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.

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.

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.

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.

Characterization of mammalian orthoreoviruses isolated from faeces of pigs in Zambia.

Mammalian orthoreovirus (MRV) has been identified in humans, livestock and wild animals; this wide host range allows individual MRV to transmit into multiple species. Although several interspecies transmission and genetic reassortment events of MRVs among humans, livestock and wildlife have been reported, the genetic diversity and geographic distribution of MRVs in Africa are poorly understood. In this study, we report the first isolation and characterization of MRVs circulating in a pig population in Zambia. In our screening, MRV genomes were detected in 19.7 % (29/147) of faecal samples collected from pigs by reverse transcription PCR. Three infectious MRV strains (MRV-85, MRV-96 and MRV-117) were successfully isolated, and their complete genomes were sequenced. Recombination analyses based on the complete genome sequences of the isolated MRVs demonstrated that MRV-96 shared the S3 segment with a different MRV isolated from bats, and that the L1 and M3 segments of MRV-117 originated from bat and human MRVs, respectively. Our results suggest that the isolated MRVs emerged through genetic reassortment events with interspecies transmission. Given the lack of information regarding MRVs in Africa, further surveillance of MRVs circulating among humans, domestic animals and wildlife is required to assess potential risk for humans and animals.

Prevalence and genotypic characterization of Giardia duodenalis isolates from asymptomatic school-going children in Lusaka, Zambia.

Giardia duodenalis is one of the most common causes of diarrhea in humans with about 250-300 million cases per year. It is considered to be a species complex comprising of eight genetic assemblages (A to H), with assemblages A and B being the major causes of human infections. In this study we carried out genotypic characterization of G. duodenalis isolates detected in asymptomatic school-going children aged 3-16 years. Between May and September 2017, a total of 329 fecal samples were collected from school-going children from Chawama compound of Lusaka City and were screened for Giardia by microscopic examination. All microscopically positive fecal samples were analyzed by semi-nested polymerase chain reaction (PCR) targeting the glutamate dehydrogenase (gdh) gene. Genotyping of amplified PCR products was conducted by restriction fragment length polymorphism (RFLP) and DNA sequence analysis. Microscopically, Giardia was found in 10% (33/329) of fecal samples. The PCR-RFLP analysis of the gdh gene revealed assemblages A and B in 27.3% (9/33) and 72.7% (24/33), respectively. Furthermore, analysis with restriction enzymes identified sub-assemblages AII (27.3%, 9/33), BIII (12.1%, 4/33), BIV (51.5%, 17/33) and mixed infections of BIII and BIV (9.1%, 3/33). Phylogenetic analysis showed the clustering of 27.6% (8/29) and 72.4% (21/29) of Zambian Giardia gdh gene sequences into assemblages A and B, respectively. This study has revealed the presence of both assemblage A and B and that spread of G. duodenalis in school-going children appears to be mostly through anthroponotic transmission. To our knowledge, this is the first report of genotypic characterization of G. duodenalis identified in Zambia.

Genetic and Biological Diversity of Porcine Sapeloviruses Prevailing in Zambia.

Porcine sapelovirus (PSV) has been detected worldwide in pig populations. Although PSV causes various symptoms such as encephalomyelitis, diarrhea, and pneumonia in pigs, the economic impact of PSV infection remains to be determined. However, information on the distribution and genetic diversity of PSV is quite limited, particularly in Africa. In this study, we investigated the prevalence of PSV infection in Zambia and characterized the isolated PSVs genetically and biologically. We screened 147 fecal samples collected in 2018 and found that the prevalences of PSV infection in suckling pigs and fattening pigs were high (36.2% and 94.0%, respectively). Phylogenetic analyses revealed that the Zambian PSVs were divided into three different lineages (Lineages 1-3) in the clade consisting of Chinese strains. The Zambian PSVs belonging to Lineages 2 and 3 replicated more efficiently than those belonging to Lineage 1 in Vero E6 and BHK cells. Bioinformatic analyses revealed that genetic recombination events had occurred and the recombination breakpoints were located in the L and 2A genes. Our results indicated that at least two biologically distinct PSVs could be circulating in the Zambian pig population and that genetic recombination played a role in the evolution of PSVs.

Molecular detection and characterization of genotype 1 bovine leukemia virus from beef cattle in the traditional sector in Zambia.

Whilst bovine leukemia virus (BLV) causes considerable economic losses to the dairy industry worldwide, information on its molecular epidemiology and economic impact in beef cattle is limited. Here, blood from 880 animals from Zambia's major cattle-rearing provinces was screened for BLV by nested PCR. Positive pools were sequenced and phylogenetically analyzed. The estimated pooled prevalence was 2.1%. All strains belonged to genotype 1 and formed a distinct phylogenetic cluster. The study suggests circulation of genotype 1 BLV in beef cattle in these regions. This is the first report on molecular detection and characterization of BLV from beef cattle in Africa.

Seroprevalence of Rift Valley fever in cattle of smallholder farmers in Kwilu Province in the Democratic Republic of Congo.

Rift Valley fever (RVF) is a zoonotic mosquito-borne disease caused by RVF virus (RVFV) that causes abortions and high mortalities in livestock and is also associated with acute and fatal disease in humans. In the Democratic Republic of Congo (DRC), information on the epidemiology of RVF is limited, particularly among cattle reared by smallholder farmers. This cross-sectional study was conducted to investigate the seroprevalence of RVF in cattle raised by smallholder farmers in Kwilu Province of DRC, which has not yet reported an RVF epidemic. A total of 677 cattle sera were collected from four territories and tested for anti-RVFV antibodies using immunofluorescent assay and enzyme-linked immunosorbent assay. The overall seroprevalence of anti-RVFV IgG was 6.5% (44/677) (95% CI 4.81-8.7). There was a statistically significant difference in the seroprevalence among the territories (χ2 = 28.79, p < 0.001). Territory seroprevalences were as follows: Idiofa 14.08% (95% CI 9.78-19.76), Bulungu 4.14% (95% CI 1.83-8.68), Gungu 3.21% (95% CI 1.41-6.78), and Masi-Manimba 1.19% (95% CI 0.06-7.37). Seroprevalence differed significantly among age categories (p = 0.0017) and ecosystem (p < 0.001). The seroprevalence of animals aged between 1 and 2 years was 20.0% (95% CI 8.4-39.13) and was higher than group aged <1 year, between 2 and 3 years, and > 3 years. Forest area (18.92% (95% CI 12.35-27.7)) had higher seropositivity than savannah area (4.06% (95% CI 2.65-6.12)). Sex difference was not significant (χ2 = 0.14, p = 0.704). These findings indicate that cattle in Kwilu Province had been exposed to RVFV, which represents a significant risk for both livestock and human health.

Publisher Correction: Discovery and genetic characterization of diverse smacoviruses in Zambian non-human primates.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Serological evidence of Zika virus infection in non-human primates in Zambia.

Zika virus (ZIKV) circulation occurs between non-human primates (NHPs) in a sylvatic transmission cycle. To investigate evidence of flavivirus infection in NHPs in Zambia, we performed a plaque reduction neutralization test (PRNT) to quantify neutralizing antibodies. PRNT revealed that sera from NHPs (African green monkeys and baboons) exhibited neutralizing activity against ZIKV (34.4%; 33/96), whereas a PRNT for yellow fever virus using NHP sera showed no neutralization activity. ZIKV genomic RNA was not detected in splenic tissues from NHPs, suggesting that the presence of anti-ZIKV neutralizing antibodies represented resolved infections. Our evidence suggests that ZIKV is maintained in NHP reservoirs in Zambia.

Molecular detection of Rickettsia felis in dogs, rodents and cat fleas in Zambia.

BACKGROUND: Flea-borne spotted fever is a zoonosis caused by Rickettsia felis, a Gram-negative obligate intracellular bacterium. The disease has a worldwide distribution including western and eastern sub-Saharan Africa where it is associated with febrile illness in humans. However, epidemiology and the public health risks it poses remain neglected especially in developing countries including Zambia. While Ctenocephalides felis (cat fleas) has been suggested to be the main vector, other arthropods including mosquitoes have been implicated in transmission and maintenance of the pathogen; however, their role in the epidemiological cycle remains to be elucidated. Thus, the aim of this study was to detect and characterize R. felis from animal hosts and blood-sucking arthropod vectors in Zambia. METHODS: Dog blood and rodent tissue samples as well as cat fleas and mosquitoes were collected from various areas in Zambia. DNA was extracted and screened by polymerase chain reaction (PCR) targeting genus Rickettsia and amplicons subjected to sequence analysis. Positive samples were further subjected to R. felis-specific real-time quantitative polymerase chain reactions. RESULTS: Rickettsia felis was detected in 4.7% (7/150) of dog blood samples and in 11.3% (12/106) of rodent tissue samples tested by PCR; this species was also detected in 3.7% (2/53) of cat fleas infesting dogs, co-infected with Rickettsia asembonensis. Furthermore, 37.7% (20/53) of cat flea samples tested positive for R. asembonensis, a member of spotted fever group rickettsiae of unknown pathogenicity. All the mosquitoes tested (n = 190 pools) were negative for Rickettsia spp. CONCLUSIONS: These observations suggest that R. felis is circulating among domestic dogs and cat fleas as well as rodents in Zambia, posing a potential public health risk to humans. This is because R. felis, a known human pathogen is present in hosts and vectors sharing habitat with humans.

Discovery and genetic characterization of diverse smacoviruses in Zambian non-human primates.

The Smacoviridae has recently been classified as a family of small circular single-stranded DNA viruses. An increasing number of smacovirus genomes have been identified exclusively in faecal matter of various vertebrate species and from insect body parts. However, the genetic diversity and host range of smacoviruses remains to be fully elucidated. Herein, we report the genetic characterization of eleven circular replication-associated protein (Rep) encoding single-stranded (CRESS) DNA viruses detected in the faeces of Zambian non-human primates. Based on pairwise genome-wide and amino acid identities with reference smacovirus species, ten of the identified CRESS DNA viruses are assigned to the genera Porprismacovirus and Huchismacovirus of the family Smacoviridae, which bidirectionally encode two major open reading frames (ORFs): Rep and capsid protein (CP) characteristic of a type IV genome organization. The remaining unclassified CRESS DNA virus was related to smacoviruses but possessed a genome harbouring a unidirectionally oriented CP and Rep, assigned as a type V genome organization. Moreover, phylogenetic and recombination analyses provided evidence for recombination events encompassing the 3'-end of the Rep ORF in the unclassified CRESS DNA virus. Our findings increase the knowledge of the known genetic diversity of smacoviruses and highlight African non-human primates as carrier animals.

First genetic detection and characterization of canine parvovirus from diarrheic dogs in Zambia.

Although canine parvovirus (CPV) causes severe gastroenteritis in dogs globally, information on the molecular epidemiology of the virus is lacking in many African countries. Here, 32 fecal samples collected from diarrheic dogs in Zambia were tested for CPV infection using molecular assays. CPV was detected in 23 samples (71.9%). Genetic characterization revealed the predominance of CPV-2c (91.3%). This finding differs from previous reports in Africa, which indicated that CPV-2a and CPV-2b were most prevalent. Phylogenetically, most Zambian CPVs formed a distinct cluster. This is the first report on the molecular characterization of CPV in Zambia.

Genetic characterisation of African swine fever virus from 2017 outbreaks in Zambia: Identification of p72 genotype II variants in domestic pigs.

African swine fever (ASF) is a contagious haemorrhagic disease associated with causing heavy economic losses to the swine industry in many African countries. In 2017, Zambia experienced ASF outbreaks in Mbala District (Northern province) and for the first time in Isoka and Chinsali districts (Muchinga province). Meanwhile, another outbreak was observed in Chipata District (Eastern province). Genetic analysis of part of the B646L gene, E183L gene, CP204L gene and the central variable region of the B602L gene of ASF virus (ASFV) associated with the outbreaks in Mbala and Chipata districts was conducted. The results revealed that the ASFV detected in Mbala District was highly similar to that of the Georgia 2007/1 isolate across all the genome regions analysed. In contrast, while showing close relationship with the Georgia 2007/1 virus in the B646L gene, the ASFV detected in Chipata District showed remarkable genetic variation in the rest of the genes analysed. These results suggest that the Georgia 2007/1-like virus could be more diverse than what was previously thought, underscoring the need of continued surveillance and monitoring of ASFVs within the south-eastern African region to better understand their epidemiology and the relationships between outbreaks and their possible origin.

Identification of group A rotaviruses from Zambian fruit bats provides evidence for long-distance dispersal events in Africa.

Group A rotavirus (RVA) is a major cause of diarrhea in children worldwide. Although RVA infects many animals, little is known about RVA in bats. The present study investigated the genetic diversity of RVA in Zambian bats. We identified RVA from two straw-colored fruit bats (Eidolon helvum) and an Egyptian fruit bat (Rousettus aegyptiacus), and analyzed the genome sequences of these strains. Genome segments of the RVA strains from Zambian E. helvum showed 97%-99% nucleotide sequence identity with those of other RVA strains from E. helvum in Cameroon, which is 2800 km from the sampling locations. These findings suggest that migratory straw-colored fruit bat species, distributed across sub-Saharan Africa, have the potential to disseminate RVA across long distances. By contrast, the RVA strain from Zambian R. aegyptiacus carried highly divergent NSP2 and NSP4 genes, leading us to propose novel genotypes N21 and E27, respectively. Notably, this RVA strain also shared the same genotype for VP6 and NSP3 with the RVA strains from Zambian E. helvum, suggesting interspecies transmission and genetic reassortment may have occurred between these two bat species in the past. Our study has important implications for RVA dispersal in bat populations, and expands our knowledge of the ecology, diversity and evolutionary relationships of RVA.

Discovery of Mwinilunga alphavirus: A novel alphavirus in Culex mosquitoes in Zambia.

Mosquito-borne alphaviruses are disseminated globally and cause febrile illness in humans and animals. Since the prevalence and diversity of alphaviruses has not been previously investigated in Zambia, reverse transcription PCR was employed as a broad-spectrum approach for the detection of alphaviruses in mosquitoes. From 552 mosquito pools, a novel alphavirus, tentatively named Mwinilunga alphavirus (MWAV), was discovered from a single Culex quinquefasciatus mosquito pool. The full genome of MWAV was subsequently determined, and pairwise comparisons demonstrated that MWAV represented a new alphavirus species. Phylogenetic analyses and a linear discriminant analysis based on the dinucleotide ratios in various virus sequences indicated that MWAV is related to a mosquito-specific alphavirus distinct from other known mosquito-borne alphaviruses due to its inability to replicate in vertebrate cell lines. Further analyses of these novel alphaviruses will help to facilitate a greater understanding of the molecular determinants of host range restriction and the evolutionary relationships of alphaviruses.

Characterization of field infectious bursal disease viruses in Zambia: evidence of co-circulation of multiple genotypes with predominance of very virulent strains.

Infectious bursal disease (IBD) is a highly contagious, immunosuppressive disease of chickens and causes substantial economic losses to the poultry industry globally. This study investigated the genetic characteristics and pathological lesions induced by IBD viruses (IBDVs) that were associated with 60 suspected outbreaks in chickens during 2015-2016 in Lusaka Province, Zambia. Nucleotide sequences of VP2 hypervariable region (VP2-HVR) (n = 38) and part of VP1 (n = 37) of Zambian IBDVs were phylogenetically analysed. Phylogenetic analysis of the VP2-HVR and VP1 revealed that most viruses (n = 31 of each genome segment) clustered with the very virulent (vv) strains. The rest of the viruses clustered with the classical strains, with two of the viruses being closely related to attenuated vaccine isolates. Two of the viruses that belonged to the vv genotype had a unique amino acid (aa) substitution Q324L whereas one virus had two unique changes, N280S and E300A in the VP2-HVR aa sequence. Although Zambian strains with a vv genotype possessed virulence marker aa within VP1 at 145T, 146D and 147N, two viruses showed unique substitutions, with one virus having 147T while the other had 147H. Pathologically, it was noted that only viruses with a vv genotype appeared to be associated with inducing pathological lesions in non-lymphoid organs (proventriculus and gizzard). Whilst documenting for the first time the presence of classical virulent IBDVs, this study demonstrates the involvement of multiple genotypes, with predominance of vvIBDVs in the epidemiology of IBD in Zambia.

First molecular detection and genetic characterization of Coxiella burnetii in Zambian dogs and rodents.

Coxiella burnetii, the causative agent of Q fever, is a zoonotic pathogen associated with sylvatic or domestic transmission cycles, with rodents being suspected to link the two transmission cycles. Infection and subsequent disease in humans has historically been associated with contact with infected livestock, especially sheep. However, recently there have been reports of Q fever outbreaks associated with contact with infected rodents and dogs. Studies exploring the potential role of these animal hosts in the epidemiology of Q fever in many developing countries in Africa are very limited. This study aimed to determine the potential role of rodents and dogs in the epidemiological cycle of C. burnetti in Zambia. Using pathogen-specific polymerase chain reaction assays targeting the 16S rRNA gene, C. burnetii was detected for the first time in 45% of rodents (9/20), in one shrew and in 10% of domestic dogs (15/150) screened in Zambia. Phylogenetic characterization of six samples based on the isocitrate synthase gene revealed that the strains were similar to a group of isolates from chronic human Q fever patients, goats and rodents reported in multiple continents. Considering the close proximity of domestic dogs and rodents to humans, especially in resource-limited communities, the presence of C. burnetii in these animals could be of significant public health importance. It is thus important to determine the burden of Q fever in humans in such resource-limited communities where there is close contact between humans, rodents and dogs.

Molecular detection and characterization of zoonotic Anaplasma species in domestic dogs in Lusaka, Zambia.

Although tick-borne pathogens, Anaplasma platys and Anaplasma phagocytophilum are recognized as zoonotic agents associated with appreciable morbidity and mortality in dogs and humans worldwide, there is limited information on these infections in many African countries, including Zambia. The purpose of this study was to detect, identify and phylogenetically characterize Anaplasma species from dogs in Chilanga District in Lusaka Province, Zambia. A total of 301 blood samples were collected from apparently healthy and semi-confined dogs. Initial screening by polymerase chain reaction with specific primers targeting the 16S rRNA gene of Anaplasma species revealed that 9% (27/301) of our samples were positive. Subsequent sequence and phylogenetic analysis of a longer fragment of the 16S rRNA and citrate synthase (gltA) genes of four positive samples showed the presence of A. platys and an Anaplasma species, which was closely related to those detected in dogs in South Africa. This is the first report on molecular identification and characterization of canine-associated zoonotic Anaplasma species in Zambia.