RaTexT®: a novel rapid tick exposure test for detecting acaricide resistance in Rhipicephalus microplus ticks in Brazil.

BACKGROUND: Acaricide resistance in cattle ticks is a significant concern in (sub)tropical regions, particularly Brazil. The Larval Packet Test (LPT) is the standard laboratory bioassay for resistance diagnosis, which requires triplicates of seven acaricidal dilutions plus controls to cover larval mortalities ranging between 0 and 100%. The value of the LPT lies in providing resistance ratios based on the ratio between the LC50 calculated with potentially resistant and susceptible ticks. However, LC50 ratios are difficult to translate into practical advice for farmers. Moreover, LPT requires laboratory facilities to maintain susceptible tick colonies, and it takes 6 weeks to obtain the larvae to be tested by LPT derived from engorged female ticks collected from cattle in the field. Our novel approach was twofold: first, we upgraded the LPT to the Resistance Intensity Test (RIT) by adopting the latest WHO guidelines for resistance detection in mosquitoes, which combines a 1 × recommended dose with 5 × and 10 × concentrated doses to reveal low, moderate and high resistance intensity, respectively. This reduced the number of test papers and tick larvae and, more importantly, provided relevant information on the resistance level. Our second innovative step was to abolish testing larvae entirely and expose partly engorged adult ticks to the same acaricidal doses immediately after removing them from cattle in the field. This resulted in the Rapid Tick exposure Test (RaTexT®), wherein partly engorged adult ticks were exposed to an acaricide-impregnated, specially designed matrix providing test results within 24 h. This approach directly compared resistance detection in tick larvae in the RIT with resistance in adult ticks in RaTexT®. METHODS: Laboratory validation was conducted in Brazil with resistant and susceptible colonies of Rhipicephalus microplus ticks. For field validation, adult R. microplus ticks collected from different cattle farms in Brazil were evaluated for resistance to RaTexT®, and the results regarding their larval progenies were compared with those for the RIT. Partly engorged adult ticks derived from cattle infested with laboratory and field strains of R. microplus were exposed to deltamethrin in RaTexT® containers, which contained six rows of four interconnected compartments, accommodating five to eight semi-engorged female ticks with a preferred size ranging between 5 and 8 mm. The corresponding larvae of each strain were exposed in the RIT to the same deltamethrin concentrations in filter papers. RESULTS: In RaTexT®, mortality in adult ticks from a resistant strain of R. microplus from Seropédica in Brazil was 38.4%, 54.2% and 75.0% at the 1 ×, 5 × and 10 × doses of deltamethrin, respectively. In RIT, mortality of larvae from the same resistant strain was 2.0%, 4.9% and 19.5% at 1 ×, 5 × and 10 × doses, respectively. The results of RaTexT® and RIT agreed since both tests identified a high level of resistance based on a cut-off of 90% mortality. In RaTexT®, mortality of adult ticks from a susceptible strain originating from Porto Alegre was 73.8%, 92.9% and 97.6% at the 1 ×, 5 × and 10 × doses, respectively. In RIT, mortality of larvae from the susceptible strain was 95.2%, 95.2% and 96.8% at the 1 ×, 5 × and 10 × doses, respectively. Interestingly, both tests identified a low number of unexpected resistant individuals in the susceptible strain since the mortality of neither larvae nor adults reached 100%. This effect remained unnoticed in the LPT, wherein a resistance ratio of 159.5 was found based on the LC50 of the resistant strain divided by the LC50 of the susceptible strain. Next, RaTexT® was compared with RIT using adult and larval ticks derived from three field strains of R. microplus in Brazil. RaTexT® detected high levels of resistance to deltamethrin in adult ticks in all strains, which was confirmed in larvae tested by the RIT. Both tests agreed on the same resistance level with significantly lower mortality rates in larvae than in adult ticks. CONCLUSIONS: RaTexT® is a novel rapid pen-site test for detecting acaricide resistance in adult livestock ticks. It potentially replaces laborious tests using larval ticks and provides results within 24 h relevant to acaricide resistance management of livestock ticks.

Day 3 parasitemia and Plasmodium falciparum Kelch 13 mutations among uncomplicated malaria patients treated with artemether-lumefantrine in Adjumani district, Uganda.

Artemisinin resistance threatens malaria control and elimination efforts globally. Recent studies have reported the emergence of Plasmodium falciparum parasites tolerant to artemisinin agents in sub-Saharan Africa, including Uganda. The current study assessed the day 3 parasite clearance and its correlation with P. falciparum K13 propeller gene (pfkelch13) mutations in P. falciparum parasites isolated from patients with uncomplicated malaria under artemether-lumefantrine (AL) treatment. This study enrolled 100 P. falciparum-positive patients to whom AL was prescribed between 09/September/2022 and 06/November/2022. Blood samples were collected in EDTA tubes before treatment initiation (day 0) and on day 3. Parasitemia was assessed by microscopy from blood smears and quantitative polymerase chain reaction (qPCR) from the DNA extracted. The day 0 parasite K13 gene was sequenced using Sanger sequencing. Sequence data were analysed using MEGA version 11 software. The data were analysed using STATA version 15, and the Mann‒Whitney U test was used to compare PCR parasite clearance on day 3 using the comparative CT value method and pfkelch13 mutations. The prevalence of day 3 parasitaemia was 24% (24/100) by microscopy and 63% (63/100) by qPCR from the AL-treated patients. P. falciparum K13-propeller gene polymorphism was detected in 18.8% (15/80) of the day 0 DNA samples. The K13 mutations found were C469Y, 12.5% (10/80); A675V, 2.5% (2/80); A569S, 1.25%, (1/80), A578S, 1.25%, (1/80) and; F491S, 1.25%, (1/80) a new allele not reported anywhere. The C469Y mutation, compared to the wild-type, was associated with delayed parasite clearance p = 0.0278, Hodges-Lehmann estimation 3.2108 on the log scale, (95%CI 1.7076, 4.4730). There was a high prevalence of day 3 P. falciparum among malaria patients treated using artemether-lumefantrine. We conclude the presence of the K13 mutation associated with artemisinin resistance by P. falciparum in Adjumani district, Uganda, necessitates regular surveillance of the effectiveness and efficacy of artemether-lumefantrine in the country.

Day 3 parasitemia and Plasmodium falciparum Kelch 13 mutations among uncomplicated malaria patients treated with artemether-lumefantrine in Adjumani district, Uganda.

Artemisinin resistance threatens malaria control and elimination efforts globally. Recent studies have reported the emergence of Plasmodium falciparum parasites tolerant to artemisinin agents in sub-Saharan Africa, including Uganda. The current study assessed the day 3 parasite clearance and its correlation with P. falciparum K13 propeller gene (pfkelch13) mutations in P. falciparum parasites isolated from patients with uncomplicated malaria under artemether-lumefantrine (AL) treatment. This study enrolled 100 P. falciparum-positive patients to whom AL was prescribed between 09/September/2022 and 06/November/2022. Blood samples were collected in EDTA tubes before treatment initiation (day 0) and on day 3. Parasitemia was assessed by microscopy from blood smears and quantitative polymerase chain reaction (qPCR) from the DNA extracted. The day 0 parasite K13 gene was sequenced using Sanger sequencing. Sequence data were analysed using MEGA version 11 software. The data were analysed using STATA version 15, and the Mann‒Whitney U test was used to compare PCR parasite clearance on day 3 using the comparative CT value method and pfkelch13 mutations. The prevalence of day 3 parasitaemia was 24% (24/100) by microscopy and 63% (63/100) by qPCR from the AL-treated patients. P. falciparum K13-propeller gene polymorphism was detected in 18.8% (15/80) of the day 0 DNA samples. The K13 mutations found were C469Y, 12.5% (10/80); A675V, 2.5% (2/80); A569S, 1.25%, (1/80), A578S, 1.25%, (1/80) and; F491S, 1.25%, (1/80) a new allele not reported anywhere. The C469Y mutation, compared to the wild-type, was associated with delayed parasite clearance p=0.0278, Hodges-Lehmann estimation 3.2108 on the log scale, (95%CI 1.7076, 4.4730). There was a high prevalence of day 3 P. falciparum among malaria patients treated using artemether-lumefantrine. We conclude that the K13 mutation associated with artemisinin resistance by P. falciparum is present in Adjumani district, Uganda. This necessitates regular surveillance of the effectiveness and efficacy of artemether-lumefantrine in the country.

Risk factors contributing to tick-acaricide control failure in communal areas of the Oliver Tambo district eastern cape province, South Africa.

Application of chemical acaricides in the control of ticks has led to the problem of tick-acaricide control failure. To obtain an understanding of the possible risk factors involved in this tick-acaricide control failure, this study investigated tick control practices on communal farms in the north-eastern part of the Eastern Cape Province (ECP) of South Africa. A semi-structured questionnaire designed to document specific farm attributes and acaricide usage practices was administered at 94 communal farms from the Oliver Tambo District municipality of the ECP. Data collected indicated that the main acaricide chemicals used at plunge dips of inland and coastal areas were synthetic pyrethroid formulations. Most (75%) farmers claimed not to have noticed a significant reduction in numbers of actively feeding and growing ticks on cattle after several acaricide treatments. Based on the farmers' perceptions, leading factors that could have led to tick-acaricide control failure included: weak strength of the dip solution (76%); poor structural state of dip tanks (42%); and irregular tick control (21%). The rearing of crossbreeds of local and exotic cattle breeds, perceived weak strength of the dip solution and high frequency of acaricide treatment, were statistically associated with proportions of farms reporting tick-acaricide control failure. Furthermore, approximately 50% of farms reported at least four tick control malpractices, which could have resulted in the emergence and spread of tick-acaricide control failure. Other sub-optimal tick control practices encountered included incorrect acaricide rotation, and failure to treat all cattle in a herd. This data will inform and guide the development of management strategies for tick-acaricide control failure and resistance in communal farming areas.

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.

Spatio-temporal analysis of sheep and goat pox outbreaks in Uganda during 2011-2022.

BACKGROUND: Sheep and goat pox (SGP) caused by sheep poxvirus (SPV) and goat poxvirus (GPV) respectively; are transboundary and World Organisation for Animal Health (WOAH)-notifiable viral diseases. There is barely any coherent information about the distribution and prevalence of SGP for Uganda. We therefore conducted this study to describe the temporal and spatial distribution of SGP suspected outbreaks in Uganda for the period 2011-2020 as well as serologically confirm presence of SGP antibodies in suspected SGP outbreaks reported in 2021-2022. RESULTS: Thirty-seven [37] SGP outbreaks were reported across the country during the study period. North-eastern region [that comprises of Karamoja region] had the highest number of outbreaks [n = 17, 45%]; followed by Central [n = 9, 2.4%], Northern [n = 8, 2.2%] and Western region [n = 3, 0.08%]. Reports from district veterinary personnel indicate that the prevalence of; and mortality rate and case fatality rate associated with SGP were 0.06%, 0.02% and 32% respectively. There was a steady increase in the number of reported SGP outbreaks [x̄ = 4] over the study period. Seropositivity of SGPV antibodies in outbreak sheep and goats that were investigated during the study period [2021-2022] was [n = 41, 27%, 95 CI;] CONCLUSION: Our analyses of SGPV passive and active reports indicate that SGP is present in Uganda with a decade long average of four outbreaks per annum. During this period, about a third of all SGPV-clinically infected animals died. SPG is therefore a major constraint to small ruminant health and productivity in Uganda. Introduction of animals from infected herds and breach in farm biosecurity were the most important predictors of SGP outbreaks. In addition to the already existing SGP commercial vaccines, small ruminant screening for SGPV before introducing them to naïve herds and ensuring on farm biosecurity should be part of the SGP control tool pack for Ugandan small ruminant farmers.

Control of ticks and tick-borne diseases in Africa through improved diagnosis and utilisation of data on acaricide resistance.

A meeting, sponsored by the Bill and Melinda Gates Foundation (BMGF) and organised by Clinglobal, was held at The International Livestock Research Institute (ILRI) in Nairobi, Kenya, from 19th - to 21st October 2022. The meeting assembled a unique group of experts on tick control in Africa. Academia, international agencies (FAO and ILRI), the private Animal Health sector and government veterinary services were represented. The significant outcomes included: (i) a shared commitment to standardisation and improvement of acaricide resistance bioassay protocols, particularly the widely used larval packet test (LPT); (ii) development of novel molecular assays for detecting acaricide resistance; (3) creation of platforms for disseminating acaricide resistance data to farmers, veterinary service providers and veterinary authorities to enable more rational evidence-based control of livestock ticks. Implementation of enhanced control will be facilitated by several recently established networks focused on control of parasites in Africa and globally, whose activities were presented at the meeting. These include a newly launched community of practice on management of livestock ticks, coordinated by FAO, an African module of the World Association for the Advancement of Veterinary Parasitology (WAAVP-AN) and the MAHABA (Managing Animal Health and Acaricides for a Better Africa) initiative of Elanco Animal Health.

Acaricide Resistance Development in Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) Populations against Amitraz and Deltamethrin on Communal Farms of the King Sabata Dalindyebo Municipality, South Africa.

Chemical acaricides are widely used to control ticks and tick-borne pathogens in cattle. However, prolonged and indiscriminate use of these chemicals inevitably leads to the selection of resistant ticks. In-vitro bioassays (adult and larval immersion tests) were conducted to assess amitraz and deltamethrin resistance in Rhipicephalus (Boophilus) microplus populations from communal farms of the King Sabata Dalindyebo municipality of South Africa. Data generated on percentage inhibition of oviposition (%IO) revealed that all the tick populations assessed showed resistance (%IO ≤ 95%) to at least one of the acaricides. All six tick populations assessed for efficacy (%IO ≥ 95%) at the DD) with deltamethrin were resistant (%IO ≤ 95%) and only one of the six tick populations assessed for efficacy with amitraz was susceptible. Based on the resistance ratios (RR), the adult immersion test detected amitraz and deltamethrin resistance in three (RR ranging from 2.30 to 3.21) and five (RR ranging from 4.10 to 14.59) of the six tick populations, respectively. With the larval immersion test, deltamethrin and amitraz resistance (larval mortality < 90% at the DD) was detected in all four and three of four R. (B.) microplus populations assessed, respectively. These data are critical for the design of an effective and sustainable tick control strategy on the communal farms.

Prevalence of gastrointestinal parasites and molecular identification of beta-tubulin mutations associated with benzimidazole resistance in Haemonchus contortus in goats from selected districts of Uganda.

Gastrointestinal parasites are among the most economically important pathogens of small ruminants causing serious economic losses and animal welfare problems for the livestock industry worldwide. The emergence of anthelmintic resistant H. contortus in small ruminants is a serious problem because it undermines effective helminth control and results in reduced productivity. Little is known about resistance to benzimidazoles (BZ) in Haemonchus in goats and sheep in Uganda. The objective of this study was to determine the prevalence of gastrointestinal parasites and to identify the presence of benzimidazole resistance associated mutations in the ß-tubulin isotype 1 gene of Haemonchus contortus in goats from selected districts of Uganda. A total of 200 goats from 10 districts of Uganda slaughtered at Kalerwe abattoir in Kampala were sampled for H. contortus adult worms. Faecal samples were also collected to detect other intestinal parasites. Faecal microscopy and analysis were performed using flotation and sedimentation techniques. DNA was extracted from adult worms and PCR and sequencing of the ITS- 2 region and ß-tubulin isotype 1 gene performed to identify H. contortus species and to determine the presence of mutations associated with anthelmintic resistance respectively. Faecal microscopy showed that the most prevalent intestinal parasites were coccidia (98%), strongyles (97.5%), Strongyloides (82%), Paramphistomum (74.5%), Moniezia (46%), Fasciola (1.5%) and Trichuris (1%). Most goats had a high intestinal burden of coccidia (≥ 5000 oocyst per gram) and strongyles (≥ 1000 egg per gram), 65% and 67.5%, respectively. The prevalence of H. contortus adult worms was 63% (126/200). Sequencing of the partial ß-tubulin isotype 1 gene of 54 Haemonchus contortus adult male isolates revealed the presence of mutations associated with anthelmintic resistance. The F200Y mutation was the most common mutation (13% of samples with good beta-tubulin sequences) followed by the E198A and E198K mutations, both found in 9% of sequenced samples. Mutation F167Y was not identified in any of the samples and there were no heterozygous individuals for any of the SNPS associated with BZ resistance that were identified in this study. These findings highlight the need for controlled use of anthelmintics especially benzimidazoles, to enable sustainable control of H. contortus in Uganda, and a need for further investigation to understand the resistance of other parasites identified in this study.

In Vitro Anti-Venom Potentials of Aqueous Extract and Oils of Toona ciliata M. Roem against Cobra Venom and Chemical Constituents of Oils.

There are high mortality and morbidity rates from poisonous snakebites globally. Many medicinal plants are locally used for snakebite treatment in Uganda. This study aimed to determine the in vitro anti-venom activities of aqueous extract and oils of Toona ciliata against Naja melanoleuca venom. A mixture of venom and extract was administered intramuscularly in rats. Anticoagulant, antiphospholipase A2 (PLA2) inhibition assay, and gel electrophoresis for anti-venom activities of oils were done. The chemical constituents of the oils of ciliata were identified using Gas chromatography-tandem mass spectroscopy (GC-MS/MS). The LD50 of the venom was 0.168 ± 0.21 µg/g. The venom and aqueous extract mixture (1.25 µg/g and 3.5 mg/g) did not cause any rat mortality, while the control with venom only (1.25 µg/g) caused death in 1 h. The aqueous extract of T. ciliata inhibited the anticoagulation activity of N. melanoleuca venom from 18.58 min. to 4.83 min and reduced the hemolytic halo diameter from 24 to 22 mm. SDS-PAGE gel electrophoresis showed that oils completely cleared venom proteins. GC-MS/MS analysis showed that the oils had sesquiterpene hydrocarbons (60%) in the volatile oil (VO) and oxygenated sesquiterpenes (48.89%) in the non-volatile oils (NVO). Some major compounds reported for the first time in T. ciliata NVOs were: Rutamarin (52.55%), ß-Himachalol (9.53%), Girinimbine (6.68%) and Oprea1 (6.24%). Most compounds in the VO were reported for the first time in T. ciliata, including the major ones Santalene (8.55%) and Himachal-7-ol (6.69%). The result showed that aqueous extract and oils of T. ciliata have anti-venom/procoagulant activities and completely neutralized the venom. We recommend a study on isolation and testing the pure compounds against the same venom.

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.

Molecular Characterization of Octopamine/Tyramine Receptor Gene of Amitraz-Resistant Rhipicephalus (Boophilus) decoloratus Ticks from Uganda.

We previously reported the emergence of amitraz-resistant Rhipicephalus (Boophilus) decoloratus ticks in the western region of Uganda. This study characterized the octopamine/tyramine receptor gene (OCT/Tyr) of amitraz-resistant and -susceptible R. (B.) decoloratus ticks from four regions of Uganda. The OCT/Tyr gene was amplified from genomic DNA of 17 R. (B.) decoloratus larval populations of known susceptibility to amitraz. The amplicons were purified, cloned and sequenced to determine mutations in the partial coding region of the OCT/Tyr gene. The amplified R. (B.) decoloratus OCT/Tyr gene was 91-100% identical to the R. (B.) microplus OCT/Tyr gene. Up to 24 single nucleotide polymorphisms (SNPs) were found in the OCT/Tyr gene from ticks obtained from high acaricide pressure areas, compared to 8 from the low acaricide pressure areas. A total of eight amino acid mutations were recorded in the partial OCT/Tyr gene from ticks from the western region, and four of them were associated with amitraz-resistant tick populations. The amino acid mutations M1G, L16F, D41G and V72A were associated with phenotypic resistance to amitraz with no specific pattern. Phylogenetic analysis revealed that the OCT/Tyr gene sequence from this study clustered into two distinct groups that separated the genotype from high acaricide pressure areas from the susceptible populations. In conclusion, this study is the first to characterize the R. (B.) decoloratus OCT/Tyr receptor gene and reports four novel amino acid mutations associated with phenotypic amitraz resistance in Uganda. However, lack of mutations in the ORF of the OCT/Tyr gene fragment for some of the amitraz-resistant R. (B.) decoloratus ticks could suggest that other mechanisms of resistance may be responsible for amitraz resistance, hence the need for further investigation.

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.

Population structure, molecular characterization, and phylogenetic analysis of Fasciola gigantica from two locations in Uganda.

Fasciola gigantica is a major pathogen that causes fasciolosis in Africa. A recent study in Uganda demonstrated that Fasciola flukes were present in 65.7% of slaughtered cattle. However, molecular identification of Fasciola species has not yet been performed in the country. In the present study, 292 Fasciola flukes were collected from Kampala and Gulu, Uganda. The samples were identified as F. gigantica using a multiplex polymerase chain reaction (PCR) assay for phosphoenolpyruvate carboxykinase (pepck) and a PCR-restriction fragment length polymorphism (RFLP) assay for DNA polymerase delta (pold). A significant genetic difference between F. gigantica obtained from cattle slaughtered at Kampala and Gulu was observed by analyzing the mitochondrial markers NADH dehydrogenase subunit 1 (nad1) and cytochrome C oxidase subunit 1 (cox1). Fasciola collected from Gulu had a more diversified population than that collected from Kampala, probably because of differences in livestock management systems. One of the possible reasons for this observation is that cattle slaughtered in Gulu were reared under an extensive communal grazing system, which is suitable for maintaining parasite diversity, whereas cattle slaughtered in Kampala mainly originated from fenced/closed farms, which limits parasite diversity. However, the cause of the difference between these two locations was not clearly defined by the results of this study. The F. gigantica population from Uganda was related to that obtained from Zambia. A star-like phylogeny was detected in a median-joining network analysis, which indicated rapid population expansion and suggested that the F. gigantica populations from both countries are maintained by domestic ruminants in eastern Africa. Interestingly, the F. gigantica population from Uganda was not related to those from Egypt and Nigeria. The results of the present study suggest that F. gigantica populations in African countries are indigenous to each country or region.

Development of acaricide resistance in tick populations of cattle: A systematic review and meta-analysis.

The development of acaricide resistance in ticks infesting cattle is a major problem in the livestock industry in tropical and subtropical regions worldwide. To determine the current global trends and prevalence of acaricide resistance development (ARD) in tick populations of cattle, a systematic review and meta-analysis with an emphasis on Rhipicephalus (Boophilus) microplus was conducted. Data searches from five English electronic databases yielded 88 journal articles published between 1992 and 2020. In total, 218 in - vitro bioassays were used to investigate 3939 tick populations of cattle; of these, the 57.6% that exhibited ARD were largely limited to South America (Brazil), Central America (Mexico), and Asia (India). A total of 3391 of these tick populations were R. (B.) microplus, of which 2013 exhibited ARD. Random effects meta-analyses indicated that the exhibition of ARD was higher in R. (B.) microplus (66.2%) than in other tick species. Global prevalence estimates of ARD in R. (B.) microplus vary as a function of geography, detection methods, and acaricide compounds. In general, high heterogeneity was noted among the studies. However, homogeneity was observed among studies from India, suggesting the establishment of acaricide resistance in Indian R. (B.) microplus populations. Current tick control interventions are urgently required to limit the evolution and implications of resistance development.

Continuing Education of Animal Health Professionals in Uganda: A Training Needs Assessment.

In Uganda, delivery of veterinary services is vital to animal health and productivity, and is heavily dependent on well-trained and skilled animal health professionals. The purpose of this study was to identify and prioritize areas for refresher training and continuous professional development of animal health professionals (veterinarians and veterinary paraprofessionals), with the overarching aim of improving veterinary service delivery in Uganda. A survey was administered electronically to 311 animal health professionals during the period November 14-30, 2019. Data were collected on relevant parameters including demographics, knowledge on preventive medicine, diagnostics, disease control and treatment, epidemiology, and One Health, as well as participants' opinions on training priorities, challenges faced, and constraints to veterinary service delivery. Most respondents were veterinarians 26-35 years old, were male, and worked in clinical practice. Lowest perceived knowledge was reported on subjects relating to laboratory diagnostics, antimicrobial resistance (AMR), and nutrition. Training topics considered to be of most benefit to respondents included laboratory diagnostics, treatment of common livestock diseases, AMR, and practical clinical skills in reproductive and preventive medicine. Participants preferred to receive training in the form of practical workshops, in-practice training, and external training. This study highlights the need to prioritize training in practical clinical skills, laboratory diagnostics, and AMR. Wet labs and hands-on practical clinical and laboratory skills should be incorporated to enhance training. Provision of targeted and successful trainings will be dependent on the allocation of adequate resources and support by relevant public and private stakeholders across the veterinary sector.

Molecular survey of cattle ticks in Burundi: First report on the presence of the invasive Rhipicephalus microplus tick.

A recent research study on prevalence of tick-borne pathogens in Burundi reported high prevalence and endemicity of Theileria parva, Anaplasma marginale and Babesia bigemina infections in cattle. Detailed information about tick species infesting animals, their distribution and genetic diversity in Burundi is outdated and limited. This study therefore assessed the prevalence and genetic diversity of tick species infesting cattle across agroecological zones (AEZs) in Burundi. A cross-sectional study on the occurrence of tick species was conducted in 24 districts of Burundi between October and December 2017. Differential identification and characterization of ticks collected was conducted using tick morphological keys and molecular tools (cox1 and 12S rRNA gene). Chi-square test was used to test for association between agroecological zones and the prevalence of tick species. Phylogenetic relationships were inferred using bayesian and maximum likelihood algorithms. A total of 483 ticks were collected from the five AEZs sampled. Six tick species comprising of Rhipicephalus appendiculatus, R. sanguineus, R. evertsi evertsi, R. microplus, R. decoloratus and Amblyomma variegatum were observed. Rhipicephalus appendiculatus were the most prevalent ticks (~45%). A total of 138 specimens (28%) were found to be Rhipicephalus microplus, suggesting an emerging threat for cattle farmers. Twelve R. appendiculatus cox1 haplotypes were obtained from 106 specimens that were sequenced. Two cox1 haplotypes of R. microplus which clustered into previously reported Clade A were observed. Rhipicephalus sanguineus and R. evertsi evertsi ticks, the vectors of numerous zoonotic pathogens, were collected from cattle, which constitute a high risk for public health. These findings reveal an overlapping distribution of tick vectors in Burundi. The design of ticks and tick-borne diseases control strategies should consider the distribution of different vectors across the AEZs particularly the presence of the highly invasive R. microplus tick in Burundi and the potential risk of introducing the pathogenic Babesia bovis.

A Survey of Priority Livestock Diseases and Laboratory Diagnostic Needs of Animal Health Professionals and Farmers in Uganda.

Background: Despite the investments made in veterinary diagnostic laboratory service delivery in Uganda, the scope and level of utilization remains low. This study aimed to determine the priority livestock diseases for which farmers and animal health professionals require veterinary diagnostic laboratory services, document the perceptions and opinions of key stakeholders on veterinary diagnostic laboratory services, and determine the factors that influence the delivery and utilization of animal disease diagnostic services in Uganda. Methods: A qualitative study approach involving a survey and key informant interviews was used to collect relevant data from four stakeholder groups: animal health workers, laboratory technologists and technicians, farmers, and key informants. The survey data were exported to excel, and descriptive statistics performed. The key informant interview recordings were transcribed, and thematic analysis performed. Results: The most reported diseases and conditions for which diagnostic services were needed were hemoparasites (including East Coast fever, anaplasmosis, babesiosis, and trypanosomosis), viral (including Foot and mouth disease, lumpy skin disease, rift valley fever, and papillomatosis), bacteria (including brucellosis, colibacillosis, anthrax, leptospirosis, and paratuberculosis) and protozoa diseases (coccidiosis), endoparasites (helminths), and mastitis. The most common diagnostic laboratory tests requested by clients, but laboratories were unable to provide included: rapid tests for contagious bovine pleuropneumonia, Foot and mouth disease, Newcastle disease, acaricide analysis, culture and antimicrobial sensitivity test, serology, and complete blood count. The most frequently reported challenges to providing diagnostic laboratory services were poor or lack of relevant equipment, insufficient or lack of supplies and reagents, high cost of reagents, inadequate or lack of laboratory staff to perform tests, and inadequate training of laboratory staff. Conclusions: This study highlighted the need to improve provision of laboratory diagnostic services to meet the prioritized diagnostic needs of farmers and animal health professionals. Increased intersectoral engagement and funding support from the private, industry, and government sectors is necessary to help address the observed challenges to provision of diagnostic laboratory services, including equipping of the laboratories, provision of supplies, and hiring and training of laboratory staff. Finally, the findings also suggest that the education of farmers and animal health workers on the value and benefits of laboratory diagnostic services may contribute to increase in sample submission and subsequent demand for diagnostic laboratory services.

Molecular detection of selected tick-borne pathogens infecting cattle at the wildlife-livestock interface of Queen Elizabeth National Park in Kasese District, Uganda.

In Uganda, ticks and tick-borne diseases (TBDs) pose a big challenge to farmers. They reduce cattle productivity and cause severe economic damage. Several studies have documented the prevalence of tick-borne pathogens in cattle; however, their genetic characteristics and the role of wildlife-livestock interaction in the epidemiology of the TBDs are not well documented. This study assessed the prevalence and genetic diversity of various tick-borne pathogens (TBPs) as well as the risk factors associated with the occurrence of TBPs in blood samples of 208 randomly selected cattle from 16 farms located around Queen Elizabeth National Park (QENP) in Kasese District in western Uganda. Farming practices, disease challenges, and the level of wildlife-livestock interactions were assessed by a questionnaire survey amongst farm owners. Polymerase chain reaction (PCR) assays revealed that 62.9% (131/208) cattle samples were positive for one or more pathogens. Using specific PCR assays, we detected Theileria spp., Theileria parva, Anaplasma marginale, Anaplasma platys-like, and Babesia bigemina at 50.5%, 27.9%, 19.2%, 11.5% and 8.7%, respectively. We also confirmed the infection of samples by Theileria velifera and Theileria mutans after sequencing the Theileria spp. 18S rRNA gene. The risk factors associated with the occurrence of TBPs included communal grazing, herd size, age, and proximity to QENP. Phylogenetic analysis of the T. parva p104 gene showed a high identity to the previous isolates from Uganda and other East African countries and clustered closer to the buffalo (Syncerus caffer) isolates, suggesting a possible cross-species transmission. The sequences of A. marginale groEL and B. bigemina RAP-1a formed well-supported clades with high identities to the previous isolates identified from central and eastern Uganda. The isolates obtained from A. phagocytophilum 16S rRNA gene sequences showed relationship with A. platys-like, Anaplasma sp., uncultured Anaplasma species and A. phagocytophilum isolates from Africa, Asia, Europe, and the USA. The findings of the present study showed that TBDs are still a burden to farmers and that management practices in this area may increase the transmission of pathogens between livestock and wildlife.

A Survey of Tick Infestation and Tick-Borne Piroplasm Infection of Cattle in Oudalan and Séno Provinces, Northern Burkina Faso.

In this study, cattle farms located in Oudalan and Séno, two provinces in the Sahel region, northern Burkina Faso, were surveyed. Cattle owners were interviewed, cattle were examined for tick infestation, and ticks as well as blood samples were collected during the dry season (October). Blood DNA samples were tested for Babesia and Theileria infections using nested PCRs and sequencing. A total of 22 herds, 174 Zebu cattle were investigated at 6 different sites. Overall, 76 cattle (43.7 %) from 18 farms (81.8%) were found infested with ticks. Cattle in Séno, adult cattle (>5 years) and those owned by the Fulani ethnic group were significantly (p < 0.05) more likely to be tick-infested. A total of 144 adult ticks belonging to five species namely: Hyalomma impeltatum, Hyalomma impressum, Hyalomma rufipes, Rhipicephalus evertsi evertsi, and Rhipicephalus guilhoni were collected from the animals. Piroplasms were detected in the blood DNA of 23 (13.2%) cattle. The cattle in Séno and adult cattle were significantly more likely to be piroplasm-positive. Five pathogens diversely distributed were identified. Theileria mutans (12/174), Babesia bigemina (5/174), Theileria annulata (3/174), and Theileria velifera (3/174) were detected for the first time in northern Burkina Faso, whereas Babesia occultans (1/174) was found for the first time in cattle in West Africa. The analysis of the sequences, including B. bigemina RAP-1a, T. annulata Tams1 genes, and the 18S rRNA genes of all the five protozoa, revealed identities ranging from 98.4 to 100% with previously published sequences. Phylogenetic analysis based on the 18S rRNA gene sequences located north Burkina Faso piroplasms in the same clade as isolates from Africa and other regions of the world. Notably, T. mutans sequences were distributed in two clades: the T. mutans Intona strain clade and the Theileria sp. (strain MSD)/ Theileria sp. B15a clade, suggesting the presence of at least two strains in the area. These findings indicate that the control of ticks and tick-borne diseases should be taken into account in strategies to improve animal health in the Sahel region.