Survival of Brucella ovis ∆abcBA in soil, bedding, and bovine urineRESUMO
Infecção por Brucella ovis afeta principalmente carneiros, causando orquite, epididimite e infertilidade, com transmissão ocorrendo sobretudo por meio de sêmen contaminado. Apesar da alta similaridade genética entre as espécies de Brucella, B. ovis não possui potencial zoonótico. Em contraste, a brucelose bovina, causada por Brucella abortus, é uma doença zoonótica amplamente distribuída, que induz abortos em bovinos e representa um risco à saúde humana. As vacinas comerciais contra a brucelose bovina utilizam cepas atenuadas que têm patogenicidade residual para animais e humanos. A cepa candidata vacinal Brucella ovis ∆abcBA (Bo∆abcBA) demonstrou segurança e eficácia em camundongos, carneiros e cães, induzindo uma resposta imune protetora com ausência de excreção ambiental. Este estudo avaliou a persistência da Bo∆abcBA no solo, na cama de animais e na urina bovina, sob condições controladas. Os resultados indicam que a cepa vacinal Bo∆abcBA tem persistência limitada na urina de animais, enquanto a contaminação da cama e do solo diminui progressivamente, atingindo o limite de detecção aos 90 dias após a inoculação. Em conclusão, os resultados fornecem característica adicional de segurança, indicando que Bo∆abcBA, além de demonstrar segurança ao hospedeiro, também apresenta indicadores de segurança ambiental.
Palavras-chave:
persistência ambiental; sobrevivência bacteriana; matriz orgânica; meio ambiente
Keywords:
environmental persistence; bacterial survival; organic matrix; environment
Palavras-chave:
persistência ambiental; sobrevivência bacteriana; matriz orgânica; meio ambiente
Keywords:
environmental persistence; bacterial survival; organic matrix; environment
Palavras-chave:
persistência ambiental; sobrevivência bacteriana; matriz orgânica; meio ambiente
Brucella ovis infection affects sheep, primarily impacting rams, in which it is associated with orchitis, epididymitis, and infertility. The predominant route of transmission is through contaminated semen. However, females can excrete the bacterium via vaginal discharge for up to 10 days following abortion (Carvalho Junior et al., 2012).
Despite the genetic similarity of approximately 97% within the genus Brucella, different species of Brucella vary in terms of virulence, preferred host, and the ability to induce disease in humans (Soler-lloréns et al., 2016). B. ovis does not have zoonotic potential (Moreno, 2014).
Bovine brucellosis is a globally distributed infectious and zoonotic disease caused by Brucella abortus, leading to abortions in cattle and potentially causing a debilitating chronic disease in humans. Abortion induced by B. abortus releases high bacterial concentrations, being a source of infection for both humans and other animals within the herd. Bovine vaccination is a primary tool for controlling bovine brucellosis. Commercially available vaccines for bovine brucellosis utilize attenuated strains of Brucella abortus, which retain residual pathogenicity for animals and humans (Brasil, 2017).
Strategies for developing safer vaccines include tools of genetic engineering. Previous studies have demonstrated that the Brucella ovis vaccine candidate ∆abcBA (Bo∆abcBA) exhibits in vitro growth kinetics identical to that of the wild-type parental strain of B. ovis (Silva et al., 2011). Furthermore, its safety has been demonstrated in mice (Silva et al., 2011; Eckstein et al., 2020), rams (Silva et al., 2013), where it has conferred protection against Brucella spp. infection (Silva et al., 2015; Eckstein et al., 2020). Additionally, it has been shown to be safe in dogs (Eckstein et al., 2020). Importantly, Bo∆abcBA protects against experimental challenge with Brucella melitensis in mice, indicating a potential for developing a multivalent vaccine (Costa et al., 2020). Importantly, Bo∆abcBA is non-pathogenic to animals and it is not excreted in detectable quantities from experimentally infected rams (Silva et al., 2015), which drastically decreases the risk of environmental contamination. These significant findings underscore Bo∆abcBA as a versatile vaccine candidate with potential applicability to other animal species, including cattle.
Therefore, considering the potential for developing a polyvalent vaccine applicable to various host species (Silva et al., 2015; Costa et al., 2020; Eckstein et al., 2020) and the relevance of bovine brucellosis in Brazil (Santos et al., 2013), this study aimed to evaluate the environmental persistence of the candidate vaccine strain Brucella ovis ∆abcBA (Bo∆abcBA) in various sterilized organic matrices. Specifically, we investigated the survival and stability of Bo∆abcBA in soil, bedding, and bovine urine under controlled conditions. By assessing these different matrices, we sought to understand the potential risks of environmental contamination and the duration for which the vaccine strain can remain viable outside the host.
Microenvironments evaluated in this study included soil, bedding, and bovine urine. Substrates for these microenvironments were collected from distinct areas individually occupied by (i) a 4 month-old female crossbred Holstein calf housed in an external paddock fully exposed to sun light, measuring 104 m2; (ii) a 2 year-old crossbred Holstein cow housed in an external paddock fully exposed to sun light, measuring 132 m2; and (iii) an 3 year-old crossbred Holstein bull housed in an external paddock fully exposed to sun light, measuring 458 m2. Soil samples were collected superficially, up to a depth of 5 cm, from locations occupied by these three animals. Similarly, bedding materials consisting of a mixture of pine shavings or vegetation remnants and dry food residues (hay and forage), with irregular particle sizes ranging from 3 to 10 cm in their largest axis were collected. A single urine sample was obtained from each animal (female calf, adult cow, and adult bull) using the manual vulvar/preputial massage technique (Figure 1A). Urine was collected into a sterile reagent glass bottle with a screw cap, and the soil and bedding were collected in clean autoclave bags.
The three soil and bedding samples from each area occupied by a single animal, were pooled and autoclaved. Pooled soil and bedding samples were weighed and distributed into three aliquots of 2.5 g each. Urine samples were filtered using a 0.45 µm filter followed by a 0.22 µm filter and divided into 2.5 mL aliquots (Figure 1B). The method for preparation of these microenvironments was adapted from Nicaogáin et al. (2018).
Generation of the candidate vaccine strain Bo∆abcBA (B. ovis ΔBOV2_A500-501::KanR) has been previously described (Silva et al., 2011). Inocula containing 3.82 x 1012 colony forming units (CFU)/mL of Bo∆abcBA were cultured in Tryptone Soy Agar (TSA) (Invitrogen, USA) with 1% hemoglobin (Becton-Dickinson, USA) and 100 µg/mL kanamycin at 37°C in 5% CO2 for 3 days. Bacterial suspensions were adjusted to the final desired concentration by spectrophotometer at an optical density of 600 nm (OD600), serially diluted (10-fold) in phosphate-buffered saline (PBS), plated on TSA plates with 1% hemoglobin incubated at 37°C in 5% CO2 for 3 days and then the number of CFU was calculated to confirm inoculum (Figure 1C). In urine, 250 µL of the original 3.82 x 1012 CFU/mL suspension was added, resulting in a bacterial concentration of 9.55 x 1011 in the spiked urine sample. For bedding and soil, a tenfold serial dilution was performed, also resulting in a bacterial concentration of 9.55 x 1011 in 2.5 mL, sufficient volume to fully moisten the dry substrates.
Inoculated matrices (soil, bedding, and urine) were kept in semi-screwed sterile bottles at 21°C, under a 12-hour artificial light and 12-hour dark cycle. At each time point (1, 7, 30, 60, and 90 days post inoculation - dpi), a representative aliquot corresponding to 10% of the total weight or volume (0.25 g of soil and bedding or 0.25 mL of urine) of the bottle containing each of the microenvironment occupied by each animal (female calf, cow, and bull) was sampled. For solid substrates, 2 mL of sterile PBS was used to aid in matrix homogenization, and for urine, in case of desiccation that occurred after 60 days of incubation, resuspension was also carried out with 2 mL of sterile PBS. Subsequently, serial dilution and plating were performed using the drop counting technique on TSA + 1% hemoglobin + 100 µg/mL kanamycin (Figure 1C). Plates were incubated at 37°C in 5% CO2 for 3 days, and then the number of CFU was calculated.
Culture of sterile matrices kept for 1 day yielded only colonies of Bo∆abcBA. Urine samples had an average of 4.68 x 108 CFU/mL, whereas soil and bedding samples had 5.13 x 109 and 7.76 x 109 CFU/g, respectively, indicating approximately 4 log decrease in CFU counts for urine and 2.7 log decrease for bedding and soil at 1 dpi (Figures 2 and 3). At 7 dpi, a marked reduction in CFU counts was observed in the urine from all animals, with an average of 1.02 x 104 CFU/mL, whereas for bedding and soil, the averages were 3.55 x 109 CFU/g and 8.51 x 109 CFU/g, respectively. At 30 dpi, CFU counts in the urine approached the detection limit. Bedding and soil samples had a decrease in CFU numbers at 30 and 60 dpi (Figures 2 and 3). Interestingly, at 60 dpi, soil and bedding had a broad variation in CFU numbers, with one of the samples near the detection limit (Figures 2 and 3). At 90 dpi, in all matrices, the Bo∆abcBA CFU numbers reached the detection limit (Figures 2 and 3).
Experimental design of this study. A. Bedding, soil, and urine matrices collected from three external paddocks, fully exposed to sunlight, occupied by a calf, a cow, or a bull. Three beddings and soil were pooled to compose a single sample per animal, while only one urine sample was collected per animal. B. Matrices were sterilized (by autoclaving or filtration), aliquoted in 2.5 g or 2.5 mL per animal. C. Samples were inoculated with 9.55 x 1011 CFU/2.5 g or 0.25 mL of Brucella ovis ∆abcBA, for bedding and soil or urine, respectively. At 1, 7, 30, 60, and 90 days post inoculation samples were serially diluted (10-fold) and plated on selective media. For dry substrates (bedding and soil) or desiccated samples (urine after 60 days), 2 mL of sterile phosphate-buffered saline (PBS) was added to allow homogenization or resuspension of urine sediment. Samples were kept at 21°C in sterile, semi-crewed bottles and subjected to 12-hour cycles of artificial light and 12-hour periods of darkness. Image created by the author using the BioRender software.
Survival of the candidate vaccine strain Brucella ovis ∆abcBA (Bo∆abcBA) in various microenvironments. Bedding, soil, and urine matrices collected from areas individually occupied by a calf, cow or bull were previously sterilized and inoculated with 1x1012 CFU/mL of Bo∆abcBA. At 1, 7, 30, 60, and 90 days post inoculation, aliquots of 10% of the total weight or volume were plated on selective media for CFU counting. Data was subjected to ANOVA and Tukey’s test. Each data point represents the bacterial quantification of the substrate from one of the animals and the bars represent the standard error of the mean. ** p = 0.0015; *** p = 0.0004; **** p < 0.0001. The dotted line represents the limit of detection.
Parametric statistical analysis using ANOVA and Tukey’s test was performed to assess the differences in Bo∆abcBA survival across different substrates over time. Additionally, the coefficient of determination (R²) was calculated to evaluate the relationship between the reduction in Bo∆abcBA bacterial survival over time, with higher values observed in bedding and soil (Figure 3). The statistical analyses were performed using GraphPad Prism 10 software.
Environmental survival of Brucella ovis ∆abcBA (Bo∆abcBA) in previously sterilized bedding, soil, and bovine urine. Each point represents the mean of bacterial quantification in substrates derived from areas occupied by a bull, a cow, or a calf, and the bars represent the standard error of the mean. Dotted line represents the limit of detection.
The Brucella genus is characterized by coccobacilli measuring 0.5-0.7 × 0.6-1.5 µm, gram-negative, occurring as isolated cells, in pairs, and rarely in short chains or groups. These bacteria are non-motile, non-spore-forming, and lack a capsule. They are classified as aerobic bacteria, with most species requiring a CO₂ supplement for growth. They grow at temperatures ranging from 20 to 40°C, with optimal growth at 37°C and a pH between 6.6 and 7.4. They do not ferment conventional carbohydrates or lyse red blood cells. Brucella species are facultative intracellular, non-toxigenic pathogens, capable of infecting a wide range of animal species, including humans (Scholz et al. 2018). In abortions caused by B. abortus, infected cows may shed up to 1010 CFU/mL in the allantoic fluid, while in placental tissues, there can be up to 1013 CFU/g of B. abortus (Alexander et al., 1981). Previous reports suggest that B. abortus survives for 4-5 hours under sunlight, 4 days in dry soil, 66 days in moist soil, and if the soil is moist and associated with low temperatures, B. abortus can survive from 151 to 185 days. In feces and sewage at low temperatures, it survives for 120 to 700 days, respectively, while in waste exposed to high temperatures, only for 2 to 4 hours. In potable water, B. abortus can survive from 5 to 114 days, and in polluted water from 30 to 150 days. In a fetus in the shade, the bacterium can remain viable for up to 180 days, and in uterine exudate in the shade, up to 200 days (Donaldson, 1978).
Blaiotta et al. (2016) assessing the persistence of zoonotic pathogens in residues containing a mixture of fresh cattle feces, bedding material, and ruminal content intended for manure production in Southern Italy, detected DNA sequences of Brucella spp., and Mycobacterium spp. in those materials. Brucella spp. was detected in samples of fresh (recently collected) material and semi-moist material with an intermediate degree of maturation. These data is in good agreement with the survival data of Bo∆abcBA for up to 90 days in soil samples. No detection of Brucella spp. was found in samples of manure ready for use after 5 months of storage (Blaitolla et al., 2016).
Donaldson (1978) reviewed factors that influence dispersion, survival, and deposition of farm animal pathogens transmitted by air. Temperature, humidity, non-ionizing radiation, and gaseous pollutants affect the survival and dispersion of these pathogens. In general, high temperatures increases bacterial death rate, and gram-negative bacteria are more sensitive to moderate humidity (40-60%). Solar radiation damages bacteria indirectly through a photodynamic effect, with the formation of photo-reactive pigments, either natural or exogenous, in the presence of oxygen, as well as the induction of lethal effects caused by ultraviolet radiation.
This is the first study to demonstrate the profile of survival of the candidate vaccine strain of Bo∆abcBA in various microenvironments. Our findings indicate that Bo∆abcBA can survive for short periods in bovine urine and a decreasing and transient persistence in bedding and soil, indicating a low potential route for environmental contamination. Previous studies by our group have shown that this vaccine strain is not shed by infected rams (Silva et al., 2013) and dogs (Eckstein et al., 2020). This study demonstrated that the persistence of this bacterium in the environment is shorter than 90 days. Importantly, Bo∆abcBA was based on a non-zoonotic Brucella species (Moreno, 2014). Furthermore, Bo∆abcBA is strongly attenuated in its preferred host or alternative host species (Silva et al., 2013; Eckstein et al., 2020), which makes it a potentially suitable tool to prevent occupational exposure, which happens with the currently available vaccines (Pereira et al., 2021), not just as an occupational hazard but also due to consumption of unpasteurized milk (Negrón et al., 2019).
Although observations under sterile conditions may not perfectly replicate the natural environment, conducting environmental persistence assays of the vaccine candidate Bo∆abcBA under laboratory conditions that simulate field situations is essential. These experimental data are critical to further demonstrate safety of Bo∆abcBA, a potential polyvalent vaccine for brucellosis (Silva et al., 2013; Eckstein et al., 2020).
In conclusion, our results add an additional layer of safety analyses on Bo∆abcBA, expanding our evaluation beyond the host safety, with additional focus on environmental safety.
ACKNOWLEDGEMENT
Work in RLS lab is supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil), FAPEMIG (Fundação de Amparo a Pesquisa do Estado de Minas Gerais, Brazil), and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil).
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