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.

Introduction

Q fever (coxiellosis in animals) is a globally distributed bacterial zoonosis caused by Coxiella burnetii, an obligate intracellular pathogen that infects multiple hosts including livestock, wildlife, humans, birds, rodents, reptiles, and arthropods such as ticks [1,2]. This disease is endemic in livestock and human populations in Africa, especially in resource-limited rural areas, where domestic species such as goats, sheep, and cattle are the primary reservoirs of C. burnetii infections in humans [3]. Infections in humans in many settings occur mainly through inhalation of contaminated aerosols or dust particles [4,5], but may also occur through direct exposure to contaminated birth products, placenta, faeces, or vaginal mucus during parturition and abortions from infected animals, or during animal slaughter [6-8]. Intake of contaminated milk or dairy products is also another source of infection in humans because these products could contain large quantities of C. burnetii [9]. The low infectious dose of 1 to 10 bacteria required to establish infections in humans and animals [10,11], together with the ability of this pathogen to live in the environment for a long time ranging from weeks to months, could contribute significantly to airborne transmission [12,13]. Ticks have also been shown to play a role in the transmission [14].

In humans, infections with C. burnetii result in a wide spectrum of clinical manifestations with about 60% of individuals with acute Q fever being asymptomatic [2,15]. Individuals with symptomatic acute Q fever commonly manifest a febrile illness associated with headache, chills, dyspnoea, myalgia, cough, chest pain, arthralgia, atypical pneumonia, hepatitis [16,17], and chronic fatigue syndrome [18]. Acute Q fever is also a significant cause of hospitalization as reported in Tanzania [19], Netherlands [20] and Kenya [21]. This could result in the loss of income and increased disease burden in endemic areas. The main causes of hospitalizations in the above-mentioned countries were pneumonia, anaemia, leukopenia, jaundice, high fever, splenomegaly, shortness of breath, hepatomegaly, fatigue, headache and dizziness. It is also estimated that less than five per cent of individuals with acute Q fever may further develop persistent focalized infections (chronic Q fever) that mainly manifest as endocarditis, especially among individuals with pre-existing heart valve diseases [15], expectant women, and those with immune deficiencies [22]. Neurological and cardiac (acute pericarditis) involvement have also been reported in patients with acute infections [2,23], while other individuals may also manifest myocarditis, a rare but severe and life-threatening condition [24]. This pathogen is also classified as a category B bioterrorism agent, even though case fatalities due to Q fever are usually low [16]. In livestock, coxiellosis is typically asymptomatic or sub-clinical. However, these infections may cause high abortion rates, stillbirths, infertility, weak calves/kids or lambs, and metritis among pregnant animals, especially cattle [7,25]. Co-infections of Q fever with other zoonotic agents such as Brucella spp. and Rift Valley fever virus (RVFV) are also common in animals; this could exacerbate the health outcomes of infected animals since these pathogens cause similar reproductive problems such as abortions [26].

Q fever was first reported in Kenya in the 1950s [27], but information on its epidemiology remains significantly low across the country. This disease is largely neglected by both medical and veterinary personnel as evidenced by a lack of data on national incidence estimates as well as an absence of surveillance and control strategies [27]. Q fever is considered a priority disease in the country due to its high epidemic potential [28]. The disease is commonly misdiagnosed at hospitals as its clinical presentation is similar to other fever-causing illnesses. Brucellosis, leptospirosis, staphylococcal infections, dengue fever, enteric fever, rickettsial infections and malaria are common causes of fever in Africa [29,30] and Latin America [31]. Studies on Q fever have been conducted in Kenya, but the majority of these studies targeted either livestock only [26,32,33], humans only [21,34], or ticks as vectors of C. burnetii [35] with very few covering both livestock with humans [36,37]. Over the last decade, One Health studies that incorporate humans and livestock and their social and environmental components are increasingly being used as they allow for better estimation of the risk of infectious disease transmission between livestock and humans, besides providing a comprehensive picture of disease epidemiology [38-40]. This approach has been applied in many countries to inform prevention and control strategies of zoonoses [41].

We therefore assessed the seroprevalence of C. burnetii in livestock (cattle, sheep, and goats) and human populations in Tana River county. We sampled only humans in Garissa county due to insecurity. This study further identified putative determinants associated with the seropositivity of C. burnetii among these hosts. Findings from this study provides a basis for the establishment of integrated livestock-human surveillance in the area.

Materials and methods

Ethics statement

The ethical and animal use clearance for livestock sampling was provided by the International Livestock Research Institute’s (ILRI) Institutional Animal Care and Use Committee (IACUC) (reference number: 2014.02). Participants also gave oral consent for livestock sampling. We obtained the ethical approval for human sampling from the African Medical and Research Foundation (AMREF) Ethics and Scientific Review Committee (reference number: P65/2013). The sampled individuals were adequately informed about the study by the clinicians and their participation was voluntary. These individuals also gave written consents for participation in the study. For children aged 5–12 years, written consents for participation were provided by their parents/guardians while for those aged between 13–17 years, we obtained consents from both the children and the parents/guardians. Adults aged >18 years provided their written consents.

Study area

This study was carried out in Bura and Hola in Tana River county and Ijara and Sangailu within Garissa county. Tana River county borders Garissa county to the northeast (Fig 1). The above study sites were purposefully selected because of good accessibility. Besides, data on the epidemiology of C. burnetii are also very limited among livestock and human populations in these rural sites. These sites have been previously described by other studies [42,43]. In brief, Hola and Bura are both settlement and irrigation schemes established respectively in 1953 and 1978. Tana River County has a binomial rainfall that ranges from 300 mm to 800 mm annually. However, areas close to the coast could receive about 1,200 mm each year [44]. The mean daily temperatures range from 32°C to 37°C. In Ijara and Sangailu, pastoral livestock production for food and income subsistence is the main economic mainstay for the local communities. The annual rainfall recorded in these sites ranges from 750 mm to 1,000 mm, while the mean daily temperatures range from 34°C to 38°C.

Fig 1

Map showing sampling sites.

The base layer of the map used to create this figure was downloaded from https://www.diva-gis.org/gdata.

Study design and sample size determination

Livestock and human data were collected between December 2013 and February 2014 through a cross-sectional study with a multistage random sampling design. The sample sizes for livestock (cattle, sheep, and goats combined) and humans in both counties were estimated using the formula; n = (1.96)2p(1-p)/d2 [45], with an allowable error (d) of 0.05. The expected seroprevalence estimates (p) of C. burnetii in livestock and humans were both taken to be 50% due to limited available data on this estimate in the study area. Given that households were the primary sampling units while animals and humans were secondary sampling units, the estimated sample sizes (n = 384) for livestock and humans were corrected for design effects due to dependence of observations within households (clusters). The design effects (DE) were calculated using the formula: DE = 1+ρ(m-1), where ρ (rho) is the intra-cluster (within-household) correlation coefficient (ICC), and m the number of livestock/humans to be sampled per household [46]. Previous similar studies have used variable ICC values in sample size calculation, for example, 0.1 [40], 0.15 [47] and 0.2 [48], but this study assumed an ICC of 0.3 for both livestock and humans. ICC values typically range between 0.05 and 0.2, although values of more than 0.3 have also been recorded for highly infectious diseases [49]. We chose 20 livestock animals to be sampled per household (cluster) a priori due to limited information on herd structure and composition in Tana River County. For the human component of this study, five subjects were to be sampled in each household as we assumed that this was the average number of subjects per household. The estimated design effects for humans and livestock were 2.2 and 6.7, respectively. These design effects yielded an adjusted sample size of 845 for humans, who were to be chosen from 169 households. The required sample size for livestock animals was 2,574; these were to be drawn from 129 households. The required minimum number of households for humans and animals sampling estimated above were derived by dividing their respective adjusted sample sizes with the number of subjects/animals to be sampled per cluster as specified above. Nevertheless, the sampling of animals in each household was proportional to herd size with more animals being sampled in households with more animals. Overall, 2,727 animals that included 466 cattle, 1,333 goats and 928 sheep were sampled from 156 households in Tana River county. A total number of 974 humans (484 in Tana River and 490 in Garissa) were also sampled from 340 households. In Garissa County, the households to be sampled were selected from a list provided by village headmen, while in Tana River, the list was prepared with the assistance of the administrators of Hola and Bura irrigation schemes. In both counties, the households were selected through simple random sampling. Households with at least one of the targeted species (cattle, sheep or goats) were included in the selection criteria since a large percentage (64.6%) of selected households for sampling did not keep all three targeted species. Both livestock and humans were sampled from the same households in Tana River County to allow the estimation of the risk of transmission of C. burnetii between these hosts. We did not sample livestock in Garissa County because of insecurity. This is because two successive sampling campaigns were always used, the first covering humans and the second livestock. In this case, the security situation in the area deteriorated soon after we completed human sampling.

Data collection

A total of 20 randomly selected animals comprising cattle, goats, and sheep were sampled in each selected household. We sampled all animals in households that had less than 20 animals. Blood (10 ml) was collected from the jugular vein of each animal into plain vacutainer tubes and then stored in cool boxes on dry ice. These samples were transported to a local field laboratory facility for serum separation. The samples were centrifuged at 3000 rpm for 5 minutes and the harvested serum from each animal was stored into 2 ml barcoded cryovials.

Additionally, five humans aged ≥5 years old were also sampled from each selected household. The sampling procedure was conducted by a qualified clinician who was recruited by the project. From each individual, 5 ml blood was taken from the median cubital vein of the left hand into plain vacutainers labelled with unique numbers. The blood samples were taken to the nearby local health centres where they were centrifuged at 3000 rpm for 5 minutes to extract serum. The obtained serum samples were stored in barcoded cryovials. All serum samples were transported on dry ice to the Biosciences eastern and central Africa (BecA) biorepository unit at the International Livestock Research Institute (ILRI) in Kenya, where they were kept at -80°C until testing for antibodies to C. burnetii was performed in 2016. These samples were kept for three years after being collected.

Additional data on the potential factors associated with the transmission of C. burnetii in livestock and humans were also collected from each sampled household using structured questionnaires developed using the Open Data Kit (ODK) application. For each sampled animal, we collected data on species (cattle, sheep, or goat), age (calf, kid or lamb, weaner or adult), and sex (male or female). Herd size (the number of cattle, sheep, and goats owned by each household at the time of sampling) was also recorded. The individual-level information collected in the case of humans included gender (male or female), age and occupation. Household-level data such as the source of water, family size and whether the sampled household kept livestock (yes or no) were also captured during sampling.

Laboratory testing

Serum samples from livestock were tested for phase I and II specific IgG antibodies against C. burnetii using a commercial indirect ELISA test kit (IDEXX laboratories, Liebefeld-Bern, Switzerland), following the manufacturers’ instructions. The serum samples and controls (negative and positive reference sera) were all tested in duplicates in each test plate and measured the optical densities (ODs) of all wells at 450 nm. The mean ODs of the tested samples and those of positive controls were then corrected by subtracting the mean OD values of the negative controls from each. The percentage positivity (PP) for each tested serum sample was then calculated as; (corrected mean OD 450 of the tested sample/corrected mean OD 450 of the positive control) × 100%. We classified animals with PP of <30% as negative, suspect (borderline) if between 30% and 40% and positive if ≥40% as recommended by the manufacturer.

Human sera were tested for antibodies against C. burnetii Phase I antigens using the SERION ELISA classic Coxiella burnetii Phase I IgA/IgG kit (Virion/Serion, Germany), following the manufacturer’s guidelines. The serum samples and reference sera (negative and positive controls) were tested in duplicates for all test plates. The ODs were measured at 450 nm and averaged. The ratio of the OD of the tested serum (S) relative to that of positive control (P) was then calculated using the formula below.

We classified humans as positive if the percentage S/P was >10% above the manufacturer’s recommended cut-off, suspect (borderline) if +/- 10% of the cut-off, and negative if >10% below the cut-off. The cut-off ranges were calculated by multiplying the average value of the measured standard OD with the numerical data on the quality control certificate. Livestock and human sera with borderline antibody titres were re-tested; samples that gave borderline results after re-testing were considered as negative during data analysis.

Statistical analyses

All the analyses were performed using R statistical environment, version 3.6.0 [50]. The questionnaire and serological data were imported and merged into one comma-separated value file (.csv). Initial descriptive statistics were generated through cross-tabulations using gmodels [51] and epiR [52] packages. These analyses included the estimation of the overall apparent seroprevalence of C. burnetii with 95% confidence intervals (CI) in livestock and humans. Further estimation of the seroprevalence by each categorical variable was also performed. The categorical variables considered for livestock included species (cattle, sheep and goats), animal sex (male and female), age (calf/kid/lamb, weaner and adult), sampling location (Bura and Hola) and land use type (pastoral and irrigated areas). Herd size (the number of cattle, sheep and goats combined for each household) being a quantitative discrete variable was first tested for the normality of residuals using the Shapiro-Wilk test before it was included in the analysis. This variable violated the assumption of linearity and was therefore converted into a categorical variable with three levels (≤ 20, 21–40 and >40 animals). However, since categorizing a quantitative variable could lead to loss of information as this assumes sudden changes in the response variable at specific values of the independent variable [45], we also compared the results obtained using this categorical variable with those generated using herd size as a log-transformed variable. In addition, due to uneven sex distribution in livestock, the combined data for all animals were also stratified by age to determine the effect of animal sex on the seroprevalence of C. burnetii. Seroprevalence estimates were also calculated for each livestock species and by the above independent variables. For human data, we estimated seroprevalence by gender (male and female), occupation (nomadic pastoralists, mixed crop-livestock farmers, students and others [e.g., nurses, housewives, drivers, chiefs, etc.]), land use type (irrigation, pastoral and riverine), source of water (boreholes, canals, dams, taps and others), sampling location (Bura, Hola, Ijara and Sangailu), and ownership of livestock (no and yes). Both age and family size had been recorded as quantitative discrete variables and were also assessed for normality of residuals before being included in the analysis. However, these two variables did not satisfy the linearity assumption. Age and family size were both converted into categorical variable with three levels of ≤17, 18–40 and >40 and ≤5, 6–10 and >10, respectively. Analysis was also performed using log-transformed formats of these two variables. The choice of the categories for all quantitative discrete variables (herd size, age and family size) was informed by a previous study implemented in the area [43].

The relationships between categorical variables and the seropositivity of C. burnetii among livestock and humans were first assessed using the χ2 test. We also used a subset of human data from Tana River county to determine whether herd-level seropositivity of C. burnetii was positively correlated with human exposure. In this analysis, we classified a herd as exposed if at least one animal (cattle, sheep, or goat) within a household had reactive antibodies against C. burnetii.

Further analyses of livestock and human data were also performed using univariable mixed-effects logistic regression models to determine the unconditional associations between the outcome variable and the independent variables for each host group. Variables with p values ≤ 0.20 by these univariable analyses (for both livestock and human data) were further selected for multivariable analysis [45,53]. Generalized linear mixed-effects models (GLMMs) were used to perform all the univariable and multivariable analyses. We fitted livestock and human data to these models using the glmer function in the lme4 package [54] and adjusted for within-household correlations of observations using the household ID as a random variable. The final multivariable mixed-effects models were selected through a backward stepwise elimination procedure. We first fitted intercept-only null models (without independent variables), each for livestock and human data, followed by maximal models using respective variables selected from the univariable analyses. The maximal models were then reduced systematically by dropping variables with p > 0.05 until final models with the lowest Akaike’s information criteria (AIC) values were selected. Final models were assessed for fit by inspecting plots of residuals versus fitted values obtained from these models [55]. We also tested for potential interaction effects among the independent variables selected in the final multivariable models by creating two product terms for these variables and determined the statistical significance of the main effects using the likelihood ratio test (LRT) [53]. The ICC estimates for within-household clustering of C. burnetii exposure among livestock animals and humans were calculated from the variance estimates of the respective final multivariable models using the icc function from the sjstats package [56].

Results

Descriptive statistics

In total, 2,727 animals (466 cattle, 1,333 goats and 928 sheep) sampled in Tana River county were tested for antibodies against C. burnetii. The percentage number of animals sampled in Hola and Bura were 79.32% (n = 2163) and 20.68% (n = 564), respectively. Overall, more females (78.14%) were sampled than males (21.86%). The overall median herd size (cattle, sheep and goats combined) was 52 (range: 2–460), while the median herd/flock sizes for cattle, goats and sheep were 35 (range: 2–200), 20 (range: 0–120) and 15 (range: 0–200), respectively. The median number of livestock animals sampled in each household was 11.5 (range: 1–77).

A total of 974 humans (484 in Tana River and 490 in Garissa) were also screened for antibodies against C. burnetii. A large proportion of the sampled humans (95.4%) were from livestock-keeping households. The median number of humans sampled in each household was 3 (range: 1–5), while the median age was 26.5 years (range: 5–90).

Seroprevalence of Coxiella burnetii

The overall animal-level seroprevalence of C. burnetii in livestock was 12.80% (95% CI: 11.57–14.11). The observed seroprevalence differed significantly by livestock species (χ2 = 48.80, df = 2, p < 0.01). Goats had the highest seroprevalence of 15.22% (95% CI: 13.34–17.27), and sheep 14.22% (95% CI: 12.04–16.64), and cattle 3.00% (95% CI: 1.65–4.99) followed in decreasing order (Table 1). From the analysis performed using combined data for all animals, female animals (14.50%, 95% CI: 13.03–16.07) had significantly (p < 0.01) higher levels of exposure compared to males (6.71%, 95% CI: 4.84–9.03). However, the results obtained from the analyses done using data stratified by age showed only significant differences in seroprevalence between adult females and adult males (χ2 = 25.32, df = 1, p < 0.02). We did not observe these differences between female and male weaners or female and male calves/kids/lambs. Adult females and males had seroprevalence estimates of 16.98% (95% CI: 15.23–18.85, n = 1708) and 11.34% (95% CI: 7.9–15.56, n = 291), respectively. Seroprevalence estimates of female and male weaners were 4.62% (95% CI: 2.55–7.63, n = 303) and 2.23% (95% CI: 0.73–5.13, n = 224), respectively. Lastly, the seroprevalence estimates for female and male calves/kids/lambs were 1.41% (95% CI: 0.04–7.60, n = 71) and 2.94% (95% CI: 0.35–10.22, n = 68), respectively. The seroprevalence estimates of C. burnetii also differed significantly among animal age groups (χ2 = 73.45, df = 2, p < 0.01). We recorded higher seroprevalence figures in adult animals (16.16%, 95% CI: 14.57–17.85) than both weaners (3.61%, 95% CI: 2.18–5.57) and calves/kids/lambs (2.16%, 95% CI: 0.46–6.18) (Table 1). The proportion of seropositive animals for C. burnetii also differed significantly by sampling locations (χ2 = 19.47, df = 1, p < 0.01). Hola had a higher seroprevalence estimate (14.24%, 95% CI: 12.79–15.78) compared to Bura (7.27%, 95% CI: 5.27–9.73). A distribution of the seroprevalence estimates for cattle, sheep and goats with each independent variable as well as results from the χ2 test are shown in Table 2.

Table 1

Results of variables assessed for their association with C. burnetii seropositivity in livestock using univariable mixed-effects logistic regression models.

Variable	Category	n	% Seroprevalence (95% CI)	Odds Ratio (95% CI)	P-value
Species	Cattle	466	3.00 (1.65–4.99)	1.00 (Ref.)
	Goats	1333	15.22 (13.34–17.27)	5.28 (2.80–9.96)	< 0.01
	Sheep	928	14.22 (12.04–16.64)	5.26 (2.73–10.15)	< 0.01
Sex	Male	596	6.71 (4.84–9.03)	1.00 (Ref.)
	Female	2131	14.50 (13.03–16.07)	2.23 (1.54–3.22)	< 0.01
Age*	Calf/kid/lamb	139	2.16 (0.45–6.18)	1.00 (Ref.)
	Weaner	527	3.61 (2.18–5.57)	1.47 (0.41–5.25)	0.55
	Adult	1999	16.16 (14.57–17.85)	7.29 (2.22–23.99)	0.01
Location	Bura	564	7.27 (5.27–9.73)	1.00 (Ref.)
	Hola	2163	14.24 (12.79–15.78)	2.11 (1.12–4.99)	0.02
Land use	Pastoral	551	9.61 (7.29–12.39)	1.00 (Ref.)
	Irrigation	2176	13.60 (12.19–15.11)	1.77 (0.89–3.49)	0.10
Herd size**	≤20	620	14.35 (11.69–17.36)	1.00 (Ref.)
	21–40	419	12.89 (9.83–16.79)	0.9 (0.4–1.9)	0.78
	>40	1688	12.20 (10.68–13.86)	0.8 (0.4–1.4)	0.41

n, number of animals sampled in each category; Ref, reference category; CI, confidence interval.

* The sum in age categories was not equal to the total number (n) of animals sampled (2,727) due to missing data (0.02%).

**Herd size comprised cattle, sheep and goats.

Table 2

Distribution of the seroprevalence of C. burnetii by livestock species.

Variable	Category	Cattle			Sheep			Goats
		n	% Seroprevalence (95% CI)	P-value	n	% Seroprevalence (95% CI)	P-value	n	% Seroprevalence (95% CI)	P-value
Sex	Male	125	0.80 (0.02–4.38)	0.12	206	8.25 (4.88–12.88)	0.01	265	8.30 (5.28–12.30)	< 0.01
	Female	341	3.81 (2.04–6.43)		722	15.93 (13.33–18.80)		1068	16.95 (14.74–19.33)
Age*	Calf/kid/lamb	77	1.30 (0.03–7.02)	0.00	25	8.00 (0.98–26.03)	< 0.01	37	0.00 (0.00–9.49)	< 0.01
	Weaner	163	0.00 (0.00–2.23)		155	3.87 (1.43–8.23)		209	6.22 (3.35–10.40)
	Adult	226	5.75 (3.10–9.64)		741	16.73 (14.11–19.62)		1032	18.02 (15.72–20.51)
Location	Bura	245	3.67 (1.69–6.86)	0.61	135	9.63 (5.23–15.90)	0.11	184	10.33 (6.33–15.66)	0.05
	Hola	221	2.26 (0.74–5.20)		793	15.01 (12.59–17.68)		1149	16.01 (13.94–18.26)
Land use	Pastoral	230	2.61 (0.96–5.59)	0.79	114	14.91 (8.93–22.80)	0.78	207	14.49 (10.00–20.04)	0.83
	Irrigation	236	3.39 (1.47–6.57)		814	14.13 (11.81–16.71)		1126	15.36 (13.31–17.60)
Herd/flock size**	≤20	39	2.56 (0.00–13.48)	0.88	141	8.51 (4.48–14.39)	0.10	440	17.27 (13.86–21.13)	0.33
	21–40	87	2.30(0.03–8.06)		145	16.55 (10.90–23.62)		187	14.97 (10.19–20.91)
	>40	340	3.24 (1.63–5.71)		642	14.95 (12.28–17.95)		706	14.02 (11.55–16.81)

n, total number of animals sampled in each category; CI, confidence interval.

* The total number of sheep (921) and goats (1278) under the age categories were not equal to their respective sampled numbers due to missing data.

** The total number of cattle, sheep and goats sampled were 466, 928 and 1333 respectively.

The overall individual-level seroprevalence of C. burnetii in humans was 24.44% (95% CI: 21.77–27.26). The seroprevalence estimates in Tana River and Garissa counties were 25.21% (95% CI: 21.40–29.32) and 23.67% (95% CI: 19.98–27.69), respectively. These estimates did not differ significantly between the two counties (p = 0.58). The results of the independent variables analysed for their associations with C. burnetii seropositivity in humans are presented in Table 3. From these analyses, gender was significantly associated with the seropositivity of C. burnetii in humans (χ2 = 4.88, df = 1, p = 0.03). More males (28.27%, 95% CI: 23.81–33.08) were found to be seropositive than females (22.03%, 95% CI: 18.75–25.60). The seroprevalence estimates for Sangailu were the highest at 28.69% (95% CI: 23.02–34.90). Hola (25.68%, 95% CI: 20.06–31.95), Bura (24.81%, 95% CI: 19.70–30.50) and Ijara (18.97%, 95% CI: 14.33–24.36) followed in decreasing order. No statistically significant difference was observed between these sites (p = 0.09). Our results also showed that herd-level seropositivity of C. burnetii in Tana River county was not a significant determinant of human exposure (p = 0.19) (Table 3).

Table 3

Results showing the seroprevalence estimates of C. burnetii in humans based on analysis performed using combined data for all animals from both counties and subset data from Tana River county.

Variable	Category	Combined data from both counties			Tana River county
		n	% Seroprevalence (95% CI)	P-value	n	% Seroprevalence (95% CI)	P-value
Gender	Male	382	28.27 (23.81–33.08)	0.03	180	26.11 (19.86–33.17)	0.74
	Female	590	22.03 (18.75–25.60)		303	24.75 (20.00–30.01)
Occupation	Pastoralist	246	26.42 (21.02–32.40)	0.32	145	26.90 (19.88–34.89)	0.53
	Mixed crop-livestock farmer	170	25.29 (18.95–32.52)		35	17.14 (6.56–33.65)
	Student	114	29.82 (21.62–39.11)		29	31.03 (15.28–50.83)
	Other*	73	17.81 (9.93–28.53)		50	22.00 (11.53–35.96)
Age	≤17	339	27.14 (22.48–32.21)	0.30	177	27.68 (21.24–34.90)	0.41
	18–40	375	22.13 (18.03–26.68)		191	22.00 (16.33–28.54)
	>40	258	24.42 (19.31–30.13)		115	26.96 (19.11–36.03)
Location	Ijara	253	18.97 (14.33–24.36)	0.09
	Sangailu	237	28.69 (23.02–34.90)
	Bura	262	24.81 (19.70–30.50)		262	24.81 (19.70–30.50)	0.83
	Hola	222	25.68 (20.06–31.95)		222	25.68 (20.06–31.95)
Own livestock	No	33	30.30 (15.59–48.71)	0.47	14	35.71 (12.76–64.86)	0.36
	Yes	691	24.75 (21.57–28.14)		455	24.84 (20.93–29.07)
Land use	Irrigation	268	26.87 (21.65–32.60)	0.54	8	0.00 (0.00–36.94)	0.25
	Pastoralism	628	23.41 (20.15–26.92)		428	25.47 (21.40–29.87)
	Riverine	74	24.32 (15.10–35.69)		48	27.08 (15.28–41.85)
Source of water	Borehole	127	20.47 (13.83–28.54)	0.36	84	15.48 (8.51–25.00)	0.18
	Canal	231	28.14 (22.43–34.41)		231	28.14 (22.44–34.41)
	Dams	336	23.81 (19.36–28.73)		127	24.41 (17.23–32.82)
	Tap	13	30.77 (9.09–61.43)		12	33.33 (9.92–65.11)
	Others e.g., rivers	17	35.29 (14.21–61.57)		15	33.33 (11.82–61.62)
Family size	≤5	212	27.83 (21.91–34.38)	0.13	96	19.79 (12.36–29.17)	0. 13
	6–10	497	25.35 (21.58–29.42)		285	29.12 (23.91–34.77)
	>10	263	20.15 (15.48–25.52)		103	19.42 (12.28–28.38)
Herd exposure	Exposed				181	28.33 (21.88–35.52)	0.19
	unexposed				138	21.58 (15.06–29.35)

n, number of humans tested; CI, confidence interval. Except for location, all the other variables in this table (gender, occupation, age, own livestock, land use, source of water and family size) had missing data. Thus, the totals within their respective categories were not equal to the sampled number of 974 for the combined data or 484 for Tana River county. Herd exposure also had missing data.

*Other occupations included community health workers, businessmen and women, housewives, chiefs, drivers and those employed such as nurses.

Analysis of factors associated with the seropositivity of C. burnetii in humans and livestock

Univariable analyses

The results of the univariable models (with households as random effects) used to analyse livestock data are shown in Table 1. These results showed that both sheep and goats had significantly higher odds of exposure to C. burnetii compared to cattle. With regards to animal sex, we found higher odds of seropositivity for C. burnetii among female animals than males. Considering the age variable, only adult animals were found to have higher odds of seropositivity compared to calves/kids/lambs. We observed significantly higher odds of seropositivity of C. burnetii among animals raised in Hola compared to those raised in Bura. Both herd size (as a categorical and log-transformed variable) and land use type were not significantly associated with C. burnetii seropositivity in animals and were excluded in the multivariable analysis. In the case of human data, the univariable mixed-effects models (results not presented) yielded comparable results to those obtained using the χ2 test in Table 3. Furthermore, we did not find significant associations between age and family sizes and the seropositivity of C. burnetii in humans. Age and family size were both included in the univariable models in turns as categorical and log-transformed variables.

Multivariable analyses

The results obtained from the final multivariable mixed-effects model used to analyse livestock data are summarized in Table 4. These results showed female animals to have 1.65 higher odds of being seropositive than males. In addition, both sheep and goats had significantly higher odds of exposure when compared with cattle (p < 0.01). We also observed adult animals having higher odds of exposure when compared to weaners and calves/kids/lambs. The estimated level of within-household clustering of C. burnetii exposures among livestock animals was 0.28 (95% CI: 0.18–0.37). The results of the LRT χ2 test used to assess the statistical significance of the two-way interactions terms of the independent variables in the final multivariable model showed that there were no statistically significant interaction effects. All the investigated two-way interaction product terms had p-values greater than 0.05.

Table 4

Variables found to be associated with C. burnetii seropositivity in livestock using multivariable mixed-effects logistic regression model.

Variable	Category	Odds Ratio (95% CI)	SE	Z	P-value
Fixed effects
Sex	Male	1.00 (Ref.)
	Female	1.65 (1.13–2.42)	0.19	2.60	0.01
Age	Calf/kid/lamb	1.00 (Ref.)
	Weaner	1.22 (0.34–4.40)	0.66	0.30	0.761
	Adult	4.88 (1.46–16.29)	0.62	2.57	0.01
Species	Cattle	1.00 (Ref.)
	Goats	3.99 (2.10–7.61)	0.33	4.21	< 0.01
	Sheep	4.02 (2.06–7.82)	0.34	4.09	< 0.01

Ref., reference category; CI, confidence intervals; SE, standard error.

Log likelihood = -908.50, number of observations = 2665, number of households = 152

The variance for the random effect variable (household ID) was 1.26 (95% CI: 0.87–1.45), SE = 0.68

The final multivariable mixed-effects model fitted for human data identified gender as the only important variable associated with C. burnetii seropositivity in humans (Table 5). Male individuals had significantly (p = 0.03) higher odds of testing positive for C. burnetii compared to females. Family size was forced in the final model as a fixed effect. Our comparison of the final model (comprised of gender and family size) and a model with gender only showed our final model had a lower AIC value (S1 Table), thereby showing the final model used in this study had a better fit. The estimated ICC for within-household correlation of C. burnetii exposures among humans was 0.03 (95% CI: 0.00–0.13). There was no significant interaction effect (LRT χ2 p = 0.53) observed for the two-way product term created for the covariates in the final model. The AIC values for all the multivariable models used to analyse livestock and human data, together with the variables included in each model are presented in S1 Table.

Table 5

Variables associated with C. burnetii seroprevalence in humans based on analysis using multivariable mixed-effects logistic regression model.

Variable	Category	Odds Ratio (95% CI)	SE	Z	P-value
Fixed effects
Gender	Male	1.00 (Ref.)
	Female	0.72 (0.53–0.98)	0.15	-2.12	0.03
Family size	≤5	1.00 (Ref.)
	6–10	0.88 (0.61–1.29)	0.19	-0.64	0.52
	>10	0.66 (0.42–1.02)	0.23	-1.86	0.06

Ref., reference category; CI, confidence intervals; SE, standard error

Log likelihood = -535.90, number of observations = 970, number of households = 339

The variance for the random effect variable (household ID) was 0.093 (95% CI: 0.00–0.71), SE = 0.18

Discussion

This study determined the serological exposure levels of C. burnetii among livestock and humans in Tana River county, and only humans in Garissa county. The detection of antibodies to C. burnetii among livestock and humans indicated that this pathogen is prevalent in the selected two counties which could significantly affect livestock and human health. The within-household (herd) correlation of C. burnetii exposure among livestock was high (ICC = 0.28). This could be due to frequent contact between infected and susceptible animals at the farm level or during grazing since this pathogen is extremely infectious. Infected animals also shed large quantities of C. burnetii in milk, faeces, urine, and vaginal discharges for months [13], which could enhance the transmission levels at the household through environmental contamination as the pathogen remains viable in the environment for a long time. For example, a previous study conducted in Washington and Montana (USA) detected large quantities of C. burnetii DNA in goats housing and birthing locations, and also small quantities in the air, a year following an outbreak [12].

In humans, we found a low ICC (0.03), an indication that human exposure to C. burnetii was not correlated among individuals within households. Furthermore, this study did not establish a positive association between herd-level seropositivity of C. burnetii and human exposure, consistent with a previous study done in Kenya [36]. Indeed, we found a large percentage of seropositive individuals (21.58%) who did not have a single animal in their herds that tested positive for C. burnetii. Whereas infected domestic animals are the primary sources for human infections [57], the lack of significant association between herd-level seropositivity of C. burnetii and human exposure in Tana River county suggests that seropositive individuals to this pathogen may have been exposed through different routes, besides the transmission from infected animals at the household level assessed in our analysis. In this study, the assessment of independent variables related to direct/indirect contact with animals and their products was also limited because data on other probable risk factors such as the consumption of raw milk and its products; contact with reproductive fluids and tissues; the slaughter of animals, working in slaughterhouses, herding of livestock, sharing of houses with livestock, management of manure [8,21,40,41] and human tick bites [14,35,37], all known to be associated with the transmission of C. burnetii in humans, were not collected. It is also likely that these individuals may have been exposed through environmental contaminations or airborne dispersal that is a common occurrence in rural areas, mostly within the radius of less than 10 km away from infected herds, depending on stocking densities, wind speed/direction, and physical barriers such as vegetation [4]. However, this study could not determine the percentage of seroconversions among humans and livestock manifesting clinical disease or when these individuals/animals were infected since we tested animals and humans for the presence of antibodies which does not discriminate between past and active infections. Further incidence studies should therefore be conducted in the area to elucidate the main transmission routes of C. burnetii among humans.

Our study showed that the seroprevalance of C. burnetii in livestock differed significantly among animal age groups, with adult animals having higher seroprevalence estimates than weaners and calves/kids/lambs. This finding has been reported in other similar studies [37,58], and it is well known that older livestock could have had repeated exposure to the pathogen over their lifetime when compared to younger ones.

This study also found significantly higher seropositivity of C. burnetii among goats (15.22%) and sheep (14.22%) compared to cattle (3.0%), in agreement with previous studies done in Ethiopia [47], Nigeria [59] and Kenya [33]. However, other studies conducted in Egypt and Ivory Coast have reported higher seroprevalence estimates among cattle than goats and sheep [60,61]. The seroprevalence found in sheep was within the estimates of between 6.7% and 51.1% reported in a recent systematic review of Q fever in Kenya [27]. However, that of goats was relatively lower compared to the range of between 20 and 46% previously reported in the country [27,32]. Nonetheless, the estimated seroprevalence figures in sheep and goats were within the ranges (11–33%) reported among small ruminants across Africa [3]. For cattle, we found a relatively lower seroprevalence (3.0, 95% CI: 1.65–4.99) compared to other studies (range: 7.4–51.1%) conducted within Kenya [27,33,36]. Although this difference could be due to many factors including the use of different diagnostic kits, this finding agreed with most studies in Africa that have reported seroprevalence estimates of less than 13% [3]. The observed seroprevalence among humans (24.44%) was comparable to a study conducted in Egypt (25.71%) [62].

Based on gender, a significantly higher seroprevalence of C. burnetii was recorded in male individuals than females. The significant variation in seroprevalence by gender could be due to the different roles performed by men and women in the livestock value chain although this is culture-dependent. In general, men are involved in various livelihood activities such as the herding of livestock, working in abattoirs/slaughterhouses, assisting animals during birthing and slaughter which could increase their risk of exposure to this pathogen due to higher chances of encountering sick animals, contaminated viscera/fluids, aborted materials, or contaminated aerosols while grazing animals. A recent study on slaughterhouse workers in Western Kenya found that this occupational risk group had a higher seroprevalence of C. burnetii [34] compared with the general community in the same area [36]. Most of these workers were also male, highlighting the gender role in the transmission of C. burnetii. In addition, a recent systematic review also reported a high pooled seropositivity of C. burnetii among people working in abattoirs with individual studies recording seroprevalence estimates of 4.7–91.7% [8]. Comparably, another study done in Togo found higher seroprevalence estimates of C. burnetii among herders compared to non-herders [40]. In contrast, a significantly higher number of female adult animals had reactive antibodies against C. burnetii compared to adult males. This finding could be due to continued exposure of female adult animals to this pathogen over the years as the market offtake of breeding females in pastoral areas tends to be lower compared to that of males. Nevertheless, caution is required in the interpretation of this finding since we sampled more female adult animals (n = 1708) than male adults (n = 291), which could have led to higher seroprevalence among female animals than males due uneven sex distribution.

Our study had several limitations including the testing of livestock/humans using ELISA rather than the complement fixation test (CFT) and immunofluorescence assay (IFA) which are considered as reference tests for diagnosis of C. burnetii infections in livestock and humans, respectively [5]. However, ELISA tests are more sensitive than both CFT and IFA [5,63]. The numbers of goats (1,333) and sheep (928) sampled were higher than those of cattle (466). This under-representation of cattle could have led to biased estimation of the overall seroprevalence of C. burnetii in livestock. Furthermore, serological cross-reactions have also been observed between C. burnetii and other bacteria such as Bartonella spp., Legionella spp., and Chlamydia spp. [64,65]. Therefore, there could have been an over-estimation of the seroprevalence estimates in humans and livestock due to false positivity. This study also collected data through a cross-sectional study design. Thus, we could not accurately determine current infections of C. burnetii in the targeted hosts since antibodies elicited against this pathogen persist for long after infection [66,67]. Therefore, we recommend future studies that have repeated sampling of individuals to obtain samples at both the acute and convalescent phases of disease progression.

Conclusion

This study established that C. burnetii was prevalent in the study area as evidenced by the considerable seropositivity detected among livestock and humans. However, herd-level seropositivity of this pathogen was not significantly associated with human exposure. Among livestock species, both goats and sheep had significantly higher seroprevalence estimates compared to cattle. The detection of antibodies against C. burnetii in livestock and humans shows the need to establish One Health active surveillance in the area to identify the potential routes of transmission of this pathogen in humans. This finding also emphasizes the need to create more public awareness about this disease in the study area to reduce transmission and burden. This is also because previous studies in Kenya [68,69] and other African Countries [70,71] have shown that the adoption of food safety and biosecurity measures against zoonosis including Q fever, to be very low or non-existent among actors in the informal livestock value chain. In addition, this finding also suggests that Q fever should be considered as part of differential diagnosis when investigating non-malarial acute febrile illnesses in the study area. Further studies in the area are required to determine the socio-economic impacts of C. burnetii on livestock production and human health.

(DOC)

Click here for additional data file.

Livestock data.

(XLSX)

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Human data.

(XLSX)

Click here for additional data file.

AIC values for all the multivariable models used to analyse livestock and human data, together with the independent variables included in each model.

(DOCX)

Click here for additional data file.

2 Aug 2021

Dear Nthiwa,

Thank you very much for submitting your manuscript "Serological epidemiological survey of Coxiella burnetii in livestock and humans in Tana River and Garissa Counties in Kenya." for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

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Associate Editor

PLOS Neglected Tropical Diseases

Fabiano Oliveira

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: -The study objectives are clearly defined and the methods in general clearly described and appropriate.

-line 120-121: Sample size calculation and sampling method is appropriate and clear, however authors state that study sites are "purposefully selected" but failed to explain why. References about the description of these counties do not clarify to the reviewers understanding, why these locations were selected for this study.

-line 234-235: Methods mention use of Multiple Correspondence Analysis (MCA), but without reference to the R package or method. Furthermore results of MCA are not discussed in results section. MCA can be useful as a variable reduction method, however, with the small number of variables in this study, multiple steps of variable reduction seems unnecessary, and most variables could have been investigated in multivariate model selection.

Reviewer #2: Line 134: Please justify the expected seroprevalences of C. burnetii in livestock and humans of 50% that were used. Same for the ICC of 0.3. Were these based on other studies? Also, please justify why you calculated the sample size for livestock in general rather than by species (i.e., separately for sheep, goats, and cattle).

Line 170: How long were the samples stored?

Laboratory testing: I suggest detailing more this section. Also, please report the inter- and intra-assay coefficients of variation for the ELISA tests, as well as the possible cross-reactivities with other pathogens.

Lines 183-184: You mention that all samples were tested in duplicates. However, did you calculate a coefficient of variation between the duplicates, and was the sample repeated when there was a discrepancy? These details should be added.

Lines 189-190: “We classified animals with PP of <30% as negative, suspect (borderline) if between 30% and 40% and positive if ≥40%”. How were these percentages chosen? Same question for the analysis of human samples.

Lines 203-204: “Samples that gave borderline results after re-testing were considered as negative during data analysis.” How many samples gave borderline results? Did you do the statistical analyses without these samples to check that you obtained the same results?

Statistical analyses: Why did you not test any interactions in the models? Did you think about potential confounding factors?

Line 217: How were the levels of this variable chosen?

Line 221: What is the difference between pastoralists and farmers?

Lines 217 and 224-226: Why did you categorize these variables, which leads to a loss of information, rather than try transforming them (e.g., log-transformation, square-root, etc.)? Also please note that the assumption of normality is that the errors are normally distributed, not necessarily that all variables in the analysis are normally distributed. Please justify the choice of categories for age and family size.

Line 235: You mention identifying non-correlated variables for multivariable analysis. Which variables were correlated?

Line 240: What you are describing is the “backward” stepwise selection method not “forward-backward”. Please correct.

Lines 243-244: Did you also check that residuals were normally distributed?

Reviewer #3: Study objectives are articulated with methods and the design is appropriate. Consider the following comments and suggestions:

Line 134. Authors must detail why they assumed a seroprevalence of 50% in both populations (humans and animals) to calculate the sample size.

Lines 139-141. Consider mentioning the rationale to select the Intra-cluster correlation coefficient (ICC) and the number of humans and animals in each household.

Lines 143-144. It is not clear the origin of the 169 and 129 selected households for human and animal sampling, respectively. Based on the experimental design, these are the selected clusters, thus is important to clarify the rationale behind these numbers.

Line 145. Is important to clarify the randomization method used (i.e., simple, systematic)

Lines 151-152. Consider explaining, briefly, how did you manage with households without one or two of the target animal species. For example, households comprised only of cattle and goats but without sheep. Is important to avoid overrepresentation of one species above the others, which can bias the final results.

Lines 173-179. It is not clear why authors did not include in the questionnaire variables related to C. burnetii transmission (e.g., raw milk consumption, contact with reproductive fluids and tissues, manure management, tick infestations, and others). Please, clarify this.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: -The results match the methods except for the lack of description of MCA results (even if briefly). Results are otherwise clear and comprehensive. Figures and tables match written results.

-line 254-257: Median number of animals per herd might be more useful instead of mean, with the mean given if very different from the median, which would indicate a skewed distribution of herd sizes. Median was actually given for human household size.

-line 259: When discussing topline numbers, it would be useful to have the response rate or know how many households refused to participate.

-tables 4 and 5: a general random effect coefficient is presented in the same column as the odds ratios. This is confusing as it is not interpreted in the same way, nor on the same scale. Individual household IDs would have a form of odds ratio, but there is no general random effect odds ratio.

Reviewer #2: Please add in the Supplementary material a table with the AIC values of all the models tested (both for livestock and humans), as well as the explanatory variables included. The explanatory variables included in the maximal and final models should be specified in the manuscript.

Descriptive results: Please note that results are typically reported as means and standard deviations (SD) for normally distributed data and medians and interquartile ranges (IQR) or ranges for non-normally distributed data (see for example the paper by Habibzadeh F. “How to report the results of public health research”).

Line 268: remove “more”.

Line 270: I suggest rephrasing to “The seroprevalence of C. burnetii differed significantly among animal age groups (χ2 = 73.45, df = 2, p < 0.01)”.

Table 1: In the column P>Z, is this the p-value?

Table 2: In the columns P>χ2, are these the p-values?

Table 5: Why is family size in the final model if its effect is not significant? Also, family size is not mentioned in the corresponding paragraph of the results.

Reviewer #3: Consider the following comments and suggestions:

Line 257. It is not clear why 77 animals were sampled in some households; based on materials and methods (lines 151-152), the maximum number of animals in each selected household must be a maximum of 20.

Lines 252 and 260. Authors mention that animal and human samples were collected from 156 and 340 households, respectively. These numbers are significantly different from those mentioned in materials and methods (i.e., 169 and 129-lines 143-144-). Considering that households are the clusters used in the sampling design, authors should explain the reasons for these differences and if it could bias the interpretation.

Line 301. The term “Risk factor” is not accurate for a cross-sectional study, as you assess the exposure and the outcome at the same point in time. Is strongly recommended to use “associated factor” instead. Review this carefully throughout the text.

Figure 1. The figure must be improved to be self-explanatory for readers. Consider the following suggestions:

• Include a panel with a map of Africa, showing Kenya's location.

• In the small panel showing regions in Kenya put the name of Kenya and the neighboring countries.

• In the big panel showing the study areas put a North Arrow, make a grid with geographical coordinates in the margins, and put a scale bar at the bottom.

• In the big panel show the exact location of Bora, Hola, Ijara, and Sangailu.

• Consider differentiating locations (e.g., different colors) where animal and human samples were obtained.

Table 1.

• Please, consider reducing the number of lines to separate columns and rows.

• Consider putting the 95% CI within brackets or parentheses next to the corresponding value (i.e., % Seroprevalence, Odds Ratio).

• Review the numbers in “Age” category. The total sum equals 2,665 and not 2,727 animals.

Table 2.

• Please, consider reducing the number of lines to separate columns and rows.

• Consider putting the 95% CI within brackets or parentheses next to the corresponding value (i.e., % Seroprevalence).

• Review the numbers in “Age” category for sheep and goats. The total sums do not correspond to other categories.

• Please, clarify the numbers in the “herd/flock size” using a footnote. When you are reviewing the total number of animals in sheep and goats, within this category, you find numbers higher than 928 and 1333 animals, respectively. Is confusing.

Table 3.

• Please, consider reducing the number of lines to separate columns and rows.

• Consider putting the 95% CI within brackets or parentheses next to the corresponding value (i.e., % Seroprevalence).

• Review the numbers in the “Combined data from both Counties” category within the following subcategories: “Gender”, “Occupation”, “Own livestock”, “Land use”, and “Source of water”, they do not sum the 974 humans mentioned in the results.

• Review the numbers in the “Tana River County” category within the following subcategories: “Gender”, “Occupation”, “Age”, “Own livestock”, “Source of water”, and “Herd exposure”, they do not sum the 484 humans mentioned in the results.

• Review if the word “Clean” is appropriate in the “Herd exposure” variable.

Table 4.

• Please, consider reducing the number of lines to separate columns and rows.

• Consider putting the 95% CI within brackets or parentheses next to the corresponding value.

Table 5.

• Please, consider reducing the number of lines to separate columns and rows.

• Consider putting the 95% CI within brackets or parentheses next to the corresponding value.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Conclusions from the manuscript are mostly supported by the data presented as are limitations. Discussion however seems to be missing some comparisons to other seroprevalence for C. burnetii studies in Kenya. A quick Web of Science search with the words (burnetii) AND (kenya) led to 10 papers on this topic, none of which are referenced in this paper. Of note among these is a study with a very similar One Health design from 2016:

Wardrop NA, Thomas LF, Cook EAJ, deGlanville WA, Atkinson PM, Wamae CN, et al.(2016)The Sero-epidemiology of Coxiella burnetii in Humans and Cattle, Western Kenya: Evidence from a Cross-Sectional Study. PLoS Negl Trop Dis10(10):e0005032.doi:10.1371/journal.pntd.0005032

Another also sampled livestock and humans:

Knobel et al. 2013:Coxiella burnetii in Humans, Domestic Ruminants, and Ticks in Rural Western Kenya

Other studies focused on seroprevalence in ruminants:

Larson et al. 2019: The sero‐epidemiology of Coxiella burnetii (Q fever) across livestock species and herding contexts in Laikipia County, Kenya

Muema et al. 2016: Seroprevalence and Factors Associated with CoxiellaburnetiiInfection in Small Ruminants in Baringo County, Kenya

The other seroprevalence studies covered other species including wildlife, camels and ticks. A more comprehensive literature search might be necessary to better discuss the results presented in full context of the current knowledge in Kenya. This would also help in explaining how this study adds to this current knowledge already in existence.

Other comments include:

-line 368-370: specifying the two countries from ref 40, 41 would be better than "elsewhere", as is done in the next sentence with ref 42, 43.

-line 387-389: The authors might consider stratifying by age when looking at the effect of sex. As mentioned, adults are more likely to be positive compared to younger animals by virtue of having lived longer and so more time for exposure. Given the very uneven sex distribution in livestock, I would suspect that few males are adults compared to females, which would explain the higher sero-prevalence in females.

Reviewer #2: Discussion

Lines 375-377: “For cattle, we found a relatively lower seroprevalence compared to other studies (ranges; 7.4-51.1%) conducted within Kenya.” What are the possible reasons for this finding?

Line 396: Please add a reference for the long-term persistence of C. burnetti antibodies.

Reviewer #3: Consider the following comments and suggestions:

Lines 346-347. When authors make the following statement: “Furthermore, this study did not establish a positive association between herd-level seropositivity of C. burnetii and human exposure.”, they must consider the limitations of a cross-sectional study to establish this kind of association. This could be also included in the discussion section.

Lines 349-354. Authors affirm that: “Whereas infected domestic animals are the primary sources for human infections [37], the lack of significant association between herd-level seropositivity of C. burnetii and human exposure in Tana River County suggests that seropositive individuals to this pathogen may have been exposed through different routes, besides the direct contacts with infected animals and/or their products assessed in our analysis”. It must be emphasized that assessment of variables related with direct/indirect contact with animals and their products were limited. Questionnaires included categories like “Occupation” but did not inquire about other probable risk factors associated with transmission (e.g., consumption of raw milk or contact with animal feces). This should be considered in the discussion.

Lines 368-389. Authors should consider including in the discussion results from two recent studies conducted in the same region in Kenya. Firstly, the study made by Koka et al. (2018) [Koka et al. Coxiella burnetii Detected in Tick Samples from Pastoral Communities in Kenya. Biomed Res Int. 2018 Jul 9;2018:8158102. doi: 10.1155/2018/8158102. PMID: 30105251; PMCID: PMC6076967] which explores the circulation of C. burnetii in ticks collected from cattle, sheep, and goats. Secondly, the study published b Nyokabi et al. (2018) [Nyokabi et al. Informal value chain actors' knowledge and perceptions about zoonotic diseases and biosecurity in Kenya and the importance for food safety and public health. Trop Anim Health Prod. 2018 Mar;50(3):509-518. doi: 10.1007/s11250-017-1460-z. Epub 2017 Nov 13. PMID: 29130123; PMCID: PMC5818561.] which evidences the low levels of knowledge about zoonoses (including Q Fever) and low adoption of food safety and biosecurity measures.

Lines 390-393. Authors make a relevant approach to the study drawbacks, nonetheless, they should consider mentioning a drawback related to an absence of inquiry of variables associated with C. burnetii transmission, as mentioned in previous comments.

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: no comments

Reviewer #2: (No Response)

Reviewer #3: Consider the following comments and suggestions:

Line 1. Review wording in the title. Consider using “Sero-epidemiological survey” instead of “Serological epidemiological survey”

Lines 14,15. “Department” instead of “Departrment”

Line 83. Consider writing “commonly manifest a febrile illness” instead of “commonly manifest with a febrile illness”

Lines 83-87. Consider including a brief description of the most common hospitalizations causes in those (or other) countries. Besides, it is recommendable to mention the neurological and cardiac compromise in some patients with acute infection. [See: Bernit, E., et al. (2002). Neurological involvement in acute Q fever. A report of 29 cases and review of the literature. Arch. Intern. Med. 162, 693– 700; Fournier, P.E., et al. (2001). Myocarditis, a rare but severe manifestation of Q fever report of 8 cases and review of the literature. Clin. Infect. Dis. 32, 1440–1447]

Lines 100-103. Probably, the list of pathogens related to febrile conditions in tropical Africa is larger. Authors are encouraged to review if other etiologies might be included.

Line 104. Consider including at least one reference to support the relevance of the “One Health approach” to prevent and control zoonotic diseases like Q Fever. [e.g., Rahaman, M. R., et al. (2019). "Is a One Health Approach Utilized for Q Fever Control? A Comprehensive Literature Review." Int J Environ Res Public Health 16(5).]

Line 108. The text “and will inform the development of control strategies” is confusing. Review wording.

Line 113. “Institutional Animal Care and Use Committee” instead of “Institutional Animal Care and Uses Committee”

Line 115. Please, explain the abbreviation “AMREF”

Line 174. Consider reorganizing categories by putting first “species” and subsequently “age”.

Lines 178-179. The text “and whether the sampled household owned livestock (yes or no) were also captured during sampling.” is confusing, consider rewording.

Lines 189-190. Detail the source (or previous experience) for this classification system.

Line 191. “Phase I” instead of “Phase 1”

Lines 199-200. Detail the source (or previous experience) for this classification system.

Line 211. “seroprevalence” instead of “Seroprevalence”

Lines 215-216. Herd size is not a continuous variable, by its nature is a quantitative discrete variable.

Line 224. It is not clear why age and family size were recorded as continuous variables; they correspond to discrete variables.

Lines 259-262. Consider including descriptive results related to occupation, and source of water as you mentioned these variables in materials and methods.

Lines 323-324. Review the term “predictor” in this phrase, as it is inaccurate. Consider using terms like “related” or “associated” based on the type of epidemiological study conducted.

Line 343. Is suggested to put USA within parentheses.

Line 364. Consider using “having higher seroprevalences than weaners…” or “having the highest seroprevalence.” instead of “having the highest seroprevalence than weaners…”.

Line 372. Please, add the citations of those previous studies with sheep in Kenya.

Line 376. “range” instead of “ranges”.

Lines 436-574. Review scientific nomenclature in all references.

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: On the whole this is a well designed and straightforward study that answers its aims and objectives. The main weakness lies in failing to address both in the introduction and discussion, the current situation of data availability in Kenya for C. burnetii and how this study differentiates from previous sero-prevalence studies, improves the already existing knowledge base and guides future studies and intervention. Authors site the lack of epidemiologic knowledge (line 98) but do not mention the epidemiologic knowledge in existence. Similarly, in the conclusion (line 398), authors conclude that this study is evidence of prevalence in both humans and cattle, however this has already been shown in other papers and should likely not be the main takeaway from this study. As is, the novelty and thus to an extant the significance of this study are not clearly explained.

This is a well designed and executed epidemiology study that would clearly improve knowledge of this disease in Kenya, but should be better presented in context of the already existing knowledge.

Reviewer #2: This study presents the results from a serological survey of Coxiella burnetti exposure in livestock and humans in two counties in Kenya. This is an interesting study, which provides important data on a pathogen that has major impacts on human and animal health. However, I have some concerns regarding the sample size calculations, as well as the laboratory and statistical analyses. Please see my detailed comments and suggestions.

Abstract

Line 37: I suggest adding the word “respectively” after humans as household ID is the random effect in both the livestock and the human models.

Lines 45-46: “The results from multivariable model showed that sex (female), age (adults), and species (goats and sheep) were significant factors associated with exposure to C. burnetii in livestock.” This sentence is unclear. I think that you meant that higher seroprevalences were found in females, adults and sheep and goats, I suggest reformulating.

Line 48: Replace “with” with “than”.

Introduction

The first two paragraphs of the introduction (lines 66-95) could be shortened.

Line 101: I suggest replacing “febrile-causing” with “fever-causing” or reformulating.

Lines 104: I suggest detailing in the introduction what a “One Health approach” is and entails, and why such approaches are important. You could even give examples (see the book “One Health Case Studies: Addressing Complex Problems in a Changing World”).

Reviewer #3: The submitted study corresponds to an observational cross-sectional survey of Coxiella burnetii in livestock and humans from two regions in Kenya. The manuscript is well written and presents comprehensively the background, methods, and results. All relevant data is provided and, in general, STROBE guidelines were followed. The obtained results are relevant for this region and complement diverse studies in this neglected disease in Kenya. Is unfortunate the absence of animal data in Garissa County to be related to human variables and results. Despite authors mentioned some drawbacks in the text, they should consider reviewing the lack of exploration and analyses of variables related to animal and human transmission. Do not discard to include a brief text to discuss probable biases in the study.

--------------------

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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9 Oct 2021

Submitted filename: Response to editorial comments.docx

Click here for additional data file.

20 Dec 2021

Dear Nthiwa,

Thank you very much for submitting your manuscript "Sero-epidemiological survey of Coxiellaburnetii in livestock and humans in Tana River and Garissa Counties in Kenya." for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Thanks for addressing the reviewer's comments. Reviewers added a few more helpful comments that will improve the clarity of the final version.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Claudia Munoz-Zanzi

Associate Editor

PLOS Neglected Tropical Diseases

Fabiano Oliveira

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Thanks for addressing the reviewer's comments. Reviewers added a few more helpful comments that will improve the clarity of the final version.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #2: No comments.

Reviewer #3: The revised version included most of comments and suggestions made for the original submission. Authors provided a detailed explanation through their responses to each reviewed item.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: No comments.

Reviewer #3: The revised version included most of comments and suggestions made for the original submission. Authors provided a detailed explanation through their responses to each reviewed item.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: No comments.

Reviewer #3: The revised version included most of comments and suggestions made for the original submission. Authors provided a detailed explanation through their responses to each reviewed item.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: No comments.

Reviewer #3: Line 63. Put a period after “Kenya”.

Line 83. Consider omitting “of the bacteria” after “tick”

Lines 126-128. Authors might consider relocating the following text: “Our study showed that C. burnetii was circulating among livestock and human populations and provides a basis for the establishment of integrated livestock-human surveillance in the area” within the discussion or conclusion sections, where it fits better

Line 188.Is confusing the mean of “unreliable security”, is that related to “safety concerns”? Consider rewording to make it clearer.

Lines 356-358. Consider including the p-value for male and female seropositivity comparison.

Lines 453-456. Review wording for this phrase, is confusing.

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The reviewer is satisfied with the responses to the previous comments.

Reviewer #2: Thank you for your detailed responses and clarifications. All your revisions were very thorough. I have just a couple minor suggested edits (line numbers correspond to the version of the manuscript with track changes):

- Line136: I suggest replacing "planned to assess" with "assessed".

- Lines 155-157: "The above study sites were purposefully selected to allow the estimation of the risk of transmission of Coxiella burnetii between livestock and humans." It is still unclear why these specific study sites were chosen rather than others. What are the characteristics that rendered them particularly relevant to study?

- Line 204: I suggest replacing "all the three-targeted species" with "all three targeted species".

- Line 228: I suggest specifying after "in 2016" the number of months/years that had passed since the samples were collected.

- Line 406: Replace "for" with "as".

- Lines 495-499: I suggest rephrasing the sentence "Further incidence studies that use multiple serological tests to improve net diagnostic performance as well as changes in antibody titres, as well as molecular tests to detect active infections should therefore be conducted in the area to elucidate the main transmission routes of C. burnetii among humans." It is unclear and difficult to follow.

- Line 519: I suggest replacing the word "study" with "difference".

Reviewer #3: I must acknowledge authors for the comprehensive review and considering most of suggestions and comments made in the original submission. Also for providing a detailed explanation through their responses to each reviewed item. The submitted revision enhanced its global quality and clearness. Also the new map and tables are self-explanatory, and they improved considerably. Some minor suggestions are listed to be considered for a final version.

--------------------

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

18 Jan 2022

Submitted filename: Response to editorial comments - R2.docx

Click here for additional data file.

28 Jan 2022

Dear Nthiwa,

We are pleased to inform you that your manuscript 'Sero-epidemiological survey of Coxiella burnetii in livestock and humans in Tana River and Garissa Counties in Kenya.' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Claudia Munoz-Zanzi

Associate Editor

PLOS Neglected Tropical Diseases

Fabiano Oliveira

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

1 Mar 2022

Dear Nthiwa,

We are delighted to inform you that your manuscript, "Sero-epidemiological survey of Coxiella burnetii in livestock and humans in Tana River and Garissa Counties in Kenya. ," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases