Introduction

Coxiella burnetii, a gram-negative intracellular zoonotic bacterium that causes the disease Q fever, is an emerging global concern due to its highly contagious nature [1, 2]. Among the varying reservoirs of C. burnetii, the most well-known are domestic and wild mammals, birds, and arthropods [1], while domestic ruminants (goats, sheep, and cattle) are considered the primary sources of infection for humans [3]. C. burnetii can be shed into the environment through the milk, feces, and urine of infected animals, while pregnant females can also release large numbers of this pathogen during abortion or parturition [3, 4]. Inhalation of contaminated aerosols is the primary route of transmission in both animals and humans [5, 6], and ingestion of unpasteurized milk or cheese is also considered a risk factor for infection [7]. Ticks also contribute to the transmission of C. burnetii between wild and domestic animals, and therefore, have been the focus of many epidemiological studies investigating Q fever [8,9,10].

Humans in direct contact with domestic ruminants, such as farm workers, slaughterhouse workers, butchers, and veterinarians, or who live close to rural locations, are at greater risk of contracting Q fever [3, 4]. Prior research has shown that the wind can contribute to the spread of C. burnetii by transporting it away from the region of primary infection [11]; this process is facilitated by hot and dry weather conditions, such as those found in northeastern Brazil [12].

Goat and sheep husbandry is common throughout the Brazilian territory, with the northeast region containing 94.6% and 68.5% of the goat and sheep populations in Brazil, respectively [13]. This practice holds great social and economic importance for the low-income population [14].

In Brazil, Q fever is defined as a disease which must be immediately reported if identified in any animal species [15], or humans since 2014 [16]. The presence of C. burnetii in small ruminants has already been reported in the semi-arid region of Pernambuco [17]; however, to date, there have been no reported cases in humans in this region. This study was designed to investigate the occurrence of C. burnetii in humans, sheep, and goats in the semi-arid region of northeast Brazil considered to be at-risk.

Methods

Ethical aspects

The study was approved by the Ethics Committee on the Use of Animals of the Universidade Federal do Vale do São Francisco (CEUA no. 0004/250319) and PROGEPE - University of Pernambuco - Plataforma Brasil (CAAE no. 98339218.0.0000.5207).

Study area

This study was carried out in the municipality of Petrolina, focusing on the Rajada District, a rural area with many goat and sheep farmers, and considered an at-risk dry area of Sequeiro (a region in the northern part of the municipality of Petrolina), which is a site of intensive sheep and goat breeding according to Souza et al. [17]. The study area is located inside the Caatinga biome of the state of Pernambuco, northeast Brazil. The climate is hot semi-arid with an average annual temperature of 25.7 °C, an area of 4,558,537 km2, representing 4.81% of the Pernambuco territory, with an estimated population of 359,372 inhabitants, and containing 269,000 heads of goats and 196,000 heads of sheep [18] (Fig. 1).

Fig. 1
figure 1

Location of the municipality of Petrolina, especially in the Rajada District, state of Pernambuco, semi-arid region of Northeastern Brazil

Full size image

Sample collection

Five farms (herein referred to as farms 1, 2, 3, 4, and 5) were investigated between March and July 2019. Blood samples were collected from an average of 50% of the goats and sheep on each farm, totaling 145 goats and 66 sheep. Sampling was performed by non-probabilistic analysis, targeting animals older than six months, irrespective of sex. The samples were sent to the laboratory for serological analysis, which was performed within 24 h of blood collection. Animals showing seropositivity to C. burnetii were resampled two days later, when a second visit was performed in the farm, specifically to collect blood, milk, and vaginal swabs, which were also sent to the laboratory for molecular analysis.

Because Farm 1 yielded the highest seropositivity rates for C. burnetii (see Results), an additional visit was carried out in September 2021 to collect samples of blood, milk, and vaginal mucus from the maximal number of small ruminants, totaling 37 goats and 28 sheep, irrespective of sex.

Blood samples were collected by venipuncture of the jugular vein. Samples were centrifuged at 3,000 × g for 10 min, after which the serum was collected in 1.5 mL tubes. Milk samples were collected, discarded from the first jet of milk, and stored in sterilized tubes. Vaginal mucus samples were collected using swabs directly from the vagina and stored in sterile tubes. All samples were frozen at -20 °C until examination.

In humans, sampling was performed by non-probabilistic analysis, and those considered at risk of infection with C. burnetii were enrolled in this study; in other words, people who reported being in direct contact with animals and/or lived in a rural environment. The individuals involved included rural workers (including those residing on farms where biological samples from small ruminants were collected) and slaughterhouse workers residing in the area considered at risk by Souza et al. [17], as well as employees of the Zoonosis Control Center (CCZ) and veterinarians from the city of Petrolina. Blood was collected by a nurse and the obtained samples were processed as described for the small ruminant samples.

Indirect immunofluorescence assay

The presence of anti-C. burnetii antibodies in sera samples were evaluated by indirect immunofluorescence assay (IFA) using crude antigens of the At12 strain of C. burnetii and then cultured in Vero cells with low passage [10], after which 15 µl of C. burnetii-infected cells were applied to each of the 12 wells on microscopic slides. Sera were diluted two-fold with PBS (0.1 M; pH 7.4), starting from a 1:64 dilution. Twenty microliters of each diluted serum sample were added to each well of the antigen slides. Slides were incubated at 37 °C for 30 min in a humid chamber. The slides were then washed twice in PBS for 10 min. Slides were then incubated at 37 °C in a humid chamber with either rabbit anti-sheep IgG (dilution 1:1,000) (Sigma, St Louis, USA), rabbit anti-human IgG (dilution 1:300) (Biomanguinhos, Fiocruz, Brazil), or rabbit anti-goat IgG (dilution 1:1,000) (Sigma, St Louis, USA) fluorescein isothiocyanate conjugate and washed as described above, with the addition of Evans blue. Slides were mounted with buffered glycerin under coverslips, and observed under a fluorescence microscope (Olympus, Tokyo, Japan) at 400x magnification. Samples were run in duplicate, and those which showed positive fluorescence at the 1:64 dilution were considered positive [10, 19]. Endpoint titers against C. burnetii were determined by testing two-fold serial dilutions of serum until the final titer was reached. Each slide contained samples containing serum previously shown to be non-reactive (negative control) and serum known to be reactive (positive control) [20].

Molecular and phylogenetic analyses

Blood, milk, and vaginal mucus samples collected from the animals were subjected to DNA extraction using the Wizard® Genomic DNA Purification kit (Promega, Madison, WI, USA). PCR targeting a 557 bp fragment of the gene that encodes the capsular polysaccharide protein (CAP) gene of Coxiella spp. was performed on extracted DNA samples, as described by Reeves et al. [19], as well as another PCR targeting a 687 bp fragment of the repetitive element IS1111 associated with the transposase gene (IS1111 gene), as described by Mares-Guia et al. [21]. C. burnetii strain At12 DNA was used as a positive control.

IS1111 amplicons were purified with ExoSAP-IT (USB Corporation) and sequenced using BigDyeTM Terminator 3.1 - Cycle Sequencing Ready Reaction (Applied Biosystems) using an ABI 3500 Genetic Analyzer (Applied Biosystems), following the manufacturer’s instructions. The sequences obtained were edited using PHED and Bioedit 7.2.5 [22] and aligned with Clustall/W in Bioedit with homologous C. burnetii sequences retrieved from the GenBank.

A neighbor-joining tree with the maximum composite likelihood model and 1,000 bootstrap replicates was built within MEGA11 [23], using a tick Coxiella endosymbiont as an outgroup.

Risk factors and statistical analysis

Two types of mixed health questionnaires were completed to assess the relevant information for each individual: one for humans and the other for animals. In the human questionnaire, the variables included sex, education, occupation, years of professional experience, hygienic precautions taken during contact with animals (including changing clothes, washing, and disinfecting hands), consumption of unpasteurized milk, and the presence or absence of respiratory, cardiac, and/or hepatic problems. The second questionnaire was answered by owners of the small ruminant farms. The variables covered included basic farm information (location, presence of other animals, and facilities), number, species, breed, age, and purpose of small ruminants; history of reproductive disease, including abortion, stillbirths, and weak newborns; and the existence of veterinary assistance.

The variables were arranged in ascending or descending order in terms of the risk scale. When necessary, these variables were normalized. The lowest-risk category was used as the baseline for comparison with other categories. An initial exploratory data bivariate analysis was performed to select variables with p-value ≤ 0.2 by the chi-square test or Fisher’s exact test. Variables with p-values < 0.05 were then subjected to logistic regression [24].

Results

Samples were collected from a total of 145 goats and 66 sheep from three farms containing both species. Among the five farms sampled from March to July 2019, 3/5 (60%) had at least one small ruminant (goat or sheep) who tested positive for anti-C. burnetii antibodies. Overall, anti-C. burnetii antibodies were identified in 4.8% (7/145) of goats and 1.5% (1/66) of sheep, with endpoint titers ranging from 64 to 4,096. PCR targeting the IS1111 gene of C. burnetii yielded amplicons only in a single goat milk sample from Farm 1, which had an IFA titer of 4,096 (Table 1). Farm 1 also had the highest seropositivity rates for goats and was the only farm with at least one seropositive sheep.

Table 1 Serological and molecular results of small ruminants from farms located in the semi-arid region of Northeastern Brazil, sampled during 2019
Full size table

In 2021, a further visit was carried out on Farm 1, due to it having the highest seropositivity among the five farms sampled in 2019. In this specific farm, blood samples were collected from 37 goats and 28 sheep, while milk and vaginal mucus was collected from 26 goats and 27 sheep. In the molecular analysis, the presence of C. burnetii DNA was observed in 18.9% (7/37) of blood samples, 7.7% (2/26) of milk samples, and 7.7% (2/26) of vaginal mucus samples, all from goats, using primers targeting the IS1111 gene. One goat tested positive for C. burnetii DNA in both blood and milk samples (Table 2). All analyzed samples were negative for the CAP gene. During this 2021 survey in Farm 1, all sampled animals (goats and sheep) were seronegative for C. burnetii; however, it is not known whether the tested blood samples of 2019 corresponded to the same individuals that were sampled during 2021, or if they were new animals never sampled before. All sheep tested negative in the analyses.

Table 2 Presence of Coxiella burnetii DNA in goat samples (blood, milk and vaginal mucus) collected in 2021 from Farm 1, where seropositivity to C. burnetii was detected in goat sera collected in 2019
Full size table

The PCR products were subjected to DNA sequencing and phylogenetic analysis; however, sequencing was successful in only one goat vaginal mucus sample. The sequence based on the IS1111 gene was identified as C. burnetii (Genbank OP503537, named strain Petrolina) and showed a nucleotide identity of 99.2–99.4% with sequences from other studies carried out in Brazil. Phylogenetic analysis of the Petrolina strain (GenBank no. OP503537) was grouped in a clade with numerous C. burnetii haplotypes from animals and humans from the states of São Paulo and Rio de Janeiro in southeastern Brazil (Fig. 2).

Fig. 2
figure 2

Phylogenetic analysis of the sequence based on the IS1111 multicopy transposase gene identified in 520 bp goat vaginal mucus (Genbank OP503537) from the semiarid region of Northeastern Brazil

Full size image

Bivariate analysis was conducted on eight animals who tested positive for anti-C. burnetii antibodies in 2019. The animals from the 2021 visit were not included in this evaluation, as they were not identified, and there was no way of knowing whether some animals had already been included in the study. The variables which passed the significance test, including farm number, contact with other animals, origin of animals, cleanliness of facilities, history of other diseases on the farm, and history of reproductive diseases on the farm, and had a positive diagnosis (p ≤ 0.2, Table 3), were selected for multivariate analysis; however, no statistically significant difference (p < 0.05) was obtained in the second analysis.

Table 3 Bivariate analysis for risk factors showing the variables associated (p ≤ 0.20) with the diagnosis of Coxiella burnetii infection in small ruminants from the semi-arid region of Northeastern Brazil
Full size table

Anti-C. burnetii antibodies were detected in 4.5% (3/66) of the sampled humans: one rural worker, one veterinarian, and one CCZ employee, with titers ranging from 128 to 256. None of the humans had any symptoms indicative of Q fever. Residents of farms where positive animals were found in the serological and/or molecular analyses entered the study; however, they were all seronegative for C. burnetii.

Discussion

The true seroprevalence in the areas of the present study may be higher because the test used was more closely related to the detection of anti-phase I antibodies. The strains of C. burnetii used for diagnosis with the IFA test were grown in Vero cells and were of a low passage, in which phase I-related antibodies are more detectable. The chronic immune response is related to the presence of high titers of anti-phase I antibodies—in contrast to the acute response that is initially related to the presence of anti-phase II antibodies—and can only be observed after several passages using cell culture or other isolation techniques [25].

From the visit of Farm 1 in 2021, C. burnetii DNA was observed in milk and vaginal mucus samples only from goats in which antibodies were not detected. This can be explained by the results of prior studies, which showed that some animals can excrete the bacteria without showing symptoms [5, 11] and before producing antibodies [26, 27]. This excretion may persist for several months in vaginal mucus and feces and for more than 40 months in milk [4].

Northeastern Brazil is responsible for approximately 70% of the goat milk production in Brazil [28], but agriculture in this region had several limitations, primarily that it is produced by small producers, without proper hygienic-sanitary care [29]. The detection of C. burnetii by real-time PCR in milk has already been performed in Goiás state, within the central region of Brazil, with 3.57% (4/112) of bovine milk samples sold directly for human consumption without undergoing official inspection or pasteurization testing positive for C. burnetii, with concentrations ranging from 125 to 404 bacteria per mL [30]. C. burnetii has also been detected in 60% (6/10) of goat milk samples from Rio de Janeiro [31]. The C. burnetii infective dose for humans from ingesting milk is unknown, but it is suggested to be higher than that from inhalation, which is linked to a dose of 1–10 bacteria [7, 32].

Of the 11 samples which tested positive on PCR, reliable DNA sequences could only be generated from one vaginal mucus sample. This may be because this sequenced sample was the only one that showed a strong band in the PCR, while nested PCR was not performed to improve DNA amplification for sequencing. The strain detected in the vaginal mucus was identified as C. burnetii, thus confirming its presence in the region. As shown in Fig. 2, this strain showed genetic divergence from strains extracted from goats in Alagoas, both in northeastern Brazil, suggesting some genotypic divergence. New genotypes were observed in some locations in Brazil: CbCbB_F2 (detected in cattle fetuses), CbG_SVB22 (goat vaginal swabs), and CbO_sn2 (sheep vaginal swabs) [33].

In the farms visited in the present study, no ticks were found, and only infestation by lice was reported by the breeders, which were not found in the animals during the collection. However, the presence of Rhipicephalus microplus ticks in cattle in the study region has previously been reported [34]. Ticks can transmit this pathogen through droppings, direct contact, or bites [35]. In addition to ticks, C. burnetii can infect mites, fleas (Xenopsylla cheopis, Ctenocephalides felis, and C. canis), humans, bed bugs, and flies [4].

The farms investigated in this study follow a semi-intensive breeding system, in which the animals are released in the morning and returned to the premises at night. These facilities are rustic, with dirt floors and no roof, as is common in the sertão of Pernambuco [36]. For the most part, the exploration of the activity in the Northeastern region takes place in extensive systems characterized by native pastures with little increase in reproductive, sanitary, and food management techniques [37].

In humans, seropositivity was detected in 4.5% (3/66) of individuals, including one rural worker, one veterinarian, and one CCZ employee, none of whom showed any symptoms related to Q fever. Rural workers residing on farms where positive animals were found showed negative serological results. These results corroborate those of other seroepidemiological studies conducted in Minas Gerais [38, 39], Rio de Janeiro [40, 41], and São Paulo [42,43,44]. In addition, in Minas Gerais state [45] patients suspected of dengue and later diagnosed as negative, yielded a 4.8% seroreactivity on IFA test for C. burnetii, with titers ranging from 16 to 128. The authors identified rural residence as a risk factor for infection. In the Northeast of Brazil, one case of Q fever diagnosis by IFA test was reported in a man with a history of dyspnea, who worked in the field with cattle and frequently consumed raw milk and its derivatives [46].

Studies on Q fever affecting domestic ruminants in Brazil are relatively scarce [17, 20, 31, 47,48,49,50], which is problematic as these animals are considered primary sources of infection in humans [11]. In addition, C. burnetii infection results in economic losses for producers, as it is linked to animal reproductive disorders and can be confused with diseases caused by other pathogens, such as Toxoplasma gondii, Chlamydophila abortus, Brucella spp., Listeria monocytogenes, Campylobacter spp., Salmonella spp., Leptospira spp., and Neospora caninum, among others [51, 52].

In conclusion, the present study confirmed the circulation of C. burnetii in Rajada district, semi-arid region of Pernambuco, located in the Northeast of Brazil, through the detection of anti-C. burnetii antibodies in small ruminants and humans from this region, in addition to confirming that the bacterium causes infection in at-risk populations. In addition, bacterial DNA was detected in milk and vaginal mucus samples without the presence of antibodies or clinical signs, demonstrating that the animals are shedding the pathogen, contributing to the dissemination of C. burnetii, and making diagnosis difficult. This study serves as a basis for alerting health professionals to implement the necessary prevention measures, as well as to train veterinarians to notify the Ministério da Agricultura, Pecuária e Abastecimento if they identify the disease in animals. It is important to improve health education regarding this disease in the municipality, particularly in rural basic health units, among individuals who present with symptoms similar to those detected with the disease, as well as to refer suspected cases for a diagnosis, as Q fever is considered an occupational disease.