Identification and distribution of Rhipicephalus microplus in selected high-cattle density districts in Uganda: signaling future demand for novel tick control approaches.

BACKGROUND: Rhipicephalus (Boophilus) microplus (Canestrini, 1888), the Asian blue tick, is a highly invasive and adaptable ectoparasite. This tick species has successfully established itself in most regions of the world, with movement of cattle being a major driver for its spread. In the recent past, R. microplus ticks have been reported in three districts of Uganda. Information on its spread and distribution are vital in deepening our understanding of the ecological scenarios that lead to tick persistence and in the formulation of control strategies. This is especially important in the cattle-dense districts. METHODS: We randomly collected tick specimens from 1,461cattle spread across seven cattle dense districts located in the Central, Karamoja and West Nile regions of Uganda from January to September 2020. The ticks were identified using standard morpho-taxonomic keys and the R. microplus tick species identities were confirmed by sequencing of the ITS2 region, 12S rRNA and 16S rRNA genes and phylogenetic analyses. RESULTS: Adult ticks (n = 13,019) were collected from 1,461 cattle. Seventeen tick species were identified based on morpho-taxonomic keys and the majority (47.4%; n=6184) of these were R. appendiculatus. In total, 257 R. microplus ticks were found infesting cattle in 18 study sites in the districts of Amudat, Kaabong, Napak (Karamoja region) and Arua (West Nile region). The identity of R. microplus was confirmed using molecular technics. No R. microplus tick was recorded in the districts of Lyantonde and Nakaseke (Central region). Arua district accounted for 82.1% (n=211) of the R. microplus ticks recorded followed by Napak district at 16.3% (n=42), while Amudat and Kaabong districts accounted for 1.5% (n=4). Rhipicephalus microplus and R. decoloratus co-existed in 6 of the 13 study sites in Arua district, while in another 6 study sites, no R. decoloratus was recorded. In the Karamoja region districts R. decoloratus co-existed with R.microplus. Of the total 618 ticks belonging to four species of the subgenus Boophilus recorded in this study, R. decoloratus accounted for 50.04% (n=334), followed by R. microplus at 41.58% (n=257), R. geigyi at 2.75% (n=17) and R. annulatus at 1.61% (n=10). In the districts of Amudat, Kaabong and Napak, R. decoloratus was more dominant (76.1%; n=179) of the three Rhipicephalus (Boophilus) tick species recorded, followed by R. microplus (19.5%; n=46) and R. geigyi (4.2%; n=10). Contrariwise, R. microplus was more dominant (84%; n=211) in Arua district followed by R. decoloratus (10.7%; n=27), R. annulatus (3.9%; n=10) and R. geigyi (1.1%; n=3). Phylogenetic analyses of the ITS2 region, 12S rRNA and 16S rRNA genes revealed subgrouping of the obtained sequences with the previously published R. microplus sequences from other parts of the world. CONCLUSION: Rhipicephalus microplus ticks were found infesting cattle in four districts of Uganda. The inability to find R. decoloratus, an indigenous tick, from six sites in the district of Arua is suggestive of its replacement by R. microplus. Rhipicephalus microplus negatively affects livestock production, and therefore, there is a need to determine its distribution and to deepen the understanding of the ecological factors that lead to its spread and persistence in an area.

A weather features dataset for prediction of short-term rainfall quantities in Uganda.

Weather data is of great importance to the development of weather prediction models. However, the availability and quality of this data remains a significant challenge for most researchers around the world. In Uganda, obtaining observational weather data is very challenging due to the sparse distribution of weather stations and inconsistent data records. This has created critical gaps in data availability to run and develop efficient weather prediction models. To bridge this gap, we obtained country-specific time series hourly observational weather data. The data period is from 2020 to 2022 of 11 weather stations distributed in the four regions of Uganda. The data was accessed from the Ogimet data repository using the "climate" R-package. The automated procedures in the R-programming language environment allowed us to download user-defined data at a time resolution from an hourly to an annual basis. However, the raw data acquired cannot be used to learn rainfall patterns because it includes duplicates and non-uniform data. Therefore, this article presents a prepared and cleaned dataset that can be used for the prediction of short-term rainfall quantities in Uganda.

Nanobodies: A Review of Generation, Diagnostics and Therapeutics.

Nanobodies, also referred to as single domain-based VHHs, are antibody fragments derived from heavy-chain only IgG antibodies found in the Camelidae family. Due to their small size, simple structure, high antigen binding affinity, and remarkable stability in extreme conditions, nanobodies possess the potential to overcome several of the limitations of conventional monoclonal antibodies. For many years, nanobodies have been of great interest in a wide variety of research fields, particularly in the diagnosis and treatment of diseases. This culminated in the approval of the world's first nanobody based drug (Caplacizumab) in 2018 with others following soon thereafter. This review will provide an overview, with examples, of (i) the structure and advantages of nanobodies compared to conventional monoclonal antibodies, (ii) methods used to generate and produce antigen-specific nanobodies, (iii) applications for diagnostics, and (iv) ongoing clinical trials for nanobody therapeutics as well as promising candidates for clinical development.

Prevalence of Crimean-Congo haemorrhagic fever in livestock following a confirmed human case in Lyantonde district, Uganda.

BACKGROUND: Crimean-Congo haemorrhagic fever (CCHF) is a tick-borne viral infection, characterized by haemorrhagic fever in humans and transient asymptomatic infection in animals. It is an emerging human health threat causing sporadic outbreaks in Uganda. We conducted a detailed outbreak investigation in the animal population following the death from CCHF of a 42-year-old male cattle trader in Lyantonde district, Uganda. This was to ascertain the extent of CCHF virus (CCHFV) circulation among cattle and goats and to identify affected farms and ongoing increased environmental risk for future human infections. METHODS: We collected blood and tick samples from 117 cattle and 93 goats, and tested these for anti-CCHFV antibodies and antigen using an enzyme-linked immunosorbent assay (ELISA), quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and target enrichment next generation sequencing. RESULTS: CCHFV-specific IgG antibodies were detected in 110/117 (94.0%) cattle and 83/93 (89.3%) goats. Animal seropositivity was independently associated with female animals (AOR = 9.42, P = 0.002), and animals reared under a pastoral animal production system (AOR = 6.02, P = 0.019] were more likely to be seropositive than tethered or communally grazed animals. CCHFV was detected by sequencing in Rhipicephalus appendiculatus ticks but not in domestic animals. CONCLUSION: This investigation demonstrated very high seroprevalence of CCHFV antibodies in both cattle and goats in farms associated with a human case of CCHF in Lyantonde. Therefore, building surveillance programs for CCHF around farms in this area and the Ugandan cattle corridor is indicated, in order to identify opportunities for case prevention and control.

ADMET profiling and molecular docking of potential antimicrobial peptides previously isolated from African catfish, Clarias gariepinus.

Amidst rising cases of antimicrobial resistance, antimicrobial peptides (AMPs) are regarded as a promising alternative to traditional antibiotics. Even so, poor pharmacokinetic profiles of certain AMPs impede their utility necessitating, a careful assessment of potential AMPs' absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties during novel lead exploration. Accordingly, the present study utilized ADMET scores to profile seven previously isolated African catfish antimicrobial peptides (ACAPs). After profiling, the peptides were docked against approved bacterial protein targets to gain insight into their possible mode of action. Promising ACAPs were then chemically synthesized, and their antibacterial activity was validated in vitro utilizing the broth dilution method. All seven examined antimicrobial peptides passed the ADMET screening, with two (ACAP-IV and ACAP-V) exhibiting the best ADMET profile scores. The ACAP-V had a higher average binding energy (-8.47 kcal/mol) and average global energy (-70.78 kcal/mol) compared to ACAP-IV (-7.60 kcal/mol and -57.53 kcal/mol), with the potential to penetrate and disrupt bacterial cell membrane (PDB Id: 2w6d). Conversely, ACAP-IV peptide had higher antibacterial activity against E. coli and S. aureus (Minimum Inhibitory Concentration, 520.7 ± 104.3 µg/ml and 1666.7 ± 416.7 µg/ml, respectively) compared to ACAP-V. Collectively, the two antimicrobial peptides (ACAP-IV and ACAP-V) are potential novel leads for the food, cosmetic and pharmaceutical industries. Future research is recommended to optimize the expression of such peptides in biological systems for extended evaluation.

Retrospective in silico mutation profiling of SARS-CoV-2 structural proteins circulating in Uganda by July 2021: Towards refinement of COVID-19 disease vaccines, diagnostics, and therapeutics.

The SARS-CoV-2 virus, the agent of COVID-19, caused unprecedented loss of lives and economic decline worldwide. Although the introduction of public health measures, vaccines, diagnostics, and therapeutics disrupted the spread of the SARS-CoV-2, the emergence of variants poses substantial threat. This study traced SARS-CoV-2 variants circulating in Uganda by July 2021 to inform the necessity for refinement of the intervention medical products. A comprehensive in silico analysis of the SARS-CoV-2 genomes detected in clinical samples collected from COVID-19 patients in Uganda revealed occurrence of structural protein variants with potential of escaping detection, resisting antibody therapy, or increased infectivity. The genome sequence dataset was retrieved from the GISAID database and the open reading frame encoding the spike, envelope, membrane, or nucleocapsid proteins was translated. The obtained protein sequences were aligned and inspected for existence of variants. The variant positions on each of the four alignment sets were mapped on predicted epitopes as well as the 3D structures. Additionally, sequences within each of the sets were clustered by family. A phylogenetic tree was constructed to assess relationship between the encountered spike protein sequences and Wuhan-Hu-1 wild-type, or the Alpha, Beta, Delta and Gamma variants of concern. Strikingly, the frequency of each of the spike protein point mutations F157L/Del, D614G and P681H/R was over 50%. The furin and the transmembrane serine protease 2 cleavage sites were unaffected by mutation. Whereas the Delta dominated the spike sequences (16.5%, 91/550), Gamma was not detected. The envelope protein was the most conserved with 96.3% (525/545) sequences being wild-type followed by membrane at 68.4% (397/580). Although the nucleocapsid protein sequences varied, the variant residue positions were less concentrated at the RNA binding domains. The dominant nucleocapsid sequence variant was S202N (34.5%, 205/595). These findings offer baseline information required for refining the existing COVID-19 vaccines, diagnostics, and therapeutics.

Risk factors for Crimean-Congo Haemorrhagic Fever (CCHF) virus exposure in farming communities in Uganda.

BACKGROUND: Crimean-Congo Haemorrhagic Fever (CCHF) is an emerging human-health threat causing sporadic outbreaks in livestock farming communities. However, the full extent and the risks associated with exposure of such communities has not previously been well-described. METHODS: We collected blood samples from 800 humans, 666 cattle, 549 goats and 32 dogs in districts within and outside Ugandan cattle corridor in a cross-sectional survey, and tested for CCHFV-specific IgG antibodies using Enzyme-Linked Immunosorbent Assays. Sociodemographic and epidemiological data were recorded using structured questionnaire. Ticks were collected to identify circulating nairoviruses by metagenomic sequencing. RESULTS: CCHFV seropositivity was in 221/800 (27·6%) in humans, 612/666 (91·8%) in cattle, 413/549 (75·2%) in goats and 18/32 (56·2%) in dogs. Human seropositivity was associated with livestock farming (AOR=5·68, p<0·0001), age (AOR=2·99, p=0·002) and collecting/eating engorged ticks (AOR=2·13, p=0·004). In animals, seropositivity was higher in cattle versus goats (AOR=2·58, p<0·0001), female sex (AOR=2·13, p=0·002) and heavy tick infestation (>50 ticks: AOR=3·52, p=0·004). CCHFV was identified in multiple tick pools of Rhipicephalus appendiculatus. INTERPRETATION: The very high CCHF seropositivity especially among livestock farmers and multiple regional risk factors associated exposures, including collecting/eating engorged ticks previously unrecognised, highlights need for further surveillance and sensitisation and control policies against the disease.

Complete Genome Sequencing of Field Isolates of Peste des Petits Ruminants Virus from Tanzania Revealed a High Nucleotide Identity with Lineage III PPR Viruses.

Peste des petits ruminants virus (PPRV) causes a highly devastating disease of sheep and goats that threatens food security, small ruminant production and susceptible endangered wild ruminants. With policy directed towards achieving global PPR eradication, the establishment of cost-effective genomic surveillance tools is critical where PPR is endemic. Genomic data can provide sufficient in-depth information to identify the pockets of endemicity responsible for PPRV persistence and viral evolution, and direct an appropriate vaccination response. Yet, access to the required sequencing technology is low in resource-limited settings and is compounded by the difficulty of transporting clinical samples from wildlife across international borders due to the Convention on International Trade in Endangered Species (CITES) of Wild Fauna and Flora, and Nagoya Protocol regulations. Oxford nanopore MinION sequencing technology has recently demonstrated an extraordinary performance in the sequencing of PPRV due to its rapidity, utility in endemic countries and comparatively low cost per sample when compared to other whole-genome (WGS) sequencing platforms. In the present study, Oxford nanopore MinION sequencing was utilised to generate complete genomes of PPRV isolates collected from infected goats in Ngorongoro and Momba districts in the northern and southern highlands of Tanzania during 2016 and 2018, respectively. The tiling multiplex polymerase chain reaction (PCR) was carried out with twenty-five pairs of long-read primers. The resulting PCR amplicons were used for nanopore library preparation and sequencing. The analysis of output data was complete genomes of PPRV, produced within four hours of sequencing (accession numbers: MW960272 and MZ322753). Phylogenetic analysis of the complete genomes revealed a high nucleotide identity, between 96.19 and 99.24% with lineage III PPRV currently circulating in East Africa, indicating a common origin. The Oxford nanopore MinION sequencer can be deployed to overcome diagnostic and surveillance challenges in the PPR Global Control and Eradication program. However, the coverage depth was uneven across the genome and amplicon dropout was observed mainly in the GC-rich region between the matrix (M) and fusion (F) genes of PPRV. Thus, larger field studies are needed to allow the collection of sufficient data to assess the robustness of nanopore sequencing technology.

Development of Nanobodies Targeting Peste des Petits Ruminants Virus: The Prospect in Disease Diagnosis and Therapy.

Peste des petits ruminants virus (PPRV) causes a highly devastating disease, peste des petits ruminants (PPR) of sheep and goats, that threatens food security, small ruminant production, and the conservation of wild small ruminants in many developing countries, especially in Africa. Robust serological and molecular diagnostic tools are available to detect PPRV infection, but they were mainly developed for domestic sheep and goats. The presence of a wide host range for PPRV does present serological diagnostic challenges. New innovative diagnostic tools are needed to detect PPRV in atypical hosts (e.g., Camelidae, Suidae, and Bovinae), in wildlife ecosystems and in complex field situations. Interestingly, single-domain antigen binding fragments (nanobodies) derived from heavy-chain-only camelid antibodies have emerged as a new hope in the development of accurate, rapid, and cost-effective diagnostic tools in veterinary and biomedical fields that are suitable for low-income countries. The main objective of this study was to construct an immune nanobody library to retrieve PPRV-reactive nanobodies that enable the development of diagnostic and therapeutic nanobodies in the future. Here, a strategy was developed whereby an alpaca (Vicugna pacos) was immunized with a live attenuated vaccine strain (PPRV/N/75/1) to raise an affinity-matured immune response in the heavy-chain-only antibody classes. The nanobody gene repertoire was engineered in pMECS-GG phagemid, whereby a ccdB gene (encoding a lethal protein) was substituted by the nanobody gene. An immune nanobody library with approximately sixty-four million independent transformants was constructed, of which 100% contained an insert with the proper size of nanobody gene. Following phage display and biopanning, nine nanobodies that specifically recognise completely inactivated PPRV were identified on enzyme-linked immunosorbent assay. They showed superb potency in rapidly identifying PPRV, which is likely to open a new perspective in the diagnosis and possible treatment of PPR infection.

Prevalence of Fascioliasis and Associated Economic Losses in Cattle Slaughtered at Lira Municipality Abattoir in Northern Uganda.

Fascioliasis (liver fluke infestation) is one of the most important parasitic diseases affecting cattle, other ruminant animals and humans. Fascioliasis causes large, but usually neglected, economic losses to cattle farmers and traders. The objectives of this study were to assess the prevalence and associated risks for fascioliasis in slaughter cattle and estimate the financial losses due to liver condemnation at the Lira Municipal abattoir in Uganda. A total of 216 cattle were sampled during the study period. Animal breed and sex were determined by observing the phenotypic characteristics of the animals. Age was determined by assessing the eruption and wearing of permanent teeth. After slaughter, the liver was examined for presence of Fasciola spp. (liver flukes) by visual inspection, palpation, and incisions. The bile ducts and gall bladder were similarly examined for presence of mature Fasciola spp. The gross weight and amount of liver trimmed-off due to fluke infestation were determined. Of the 216 liver examined, 65.7% (n = 142) were infested with Fasciola spp. Cattle that were aged 4-5 years old at the time of slaughter had significantly greater odds (OR = 5.84; CI [2.79-12.22]) of being infested with Fasciola spp. compared to those that were younger than 3.5 years old. In contrast, cattle that had a body condition score of 3.5 or 4 had lower odds (OR= 0.42; CI [0.21-0.88] and OR = 0.22; CI [0.04-1.10]) of fascioliasis than those with a BCS of 3. Other tested variables including animal origin, breed, sex, and gross weight of the liver had no significant effect on the prevalence of fascioliasis. This study also revealed that the abattoir loses an estimated 38 million UGX annually due to condemnation of Fasciola-infested liver (one UGX= 0.00027 USD; July 2016). Our study showed that the prevalence of fascioliasis was high in Lira District, Uganda, which results in a large amount of liver being condemned and destroyed, leading to financial losses for affected farmers in the area. Therefore, there is a need to take the necessary preventive measures to control the disease and increase awareness among farmers and medical personnel in the area due to the zoonotic nature of fascioliasis.

Identification of Antimicrobial Peptides Isolated From the Skin Mucus of African Catfish, Clarias gariepinus (Burchell, 1822).

Antimicrobial peptides (AMPs) constitute a broad range of bioactive compounds in diverse organisms, including fish. They are effector molecules for the innate immune response, against pathogens, tissue damage and infections. Still, AMPs from African Catfish, Clarias gariepinus, skin mucus are largely unexplored despite their possible therapeutic role in combating antimicrobial resistance. In this study, African Catfish Antimicrobial peptides (ACAPs) were identified from the skin mucus of African Catfish, C. gariepinus. Native peptides were extracted from fish mucus scrapings in 10% acetic acid (v/v) and ultra-filtered using 5 kDa molecular weight cut-off membrane. The extract was purified using C18 Solid-Phase Extraction. The antibacterial activity was determined using the Agar Well Diffusion method and broth-dilution method utilizing Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922). Thereafter, Sephadex G-25 gel filtration was further utilized in bio-guided isolation of the most active fractions prior to peptide identification using Orbitrap Fusion Lumos Tribrid Mass Spectrometry. The skin mucus extracted from African Catfish from all the three major lakes of Uganda exhibited antimicrobial activity on E. coli and S. aureus. Lake Albert's C. gariepinus demonstrated the best activity with the lowest MIC of 2.84 and 0.71 µg/ml on S. aureus and E. coli, respectively. Sephadex G-25 peak I mass spectrometry analysis (Data are available via ProteomeXchange with identifier PXD029193) alongside in silico analysis revealed seven short peptides (11-16 amino acid residues) of high antimicrobial scores (0.561-0.905 units). In addition, these peptides had a low molecular weight (1005.57-1622.05 Da) and had percentage hydrophobicity above 54%. Up to four of these AMPs demonstrated α-helix structure conformation, rendering them amphipathic. The findings of this study indicate that novel AMPs can be sourced from the skin mucus of C. gariepinus. Such AMPs are potential alternatives to the traditional antibiotics and can be of great application to food and pharmaceutical industries; however, further studies are still needed to establish their drug-likeness and safety profiles.

Genetic diversity of aflatoxin-producing Aspergillus flavus isolated from selected groundnut growing agro-ecological zones of Uganda.

BACKGROUND: Groundnut pre- and post-harvest contamination is commonly caused by fungi from the Genus Aspergillus. Aspergillus flavus is the most important of these fungi. It belongs to section Flavi; a group consisting of aflatoxigenic (A. flavus, A. parasiticus and A. nomius) and non-aflatoxigenic (A. oryzae, A. sojae and A. tamarii) fungi. Aflatoxins are food-borne toxic secondary metabolites of Aspergillus species associated with severe hepatic carcinoma and children stuntedness. Despite the well-known public health significance of aflatoxicosis, there is a paucity of information about the prevalence, genetic diversity and population structure of A. flavus in different groundnut growing agro-ecological zones of Uganda. This cross-sectional study was therefore conducted to fill this knowledge gap. RESULTS: The overall pre- and post-harvest groundnut contamination rates with A. flavus were 30.0 and 39.2% respectively. Pre- and post-harvest groundnut contamination rates with A. flavus across AEZs were; 2.5 and 50.0%; (West Nile), 55.0 and 35.0% (Lake Kyoga Basin) and 32.5 and 32.5% (Lake Victoria Basin) respectively. There was no significant difference (χ2 = 2, p = 0.157) in overall pre- and post-harvest groundnut contamination rates with A. flavus and similarly no significant difference (χ2 = 6, p = 0.199) was observed in the pre- and post-harvest contamination of groundnut with A. flavus across the three AEZs. The LKB had the highest incidence of aflatoxin-producing Aspergillus isolates while WN had no single Aspergillus isolate with aflatoxin-producing potential. Aspergillus isolates from the pre-harvest groundnut samples had insignificantly higher incidence of aflatoxin production (χ2 = 2.667, p = 0.264) than those from the post-harvest groundnut samples. Overall, A. flavus isolates exhibited moderate level (92%, p = 0.02) of genetic diversity across the three AEZs and low level (8%, p = 0.05) of genetic diversity within the individual AEZs. There was a weak positive correlation (r = 0.1241, p = 0.045) between genetic distance and geographic distance among A. flavus populations in the LKB, suggesting that genetic differentiation in the LKB population might be associated to geographic distance. A very weak positive correlation existed between genetic variation and geographic location in the entire study area (r = 0.01, p = 0.471), LVB farming system (r = 0.0141, p = 0.412) and WN farming system (r = 0.02, p = 0.478). Hierarchical clustering using the unweighted pair group method with arithmetic means (UPGMA) revealed two main clusters of genetically similar A. flavus isolates. CONCLUSIONS: These findings provide evidence that genetic differentiation in A. flavus populations is independent of geographic distance. This information can be valuable in the development of a suitable biocontrol management strategy of aflatoxin-producing A. flavus.

Paradigm shift in the diagnosis of peste des petits ruminants: scoping review.

Peste des petits ruminants virus causes a highly contagious disease, which poses enormous economic losses in domestic animals and threatens the conservation of wild herbivores. Diagnosis remains a cornerstone to the Peste des petits ruminants Global Control and Eradication Strategy, an initiative of the World Organisation for Animal Health and the Food and Agriculture Organisation. The present review presents the peste des petits ruminants diagnostic landscape, including the practicality of commercially available diagnostic tools, prototype tests and opportunities for new technologies. The most common peste des petits ruminants diagnostic tools include; agar gel immunodiffusion, counter-immunoelectrophoresis, enzyme-linked immunosorbent assays, reverse transcription polymerase chain reaction either gel-based or real-time, reverse transcription loop-mediated isothermal amplification, reverse transcription recombinase polymerase amplification assays, immunochromatographic lateral flow devices, luciferase immunoprecipitation system and pseudotype-based assays. These tests vary in their technical demands, but all require a laboratory with exception of immunochromatographic lateral flow and possibly reverse transcription loop-mediated isothermal amplification and reverse transcription recombinase polymerase amplification assays. Thus, we are proposing an efficient integration of diagnostic tests for rapid and correct identification of peste des petits ruminants in endemic zones and to rapidly confirm outbreaks. Deployment of pen-side tests will improve diagnostic capacity in extremely remote settings and susceptible wildlife ecosystems, where transportation of clinical samples in the optimum cold chain is unreliable.

Structural basis for the high specificity of a Trypanosoma congolense immunoassay targeting glycosomal aldolase.

BACKGROUND: Animal African trypanosomosis (AAT) is a neglected tropical disease which imposes a heavy burden on the livestock industry in Sub-Saharan Africa. Its causative agents are Trypanosoma parasites, with T. congolense and T. vivax being responsible for the majority of the cases. Recently, we identified a Nanobody (Nb474) that was employed to develop a homologous sandwich ELISA targeting T. congolense fructose-1,6-bisphosphate aldolase (TcoALD). Despite the high sequence identity between trypanosomatid aldolases, the Nb474-based immunoassay is highly specific for T. congolense detection. The results presented in this paper yield insights into the molecular principles underlying the assay's high specificity. METHODOLOGY/PRINCIPAL FINDINGS: The structure of the Nb474-TcoALD complex was determined via X-ray crystallography. Together with analytical gel filtration, the structure reveals that a single TcoALD tetramer contains four binding sites for Nb474. Through a comparison with the crystal structures of two other trypanosomatid aldolases, TcoALD residues Ala77 and Leu106 were identified as hot spots for specificity. Via ELISA and surface plasmon resonance (SPR), we demonstrate that mutation of these residues does not abolish TcoALD recognition by Nb474, but does lead to a lack of detection in the Nb474-based homologous sandwich immunoassay. CONCLUSIONS/SIGNIFICANCE: The results show that the high specificity of the Nb474-based immunoassay is not determined by the initial recognition event between Nb474 and TcoALD, but rather by its homologous sandwich design. This (i) provides insights into the optimal set-up of the assay, (ii) may be of great significance for field applications as it could explain the potential detection escape of certain T. congolense strains, and (iii) may be of general interest to those developing similar assays.

MIF-Mediated Hemodilution Promotes Pathogenic Anemia in Experimental African Trypanosomosis.

Animal African trypanosomosis is a major threat to the economic development and human health in sub-Saharan Africa. Trypanosoma congolense infections represent the major constraint in livestock production, with anemia as the major pathogenic lethal feature. The mechanisms underlying anemia development are ill defined, which hampers the development of an effective therapy. Here, the contribution of the erythropoietic and erythrophagocytic potential as well as of hemodilution to the development of T. congolense-induced anemia were addressed in a mouse model of low virulence relevant for bovine trypanosomosis. We show that in infected mice, splenic extramedullary erythropoiesis could compensate for the chronic low-grade type I inflammation-induced phagocytosis of senescent red blood cells (RBCs) in spleen and liver myeloid cells, as well as for the impaired maturation of RBCs occurring in the bone marrow and spleen. Rather, anemia resulted from hemodilution. Our data also suggest that the heme catabolism subsequent to sustained erythrophagocytosis resulted in iron accumulation in tissue and hyperbilirubinemia. Moreover, hypoalbuminemia, potentially resulting from hemodilution and liver injury in infected mice, impaired the elimination of toxic circulating molecules like bilirubin. Hemodilutional thrombocytopenia also coincided with impaired coagulation. Combined, these effects could elicit multiple organ failure and uncontrolled bleeding thus reduce the survival of infected mice. MIF (macrophage migrating inhibitory factor), a potential pathogenic molecule in African trypanosomosis, was found herein to promote erythrophagocytosis, to block extramedullary erythropoiesis and RBC maturation, and to trigger hemodilution. Hence, these data prompt considering MIF as a potential target for treatment of natural bovine trypanosomosis.

Comparative evaluation of the nested ITS PCR against the 18S PCR-RFLP in a survey of bovine trypanosomiasis in Kwale County, Kenya.

We compared the nested internal transcribed spacer (ITS) PCR and the 18S PCR-RFLP (restriction-fragment length polymorphism) pan-trypanosome assays in a cross-sectional survey of bovine trypanosomiasis in 358 cattle in Kwale County, Kenya. The prevalence of trypanosomiasis as determined by the nested ITS PCR was 19.6% (70/358) and by 18S PCR-RFLP was 16.8% (60/358). Of the pathogenic trypanosomes detected, the prevalence of Trypanosoma congolense and Trypanosoma vivax was greater than that of Trypanosoma simiae The nested ITS PCR detected 83 parasite events, whereas the 18S PCR-RFLP detected 64; however, overall frequencies of infections and the parasite events detected did not differ between the assays (χ(2) = 0.8, df = 1, p > 0.05 and χ(2) = 2.5, df = 1, p > 0.05, respectively). The kappa statistic (0.8) showed good agreement between the tests. The nested ITS PCR and the 18S PCR-RFLP had comparable sensitivity, although the nested ITS PCR was better at detecting mixed infections (χ(2) = 5.4, df = 1, p < 0.05).

An Anti-proteome Nanobody Library Approach Yields a Specific Immunoassay for Trypanosoma congolense Diagnosis Targeting Glycosomal Aldolase.

BACKGROUND: Infectious diseases pose a severe worldwide threat to human and livestock health. While early diagnosis could enable prompt preventive interventions, the majority of diseases are found in rural settings where basic laboratory facilities are scarce. Under such field conditions, point-of-care immunoassays provide an appropriate solution for rapid and reliable diagnosis. The limiting steps in the development of the assay are the identification of a suitable target antigen and the selection of appropriate high affinity capture and detection antibodies. To meet these challenges, we describe the development of a Nanobody (Nb)-based antigen detection assay generated from a Nb library directed against the soluble proteome of an infectious agent. In this study, Trypanosoma congolense was chosen as a model system. METHODOLOGY/PRINCIPAL FINDINGS: An alpaca was vaccinated with whole-parasite soluble proteome to generate a Nb library from which the most potent T. congolense specific Nb sandwich immunoassay (Nb474H-Nb474B) was selected. First, the Nb474-homologous sandwich ELISA (Nb474-ELISA) was shown to detect experimental infections with high Positive Predictive Value (98%), Sensitivity (87%) and Specificity (94%). Second, it was demonstrated under experimental conditions that the assay serves as test-of-cure after Berenil treatment. Finally, this assay allowed target antigen identification. The latter was independently purified through immuno-capturing from (i) T. congolense soluble proteome, (ii) T. congolense secretome preparation and (iii) sera of T. congolense infected mice. Subsequent mass spectrometry analysis identified the target as T. congolense glycosomal aldolase. CONCLUSIONS/SIGNIFICANCE: The results show that glycosomal aldolase is a candidate biomarker for active T. congolense infections. In addition, and by proof-of-principle, the data demonstrate that the Nb strategy devised here offers a unique approach to both diagnostic development and target discovery that could be widely applied to other infectious diseases.