Molecular and serological diagnosis of multiple bacterial zoonoses in febrile outpatients in Garissa County, north-eastern Kenya.

Bacterial zoonoses are diseases caused by bacterial pathogens that can be naturally transmitted between humans and vertebrate animals. They are important causes of non-malarial fevers in Kenya, yet their epidemiology remains unclear. We investigated brucellosis, Q-fever and leptospirosis in the venous blood of 216 malaria-negative febrile patients recruited in two health centres (98 from Ijara and 118 from Sangailu health centres) in Garissa County in north-eastern Kenya. We determined exposure to the three zoonoses using serological (Rose Bengal test for Brucella spp., ELISA for C. burnetti and microscopic agglutination test for Leptospira spp.) and real-time PCR testing and identified risk factors for exposure. We also used non-targeted metagenomic sequencing on nine selected patients to assess the presence of other possible bacterial causes of non-malarial fevers. Considerable PCR positivity was found for Brucella (19.4%, 95% confidence intervals [CI] 14.2-25.5) and Leptospira spp. (1.7%, 95% CI 0.4-4.9), and high endpoint titres were observed against leptospiral serovar Grippotyphosa from the serological testing. Patients aged 5-17 years old had 4.02 (95% CI 1.18-13.70, p-value = 0.03) and 2.42 (95% CI 1.09-5.34, p-value = 0.03) times higher odds of infection with Brucella spp. and Coxiella burnetii than those of ages 35-80. Additionally, patients who sourced water from dams/springs, and other sources (protected wells, boreholes, bottled water, and water pans) had 2.39 (95% CI 1.22-4.68, p-value = 0.01) and 2.24 (1.15-4.35, p-value = 0.02) times higher odds of exposure to C. burnetii than those who used unprotected wells. Streptococcus and Moraxella spp. were determined using metagenomic sequencing. Brucellosis, leptospirosis, Streptococcus and Moraxella infections are potentially important causes of non-malarial fevers in Garissa. This knowledge can guide routine diagnosis, thus helping lower the disease burden and ensure better health outcomes, especially in younger populations.

Cross-sectional serosurvey of Leptospira species among slaughter pigs, goats, and sheep in Uganda.

INTRODUCTION: Leptospira are a group of bacteria, including pathogenic types that cause leptospirosis. In Uganda, Leptospira exposure has been reported in humans, with domesticated animals being speculated as the source. However, comparable evidence of Leptospira prevalence and circulating serovars/serogroups in animals is only documented for cattle, and dogs. Our study determined Leptospira seroprevalence, associated risk factors and serogroups circulating among slaughtered pigs, goats, and sheep in Uganda. METHODS: During an 11-month cross-sectional survey in selected slaughter facilities in three regions of Uganda, we collected blood from 926 pigs, 347 goats, and 116 sheep. The age, sex, breed, and origin of each sampled animal were noted. The samples were tested for anti-Leptospira antibodies using the microscopic agglutination test, based on a panel of 12 serovars belonging to 12 serogroups. RESULTS: Leptospira seroprevalence was 26.67% (247/926, 95%CI 23.92-29.61) among pigs, and 21.81% (101/463, 95%CI 18.29-25.80) in goats and sheep (small ruminants). L. interrogans Australis and L. kirschneri Grippotyphosa were the commonest serovars among pigs, as was L. borgpetersenii Tarassovi in small ruminants. Pigs sourced from the Eastern (Odds Ratio [OR] = 2.82, 95%CI 1.84-4.30) and Northern (OR = 3.56, 95%CI 2.52-5.02) regions were more likely to be seropositive, compared to those from the Central region. For small ruminants, being female (OR 2.74, 95% CI 1.69-4.57) and adult (OR 4.47, 95% CI 1.57-18.80) was significantly more associated with Leptospira seropositivity. Conclusion/significance: Detection of a moderate seroprevalence, and several Leptospira serogroups among pigs, sheep, and goats from all regions of Uganda, supports existing reports in cattle and dogs, and implies widespread Leptospira exposure in domestic animals in Uganda. These findings may inform future programs for the control of leptospirosis in livestock in Uganda.

Longitudinal Study of Selected Bacterial Zoonoses in Small Ruminants in Tana River County, Kenya.

Brucellosis, Q fever, and leptospirosis are priority zoonoses worldwide, yet their epidemiology is understudied, and studies investigating multiple pathogens are scarce. Therefore, we selected 316 small ruminants in irrigated, pastoral, and riverine settings in Tana River County and conducted repeated sampling for animals that were initially seronegative between September 2014 and June 2015. We carried out serological and polymerase chain reaction tests and determined risk factors for exposure. The survey-weighted serological incidence rates were 1.8 (95% confidence intervals [CI]: 1.3-2.5) and 1.3 (95% CI: 0.7-2.3) cases per 100 animal-months at risk for Leptospira spp. and C. burnetii, respectively. We observed no seroconversions for Brucella spp. Animals from the irrigated setting had 6.83 (95% CI: 2.58-18.06, p-value = 0.01) higher odds of seropositivity to C. burnetii than those from riverine settings. Considerable co-exposure of animals to more than one zoonosis was also observed, with animals exposed to one zoonosis generally having 2.5 times higher odds of exposure to a second zoonosis. The higher incidence of C. burnetii and Leptospira spp. infections, which are understudied zoonoses in Kenya compared to Brucella spp., demonstrate the need for systematic prioritization of animal diseases to enable the appropriate allocation of resources.

Sero-epidemiological survey of Coxiella burnetii in livestock and humans in Tana River and Garissa counties in Kenya.

BACKGROUND: Coxiella burnetii is a widely distributed pathogen, but data on its epidemiology in livestock, and human populations remain scanty, especially in developing countries such as Kenya. We used the One Health approach to estimate the seroprevalance of C. burnetii in cattle, sheep, goats and human populations in Tana River county, and in humans in Garissa county, Kenya. We also identified potential determinants of exposure among these hosts. METHODS: Data were collected through a cross-sectional study. Serum samples were taken from 2,727 animals (466 cattle, 1,333 goats, and 928 sheep) and 974 humans and screened for Phase I/II IgG antibodies against C. burnetii using enzyme-linked immunosorbent assay (ELISA). Data on potential factors associated with animal and human exposure were collected using a structured questionnaire. Multivariable analyses were performed with households as a random effect to adjust for the within-household correlation of C. burnetii exposure among animals and humans, respectively. RESULTS: The overall apparent seroprevalence estimates of C. burnetii in livestock and humans were 12.80% (95% confidence interval [CI]: 11.57-14.11) and 24.44% (95% CI: 21.77-27.26), respectively. In livestock, the seroprevalence differed significantly by species (p < 0.01). The highest seroprevalence estimates were observed in goats (15.22%, 95% CI: 13.34-17.27) and sheep (14.22%, 95% CI: 12.04-16.64) while cattle (3.00%, 95% CI: 1.65-4.99) had the lowest seroprevalence. Herd-level seropositivity of C. burnetii in livestock was not positively associated with human exposure. Multivariable results showed that female animals had higher odds of seropositivity for C. burnetii than males, while for animal age groups, adult animals had higher odds of seropositivity than calves, kids or lambs. For livestock species, both sheep and goats had significantly higher odds of seropositivity than cattle. In human populations, men had a significantly higher odds of testing positive for C. burnetii than women. CONCLUSIONS: This study provides evidence of livestock and human exposure to C. burnetii which could have serious economic implications on livestock production and impact on human health. These results also highlight the need to establish active surveillance in the study area to reduce the disease burden associated with this pathogen.

A systematic review and meta-analysis of the aetiological agents of non-malarial febrile illnesses in Africa.

BACKGROUND: The awareness of non-malarial febrile illnesses (NMFIs) has been on the rise over the last decades. Therefore, we undertook a systematic literature review and meta-analysis of causative agents of non-malarial fevers on the African continent. METHODOLOGY: We searched for literature in African Journals Online, EMBASE, PubMed, Scopus, and Web of Science databases to identify aetiologic agents that had been reported and to determine summary estimates of the proportional morbidity rates (PMr) associated with these pathogens among fever patients. FINDINGS: A total of 133 studies comprising 391,835 patients from 25 of the 54 African countries were eligible. A wide array of aetiologic agents were described with considerable regional differences among the leading agents. Overall, bacterial pathogens tested from blood samples accounted for the largest proportion. The summary estimates from the meta-analysis were low for most of the agents. This may have resulted from a true low prevalence of the agents, the failure to test for many agents or the low sensitivity of the diagnostic methods applied. Our meta-regression analysis of study and population variables showed that diagnostic methods determined the PMr estimates of typhoidal Salmonella and Dengue virus. An increase in the PMr of Klebsiella spp. infections was observed over time. Furthermore, the status of patients as either inpatient or outpatient predicted the PMr of Haemophilus spp. infections. CONCLUSION: The small number of epidemiological studies and the variety of NMFI agents on the African continent emphasizes the need for harmonized studies with larger sample sizes. In particular, diagnostic procedures for NMFIs should be standardized to facilitate comparability of study results and to improve future meta-analyses. Reliable NMFI burden estimates will inform regional public health strategies.

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.

Seroprevalence of foot-and-mouth disease virus in cattle herds raised in Maasai Mara ecosystem in Kenya.

A cross-sectional study was carried out to determine foot-and-mouth disease (FMD) seroprevalence and identify risk factors of exposure among cattle herds raised in three zones with different types of land use and progressively distant from the Maasai Mara National Reserve (MMNR) boundary. We selected five villages purposively; two in zone 1 (area < 20 km from the MMNR), another two in zone 2 (area between 20-40 km away from the MMNR) and one in zone 3 (area >40 km away from the MMNR). A total of 1170 cattle sera were collected from 390 herds in all the zones and tested for antibodies against the non-structural proteins (NSPs) of FMD virus (FMDV) using two 3ABC-based Enzyme-Linked Immunosorbent Assay ELISA kits. All sera samples were also screened for serotype-specific antibodies using Solid Phase Competitive ELISA (SPCE) kits (IZSLER, Italy). We targeted FMDV serotypes A, O, South African Territory [SAT] 1 and SAT 2, known to be endemic in East Africa including Kenya. Data on putative risk factors for FMD seropositivity in cattle were collected using a questionnaire. The overall apparent animal-level FMD seroprevalence based on the parallel comparison of the two anti-NSPs ELISA kits was 83.8 % (95 % CI; 81.8-85.9), and differed significantly across zones. Zone 1 had a higher seroprevalence than zones 2 and 3 (χ2 = 116.1, df = 2, p < 0.001). In decreasing order, the overall seroprevalences of FMDV serotypes A, SAT 2, O and SAT 1 were 26.3 % (95 % CI; 23.5-29.2), 21.4 % (95 % CI; 18.8-24.0), 21.2 % (95 % CI; 18.7-23.9) and 13.1 % (95 % CI; 11.1-15.3), respectively. The distribution of these serotypes differed significantly between zones (p < 0.05) except for SAT 2 serotype (χ2 = 0.90, df = 2, p = 0.639). Both serotypes A and O were more prevalent in zones 1 and 2 than zone 3 while serotype SAT 1, was higher in zone 3 compared to other zones. The results of multivariable analyses identified animal sex (i.e., female), raising of cattle in zones 1 and 2 (areas < 40 km away from the MMNR); mixing of cattle from multiple herds at watering points, and pastoral husbandry practices, as significant predictors of animal-level FMD seropositivity. This study established that FMD seroprevalence declined with distance from the MMNR.

Positive association between Brucella spp. seroprevalences in livestock and humans from a cross-sectional study in Garissa and Tana River Counties, Kenya.

BACKGROUND: Brucella spp. is a zoonotic bacterial agent of high public health and socio-economic importance. It infects many species of animals including wildlife, and people may get exposed through direct contact with an infected animal or consumption of raw or undercooked animal products. A linked livestock-human cross-sectional study to determine seroprevalences and risk factors of brucellosis in livestock and humans was designed. Estimates were made for intra-cluster correlation coefficients (ICCs) for these observations at the household and village levels. METHODOLOGY: The study was implemented in Garissa (specifically Ijara and Sangailu areas) and Tana River (Bura and Hola) counties. A household was the unit of analysis and the sample size was derived using the standard procedures. Serum samples were obtained from selected livestock and people from randomly selected households. Humans were sampled in both counties, while livestock could be sampled only in Tana River County. Samples obtained were screened for anti-Brucella IgG antibodies using ELISA kits. Data were analyzed using generalized linear mixed effects logistic regression models with the household (herd) and village being used as random effects. RESULTS: The overall Brucella spp. seroprevalences were 3.47% (95% confidence interval [CI]: 2.72-4.36%) and 35.81% (95% CI: 32.87-38.84) in livestock and humans, respectively. In livestock, older animals and those sampled in Hola had significantly higher seroprevalences than younger ones or those sampled in Bura. Herd and village random effects were significant and ICC estimates associated with these variables were 0.40 (95% CI: 0.22-0.60) and 0.24 (95% CI: 0.08-0.52), respectively. In humans, Brucella spp. seroprevalence was significantly higher in older people, males, and people who lived in pastoral areas than younger ones, females or those who lived in irrigated or riverine areas. People from households that had at least one seropositive animal were 3.35 (95% CI: 1.51-7.41) times more likely to be seropositive compared to those that did not. Human exposures significantly clustered at the household level; the ICC estimate obtained was 0.21 (95% CI: 0.06-0.52). CONCLUSION: The presence of a Brucella spp.-seropositive animal in a household significantly increased the odds of Brucella spp. seropositivity in humans in that household. Exposure to Brucella spp. of both livestock and humans clustered significantly at the household level. This suggests that risk-based surveillance measures, guided by locations of primary cases reported, either in humans or livestock, can be used to detect Brucella spp. infections in livestock or humans, respectively.

A multiplex fluorescence microsphere immunoassay for increased understanding of Rift Valley fever immune responses in ruminants in Kenya.

Rift Valley fever virus (RVFV) is an important mosquito-borne pathogen with devastating impacts on agriculture and public health. With outbreaks being reported beyond the continent of Africa to the Middle East, there is great concern that RVFV will continue to spread to non-endemic areas such as the Americas and Europe. There is a need for safe and high throughput serological assays for rapid detection of RVFV during outbreaks and for surveillance. We evaluated a multiplexing fluorescence microsphere immunoassay (FMIA) for the detection of IgG and IgM antibodies in ruminant sera against the RVFV nucleocapsid Np, glycoprotein Gn, and non-structural protein NSs. Sheep and cattle sera from a region in Kenya with previous outbreaks were tested by FMIA and two commercially available competitive ELISAs (BDSL and IDvet). Our results revealed strong detection of RVFV antibodies against the Np, Gn and NSs antigen targets. Additionally, testing of samples with FMIA Np and Gn had 100% agreement with the IDvet ELISA. The targets developed in the FMIA assay provided a basis for a larger ruminant disease panel that can simultaneously screen several abortive and zoonotic pathogens.