Detection of Extended-Spectrum Beta-Lactamase (ESBL)-Producing Enterobacteriaceae from Diseased Broiler Chickens in Lusaka District, Zambia.

Poultry products in Zambia form an integral part of the human diet in many households, as they are cheap and easy to produce. The burden of poultry diseases has, however, remained a major challenge. Growing consumer demand for poultry products in Zambia has resulted in non-prudent antimicrobial use on farms, intending to prevent and treat poultry diseases for growth optimisation and maximising profits. This cross-sectional study aimed to identify the different types of bacteria causing diseases in chickens in Lusaka and to detect the extended-spectrum lactamase (ESBL)-encoding genes. We collected 215 samples from 91 diseased chickens at three post-mortem facilities and screened them for Gram-negative bacteria. Of these samples, 103 tested positive for various clinically relevant Enterobacteriaceae, including Enterobacter (43/103, 41.7%), Escherichia coli (20/103, 19.4%), Salmonella (10/103, 9.7%), and Shigella (8/103, 7.8%). Other isolated bacteria included Yersinia, Morganella, Proteus, and Klebsiella, which accounted for 21.4%. E. coli, Enterobacter, Salmonella, and Shigella were subjected to antimicrobial susceptibility testing. The results revealed that E. coli, Enterobacter, and Shigella were highly resistant to tetracycline, ampicillin, amoxicillin, and trimethoprim-sulfamethoxazole, while Salmonella showed complete susceptibility to all tested antibiotics. The observed resistance patterns correlated with antimicrobial usage estimated from sales data from a large-scale wholesale and retail company. Six (6/14, 42.9%) E. coli isolates tested positive for blaCTX-M, whilst eight (8/14, 57.1%) Enterobacter samples tested positive for blaTEM. Interestingly, four (4/6, 66.7%) of the E. coli isolates carrying blaCTX-M-positive strains were also positive for blaTEM. Sanger sequencing of the PCR products revealed that five (5/6, 83.3%) of the abovementioned isolates possessed the blaCTX-M-15 allele. The results suggest the presence of potentially pathogenic ESBL-producing Enterobacteriaceae in poultry, threatening public health.

Phylogenetic Analysis of Newcastle Disease Virus Isolated from Poultry in Live Bird Markets and Wild Waterfowl in Zambia.

Poultry production is essential to the economy and livelihood of many rural Zambian households. However, the industry is threatened by infectious diseases, particularly Newcastle disease virus (NDV) infection. Therefore, this study employed next-generation sequencing to characterise six NDV isolates from poultry in Zambia's live bird markets (LBMs) and wild waterfowl. Four NDV isolates were detected from 410 faecal samples collected from chickens in LBMs in Lusaka and two from 2851 wild birds from Lochinvar National Park. Phylogenetic analysis revealed that the four NDVs from LBM clustered in genotype VII and sub-genotype VII.2 were closely related to viruses previously isolated in Zambia and other Southern African countries, suggesting possible local and regional transboundary circulation of the virus. In contrast, the two isolates from wild birds belonged to class I viruses, genotype 1, and were closely related to isolates from Europe and Asia, suggesting the possible introduction of these viruses from Eurasia, likely through wild bird migration. The fusion gene cleavage site motif for all LBM-associated isolates was 112RRQKR|F117, indicating that the viruses are virulent, while the isolates from wild waterfowl had the typical 112ERQER|L117 avirulent motif. This study demonstrates the circulation of virulent NDV strains in LBMs and has, for the first time, characterised NDV from wild birds in Zambia. The study further provides the first whole genomes of NDV sub-genotype VII.2 and genotype 1 from Zambia and stresses the importance of surveillance and molecular analysis for monitoring the circulation of NDV genotypes and viral evolution.

Prevalence and Genomic Characterization of Rotavirus A from Domestic Pigs in Zambia: Evidence for Possible Porcine-Human Interspecies Transmission.

Rotavirus is a major cause of diarrhea globally in animals and young children under 5 years old. Here, molecular detection and genetic characterization of porcine rotavirus in smallholder and commercial pig farms in the Lusaka Province of Zambia were conducted. Screening of 148 stool samples by RT-PCR targeting the VP6 gene revealed a prevalence of 22.9% (34/148). Further testing of VP6-positive samples with VP7-specific primers produced 12 positives, which were then Sanger-sequenced. BLASTn of the VP7 positives showed sequence similarity to porcine and human rotavirus strains with identities ranging from 87.5% to 97.1%. By next-generation sequencing, the full-length genetic constellation of the representative strains RVA/pig-wt/ZMB/LSK0137 and RVA/pig-wt/ZMB/LSK0147 were determined. Genotyping of these strains revealed a known Wa-like genetic backbone, and their genetic constellations were G4-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H1 and G9-P[13]-I5-R1-C1-M1-A8-N1-T1-E1-H1, respectively. Phylogenetic analysis revealed that these two viruses might have their ancestral origin from pigs, though some of their gene segments were related to human strains. The study shows evidence of reassortment and possible interspecies transmission between pigs and humans in Zambia. Therefore, the "One Health" surveillance approach for rotavirus A in animals and humans is recommended to inform the design of effective control measures.

Genome Sequence of a Clinical Isolate of the Human Pathogenic Strain "Candidatus Borrelia fainii" Qtaro.

We report sequences of the complete linear chromosome and five linear plasmids of the relapsing fever spirochete "Candidatus Borrelia fainii" Qtaro. The chromosome sequence of 951,861 bp and the 243,291 bp of plasmid sequences were predicted to contain 852 and 239 protein-coding genes, respectively. The predicted total GC content was 28.4%.

Prevalence and Association of Trypanosomes and Sodalis glossinidius in Tsetse Flies from the Kafue National Park in Zambia.

Tsetse flies are obligate hematophagous vectors of animal and human African trypanosomosis. They cyclically transmit pathogenic Trypanosoma species. The endosymbiont Sodalis glossinidius is suggested to play a role in facilitating the susceptibility of tsetse flies to trypanosome infections. Therefore, this study was aimed at determining the prevalence of S. glossinidius and trypanosomes circulating in tsetse flies and checking whether an association exists between trypanosomes and Sodalis infections in tsetse flies from Kafue National Park in Zambia. A total of 326 tsetse flies were sampled from the Chunga and Ngoma areas of the national park. After DNA extraction was conducted, the presence of S. glossinidius and trypanosome DNA was checked using PCR. The Chi-square test was carried out to determine whether there was an association between the presence of S. glossinidius and trypanosome infections. Out of the total tsetse flies collected, the prevalence of S. glossinidius and trypanosomes was 21.8% and 19.3%, respectively. The prevalence of S. glossinidius was 22.2% in Glossina morsitans and 19.6% in Glossina pallidipes. In relation to sampling sites, the prevalence of S. glossinidius was 26.0% in Chunga and 21.0% in Ngoma. DNA of trypanosomes was detected in 18.9% of G. morsitans and 21.4% of G. pallidipes. The prevalence of trypanosomes was 21.7% and 6.0% for Ngoma and Chunga, respectively. The prevalences of trypanosome species detected in this study were 6.4%, 4.6%, 4.0%, 3.7%, 3.1%, and 2.5% for T. vivax, T. simiae, T. congolense, T. godfreyi, T. simiae Tsavo, and T. b. brucei, respectively. Out of 63 trypanosome infected tsetse flies, 47.6% of the flies also carried S. glossinidius, and the remaining flies were devoid of S. glossinidius. A statistically significant association was found between S. glossinidius and trypanosomes (p < 0.001) infections in tsetse flies. Our findings indicated that presence of S. glossinidius increases the susceptibility of tsetse flies to trypanosome infections and S. glossinidius could be a potential candidate for symbiont-mediated vector control in these tsetse species.

Detection of Old and New World Relapsing Fever Borreliae in Ornithodoros Ticks Collected from Warthog Burrows in Zambia.

Relapsing fever (RF) is an arthropod-borne disease caused by Borrelia spirochete, which is one of the major public health concerns in endemic regions including Africa. However, information on Borrelia spirochetes is limited in Zambia. Here, we investigate the Borrelia spirochetes harbored by Ornithodoros ticks in Zambian National Parks. We analyzed 182 DNA samples pooled from 886 Ornithodoros ticks. Of these, 43 tested positive, and their sequence revealed that the ticks harbored both Old and New World RF borreliae. This research presents the first evidence of Old-World RF borreliae in Zambia. The New World RF borreliae detected herein differed from the Candidatus Borrelia fainii previously reported in Zambia and were closely related to the pathogenic Borrelia sp. VS4 identified in Tanzania. Additionally, Borrelia theileri was recently reported in Zambia. Hence, at least four different Borrelia species occur in Zambia, and the organisms causing relapsing fever there might be more complex than previously thought. We empirically confirmed that real-time PCR with TaqMan minor groove binder probes accurately and simultaneously detected both Old and New World RF. In this manner, they could facilitate quantitative analyses of both types of RF borreliae. Subsequent investigations should endeavor to isolate the aforementioned Borrelia spp. and perform serosurveys on patients with RF.

Luna Virus and Helminths in Wild Mastomys natalensis in Two Contrasting Habitats in Zambia: Risk Factors and Evidence of Virus Dissemination in Semen.

Transmission dynamics and the maintenance of mammarenaviruses in nature are poorly understood. Using metagenomic next-generation sequencing (mNGS) and RT-PCR, we investigated the presence of mammarenaviruses and co-infecting helminths in various tissues of 182 Mastomys natalensis rodents and 68 other small mammals in riverine and non-riverine habitats in Zambia. The Luna virus (LUAV) genome was the only mammarenavirus detected (7.7%; 14/182) from M. natalensis. Only one rodent from the non-riverine habitat was positive, while all six foetuses from one pregnant rodent carried LUAV. LUAV-specific mNGS reads were 24-fold higher in semen than in other tissues from males. Phylogenetically, the viruses were closely related to each other within the LUAV clade. Helminth infections were found in 11.5% (21/182) of M. natalensis. LUAV-helminth co-infections were observed in 50% (7/14) of virus-positive rodents. Juvenility (OR = 9.4; p = 0.018; 95% CI: 1.47-59.84), nematodes (OR = 15.5; p = 0.001; 95% CI: 3.11-76.70), cestodes (OR = 10.8; p = 0.025; 95% CI: 1.35-86.77), and being male (OR = 4.6; p = 0.036; 95% CI: 1.10-18.90) were associated with increased odds of LUAV RNA detection. The role of possible sexual and/or congenital transmission in the epidemiology of LUAV infections in rodents requires further study, along with the implications of possible helminth co-infection.

Detection of Tick-Borne Bacterial and Protozoan Pathogens in Ticks from the Zambia-Angola Border.

Tick-borne diseases (TBDs), including emerging and re-emerging zoonoses, are of public health importance worldwide; however, TBDs tend to be overlooked, especially in countries with fewer resources, such as Zambia and Angola. Here, we investigated Rickettsia, Anaplasmataceae, and Apicomplexan pathogens in 59 and 96 adult ticks collected from dogs and cattle, respectively, in Shangombo, a town at the Zambia-Angola border. We detected Richkettsia africae and Rickettsia aeschilimannii in 15.6% of Amblyomma variegatum and 41.7% of Hyalomma truncatum ticks, respectively. Ehrlichia minasensis was detected in 18.8% of H. truncatum, and Candidatus Midichloria mitochondrii was determined in Hyalomma marginatum. We also detected Babesia caballi and Theileria velifera in A. variegatum ticks with a 4.4% and 6.7% prevalence, respectively. In addition, Hepatozoon canis was detected in 6.5% of Rhipicephalus lunulatus and 4.3% of Rhipicephalus sanguineus. Coinfection of R. aeshilimannii and E. minasensis were observed in 4.2% of H. truncatum. This is the first report of Ca. M. mitochondrii and E. minasensis, and the second report of B. caballi, in the country. Rickettsia africae and R. aeschlimannii are pathogenic to humans, and E. minasensis, B. caballi, T. velifera, and H. canis are pathogenic to animals. Therefore, individuals, clinicians, veterinarians, and pet owners should be aware of the distribution of these pathogens in the area.

Seroprevalence and Risk Factors of Crimean-Congo Hemorrhagic Fever in Cattle of Smallholder Farmers in Central Malawi.

Crimean-Congo hemorrhagic fever virus (CCHFV) is endemic in Africa, Asia, and Eastern Europe where it circulates among animals and ticks causing sporadic outbreaks in humans. Although CCHF is endemic in sub-Saharan Africa, epidemiological information is lacking in many countries, including Malawi. To assess the risk of CCHF in Malawi, we conducted an epidemiological study in cattle reared by smallholder livestock farmers in central Malawi. A cross-sectional study was conducted in April 2020 involving seven districts, four from Kasungu and three from Lilongwe Agriculture Development Divisions. A structured questionnaire was administered to farmers to obtain demographic, animal management, and ecological risk factors data. Sera were collected from randomly selected cattle and screened for CCHF virus (CCHFV) specific antibodies using a commercial ELISA kit. Ticks were collected from cattle and classified morphologically to species level. An overall CCHFV seropositivity rate of 46.9% (n = 416; 95% CI: 42.0-51.8%) was observed. The seropositivity was significantly associated with the age of cattle (p < 0.001), sex (p < 0.001), presence of ticks in herds (p = 0.01), district (p = 0.025), and type of grazing lands (p = 0.013). Five species of ticks were identified, including Hyalomma truncatum, a known vector of CCHFV. Ticks of the species Hyalomma truncatum were not detected in two districts with the highest seroprevalence for CCHF and vector competency must be further explored in the study area. To our knowledge, this is the first report of serologic evidence of the presence of CCHV among smallholder cattle in central Malawi. This study emphasizes the need for continued monitoring of CCHFV infection among livestock, ticks, and humans for the development of data-based risk mitigation strategies.

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.

Identification of Escherichia coli and Related Enterobacteriaceae and Examination of Their Phenotypic Antimicrobial Resistance Patterns: A Pilot Study at A Wildlife-Livestock Interface in Lusaka, Zambia.

A cross-sectional study was used to identify and assess prevalence and phenotypic antimicrobial resistance (AMR) profiles of Escherichia coli and other enterobacteria isolated from healthy wildlife and livestock cohabiting at a 10,000 acres game ranch near Lusaka, Zambia. Purposive sampling was used to select wildlife and livestock based on similarities in behavior, grazing habits and close interactions with humans. Isolates (n = 66) from fecal samples collected between April and August 2018 (n = 84) were examined following modified protocols for bacteria isolation, biochemical identification, molecular detection, phylogenetic analysis, and antimicrobial susceptibility testing by disc diffusion method. Data were analyzed using R software, Genetyx ver.12 and Mega 6. Using Applied Profile Index 20E kit for biochemical identification, polymerase chain reaction assay and sequencing, sixty-six isolates were identified to species level, of which Escherichia coli (72.7%, 48/66), E. fergusonii (1.5%, 1/66), Shigella sonnei (22.7%, 14/66), Sh. flexinerri (1.5%, 1/66) and Enterobacteriaceae bacterium (1.5%, 1/66), and their relationships were illustrated in a phylogenetic tree. Phenotypic antimicrobial resistance or intermediate sensitivity expression to at least one antimicrobial agent was detected in 89.6% of the E. coli, and 73.3% of the Shigella isolates. The E. coli isolates exhibited the highest resistance rates to ampicillin (27%), ceftazidime (14.3%), cefotaxime (9.5%), and kanamycin (9.5%). Multidrug resistance (MDR) was detected in 18.8% of E. coli isolates while only 13.3% Shigella isolates showed MDR. The MDR was detected among isolates from impala and ostrich (wild animals in which no antimicrobial treatment was used), and in isolates from cattle, pigs, and goats (domesticated animals). This study indicates the possible transmission of drug-resistant microorganisms between animals cohabiting at the wildlife-livestock interface. It emphasizes the need for further investigation of the role of wildlife in the development and transmission of AMR, which is an issue of global concern.

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.

Molecular characterization and phylogenetic analysis of Trypanosoma spp. detected from striped leaf-nosed bats (Hipposideros vittatus) in Zambia.

Bat trypanosomes consist of more than 30 trypanosome species from over 70 species of bats. Recent studies suggest that bats play a role in disseminating trypanosomes from African continent to the terrestrial mammals both in the Afrotropic-Palearctic Ecozones and Nearctic Ecozone. However, the diversity, distribution, and evolution of bat trypanosomes are still unclear. To better understand their evolution, more genetic data of bat trypanosomes from a variety of locations are required. During a survey of Borrelia spp. of bats inhabiting a cave in Zambia, we observed flagellate parasites from 5 of 43 hemocultures. Sequence and phylogenetic analyses of the glycosomal glyceraldehyde 3-phosphate dehydrogenase gene (gGAPDH; 572 bp) and the 18S ribosomal RNA gene (18S rRNA gene; 1,079-1,091 bp) revealed that all were Trypanosoma spp. belonged to the Trypanosoma cruzi clade. Three and two of them exhibited the similarity with T. conorhini and T. dionisii, respectively. The present study provides the first genetic data on Trypanosoma spp. of bats inhabiting Zambia.

Human Borreliosis Caused by a New World Relapsing Fever Borrelia-like Organism in the Old World.

BACKGROUND: Relapsing fever is an infectious disease previously neglected in Africa, which imposes a large public health burden in the country. We aimed to investigate and report on a case of relapsing fever borreliosis in Zambia. METHODS: A previously unknown Borrelia species was isolated from the blood of a febrile patient. Investigations of the presumptive vector ticks and natural hosts for the Borrelia species were conducted by culture isolation and/or DNA detection by Borrelia-specific polymerase chain reaction. Using culture isolates from the patient and bat specimens, genetic characterization was performed by multilocus sequence analysis based on the draft genome sequences. RESULTS: The febrile patient was diagnosed with relapsing fever. The isolated Borrelia species was frequently detected in Ornithodoros faini (n = 20/50 [40%]) and bats (n = 64/237 [27%]). Multilocus sequence analysis based on a draft genome sequence revealed that the Borrelia species isolates from the patient and presumptive reservoir host (bats) formed a monophyletic lineage that clustered with relapsing fever borreliae found in the United States. CONCLUSIONS: A febrile illness caused by a Borrelia species that was treatable with erythromycin was identified in Zambia. This is the first study to report on relapsing fever Borrelia in Zambia and suggesting the likely natural reservoir hosts of the isolated Borrelia species. Interestingly, the isolated Borrelia species was more closely related to New World relapsing fever borreliae, despite being detected in the Afrotropic ecozone.

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.

Seroprevalence of Filovirus Infection of Rousettus aegyptiacus Bats in Zambia.

Bats are suspected to play important roles in the ecology of filoviruses, including ebolaviruses and marburgviruses. A cave-dwelling fruit bat, Rousettus aegyptiacus, has been shown to be a reservoir of marburgviruses. Using an enzyme-linked immunosorbent assay with the viral glycoprotein antigen, we detected immunoglobulin G antibodies specific to multiple filoviruses in 158 of 290 serum samples of R aegyptiacus bats captured in Zambia during the years 2014-2017. In particular, 43.8% of the bats were seropositive to marburgvirus, supporting the notion that this bat species continuously maintains marburgviruses as a reservoir. Of note, distinct peaks of seropositive rates were repeatedly observed at the beginning of rainy seasons, suggesting seasonality of the presence of newly infected individuals in this bat population. These data highlight the need for continued monitoring of filovirus infection in this bat species even in countries where filovirus diseases have not been reported.

Direct detection of falciparum and non-falciparum malaria DNA from a drop of blood with high sensitivity by the dried-LAMP system.

BACKGROUND: Because of the low sensitivity of conventional rapid diagnostic tests (RDTs) for malaria infections, the actual prevalence of the diseases, especially those caused by non-Plasmodium falciparum (non-Pf) species, in asymptomatic populations remain less defined in countries lacking in well-equipped facilities for accurate diagnoses. Our direct blood dry LAMP system (CZC-LAMP) was applied to the diagnosis of malaria as simple, rapid and highly sensitive method as an alternative for conventional RDTs in malaria endemic areas where laboratory resources are limited. RESULTS: LAMP primer sets for mitochondria DNAs of Plasmodium falciparum (Pf) and human-infective species other than Pf (non-Pf; P. vivax, P. ovale, P. malariae) were designed and tested by using human blood DNA samples from 74 residents from a malaria endemic area in eastern Zambia. These malaria dry-LAMPs were optimized for field or point-of-care operations, and evaluated in the field at a malaria endemic area in Zambia with 96 human blood samples. To determine the sensitivities and specificities, results obtained by the on-site LAMP diagnosis were compared with those by the nested PCR and nucleotide sequencing of its product. The dry LAMPs showed the sensitivities of 89.7% for Pf and 85.7% for non-Pf, and the specificities of 97.2% for Pf and 100% for non-Pf, with purified blood DNA samples. The direct blood LAMP diagnostic methods, in which 1 µl of anticoagulated blood were used as the template, showed the sensitivities of 98.1% for Pf, 92.1% for non-Pf, and the specificities of 98.1% for Pf, 100% for non-Pf. The prevalences of P. falciparum, P. malariae and P. ovale in the surveyed area were 52.4, 25.3 and 10.6%, respectively, indicating high prevalence of asymptomatic carriers in endemic areas in Zambia. CONCLUSIONS: We have developed new field-applicable malaria diagnostic tests. The malaria CZC-LAMPs showed high sensitivity and specificity to both P. falciparum and non-P. falciparum. These malaria CZC-LAMPs provide new means for rapid, sensitive and reliable point-of-care diagnosis for low-density malaria infections, and are expected to help update current knowledge of malaria epidemiology, and can contribute to the elimination of malaria from endemic areas.

Clinical and subclinical bovine leukemia virus infection in a dairy cattle herd in Zambia.

Bovine leukemia virus (BLV) causes enzootic bovine leucosis (EBL) and is responsible for substantial economic losses in cattle globally. However, information in Africa on the disease is limited. Here, based on clinical, hematological, pathological and molecular analyses, two clinical cases of EBL were confirmed in a dairy cattle herd in Zambia. In contrast, proviral DNA was detected by PCR in five apparently healthy cows from the same herd, suggesting subclinical BLV infection. Phylogenetic analysis of the env gene showed that the identified BLV clustered with Eurasian genotype 4 strains. This is the first report of confirmed EBL in Zambia.

Determination of the prevalence of African trypanosome species in indigenous dogs of Mambwe district, eastern Zambia, by loop-mediated isothermal amplification.

BACKGROUND: Dogs have been implicated to serve as links for parasite exchange between livestock and humans and remain an important source of emerging and re-emerging diseases including trypanosome infections. Yet, canine African trypanosomosis (CAT), particularly in indigenous dogs (mongrel breed) remains under- reported in literature. This study evaluated the performance of loop-mediated isothermal amplification (LAMP) in detecting trypanosomes in blood from indigenous dogs of tsetse-infested Mambwe district in eastern Zambia. METHODS: A cross sectional survey of CAT was conducted within 5 chiefdoms (Msoro, Kakumbi, Munkanya, Nsefu, Malama) of Mambwe district, eastern Zambia, during October 2012. Blood samples from 237 indigenous hunting dogs were collected and screened by microscopy and LAMP. RESULTS: Of the 237 dogs screened for CAT, 14 tested positive by microscopy (5.9%; 95% CI: 2.9 - 8.9%), all of which also tested positive by LAMP. In addition, LAMP detected 6 additional CAT cases, bringing the total cases detected by LAMP to 20 (8.4%; 95% CI: 4.9 - 12.0%). Irrespective of the detection method used, CAT was only recorded from 3 chiefdoms (Munkanya, Nsefu, Malama) out of the 5. According to LAMP, these infections were caused by Trypanosoma congolense, Trypanosoma brucei brucei and the zoonotic Trypanosoma brucei rhodesiense. Although these CAT cases generally did not manifest clinical illness, an association was observed between infection with Trypanosoma brucei subspecies and occurrence of corneal opacity. CONCLUSIONS: This communication reports for the first time the occurrence of CAT in indigenous Zambian dogs. Our study indicates that LAMP is a potential diagnostic tool for trypanosome detection in animals. LAMP was more sensitive than microscopy and was further capable of distinguishing the closely related T. b. brucei and T. b. rhodesiense. In view of the sporadic cases of re-emerging HAT being reported within the Luangwa valley, detection of the human serum resistant associated (SRA) gene in trypanosomes from mongrels is intriguing and indicative of the risk of contracting HAT by local communities and tourists in Mambwe district. Consequently, there is a need for continuous trypanosome surveillances in animals, humans and tsetse flies using sensitive and specific tests such as LAMP.