Phenotypic and Genotypic Characterization of Extended Spectrum Beta-Lactamase-Producing Clinical Isolates of Escherichia coli and Klebsiella pneumoniae in Two Kenyan Facilities: A National Referral and a Level Five Hospital.

Background: The emergence of antimicrobial resistance (AMR) and multidrug resistance (MDR) among Escherichia coli and Klebsiella pneumoniae, especially through the production of extended spectrum ß-lactamases (ESBLs), limits therapeutic options and poses a significant public health threat. Objective: The aim of this study was to assess the phenotypic and genetic determinants of antimicrobial resistance of ESBL-producing Escherichia coli and Klebsiella pneumoniae isolates from patient samples in two Kenyan Hospitals. Methods: We collected 138 E. coli and 127 K. pneumoniae isolates from various clinical specimens at the two health facilities from January 2020 to February 2021. The isolates' ESBL production and antibiotic susceptibility were phenotypically confirmed using a standard procedure. Molecular analysis was done through conventional polymerase chain reaction (PCR) with appropriate primers for gadA, rpoB, blaTEM, blaSHV, blaOXA, blaCTX-M-group-1, blaCTX-M-group-2, blaCTX-M-group-9, and blaCTX-M-group-8/25 genes, sequencing and BLASTn analysis. Results: Most E. coli (82.6%) and K. pneumoniae (92.9%) isolates were ESBL producers, with the highest resistance was against ceftriaxone (69.6% among E. coli and 91.3% among K. pneumoniae) and amoxicillin/clavulanic acid (70.9% among K. pneumoniae). The frequency of MDR was 39.9% among E. coli and 13.4% among K. pneumoniae isolates. The commonest MDR phenotypes among the E. coli isolates were CRO-FEP-AZM-LVX and CRO-AZM-LVX, while the FOX-CRO-AMC-MI-TGC-FM, FOX-CRO-FEP-AMC-TZP-AZM-LVX-MI and CRO-AMC-TZP-AZM-MI were the most frequent among K. pneumoniae isolates. Notably, the FOX-CRO-FEP-AMC-TZP-AZM-LVX-MI phenotype was observed in ESBL-positive and ESBL-negative K. pneumoniae isolates. The most frequent ESBL genes were blaTEM (42%), blaSHV (40.6%), and blaOXA (36.2%) among E. coli, and blaTEM (89%), blaSHV (82.7%), blaOXA (76.4%), and blaCTX-M-group-1 (72.5%) were most frequent ESBL genes among K. pneumoniae isolates. The blaSHV and blaOXA and blaTEM genotypes were predominantly associated with FOX-CRO-FEP-MEM and CRO-FEP multidrug resistance (MDR) and CRO antimicrobial resistance (AMR) phenotypes, among E. coli isolates from Embu Level V (16.7%) and Kenyatta National Hospital (7.0%), respectively. Conclusions: The high proportion of ESBL-producing E. coli and K. pneumoniae isolates increases the utilization of last-resort antibiotics, jeopardizing antimicrobial chemotherapy. Furthermore, the antimicrobial resistance patterns exhibited towards extended-spectrum cephalosporins, beta-lactam/beta-lactamase inhibitor combinations, fluoroquinolones, and macrolides show the risk of co-resistance associated with ESBL-producing isolates responsible for MDR. Hence, there is a need for regular surveillance and implementation of infection prevention and control strategies and antimicrobial stewardship programs.

Antimicrobial Usage, Susceptibility Profiles, and Resistance Genes in Campylobacter Isolated from Cattle, Chicken, and Water Samples in Kajiado County, Kenya.

Campylobacter organisms are the major cause of bacterial gastroenteritis and diarrhoeal illness in man and livestock. Campylobacter is growingly becoming resistant to critically crucial antibiotics; thereby presenting public health challenge. This study aimed at establishing antimicrobial use, susceptibility profiles, and resistance genes in Campylobacter isolates recovered from chicken, cattle, and cattle-trough water samples. The study was conducted between October 2020 and May 2022 and involved the revival of cryopreserved Campylobacter isolates confirmed by PCR from a previous prevalence study in Kajiado County, Kenya. Data on antimicrobial use and animal health-seeking behaviour among livestock owners (from the same farms where sampling was done for the prevalence study) were collected through interview using a pretested semistructured questionnaire. One hundred and three isolates (29 C. coli (16 cattle isolates, 9 chicken isolates, and 4 water isolates) and 74 C. jejuni (38 cattle isolates, 30 chicken isolates, and 6 water isolates)) were assayed for phenotypic antibiotic susceptibility profile using the Kirby-Bauer disk diffusion method for ampicillin (AX), tetracycline (TE), gentamicin (GEN), erythromycin (E), ciprofloxacin (CIP), and nalidixic acid (NA). Furthermore, detection of genes conferring resistance to tetracyclines (tet (O), ß-lactams (bla OXA-61), aminoglycosides (aph-3-1), (fluoro)quinolones (gyrA), and multidrug efflux pump (cmeB) encoding resistance to multiple antibiotics was detected by mPCR and confirmed by DNA sequencing. The correlation between antibiotic use and resistance phenotypes was determined using the Pearson's correlation coefficient (r) method. Tetracyclines, aminoglycosides, and ß-lactam-based antibiotics were the most commonly used antimicrobials; with most farms generally reported using antimicrobials in chicken production systems than in cattle. The highest resistance amongst isolates was recorded in ampicillin (100%), followed by tetracycline (97.1%), erythromycin (75.7%), and ciprofloxacin (63.1%). Multidrug resistance (MDR) profile was observed in 99 of 103 (96.1%) isolates; with all the Campylobacter coli isolates displaying MDR. All chicken isolates (39/39, 100%) exhibited multidrug resistance. The AX-TE-E-CIP was the most common MDR pattern at 29.1%. The antibiotic resistance genes were detected as follows: tet (O), gyrA, cmeB, bla OXA-61 , and aph-3-1 genes were detected at 93.2%, 61.2%, 54.4%, 36.9%, and 22.3% of all Campylobacter isolates, respectively. The highest correlations were found between tet (O) and tetracycline-resistant phenotypes for C. coli (96.4%) and C. jejuni (95.8%). A moderate level of concordance was observed between the Kirby-Bauer disk diffusion method (phenotypic assay) and PCR (genotypic assay) for tetracycline in both C. coli (kappa coefficient = 0.65) and C. jejuni (kappa coefficient = 0.55). The study discloses relatively high resistance profiles and multidrug resistance to antibiotics of critical importance in humans. The evolution of the multidrug-resistantCampylobacter isolates has been linked to the use and misuse of antimicrobials. This poses a potential hazard to public and animal health, necessitating need to reduce the use of antibiotics in livestock husbandry practice coupled with stringent biosecurity measures to mitigate antimicrobial resistance.

Molecular Prevalence and Risk Factors of Campylobacter Infection in Puppies in the Nairobi Metropolitan Region, Kenya.

Campylobacter species are widely distributed pathogens; however, data on its epidemiology in puppies remain scanty, especially in Kenya. A cross-sectional study was conducted in the Nairobi Metropolitan Region to determine molecular prevalence and associated risk factors of Campylobacter species infection in puppies. A total of 260 rectal swabs were collected from puppies from breeding kennels, shelters, and the University of Nairobi Veterinary Teaching and Referral Hospital. The samples were subjected to polymerase chain reaction (PCR) assays for identification of Campylobacter species. Data on potential risk factors associated with puppy exposure were collected using a semistructured questionnaire. Multivariable mixed effects logistic regression analyses were performed with kennels as random effects. Campylobacter species were detected in 64 of the 260 sampled puppies yielding an overall prevalence of 24.6%. Multivariable results showed that puppies from shelters, puppies from kennels that are washed daily, puppies with a recent history of vomiting, and those treated with antibiotics in the past month were significantly associated with the presence of Campylobacter species. Being a kenneled puppy and having had concurrent bacterial infections were identified as protective factors. This study provides molecular evidence of puppy exposure to Campylobacter species which could have impact on puppy health and highlights the need to develop awareness and management strategies to potentially reduce the risk of transmitting this pathogen among puppies, to humans, and other animals.

Seroprevalence and spatial distribution of livestock brucellosis using three serological tests in Kajiado County, Kenya.

Background: Brucellosis is a serious zoonotic infection with a global socioeconomic impact on both the livestock industry and human health. In Kenya, brucellosis is endemic but there is limited information on the true burden of the disease due to weak or peace-meal surveillance. The true burden and spread of animal brucellosis in Kajiado County is not known. Aim: The aim of the study was to determine the current seroprevalence and spatial distribution of livestock brucellosis in Kajiado County and also to compare the three serological tests, namely; Rose Bengal plate test (RBPT), indirect enzyme-linked immunosorbent assay (i-ELISA), and competitive ELISA (c-ELISA) in the detection of seropositive animals. Methods: A cross-sectional study was undertaken in 5 sub-counties and 13 wards, where a total of 782 serum samples from unvaccinated bovine (n = 278; 34 herds), ovine (n = 256; 25 flocks), and caprine (n = 248; 28 flocks), were screened for anti-Brucella antibodies using RBPT, i-ELISA, and c-ELISA tests, in parallel. Results: Overall animal seroprevalence was 6.91% (54/782); while that for bovine, ovine, and caprine was 18.35% (51/278), 0.78% (2/256), and 0.4% (1/248), respectively. Bovine seroprevalence was 2.2% (6/278), 14.4% (40/278), and 4.7% (13/278) in RBPT, i-ELISA, and c-ELISA tests, respectively; while ovine 0.78% (2/256) and caprine 0.4% (1/248) were positive only in c-ELISA. Bovine herd seropositivity was 67.65% (23/34), whereas ovine and caprine flock seropositivities were 8% (2/25) and 3.6% (1/28), respectively. Conclusion: The findings indicate a moderate seroprevalence of brucellosis in bovine, while that of ovine and caprine was low in Kajiado County. Indirect ELISA was found superior to both c-ELISA and RBPT in detecting bovine seropositive animals, while c-ELISA was superior to both RBPT and i-ELISA in detecting seropositive ovines and caprines. These results will contribute to baseline data for further study of Brucella infection and a starting point for the formulation of a strategy for the control of brucellosis in Kajiado County.

Seasonal Prevalence and Molecular Identification of Thermophilic Campylobacter from Chicken, Cattle, and Respective Drinking Water in Kajiado County, Kenya.

Thermophilic Campylobacter species are a leading cause of human gastroenteritis throughout the world and have been implicated in reproductive disorders (abortion), mastitis, enteritis, and/or diarrhoea in livestock. A cross-sectional survey was conducted in Kajiado County to determine prevalence, seasonality, and molecular detection of thermophilic Campylobacter species (with emphasis on C. jejuni, C. coli, and other thermophilic Campylobacter species) in chicken, cattle, and respective pooled drinking water. A total of 457 samples comprising 265 cattle rectal swabs, 142 chicken cloacal swabs, and 50 trough water samples were collected from 55 randomly selected smallholder farms. Individual samples were subjected to standard techniques for isolation and biochemical tests, followed by singleplex polymerase chain reaction (sPCR) assays for identification and confirmation of genus and species. Overall, thermophilic Campylobacter prevalence was 35.4% (95% confidence interval (95% CI) = 31.0-39.8), with C. jejuni dominating at 55.6% (95% CI = 47.9-63.3%) over C. coli in all sample types. The highest thermophilic Campylobacter prevalence was observed in cloacal swabs of live chicken at 44.4% (95% CI = 36.2-52.6%), followed by rectal swabs from live cattle at 30.9% (95% CI = 25.3-36.5%). Water samples from cattle drinkers/trough were found to be contaminated at 34% (95% CI = 20.9-47.1%). The isolation rate was higher in cattle under the confinement system (44.3%) (95% CI = 36.1-52.5%) than in those under the free-roaming grazing system. Thermophilic Campylobacter species were isolated in both seasons, with higher prevalence (39.8% (95% CI = 33.6-45.9)) recorded during rainy and cold season in all sample types except for water. There was significant (P < 0.05) association between season and thermophilic Campylobacter occurrence, even though there were no statistical differences in the prevalence values across the two seasons. Results of this study demonstrate that cattle, chicken, and respective drinking water harbour potentially pathogenic thermophilic campylobacters, with C. jejuni being widely distributed among farms. It is possible that seasonal variations and cattle confinement result in differences in thermophilic Campylobacter carriage. Further epidemiological and phylogenetic studies comparing distribution of thermophilic Campylobacter spp. isolates in livestock, environmental, and human samples are recommended to establish source attribution to reduce the impact of resultant diseases for the wellbeing of public and livestock.

Molecular Detection and Characterization of Pasteurella multocida Infecting Camels in Marsabit and Turkana Counties, Kenya.

Pasteurella multocida infection is common in Kenya though there is little knowledge of the genetic diversity of the pathogen. P. multocida is part of the normal flora in the respiratory tract of camels, but it becomes pathogenic when the resistance of the camel body is diminished by bad ecological conditions. This study was conducted to detect, characterize, and determine the genetic diversity of P. multocida infecting camels in Marsabit and Turkana Counties. The KMT1 gene was targeted as the marker gene for P. multocida and hyaD-hyaC, bcbD, dcbF, ecbJ, and fcbD as marker genes for capsular serogroups A, B, D, E, and F, respectively. Out of 102 blood and 30 nasal swab samples, twenty-one samples (16%) were confirmed to be positive for P. multocida and only capsular group E was detected in both counties. The P. multocida sequences were highly conserved and were related to strains from other parts of the world. Our study has confirmed that camels in Marsabit and Turkana Counties of Kenya are infected by P. multocida of capsular type E. Farmers should not underfeed camels, ensure appropriate medication and vaccination programs, and minimize herding of camels in crowded areas especially in wet conditions in order to slow the spread of P. multocida infection.

Molecular Characterization of ESBLs and QnrS Producers From Selected Enterobacteriaceae Strains Isolated From Commercial Poultry Production Systems in Kiambu County, Kenya.

Background: The emergence and spread of Extended-spectrum ß-lactamases (ESBLs) in Enterobacteriaceae through the plasmid-mediated exchange have become a major threat to public health by complicating the treatment of severe infections in both animals and humans. Therefore, the current study focused on evaluating the manifestation of ESBLs production from the fecal isolates of E. coli, Shigella spp, Salmonella spp, and Klebsiella spps in commercial poultry production systems of Kiambu County, Kenya. Materials and methods: Out of 591 isolates identified as E. coli, Shigella spp, Salmonella spp, and Klebsiella spps from 437 fecal samples, only 78 were phenotypically suggestive to be ESBL producers. The possible ESBL producers were screened for the presence of blaTEM, blaCTX-M, blaOXA, and blaSHV using the PCR technique. These isolates were also screened for carriage of the QnrS gene that confers resistance to the fluoroquinolone class of drugs. Results: The most detected ESBL gene from the isolates was blaOXA (n = 20; 26%), followed by blaTEM (n = 16, 21%), with the majority of them detected in E. coli. The blaCTX-M was identified in all the 4 enteric's bacteria-type isolates tested. Three E. coli and Salmonella spp respectively were found to harbor all the 5 antimicrobial resistance (AMR) gene types. The blaTEM, blaOXA, blaSHV, and QnrS genes were not detected from Klebsiella and Shigella spps. Additionally, most of the AMR gene co-carriage was detected in both E. coli and Salmonella spps as follows blaTEM + blaOXA (n = 4); blaTEM + QnrS (n = 3); blaTEM + blaOXA + QnrS (n = 3), concurrently. Conclusion: Our findings highlight the significance of commercial poultry production in disseminating transferable antibiotic resistance genes that act as potential sources of extensive drug resistance in livestock, humans, and the environment, leaving limited therapeutic options in infection management.

Global research alliance in infectious disease: a collaborative effort to combat infectious diseases through dissemination of portable sequencing.

OBJECTIVE: To disseminate the portable sequencer MinION in developing countries for the main purpose of battling infectious diseases, we found a consortium called Global Research Alliance in Infectious Diseases (GRAID). By holding and inviting researchers both from developed and developing countries, we aim to train the participants with MinION's operations and foster a collaboration in infectious diseases researches. As a real-life example in which resources are limited, we describe here a result from a training course, a metagenomics analysis from two blood samples collected from a routine cattle surveillance in Kulan Progo District, Yogyakarta Province, Indonesia in 2019. RESULTS: One of the samples was successfully sequenced with enough sequencing yield for further analysis. After depleting the reads mapped to host DNA, the remaining reads were shown to map to Theileria orientalis using BLAST and OneCodex. Although the reads were also mapped to Clostridium botulinum, those were found to be artifacts derived from the cow genome. An effort to construct a consensus sequence was successful using a reference-based approach with Pomoxis. Hence, we concluded that the asymptomatic cow might be infected with T. orientalis and showed the usefulness of sequencing technology, specifically the MinION platform, in a developing country.

Beta hemolysin gene of Staphylococcus phage 3AJ_2017 genome is a suitable molecular marker for identification and characterization of pathogenic Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus cause diseases both in humans and animals. These diseases range from mild to fatal infections thus necessitating development of a specific molecular method for detection of pathogenic S. aureus. OBJECTIVES: To identify and analyze genetic profile of pathogenic S. aureus using bacteriophage based genetic biomarkers. METHODS: Using culture and biochemical methods, 148 S. aureus (87 %) were isolated from 170 raw milk samples taken from 10 dairy farms in Marsabit and Isiolo counties in Northern Kenya between June 2016 and February 2017. The samples were collected directly from dairy lactating cows previously diagnosed with S. aureus in a follow-up study. The isolates were analyzed by PCR and sequencing of beta hemolysin (hlb) gene. The genetic relationship between five Kenyan S. aureus isolates and five isolates previously identified was inferred. RESULTS: From the 96 isolates screened for hlb gene, 75 (78.1%) tested positive. Some of the positive isolates yielded a band size of 975 bp, while others 1100 bp. Through Basic Local Alignment Search Tool (BLAST) search analysis, the two different band sizes (975 bp and 1100 bp) were both confirmed to be hlb gene from S. aureus isolates indicating that the difference in band size may have been due to deletions that were detected in the 975 bp hlb gene. Some S. aureus isolates from Kenya appeared to be closely related to isolates from other parts of the world, while some showed a distant relationship. CONCLUSIONS: Phage-derived hlb gene is a suitable molecular marker for detection of pathogenic S. aureus.

Antimicrobial Usage and Detection of Multidrug-Resistant Staphylococcus aureus, Including Methicillin-Resistant Strains in Raw Milk of Livestock from Northern Kenya.

The association of antimicrobial usage (AMU) with prevalence of antimicrobial-resistant (AMR) Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA) in livestock raw milk consumed by pastoralists in Kenya remains unclear. We investigated the relationship between AMU and emergence of multidrug-resistant (MDR) S. aureus, including MRSA in raw milk of livestock. AMU data were obtained using sales records from veterinary pharmacies. S. aureus was isolated from 603 milk samples from various livestock species, including sheep, goat, cow, and camel reared in Isiolo and Marsabit counties in Kenya. Resistant phenotypes and genotypes were determined by disc diffusion and molecular methods, respectively. Correlation between AMU and occurrence of resistance was determined by Pearson's correlation coefficient (r) method. The consumption of various antimicrobial classes were as follows; 4,168 kg of oxytetracycline, 70 kg of sulfonamides, 49.7 kg of aminoglycosides, 46 kg of beta-lactams, 39.4 kg of macrolides, and 0.52 kg for trimethoprim. The S. aureus isolates were mainly resistant to tetracycline (79%), ampicillin (58%), and oxacillin (33%), respectively. A few isolates (5-18%) were resistant to clindamycin, cephalexin, erythromycin, kanamycin, and ciprofloxacin. Most of the MDR-S. aureus isolates were MRSA (94%). The genetic determinants found in the AMR isolates included tetK/tetM (96.5%/19%) for tetracycline, blaZ (79%) for penicillin, aac (6')/aph (2'')/aph (3')-IIIa (53%) for aminoglycosides, mecA (41%) for oxacillin, and msrA/ermA (24%/7%) for macrolides. Oxytetracycline usage was correlated to tetK/tetM (r = 0.62/1) detection, penicillins to mecA/blaZ (r = 0.86/0.98), aminoglycoside to aac (6')/aph (2'')/aph (3')-IIIa (r = 0.76/-13), and macrolide usages for detection of ermA/msrA (r = 0.94/0.77). AMU appeared to be associated with occurrence of MDR-SA and the tetM detection. Consumption of raw milk contaminated with MRSA could pose a serious public health risk in pastoral communities in northern Kenya.

Detection of Brucella spp. in raw milk from various livestock species raised under pastoral production systems in Isiolo and Marsabit Counties, northern Kenya.

INTRODUCTION: Brucellosis is an important zoonotic disease in Kenya, and identifying the bacteria in milk is important in assessing the risk of exposure in people. METHODS: A cross-sectional study that involved 175 households was implemented in the pastoral counties of Marsabit and Isiolo in Kenya. Pooled milk samples (n = 164) were collected at the household level, and another 372 were collected from domesticated lactating animals (312 goats, 7 sheep, 50 cattle and 3 camels). Real-time polymerase chain reaction (qPCR) testing of the milk samples was performed to identify Brucella species. Brucella anti-LPS IgG antibodies were also detected in bovine milk samples using an indirect enzyme-linked immunosorbent assay (ELISA). RESULTS: Based on the qPCR, the prevalence of the pathogen at the animal level (considering samples from individual animals) was 2.4% (95% confidence interval (CI) 1.1-4.5) and 3.0% (CI: 1.0-7.0) in pooled samples. All 14 samples found positive by qPCR were from goats, with 10 contaminated with B. abortus and 4 with B. melitensis. The Brucella spp. antibody prevalence in bovine milk using the milk ELISA was 26.0% (95% CI: 14.6-40.3) in individual animal samples and 46.3% (95% CI: 30.7-62.6) in pooled samples. CONCLUSION: The study is the first in Kenya to test for Brucella spp. directly from milk using qPCR without culturing for the bacteria. It also detected B. abortus in goats, suggesting transmission of brucellosis between cattle and goats. The high prevalence of Brucella spp. is a significant public health risk, and there is a need for intervention strategies necessary in the study area.

Investigation of the governance structure of the Nairobi dairy value chain and its influence on food safety.

The dairy value chain of Nairobi is comprised, in its majority, of small-scale independent enterprises that operate within a complex interlinked system. In this complexity, the coordination and power structures of the system may have major influences on the management of dairy food safety. Therefore, the aim of this study was to investigate the governance structure and challenges faced by stakeholders throughout the Nairobi dairy value chain and assess their potential implications on food safety. Qualitative data were collected through focus group discussions and key informant interviews based on a dairy value chain mapping framework previously developed. Thematic analysis enabled identification of governance themes, key challenges and analysis of their implications on food safety. Themes were organized depending on their association with farmers (informal settlement or peri-urban), dairy cooperatives, dairy traders, processing companies, retailers or government officers. The identified governance themes included: i) weak linkage between government and farmers, ii) inadequate compliance with government regulations by traders and retailers, iii) emphasis on business licenses and permits for revenue rather than for food safety, iv) multiple licensing resulting in high business cost and lack of compliance, v) fragmented regulation, vi) unfair competition and vii) sanctions that do not always result in compliance. The key challenges identified included, among others: i) inadequate farmer support, ii) harassment of traders and retailers and iii) high business costs for traders, retailers, dairy cooperatives and large processors. The implication of governance and challenges of food safety were, among others: i) inadequate extension services, ii) insufficient cold chain, iii) delivery of adulterated and low milk quality to bulking centers, iv) inadequate food safety training and v) lack of policies for management of waste milk. The range of issues highlighted are based on stakeholders' perceptions and reflects the complexity of the relationships between them. Many of the governance themes demonstrate the linkages that are both beneficial or confrontational between the formal and informal sectors, and between industry and regulatory authorities, with possible direct food safety consequences. Findings obtained provide indications to decision-makers of potential governance areas that could help improve efficiency and food safety along the dairy value chain.

Efficacy of Ivermectin, Liquid Paraffin, and Carbaryl against Mange of Farmed Rabbits in Central Kenya.

Mange is a common disease of rabbits globally, and knowledge of efficacy of drugs used in its treatment is critical for effective disease control. The current study evaluated the efficacy of three commonly used therapeutic agents in Kenya against mange. In a controlled laboratory trial, 20 adult rabbits were recruited for the study (16 of which were infested with mange, while 4 were mange-free). The 16 mange-infested rabbits were randomly allocated into 4 treatment groups each consisting of 4 rabbits, while 4 mange-free rabbits formed the negative control group. Treatments were administered as follows: group 1 (G1) received two ivermectin injections at an interval of 14 days, group 2 (G2) was treated with a combination of carbaryl and liquid paraffin applied every other day up to the end of the experiment, group 3 (G3) was treated with liquid paraffin droplets applied daily until the lesion cleared, while group 4 (G4, infected-untreated) received distilled water applied topically on their ears and group 5 (G5, uninfected-untreated negative control) was not treated with any preparation. The lesions were scored and sampled daily to check the viability of the mites. A field efficacy trial of the test compounds was performed using 105 mange-infested rabbits. The results revealed that all the test agents: ivermectin, liquid paraffin, carbaryl-water, and carbaryl-liquid paraffin combination were effective against mange, recording the lesion score of zero for psoroptic mange by day 21 in the laboratory and field trials. Lesion scores in the treated groups were significantly reduced (p < 0.05) at the termination of study compared with those of the positive control group in the laboratory trial. A point-biserial correlation revealed a strong association (r pb = 0.79, p < 0.05) between the presence of viable mites and degree of psoroptic lesions in the field trial.

Molecular detection and genetic characterisation of pathogenic Theileria, Anaplasma and Ehrlichia species among apparently healthy sheep in central and western Kenya.

Tick-borne diseases (TBDs) caused by Theileria, Babesia, Anaplasma and Ehrlichia species are common in tropical and subtropical regions. In this study, we investigated the presence and genetic diversity of Theileria spp., Anaplasma ovis, B. ovis, E. ruminantium and Anaplasma spp. in sheep from the Machakos and Homa Bay counties of Kenya. In order to improve the diagnosis and control of ovine TBDs, a total of 76 blood samples from apparently healthy sheep were screened using a polymerase chain reaction (PCR). The assays were conducted using primers based on Theileria spp. 18S rRNA, Anaplasma ovis Major surface protein-4 (AoMSP4), B. ovis 18S rRNA, E. ruminantium pCS20 and Anaplasma spp. 16S rRNA. The overall infection rates for Theileria spp., A. ovis, E. ruminantium and Anaplasma spp. were 39/76 (51.3%), 26/76 (34.2%), 6/76 (7.9%) and 31/76 (40.8%), respectively. The overall co-infection was 47/76 (61.8%). All Theileria spp. positive samples were confirmed to be of Theileria ovis on sequencing. A phylogenetic analysis of the 18S rRNA gene sequences of T. ovis revealed that all isolates of this study clustered with T. ovis sequences extracted from the GenBank suggesting this gene is highly conserved. E. ruminantium pCS20 sequences were in the same clade on the phylogenetic tree. However, three AoMSP4 sequences from this study appeared in the same clade, while one sequence formed a separate branch revealing genetic divergence. The 16S rRNA sequencing revealed uncharacterised Anaplasma spp. and A. ovis. The phylogenetic analyses of the uncharacterised Anaplasma spp. revealed that the two sequences from this study appear in an independent clade from other sequences extracted from the GenBank. This study provides important information regarding the occurrence of tick-borne pathogens and their degree of genetic diversity among sheep in Kenya, which is useful for the diagnosis and control of TBDs.

Toxoplasma gondii: sensitive and rapid detection of infection by loop-mediated isothermal amplification (LAMP) method.

Loop-mediated isothermal amplification (LAMP) method amplifies DNA with high specificity, sensitivity and rapidity. In this study, we used a conserved sequence in the 200- to 300-fold repetitive 529 bp gene of Toxoplasma gondii to design primers for LAMP test. Detection limit of T. gondii LAMP assay with the primers is 1 pg/microL of T. gondii DNA, which was evaluated using 10-fold serially diluted DNA of cultured parasites. Furthermore, LAMP and conventional PCR methods were applied for amplification of the T. gondii DNA extracted from the lymph nodes taken from pigs which were suspected to be Toxoplasma infection. As a result, 76.9% (70/91) and 85.7% (78/91) of the samples were positive on PCR and LAMP analyzes, respectively. Therefore, the LAMP has a potential to be applied as an alternative molecular diagnostic tool for detection of T. gondii infection from veterinary samples. This is the first study, which applies the LAMP method to diagnose Toxoplasma from veterinary samples.

Cloning, expression, and characterization of Babesia gibsoni dihydrofolate reductase-thymidylate synthase: inhibitory effect of antifolates on its catalytic activity and parasite proliferation.

Dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a well-validated antifolate drug target in certain pathogenic apicomplexans, but not in the genus Babesia, including Babesia gibsoni. Therefore, we isolated, cloned, and expressed the wild-type B. gibsoni dhfr-ts gene in Escherichia coli and evaluated the inhibitory effect of antifolates on its enzyme activity, as well as on in vitro parasite growth. The full-length gene consists of a 1,548-bp open reading frame encoding a 58.8-kDa translated peptide containing DHFR and TS domains linked together in a single polypeptide chain. Each domain contained active-site amino acid residues responsible for the enzymatic activity. The expressed soluble recombinant DHFR-TS protein was approximately 57 kDa after glutathione S-transferase (GST) cleavage, similar to an approximately 58-kDa native enzyme identified from the parasite merozoite. The non-GST fusion recombinant DHFR enzyme revealed K(m) values of 4.70 +/- 0.059 (mean +/- standard error of the mean) and 9.75 +/- 1.64 microM for dihydrofolic acid (DHF) and NADPH, respectively. Methotrexate was a more-potent inhibitor of the enzymatic activity (50% inhibition concentration [IC(50)] = 68.6 +/- 5.20 nM) than pyrimethamine (IC(50) = 55.0 +/- 2.08 microM) and trimethoprim (IC(50) = 50 +/- 12.5 microM). Moreover, the antifolates' inhibitory effects on DHFR enzyme activity paralleled their inhibition of the parasite growth in vitro, indicating that the B. gibsoni DHFR could be a model for studying antifolate compounds as potential drug candidates. Therefore, the B. gibsoni DHFR-TS is a molecular antifolate drug target.