seroprevalence and Associated Risk Factors in Occupationally Exposed Humans in Selected Districts of Southern Province, Zambia.

Background: Brucellosis is a neglected debilitating zoonosis widely recognized as an occupational health hazard. The seroprevalence of human anti-Brucella antibodies in high-risk populations, as well as their risk factors, have not been well-documented in Zambia. This study aimed at estimating the Brucella seroprevalence in herdsmen and abattoir workers and assess the associated risk factors. Methods: A cross-sectional seroepidemiological study was carried out between May and December 2020 among abattoir workers and herdsmen in Namwala, Monze and Choma districts of Southern Province in Zambia. Seroprevalence was assessed by indirect enzyme-linked immunosorbent assay (i-ELISA) or competitive enzyme-linked immunosorbent assay (c-ELISA) while a questionnaire was administered to obtain epidemiological data. Results: A total of 153 individuals were recruited in the study. The overall Brucella seroprevalence was 20.3% (95% CI: 14.6-27.5). Seropositivity among herdsmen and abattoir workers was 14.4% (95% CI: 9.2-21.8) and 46.4%, (95% CI: 28.8-65.0), respectively. Comparable seropositive results among districts showed Namwala with 26.9%, which was the highest, seconded by Monze 19.0%, and the least was Choma with 11.36%, seropositivity. The multivariate logistic regression model showed that occupation, age category, and district of residence were predictors of being seropositive to Brucella spp. antibodies. The odds of abattoir workers being seropositive to Brucella antibodies were 8.6 (95% CI: 2.6-28.2) higher than that of herdsmen being the reference group. The odds of age category 17-50 years being seropositive to Brucella antibodies were 7.0 (95% CI: 0.7-72.2) higher than being <16 years as the reference group. The odds of one having attained primary level of education being seropositive to Brucella were 1.3 (95% CI: 0.1-14.7) or secondary level of education were 6.2 (95% CI: 0.5-72.6) or tertiary level of education were 5.1 (95% CI: 0.2, 113.3) higher than that of no level of education as the reference group. Furthermore, the odds of a respondent being seropositive to Brucella antibodies were 4.5 (95% CI: 1.3-15.7) for Namwala and 4.9 (95% CI: 1.1-21.7) for Monze higher than that of Choma as the reference group. Conclusion: Anti-Brucella antibodies are prevalent among herdsmen and abattoir workers in the study areas of Zambia (20.26%), a sign of exposure to Brucella pathogens. Type of profession, age and level of education seem to influence the exposure to Brucella pathogens. This zoonosis should be considered as one of the differential diagnosis in humans presenting intermittent fever, malaria-like signs and general pain in humans.

Attenuated infection by a Pteropine orthoreovirus isolated from an Egyptian fruit bat in Zambia.

BACKGROUND: Pteropine orthoreovirus (PRV) is an emerging bat-borne zoonotic virus that causes severe respiratory illness in humans. Although PRVs have been identified in fruit bats and humans in Australia and Asia, little is known about the prevalence of PRV infection in Africa. Therefore, this study performed an PRV surveillance in fruit bats in Zambia. METHODS: Egyptian fruit bats (Rousettus aegyptiacus, n = 47) and straw-colored fruit bats (Eidolon helvum, n = 33) captured in Zambia in 2017-2018 were screened for PRV infection using RT-PCR and serum neutralization tests. The complete genome sequence of an isolated PRV strain was determined by next generation sequencing and subjected to BLAST and phylogenetic analyses. Replication capacity and pathogenicity of the strain were investigated using Vero E6 cell cultures and BALB/c mice, respectively. RESULTS: An PRV strain, tentatively named Nachunsulwe-57, was isolated from one Egyptian fruit bat. Serological assays demonstrated that 98% of sera (69/70) collected from Egyptian fruit bats (n = 37) and straw-colored fruit bats (n = 33) had neutralizing antibodies against PRV. Genetic analyses revealed that all 10 genome segments of Nachunsulwe-57 were closely related to a bat-derived Kasama strain found in Uganda. Nachunsulwe-57 showed less efficiency in viral growth and lower pathogenicity in mice than another PRV strain, Miyazaki-Bali/2007, isolated from a patient. CONCLUSIONS: A high proportion of Egyptian fruit bats and straw-colored fruit bats were found to be seropositive to PRV in Zambia. Importantly, a new PRV strain (Nachunsulwe-57) was isolated from an Egyptian fruit bat in Zambia, which had relatively weak pathogenicity in mice. Taken together, our findings provide new epidemiological insights about PRV infection in bats and indicate the first isolation of an PRV strain that may have low pathogenicity to humans.

Documenting the absence of bovine brucellosis in dairy cattle herds in the southern region of Malawi and the associated knowledge, attitudes and practices of farmers.

There is paucity of Brucella prevalence data in Malawi. For this reason, a cross-sectional study was conducted, from 06 January 2020 to 27 February 2020, to estimate the seroprevalence of brucellosis in dairy cattle herds amongst smallholder farmers, government and private dairy farms in the southern region. A total of 529 serum samples were screened for anti-Brucella antibodies using the Rose Bengal test (RBT) and a competitive enzyme-linked immunosorbent assay (cELISA). A pre-tested electronic (Epicollect tool, Wellcome Sanger Institute, United Kingdom) questionnaire was administered to 378 smallholder farmers to assess their knowledge, attitudes and practices towards brucellosis. Descriptive statistics were used to analyse the data in Microsoft Excel® and Statistical Package for Social Sciences (SPSS®) version 21. No animal tested positive for presence of anti-Brucella antibodies, indicating 0% prevalence (individual and herd levels). The majority (94.2%; 95% confidence interval [CI]: 91.8-96.5) of smallholder farmers had never heard about brucellosis. Furthermore, assisting during parturition without protective equipment (41.3%; 95% CI: 36.3-46.2) and using bulls for breeding (75%; 95% CI: 70.2-78.9) were amongst the common risk practices that were identified. We could not detect brucellosis in this study that indicates the disease could be very rare or even absent in the dairy cattle herds of the southern region of Malawi. However, further Brucella studies need to be conducted in cattle, small livestock, wildlife and humans to document the true status of brucellosis in the country. Brucellosis surveillance, monitoring, awareness and preventive measures are required to maintain this favourable situation.

Immunization Coverage and Antibody Retention against Rabies in Domestic Dogs in Lusaka District, Zambia.

Rabies remains endemic in Zambia. Despite conducting canine vaccinations in Lusaka district, the vaccination coverage and actual seropositivity in the dog population in Lusaka district are rarely evaluated. This study estimated the seropositivity-based immunization coverage in the owned dog population in Lusaka district using the expanded program on immunization cluster survey method. The time-series trend of neutralizing antibodies against rabies in vaccinated dogs was also evaluated. Of 366 dogs in 200 dog-owning households in Lusaka district, blood samples were collected successfully from 251 dogs. In the sampled dogs, 42.2% (106/251) had an antibody titer ≥0.5 IU/mL. When the 115 dogs whose blood was not collected were assumed to be seronegative, the minimum immunization coverage in Lusaka district's owned dog population was estimated at 29.0% (95% confidence interval: 22.4-35.5). It was also found that a single vaccination with certified vaccines is capable of inducing protective levels of antibodies. In contrast, higher antibody titers were observed in multiple-vaccinated dogs than in single-vaccinated dogs, coupled with the observation of a decline in antibody titer over time. These results suggest the importance of continuous booster immunization to maintain herd immunity and provide useful information to plan mass vaccination against rabies in Zambia.

Serological Evidence of Filovirus Infection in Nonhuman Primates in Zambia.

Ebolaviruses and marburgviruses are filoviruses that are known to cause severe hemorrhagic fever in humans and nonhuman primates (NHPs). While some bat species are suspected to be natural reservoirs of these filoviruses, wild NHPs often act as intermediate hosts for viral transmission to humans. Using an enzyme-linked immunosorbent assay, we screened two NHP species, wild baboons and vervet monkeys captured in Zambia, for their serum IgG antibodies specific to the envelope glycoproteins of filoviruses. From 243 samples tested, 39 NHPs (16%) were found to be seropositive either for ebolaviruses or marburgviruses with endpoint antibody titers ranging from 100 to 25,600. Interestingly, antibodies reactive to Reston virus, which is found only in Asia, were detected in both NHP species. There was a significant difference in the seropositivity for the marburgvirus antigen between the two NHP species, with baboons having a higher positive rate. These results suggest that wild NHPs in Zambia might be nonlethally exposed to these filoviruses, and this emphasizes the need for continuous monitoring of filovirus infection in wild animals to better understand the ecology of filoviruses and to assess potential risks of outbreaks in humans in previously nonendemic countries.

Serologic and molecular evidence for circulation of Crimean-Congo hemorrhagic fever virus in ticks and cattle in Zambia.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonosis with a high case fatality rate in humans. Although the disease is widely found in Africa, Europe, and Asia, the distribution and genetic diversity of CCHF virus (CCHFV) are poorly understood in African countries. To assess the risks of CCHF in Zambia, where CCHF has never been reported, epidemiologic studies in cattle and ticks were conducted. Through an indirect immunofluorescence assay, CCHFV nucleoprotein-specific serum IgG was detected in 8.4% (88/1,047) of cattle. Among 290 Hyalomma ticks, the principal vector of CCHFV, the viral genome was detected in 11 ticks. Phylogenetic analyses of the CCHFV S and M genome segments revealed that one of the detected viruses was a genetic reassortant between African and Asian strains. This study provides compelling evidence for the presence of CCHFV in Zambia and its transmission to vertebrate hosts.

Pigs as a potential source of emerging livestock-associated Staphylococcus aureus in Africa: a systematic review.

OBJECTIVE: To assess the emergence of livestock-associated Staphylococcus aureus including methicillin-resistant S. aureus (MRSA) in the pig and pork production systems in Africa for the past two decades. METHODS: PubMed and African Journals OnLine were searched for relevant primary studies from 2000 to 2019 using standardized key words. In total, 19 eligible articles were included in this review. RESULTS: The prevalence of S. aureus including MRSA ranged from 0% to 55% among live pigs and raw pork, and from 9.4% to 30.8% among pig farm and abattoir workers. Risk factors associated with S. aureus carriage among workers were: male gender, working in an abattoir, and medical-related occupation of a household member. S. aureus and MRSA from pigs and pork production systems in Africa are potentially pathogenic with diverse spa types and clonal complexes, with genes encoding antimicrobial resistance, heavy metal resistance, and virulence factors including secreted and enterotoxins, proteases and immune evasion cluster. The typical livestock-associated S. aureus CC398 and mecC genes were reported in two studies. CONCLUSION: Pigs are a potential source of the emerging livestock-associated S. aureus in Africa. Continued monitoring using a 'One Health' approach is recommended for effective infection prevention and control of these infections in Africa.

Domestic dog demographics and estimates of canine vaccination coverage in a rural area of Zambia for the elimination of rabies.

BACKGROUND: An estimated 75% or more of the human rabies cases in Africa occur in rural settings, which underscores the importance of rabies control in these areas. Understanding dog demographics can help design strategies for rabies control and plan and conduct canine mass vaccination campaigns effectively in African countries. METHODOLOGY/PRINCIPAL FINDINGS: A cross-sectional survey was conducted to investigate domestic dog demographics in Kalambabakali, in the rural Mazabuka District of Zambia. The population of ownerless dogs and the total achievable vaccination coverage among the total dog population was estimated using the capture-recapture-based Bayesian model by conducting a canine mass vaccination campaign. This study revealed that 29% of the domestic dog population was under one year old, and 57.7% of those were under three months old and thus were not eligible for the canine rabies vaccination in Zambia. The population growth was estimated at 15% per annum based on the cross-sectional household survey. The population of ownerless dogs was estimated to be small, with an ownerless-to-owned-dog ratio of 0.01-0.06 in the target zones. The achieved overall vaccination coverage from the first mass vaccination was estimated 19.8-51.6%. This low coverage was principally attributed to the owners' lack of information, unavailability, and dog-handling difficulties. The follow-up mass vaccination campaign achieved an overall coverage of 54.8-76.2%. CONCLUSIONS/SIGNIFICANCE: This paper indicates the potential for controlling canine rabies through mass vaccination in rural Zambia. Rabies education and responsible dog ownership are required to achieve high and sustainable vaccination coverage. Our findings also propose including puppies below three months old in the target population for rabies vaccination and emphasize that securing an annual enforcement of canine mass vaccination that reaches 70% coverage in the dog population is necessary to maintain protective herd immunity.

An African tick flavivirus forming an independent clade exhibits unique exoribonuclease-resistant RNA structures in the genomic 3'-untranslated region.

Tick-borne flaviviruses (TBFVs) infect mammalian hosts through tick bites and can cause various serious illnesses, such as encephalitis and hemorrhagic fevers, both in humans and animals. Despite their importance to public health, there is limited epidemiological information on TBFV infection in Africa. Herein, we report that a novel flavivirus, Mpulungu flavivirus (MPFV), was discovered in a Rhipicephalus muhsamae tick in Zambia. MPFV was found to be genetically related to Ngoye virus detected in ticks in Senegal, and these viruses formed a unique lineage in the genus Flavivirus. Analyses of dinucleotide contents of flaviviruses indicated that MPFV was similar to those of other TBFVs with a typical vertebrate genome signature, suggesting that MPFV may infect vertebrate hosts. Bioinformatic analyses of the secondary structures in the 3'-untranslated regions (UTRs) revealed that MPFV exhibited unique exoribonuclease-resistant RNA (xrRNA) structures. Utilizing biochemical approaches, we clarified that two xrRNA structures of MPFV in the 3'-UTR could prevent exoribonuclease activity. In summary, our findings provide new information regarding the geographical distribution of TBFV and xrRNA structures in the 3'-UTR of flaviviruses.

Lactococcus garvieae isolated from Lake Kariba (Zambia) has low invasive potential in Nile tilapia (Oreochromis niloticus).

The pathogenesis of Lactococcus garvieae (L. garvieae) was assessed in Nile tilapia (Oreochromis niloticus) following administration by two different routes of infection (intraperitoneal versus immersion), using 180 fish divided into three groups. The first group of fish was injected intraperitoneally (IP) with 3 × 105 colony-forming units (cfu) of L. garvieae; the second group was infected by immersion (IMM) into water containing 9.6 × 105  cfu/ml L. garvieae, and in group 3 (Control), the fish were injected IP with sterile normal saline. Mortalities were recorded daily, and on 3, 5, 7, and 13 days post-infection (dpi), liver, kidney, spleen, brain and eyes were sampled. The level of infection between groups was assessed by number of mortalities that occurred, pathology/histopathology of internal organs, bacterial re-isolation and presence of bacteria in situ determined using immunohistochemistry. A significant difference (p < .0001) was observed between L. garvieae re-isolation from tilapia following administration by IP injection and IMM. Similarly, more clinical signs and mortalities (p < .001) were observed in the IP group compared to the IMM group where no mortalities were observed. These findings suggest that L. garvieae has a low invasive potential in Nile tilapia with intact skin/external barriers and highlights the importance of maintaining fish without cuts or abrasions under field conditions.

Mastomys natalensis is a possible natural rodent reservoir for encephalomyocarditis virus.

Encephalomyocarditis virus (EMCV) infects a wide range of hosts and can cause encephalitis, myocarditis, reproductive disorders and diabetes mellitus in selected mammalian species. As for humans, EMCV infection seems to occur by the contact with animals and can cause febrile illnesses in some infected patients. Here we isolated EMCV strain ZM12/14 from a natal multimammate mouse (Mastomys natalensis: M. natalensis) in Zambia. Pairwise sequence similarity of the ZM12/14 P1 region consisting of antigenic capsid proteins showed the highest similarity of nucleotide (80.7 %) and amino acid (96.2%) sequence with EMCV serotype 1 (EMCV-1). Phylogenetic analysis revealed that ZM12/14 clustered into EMCV-1 at the P1 and P3 regions but segregated from known EMCV strains at the P2 region, suggesting a unique evolutionary history. Reverse transcription PCR (RT-PCR) screening and neutralizing antibody assays for EMCV were performed using collected tissues and serum from various rodents (n=179) captured in different areas in Zambia. We detected the EMCV genome in 19 M. natalensis (19/179=10.6 %) and neutralizing antibody for EMCV in 33 M. natalensis (33/179=18.4 %). However, we did not detect either the genome or neutralizing antibody in other rodent species. High neutralizing antibody litres (≧320) were observed in both RT-PCR-negative and -positive animals. Inoculation of ZM12/14 caused asymptomatic persistent infection in BALB/c mice with high antibody titres and high viral loads in some organs, consistent with the above epidemiological results. This study is the first report of the isolation of EMCV in Zambia, suggesting that M. natalensis may play a role as a natural reservoir of infection.

Estimating the basic reproduction number for the 2015 bubonic plague outbreak in Nyimba district of Eastern Zambia.

BACKGROUND: Plague is a re-emerging flea-borne infectious disease of global importance and in recent years, Zambia has periodically experienced increased incidence of outbreaks of this disease. However, there are currently no studies in the country that provide a quantitative assessment of the ability of the disease to spread during these outbreaks. This limits our understanding of the epidemiology of the disease especially for planning and implementing quantifiable and cost-effective control measures. To fill this gap, the basic reproduction number, R0, for bubonic plague was estimated in this study, using data from the 2015 Nyimba district outbreak, in the Eastern province of Zambia. R0 is the average number of secondary infections arising from a single infectious individual during their infectious period in an entirely susceptible population. METHODOLOGY/PRINCIPAL FINDINGS: Secondary epidemic data for the most recent 2015 Nyimba district bubonic plague outbreak in Zambia was analyzed. R0 was estimated as a function of the average epidemic doubling time based on the initial exponential growth rate of the outbreak and the average infectious period for bubonic plague. R0 was estimated to range between 1.5599 [95% CI: 1.382-1.7378] and 1.9332 [95% CI: 1.6366-2.2297], with average of 1.7465 [95% CI: 1.5093-1.9838]. Further, an SIR deterministic mathematical model was derived for this infection and this estimated R0 to be between 1.4 to 1.5, which was within the range estimated above. CONCLUSIONS/SIGNIFICANCE: This estimated R0 for bubonic plague is an indication that each bubonic plague case can typically give rise to almost two new cases during these outbreaks. This R0 estimate can now be used to quantitatively analyze and plan measurable interventions against future plague outbreaks in Zambia.