Autochthonous Leishmania infantum in Dogs, Zambia, 2021.

Leishmaniases are neglected tropical diseases of humans and animals. We detected Leishmania infantum in 3 mixed-breed dogs in Zambia that had no travel history outside the country. Our findings suggest presence of and probable emergence of leishmaniasis in Zambia, indicating the need for physicians and veterinarians to consider the disease during diagnosis.

Detection of B.1.351 SARS-CoV-2 Variant Strain - Zambia, December 2020.

The first laboratory-confirmed cases of coronavirus disease 2019 (COVID-19), the illness caused by SARS-CoV-2, in Zambia were detected in March 2020 (1). Beginning in July, the number of confirmed cases began to increase rapidly, first peaking during July-August, and then declining in September and October (Figure). After 3 months of relatively low case counts, COVID-19 cases began rapidly rising throughout the country in mid-December. On December 18, 2020, South Africa published the genome of a SARS-CoV-2 variant strain with several mutations that affect the spike protein (2). The variant included a mutation (N501Y) associated with increased transmissibility.†,§ SARS-CoV-2 lineages with this mutation have rapidly expanded geographically.¶,** The variant strain (PANGO [Phylogenetic Assignment of Named Global Outbreak] lineage B.1.351††) was first detected in the Eastern Cape Province of South Africa from specimens collected in early August, spread within South Africa, and appears to have displaced the majority of other SARS-CoV-2 lineages circulating in that country (2). As of January 10, 2021, eight countries had reported cases with the B.1.351 variant. In Zambia, the average number of daily confirmed COVID-19 cases increased 16-fold, from 44 cases during December 1-10 to 700 during January 1-10, after detection of the B.1.351 variant in specimens collected during December 16-23. Zambia is a southern African country that shares substantial commerce and tourism linkages with South Africa, which might have contributed to the transmission of the B.1.351 variant between the two countries.

Prevalence and genetic diversity of Shibuyunji virus, a novel tick-borne phlebovirus identified in Zambia.

Tick-borne pathogens are an emerging public health threat worldwide. However, information on tick-borne viruses is scanty in sub-Saharan Africa. Here, by RT-PCR, 363 ticks (Amblyomma, Hyalomma and Rhipicephalus) in the Namwala and Livingstone districts of Zambia were screened for tick-borne phleboviruses (TBPVs). TBPVs (L gene) were detected in 19 (5.2%) Rhipicephalus ticks in Namwala. All the detected TBPVs were Shibuyunji viruses. Phylogenetically, they were closely related to American dog tick phlebovirus. This study highlights the possible role of Rhipicephalus ticks as the main host of Shibuyunji virus and suggests that these viruses may be present outside the area where they were initially discovered.

West Nile Virus in Farmed Crocodiles, Zambia, 2019.

We detected West Nile virus (WNV) nucleic acid in crocodiles (Crocodylus niloticus) in Zambia. Phylogenetically, the virus belonged to lineage 1a, which is predominant in the Northern Hemisphere. These data provide evidence that WNV is circulating in crocodiles in Africa and increases the risk for animal and human transmission.

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.

Detection of Human Adenovirus and Rotavirus in Wastewater in Lusaka, Zambia: Demonstrating the Utility of Environmental Surveillance for the Community.

Enteric infections due to viral pathogens are a major public health concern. Detecting the risk areas requires a strong surveillance system for pathogenic viruses in sources such as wastewater. Towards building an environmental surveillance system in Zambia, we aimed to identify group A rotavirus (RVA) and human adenovirus (HAdV) in wastewater. Convenient sampling was conducted at four study sites every Tuesday for five consecutive weeks. The research team focused on three different methods of viral concentration to determine the suitability in terms of cost and applicability for a regular surveillance system: the bag-mediated filtration system (BMFS), polyethylene glycol-based (PEG) precipitation, and skimmed milk (SM) flocculation. We screened 20 wastewater samples for HAdV and RVA using quantitative polymerase chain reaction (qPCR) and conventional polymerase chain reaction (cPCR). Of the 20 samples tested using qPCR, 18/20 (90%) tested positive for HAdV and 14/20 (70%) tested positive for RVA. For the genetic sequencing, qPCR positives were subjected to cPCR, of which 12 positives were successfully amplified. The human adenovirus was identified with a nucleotide identity range of 98.48% to 99.53% compared with the reference genome from GenBank. The BMFS and SM flocculation were the most consistent viral concentration methods for HAdV and RVA, respectively. A statistical analysis of the positives showed that viral positivity differed by site (p < 0.001). SM and PEG may be the most appropriate options in resource-limited settings such as Zambia due to the lower costs associated with these concentration methods. The demonstration of HAdV and RVA detection in wastewater suggests the presence of the pathogens in the communities under study and the need to establish a routine wastewater surveillance system for the identification of pathogens.

Challenges of controlling contagious bovine pleuropneumonia in sub-Saharan Africa: a Zambian perspective.

Contagious bovine pleuropneumonia (CBPP) is a disease of economic importance that is widely distributed in sub-Saharan African and contributes significantly to cattle morbidity and mortality. Control of CBPP offers a number of challenges as a result many developing countries in Africa are still struggling with this disease. In this study, we look at the challenges encountered in CBPP control in sub-Saharan Africa from the Zambian perspective. In conducting this study, we reviewed scientific literature and reports from the Ministry of Agriculture and Livestock and related animal institutions, and also made interviews with experts and key government officials involved in CBPP control in Zambia. Among the challenges identified for the successful control of CBPP were as follows: failure in the delivery of veterinary services, lack of a cattle identification system, natural phenomenon, livestock husbandry systems in the traditional sector, human movements, traditional practices among cattle farmers and cattle marketing systems. It was seen that the epidemiology of CBPP in Zambia is influenced by both ecological and anthological factors. Therefore, design and implementation of any control or eradication programme should be area/regional-dependent taking into account the different factors influencing disease transmission and maintenance.

High throughput analysis of MHC class I and class II diversity of Zambian indigenous cattle populations.

The classical MHC class I and class II molecules play key roles in determining the antigenic-specificity of CD8+ and CD4+ T-cell responses-as such characterisation of the repertoire of MHCI and MHCII allelic diversity is fundamental to our ability to understand, and potentially, exploit how genetic diversity influences the outcome of immune responses. Cattle remain one of the most economically livestock species, with particular importance to many small-holder farmers in low-and-middle income countries (LMICs). However, our knowledge of MHC (BoLA) diversity in the indigenous breeds that form the mainstay of cattle populations in many LMICs remains very limited. In this study we develop a MiSeq-based platform to enable the rapid analysis of BoLA-DQA and BoLA-DQB, and combine this with similar platforms to analyse BoLA-I and BoLA-DRB repertoires, to study a large cohort of cattle (~800 animals) representing the 3 major indigenous breeds (Angoni, Barotse, Tonga) in Zambia. The data presented confirms the capacity of this high-throughput and high-resolution approach to provide a full characterisation of the MHCI-MHCII genotypes of cattle for which little previous MHC sequence data has been obtained. The cattle in Zambia were found to express a diverse range of MHCI, MHCII and extended MHCI-MHCII haplotypes. The combined MHCI-MHCII genotyping now possible opens new opportunities to rapidly expand our knowledge of MHC diversity in cattle that could find applications in a related translational disciplines such as vaccine development.

Molecular detection and characterization of Anaplasma spp. in cattle and sable antelope from Lusaka and North-Western provinces of Zambia.

Rickettsiales of the genus Anaplasma are globally distributed tick-borne pathogens of animals and humans with complex epidemiological cycles. Anaplasmosis is an important livestock disease in Zambia but its epidemiological information is inadequate. This study aimed to detect and characterize the species of Anaplasma present in domestic and wild ruminants in Zambia with a focus on the infection risk posed by the translocation of sable antelope (Hippotragus niger) from North-Western Province to Lusaka Province. Archived DNA samples (n = 100) extracted from whole blood (sable n = 47, cattle n = 53) were screened for Anaplasmataceae using 16S rRNA partial gene amplification followed by species confirmation using phylogenetic analysis. Out of the 100 samples, Anaplasma species were detected in 7% (4/57) of the cattle and 24% (10/43) of the sable antelope samples. Of the 14 positive samples, five were determined to be A. marginale (four from cattle and one from sable), seven were A. ovis (sable) and two were A. platys (sable). Phylogenetic analysis of the 16S rRNA partial gene sequences revealed genetic proximity between A. ovis and A. marginale, regardless of host. The detection of Anaplasma in wildlife in Zambia shows the risk of transmission of Anaplasma species associated with wildlife translocation.

Prevalence and Molecular Identification of Schistosoma haematobium among Children in Lusaka and Siavonga Districts, Zambia.

Schistosomiasis remains a public health concern in Zambia. Urinary schistosomiasis caused by Schistosoma haematobium is the most widely distributed infection. The aim of the current study was to determine the prevalence and risk factors of urinary schistosomiasis and identify the strain of S. haematobium among children in the Siavonga and Lusaka districts in Zambia. Urine samples were collected from 421 primary school children and S. haematobium eggs were examined under light microscopy. A semi-structured questionnaire was used to obtain information on the socio-demographic characteristics and the potential risk factors for urinary schistosomiasis. DNA of the parasite eggs was extracted from urine samples and the internal transcribed spacer gene was amplified, sequenced and phylogenetically analysed. The overall prevalence of S. haematobium was 9.7% (41/421) (95% CI: 7.16-13.08), male participants made up 6.2% (26/232) (95% CI: 4.15-9.03), having a higher burden of disease than female participants who made up 3.5% (15/421) (95% CI: 2.01-5.94). The age group of 11-15 years had the highest overall prevalence of 8.3% (35/421) (5.94-11.48). Participants that did not go fishing were 0.008 times less likely to be positive for schistosomiasis while participants whose urine was blood-tinged or cloudy on physical examination and those that lived close to water bodies were 9.98 and 11.66 times more likely to test positive for schistosomiasis, respectively. A phylogenetic tree analysis indicated that S. haematobium isolates were closely related to pure S. haematobium from Zimbabwe and hybrids of S. haematobium × S. bovis from Benin, Senegal and Malawi. The current study shows that urinary schistosomiasis is endemic in the study areas and is associated with water contact, and S. haematobium isolated is closely related to hybrids of S. bovis × S. haematobium strain, indicating the zoonotic potential of this parasite.

Detection of Babesia spp. in free-ranging Pukus, Kobus vardonii, on a game ranch in Zambia.

Babesia spp. were detected from 4 asymptomatic pukus captured on a game ranch in central Zambia in October 2008. Blood smears were examined in 4 species of aymptomatic free-ranging antelopes, namely the puku (Kobus vordanii), reedbuck (Redunca arundinum), bushbuck (Tragelaphus sylvaticus), and kudu (Tragelaphus strepsiceros), and showed the presence of Babesia parasites only in the puku. In the puku, the prevalence of babesiosis was estimated at 33.3% (n = 12), while the overall prevalence in all examined animals was 8.5% (n = 47). The parasites showed morphological characteristics of paired ring-like stages with the length varying between 1.61 µm and 3.02 µm (mean = 2.12 µm, n = 27; SD = 0.76 µm). Both the infected and non-infected pukus showed good body condition scores (BCS), while the dominant tick species detected from all animals were Rhipicephalus appendiculatus, Rhipicephalus spp., and Boophilus spp. To our knowledge this is the first report of Babesia spp. infection in pukus in Zambia. These findings suggest that wildlife could play an important role in the epidemiology of babesiosis in Zambia.

Comparison of complement fixation test, competitive ELISA and LppQ ELISA with post-mortem findings in the diagnosis of contagious bovine pleuropneumonia (CBPP).

The complement fixation test (CFT), the c-ELISA and an indirect LppQ ELISA were compared to post-mortem (PM) inspection for the diagnosis of contagious bovine pleuropneumonia (CBPP). Sera from 797 cattle in the CBPP affected area of Kazungula, Zambia and 202 sera from Lusaka, Zambia, a CBPP-free area were used. The clinical history of CBPP was recorded and all the cattle from Kazungula were slaughtered and PM inspections conducted. The prevalence of CBPP in Kazungula was 67.5% (95%CI 67.2%, 70.8%), 52.6% (95%CI 49.2%, 56.2%), 59.0% (95%CI 55.5%, 62.4%) and 44.4% (95%CI 41.0%, 47.9%) using PM inspection, CFT, c-ELISA and LppQ ELISA, respectively. Three of the 202 negative control animals tested positive on the c-ELISA although they were from a known CBPP negative zone. In this study, the c-ELISA was more sensitive in detecting cattle with lesions in the chronic stage than any other test whilst the CFT detected more during the onset stage. No single serological test could detect all stages of CBPP infection, therefore the use of more than one test is advised.

Molecular Detection and Characterization of Rickettsia Species in Ixodid Ticks Collected From Cattle in Southern Zambia.

Tick-borne zoonotic pathogens are increasingly becoming important across the world. In sub-Saharan Africa, tick-borne pathogens identified include viruses, bacteria and protozoa, with Rickettsia being the most frequently reported. This study was conducted to screen and identify Rickettsia species in ticks (Family Ixodidae) infesting livestock in selected districts of southern Zambia. A total of 236 ticks from three different genera (Amblyomma, Hyalomma, and Rhipicephalus) were collected over 14 months (May 2018-July 2019) and were subsequently screened for the presence of Rickettsia pathogens based on PCR amplification targeting the outer membrane protein B (ompB). An overall Rickettsia prevalence of 18.6% (44/236) was recorded. Multi-locus sequencing and phylogenetic characterization based on the ompB, ompA, 16S rRNA and citrate synthase (gltA) genes revealed the presence of Rickettsia africae (R. africae), R. aeschlimannii-like species and unidentified Rickettsia species. While R. aeschlimannii-like species are being reported for the first time in Zambia, R. africae has been reported previously, with our results showing a wider distribution of the bacteria in the country. Our study reveals the potential risk of human infection by zoonotic Rickettsia species and highlights the need for increased awareness of these infections in Zambia's public health systems.

Rickettsia lusitaniae in Ornithodoros Porcinus Ticks, Zambia.

Rickettsial pathogens are amongst the emerging and re-emerging vector-borne zoonoses of public health importance. Though traditionally considered to be transmitted by ixodid ticks, the role of argasid ticks as vectors of these pathogens is increasingly being recognized. While bat-feeding (Ornithodoros faini) and chicken-feeding (Argas walkerae) argasid ticks have been shown to harbor Rickettsia pathogens in Zambia, there are currently no reports of Rickettsia infection in southern Africa from warthog-feeding (Phacochoerus africanus) soft ticks, particularly Ornithodoros moubata and Ornithodoros porcinus. Our study sought to expand on the existing knowledge on the role of soft ticks in the epidemiology of Rickettsia species through screening for Rickettsia pathogens in warthog burrow-dwelling soft ticks from two national parks in Zambia. The tick species from which Rickettsia were detected in this study were identified as Ornithodoros porcinus, and an overall minimal Rickettsia infection rate of 19.8% (32/162) was observed. All of the sequenced Rickettsia were identified as Rickettsia lusitaniae based on nucleotide sequence similarity and phylogenetic analysis of the citrate synthase (gltA) and 17kDa common antigen (htrA) genes. Utilizing all of the gltA (n = 10) and htrA (n = 12) nucleotide sequences obtained in this study, BLAST analysis showed 100% nucleotide similarity to Rickettsia lusitaniae. Phylogenetic analysis revealed that all of the Zambian gltA and htrA gene sequences could be grouped with those of Rickettsia lusitaniae obtained in various parts of the world. Our data suggest that Rickettsia lusitaniae has a wider geographic and vector range, enhancing to our understanding of Rickettsia lusitaniae epidemiology in sub-Saharan Africa.

First COVID-19 case in Zambia - Comparative phylogenomic analyses of SARS-CoV-2 detected in African countries.

Since its first discovery in December 2019 in Wuhan, China, COVID-19, caused by the novel coronavirus SARS-CoV-2, has spread rapidly worldwide. While African countries were relatively spared initially, the initial low incidence of COVID-19 cases was not sustained for long due to continuing travel links between China, Europe and Africa. In preparation, Zambia had applied a multisectoral national epidemic disease surveillance and response system resulting in the identification of the first case within 48 h of the individual entering the country by air travel from a trip to France. Contact tracing showed that SARS-CoV-2 infection was contained within the patient's household, with no further spread to attending health care workers or community members. Phylogenomic analysis of the patient's SARS-CoV-2 strain showed that it belonged to lineage B.1.1., sharing the last common ancestor with SARS-CoV-2 strains recovered from South Africa. At the African continental level, our analysis showed that B.1 and B.1.1 lineages appear to be predominant in Africa. Whole genome sequence analysis should be part of all surveillance and case detection activities in order to monitor the origin and evolution of SARS-CoV-2 lineages across Africa.

Co-Circulation of Multiple Serotypes of Bluetongue Virus in Zambia.

Bluetongue (BT) is an arthropod-borne viral disease of ruminants with serious trade and socio-economic implications. Although the disease has been reported in a number of countries in sub-Saharan Africa, there is currently no information on circulating serotypes and disease distribution in Zambia. Following surveillance for BT in domestic and wild ruminants in Zambia, BT virus (BTV) nucleic acid and antibodies were detected in eight of the 10 provinces of the country. About 40% (87/215) of pooled blood samples from cattle and goats were positive for BTV nucleic acid, while one hartebeest pool (1/43) was positive among wildlife samples. Sequence analysis of segment 2 revealed presence of serotypes 3, 5, 7, 12 and 15, with five nucleotypes (B, E, F, G and J) being identified. Segment 10 phylogeny showed Zambian BTV sequences clustering with Western topotype strains from South Africa, intimating likely transboundary spread of BTV in Southern Africa. Interestingly, two Zambian viruses and one isolate from Israel formed a novel clade, which we designated as Western topotype 4. The high seroprevalence (96.2%) in cattle from Lusaka and Central provinces and co-circulation of multiple serotypes showed that BT is widespread, underscoring the need for prevention and control strategies.

Differential expression of pattern recognition receptors in the three pathological forms of sheep paratuberculosis.

Paratuberculosis is a chronic inflammatory disease of the gut caused by Mycobacterium avium subspecies paratuberculosis. Three forms have been described in sheep--paucibacillary, multibacillary and asymptomatic. The pauci- and multibacillary forms are characterized by type 1 and type 2 immune responses respectively; asymptomatic animals have no clinical signs or pathology. What determines this polarization is unknown, although pattern recognition receptors (PRR) have been implicated in other mycobacterial diseases. To investigate this in sheep paratuberculosis we used real-time RT-PCR to quantify the expression of fifteen PRR and adaptor genes from forty infected and nine control animals. These data show that there is a relationship between the different pathological forms and PRR transcript profiles. Nine PRRs were up-regulated in asymptomatic animals; with TLR9 being significantly raised in relation to the other three groups. Comparison of the three infected groups showed increases in many PRRs, with CARD15 and Dectin-2 being particularly high in both diseased groups. Significant differences between the pauci- and multibacillary animals included TLR2, CD14 and Dectin-1. Sequence analysis of TLR2 exon 2 and CARD15 exon 11 in the forty animals failed to identify any relationship between SNPs and pathological form.

Differential expression of pattern recognition receptors during the development of foetal sheep.

Pattern recognition receptors (PRRs) play a crucial role in the initiation of the adaptive immune response. Immunological competence of foetal lambs occurs progressively throughout gestation, and in order to understand the role played by PRRs in foetal immunological competence, we quantified transcript expression, in the skin and spleen, of the TLRs, key C-type lectins and CARD15 during the critical second trimester. These data show that lambs express the same spectrum of PRRs as the adult but that the level of expression for most is dependent on developmental age. Key findings include: TLR1 and TLR5 are expressed at high levels in the foetus but are low in the adult; in contrast, TLR4, CD14 and CARD15 increase with age. In addition, TLR9 and TLR10 are expressed by the spleen and not the skin, while CARD15 is low in the spleen and high in the skin.

Differential expression of pattern recognition receptors in sheep tissues and leukocyte subsets.

The various members of the different pattern recognition receptor families are now recognized as playing a crucial role in the initial interactions between a pathogen and the host. This paper identifies all 10 members of the TLR family in sheep as well as key members of the C-type lectin and NLR families. Our data show that sheep possess the 'human' and not the 'mouse' pattern of TLRs and confirm the high degree of sequence identity between orthologous genes in the different species. In the absence of definitive antibodies, qRT-PCR assays were developed to quantify PRR transcript expression patterns in a range of normal sheep tissues as well as isolated dendritic cell (DC) and leukocyte subsets. These data show that the lymphoid organs (spleen and lymph nodes) express the widest range of PRRs and that organs such as the lung and kidney have distinctive arrays of PRRs that reflect their potential risk of pathogen exposure. In addition we show that the two DC subsets, defined by the differential expression of CD172a/CD45RA and their cytokine expression profiles, have different and characteristic PRR complements again possibly reflecting their distinctive function. These data are important for future studies on the role of PRRs in disease pathogenesis and control.

The Epidemiology of African Swine Fever in "Nonendemic" Regions of Zambia (1989-2015): Implications for Disease Prevention and Control.

African swine fever (ASF) is a highly contagious and deadly viral hemorrhagic disease of swine. In Zambia, ASF was first reported in 1912 in Eastern Province and is currently believed to be endemic in that province only. Strict quarantine measures implemented at the Luangwa River Bridge, the only surface outlet from Eastern Province, appeared to be successful in restricting the disease. However, in 1989, an outbreak occurred for the first time outside the endemic province. Sporadic outbreaks have since occurred almost throughout the country. These events have brought into acute focus our limited understanding of the epidemiology of ASF in Zambia. Here, we review the epidemiology of the disease in areas considered nonendemic from 1989 to 2015. Comprehensive sequence analysis conducted on genetic data of ASF viruses (ASFVs) detected in domestic pigs revealed that p72 genotypes I, II, VIII and XIV have been involved in causing ASF outbreaks in swine during the study period. With the exception of the 1989 outbreak, we found no concrete evidence of dissemination of ASFVs from Eastern Province to other parts of the country. Our analyses revealed a complex epidemiology of the disease with a possibility of sylvatic cycle involvement. Trade and/or movement of pigs and their products, both within and across international borders, appear to have been the major factor in ASFV dissemination. Since ASFVs with the potential to cause countrywide and possibly regional outbreaks, could emerge from "nonendemic regions", the current ASF control policy in Zambia requires a dramatic shift to ensure a more sustainable pig industry.