Acta Tropica 240 (2023) 106841 Contents lists available at ScienceDirect Acta Tropica journal homepage: www.elsevier.com/locate/actatropica Anthrax bio-surveillance of livestock in Arua District, Uganda, 2017–2018 Michael Omodo a, 1, Jaume Gardela b, 1, *, Alice Namatovu c, Rose Ademun Okurut a, Martin Esau a, Merab Acham a, Maria Flavia Nakanjako a, Mugezi Israel a, Emmauel Isingoma a, Mwanja Moses a, Lumu Paul a, Ben Ssenkeera a, Stella A. Atim a, Doreen N. Gonahasa d, Musa Sekamatte d, Meriadeg Ar Gouilh e, f, Jean Paul Gonzalez g a National Animal Disease Diagnostics and Epidemiology Center (NADDEC), Ministry of Agriculture, Animal Industry and Fisheries, Kampala, Uganda b Department of Animal Health and Anatomy, Faculty of Veterinary Medicine, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain c College of Veterinary Medicine, Animal Resources and Biosecurity (COVAB), Makerere University, Uganda d Ministry of Health, National One Health Platform, Kampala, Uganda e Normandy University, DYNAMYCURE U1311 INSERM, UNICAEN, UNIROUEN, Caen University, 14000 Caen, France f University Hospital Center of Caen, Virology Department, 14000 Caen, France g School of Medicine, Department of Microbiology and Immunology, Georgetown University, Medical Center, Washington DC, USA A R T I C L E I N F O A B S T R A C T Keywords: Anthrax, caused by Bacillus anthracis, is a widespread zoonotic disease with many human cases, especially in B. anthracis developing countries. Even with its global distribution, anthrax is a neglected disease with scarce information Detection about its actual impact on the community level. Due to the ecological dynamics of anthrax transmission at the One health wildlife-livestock interface, the Sub-Saharan Africa region becomes a high-risk zone for maintaining and Sub-saharan Africa region Zoonosis acquiring the disease. In this regard, some subregions of Uganda are endemic to anthrax with regular seasonal Cattle trends. However, there is scarce data about anthrax outbreaks in Uganda. Here, we confirmed the presence of B. anthracis in several livestock samples after a suspected anthrax outbreak among livestock and humans in Arua District. Additionally, we explored the potential risk factors of anthrax through a survey within the community kraals. We provide evidence that the most affected livestock species during the Arua outbreak were cattle (86%) compared to the rest of the livestock species present in the area. Moreover, the farmers’ education level and the presence of people’s anthrax cases were the most critical factors determining the disease’s knowledge and awareness. Consequently, the lack of understanding of the ecology of anthrax may contribute to the spread of the infection between livestock and humans, and it is critical to reducing the presence and persistence of the B. anthracis spores in the environment. Finally, we discuss the increasingly recognized necessity to strengthen global capacity using a One Health approach to prevent, detect, control, and respond to public threats in Uganda. 1. Introduction B. anthracis spores are known to survive for decades in alkaline soil rich in calcium ions which are believed to facilitate sporulation hence Anthrax is a zoonotic disease caused by the Gram-positive bacterium driving landscape-level patterns of spore persistence (Carlson et al., Bacillus anthracis rods, a soil-transmitted pathogen found on all conti­ 2018; Hugh-Jones and Blackburn, 2009). Both livestock and wild her­ nents and reported in several islands like Haiti, the Philippines, and bivores are exposed to B. anthracis spores from the soil while grazing. Indonesia (Carlson et al., 2018). Globally, an estimated 20,000 to 100, They become infected and usually return spores to the soil when dead 000 human anthrax cases occur annually, particularly in developing and decomposed (Turner et al., 2014). countries with poor rural settings (Swartz, 2001). Naturally, the enzo­ Anthrax is a neglected disease, and its global distribution is still otic cycle of anthrax is characterized by a combination of long-term poorly characterized (Carlson et al., 2018). It has been shown that spore persistence in soil and an obligate-lethal transmission route of wildlife epizootics may lead to downstream infections in both humans ingestion, primary in herbivorous mammals (Carlson et al., 2018). and livestock (Mukarati et al., 2020). Sub-Saharan Africa is a high-risk * Correspondence author. E-mail address: jaume.gardela@uab.cat (J. Gardela). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.actatropica.2023.106841 Received 25 November 2022; Received in revised form 16 January 2023; Accepted 21 January 2023 Available online 21 January 2023 0001-706X/© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). M. Omodo et al. Acta Tropica 240 (2023) 106841 zone because wildlife and livestock frequently share grazing fields in countries like Zambia, in which a total of 1216 bovine cases were re­ National Parks (Mwakapeje et al., 2018). One particular example is the ported in the Zambezi flood plain between 1999 and 2007 (Munan­ Queen Elisabeth National Park in the Western Region of Uganda where g’andu et al., 2012). The lack of information associated with anthrax hundreds of hippopotamus scammed to deadly anthrax in 2004–2005 outbreaks is attributed to farmers who do not report suspected cases to (Driciru et al., 2018). The sub-Saharan Africa has an estimated popu­ the veterinary department in Uganda, making it more difficult to un­ lation of 16.2 million poor livestock keepers (Carlson et al., 2019), who derstand the magnitude, impact, and control of the disease’s spread stand at high risk of acquiring the disease by occupational exposure. (personal communication). Human clinical presentations of anthrax, case-fatality rates, and mor­ This study aims to identify and confirm the presence of anthrax in tality are a function of the exposure pathway determined by the biological samples and determine case fatalities among livestock after a ecological dynamics at the wildlife–livestock interface (Alexander et al., suspected outbreak of anthrax in Arua between 2017 and 2018, a north- 2012). In some regions, such as the West Nile sub-region in western district of Uganda located in the West Nile sub-region. We also north-western Uganda, anthrax is hyperendemic, and cases follow reg­ aim to establish the potential risk factors of anthrax that affect the cattle ular seasonal trends (Ntono et al., 2021). population in Arua District at the community level. Understanding the In Uganda, anthrax outbreaks occur sporadically and continuously risk factors has important implications for preventing, detecting, con­ from different subregions of the country (Coffin et al., 2015). As a result, trolling, and responding to future anthrax outbreaks in the region, the disease has been ranked as one of the most critical zoonosis along­ meeting the requirements of the International Health Regulations side Ebola, brucellosis, African sleeping sickness, plague, and rabies, (World Health Organization, 2008) and Global Health Security Agenda which are prioritized zoonotic diseases selected to be controlled and (Wolicki et al., 2016). maintained at minimum levels (Sentumbwe et al., 2018). Therefore, control of anthrax requires a One Health approach involving both 2. Materials and methods human and animal health stakeholders for cross-sectoral integration and coordination of activities to prevent, detect, and respond to endemic 2.1. Study area anthrax in Uganda. Anthrax still remains a challenge to many farming production sys­ Arua District (Fig. 1) is located in the West Nile sub-region of tems in Uganda (Vudriko et al., 2021). Opening infected carcasses by northwest Uganda. In 2020, the population of the Arua District was community members in all anthrax hot spot areas and leakage of in­ estimated at 750,000 habitants (Citypopulation.de, n.d.). It is bordered ternal discharges in the grazing ground occurs, putting at risk the health by other districts of Uganda (Yumbe, Moyo, Maracha, Koboko, and of a large population of ruminants and humans in the region. Such ac­ Adjumani) and the Democratic Republic of the Congo (DRC) to the west. tivities contribute to spore transformation, transmission as well as Because of its localization in a corner of Uganda that borders DRC and environmental contamination. Uganda faces a scarcity of statistics about South Sudan, the most significant amount of local community activity bovine data associated with anthrax outbreaks compared to other results from cross-border trade. Agriculture is the mainstay of the Arua’s Fig. 1. Location of Arua District (purple) in Uganda and detail of hot spots Sub-Counties (pink) of anthrax outbreaks in Arua District in 2018 (Rigbo, Rhino Camp, Ogoko, and Okollo). 2 M. Omodo et al. Acta Tropica 240 (2023) 106841 District economy, with approximately 117,000 head of local Zebu cattle. 82% and sensitivity of 98%, showing better sensitivity than the gold Rigbo, Rhino Camp, Ogoko, and Okollo Sub-Counties were the centres of standard culture, Laboratory Response Network real-time PCR, or anthrax outbreaks reported in 2018 (Fig. 1). immunohistochemistry (Kolton et al., 2019). However, additional testing (microscopy) was performed to confirm the cases. 2.2. Data collection from Arua District 2.4. Collection of biological specimens from the Arua District To better understand the status of anthrax outbreaks and the dy­ namics among domestic animals in Arua District, data was collected In the Arua District, twenty-four (24) biological samples from sus­ from community herds of livestock (kraal) through surveys in the Arua pected anthrax cases were collected from community herds of cattle Sub-Counties with elevated mortality reports of suspected anthrax out­ during the outbreak seasons. Samples comprised of fixed blood smears, breaks in livestock between 2017 and 2018 (Rigbo, Rhino Camp, Ogoko, pooled unclotted blood, smoked meat, fresh meat, bovine skin scrapings, and Okollo Sub-Counties). Data sources were collected from 57 com­ ear notch swabs, human skin lesions, and environmentally contami­ munity herds of livestock from responsible kraal leaders. The ques­ nated material. Human samples from Arua health centres were sub­ tionary was designed and previously pretested before its usage. mitted to the National Animal Disease Diagnostics and Epidemiology Additionally, the district veterinary officer and the Sub-County chief Centre (NADDEC) to test and confirm zoonotic anthrax in the affected were interviewed about the anthrax situation. districts under the One Health arrangement. The AAD rapid test lateral Data collected comprised both qualitative and quantitative variables flow immunoassay and microbiological methods such as Gram staining of demographic information of respondents (name, age, sex, and size of and polychrome methylene blue staining (M’Fadyean stain) were used their family), localization (Sub-County, parish, and village), knowledge to detect and confirm B. anthracis. about anthrax disease, education received (none, elementary, second­ ary, or higher education), ownership of companion animals, and herd 2.5. Active Anthrax Detect rapid test size. The data frame also recorded the species affected, the number of domestic animals and livestock suspected to be infected with anthrax Detection of B. anthracis in animal tissues was performed using a during the outbreaks, and clinical signs, if any (loss of appetite, fever, simple, rapid, and field-deployable kit provided by InBios to purposely recumbency, external bleeding, sudden death, blotting, and rigor mortis). screen and respond to the outbreak faster and reduce the burden of Information about human activities that facilitate disease dispersion was livestock disease. The AAD is a screening assay designed to identify the also recorded (i.e., management of the dead carcasses, opening and B. anthracis capsular polypeptide (polyglutamic acid) antigen (Gate­ skinning the dead animals in the field to consume and sell the meat, s-Hollingsworth et al., 2022, 2015; Kolton et al., 2019). Samples were carcass disposal and soil treatment methods, transportation of animal tested following the manufacturer’s instructions. Positive (the wild products with dripping blood discharges to trading centers for sell, and strain of encapsulated B. anthracis) and negative quality controls transport means used to carry meat to the selling points). (B. subtilis) were run alongside the test samples based on immunochro­ Interviews with the Veterinary Department included records about matographic principles. Test results were read after 15 min. Each car­ vaccination status, if any, species affected, physical control actions (i.e., tridge developed test and control lines. For a positive result, both lines quarantine and other events which contributed to the persistence or appeared. For a negative result, only the control line was visible. Sam­ control of the disease in the area), management or disposal of carcasses, ples that did not show any of the lines were repeated. Samples were availability of personal protective equipment, biosecurity measures if further confirmed using Gram and M’Fydean stains. any, capacity to the regional veterinary laboratory to handle the diag­ nosis of anthrax samples, the level of community reporting of suspected 2.6. Gram staining method cases, number of reported dead animals that succumbed to suspected anthrax, and availability of chemicals to disinfect carcasses and burial The samples were tested following a previously validated protocol sites as well as the availability of emergency fuel for burning dead for the Gram staining method. Both thick and thin smears were prepared animals. on labelled clean glass slides and allowed to dry by air and on a heating block for 2 min. With the addition of crystal violet stain for 1 min, then 2.3. Anthrax standard case definitions in livestock flooded with mordant iodine for 1 min. Followed by decolorizing the sample with 70% acetone-alcohol, the tissue was stained with dilute During the study, outbreaks were still occurring in which animals carbol-fuchsin as a counterstain. All excess stains were washed off with were sick, and others died suddenly in the kraals and the field. There­ clean tap water, dried, and examined at room temperature under fore, the team decided to collect biological specimens, observe several magnification × 100 with a light microscope using immersion oil. For cases’ conditionality, and develop clinical descriptions for the target quality control, known internal quality controls strains of wild population as described below. B. anthracis and gram-negative Escherichia coli were examined alongside A suspected case of herbivore anthrax was defined as any case of the samples. sudden death of animals with/without bleeding from natural orifices such as anus, eyes, and ears (unclotted dark tarry blood), absence of or 2.7. The M’Fadyean stain incomplete rigor mortis, and rapid bloating of carcasses. In pigs and carnivores, the main symptoms suspected of anthrax were local edema Samples were also assessed by the M’Faydean staining method and swelling of the face and neck. (M’Fadyean, 1903). Briefly, thin and thick smears were prepared using A confirmed animal anthrax case of required laboratory detection of coverslips on a clean glass slide. The specimens were dried at room B. anthracis either in smears, rapid screening test, or bacterial isolation. temperature and fixed with potassium permanganate (40 g/L) solution As a criterion for laboratory diagnosis confirmation, we used interna­ for 10 min. Then, the smears were covered with Loffler’s polychrome tionally recognized standard diagnostic techniques. The interpretation methylene blue for 1 min, and the excess stain was removed using tap for anthrax diagnosis in animals is reported in the Manual of Standards water. The slides were wiped with soft tissue and allowed to dry at room for Diagnostic Tests and Vaccine for Terrestrial Animals of the Office temperature under a biosafety cabinet. Samples were examined with a International des Épizooties (World Organization for Animal Health, light microscope using immersion oil at × 100 magnification. For 2021). The samples were subjected to the Active Anthrax Detect (AAD) quality control, known prepared positive control smears of a wild strain rapid test lateral flow immunoassay, which has been previously tested in of B. anthracis and negative controls (B. subtilis Sterne strain) were animal suspected anthrax tissue samples in Namibia with a specificity of examined. 3 M. Omodo et al. Acta Tropica 240 (2023) 106841 2.8. Statistical methods and data analyses received secondary education (13/21). The remaining 7.1% of the people knowing about anthrax received primary education (1/14), Kraals’ survey data were analyzed using the survey package (Lumely, representing the 8.3% of the total population that received primary 2020) in R Core Team, (2019). Descriptive statistics for continuous and education (1/12). Receiving secondary education was statistically sig­ categorical variables were determined. Additionally, subpopulation nificant to knowing about anthrax disease (p < 0.001). Additionally, analyses were performed, associations between variables were deter­ higher knowledge about anthrax disease was found in the Payawe and mined using the Wald test, and multiple linear regression models were Ndara villages (located in the Rhino Camp and Ogoko Sub-Counties, conducted, including several predictors to the variable “knowledge of respectively; p < 0.05) and, interestingly enough, were the villages in anthrax” and “anthrax outbreaks in people”. Statistical significance was which more cases of affected people were reported (20 and 12 people, set at p < 0.05. Data were expressed as mean ± standard deviation respectively; p < 0.01). unless otherwise stated. The practice of eating dead animals is widely spread in these com­ munities-89.7% of the communities consumed the meat of dead animals 3. Results (51/57). More than half of the dead carcasses were opened for meat consumption (57.3% - 129/225), and 12.9% were skinned (29/225). 3.1. Arua District kraal surveys findings The source of infection in people was contact with livestock (50%) and contact with contaminated meat or animal products in local markets Data from kraals surveys were obtained in 6 villages (Agera, Ndara, (50%). Villagers transported contaminated meat to the local markets Owodromati, Pasumu, Payawe, and Tumawele) located in the Rigbo, shipping beef on their backs (21.7%) and motorbikes (78.3%) without Rhino Camp, Ogoko, and Okollo Sub-Counties of the Arua District. Some any protective clothing. Type of transportation means of contaminated community members concealed information for fear of restriction from meat was not associated with anthrax outbreaks in people (p > 0.05). trade and consumption of their beef. All the responsible kraal leaders (57 individuals) were males with an average of 34.6 ± 12.1 years (minimum 3.2. Arua District Veterinary Department interviews 19 years, maximum 70 years). We recorded 6781 cattle, with a herd average number of 119 ± 71.5 Arua District Veterinary Department is faced with numerous chal­ animals. Additionally, the number of sick and dead goats, sheep, pigs, lenges attributed to a lack of resources to contain the spread of the and dogs were recorded. A total of 339 animals were reported sick with disease in the region. The district lacked information, education, and clinical signs compatible with anthrax, with a case fatality rate of 66.4% communication materials for educating communities about the signs (225/339). Clinical signs compatible with anthrax include sudden death and risks of disease and preventative measures. Records from the (49%), rigor mortis (44%), fever (44%), external bleeding (39%), loss of Department revealed numerous challenges surrounding the outbreak in appetite (37%), blotting (32%), and recumbency (26%). More than a 2017–2018. Vaccines were not available to prevent and protect com­ quarter of the dead animals were abandoned in the bush (31.1% - 70/ munity livestock from spreading anthrax in the district. Additionally, 225) without any disinfection or proper elimination method of the quarantine and law enforcement procedures to limit the movement of carcasses. Of the total animals reported dead, 86.2% were cattle (194/ livestock in and out of the district were weak. From the community, 225), 8% were goats (18/225), and 5.8% were sheep (13/225). Mor­ carcasses were opened for consumption. During the outbreak, the dis­ tality in cattle was 2.9% (194/6781). Pigs and dogs were not affected by trict technical response team lacked personal protective equipment to the anthrax outbreak. However, 40 people were affected: 20 people in handle carcasses, appropriate chemicals such as soda ash and fuel to Payawe, 12 people in Ndara, 5 people in Owodromati, 2 people in Agera, burn the carcasses. The wrong depth of the pit dug to bury carcasses 1 person in Tamuwele, and 0 people in Pasumu. (about less than 6 feet), and no biosecurity measures taken to contain the A quarter of the responsible kraal leaders knew about anthrax spread of spores enabled scavengers like dogs to disturb the burial sites (24.6% - 14/57), whereas 75.4% lacked knowledge of the disease (43/ in the search for beef. 57) (Fig. 2). More than one third of the responsible kraal leaders were The community’s level of reporting diseased animals was very poor, illiterates (38.6% - 22/57), 21.1% received primary education (12/57), and the data provided was not significant enough for proper planning. It 36.8% received secondary education (21/57), and 3.5% received higher is believed that more animals died due to suspected anthrax, but reports education (2/57). The statistical analysis showed that the knowledge at the center show that only 125 cattle, 9 sheep, and 16 goats died of about anthrax disease was associated with the education level (p< suspected anthrax. In addition, because of logistical challenges in the 0.001). Almost the totality of the responsible kraal leaders within the Veterinary Department, no real-time surveillance was conducted to population knowing about anthrax received secondary education establish the scope of infection and respond timely. The regional vet­ (92.9% - 13/14), representing the 61.9% of the total population that erinary laboratory in Arua lacked laboratory reagents to test, detect, and Fig. 2. Knowledge about anthrax associated with the education level of responsible kraal leaders. Asterisk indicates the education level statistically significant to knowing about anthrax disease. 4 M. Omodo et al. Acta Tropica 240 (2023) 106841 confirm anthrax in real-time. Samples were collected and shipped to and decomposing livestock carcasses. Additionally, political and war NADDEC and Uganda Virus Research Institute for a conclusive labora­ pressures in the Republic of South Sudan triggered the movement of tory diagnosis. In Pawor Sub-County, Odupi, and Rhino Camp 65, 30, people with their livestock to settle in Rhino Camp, in Arua District. This and 29 cattle were reported dead, respectively. may have contributed to the unquantified risk of introducing animal diseases in Uganda. Moreover, no measures were put in place to screen 3.3. Tested samples the foreign livestock for possible epidemics, which may increase the risk of animal diseases, including anthrax outbreaks. Similarly, the risk of From the 24 samples submitted to NADDEC during the outbreak disease transmission between the indigenous and foreign livestock is (Table 1), 19 samples tested positive (79.2%) on Gram and polychrome likely to occur because all livestock share one drinking water point: river methylene blue stains. Positive samples demonstrated Gram-positive Nile banks and grazing fields. dark blue rods with a capsule on gram stain and polychrome methy­ Particularly, cattle were mentioned and linked to cutaneous anthrax lene blue-stained large blue rods surrounded by red amorphous capsules human infections in the district. The 69 human cases reported with characteristic of B. anthracis and numerous chains of non-sporulated cutaneous anthrax in Olujobo health center were linked to livestock rods. Under the light microscope, the observed positive smears farming and behavioral risks of shipping beef on their backs and mo­ demonstrated an uneven distribution of square-ended Gram-positive torbikes to trading centers without the protection of their bodies using rods ranging from low to high intensity (+ to +++) of sporulated and any protective clothing, except using their bare hands. The susceptibility non-sporulated Gram-positive rods. For the ADD rapid test (Table 1), 12 to anthrax infection varies depending on the host species, where cattle samples tested positive (50%). are known to be the most devastated species, followed by sheep and goats (Fasanella et al., 2010). A similar situation was noticed in Arua 4. Discussion District between 2017 and 2018. According to the district veterinary officer and the community surveys, more heads of cattle died of anthrax In domestic animals and people, anthrax is particularly common in than goats and sheep. parts of Asia, the Middle East, and Africa, where control measures in the Cattle tend to pull pasture directly from the ground with their roots, livestock sector remain inadequate (Antonation et al., 2016). Anthrax is unlike sheep or goats, which browse on shrubs on off the ground level a notifiable disease in Uganda, and it is a disease of public health and (Hornitzky and Muller, 2010). Therefore, cattle are most likely to ingest veterinary importance (Republic of Uganda - Ministry of Health, 2021). high doses of the encapsulated bacteria from potentially contaminated However, Uganda has a national surveillance plan for anthrax developed soil compared to browsing ruminants. Humans have a moderate sus­ in 2018 with the support of the Food and Agricultural Organization of ceptibility to the infection, while pigs and carnivores are more resistant the United Nations - Uganda purposely to control anthrax in the country to the deadly spores (Epp et al., 2010). According to these results, our (Food and Agriculture Organization of the United Nations, 2018). survey data showed that pigs and dogs were not reported to be sick or The disease remains endemic with high incidences of anthrax in dead due to anthrax outbreaks. However, dogs may act as instrumental livestock and is one of the listed priority zoonotic diseases of public in scavenging on the carcasses and spreading the infectious agents me­ health and veterinary importance in Uganda (Vudriko et al., 2021). chanically to clean areas. Despite annual anthrax outbreaks in animals, there is a weak surveil­ The most accepted philosophy for browsing animal infection in Af­ lance system in the animal sector, which has led to underreporting and rica is that blowflies feeding off the carcasses move to rest on the leaves failure to detect early cases for immediate intervention. Therefore, a of nearby trees and shrubs and deposit anthrax spores on these leaves reliable and sensitive diagnostic method is of higher value to reduce the (De Vos, 1994). Browsing animals become infected after feeding on chance of zoonotic transmission and understand the burden diffusion contaminated leaves. Additionally, biting flies are suspected of trans­ and prevalence in time. In 2019, due to the lack of a rapid and reliable mitting anthrax amongst wild animals and playing important roles in the diagnostic method and other factors, the community herds surveyed in wild epidemics (Hugh-Jones, 2016), making it difficult to halt the the Arua District reported 339 livestock infected with anthrax, 225 an­ infection in the susceptible population if flies are heavily involved in the imals were reported dead, and 40 people were affected by B. anthracis. spread of spores. In Arua District, a similar scenario of decomposing Keeping livestock for livelihood in agricultural communities is a bodies of livestock on the scene was heavily surrounded by active flies, common practice in Arua District. However, animal husbandry practices making it difficult to halt the spread of spores to the clean environments. in this community do not consider preventing animal diseases like Furthermore, seasonal variations of anthrax transmission and unre­ anthrax. In Arua District, animals graze extensively, and others spend ported livestock deaths in the district may also contribute to the 24 h in the field, where they may frequently contact contaminated soil persistence of the outbreaks (Ashkenazi-Hoffnung et al., 2009). A dis­ cussion with a local area leader in Pasumu village revealed that many Table 1 sudden deaths of animals in the region occurred but were not reported to Summary of human and animal tested samples using three diagnostic methods the veterinary authorities. Animals fell sick, lost appetite, failed to during the suspected anthrax outbreak in Arua District (2018). respond to treatment, blood started oozing out of the nostrils, and finally Sample Type Tested Positive cases died. In Ndara village, 13 animals were lost in December 2018, and one samples of the handlers of beef, a 17-year-old boy, developed skin lesions 4 days GS PMB AAD after carrying meat on his back. Bovine fixed blood smears 9 6 6 0 Due to ignorance of the risk and custom, some community members Bovine pooled unclotted 3 3 3 3 exhumed dead animals and admitted that the common method of car­ blood Smoked meat bovine 1 1 1 1 casses disposal was consumption since it is taboo to bury animals. As a Fresh meat bovine 2 0 0 0 risk factor, the digging process may expose the anthrax bacterium, Bovine skin swabs 1 1 1 1 which can further cause a spread of the contaminated soil extensively by Bovine ear notch swabs 5 5 5 4 blowing wind and lead to fresh outbreaks in the area (Ravenel, 2008). Environmental samples 2 2 2 2 (blood on grass) Due to a lack of proper disposal practices and resources, other cattle Human cutaneous swab 1 1 1 1 keepers decided to abandon dead animals in the bush. These carcass Total 24 19 19 12 disposal methods were contrary to appropriate burning procedures, (79.2%) (79.2%) (50%) followed by disinfection of the site, which are better and more efficient GS: Gram staining; PMB: polychrome methylene blue stain; AAD: Active Anthrax mechanisms to reduce the chances of spore survival and further out­ Detect rapid test. breaks in the affected areas (Mwakapeje et al., 2017). 5 M. Omodo et al. Acta Tropica 240 (2023) 106841 The community’s lack of awareness and knowledge is a risk factor environments in which regular anthrax cases occur. In this regard, that contributes to the spread of diseases. Our data identified that real-time polymerase chain reaction is a preferred method for detecting educational status and previous contact with the disease are associated B. anthracis in animal tissue and blood samples (Avberšek et al., 2021; with the knowledge of anthrax. This could be because education impacts Berg et al., 2006). Awareness in the community is vital for the locals to on information access and capacity to comprehend health messaging. understand forms of anthrax, species affected, impact and disease pre­ Furthermore, the previous contact with the disease may increase sentation. The importance of reporting to the Veterinary Departments is awareness of avoiding contracting anthrax. Therefore, education is the paramount in controlling the spread of the disease and reducing human cornerstone to expanding the knowledge of anthrax among the farmers’ infections as well as livestock mortalities. population and preventing its spread. These findings are supported by a study conducted in Ethiopia, in which education background and 5. Conclusions knowledge of anthrax were associated with better anthrax prevention practices (Mesfin et al., 2021). Other factors include sex (females had This study identified and confirmed the presence of anthrax in bio­ better anthrax prevention practice than males), time spent arriving at logical samples after a suspected outbreak in Arua District. The cattle the nearby clinic, and attitude towards anthrax prevention (Mesfin et al., population was more affected by anthrax than any other reared species 2021). included in the study, such as goats and sheep. Lack of awareness among In the wildlife sector, sporadic anthrax outbreaks occurred in and cattle owners and community concerning the nature, the transmission of around Queen Elizabeth National Park, where anthrax collectively killed anthrax from animals to humans, scarcity of the livestock vaccine, social over 500 wildlife, particularly hippopotamus and small herbivores and norms, cultures, environmental factors, and poverty were the key 400 domestic animals (Coffin et al., 2015). Comprehensive studies in the drivers that may have contributed to the spread of anthrax in livestock, tropical rainforest of Africa revealed that B. anthracis is accountable for as noted in this study. The grazing system where cattle are not controlled widespread incidences and persistent mortality among domestic herbi­ and monitored may easily promote the transmission and maintenance of vores and wild mammals for 3 decades with significant losses (Hoff­ anthrax outbreak cycles in the Arua District’s grazing field and other mann et al., 2017). With the presence of Ajai Game Reserve in the Arua Uganda districts. District and zero control of environmental contamination, there is a likelihood of anthrax spilling over to the wildlife herbivores in the Ethics approval future. In this case, grazing animals may acquire the disease by ingesting spores when grazing over sites where previous victims died and depos­ The field and laboratory outbreak investigations were conducted in ited the spores in the environment. For instance, licking of bones (pica or accordance with the Ministry of Agriculture, Animal Industry and osteophagia) in search for minerals like calcium from animals that died Fisheries (MAAIF) and World Oragnisation for Animal Health (WOAH) of anthrax may result in cases or outbreaks in the wildlife (Lafferty and requirements for responding to and managing Animal disease outbreaks Chapman, 2014). in Uganda. The primary intent of the investigation was public health Failure of livestock vaccination programs in enzootic districts is a control, as did not require ethics committee approval or written consent risk factor for livestock and wildlife herbivores (Rao et al., 2019). of the farmers. Vaccination of susceptible animals is one of the options to confer pro­ tective immunity and preserve animal health (Rao et al., 2019). From CRediT authorship contribution statement 2015–2019, no livestock vaccination was conducted as a predisposing factor, which may also have contributed to the growing number of Michael Omodo: Conceptualization, Data curation, Funding acqui­ anthrax fatalities among cattle. Due to poverty, livestock owners could sition, Methodology, Project administration, Resources, Validation, not afford to buy their own vaccines because of a high cost of a single Writing – original draft, Writing – review & editing. Jaume Gardela: dose, which ranges from US$8 to US$19, which is another risk factor Data curation, Formal analysis, Methodology, Software, Validation, that paves the way for continuous anthrax outbreaks in the region unless Visualization, Writing – original draft, Writing – review & editing. Alice government or donor organizations step in to support the district. Namatovu: Supervision. Rose Ademun Okurut: Supervision. Martin Only in 2020, the Food and Agricultural Organization of United Esau: Investigation. Merab Acham: Investigation. Maria Flavia Nations - Uganda, supported the district of Arua with 30,000 doses of Nakanjako: Investigation. Mugezi Israel: Investigation. Emmauel anthrax vaccine (personal communication). Livestock, including cattle, Isingoma: Investigation. Mwanja Moses: Investigation. Lumu Paul: goats, sheep, and pigs, were vaccinated against anthrax and black Investigation. Ben Ssenkeera: Investigation. Stella A. Atim: Investi­ quarter. The vaccination targeted animals in high-risk areas where ref­ gation. Doreen N. Gonahasa: Investigation, Writing – review & editing. ugees and host communities reside, including Omugo, Odupi, Invepi, Musa Sekamatte: Investigation, Writing – review & editing. Meriadeg and Uriama, all in Terego County. Due to high population livestock Ar Gouilh: Writing – review & editing. Jean Paul Gonzalez: Writing – density, many animals missed out on the vaccination due to inadequate review & editing. quantity of the vaccine doses provided. Another category of farmers ignored taking their animals for the vaccination exercise. According to Declaration of Competing Interest the Veterinary District officer, the vaccine served only 50% of the tar­ geted livestock population, leaving another 50% of the population The authors declare that they have no known competing financial without protective immunity against future anthrax outbreaks. interests or personal relationships that could have appeared to influence Due to quarantine and trade barriers associated with livestock dis­ the work reported in this paper. eases, including anthrax, animal keepers were afraid to disclose house­ holds with infected livestock. Therefore, prompt identification and early Data availability response interventions were affected. Secondly, concrete data to estab­ lish the actual burden of the disease was next to impossible. Strength­ Data will be made available on request. ening regional level laboratory capacity for anthrax is critical for the early identification of an outbreak. There is a need to strengthen labo­ ratory surveillance at the regional level and timely detection of anthrax. Acknowledgements Quick and reliable disease detection and confirmation methods are essential to correctly identify B. anthracis and understand its real diffu­ The authors would like to express their gratitude to the local Sub- sion and prevalence (Zasada, 2020), particularly in remote County chiefs, district veterinary officers, community elders, and kraal 6 M. Omodo et al. Acta Tropica 240 (2023) 106841 leaders for being supportive during data collection. Author JG has Wieler, L.H., Boesch, C., Klee, S.R., Wittig, R.M., Calvignac-Spencer, S., Leendertz, F. H., 2017. Persistent anthrax as a major driver of wildlife mortality in a tropical received mobility support from Universitat Autònoma de Barcelona rainforest. Nature 548, 82–85. https://doi.org/10.1038/nature23309. (action Erasmus+ KA107 Mobility Fellowship) and was supported by Hornitzky, M., Muller, J., 2010. Anthrax - part 1. Diagnostic overview. Australia and the Generalitat de Catalunya, Agency for Management of University and New Zealand Standard Diagnostic Procedure, pp. 1–15. Research Grants co-financed with the European Social Found (grants for Hugh-Jones, M., Blackburn, J., 2009. The ecology of Bacillus anthracis. Mol. Aspects Med. 30, 356–367. https://doi.org/10.1016/j.mam.2009.08.003. the recruitment of new research staff 2018 FI_B 00236). Authors RAO, Hugh-Jones, M.E., 2016. Overview of Anthrax [WWW Document]. MSD Man. URL ME, MA, MFN, MI, EI, MM, LP, BS, and SAA were funded by Livestock https://www.msdvetmanual.com/generalized-conditions/anthrax/overview-of-anth Disease Control project II. rax (accessed 5.24.22). Kolton, C.B., Marston, C.K., Stoddard, R.A., Cossaboom, C., Salzer, J.S., Kozel, T.R., Gates-Hollingsworth, M.A., Cleveland, C.A., Thompson, A.T., Dalton, M.F., References Yabsley, M.J., Hoffmaster, A.R., 2019. Detection of Bacillus anthracis in animal tissues using InBios active anthrax detect rapid test lateral flow immunoassay. Lett. Alexander, K.A., Lewis, B.L., Marathe, M., Eubank, S., Blackburn, J.K., 2012. Modeling of Appl. Microbiol. 68, 480–484. https://doi.org/10.1111/lam.13134. wildlife-associated zoonoses: applications and caveats. Vector-Borne Zoonotic Dis. Lafferty, K., Chapman, S., 2014. Advancing strategic work and accelerating leadership 12, 1005–1018. https://doi.org/10.1089/vbz.2012.0987. talent at GlaxoSmithKline. Using Exp. Dev. Leadersh. Talent How Organ. Leverage Antonation, K.S., Grützmacher, K., Dupke, S., Mabon, P., Zimmermann, F., Lankester, F., Job Dev. 21, 188–204. https://doi.org/10.1002/9781118829417.ch9. Peller, T., Feistner, A., Todd, A., Herbinger, I., de Nys, H.M., Muyembe-Tamfun, J.-J., Lumely, T., 2020. survey: analysis of complex survey samples. R Package Vesion 4.0. Karhemere, S., Wittig, R.M., Couacy-Hymann, E., Grunow, R., Calvignac-Spencer, S., M’Fadyean, J., 1903. A peculiar staining reaction of the blood of animals dead of Corbett, C.R., Klee, S.R., Leendertz, F.H., 2016. Bacillus cereus biovar anthracis anthrax. J. Comp. Pathol. Ther. 16, 35–IN1. https://doi.org/10.1016/S0368-1742 causing anthrax in Sub-Saharan Africa—chromosomal monophyly and broad (03)80006-1. geographic distribution. PLoS Negl. Trop. Dis. 10, e0004923 https://doi.org/ Mesfin, D., Mulatu, K., Birara, A., Shibabaw, T., Birhanu, D., Yalew, W., 2021. Anthrax 10.1371/journal.pntd.0004923. prevention practice and associated factors among farmers in Farta district, South Ashkenazi-Hoffnung, L., Kaufman, Z., Bromberg, M., Block, C., Keller, N., Dictar, R., Gondar, Amhara region, Northwest Ethiopia. Heliyon 7, 2009–2013. doi:10.1016/j. Goldberg, A., Green, M.S., 2009. Seasonality of Bacillus species isolated from blood heliyon.2021.e08531. cultures and its potential implications. Am. J. Infect. Control 37, 495–499. https:// Mukarati, N.L., Matope, G., de Garine-Wichatitsky, M., Ndhlovu, D.N., Caron, A., doi.org/10.1016/j.ajic.2008.08.008. Pfukenyi, D.M., 2020. The pattern of anthrax at the wildlifelivestock-human Avberšek, J., Mićunović, J., Cociancich, V., Paller, T., Kušar, D., Zajc, U., Ocepek, M., interface in Zimbabwe. PLoS Negl. Trop. Dis. 14, 1–20. https://doi.org/10.1371/ Špičić, S., Duvnjak, S., Pate, M., 2021. A suggested diagnostic approach for sporadic journal.pntd.0008800. anthrax in cattle to protect public health. Microorganisms 9, 1567. https://doi.org/ Munang’andu, H.M., Banda, F., Siamudaala, V.M., Munyeme, M., Kasanga, C.J., 10.3390/microorganisms9081567. Hamududu, B., 2012. The effect of seasonal variation on anthrax epidemiology in the Berg, T., Suddes, H., Morrice, G., Hornitzky, M., 2006. Comparison of PCR, culture and upper Zambezi floodplain of western Zambia. J. Vet. Sci. 13, 293–298. https://doi. microscopy of blood smears for the diagnosis of anthrax in sheep and cattle. Lett. org/10.4142/jvs.2012.13.3.293. Appl. Microbiol. 43, 181–186. https://doi.org/10.1111/j.1472-765X.2006.01931.x. Mwakapeje, E.R., Assenga, J.A., Kunda, J.S., Mjingo, E.E., Makondo, Z.E., Nonga, H.E., Carlson, C.J., Getz, W.M., Kausrud, K.L., Cizauskas, C.A., Blackburn, J.K., Bustos Mdegela, R.H., Skjerve, E., 2017. Prevention, detection, and response to anthrax Carrillo, F.A., Colwell, R., Easterday, W.R., Ganz, H.H., Kamath, P.L., Økstad, O.A., outbreak in Northern Tanzania using one health approach: a case study of Selela Turner, W.C., Kolstø, A., Stenseth, N.C., 2018. Spores and soil from six sides: ward in Monduli district. Int. J. One Heal. 3, 66–76. https://doi.org/10.14202/ interdisciplinarity and the environmental biology of anthrax (Bacillus anthracis). IJOH.2017.66-76. Biol. Rev. 93, 1813–1831. https://doi.org/10.1111/brv.12420. Mwakapeje, E.R., Høgset, S., Fyumagwa, R., Nonga, H.E., Mdegela, R.H., Skjerve, E., Carlson, C.J., Kracalik, I.T., Ross, N., Alexander, K.A., Hugh-Jones, M.E., Fegan, M., 2018. Anthrax outbreaks in the humans - livestock and wildlife interface areas of Elkin, B.T., Epp, T., Shury, T.K., Zhang, W., Bagirova, M., Getz, W.M., Blackburn, J. Northern Tanzania: a retrospective record review 2006-2016. BMC Public Health 18, K., 2019. The global distribution of Bacillus anthracis and associated anthrax risk to 106. https://doi.org/10.1186/s12889-017-5007-z. humans, livestock and wildlife. Nat. Microbiol. 4, 1337–1343. https://doi.org/ Ntono, V., Eurien, D., Bulage, L., Kadobera, D., Harris, J., Ario, A.R., 2021. Cutaneous 10.1038/s41564-019-0435-4. anthrax outbreak associated with handling dead animals, Rhino Camp sub-county: Citypopulation.de, n.d. Estimated population of Arua District in 2020 [WWW Arua District, Uganda, January–May 2018. One Heal. Outlook 3. https://doi.org/ Document]. URL https://www.citypopulation.de/en/uganda/admin/northern/00 10.1186/s42522-021-00040-z. 2__arua/ (accessed 5.18.22). R Core Team, 2019. R: a language and environment for statistical computing. Coffin, J.L., Monje, F., Asiimwe-Karimu, G., Amuguni, H.J., Odoch, T., 2015. A One R Foundation for Statistical Computing. https://www.r-project.org/. Health, participatory epidemiology assessment of anthrax (Bacillus anthracis) Rao, S., Traxler, R., Napetavaridze, T., Asanishvili, Z., Rukhadze, K., Maghlakelidze, G., management in Western Uganda. Soc. Sci. Med. 129, 44–50. https://doi.org/ Geleishvili, M., Broladze, M., Kokhreidze, M., Reynolds, D., Shadomy, S., 10.1016/j.socscimed.2014.07.037. Salman, M., 2019. Risk factors associated with the occurrence of anthrax outbreaks De Vos, V., Coetzer, J., Thomson, G., Tustin, R., 1994. Anthrax. Infectious Diseases of in livestock in the country of Georgia: a case-control investigation 2013-2015. PLoS Livestock with Special Reference to Southern Africa. Oxford University Press, Cape ONE 14, 1–16. https://doi.org/10.1371/journal.pone.0215228. Town, pp. 1262–1289. Vol. 2. Ravenel, M.P., 2008. Anthrax in man and animals. Am. J. Public Heal. Nations Heal. 30, Driciru, M., Rwego, I.B., Asiimwe, B., Travis, D.A., Alvarez, J., VanderWaal, K., 299–300. Pelican, K., 2018. Spatio-temporal epidemiology of anthrax in Hippopotamus Republic of Uganda - Ministry of Health, 2021. National Technical Guidelines for amphibious in Queen Elizabeth protected area, Uganda. PLoS ONE 13, 1–21. https:// Integrated Disease Surveillance and Response, 3rd Ed. doi.org/10.1371/journal.pone.0206922. Sentumbwe, J., Mbolanyi, B., Aruho, R., Kaboyo, W., Mutonga, D., Basler, C., Paige, S., Epp, T., Waldner, C., Argue, C.K., 2010. Case-control study investigating an anthrax Behravesh, C.B., 2018. Multisectoral prioritization of zoonotic diseases in Uganda, outbreak in Saskatchewan, Canada - Summer 2006. Can. Vet. J. 51, 973–978. 2017: a one health perspective. PLoS ONE 13, 1–11. https://doi.org/10.1371/ Fasanella, A., Galante, D., Garofolo, G., Jones, M.H., 2010. Anthrax undervalued journal.pone.0196799. zoonosis. Vet. Microbiol. 140, 318–331. https://doi.org/10.1016/j. Swartz, M.N., 2001. Recognition and management of anthrax — an update. N. Engl. J. vetmic.2009.08.016. Med. 345, 1621–1626. https://doi.org/10.1056/NEJMra012892. Food and Agriculture Organization of the United Nations, 2018. FAO, Government of Turner, W.C., Kausrud, K.L., Krishnappa, Y.S., Cromsigt, J.P.G.M., Ganz, H.H., Uganda launch a new surveillance evaluation tool to support government in Mapaure, I., Cloete, C.C., Havarua, Z., Küsters, M., Getz, W.M., Stenseth, N.C., 2014. prevention and control zoonotic diseases [WWW Document]. URL https://www.fao. Fatal attraction: vegetation responses to nutrient inputs attract herbivores to org/uganda/news/detail-events/en/c/1111845/ (accessed 11.8.21). infectious anthrax carcass sites. Proc. R. Soc. B Biol. Sci. 281, 20141785 https://doi. Gates-Hollingsworth, M.A., Kolton, C.B., Hoffmaster, A.R., Meister, G.T., Moore, A.E., org/10.1098/rspb.2014.1785. Green, H.R., Pogoda, J.M., Pillai, S.P., Kozel, T.R., 2022. Rapid capsular antigen Vudriko, P., Ekiri, A.B., Endacott, I., Williams, S., Gityamwi, N., Byaruhanga, J., immunoassay for diagnosis of inhalational anthrax: preclinical studies and Alafiatayo, R., Mijten, E., Tweyongyere, R., Varga, G., Cook, A.J.C., 2021. A survey evaluation in a nonhuman primate model. MBio 13. https://doi.org/10.1128/ of priority livestock diseases and laboratory diagnostic needs of animal health mbio.00931-22. professionals and farmers in Uganda. Front. Vet. Sci. 8, 1–20. https://doi.org/ Gates-Hollingsworth, M.A., Perry, M.R., Chen, H., Needham, J., Houghton, R.L., 10.3389/fvets.2021.721800. Raychaudhuri, S., Hubbard, M.A., Kozel, T.R., 2015. Immunoassay for capsular Wolicki, S.B., Nuzzo, J.B., Blazes, D.L., Pitts, D.L., Iskander, J.K., Tappero, J.W., 2016. antigen of Bacillus anthracis enables rapid diagnosis in a rabbit model of inhalational Public health surveillance: at the core of the global health security agenda. Heal. anthrax. PLoS ONE 10, e0126304. https://doi.org/10.1371/journal.pone.0126304. Secur. 14, 185–188. https://doi.org/10.1089/hs.2016.0002. Hoffmann, C., Zimmermann, F., Biek, R., Kuehl, H., Nowak, K., Mundry, R., Agbor, A., World Health Organization, 2008. International Helath Regulations, 2nd Ed. (2005). Angedakin, S., Arandjelovic, M., Blankenburg, A., Brazolla, G., Corogenes, K., World Organization for Animal Health, 2021. Manual of Diagnostic Tests and Vaccines Couacy-Hymann, E., Deschner, T., Dieguez, P., Dierks, K., Düx, A., Dupke, S., for Terrestrial Animals 2021. https://www.oie.int/en/what-we-do/standards/co Eshuis, H., Formenty, P., Yuh, Y.G., Goedmakers, A., Gogarten, J.F., Granjon, A.C., des-and-manuals/terrestrial-manual-online-access/. McGraw, S., Grunow, R., Hart, J., Jones, S., Junker, J., Kiang, J., Langergraber, K., Zasada, A.A., 2020. Detection and identification of Bacillus anthracis: from conventional Lapuente, J., Lee, K., Leendertz, S.A., Léguillon, F., Leinert, V., Löhrich, T., to molecular microbiology methods. Microorganisms 8, 125. https://doi.org/ Marrocoli, S., Mätz-Rensing, K., Meier, A., Merkel, K., Metzger, S., Murai, M., 10.3390/microorganisms8010125. Niedorf, S., De Nys, H., Sachse, A., Van Schijndel, J., Thiesen, U., Ton, E., Wu, D., 7