Calf Sentinels for Diarrheal Disease - Master's thesis by David Wolking

Calves as Sentinels for Diarrheal Disease Households Practicing Traditional Livestock Husbandry

David J. Wolking International Agriculutural Development University of California, Davis David J. Wolking

Masters Thesis Calves as Sentinels for Diarrheal Disease in Households Practicing Traditional Livestock Husbandry Author: David J. Wolking Unofficial THESIS submitted in partial satisfaction of the requirements for the degree of MASTER OF SCIENCE in International Agricultural Development in the Office of Graduate Studies of the Unviersity of California, Davis, November 2009. Committee: Dr. Jonna A.K. Mazet (Chair), Dr. Johan W. Six, Dr. Montague W. Demment, and Dr. Deana L. Clifford University of California Davis One Shields Avenue Davis, CA 95616 Cover Photo: A Barabaig boy holds one of his prize cavles. Calves are highly valued cultural and economic assets among pastoralists, and close bonds are often formed between calves and their caretakers, typically women and children (Photo by D. Wolking).

ACKNOWLEDGEMENTS This project was permitted to conduct research in Tanzania under the authority of the Tanzanian Commission for Science and Technology (COSTECH), Dar es Salaam, Tanzania (RCA 2008/129). Additionally, human subjects and animal handling protocols were permitted by University of California Davis Institutional Review Board Protocol # 200816605-1 and Animal Care and Use Protocol #12394. This research was made possible by a Jim Ellis Mentorship Program for Graduate Students Award, and with the support of the Health for Animals and Livelihood Improvement in the Ruaha Ecosystem (HALI) project through the Global Livestock Collaborative Research Support Program (GL-CRSP), which is funded by the Office of Agriculture, Bureau for Economic Growth, Agriculture and Trade, USAID, under terms of Grant No. PCE-G-00-98-00036-00. The opinions expressed herein are those of the authors and do not necessarily reflect the views of USAID. I am grateful to the households and communities that participated in this study. Thanks also to contributors from the Sokoine University of Agriculture, Faculty of Veterinary Medicine, Dr. Rudovick Kazwala, Ally Kitime and Enos Kamani, and team members of the HALI project, Erasto Katowo, Howard Kombe, Asha Makweta, Dr. Woutrina Miller, and Dr. Harrison Sadiki. Special thanks to Dr. Jon Erickson and Michel Masozera of the HALI project and University of Vermont, for sharing socioeconomic data and advice. This project would not have been possible without the patience, guidance, mentorship, and support of Dr. Jonna Mazet and Dr. Deana Clifford of the Wildlife Health Center, University of California, Davis. I would like to thank the GL-CRSP of the University of California, Davis, in particular Dr. Montague Demment and Susan Johnson, who introduced me to livestock and to HALI, and who have made my experience at UC Davis truly memorable. Finally, I would like to acknowledge my family, in particular Misty Richmond and my parents, Mary and Edward Wolking, who have been subjected to countless hours of rambling on soil science, epidemiology, statistics, and development over the past four years, and to whom I am deeply thankful. 1

University of California Davis ABSTRACT For many in the developing world, diarrheal disease is a constant and debilitating part of life, especially for children. But humans do not bear the burden of diarrheal disease alone; young livestock, especially neonates are highly susceptible, and diarrhea is a leading cause of calf morbidity and mortality worldwide. In the arid and semi-arid areas of Sub-Saharan Africa, pastoral livestock production represents a viable subsistence and livelihood strategy. Because the pastoral livestock production system is characterized by close interaction and contact between human and animal populations, it may be associated with higher rates of infection with and exposure to Cryptosporidium and other zoonotic diarrheal disease causing pathogens. On the positive side, this close interaction also encourages high visibility and awareness of herd condition, a factor with considerable potential for early assessment of herd health. Due to their susceptibility and high visibility, calves could function as a practical surveillance and early warning system for the presence of diarrheagenic pathogens, allowing livestock keepers to implement preventive measures and reduce the risk of disease transmission to people. Following the detection of Cryptosporidium and Giardia in water and fecal samples in the Ruaha Landscape of Tanzania, this study was developed to investigate the prevalence of diarrheal disease and the prevalence and risk factors associated with Cryptosporidium and Giardia in calves. Fifty-two pastoral households were interviewed and their calf herds sampled to determine the potential for a calf-based diarrheal disease early warning system for pastoral areas. Results show that 60% of households reporting cases of human diarrhea also have diarrhea in their calf herds, and that calf herds shedding Cryptosporidium occysts are six times more likely to be diarrheic. Because Cryptosporidium shares a similar transmission ecology to a wide range of fecal-oral diarrheagenic organisms, and typical calf diarrhea outbreaks often involve multiple pathogens with mixed infections, diarrhea observed in calves may be indicative of protozoal and other pathogenic presence, resulting in the contamination of the local environment with a wide variety of infectious organisms. Calf diarrhea therefore, poses a serious health risk to households, particularly to women and children who are both more susceptible to infection, and more intimately involved with the care and management of younger animals. Increase in calf diarrheal incidence should be used as a warning sign to trigger interventions designed to minimize transmission of diarrhea causing agents, especially zoonotic protozoal pathogens. The use of calves as sentinel animals for diarrheal disease, therefore, is a potentially viable monitoring and surveillance option for resource constrained livestock producers in rural areas, especially among pastoralists.

INTRODUCTION developing countries6,7. Yet diarrhea is a preventable and treatable condition, given adequate hydration, sanitation, and hygiene, along with improvements in overall health and nutrition5,8. In many rural areas, however, diarrheal disease prevention and treatment options in people and livestock are limited by water quality and availability, sanitation infrastructure, and medical/veterinary services5,9. Livestock disease treatment options and access to veterinary extension services are often rare or non-existent in rural communities in developing countries, resulting in heavy reliance on disease prevention strategies by livestock keepers to optimize herd health10.

For many in the developing world, diarrheal disease is a constant and debilitating part of life, with a devastating impact on health, nutrition, and economic productivity 1,2,3,4. For children, it is especially threatening. Second only behind pneumonia in causes of death among children under five worldwide, diarrhea kills more young people than AIDS, malaria, and measles combined, with approximately 46% of these cases occurring in Africa5. Humans do not bear the burden of diarrheal disease alone; young livestock, especially neonates are highly susceptible, and diarrhea is a leading cause of calf morbidity and mortality in both developed and 2

Masters Thesis In the arid and semi-arid areas of Sub-Saharan Africa, pastoral and agro-pastoral livestock production represents a viable subsistence and livelihood strategy11 characterized by close contact between human and animal populations, a practice that could increase rates of infection with Cryptosporidium parvum and other zoonotic pathogens12,13,14. Yet this close interaction also encourages high visibility and awareness of herd condition, a factor with considerable potential for assessment of herd health and early disease intervention. As the viability of any livestock production system is dependent Figure 1. A baby plays with a newborn goat. Children and young animals, two upon the successful raising of highly suceptible populations for diarrheal disease, are closely connected in pastoral households, as women and children are the primary caretakers of young and small calves for herd replacement, calf stock (Photo by H. Sadiki). health is a critical component of traditional husbandry practices. wildlife, livestock, and human populations globally, Despite this importance, however, little attention and can contribute to poor growth and nutritional has been paid to calf illness in pastoral disease disorders, particularly in young children 23,24. surveillance activities, especially with regard to calf Cryptosporidium and Giardia frequently act alone, diarrhea. but often have more pronounced effects when concurrent enteropathogens are present25. Though Diarrhea in both humans and livestock has a human infection with both protozoal pathogens has complex, multifactorial etiology, involving pathogen been associated with close contact with livestock exposure along with a wide range of environmental, and farm animals12,13,14,20, their zoonotic potential 15,16 management, and nutritional factors . While can be drastically minimized through management several protozoal pathogens have been identified practices targeted at reducing fecal-oral transmission in clinical cases of human and calf diarrhea17,18, including the isolation of sick animals, reduced Cryptosporidium and Giardia are two of the most contact with manure, water treatment/filtration, common causes of protozoan diarrhea in humans and good general hygiene15. and calves worldwide, leading to significant morbidity and mortality in both the developed Disease control and prevention strategies can and developing world19. C. parvum is a zoonotic be drastically improved through disease surveillance protozoal pathogen of public health significance20, that enables the understanding of transmission associated with intense but short-lived diarrhea, risks, the evaluation of an appropriate response, and nausea, abdominal pain, and severe long-lasting assessments of the effectiveness of control efforts26. infection that can be life-threatening in individuals But disease-monitoring programs are often costly with compromised immune systems, especially and impractical for areas with underdeveloped those with HIV/AIDS and young children21,22. In surveillance and reporting infrastructure27, especially addition, Giardia lamblia has infected a wide range of pastoral areas lacking access to medical and 3

University of California Davis veterinary services. Animal sentinels have been proposed as substitutes for other more resource intensive surveillance programs to detect the presence of pathogens, to detect changes in the prevalence or incidence of disease/pathogens over time, and to evaluate the efficacy of disease control interventions28. Furthermore, the use of animal sentinels for the early detection and identification of pathogens may enhance disease prevention efforts and minimize the risk of disease transmission from animals to humans29. Due to the intimate interaction of humans and animals in pastoral and other traditional husbandry systems, the visibility of young livestock in and around communities, and their susceptibility to both diarrheal disease and protozoal pathogens, calves have the potential to function as practical surveillance and early warning sentinels for the presence of diarrheagenic

pathogens in rural areas. In addition, the lack of taboo associated with discussing calf diarrhea relative to diarrhea in people, may allow livestock managers and public health professionals to respond more easily with appropriate control measures to reduce both the risk of disease transmission and the levels of morbidity in calves and children. Based on the detection of Cryptosporidium parvum and Giardia lamblia in water sources and wildlife and livestock fecal samples in the Ruaha Landscape of Tanzania30, and the expected high susceptibility of young livestock to infection, this study was developed with two objectives: (1) to investigate the prevalence of diarrheal disease in young livestock populations in the pastoral areas, along with the prevalence of Cryptosporidium and Giardia, and risk factors associated with their infection in calves; and (2) to assess the potential for calves to serve as early warning sentinels to assist in diarrheal disease surveillance, prevention, and control at the household and herd level.

MATERIALS AND METHODS

Figure 2. Map of the study area and HALI project survey and sampling sites (Mazet et al., 2009) Reproduced with authorsâ&#x20AC;&#x2122; permission. 4

St u d y Are a . T h e Ru a h a Landscape, part of the broad valley of the Great Ruaha River located in the Southern Highlands of Tanzania in the eastern rift, is one of Tanzaniaâ&#x20AC;&#x2122;s largest wild areas, covering a region larger than Denmark (>45,000 km2). In addition to the conservation significance of the region, Ruaha is home to a substantial rural population, dependent entirely on the natural resource base with agriculture accounting for 80% of rural livelihoods31. Due to a lack of irrigation infrastructure and variable rainfall (between 400-600 mm p.a.), pastoral and agro-pastoral livestock production is a significant contributor to rural livelihoods.

Masters Thesis Study Population and Sampling Frame. Households were interviewed and cattle sampled during the dry season between August and October of 2008 in Pawaga and Idodi divisions of Iringa region, which border Ruaha National Park and the Pawaga-Idodi Wildlife Management Area. Settlements in Pawaga Division follow the course of the Little Ruaha River, a tributary of the Great Ruaha River, while the majority of settlements in Idodi division are at the foot of the rift escarpment southeast of the Great Ruaha River and boundary to Ruaha National Park (Figure 2). Study households consisted of agro-pastoralists and pastoralists from the Barabaig, Maasai, and Sukuma tribes participating in a larger research program investigating the impact of water scarcity and disease on wildlife, livestock, and human health and livelihoods, conducted by the Health for Animals and Livelihood Improvement (HALI) project. Participants in the HALI project were selected to obtain a representative sample of pastoralist households according to ethnicity, socio-economic status, and geographic location. Response data from a cross-sectional socio-economic and disease survey conducted by HALI between December 2006 and November 2007 was reviewed to generate a list of households with reported occurrence of diarrheal disease episodes at the herd and household levels32. Study Design. Along with a review of the HALI response data, a cross-sectional study design combining calf sampling with a questionnaire was used to investigate the prevalence of and risk factors associated with diarrheal disease and the protozoal pathogens Cryptosporidium and Giardia in calf herds during the height of the dry season.

60 households to reach the targeted sample size of 300 calves, as well as to ensure representation of a wide range of management and husbandry practices in the study. As an end objective was to investigate the prevalence of Cryptosporidium and Giardia among young livestock at the herd level, a sample size of five animals per household was held to be sufficient; however, as the range of animals in the calf herds varied from a single calf to over 100, calf herds were sampled proportionately, with every animal sampled in herds with five or less calves (n = 11), and a mean of 54% of calves sampled in larger herds, ranging from 6-10 calves. Of the 312 fecal samples collected, only 262 were available for analysis due to sample destruction during sample storage by pests. The household sample size of 60 was consequently reduced to 52. Sample Collection. Calf fecal samples were collected directly from the rectum and preserved in 2% buffered formalin until testing. The presence (or absence) of clinically apparent diarrhea was recorded for each sampled calf. The determination of which calves to sample in larger calf herds was made by the selection of a random calf, followed by the sampling of every other calf in the boma (livestock pens surrounded by acacia thorn fencing) until approximately 50% of the calves present had been sampled. In several herds, convenience sampling was utilized, as animals could not be moved into the boma for sampling. In such instances, calves were semi-randomly selected based on the teamâ&#x20AC;&#x2122;s ability to capture the animal. To allow for cross-herd comparisons and assessment of wealth, livestock holdings for each household were converted to tropical livestock units (TLUs) following convention, where one TLU is the equivalent of 250 kilograms of livestock weight33. As the weights of the animals were not measured, TLUs were calculated as small ruminants equal to 0.1 TLU, and cattle equal to 0.8 TLU following Nduma et al.34. HALI project water sampling data was investigated to better appreciate the potential role of water on elevated risk for diarrheal disease and protozoal shedding. HALI water samples were collected quarterly at 11 locations throughout the Ruaha landscape and study area from February 2007 to March 2009.

A two-stage sampling procedure was used to select study households and animals from the HALI sampling frame. The prevalence of Cryptosporidium and Giardia, as well as the prevalence of general diarrheal disease in the calf population was unknown. Based on local herd size estimates, a herd level sample size of approximately five calves per household was estimated to be adequate to determine whether or not Cryptosporidium and Giardia were present or absent in the herd. Accordingly we aimed to sample 5

University of California Davis Laboratory Analysis. Prepared fecal samples were examined for Cryptosporidium oocysts and Giardia cysts by direct fluorescent-labeled antibody Giardia cyst (DFA) staining using the A100FLK Aqua-Glo™ Giardia/Cryptosporidium Direct Comprehensive Kit (Waterborne Inc., New Orleans, LA, USA) following the manufacturer’s instructions. A single trained laboratory technician Cryptosporidium oocyst (E. Kamani) at the Sokoine University of Agriculture’s Faculty of Veterinary Medicine in Tanzania who was unaware of the clinical diarrhea history of each animal examined and counted all samples. Relative Cryptosporidium Figure 3. Under the fluorescent microscope (1000X), oocysts and cysts, when present, appear apple green in color and shiny (Photo by E. Kamani). oocyst and Giardia cyst shedding was quantified by visually counting Water samples were analyzed for Cryptosporidium fluorescing oocysts and cysts in all fields (1000x) of and Giardia using the US Environmental Protection the slide well. Samples were recorded as negative Agency Method 1623 (IMS concentration and DFA when no oocysts or cysts were found. Estimates staining for visualization of cysts and oocysts). for the shedding intensity of Cryptosporidium and Giardia by an individual animal were obtained Household Surveys. At each household, following through visual quantification of oocysts and cysts sampling of the calf herd, the head of household or on the slide well and adjusted using methodology main caretaker of young livestock was interviewed developed by Xiao et al.35 to approximate the level at the site of the calf herd using a standardized of fecal loading and subsequent environmental 35-question questionnaire administered by a trained contamination and public health risk associated with interviewer in Kiswahili. The questionnaire included the pathogens. questions about herd health; general livestock management practices; management practices Statistical Analysis. Data were analyzed using specific to the calf herd; and potential risk factors for JMP statistical software (Version 7.0 for Mac, SAS disease, such as calf housing, water sources, mixing Institute Inc., Cary, NC, USA). Bivariate analyses of animals, and approximate age of weaning. In using the Pearson’s chi-square and Fischer’s exact addition, questions regarding dangerous diseases tests were used to test for independence and to begin for young livestock and related veterinary practices to identify risk factors associated with each of the for treatment and prevention of disease, household outcome variables (shedding of Cryptosporidium, interaction and contact with the animals, and shedding of Giardia, and clinically apparent household hygiene and sanitation practices were diarrhea), with a P-value of <0.05 considered posed to obtain a picture of the reported disease significant36. Biologically plausible variables were burden in calves and other potential zoonotic disease included in multivariate logistic regression models transmission risks. Where possible, responses were specific to each of the outcome variables to identify confirmed through visual observation and recorded risk factors, with the final model fit evaluated using using a standardized checklist. Using HALI project likelihood ratio with a P-value of < 0.05 considered survey data from 2006-2007, the prevalence of statistically significant37,38. Odds ratios (OR) and reported human diarrhea among study households 95% confidence intervals were used to evaluate the was also explored. strength of associations for potential risk factors39.

Masters Thesis RESULTS

21 reported cases of diarrhea among their calves (40.4%). Diarrhea was observed by the trained interviewer in 53.9% of the calf herds. Calf herds containing at least one diarrheic animal were identified at three of five households reporting cases of human diarrhea and at two of three households that reported visits by household members to medical clinics for treatment of diarrhea (Table 1). In addition, calves shedding Cryptosporidium and Giardia were identified in herds of households reporting human diarrhea and among households reporting the sheltering of sick animals within the human living quarters. Calf diarrhea was detected in 38.9% of herds from households that reported having cases of diarrhea in their adult herds, with the majority of these calf herds (77.8%)

Diarrheal disease. Table 1 presents the results for cases of human and livestock diarrhea along with interview results. Of the five cases of reported human diarrhea among HALI project households in the current study (n=45), 60% involved children under the age of seven. A further 8.1% (n=37) of HALI project households reported a family member having to travel to a local health clinic for treatment of diarrhea, a journey ranging from 20 minutes to four hours on foot (mean = 90.7 minutes). Diarrhea emerged as the third most frequent symptomatic cause for clinic visitation, behind fevers (40.5%), and malaria (21.6%). Of the 52 households surveyed during calf sampling, 18 reported cases of diarrhea occurring in their adult cattle herds (34.6%), while

Table 1. Household and herd-level cases of human (reported) and livestock (reported and observed) diarrhea and interview results among study households. Case Description

No. of Households (Prevalence)

Corresponding No. of Households with Calf Diarhea

Cryptosporidium Shedding

Giardia Shedding

Reported visitation of a medical clinic by a household member for diarrhea*

3 (8.3%)

2/3 (66.7%)

1/3 (33.3%)

Household member reporting diarrhea*

5 (11.1%)

3/5 (60.0%)

1/5 (20.0%)

3/5 (60.0)

Reported diarrhea in adult cattle herd+

18 (34.6%)

7/18 (38.9%)

6/18 (33.3%)

14/18 (77.8%)

Reported diarrhea in calf herd+

21 (40.4%)

9/21 (42.9%)

17/21 (81.0%)

Diarrhea observed in at least one calf in the herd+

28 (53.9%)

17/28 (60.7%)

24/28 (85.7%)

Households sheltering young and sick animals within the human living quarters+

42 (82.3%)

22/42 (52.4%)

17/42 (40.5%)

31/42 (73.8%)

Households reporting women and children as the primary caretakers for young and small stock+

45 (86.5%)

26/45 (57.8%)

20/45 (44.4%)

36/45 (80.0%)

*HALI Project interviews conducted between December 2006 and November 2007. Reported diarrhea was for the two-week period preceding the interview only. +Results obtained from calf sampling and interview period between August 2008 and January 2009.

University of California Davis Table 2. Frequency and Odds Ratios (OR) of clinical diarrhea in calf herds by risk factor (n= 52 households). Risk Factor

Overall (%)

95% CI for OR (lower, upper)

P-value

Washing newborns

11.5

6.3

(0.70-56.4)

0.08

Small adult herds

40.4

9.0

(2.6-31.7)

0.0004

Small calf herds

42.3

13.9

(3.7-53.0)

<0.0001

Poor households

30.8

3.2

(1.0-10.3)

0.04

Cryptosporidium

32.8

5.9

(1.8-20.4)

0.004

Giardia 46.2 3.6 (0.9-13.9) *Wealth status determined by livestock holdings evaluated through tropical livestock units (TLUs)34.

shedding Giardia cysts. Cryptosporidium shedding was detected in 33.3% and 42.9% of calf herds from households reporting diarrhea in their adult and young animals, respectively.

0.05

On the individual calf level, 9.9% of sampled calves were shedding Cryptosporidium oocysts and 22.9% were shedding Giardia cysts. Similar to diarrhea, the prevalences of Cryptosporidium (OR = 4.4, 95% CI = 1.5-13.1) and Giardia (OR = 2.9, 95% CI = 1.5-5.6) were significantly higher in neonates than in older calves. Fecal shedding intensity estimates for Cryptosporidium ranged from 15 to 121,625 oocysts per gram feces (mean = 8,578.5), while Giardia ranged significantly higher, from 262 to 217,188 cysts per gram (mean = 19,837.5). Mean fecal loading was estimated at 19,837,500 oocysts/calf and 43,642,500 cysts/calf per day for Cryptosporidium and Giardia, respectively.

Herd management and livestock husbandry factors were strongly associated with increased risk of calf diarrhea in households with small calf herds (OR = 13.9, 95% CI = 3.7- 53.0) and small adult cattle herds (OR = 9.0, 95% CI = 2.6-31.7; Table 2). Using wealth status represented by Tropical Livestock Units (TLUs), poorer households (e.g. those with less than 35 TLUs) were at higher risk (OR = 3.2, 95% CI = 1.02-10.3) of calf diarrhea, which was consistent with the higher risk in small-herd households. Of the protozoal pathogens, Cryptosporidium was most strongly associated with increased risk for calf diarrhea (OR = 5.9, 95% CI = 1.7-20.4).

At the herd level, 42.3% and 75.0% of the 52 herds participating in the study contained at least one animal shedding Cryptosporidium oocysts or Giardia cysts, respectively. Moving the boma or livestock pen (OR = 16.1, 95% CI = 1.9-135.4), smaller calf bomas (OR = 26.7, P = 0.002 estimated from Fischerâ&#x20AC;&#x2122;s exact test using 0.3 herds with large bomas and diarrhea when actually none were counted), and clinically apparent diarrhea (OR = 5.9, 95% CI = 1.7-20.4) were positively associated with calves in the herd shedding Cryptosporidium oocysts. Multivariate logistic regression risk modeling for Cryptosporidium shedding support several

Of the 262 sampled animals, 17.2% of individual calves had clinically apparent diarrhea, with 13.3% and 28.9% of these animals identified as shedding Cryptosporidium oocysts or Giardia cysts at the time of sampling, respectively. The prevalence of diarrhea was significantly higher in calves under three months of age (OR = 2.5, 95% CI = 1.2-5.2). Protozoa. All 262 calf fecal samples were screened for Cryptosporidium parvum and Giardia lamblia.

Table 3. Factors best predicting Cryptosporidium shedding by multivariate logistic regression in 52 pastoral calf herds. Risk Factor

95% CI

Movement of boma

4.65

1.3-16.1

High calf stocking density

2.13

0.95-4.8

Distance from nearest village

2.19

0.96-5.0

Masters Thesis management factors highlighted in the bivariate analysis (Table 3). Results reveal t h a t h e rd i n f e c t i o n with Cryptosporidium is best predicted by a combination of moving the boma, high calf stocking density, and increasing distance from the nearest village. Several hygiene and water-related diarrheal disease and protozoal risk factors emerged during the bivariate and multivariate analyses. Washing udders prior to milking, practiced by 64.3% of respondents (n=24), was strongly associated as a protective measure to reduce the risk associated with Cryptosporidium (OR = 0.17, 95% CI = 0.03- Figure 4. Location of calf herds with clinically apparent diarrhea and shedding protozoal pathogens (Cryptosporidium and/or Giardia) in the Ruaha Landscape, Tanzania. 0.9), and was associated with lower prevalences were marginally associated with increased risk of of diarrhea and shedding of both protozoa. NinetyGiardia. Based on HALI project data, only 17 of eight percent of respondents (n=50) reported hand 46 households (37%) involved in this study reported washing as a consistent household practice before treating water for household consumption. working with animals, while slightly less (84.0%), reported hand washing as an activity practiced prior DISCUSSION to milking. Both Cryptosporidium and Giardia were detected at multiple water sites, including the The reported presence of diarrhea in the human Idodi River near Idodi Village and the Great Ruaha population, especially among children, along with the River within Ruaha National Park. Surface water fact that individuals do seek treatment for diarrhea was reported by 84.4% of households (n=45) as at local health clinics despite long walking distances the primary water source for human consumption, indicates that diarrhea is a recognized public health with 90.2% (n=51) of households reporting surface issue among pastoralists in Ruaha. Furthermore, the water use as the primary water source for livestock. reported high prevalence for diarrhea in livestock, Households relying on surface water for watering combined with our observations that 54% of livestock (OR = 3.2, 95% CI = 0.03-1.2), and sampled herds had one or more calves with clinical those providing water for livestock in containers diarrhea shows that diarrheal disease is a veterinary near the household (OR = 5.6, 95% CI = 0.08-1.2) 9

University of California Davis concern among livestock keepers, 21% of whom actively treat animals with visible signs of diarrhea. Reported calf diarrhea underestimated the actual prevalence determined by our trained interviewer by approximately 14%, likely because the trained interviewer recorded all cases of clinically apparent diarrheal disease. As calf diarrhea appears relatively common in these herds, attention to calves with diarrhea may not be a common practice. It is likely that reported prevalence for human diarrhea is also lower than the actual prevalence, as individuals may be resistant to sharing personal health information, especially with regard to defecation, and may be unaware of the status of family members for similar reasons. Because of the time interval between the collection of HALI project data on human diarrheal disease and the survey and sampling activities on calves, no attempt was made to link risk factors between the two populations. It must be noted, however, that the majority of households reporting cases of human diarrhea were also shown to have clinically apparent diarrhea in their calf herds, along with animals shedding both Cryptosporidium and Giardia. In this pastoral system, small stock and young animals are managed in close proximity to adult livestock enclosures, which are typically located adjacent to household structures. The reported sheltering of young and sick livestock within the human living quarters for protection and observation, along with the frequent care of these same animals by women and young children demonstrates the intimate connection between calves and susceptible household members. Furthermore, it was demonstrated that animals shedding Cryptosporidium are six times more likely to be diarrheic. These diarrheic animals, especially calves, pose a serious risk for the transmission of fecal-oral pathogens like Cryptosporidium and Giardia, which due to (1) a low infective dose (as few as 10 oocysts or cysts), (2) the relatively high estimated fecal loading potential of a shedding calf, and (3) the high survival and resiliency of oocysts and cysts in the environment, must be recognized as a zoonotic health threat40,41.

A second objective of the study was to determine the sensitivity of calves to infection by Cryptosporidium and Giardia to assess the potential for calves as a sentinel animal for the surveillance of diarrheal disease-causing pathogens. According to a conceptual framework developed by Halliday et al.27, animal sentinels must be susceptible and responsive to a zoonotic pathogen that is spatially or ecologically related to the target population, in this case the household family. Calves, due to their inherent value and economic importance in the pastoral production system, are a highly visible group from a management perspective and are closely monitored by household members. In addition, the prevalence of diarrhea in calf herds was higher than that reported for adult animals, demonstrating a calf herdsâ&#x20AC;&#x2122; greater sensitivity to an observable response by household members. Because both Cryptosporidium and Giardia were detected in calf herds from households reporting diarrhea in their animals, monitoring of calf diarrhea has potential for the prevention and control of diarrheal disease. As the utility of an animal sentinel system is determined by the ability of the animal to provide early warning signs of the presence of pathogens27, both Cryptosporidium and Giardia were evaluated for use in the calf sentinel system due to their similar fecal-oral transmission ecology, zoonotic potential, and diarrheic effect on susceptible animals. While both were detected at relatively high prevalences at the herd level compared to other studies conducted in the region31,42,43, only Cryptosporidium shedding was associated with an increased risk of diarrheal disease in calves. It appears that Giardia shedding, with a high herd level prevalence of 75% may be endemic, common, and most often asymptomatic in calves, limiting its potential as a sentinel pathogen. Due to its significantly higher prevalence among neonates and greater sensitivity to diarrhea as an observable response mechanism, Cryptosporidium emerged as the more suitable protozoal pathogen for a calf sentinel system (Box 1). The use of a sentinel system for diarrheal disease in pastoral areas is practical only if its economical application results in early detection and preventive 10

Masters Thesis Box 1. Components of the proposed calf sentinel system for diarrheal disease. Target population

Pastoral and agro-pastoral livestock producers

• Isolated from surveillance and disease reporting structure. • Diarrheal disease is a reported public health and veterinary concern. • Lack of access to veterinary and medical care. • The majority of households reporting cases of diarrhea in humans also have diarrheic calf herds.

Sentinel

Calves

• Important culturally and economically to target population. • Highly visible to managers and household members. • Highly susceptible to both diarrhea and Cryptosporidium (low infectious dose, high shedding potential). • Logistically easy to observe and sample.

Pathogen*

Cryptosporidium

• Zoonotic protozoa pathogen of public health interest. • High specificity for laboratory detection. • High sensitivity to diarrheal disease/easily observable response (diarrhea). • High prevalence among sentinel population.

*Cryptosporidium is intended as a proxy pathogen: its association with diarrheic calves is considered indicative of the presence of a variety of diarrheagenic and infectious organisms.

response. Because Cryptosporidium shares a similar transmission ecology to a wide range of fecal-oral diarrheagenic organisms, its proposed integration into a sentinel system is intended to serve as a proxy. Typically, in outbreaks involving calf diarrhea, multiple pathogens can be identified and mixed infections often occur21,44. As demonstrated in this study, diarrhea observed in calves may be indicative of protozoal and other pathogenic presence, resulting in the contamination of the local environment with a wide variety of infectious organisms. Actively surveying for diarrheic animals and an increase in the incidence of diarrheal disease among calves could allow livestock keepers to rapidly respond with preventive control measures and minimize transmission risks to the household. A calf sentinel system for diarrheal disease is therefore proposed as a rudimentary yet potentially effective surveillance system for isolated and resource restrained areas.

that could serve as complimentary disease prevention and control practices when combined with the calf sentinel approach. Several livestock husbandry factors, including smaller cattle, calf, and small ruminant herd sizes were significantly associated with increased risk for diarrhea. These associations may be partially explained by the positive associations between small bomas and diarrhea, also supporting the assumption that stocking density plays a role in elevated disease risk. While not significant for diarrhea, high stocking density was a predictor for shedding of Cryptosporidium oocysts in the multivariate model. This is consistent with the literature supporting the association between high density of animals and Cryptosporidium infection45. Boma size and the number of bomas managed by a household is partly determined by the size of the herd, and the amount of time animals spend in a boma may reflect holding capacity; smaller herds may spend more time in bomas and in the immediate vicinity of the household, allowing for more exposure to manure and fecal pathogens. Likewise, it is possible that larger herds are allowed more time outside of bomas reducing their exposure. Young and sick animals were more likely to be sheltered inside the

Utilizing calf sentinel response as an early warning mechanism along with practically implementable preventive measures may help reduce the risks associated with herd-household disease transmission in pastoral and rural areas, and vice versa. Study results have identified several intriguing risk factors associated with diarrheal disease and the protozoa 11

University of California Davis household if the calf herd was large (OR = 9.7, 95% CI = 1.1-84.5), suggesting that larger herds are further protected from potential exposure due to the isolation of sick animals. This practice, however, is especially risky for household health. Movement of the boma, a factor with more shortterm management implications, was significantly associated with Cryptosporidium shedding. In our surveys, movement of the boma was a practice associated with manure management, often implemented when the boma became too full of manure. As the boma fills with manure, the floor of the boma elevates in height relative to the perimeter fencing, thus reducing the penâ&#x20AC;&#x2122;s ability to prevent livestock escapes and protect against predator attacks. Boma movement is also implemented as a response to precipitation and for this reason has been considered a preventive disease measure. If preventive, this management practice should have been associated with diminished risk of diarrhea

and shedding, yet in our study it was associated with increased likelihood of shedding for Cryptosporidium. It is therefore suggested that movement of the boma may be more of a practical response to disease than a preventive measure, as movement usually occurs just after the fecal deposition has reached a maximally tolerated level. Despite the emergence of boma movement as predictive for Cryptosporidium shedding, manure management is still recommended as a potential prevention strategy, including the removal of manure from livestock pens or the more frequent movement of pens from fecally contaminated areas to minimize transmission. Managing for lower stocking densities to reduce exposure, by either decreasing numbers of animals per boma or increasing the size of livestock enclosures, and isolation of symptomatic animals to contain potentially contaminated fecal material from the remainder of the herd and household members are also strongly recommended. However, traditional

Figure 5. Bomas (livestock enclosures surrounded by thorn fencing) and households are often located in close proximity, sometimes within a few meters of one another. Young and sick animals are often sheltered inside the household as well (Photo by D. Wolking). 12

Masters Thesis isolation practices, especially those involving sheltering of sick animals inside the household should be discouraged, as they increase the risk for pathogen exposure to household members. The vast majority of households in this study relied primarily on surface water for both herd and household use. Given that both Cryptosporidium and Giardia were identified in water sources utilized by livestock, humans, and wildlife, the potential for waterborne transmission is Figure 6. A young girl milks a cow as a calf waits for its turn to suckle in the boma. Washing significant. Combined of the udders prior to milking can help to reduce the risk for fecal-oral disease transmission w i t h t h e c o m m o n between humans and livestock (Photo by D. Wolking). practice of sharing of reported hand washing as an activity practiced prior water with livestock at the household through to milking. It is possible that udder washing prior containers, water as a potential transmission route to milking may be a more reliable measure for the appears increasingly risky, for both humans and interaction between hand washing and milking. livestock. As these water-related factors can play a distinct role in diarrheal disease and the transmission To minimize household risk associated with ecology of the protozoa, they are especially critical to water and hygiene related factors of disease address from a public health perspective46,47. transmission, it is recommended that water for household consumption be treated in a practical and appropriate manner for household use; water supplied for the household and livestock should be kept separate to prevent contamination; and hand washing with soap should be practiced prior to and following contact with animals, especially during milking.

Hygiene, shown by washing of the udder prior to milking, seems to play a distinct role in reduction of disease risk. Cow to calf transmission of protozoa can be a major transmission route for infection48, and first washing the udders, as milk is harvested for human consumption, may also remove diseasecausing organisms from the udder for the calf, which typically drinks immediately after. Washing udders also suggests an attention to hygiene that may be affiliated with other preventive measures not assessed by this study. While nearly all respondents reported hand washing as a consistent household practice before working with animals, slightly less 13

University of California Davis CONCLUSION The use of calves as sentinel animals for diarrheal disease is a potentially viable monitoring and surveillance option for resource constrained livestock producers in rural areas, especially in pastoral production systems. Increase in calf diarrheal incidence can be used as a warning sign to trigger interventions to minimize transmission of diarrhea causing agents, especially zoonotic protozoal pathogens. Livestock keepers recognizing these early warning signs could then implement appropriate disease prevention and control practices to protect the herd and household, through the isolation of sick animals, the movement of livestock, storage of manure, hand washing before and after contact with animals (especially during milking), separation of herd and household water supplies, and appropriate water treatment.

REFERENCES 8. Richards L., Claeson M., and Pierce N.F., 1993: Management of acute diarrhea in children: lessons learned. Pediatr Infect Dis J. 12(1): 5-9.

1. Everhart, J.E., (ed.), 1994: Digestive Disease in the United States: epidemiology and impact. NIH Publ 94-1447. Bethesda, MD: National Institutes of Health.

9. World Health Organization/United Nations Children’s Fund, 2008: Progress on Drinking Water and Sanitation: Special focus on sanitation. UNICEF: New York, NY.

2. Lubeck, D.P., Bennett C.L., Mazonson, P.D., Fifer, S.K., and Fries J.F., 1993: Quality of life and health service use among HIV-infected patients with chronic diarrhea. J Acquir Immune Defic Syndr Hum Retrovirol 6:478-484.

10. Cheneau Y., El Idrissi A.H., and Ward D., 2004: An assessment of the strengths and weaknesses of current veterinary systems in the developing world. Rev Sci Tech 23: 351-9.

3. Watson A., Samore M.H., and Wanke C.A., 1996: Diarrhea and quality of life in ambulatory HIV-infected patients. Dig Dis Sci 41:1794-1800.

11. Thornton P.K., Kruska R.L., Henninger N., Kristjanson P.M., Reid R.S., Atieno F., et al. 2002: Mapping poverty and livestock in the developing world. Nairobi: International Livestock Research Institute.

4. --, 2008: Maternal and Child Undernutrition: Global and regional exposures and health consequences’. The Lancet 371(9608), pp 243-260.

12. Hunter, P.R., Hughes, S., Woodhouse, S., Syed, Q., Verlaner, N.Q., Chalmers, R.M., Morgan, K., Nichols, G., Beeching, N., and Osborn, K., 2004: Sporadic cryptosporidiosis case control study with genotyping. Emerg. Infect. Dis. 10: 1241-1249.

5. UNICEF/WHO. 2009: Diarrhoea: Why children are dying and what can be done. The United Nations Children’s Fund (UNICEF)/World Health Organization (WHO): New York, NY. 6. Kasle, M., 2008: Neonatal diarrhea in the calf – etiology and pathophysiology. Praktische Tierarzt 89(10): 852.

13. Lengerich, E.J., Addiss, D.G., Marx, J.J., Ungar, B.L.P., and Juranek, D.D., 1993: Increased exposure to cryptosporidia among dairy farmers in Wisconsin. J. Infect. Dis. 167: 1252-1255.

7. Wudu, T., Kelay, B., Mekonnen, H.M., and Tesfu, K., 2008: Calf morbidity and mortality in smallholder dairy farms in Ada’a Liben district of Oromia, Ethiopia. Tropical Animal Health and Production 40(5): 369-376.

14. Siwila, J., Phiri, I.G.K., Vercruysse, J., Goma, F., Gabriel, S., Claerebout, E., and Geurden, T., 2007: Asymptomatic cryptosporidiosis in Zambian 14

Masters Thesis dairy farm workers and their household members. Trans. R. Soc. Trop. Med. Hyg. 101: 733-734.

23. Hunter, P.R., and Thompson, R.C., 2005: The zoonotic transmission of Giardia and Cryptosporidium. International Journal for Parasitology 35, pp. 1181-1190.

15. Barrington, G.M., Gay, J.M, and Evermann, J.F., 2002: Biosecurity for neonatal gastrointestinal diseases. Vet. Clin. North Am. Food Anim. Pract. 18: 7-34.

24. Thompson, R.C.A, and Monis, P.T., 2004: Variation in Giardia: Implications for taxonomy and epidemiology. Adv. Parasitol. 58, pp. 69-137.

16. Donowitz, M., Barnett, J.L., and Powell, D.W., 1999: AGA Technical Review on the Evaluation and Management of Chronic Diarrhea. Gastroenterology 116: 1464-1486.

25. DeGraaf, D. C., Vabopdenbosch, E., OrtegaMora, L. M., Abbassi, H. and Peeters, J. E., 1999: A review of the importance of cryptosporidiosis in farm animals. Int. J. Parasitol. 29: 1269-1287.

17. Fayer, R., Basbarre, L., Pasquali, P., Canals, A., Almeria, S., and Zarlenga, D., 1998: Cryptosporidium parvum infection in bovine neonates: dynamic clinical, parasitic and immunologic patterns. Int. J. Parasitol. 28: 49-56.

26. Berkelman, R.L., Bryan, R.T. Osterholm, M.T., LeDuc, J.W., & Hughes, J.M. 1994: Infectious Disease Surveillance: A Crumbling Foundation. Science 264: 368-370.

18. Tzipori, S., Smith, M., Halpin, C., Angus, K.W., Sherwood, D., and Campbell, I., 1983: Experimental cryptosporidiosis in calves: clinical manifestations and pathological findings. Vet. Rec. 112: 116-120.

27. Halliday, J.E.B., Meredith, A.L., Knobel, D.L., Shaw, D.J., Bronsvoort, T.B., and Cleaveland, S., 2007: A framework for evaluating animals as sentinels for infectious disease surveillance. Journal of the Royal Society Interface 4: 973-984.

19. Snel, S.J., Baker, M.G., Kamalesh, V., French, N., and Learmonth, J., 2009: A tale of two parasites: the comparative epidemiology of cryptosporidiosis and giardiasis. Epidemiol. Infect. 137: 1641-1650.

28. McCluskey, B.J., 2003: Use of sentinel herds in monitoring and surveillance systems. In Animal disease surveillance and survey systems: methods applications (ed. M.D. Salman), pp. 119-133. Iowa, IA: Iowa State Press.

20. Preiser, G., Preiser, L., and Madeo, L., 2003: An outbreak of cryptosporidiosis among veterinary science students who work with calves. J. Am. Coll. Health 51: 213-215.

29. Ferguson, N.M., Cummings, D.A.T., Cauchemez, S., Fraser, C., Riley, S., Meeyai, A., Iamsirithaworn, S., and Burke, D.S., 2005: Strategies for containing an emerging influenza pandemic in Southeast Asia. Nature 437: 209-214 (doi: 10.1038/nature04017.)

21. Oâ&#x20AC;&#x2122;Handley, R.M., Cockwill, C. McAllister, T.A., Jelinski, M., Morck, D.W., and Olson, M.E., 1999: Duration of naturally acquired giardiosis and cryptosporidiosis in dairy calves and their association with diarrhea. J. Am. Vet. Med. Assoc. 214: 391-396.

30. Miller, W.A., Clifford, D., Kazwala, R., Kitime, A., Kitambi, A., and Mazet, J., Forthcoming: Fecal Pathogen Pollution in Surface Waters of the Ruaha Ecosystem, Tanzania. Research Brief 10-03HALI. Global Livestock Collaborative Research Support Program (GL-CRSP), University of California Davis. Davis, CA.

22. Naciri, M., Lefay, M.P., Mancassola, R., Poirier, P., and Chermette, R., 1999: Role of Cryptosporidium parvum as a pathogen in neonatal diarrhoea complex in suckling and dairy calves in France. Vet. Parastiol. 85, 245-257.

31. Tanzania Ministry of Agriculture and Food 15

University of California Davis Security, 2002: Agriculture Sector Development Strategy. Dar es Salaam: Ministry of Agriculture and Food Security Tanzania. Available: http://www.fao. org/tc/tca/docs/tza_asds.pdf. [Accessed 25 October 2009.]

40. DuPont, H.L., Chappell, C.L., Sterling, C.R., Okhuysen, P.C., Rose, J.B., and Jakubowski, W., 1995: The infectivity of Cryptosporidium parvum in healthy volunteers. N Engl J Med 332: 855-9.

32. Masozera, M., In Prep: Water Management and Rural Livelihoods in the Ruaha Landscape, Tanzania. In: Ecosystem Services and Protected Areas Management in Africa. Doctoral Dissertation. University of Vermont.

41. Ortega, Y.R., and Adam, R.D., 1997: Giardia: overview and update. Clin Infect Dis 25: 545-50. 42. Kusiluka, L.J.M., Karimuribo E.D., Mdegela, R.H., Luoga, E.J., Munishi, P.K.T., Mlozi, M.R.S., and Kambarage, D.M., 2005: Prevalence and impact of water-borne zoonotic pathogens in water, cattle, and humans in selected villages in Dodoma Rural and Bagamoyo districts, Tanzania. Physics and Chemistry of the Earth 30: 818-825.

33. Bekure, S. de Leeuw, P.N., Grandin, B.E., and Neate, P.J.H., 1991: An analysis of the livestock production system of Maasai pastoralists in eastern Kajiado District, Kenya. International Livestock Center for Africa Systems Study. Addis Ababa, Ethiopia: International Livestock Center for Africa (now ILRI).

43. Bendali, F., Bichet, H., Schelcher, F., and Sanaa, M., 1999: Pattern of diarrhoea in newborn beef calves in south-west France. Vet. Res. 30: 61-74.

34. Nduma, I., Kristjanson, P., and McPeak, J., 2001: Diversity in income-generating activities for sedentarized pastoral women in northern Kenya. Human Organization, 60: 319-325.

44. Oâ&#x20AC;&#x2122;Handley, R.M., 2007: Cryptosporidium parvum infections: are current perceptions accurate? TRENDS in Parasitology 23(10): 477-480.

35. Xiao, L., and Herd, R.P., 1993: Quantification of Giardia Cysts and Cryptosporidium Oocysts in Fecal Samples by Direct Immunoflourescence. J. Clin. Microbio. 33: 2944-2946.

45. Quigley, J.D., Martin, K.R., Bemis, D.A., Potgieter, L.N.D., Reinemeyer, C.R., Rorhbach, B.W., Dowlen, H.H., and Lamar, K.C., 1994: Effects of housing and colostrum feeding on the prevalence of selected infectious organisms in feces of Jersey Calves. J. Dairy Sci. 77:3124-3131.

36. Agresti, A., 2002: Categorical Data Analysis, Second Edition. John Wiley & Sons, Inc., Hoboken, NJ, 710 pp.

46. Graczyk, T.K., Fayer, R., and Cranfield, M.R., 1997: Zoonotic transmission of Cryptosporidium paruvm: Implications for waterborne cryptosporidiosis. Parasitology Today 13(9): 348-351.

37. Kleinbaum, D.G., Kupper, L.L., and Morgan, H., 1982: Epidemiologic Research: Principles and Quantitative Methods. Lifetime Learning Publications, Belmont, CA, 527 pp. 38. Hosmer, D.W., and Lemeshow, S.W., 1989: Applied Logistic Regression. Wiley, New York, NY, 307 pp.

47. Tumwine, J.K., Thompson, J., Katua-Katua, M., Mujwajuzi, M., Johnstone, N., Wood, E., and Porras, I., 2002: Diarrhoea and effects of different water sources, sanitation, and hygiene behaviour in East Africa. Trop. Med. and Int. Health 7(9): 750-756.

39. Kahn, H.A., and Sempos, C.T., 1989: Statistical Methods in Epidemiology: Volume 12 of Monographs in Epidemiology and Biostatistics. Oxford University Press, USA, 292 pp.

48. Huetink, R.E.C., van der Giessen, J.W.B., 16

Masters Thesis Noordhuizen, J.P.T.M, and Ploeger, H.W., 2001: Epidemiology of Cryptosporidium spp. and Giardia duodenalis on a dairy farm. Vet. Parasitology 102(12): 53-67. Figure 2 reprinted with author permission from: Mazet, J.A.K., Clifford, D.L., Coppolillo, P.B., Erickson, J.D., and Kazwala, R.R., 2010: New medicine for emerging zoonoses: Reducing disease risk at human-animal-envioronment interfaces in Tanzania using a One Health approach. Research Brief 09-04-HALI. Global Livestock Collaobrative Research Support Program (GL-CRSP), University of California, Davis. Davis, CA.

This is an author generated publication. It is not an official publication of the University of California, Davis, the Global Livestock CRSP, or the US Agency for International Development. This document is intended as a display copy. For more information, please contact the author, David Wolking (Email: djwolk@gmail.com).

This is an author generated publication and is not an official publication of University of California, Davis, the Global Livestock CRSP, or USAID. This document is intended as a prototype and display copy only.

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