Reflections on Drivers for the Emergence and Spread of Antimicrobial Resistant Bacteria Detected from Chickens reared on Commercial Layer Farms in Mukono District, Uganda.

Purpose: We investigated the fecal carriage of antimicrobial resistant Escherichia coli and potential practices influencing antimicrobial resistance (AMR) dynamics among poultry farm settings in Mukono District, Uganda. Methods: Twenty-nine commercial layer farms were visited and samples collected from 10 birds. The samples were then subjected to culture and sensitivity testing. The investigative framework for antimicrobial stewardship practices (IFAP) was used as a participatory tool to generate data through interviews and observations on antimicrobial use, drivers for use, players, and actions following non-responsive treatment outcomes. Results: The cultures done on 290 cloacal swabs yielded a total of 273 Escherichia coli isolates (94.1% recovery rate) which were tested in vitro for their sensitivity to different antibiotics. The prevalence of multi-drug resistant E. coli was 59.3% (162/273). A high prevalence of resistance to tetracycline (91.6%, n = 250) and trimethoprim sulphamethoxazole (70.3%, n = 192) was noted. In this collection of isolates, the prevalence of molecular determinants associated with the predominant phenotypes was; tetA (79.3%; 138/174), tetB (17.2%; 30/174), tetC (7.5%; 13/174), sul1 (11.5%; 20/174), and sul2 (60.3%; 105/174). Responses derived using the IFAP revealed several vices related to misuse and overuse of antibiotics, a threat to the poultry industry. The farmers also reported habits of selling off sick birds for slaughter when treatment outcomes were non-responsive. Such a practice could drive dissemination of antimicrobial resistant organisms and antibiotic residues to the consumers of those poultry products. Conclusion: The IFAP tool was useful and can be modified, and adopted for use in engaging agricultural communities in participatory AMR surveillance. A high carriage of multi-drug resistant E. coli was detected in the birds. On these farms, the worrying antimicrobial stewardship practices discovered could be sponsoring the emergence and spread of antimicrobial resistant bacteria in the Ugandan context.

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.

A community approach for pathogens and their arthropod vectors (ticks and fleas) in cats of sub-Saharan Africa.

BACKGROUND: Arthropod-borne pathogens and their vectors are present throughout Africa. They have been well studied in livestock of sub-Saharan Africa, but poorly studied in companion animals. Given their socioeconomic importance, the African Small Companion Animal Network (AFSCAN), as part of the WSAVA Foundation, initiated a standardized multi-country surveillance study. METHODS: In six countries (Ghana, Kenya, Nigeria, Tanzania, Uganda, and Namibia) in both rural and urban settings, 160 infested cats were sampled to assess their ectoparasite community (ticks and fleas), as well as the micro-parasite prevalence within those ectoparasites (60 and 118 pools of ticks and fleas, respectively) and blood (276 cats, including 116 non-infested). RESULTS: Almost two thirds of all infested cats originated from Tanzania and Kenya. Despite the large macro-geographical variation, no consistent difference was found in ectoparasite diversity and numbers between East and West Africa. Far more flea-infested than tick-infested cats were found. The most dominant ectoparasite was Ctenocephalides felis. Among the ticks, the exophilic Haemaphysalis spp. were the commonest, including species that are not typically linked with companion animals (Haemaphysalis spinulosa and Haemaphysalis elliptica). The most prevalent pathogens found in the blood and fleas were Bartonella henselae and Mycoplasma haemofelis. In the ticks, the dog-associated Hepatozoon canis was most commonly found. A high degree of co-parasitism was found in all countries and habitats. CONCLUSIONS: Our continent-wide standardized field study highlights the cat's potential to serve as a reservoir of pathogens that can be transmitted to humans or livestock, especially when cats are expected to become more commonly kept in African villages and towns.

A community approach of pathogens and their arthropod vectors (ticks and fleas) in dogs of African Sub-Sahara.

BACKGROUND: Arthropod-borne pathogens and their vectors are present throughout Africa. They have been well-studied in livestock of sub-Saharan Africa, but poorly in companion animals. Given the socio-economic importance of companion animals, the African Small Companion Animal Network (AFSCAN), as part of the WSAVA Foundation, initiated a standardized multi-country surveillance study. METHODS: Macro-geographic variation in ectoparasite (ticks and fleas) and pathogen communities in dogs was assessed through molecular screening of approximately 100 infested dogs in each of six countries (Ghana, Kenya, Nigeria, Tanzania, Uganda and Namibia), both in rural and urban settings. The most important intrinsic and extrinsic risk factors within the subpopulation of infested dogs were evaluated. RESULTS: Despite the large macro-geographic variation in the dogs screened, there was no consistent difference between East and West Africa in terms of the diversity and numbers of ticks. The highest and lowest numbers of ticks were found in Nigeria and Namibia, respectively. Most often, there was a higher diversity of ticks in rural habitats than in urban habitats, although the highest diversity was observed in an urban Uganda setting. With the exception of Namibia, more fleas were collected in rural areas. We identified tick species (including Haemaphysalis spinulosa) as well as zoonotic pathogens (Coxiella burnetti, Trypanosoma spp.) that are not classically associated with companion animals. Rhipicephalus sanguineus was the most abundant tick, with a preference for urban areas. Exophilic ticks, such as Haemaphysalis spp., were more often found in rural areas. Several multi-host ticks occurred in urban areas. For R. sanguineus, housing conditions and additional pets were relevant factors in terms of infestation, while for a rural tick species (Haemaphysalis elliptica), free-roaming dogs were more often infested. Tick occurrence was associated to the use of endoparasiticide, but not to the use of ectoparasiticide. The most prevalent tick-borne pathogen was Hepatozoon canis followed by Ehrlichia canis. High levels of co-parasitism were observed in all countries and habitats. CONCLUSIONS: As dogs share a common environment with people, they have the potential to extend the network of pathogen transmission to humans. Our study will help epidemiologists to provide recommendations for surveillance and prevention of pathogens in dogs and humans.

Epidemiological Dynamics of Extended-Spectrum ß-Lactamase- or AmpC ß-Lactamase-Producing Escherichia coli Screened in Apparently Healthy Chickens in Uganda.

The dynamics of extended-spectrum ß-lactamase- (ESBL-) and AmpC ß-lactamase-producing bacteria (which are deadly groups of antimicrobial-resistant bacteria) have not been well understood in developing countries. This raises major concerns to antimicrobial resistance (AMR) control. We investigated the prevalence and factors linked to the fecal carriage of ESBL- or AmpC-producing Escherichia coli (ESBL-/AmpC-EC) in commercial chickens. Cloacal swabs from 400 birds were sampled and submitted to the Central Diagnostic Laboratory for ESBL-/AmpC-EC screening by culture methods using MacConkey agar supplemented with cefotaxime. Epidemiological data were collected using a structured questionnaire and plausible risk factor analyses prepared by R software using X 2 test and logistic regression modeling. Results showed that the prevalence of ESBL-/AmpC-EC was 17.5%. Univariable screening hypothesized that carriage was probably influenced by a type of commercial chicken, geographical location, age group, flock size, and housing system (p < 0.05). Modeling exposed that broiler birds were at a higher risk of being ESBL-/AmpC-EC carriers (COR = 9.82, CI = 3.85-25.07). Birds from Wakiso Town Council (COR = 4.89, CI = 2.04-11.72) and flocks of 700-1200 birds were also at a higher risk of harboring ESBL-/AmpC-EC (COR = 2.41, CI = 1.11-5.23). Birds aged 4 months and below were more susceptible to ESBL-/AmpC-EC carriage compared with those aged 1 month and below being 6.33 times (CI = 1.65-24.35) likely to be carriers. The occurrence of ESBL-/AmpC-EC in flocks suggests possible treatment failures while managing colibacillosis. Consequently, injudicious antimicrobial use should be replaced with an accurate diagnosis by bacterial culture and sensitivity testing so as to circumvent AMR emergence, spread, and associated losses.

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 retrospective analysis of antimicrobial resistance in pathogenic Escherichia coli and Salmonella spp. isolates from poultry in Uganda.

There are increasing reports of antimicrobial treatment failures for bacterial diseases of poultry in Uganda. The paucity of data on antimicrobial resistance (AMR) of pathogenic bacteria in Uganda is a major setback to AMR control. This study investigated the occurrence of fowl typhoid, colibacillosis, and AMR in associated pathogens from 2012 to 2018. Laboratory records from the Central Diagnostic Laboratory (CDL), a National Veterinary Diagnostic Facility located at Makerere University, were reviewed. Archived isolates of the causative bacteria for the two diseases were also evaluated for AMR. The frequencies of the two disease conditions, their clinical and necropsy presentations and the demographic data of the diagnostic samples were summarized from the records. Archived bacterial isolates were revived before antimicrobial susceptibility testing. This was done on Mueller Hinton agar using the disk diffusion method, against 16 antimicrobials of medical and veterinary importance according to the Clinical Laboratory Standards Institute guidelines. A total of 697 poultry cases were presented for bacteriological investigations in the review period. Colibacillosis and salmonellosis had prevalence rates of 39.7% (277/697) and 16.2% (113/697), respectively. A total of 63 and 92 isolates of Escherichia coli and Salmonella spp., respectively, were archived but 43 (68.3%) E. coli and 47 (51.1%) Salmonella spp. isolates were recovered and evaluated for AMR. Multidrug resistance was more frequent in E. coli (38; 88.4%) than salmonellae (25; 53.2%), (p < 0.001). The high prevalence of colibacillosis, salmonellosis and the AMR of associated pathogens warrants immediate institution of appropriate disease control measures.

Molecular epidemiology of anaplasmosis in small ruminants along a human-livestock-wildlife interface in Uganda.

BACKGROUND: Information as regards the epidemiology of the Anaplasmataceae in small ruminants in several low- and middle-income countries is scarce. METHODS: In this study a total of 712 DNA samples collected from small ruminants were analyzed for Anaplasmataceae and Anaplasma ovis using the 16S rRNA and MSP4 genes respectively. Infection risk was assessed by location, sex and age of the animals and qGIS® was used to construct spatial maps. RESULTS: The prevalence of Anaplasmataceae spp was 89.1% (95% CI: 77.5-95.9) and 79.1% (95% CI: 75.9-82.1) in ovines and caprines respectively (RR = 1.1, 95% CI: 1.0-1.3); higher than those previously reported in other eastern African countries. The prevalence of A. ovis was 26.1% and 25.4% for both ovines and caprines respectively with ovines showing significantly higher levels of infection than caprines (P < 0.05). The risk of Anaplasma ovis infections was not affected by age (OR = 1.2, 95% CI: 0.9-1.7) or sex (OR = 1.1, 95% CI: 0.6-2.0). Small ruminants located at the forest edge (<0.3 km) showed higher A. ovis prevalence than those found inland with infections present in the midland regions associated with increased agricultural activity. CONCLUSION: Anaplasma ovis remains a major challenge for small ruminant husbandry in Uganda and infections are under-reported. Policy efforts to prioritize management of Anaplasmataceae for small ruminant health would promote livestock productivity in vulnerable communities, improving livelihoods and ecosystem health.

First Molecular Detection of Babesia ovis, Theileria spp., Anaplasma spp., and Ehrlichia ruminantium in Goats from Western Uganda.

Ticks and tick-borne diseases are major impediments to livestock production. To date, there have been several studies on the prevalence of tick-borne pathogens (TBPs) in cattle, but very few studies have documented TBPs in goats in Uganda. In this study, polymerase chain reaction assays and sequence analysis of different molecular markers were used to assess the presence and genetic characteristics of TBPs in 201 goats from Kasese district in western Uganda. The risk factors associated with TBP infections were also analyzed. We detected Theileria spp. (13.4%), Anaplasma phagocytophilum (10.9%), Anaplasma ovis (5.5%), Babesia ovis (5.5%), and Ehrlichia ruminantium (0.5%). The sequences of B. ovis ssu rRNA and A. ovismsp4 genes showed some degree of diversity among the parasite isolates in this study. The E. ruminantium pCS20 sequence formed a well-supported clade with isolates from Amblyomma variegatum ticks from Uganda. Wildlife interaction, sampling location, low body condition score, tick infestation, and herd size were significantly associated with TBP infections in the goats. The findings in this study provide important information on the epidemiology of tick-borne pathogens in Uganda, and show that goats could be potential reservoirs for tick-borne pathogens.

First Molecular Detection and Characterization of Hemotropic Mycoplasma Species in Cattle and Goats from Uganda.

Hemoplasmas (hemotropic mycoplasmas) are small pleomorphic bacteria that parasitize the surface of red blood cells of several mammalian species including cattle, goats, and humans, causing infectious anemia. However, studies on hemoplasmas have been neglected and to date, there are no studies on bovine and caprine hemoplasmas in Uganda or the entire East African region. In this study, a polymerase chain reaction (PCR) assay targeting the 16S rRNA gene was used to investigate the presence of hemoplasma in 409 samples (cattle = 208; goats = 201) collected from Kasese district, western Uganda. Results showed that 32.2% (67/208) of cattle samples and 43.8% (88/201) of goat samples were positive for hemoplasmas. Sequencing analysis identified Candidatus Mycoplasma haemobos and Mycoplasma wenyonii in cattle, while Candidatus Mycoplasma erythrocervae and Mycoplasma ovis were identified in goats. Statistical analysis showed that goats were at a higher risk of infection with hemoplasmas compared with cattle. To the best of our knowledge, this is the first molecular evidence of hemoplasmas in bovine and caprine animals in Uganda and the entire east African region.

Comparison of Tick Control and Antibiotic Use Practices at Farm Level in Regions of High and Low Acaricide Resistance in Uganda.

Uganda has experienced tick acaricide resistance in the livestock sector. With increase in incidence of tick-borne diseases (TBDs), use of antibiotics for control of TBDs and other opportunistic diseases has raisedserious concerns. The purpose of this study was to compare the farmers' tick control and antibiotic use practices on farms in regions of low (LARA) and high (HARA) tick acaricide resistance in Uganda, determine the prevalence of antibiotic residues in milk from both regions, and identify factors associated with antibiotic residues in milk. One representative district was selected from each region from which 10 farms were randomly selected. Delvotest SP-NT® test kit was used to detect antibiotic residues in milk. Half-body tick counts and acaricide efficacy tests were performed. Majority (70%) of HARA's respondents reported a corresponding increase in a monthly incidence of TBDs with an average of 3.2 cases of TBDs treated per farm compared to 0.2 cases in LARA. East Coast fever (ECF) was identified as the most common TBD in both regions, though cases of coinfection were more common in HARA. Half of HARA's respondents reported a corresponding increase in the use of antibiotics on their farms due to tick resistance compared to LARA. Antibiotics were the most used drugs on farms in both regions with oxytetracycline being the commonly used antibiotic. Ticks from HARA were resistant to deltamethrin, amitraz, and coformulation (chlorpyriphos and cypermethrin) while resistance against deltamethrin was confirmed in LARA. HARA farms had a significantly higher prevalence of antibiotic residues (21.25%) in raw milk than in LARA (4%) farms (p < 0.05). Acaricide resistance and practice of reading drug use instructions were significantly associated with antibiotic residues in milk at farm level. Overall, the study provides vital information linking acaricide resistance to antibiotic use practices, consequently leading to antibiotic residues in milk.

Inhibitory effects of novel ciprofloxacin derivatives on the growth of four Babesia species and Theileria equi.

The problems of parasite resistance, as well as the toxic residues to most of the commercially available antipiroplasmic drugs severely weaken their effective, curative, and environmental safe employment. Therefore, it is clear that the development of treatment options for piroplasmosis is vital for improving disease treatment and control. Ciprofloxacin is a broad-spectrum antibiotic that targets mainly the DNA replication machinery by inhibiting DNA gyrase and topoisomerase enzymes. As a result, ciprofloxacin is used for treating several bacterial and parasitic infections. In this study, the efficacy of 15 novel ciprofloxacin derivatives (NCD) that had been developed against drug-resistant Mycobacterium tuberculosis was evaluated against piroplasm parasite multiplication in vitro. The half-maximal inhibitory concentration (IC50) values of the most effective five compounds of NCD (No. 3, 5, 10, 14, 15) on Babesia bovis, Babesia bigemina, Babesia caballi, and Theileria equi were 32.9, 13.7, 14.9, and 30.9; 14.9, 25.8, 13.6, and 27.5; 34.9, 33.9, 21.1, and 22.3; 26.7, 28.3, 34.5, and 29.1; and 4.7, 26.6, 33.9, and 29.1 µM, respectively. Possible detrimental effects of tested NCD on host cells were assessed using mouse embryonic fibroblast (NIH/3T3) and Madin-Darby bovine kidney (MDBK) cell lines. Tested NCD did not suppress NIH/3T3 and MDBK cell viability, even at the highest concentration used (500 µM). Combination treatments of the identified most effective compounds of NCD/diminazene aceturate (DA), /atovaquone (AQ), and /clofazimine (CF) showed mainly synergistic and additive effects. The IC50 values of NCD showed that they are promising future candidates against piroplasmosis. Further in vivo trials are required to evaluate the therapeutic potential of NCD.

Evaluation of the inhibitory effect of ivermectin on the growth of Babesia and Theileria parasites in vitro and in vivo.

BACKGROUND: Treatment is the principle way to control and eliminate piroplasmosis. The search for new chemotherapy against Babesia and Theileria has become increasingly urgent due to parasite resistance to current drugs. Ivermectin (IVM) was the world's first endectocide, capable of killing a wide variety of parasites and vectors, both inside and outside the body. It is currently authorized to treat onchocerciasis, lymphatic filariasis, strongyloidiasis, and scabies. The current study documented the efficacy of IVM on the growth of Babesia and Theileria in vitro and in vivo. METHODS: The fluorescence-based assay was used for evaluating the inhibitory effect of IVM on four Babesia species, including B. bovis, B. bigemina, B. divergens, B. caballi, and Theileria equi, the combination with diminazene aceturate (DA), clofazimine (CF), and atovaquone (AQ) on in vitro cultures, and on the multiplication of a B. microti-infected mouse model. The cytotoxicity of compounds was tested on Madin-Darby bovine kidney (MDBK), mouse embryonic fibroblast (NIH/3 T3), and human foreskin fibroblast (HFF) cell lines. RESULTS: The half-maximal inhibitory concentration (IC50) values determined for IVM against B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi were 53.3 ± 4.8, 98.6 ± 5.7, 30.1 ± 2.2, 43.7 ± 3.7, and 90.1 ± 8.1 µM, respectively. Toxicity assays on MDBK, NIH/3 T3, and HFF cell lines showed that IVM affected the viability of cells with a half-maximal effective concentration (EC50) of 138.9 ± 4.9, 283.8 ± 3.6, and 287.5 ± 7.6 µM, respectively. In the in vivo experiment, IVM, when administered intraperitoneally at 4 mg/kg, significantly (p < 0.05) inhibited the growth of B. microti in mice by 63%. Furthermore, combination therapies of IVM-DA, IVM-AQ, and IVM-CF at a half dose reduced the peak parasitemia of B. microti by 83.7%, 76.5%, and 74.4%, respectively. Moreover, this study confirmed the absence of B. microti DNA in groups treated with combination chemotherapy of IVM + DA and IVM + AQ 49 days after infection. CONCLUSIONS: These findings suggest that IVM has the potential to be an alternative remedy for treating piroplasmosis.

Screening the Medicines for Malaria Venture Pathogen Box against piroplasm parasites.

Diminazene aceturate (DA) and imidocarb dipropionate are commonly used in livestock as antipiroplasm agents. However, toxic side effects are common in animals treated with these two drugs. Therefore, evaluations of novel therapeutic agents with high efficacy against piroplasm parasites and low toxicity to host animals are of paramount importance. In this study, the 400 compounds in the Pathogen Box provided by the Medicines for Malaria Venture foundation were screened against Babesia bovis, Babesia bigemina, Babesia caballi, and Theileria equi. A fluorescence-based method using SYBR Green 1 stain was used for initial in vitro screening and determination of the half maximal inhibitory concentration (IC50). The initial in vitro screening performed using a 1 µM concentration as baseline revealed nine effective compounds against four tested parasites. Two "hit" compounds, namely MMV021057 and MMV675968, that showed IC50 < 0.3 µM and a selectivity index (SI)> 100 were selected. The IC50s of MMV021057 and MMV675968 against B. bovis, B. bigemina, T. equi and B. caballi were 23, 39, 229, and 146 nM, and 2.9, 3, 25.7, and 2.9 nM, respectively. In addition, a combination of MMV021057 and DA showed additive or synergistic effects against four tested parasites, while combinations of MMV021057 with MMV675968 and of MMV675968 with DA showed antagonistic effects. In mice, treated with 50 mg/kg MMV021057 and 25 mg/kg MMV675968 inhibited the growth of Babesia microti by 54 and 64%, respectively, as compared to the untreated group on day 8. Interestingly, a combination treatment with 6.25 mg/kg DA and 25 mg/kg MMV021057 inhibited B. microti by 91.6%, which was a stronger inhibition than that by single treatments with 50 mg/kg MMV021057 and 25 mg/kg DA, which showed 54 and 83% inhibition, respectively. Our findings indicated that MMV021057, MMV675968, and the combination treatment with MMV021057 and DA are prospects for further development of antipiroplasm drugs.

Inhibitory effects of Syzygium aromaticum and Camellia sinensis methanolic extracts on the growth of Babesia and Theileria parasites.

Currently, chemotherapeutics against piroplasmosis are also associated with toxicity and the emergence of drug-resistant parasites. Therefore, the discovery of new drug compounds is necessary for the effective control of bovine and equine piroplasms. Syzygium aromaticum (clove) and Camellia sinensis (green tea) have several documented medicinal properties. In the present study, the growth-inhibiting effects of S. aromaticum and C. sinensis methanolic extracts were evaluated in vitro and in vivo. The half-maximal inhibitory concentration (IC50) values for methanolic S. aromaticum against Babesia bovis, B. bigemina, B. divergens, B. caballi, and Theileria equi were 109.8 ± 3.8, 8.7 ± 0.09, 76.4 ± 4.5, 19.6 ± 2.2, and 60 ± 7.3 µg/ml, respectively. Methanolic C. sinensis exhibited IC50 values of 114 ± 6.1, 71.3 ± 3.7, 35.9 ± 6.8, 32.7 ± 20.3, and 60.8 ± 7.9 µg/ml against B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi, respectively. The toxicity assay on Madin-Darby bovine kidney (MDBK), mouse embryonic fibroblast (NIH/3T3), and human foreskin fibroblast (HFF) cell lines showed that methanolic S. aromaticum and methanolic C. sinensis affected only the viability of the MDBK cell line with half-maximal effective concentrations (EC50) of 894.7 ± 4.9 and 473.7 ± 7.4 µg/ml, respectively, while the viability of NIH/3T3 and HFF cell lines was not affected even at 1000 µg/ml. In the in vivo experiment, methanolic S. aromaticum and methanolic C. sinensis oral treatments at 150 mg/kg inhibited the growth of Babesia microti in mice by 69.2% and 42.4%, respectively. These findings suggest that methanolic S. aromaticum and methanolic C. sinensis extracts have the potential as alternative remedies for treating piroplasmosis.

Low concentrations of Lactobacillus rhamnosus GG (Yoba®) are safe in male Drosophila melanogaster.

OBJECTIVE: The purpose of the study was to generate information on the safety of probiotics, thus the study objectives were to evaluate the effects of Yoba® on basic physiochemical properties. The study assessed male w1118 Drosophila melanogaster which were provided food supplemented with Yoba® at 1%, 3%, 6%, and 12% on motor function, total protein, catalase activity, and hydrogen peroxide scavenging activity and lifespan. RESULTS: Yoba® at high concentration (≥ 6%) increased locomotor activity in Drosophila melanogaster, however, total protein, catalase, and hydrogen peroxide scavenging activity were significantly higher at 1% Yoba® compared to 3%, 6%, and 12% Yoba®. Yoba consumed at 1% was associated with greater physiological benefits in Drosophila melanogaster. Findings in the study offer a rationale for the consumption of Yoba® at 1% in humans as is currently being promoted by the Yoba for Life consortium, however, high concentrations of Yoba® would disrupt physiological function as shown by this study.

The effects of trans-chalcone and chalcone 4 hydrate on the growth of Babesia and Theileria.

BACKGROUND: Chemotherapy is a principle tool for the control and prevention of piroplasmosis. The search for a new chemotherapy against Babesia and Theileria parasites has become increasingly urgent due to the toxic side effects of and developed resistance to the current drugs. Chalcones have attracted much attention due to their diverse biological activities. With the aim to discover new drugs and drug targets, in vitro and in vivo antibabesial activity of trans-chalcone (TC) and chalcone 4 hydrate (CH) alone and combined with diminazene aceturate (DA), clofazimine (CF) and atovaquone (AQ) were investigated. METHODOLOGY/PRINCIPAL FINDINGS: The fluorescence-based assay was used for evaluating the inhibitory effect of TC and CH on four Babesia species, including B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi, the combination with DA, CF, and AQ on in vitro cultures, and on the multiplication of a B. microti-infected mouse model. The cytotoxicity of compounds was tested on Madin-Darby bovine kidney (MDBK), mouse embryonic fibroblast (NIH/3T3), and human foreskin fibroblast (HFF) cell lines. The half maximal inhibitory concentration (IC50) values of TC and CH against B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi were 69.6 ± 2.3, 33.3 ± 1.2, 64.8 ± 2.5, 18.9 ± 1.7, and 14.3 ± 1.6 µM and 138.4 ± 4.4, 60.9 ± 1.1, 82.3 ± 2.3, 27.9 ± 1.2, and 19.2 ± 1.5 µM, respectively. In toxicity assays, TC and CH affected the viability of MDBK, NIH/3T3, and HFF cell lines the with half maximum effective concentration (EC50) values of 293.9 ± 2.9, 434.4 ± 2.7, and 498 ± 3.1 µM and 252.7 ± 1.7, 406.3 ± 9.7, and 466 ± 5.7 µM, respectively. In the mouse experiment, TC reduced the peak parasitemia of B. microti by 71.8% when administered intraperitoneally at 25 mg/kg. Combination therapies of TC-DA and TC-CF were more potent against B. microti infection in mice than their monotherapies. CONCLUSIONS/SIGNIFICANCE: In conclusion, both TC and CH inhibited the growth of Babesia and Theileria in vitro, and TC inhibited the growth of B. microti in vivo. Therefore, TC and CH could be candidates for the treatment of piroplasmosis after further studies.

Safety of Drinking Water from Primary Water Sources and Implications for the General Public in Uganda.

Background: There is scarcity of information about the quality and safety of drinking water in Africa. Without such vital information, sustainable development goal number 6 which promotes availability and sustainable management of water and sanitation remains elusive especially in developing countries. The study aimed at determining concentrations of inorganic compounds, estimated daily intake (EDI), target hazard quotient (THQ), hazard index (HI), incremental lifetime cancer risk (ILCR), and identify safe drinking water source sources in Southwestern Uganda. Methods: This was an observational study in which 40 drinking water samples were collected from georeferenced boreholes, springs, open wells, bottled, and taps within Bushenyi district of Southwestern Uganda. Water samples were analyzed for copper (Cu), iron (Fe), zinc (Zn), lead (Pb), cadmium (Cd), and chromium (Cr) levels using atomic absorption spectrometry (AAS). Water safety measures (EDI, HI, and ILCR) were established for each water source and compared with local and international water permissible standards for each analyte. A spatial map was drawn using qGIS®, and analysis of quantitative data was done using MS Excel 2013 at 95% significance. Results: Heavy metals were present in the following order: 11.276 ppm > 4.4623 ppm > 0.81 ppm > 0.612 ppm > 0.161 ppm for Fe, Zn, Pb, Cu, and Cd, respectively, while Cr was not detected. Fe was the primary water heavy metal in the order of open well > borehole > tap > spring > bottled water. This was followed by Zn levels in the order of tap > bottled > spring > borehole > open well. All compounds were within international water safety standards except Pb. Hence, there is need for the government of Uganda to establish water filtration systems, particularly for Pb to improve the quality of water for the general public. The EDI was similar (P > 0.05) for water consumed from spring, bottled, and tap sources for Fe and Zn levels. Similarly, no differences were found in the EDI for children and adults (P > 0.05). Furthermore, the HI showed an absence of noncarcinogenic risk associated (HI < 1), although the ILCR was higher in adults than children (P < 0.05) due to high Cd concentrations. Conclusion: The current identified Fe is a major heavy metal in drinking water of Uganda, and boreholes were the major safest sources of drinking water identified in this study.

Food Safety Analysis of Milk and Beef in Southwestern Uganda.

Background: Inorganic pollutants in milk and beef are of major public health concern; however, information in Africa is still limited due to low food safety monitoring practices. In this study, we established levels of lead (Pb), zinc (Zn), cadmium (Cd), copper (Cu), and iron (Fe) in milk and beef and obtained the estimated daily intake (EDI) and incremental lifetime cancer risk (ILCR) as measures of risk to the Ugandan population. Materials and Methods: This was a cross-sectional study in which a total of 40 samples of milk and beef were collected from Bushenyi district in southwestern Uganda. Samples were analyzed by atomic absorbance spectrophotometer, and the EDI and ILCR were computed using the US EPA reference values. Results and Discussion: Heavy metal concentrations were highest in the order of Zn > Fe > Pb > Cu in milk samples, while in beef samples, concentrations were highest in the order of Zn > Pb > Fe > Cu and no Cd was detected. Furthermore, beef had significantly higher (P < 0.05) Pb and Fe concentrations than milk. The EDI was highest in children, and this was followed by very high ILCR levels, showing that milk and beef are not safe for children in Uganda. Bearing in mind that a high HI was shown, beef and milk from these regions are not recommended for consumption especially by children although more studies remain to be conducted. Conclusion: Heavy metals in milk and beef of Uganda may predispose the indigenous community to cancer and other health-related illnesses, showing a need for improved food safety screening to promote food safety.

C190A knockdown mutation in sodium channel domain II of pyrethroid-resistant Rhipicephalus appendiculatus.

This study investigated the target site mutations in the partial sequence of voltage-sensitive sodium channel (VSSC) domain II of synthetic pyrethroid (SP)-resistant Rhipicephalus appendiculatus. Genomic DNA was extracted from seven tick populations (two susceptible and five resistant) collected from central, eastern and southwestern Uganda. The PCR amplicons of the VSSC domain II were cloned and sequenced to determine novel single nucleotide polymorphisms (SNP). A non-synonymous mutation C78 A corresponding to C190 A was found in all the five SP-resistant ticks. The C78 A mutation led to amino acid substitution from leucine to isoleucine (L21I) which was previously reported to confer knockdown (kdr) mutation in R. (Boophilus) microplus. The genetic confirmation of SP-resistant R. appendiculatus in central and southwestern Uganda calls for an urgent strategy for controlling the ticks.