Leptospirosis in sugarcane plantation and fishing communities in Kagera northwestern Tanzania

Background Leptospirosis is a bacterial zoonotic disease of worldwide importance, though relatively neglected in many African countries including sub Saharan Africa that is among areas with high burden of this disease. The disease is often mistaken for other febrile illnesses such as dengue, malaria, rickettsioses and enteric fever. Leptospirosis is an occupational disease likely to affect people working in environments prone to infestation with rodents which are the primary reservoir hosts of this disease. Some of the populations at risk include: sugarcane plantation workers, wetland farmers, fishermen and abattoir workers. In this study we investigated the prevalence of antibodies against Leptospira among sugarcane plantation and factory workers, fishing communities as well as among rodents and shrews in domestic and peridomestic environments within the study areas. Methods The study was conducted in Kagera region, northwestern Tanzania and it involved sugarcane plantation workers (cutters and weeders), sugar factory workers and the fishing community at Kagera Sugar Company in Missenyi district and Musira island in Lake Victoria, Kagera, respectively. Blood was collected from consenting human adults, and from rodents and shrews (insectivores) captured live using Sherman traps. Serological detection of leptospiral antibodies in blood serum was carried out by the microscopic agglutination test (MAT). Results A total of 455 participants were recruited from the sugarcane plantation (n = 401) and fishing community (n = 54) while 31 rodents and shrews were captured. The overall prevalence of antibodies against Leptospira in human was 15.8%. Sugarcane cutters had higher seroprevalence than other sugar factory workers. Prevalent antibodies against Leptospira serovars in humans were against serovars Lora (6.8%), Sokoine (5.3%), Pomona (2.4%), Hebdomadis (1.1%) and Kenya (0.2%). Detected leptospiral serovars in reservoir hosts were Sokoine (12.5%) and Grippotyphosa (4.2%). Serovar Sokoine was detected both in humans and small mammals. Conclusion Leptospirosis is a public health threat affecting populations at risk, such as sugarcane plantation workers and fishing communities. Public awareness targeting risk occupational groups is much needed for mitigation of leptospirosis in the study areas and other vulnerable populations in Tanzania and elsewhere.


Introduction
Leptospirosis is a public health concern especially in the tropical and subtropical countries where the environment is optimal for survival of pathogenic leptospires [1]. The annual morbidity and mortality caused by leptospirosis worldwide is estimated to be 14.7 cases per 100,000 population [2]. Globally, Oceania region has the highest disease burden (150.6 cases/ 100,000 population), South east Asia (55.5), Caribbean (50.6) and East Sub Saharan Africa (25.6) [2,3]. In Tanzania the annual incidence is 75-102 cases per 100,000 population [4]. Rodents are considered major reservoirs of Leptospira [5] and other wild animals and birds found in wetland areas may also carry and spread leptospires into the surroundings [6]. The disease is associated with certain occupational activities such as rice and sugarcane farming, fishing and fish farming, livestock keeping, handling animal products and water sports [7,8].
Males are most affected than females contributing to 80% of the total burden [3]. Humans can be infected through contact with urine or other materials from infected animals or contaminated water and soil [9]. In Tanzania, leptospirosis has been reported in patients with nonmalaria fevers [10,11] and in animals including rodents and domestic animals [12][13][14][15]. Antibodies against Leptospira have been demonstrated also in freshwater fishes [6] in Tanzania suggesting potential risk to fishermen and people undertaking irrigation activities such as, rice farming and sugarcane plantation. Studies on leptospirosis in these at risk populations are lacking, hence the burden of leptospirosis in fishing communities and sugarcane plantations is Competing interests: Co-author (IA) is a chief medical officer at Kagera Sugar Company Ltd, that is one of the study location. However, this did not influence site selection. Employment to this company have no competing interest and did not influence the study design nor reporting of study findings.
not known. Sugarcane plantation and rice farming are important agricultural sectors in Tanzania, which engage permanent and seasonal workers from different parts of the country. Understanding the burden of leptospirosis in these occupational groups could provide baseline information needed for informing policy, especially because the disease is neglected and rarely considered for diagnosis in the health system [16]. In this study we investigated the serological prevalence of leptospirosis in selected risk populations of sugarcane plantation workers and fishing in northwestern Tanzania Also, we identified potential Leptospira serovars circulating in the region, which would serve as important antigens for diagnostic purposes.

Study location
This study was conducted in Kagera region northwestern Tanzania at Musira island (S 011 9.914', E 031˚49.772' with elevation of 1120 meter above sea level, and at Kagera sugar company (S 01˚12.807, E 031˚16.510') with elevation of 1157 meter above sea level. Kagera region receives bi-modal rainfall pattern ranging between 900-2,000 mm per annum, temperatures range between 20˚C and 28˚C. Kagera region is located along Lake Victoria hence fishing is among major socio-economic activity apart from large scale sugarcane plantation. Kagera Sugar Company is one of the biggest sugarcane plantations in the country. The two study sites (fishing community and Musira and sugarcane plantation community at Kagera sugar company are 76.2 km apart (Fig 1).

Study populations and sample size
Human subjects. Human leptospirosis is estimated to be 10% in Tanzania [10,13,15]. Sample size estimation formula used was N = Z 2 XP (1-P)/δ 2 , whereby; N = Estimated sample size, Z = Standard normal variate (1.96) for 95% confidence level, P = Proportion (prevalence), δ = Precision or absolute error (~0.05) for 95% confidence level. N = 1.96 2 x0.1 (1-0.1)/0.05 2 = 138 + 15% of 138 = 160 To increase the power of the study to 80%, 455 of the human study population were sampled from sugarcane plantation workers/cutters and fishermen along Lake Victoria in Kagera region. Sugarcane cutters make the largest proportion of the Kagera sugar company staff. Other employees include those engaged with management, sugarcane planting, weeding, fertilizer application, herbicides application, health services and security. There were 924 sugarcane cutters (seasonal employees) eligible for the study whereas 401 (43.4%) agreed to participate in the study following sensitization about leptospirosis disease. Encampment residents including petty traders in the sugarcane plantation community as well as hospitalized patients were also studied.
Other study participants were from the fishing community at Musira island located near Bukoba town, which were predominantly fishermen.
Inclusion and exclusion criteria for human subjects. The study included adult sugarcane plantation workers/cutters, weeders, and factory workers working in the sugarcane plantation or encampment residents residing in Kagera sugar company compounds; fishermen and other willing individuals living in the Musira island fishing community. Individuals under 18 year old and those who did not agree to sign informed consent were excluded from the study. Participants were informed about the study at gatherings held in residential camps of Kagera Sugar Company while in the fishing community, individuals were informed and at a public gathering called by the village authority/ chairman. Information about leptospirosis disease and how it is transmitted to humans was communicated to villagers at this gathering to sensitize and increase the participation rate in the study. No interviews were conducted with participants to ascertain whether recalled history of clinical illness related with leptospirosis since majority were assumed likely to agree to this because fevers are common in this tropical region.
Hospitalized participants. There were 58 patients hospitalized at Kagera sugar hospital during the sampling period. These were informed about leptospirosis whereby 13 (22.4%) patients with fevers agreed to provide blood for testing antibodies against leptospirosis.
Blood samples (2-4 ml) were aseptically collected by medical personnel using sterile 5 ml vacutainer tubes and kept in a refrigerator overnight to separate serum. Un-separated blood was centrifuged at 3000g for 10 min to obtain the serum for microscopic agglutination test (MAT).
Rodents and shrews (Crocidura spp.) were captured live using Sherman live traps. The rodents and shrews were then anaesthetized using di-ethyl ether. Blood (1 to 2 ml) was aseptically collected from the supraorbital vein and/ or through heart puncture using capillary tubes and sterile syringes and needles, respectively. The blood was immediately transferred into plain 2.5 ml Eppendorf tubes and allowed to clot to obtain serum samples which were then shipped in cool boxes to the Leptospirosis Research Laboratory at Pest Management Centre, Sokoine University of Agriculture where was kept frozen at -20˚C until used.
Serological detection of leptospiral antibodies. Antibodies against Leptospira in humans and animals were detected using MAT as previously described [17,18]. Live leptospiral antigens consisted of six serovars were used, four of which are local isolates, namely L. kirschneri serovar Sokoine, L. borgpetersenii serovar Kenya, L. interrogans serovar Lora and L. kirschneri serovar Grippotyphosa [1,14,15,19]. The remaining two (L. interrogans serovars Hebdomadis and L. interrogans serovar Pomona) were reference isolates initially supplied by the Royal Tropical Institute (KIT Biomedical Research, WHO/FAO/OIE Leptospirosis Reference Centre), Amsterdam, the Netherlands. These leptospires were grown in Ellinghausen and McCullough-Johnson and Harris, (EMJH) culture medium for 5 to 8 days while monitoring for growth density and purity using dark field microscope. Live leptospiral antigens with density 3×10 8 leptospires/ml were used in MAT as recommended elsewhere [17,18]. Briefly, 10μl of serum was mixed with 90μl of phosphate buffered saline (PBS) (pH 7) and then serially double diluted with PBS diluted further to obtain initial dilutions of 1:10, 1:20, 1:40 and 1:80. Volume of 50μl of well grown live leptospires antigen were then added to each well to obtain final dilutions of 1:20, 1:40, 1:80 and 1:160 recommended for initial screening. Positive samples at � 1:20 were diluted further and retested to determine cut-off agglutination titres, which were those in which 50% of the leptospires agglutinated compared with the negative control in which only PBS and the live cultures were mixed [17,18]. The highest dilution in screening was 1:160 whereas in titration was 1:20480.
Isolation of Leptospira from rodents and insectivores. The isolation of Leptospira from rodents and insectivores/shrews was done by aseptically culturing the kidney homogenate and urine into Fletcher culture media as previously described [13,15]. Briefly, two drops of fresh urine were inoculated into a screw capped tube containing sterile semisolid Leptospira Fletcher's culture medium prepared according to manufacturer's instructions and containing 50mg/ ml of 5-Flourouracil selective inhibitor. Kidney specimens were placed into a sterile vials containing sterile phosphate buffered saline (pH 7.0) and homogenized using sterile syringe needle cap as previously described [13,15]. Two drops of kidney homogenate were aseptically inoculated into Fletcher medium and incubated at room temperature while in field and at 30˚C in the laboratory for 8 up to 20 weeks. Cultures were observed for leptospiral growth under dark field microscope every 7 days.
Data analysis. Prevalence of antibodies against Leptospira serovars tested were used to compare various at-risk groups and whether gender, occupation and age were risk factors. Various age groups were compared as well as occupation groups. Statistical significance of differences between proportions of prevalence of antibodies against Leptospira was determined using chi-test and associations were considered statistically significant when P-values were � 0.05. Statistical analyses were conducted using MedCalc [20][21][22]. MedCalc uses the "N-1" Chi-squared test according to Campbell [20] and Richardson [21], whereas confidence interval is calculated according to Altman and co-workers [22].

Humans-sugarcane plantation workers and fishing community
A total of 455 participants were sampled of which 401 (132 females and 269 males) were from sugarcane plantation and 54 (16 females and 38 males were from the Musira fishing island in Lake Victoria. The demographic profile of human participants was as shown in Table 1.

Rodents and insectivores
The majority of rodents were Rattus spp. (55%) trapped indoors. Other rodents trapped included forest species (Lophuromys spp.) captured in the bushes near the sugarcane plantation and Arvicanthis spp. found in fallow land near sugarcane fields (Table 2).

Leptospirosis seroprevalence in humans
Prevalence of human leptospirosis in the two study populations of sugarcane plantation workers and fishing community was 15.8%. Fifty eight of the 317 (18.3%) sugarcane cutters were seropositive compared to 8 out of 54 (14.8%) of the fishing community subjects. Two of the 13 (15.4%) hospitalized patients were seropositive while other participants including office cleaners, petty traders and security guards contributed to 7.0% seropositivity (Table 1).
Antibodies against tested Leptospira were relatively lower in rodents than in humans. The highest titre (1:2560) was observed in two individuals against serovar Pomona (Fig 2).

Comparison of antibody prevalence in different groups
Prevalence of antibodies against Leptospira among different occupational groups, populations, gender and age groups were compared to determine whether certain groups were at more risk year old also showed significant difference in percentage of positives (37.8%) that was also statistically significant (p = 0.0044, 95% CI = 9.5238 to 62.8984, χ2 = 8.105, df = 1). The prevalence of antileptospiral antibodies between fishing community and sugarcane cutters was 3.6% that is not statistically significant (p = 0.5241, 95% CI = -8.8082 to 12.0938, χ2 = 0.406, df = 1). Comparison of positive rate found in fishing community and unexposed group (others) showed 9.3%, which was not statistically significant Anti-leptospiral antibodies prevalence in rodents and shrews. Twenty-four rodents and shrews (Crocidura spp.) of the 31 animals collected around sugarcane plantations, forest and houses at Musira island and Kagera sugar company were tested for leptospiral antibodies. Four of the 24 animals (16.7%) were seropositive. Antibodies against Leptospira serovar Gripptyphosa was detected in one Rattus rattus rodent species (4.2%) whereas antibodies against L. serovar Sokoine was detected in two Rattus rattus and one insectivore (Crocidura spp.) (13%). Comparison of positive rates found in rodents and insectivores was not statistically significant (difference = 14.6%, 95% CI = -21.8152 to 62.0923, X 2 = 0.307, df = 1, p = 0.5792). Antibodies against L. serovar Kenya, Lora, Pomona and Hebdomadis were not detected in rodents nor insectivores.
The seropositivity shows that antibodies against certain Leptospira serovars were detected either in humans or animals, while some were found in both humans and animals (Table 3). Leptospira serovar Sokoine had higher seropositivity rate in both humans (5.3%) and animals (12.5%) unlike the other serovars (Pomona, Grippotyphosa, Hebdomadis and Lora) detected in humans or animals only ( Table 3). The overall prevalence of leptospirosis in rodents and insectivores was 16.7%, of which rodents contributed 12.5% (3/24) and insectivores 4.2% (1/24) (Table 3) The overall difference in prevalence of antibodies against Leptospira between humans and rodents was 0.9% (95% CI = -9.7420 to 20.3338, χ2 = 0.014, df = 1, p = 0.9064) whereas the prevalence of antibodies against Leptospira serovar Sokoine which occurred in both humans and rodents differed between the two groups by 7.2% (95% CI = -1.3180 to 25.7827, χ2 = 2.208, df = 1, p = 0.1373). The differences in seroprevalence of leptospiral antibodies in humans and rodents as well as of serovar Sokoine were not statistically significant (Table 3).
Leptospira isolation from rodents and insectivores' urine and kidney homogenates did not yield positive cultures.

Discussion
This study shows high prevalence of antibodies against Leptospira in humans involved in sugar production and fishing in the Kagera region, northwestern Tanzania. Leptospirosis in rodents and shrews captured in the areas is also reported. Findings suggests that sugarcane plantation workers especially sugarcane cutters and fishing communities are potentially at risk. A prevalence of 15.8% was found in sugarcane plantation workers, with cane cutters having the higher prevalence of 18.4%, followed by other plantation workers and hospitalized patients. Prevalence of anti-leptospiral antibodies was also high (14.8%) in fishermen and other individuals living on the Musira island, which is a fishing island. This suggests that fishing communities can get leptospirosis following contact with water contaminated with urine of the reservoir hosts. The prevalence of human leptospirosis in sugarcane plantation workers reported in this study (18.4%) is lower than that reported in sugarcane plantation workers in central America (59%) [19] but higher than the 0.7% prevalence reported from Trinidad and Tobago [23].
Prevalence of antibodies against Leptospira among different occupational groups, populations, gender and age groups showed variations suggesting that individuals belonging to certain groups and occupation groups have different levels of risk of contracting leptospirosis. For example, while there was no significant difference in the prevalence of leptospiral antibodies between male and females despite that the study had more males than females due to the nature of the occupation of the study populations, there was a significant difference in prevalence of antibodies against Leptospira found in participants in two age groups of 18-37; 38-57 year old versus participants with age above 58 year old. Findings show that participants with over 58 year old have significantly higher proportion of antibodies against Leptospira than those with age below 58 year old (i.e. 18-37; 38-57 year old). This could be probably associated with potential prolonged exposure to risk environment such as sugarcane cutting for many years than newer entrants. The fishing community and sugarcane plantations appear to have similar risk levels since the prevalence of antibodies in these two populations was not statistically significant. However, comparison of fishing community and sugarcane cutters considered risk populations with unexposed groups consisting of participants engaged with less risk activities such as office work, security and petty traders show that fishing community does not differ to the unexposed group while sugarcane cutters show more risk than unexposed group. This can be explained with fact that fishing community included the general population of the fishing island including school pupils and other residents likely to have various levels of risk of contracting leptospirosis while sugarcane cutters consisted a uniform group of individuals engaged with same activity of cutting sugar hence likely to have same level of risk higher than the general population.
These findings shows that the roof rat (Rattus spp.) is an important reservoir of leptospirosis in Kagera region as demonstrated by high positivity rate among the house rats collected in different localities in the study areas. Comparison of positive rates found in the roof rats and an insectivore showed no statistically significant difference due to small sample size of rodents and shrews collected. A larger sample size estimated for this study was not achieved due to seasonal variations in rodent populations hence suggesting further sampling to enhance robust determination of the major reservoir of Leptospira in this region. Antibodies against L. serovar Kenya, Lora, Pomona and Hebdomadis were not detected in rodents nor insectivores. The rats were seropositive against L. kirschneri serovars Sokoine and L. kirschneri serovar Grippotyphosa. Rodents had lower antibody titres (1:20-1:40) than humans in which higher titres up to 1:2560 were determined by MAT which is the gold standard test for leptospirosis diagnosis [9,24]. High antibody titres against Leptospira serovars detected in humans suggest the existence of recent infections.
The predominant circulating Leptospira serovars which antibodies against was detected in humans, namely Leptospira interrogans serovar Lora, L. kirschneri serovar Sokoine, L. interrogans serovar Pomona, L. interrogans serovar Hebdomadis and L. borgpetersenii serovar Kenya have been previously reported in humans, rodents and domestic animals [10,15,25]. Leptospira kirschneri serovar Sokoine was mainly found in both humans and animals in Tanzania whereas L. interrogans serovar Grippotyphosa was mainly detected in the reservoir hosts. Leptospira interrogans serovar Lora was not detected in rodents, indicating potential diversity of sources of human infection. It is known that certain Leptospira serovars demonstrate hostspecificity and might be absent in certain rodent species [15]. Further investigations are needed to establish the source or reservoir hosts of serovar Lora in the study areas and to determine whether the plantation workers who also come from outside Kagera had leptospirosis exposure prior to their recruitment at the sugarcane plantation. This could be achieved by including leptospirosis screening during general health examinations performed before recruiting cane cutters.
The observed high prevalence of leptospirosis in the fishing community corroborate previous report of high seropositivity/leptospiral antibodies in freshwater fishes and thus potential risk of leptospirosis in fishing communities and in people working in the fishing industry [15,19,23]. It is recommended that leptospirosis control should include rodent management, and public awareness. Furthermore, leptospirosis screening should be introduced in risk occupational groups in Tanzania and elsewhere where the disease is neglected [16]. Detection of leptospiral antibodies in hospitalized patients during this study indicates further the importance of considering leptospirosis among febrile illnesses that are non-malarial. The prevalence of 15.4% of leptospirosis in hospitalized patients corroborates previous reports from northern Tanzania and Morogoro among hospitalized patients with febrile illness [10,11,26]. This further emphasizes the need to include leptospirosis in differential diagnosis of febrile illnesses.
Further surveillance studies are needed to isolate and characterize the disease causative Leptospira serovars beyond serological surveillance. These should include cross agglutination absorption test, and molecular typing [25]. The major limitations of this study were failure to isolate the causal agent, which would have enabled its characterization. Similarly, future studies should include larger populations of potential reservoirs.
Leptospirosis is a public health threat in sugarcane plantation workers and the fishing communities. Preventive measures are needed to mitigate risks of leptospirosis. These should include rodent control, public awareness and screening for leptospirosis in individuals with non-malarial fevers [16] and vulnerable occupational groups such as sugarcane cutters. Leptospira serovars Lora, Sokoine, Pomona, Hebdomadis, Kenya and Grippotyphosa should be included as antigens for broader leptospirosis screening in humans and animals from this region.