Essay: Seroprevalence and risk factors for Brucella and Leptospira antibodies in cattle in Zirobwe beef producers cooperative society in Luwero district, Central Uganda

Abstract
A cross sectional study was conducted in Zirobwe meat producers’ cooperative society in Zirobwe sub county, Luwero district in central Uganda to determine the seroprevalence of brucellosis and leptospirosis and study the risk factors associated with the diseases. A total of 140 serum samples were randomly selected from 28 member farms using the automatic random number generator version 4. The serum samples were analyzed in series by using Rose Bengal plate test and the Brucella immunocomb test kit?? (Dot ELISA) for determining the seroprevalence of brucella and leptospira immunocomb test kit?? for determining the seroprevalence of Leptospira antibodies. A total of 28 risk factor questionnaire were filled by farmers and results analyzed using Microsoft excel??, Winipep statistical software. The seroprevalence of Brucella antibodies was 32% by RBPT, 23% by Brucella immunocomb kit?? and a kappa index of 0.042 and 49% by Leptospira immunocomb kit??. A map showing the distribution of Brucella and Leptospira antibodies was drawn using ESRI-ArcGIS 9.1 software. New animals, breeding method, grazing type, history of previous abortion at the farm as well as throwing of aborted fetus in bushes were contributed significantly to the seroprevalence of both brucellosis and leptospirosis. The author concluded that the seroprevalence of leptospirosis is higher than brucellosis in cattle and could also be contributing greatly top the infectious abortion in cattle and that there was an eminent public health threat posed by both diseases. A comprehensive national study to establish the extent of leptospirosis and brucellosis in both cattle, humans including canines in order to design control strategies is highly recommended.
Chapter 1
1.0 Background
Livestock (cattle, goats, sheep) is important in Uganda and contributes greatly to household incomes, food security and employment, livestock alone contributes about 9% to the total agricultural output and 2% to total gross domestic product (GDP) (Behnke and Nakirya, 2012) The national livestock population is estimated at 12.8 million cattle, 12.5 million goats, 3.4 million sheep, 3.2 million pigs (MAAIF, 2012). Agriculture supports about 79% of the Uganda’s human population with the majority being in the cattle keeping communities (cattle corridor) (statistical abstract, 2011). Productivity is hampered by increasing threats of infectious disease of which zoonotic diseases including brucellosis, leptospirosis which do not only affect the health of animals but also the farmers, their families and consumers are of great concern (FAO, 2009). Brucellosis affects humans; livestock and wildlife, in humans the impacts are severe and quiet often misdiagnosed by medical professionals to the extent of confusing it with malaria because of almost the same clinical signs and symptoms (Cadmus et al, 2006)
According to the agricultural census of 2008, livestock ownership was spread throughout the country with high concentration in pastoral communities like Eastern (Soroti and Serere districts) and parts of central region (Nakaseke and Luwero districts).
In Uganda, brucellosis is an important zoonotic disease estimated at 14 % (Nabukenya et al, 2013) it is widespread in both humans (Makita et al. 2011, Miller et al. 2015) and animals (Mwebe et al. 2011) affecting humans mainly who deal with animals and animal products including consumption of poorly cooked contaminated meat, milk and milk products from infected animals and animal handling through contact with aborted materials (Nabukenya et al, 2013).
In animals, brucellosis is characterized by storm abortions (premature expulsion of fetus), Kungu et al. 2010), retained placentas, hygromas and birth of weak calves with a negative impact on the ‘replenishment stock and production (Hassan et al, 2006) that are critical in the beef sector because ‘today’s calf is tomorrow’s cow’ and or meat and subsequently a source of income for beef farmers. Abortion leads to huge economic losses through decreased animal productivity and infertility in cattle up to the tune of 20% (Mwiine, 2004; FAO, 2009).
Uganda is recognized as one of the hotspots for zoonotic diseases such as brucellosis and lately leptospirosis (Artherstone et al , 2014) although it has not been studied in humans , particularly favored by the host of risk factors like traditional farming practices characterized by limited biosecurity, poor farm management systems, regular exchange of breeding materials and limited to no screening for brucellosis, abundance of wildlife that freely graze with livestock being responsible for the ever increasing prevalence of brucellosis in the country ((Kabagambe et al. 2001).
The beef value chain in Uganda is fragmented, each stage is independent of the other with no coordination and the producer is detached from the trader, the processor and the consumer’. As a response to this, the government of Uganda designed the Uganda Meat Export Development of 2008, where a number of cooperatives including Zirobwe meat producers were formed that eventually made up the Uganda meat producers’ cooperative union. Formation of these cooperatives had been envisaged as best vehicle to improve animal husbandry practices, improve animal health services delivery since the farmers would be in organized groups. This was, however, short lived as the program was closed in 2013 before any improvements could be evaluated. In same program, farmers had been promised better markets including export markets for better quality animals and as a result, farmers started cross breeding the local animals ,sharing bulls, bought animals from all over the country but with limited knowledge on biosecurity. This meant that every farmer who had capacity bought a bull or animal from other farms but the capacity of the government structures to ascertain the health status is limited. In one of the cooperative visits to Zirobwe, there were several complaints from the cattle producers of animals aborting towards term and some were observant enough to say that new animals were aborting more than the ones that had stayed on the farm for more than 5 years. This is consistent with the findings of study by ( Sekitoleko et al,2008) that found out that the annual cattle abortion rate in Uganda had risen past the acceptable range of 2-5 % to 6.2 %. The complaints of farmers not withstanding hence this study with the overall objective to study abortion, its possible infectious causes, associated risk factors and control practices by farmers of Zirobwe meat producers’ cooperative society in Luwero District in order to generate data and information necessary for its control .The findings could be a baseline for the livestock cooperative movement in Uganda both for the country and the Uganda meat producers’ cooperative union and applicable in control of abortion
1.2 Justification
The increasing need for foodstuff combined with increasing competition for farmland from the growing population, forces farmers to adjust and intensify their farming practices. As of 2012, the Ugandan national livestock population was projected at 12.8 million cattle; this reflects a growth rate of 3% per annum- a rate believed to be lower than the growth in demand for livestock products (human population growth rate 3.3%) possibly due to a number of production diseases like brucellosis, leptospirosis. Approximately 4.5 million of Ugandan families rear at least cattle livestock (Livestock census, 2008). A previous study in Zirobwe by Nizeyimana et al. (2012) indicated that the prevalence of brucellosis was 5% (95%), increasing annual rate of abortion 6.2 % (Sekitoleko et al, 2008) and several complaints of farmers about abortion during several Uganda meat producers trainings to Zirobwe primary society despite the interventions influenced the study to quantify the problem and its potential infectious causes like brucellosis and leptospirosis that could be responsible for these abortions.
1.3 Overall Objective
The overall objective was to study abortion, its possible infectious causes, associated risk factors and control practices by farmers of Zirobwe meat producers’ cooperative society in Luwero District in order to generate data and information necessary for its control .
1.4 Specific Objectives
1. To determine the prevalence of brucellosis and leptospirosis in Zirobwe beef producers’ Cooperative society.
2. To study the risk factors associated with brucellosis, leptospirosis in Zirobwe beef producers’ cooperative society.
1.5 Hypothesis
1. Prevalence of Brucella antibodies is higher than that of Leptospira in Zirobwe Sub County.
2. Cattle abortions in Zirobwe are due to brucellosis infections only?
Chapter 2
Literature review
1.0 Bovine brucellosis
Brucellosis is a bacterial zoonosis that exists worldwide and endemic in most countries of Africa (Godfroid et al 2013).It is named after Sir Edward Bruce who in 1886 isolated a causative agent from a soldier in Malta where the disease had caused a considerable morbidity and mortality among military personnel (Mangen et al, 2002). In 19th century brucellosis was thus called Malta fever because of the location where the disease was found (Mangen et al, 2002). In Uganda a study in cattle and humans in western Uganda (Miller et al, 2015) indicated a prevalence of 14 and 11 % respectively, in central Uganda, (Mwebe et al, 2011) indicated wide distribution of brucellosis in the country. Brucellosis is called ‘ubutorogye’ in Runyakitara (language of western Uganda) in animals and Muhinyo in human beings.
2.1 Etiology
Bovine brucellosis is caused by a coco bacillary organism of the genus Brucellae, specie brucella abortus. Bovines are the preferred natural host and thus serve as reservoirs of infection although quite often is isolated other hosts (Aparicio, 2013). B. abortus has many biotypes and at least nine biotypes have been isolated, the common ones include biotypes 1 and 2 (Aparicio, 2013). In India a study carried out to estimate prevalence of bovine brucellosis revealed that 55% of the infections were due to biotype 1 followed by biotype 2 (Renukaradya et al, 2002).
2.2 Characteristics of Brucella abortus
Brucella abortus is a short, small, gram negative non spore forming rod occurring singly, in pairs or in short chains . It’s a facultative intracellular parasite capable of multiplying within the hosts phagocytes, aerobic with little or no fermentation on carbohydrates in conventional media, catalase and oxidase positive. B. abortus grows fairly well in an ordinary infusion agar with no enrichment; grows in a number of agars like glucose, liver and glycerol agar. In liver agar growth occurs at 37oC, 10% carbon dioxide for only three days (Laing et al, 1988).
2.3 Epidemiology
Bovine brucellosis is distributed worldwide with highest prevalence in developing countries; it has been eliminated in some countries like Australia and Great Britain (Andrews et al, 2004). As of 2008, only 15 European countries were reported free from brucellosis (Aparicio, 2013). In tropical Africa, an estimate of the average prevalence of 22.5 ?? 1.1 % has been reported, although it varies between countries 10.4??2 to 40.9??3 (Nakavuma, 1994). In clean herds, occurrence of brucellosis is due to introduction of new animals. Sero-prevalence of bovine brucellosis in countries like Ivory Coast was at 10.3%, Nigeria 13.5%, Tanzania 11.7%, and Kenya 13.7% (Nakavuma, 1994). Previous serological surveys in Uganda have reported an increasing trend of bovine brucellosis in the different parts of the country; Nakavuma, 1994 (14.7%) in central region, Nabaasa, 2002 (7%) in Mbarara District, Nizeyimana, et al, 2012 (5%) in Luwero and Nakasongola Districts, Magona et al 2009; 100% and 5.5% under pastoral system and zero grazing respectively, Kungu et al 2010 48.9%and (46.8%) in Amuru and Gulu districts.
2.4 Transmission
Following an infection, the incubation period of brucella abortus lasts between 14-20 days and after this, cattle remain chronically infected, young animals become latent carriers until they become sexually mature (Andrews et al, 2004). Transmission is both direct and indirect. In direct transmission, there is contact between uninfected animals and aborted materials, discharges of the genital tract of infected animals while in indirect transmission, there is contact with contaminated materials like pasture, commercial feeds and water.
2.5 Sources of infection
The primary sources of infection for brucellosis are reservoirs (infected cattle) that discharge large quantities of brucella abortus organisms with fetus, placenta and uterine fluids mainly at calving or abortion. Brucella abortus are also discharged in milk of infected cows after abortion or calving. Semen from infected bulls can also be a source of infection making the importation of semen from infected bulls and or bulls for natural service equally an important source (Mangen et al, 2002). Pastures contaminated by wild animals, aborted materials thrown in bush, latent carriers (reservoirs) are equally an important source. Other sources include contaminated commercial feed, water supplies veterinary equipment as well animal behaviors like licking new born, genital area of infected animal may also facilitate spread (Godfroid et al, 2005).
2.6 Routes of infection
Infection with brucellosis occurs through the oral route by ingestion of contaminated material ranging from pasture, water, milk and licking discharges of the genital tract, newborn and aborted material (Godfroid et al, 2005). The other route of infection is penetration of injured skin and mucous membranes by B. abortus organisms (Radostits et al, 2000). Coitus is not a common route of infection and occurs when the bull’s semen is infected.
2.6 Factors affecting transmission of bovine brucellosis
Transmission of bovine brucellosis is affected by several factors that include: host factors like age, sex, reproductive status of the animal and the immunological status. Mature animals are highly susceptible compared to younger ones (Blood et al, 1994). Pregnant animals have reduced resistance to B. abortus compared to heifers that have not been bred due to increased levels of erythritol produced by uterus that stimulates growth of Brucella abortus thus the localization in the uterus and placenta (Peterson et al, 2013). Na??ve (un exposed) cattle are more susceptible than exposed animals (vaccinated and or natural infection), naive (unexposed) animals are at high risk of brucellosis infection when exposed through natural infection than exposed animals (vaccinated).
2.7 Risk factors
The spread and infection of cattle with brucellosis is facilitated by a number of predisposing factors in cattle and include:
a) Herd size: ‘A big herd size interferes with regular screening as well as control of brucellosis; it usually involves regular purchases for replacement that together with keeping high stocking rate increases chances of contact of clean herd with infected animals’.
b) Management practices. Communal grazing facilitates contact between contact between infected and un infected herds, this makes segregation of reservoirs difficult because of the different herds from different areas (Blood et al, 1994). Extensive grazing like cattle ranching and game ranching with buffaloes increases spread of bovine brucellosis.
2.8 Pathogenesis
Predilection sites for B. abortus are the uterus, udder, testicle, accessory male sex glands, lymph nodes and joint capsule. Following invasion of the body localization occurs in the lymph nodes draining the area and spreads to other lymphoid tissues including the spleen and mammary glands (Blood et al, 1994). In bulls, B. abortus settles in one or both testicles, accessory glands causing chronic inflammation with sero-fibrinous exudates and caseous necrosis of the testicles and arthritis when it affects the joints. In non-pregnant animals localization occurs in the udder and uterus (Arthur et al, 1992), when an animal is pregnant, periodic phase of infection originates from the udder a source considered important for calves and humans as well.
In pregnant animals, brucellosis causes lesions within the lumen of the uterus causing severe ulcerative endometritis of the inter cotolydenoly space, placentatitis with invasion of the allantochorion and villi destruction leading to abortion that normally occurs in the 3rd trimester and if parturition occurs, calves are born weak with retention of the placenta because of placentatitis (Arthur et al, 1992).
2.9 Clinical manifestation of bovine brucellosis
Manifestation of bovine brucellosis depends mainly on the immune status of the animal; the major clinical signs in non-vaccinated susceptible animals are abortion, birth of weak calves, placenta retention, repeat breeding, metritis, still birth and eventual infertility in females (Ghodasara et al, 2010). Signs in males include unilateral and or bilateral orchitis, epididymitis, pain full swelling of the scrotal sac. Other signs include inflammation of joints with formation of hygromas on knees.
2.2.0 Impacts of bovine brucellosis
In infected cattle population, brucellosis leads to reduced calving rate due to infertility and abortion. Abortion reduces the number of calves born and infertility due to non-conception and repeat breeding too affects the calving rate. This directly leads to reduced milk production, increased cattle replacement costs and reduced market value of infected cows and products (Mangen et al, 2002). In developing countries like Uganda where the prevalence of brucellosis is still high, a lot of economic losses are suffered mainly due to loss of calves because of abortion in infected cattle population, reduced milk production, culling and condemnation of infected animals and interference with the national herd population, at national level, it threatens export of live animals and animals products (Mwiine, 2004). Infection in humans causes a debilitating disease that reduces work force and is also expensive to treat.
2.2.1 Diagnosis
Confirmation of brucellosis is by culture of contaminated fluids and tissues, particularly from aborted fetuses, this is, however, very slow and requires a suitable diagnostic laboratory to the level of biosafety level three due to the potential risk the organism poses to human handlers. In addition to being slow, convenience while screening large herds in developing countries is not very practical and thus serological tests are always used. Diagnosis of brucellosis employs a range of techniques, ranging from observation of clinical signs, post-mortem findings, serological and bacteriological examinations, other than bacteriological identification through culture, other tests need to be combined for a thorough diagnosis and thus laboratory diagnosis is an absolute prerequisite for a proper diagnosis since many other diseases manifest with similar clinical or pathological signs (Henk and Sally, 2004).
Clinical diagnosis is based on cardinal signs like abortion storm in 3rd trimester with epidemiological factors like introduction of new animals in the herd or a bull to the herd for mating with subsequent abortion suggesting a tentative diagnosis.
2.2.2 Serological tests.
Serological tests detect antibodies against B.abortus in serum, milk, whey, vaginal discharges and seminal plasma. There are a number of serological tests with varying degrees of sensitivities and specificities. None of them is absolutely accurate, they often complement each other, they are fast to perform and important in large herd screening and diagnosis.
2.2.3 Rose Bengal plate test (RBPT)
Rose Bengal plate test is a simple rapid test that detects early brucellosis infection and is qualitative in nature. It is Important in screening large herds. It is a single dilution serum agglutination test with high specificity and sensitivity (Odru, 1979). Equal volumes of test serum and Rose Bengal stained antigen is put on ceramic tile and mixed together well using a wood application stick (Alton et al. 1988) and the tile rocked gently for 4 minutes to allow for agglutination and results observed. Cross reaction between Brucella and other organism like Yersinia species puts the specificity of this test to question. Rose Bengal test detects specific antibodies of the IgM and IgG types and it is more effective in detecting antibodies of the IgG1 type than IgM and IgG2 types (Levieux, 1974). False positives in Rose Bengal plate test are usually due to residual antibody from vaccination, colostral antibodies in calves, cross reaction with certain bacteria and laboratory errors (Blood et al, 1994).
2.2.4 Enzyme linked immunosorbant assay (ELISA)
This is a useful test in diagnosis of brucellosis because of its high sensitivity and important in screening. ELISA has ability to detect antibodies of the different isotypes of B. abortus (Blood et al, 1994). It involves use of a whole cell and purified liposaccharide antigens and a variety of antiglobulin conjugates and substrates (Duncan, 1990). This assay is ideal for detecting antibodies to Brucella abortus in milk and serum.
2.2.5 Dot ELISA
This is the newest serological technique developed by Biogal-Galed labs, Israel and is called immunocomb?? bovine Brucella antibody test kit designed for conducting Dot ELISA with serum, milk and plasma samples. It is a portable self-contained, quick to perform taking 38 minutes to provide results.
This test is performed on a developing plate with multiple chambers and is based on solid phase immune assay principle. The multiple chambers have rows A to F, test sera is deposited in row A of the developing plate where a comb is inserted into the well so that the antibodies from the samples can react with antigen on the comb’s teeth. Non bound antibodies are washed in row B, Row C contains a conjugate Ig G labelled with the enzyme so that when the comb is immersed the bound antibodies are labelled followed by subsequent washing in row D and E and the comb inserted in row F with a chromogen where enzyme reaction takes place generating a color change.
The level of antibodies is determined according to the intensity of the test color result. Thus, no or a light grey color indicates no (negative) or low level of antibodies. Higher levels of antibodies are indicated by darker color results. The positive control develops a distinct grey color that should be scored S3.Specimens with identical or darker grey color results (S3 ‘ S5) are considered positive. Results may be seen in the acute phase of infection. A dark grey color result (S5 or greater) indicates a high antibody titer. Specimens with colorless (white) or faint color result (S0 ‘ S2) are considered negative or low positive (suspicious).
2.2.7 Differential diagnosis of brucellosis
In Uganda, most of the abortions are attributed to brucellosis because most of the other infectious agents have rarely been studied; a recent study by Artherstone et al. (2014) in Uganda indicated a high sero-positivity against Leptospira serovars in buffalos (42%) and cattle (29%). This prevalence is high and such is a time bomb since no study has related these findings to abortions in cattle, they are several other infectious agents that cause abortion in 3rd trimester with leptospirosis reported to be responsible for up to 50 %, Listeriosis and compylobacteriosis. These agents need to be studied to evaluate their contribution to abortions.
1.1 Leptospirosis
Leptospirosis is an emerging, widely distributed bacterial zoonotic disease that affects all mammals, including humans, livestock and wildlife and caused by different serovars of pathogenic species of the genus Leptospira (Sophia et al, 2014, Givens M.D., 2001.). It is common in both the tropical and subtropical regions with favorable environmental conditions for survival and transmission of the pathogen. First identified and described by Weil in 1886 as the causative organism of Weil’s disease in Japan in 1908 (Levett, 2001). In East and central Africa, leptospirosis was reported as far as three decades ago (Justine et al. 2015) and as an occupational disease affecting veterinarians, abattoir workers, farmers that are in constant exposure to contaminated materials.
In Tanzania, studies have indicated that leptospiral infections are prevalent in humans, livestock, and rodents in some parts of the country (Justine et al. 2015). Leptospira organisms are thin gram negative facultative aerobes that survive in moderate temperatures, alkaline PH ranging from 6.8 to 8.0 and stagnant water but are susceptible to dry conditions.
2.4. Transmission of Leptospirosis
Leptospirosis is spread by both direct and indirect means, in direct contact in humans, contact with urine from an infected animals and or infected biological materials. Cattle contract the disease by eating and drinking urine and water contaminated with leptospira organisms or directly through the injured skin, mucous membranes and aborted fetuses of infected cattle or carrier cattle. Recovered animals and animals with in apparent leptospirosis frequently excrete billions of leptospiras in their urine for several months. Leptospirosis is maintained in nature by chronic infection of renal tubules of maintenance host.in the whole world; livestock farming presents the major risk factor for leptospirosis infections. Dairy farming equally presents the highest risk usually associated with leptospira Hardjo (Levett, 2001).
2.5 Clinical signs of Leptospirosis
Leptospirosis affects both humans and animals and presents differently; in cattle, Leptospirosis is mainly associated with production and reproductive losses causing abortion in both early and late stages of pregnancy. In acute infections in early gestation, leptospirosis causes early embryonic death and subsequent resorption due to the extreme septicemia.
In late pregnancy, infection cause abortion resulting from placental invasion and transmission to the fetus, at times delivery of dead or weak calves is observed, other signs include lethargy, loss of appetite, fever, mastitis in lactating cows, agalactia, production of yellow or blood stained milk aneamia and pneumonia.
At slaughter jaundice, Subserosal and sub mucosal hemorrhage and septicemia are very significant findings. In humans, leptospirosis presents with headache, frequent chills, muscle pains, vomiting, jaundice, abdominal pain, diarrhea and reddening of eyes, signs that are often mistaken for malaria in the first phase of infection, in the second phase, persons may have kidney, liver failure and meningitis, a phase called Weil’s disease (WHO, 2003).
2.6 Epidemiology of Leptospirosis
Bovine leptospirosis is endemic in Sub-Saharan Africa despite the scarcity of epidemiological data ‘in most African countries including Uganda’. Prevalence and incidence information in humans is also scarce in many countries. In central African countries available data is even outdated save for the scarcity (Justine et al. 2015). On comparison, there is actually more data available from animals than humans about leptospirosis possibly because the disease has not been very well studied, understood and diagnosed in humans probably due to the larger economic losses caused by leptospirosis in livestock compared to losses due to human morbidity and mortality led to the studies in animals.
The transmission of leptospirosis seem to be associated with rain fall, livestock keeping and handling with rodents being responsible for urban and usually associated with poor hygiene and waste disposal. In East Africa, Kenya has more epidemiological data about leptospirosis than Tanzania and Uganda dating back as far as 1950 and has thus been key in establishment of epidemiology of leptospirosis on the African continent. Tanzania started in 1997 to conduct leptospirosis studies (Sophia et al 2014).
Leptospirosis is a zoonosis of global importance that is still under-recognized, reported in some parts of the world especially Sub-Saharan Africa reflected by the fact that only observational studies have been identified and interventional, control studies lacking .
The incubation period for leptospira is between three to twenty days following invasion through mucous membranes or broken skin of cattle followed by entry into the blood stream where they replicate in the liver, kidney , spleen , reproductive tract and sometimes the nervous system.
Transmission to cattle is by the maintenance and incidental hosts with maintenance hosts being efficient at transmitting disease from one animal to another due to colonization of organs poorly accessed by the immune system like the renal tubules of the kidney, the genital tract and are able to shed leptospira organisms without showing clinical signs there by becoming constant source of infection for most of the animal’s life time (Lilenbaum et, al 2014).
Transmission of leptospira organisms depends on the host population density, the degree of contact between maintenance and accidental hosts. Human infections are mainly occupational and or recreational through direct contact with infected animals is the most important risk factor in farmers, veterinarians, abattoir workers and meat inspectors.
2. 7 Control and eradication of brucellosis
Effective control and eradication of bovine brucellosis depends on accurate diagnosis, in developing countries like Uganda, control is the only feasible option unlike developed countries where eradication using test and slaughter program has been possible because of the capacity to compensate affected farmers. Brucellosis control methods include;
a) Test and reduction of reservoirs of infection. This involves testing for positive reactors that are culled and slaughtered to reduce on the number of infected cows from herds and subsequent exposure to un affected animals.
b) Quarantine. Restricted cattle movement of suspected for a period sufficient so that the suspects can have time to suffer brucellosis or not and assurance given that the negative cattle will not be a source for inter herd transmission. This prevents inter herd transmission by infected cattle especially false negatives and those incubating the diseases. Average incubation period is between 120 days-1 year. (Blood et al, 1994)
c) Depopulation. Slaughter of all cattle in herd on presumption that all of them have been exposed and are capable of becoming infected and acting as a source of infection. This is not a popular method in developing countries.
d) Education. Awareness campaigns to all stakeholders in the brucellosis control program to understand the epidemiology, risk factors and control methods (Nielsen and Duncan, 1990).
e) Herd management. This includes measure like isolation of pregnant animals prior to calving, purchase of replacement stock from brucellosis free herds and artificial insemination to prevent spread by infected bulls during natural mating and contact.
f) Vaccination. This is the most feasible and practical in developing countries (Nabaasa, 2002). Vaccination against B. abortus in cattle can be done using specific vaccines for a given category of cattle, giving varying degrees of protection and side effects. These vaccines include: B. abortus strain-19, which is a live vaccine and the whole cell killed adjuvant B. abortus 45/20 vaccine (FAO, 2006). Calves and non-pregnant heifers are vaccinated mainly using B. abortus strain-19 but B. abortus 45/20 vaccine can also be used. This is enough to provide along life immunity with individual effectiveness following administration is quoted at 65-75 % (Howard, 1981). However B. abortus strain 19 has a disadvantage of causing abortion in pregnant heifers that are vaccinated un-knowingly. It also causes persistent antibody titres that may be hard to distinguish from infection (Plommet, 1991). B. abortus strain 45/20 vaccine is a killed vaccine in an oil adjuvant and is usually given in two doses at twelve weeks interval period. This vaccine, however, needs an annual booster and can be used on all ages regardless of the pregnancy status (Andrews et al, 2004)
Chapter 3
Methods and Materials
3.0. Study area description
A cross sectional epidemiological survey was conducted among the cattle producers in Zirobwe beef cooperative society in Zirobwe sub county, Luwero District in central Uganda located at approximately 50 kilometers north of Kampala at 00 40 59N, 32 42 04E and 0.6830 Latitude, 32.7010 Longitude. The area was selected based on previous studies and farmers’ complaints on increasing number of abortions and the beef value chain model adopted by the society under the Uganda meat export development program (UMEDP, 2008).
Zirobwe cooperative society is a member based farmers’ group with a total of 30 members who all own farms, it is fully registered with the registrar of cooperatives in Uganda and is a member of Uganda meat producers cooperative union limited.
3.1 Study Design
A cross sectional epidemiological survey among the cattle producers in the Zirobwe cooperative society involved random blood sample collection from cattle for serological analysis and systematic use of questionnaires administered to cattle farmers of the society purposely to study the seroprevalence and risk factors for Brucella and Leptospira antibodies in cattle in Zirobwe beef producers cooperative society in Luwero district, Central Uganda.
3.2 Sample size determination
Sample size was calculated by statistical software WINPEPI (Abramson, 2004), based on the a 5% desired precision 95% confidence interval and a crude prevalence of 5% estimated from previous study by Nizeyimana et al, 2012. The sample size was adjusted to take into account sensitivity and specificity of Rose Bengal plate test of 94.7 and 99% respectively.
By substitution, based on cattle population of 600 cattle in Zirobwe cooperative society membership of 30 individual cattle producers and then using RBT of sensitivity of 94.7 and a specificity of 99%, a total of 81 samples equivalent to about 3 samples per farm were to be sampled and since the sampling frame was small, a total of 140 samples in 28 farms were sampled as farmers preferred to have more animals of their small herds screened, 2 farms refused to participate. In this study only sexually mature animals and in particular females qualified to be in the study, Males were only sampled on farms that were sharing a bull for breeding.
3.3 Sampling
Sampling was done at each of the farms that belonged to the membership of the cooperative society and as such the sampling unit was a farm /herd that belong to a farmer of Zirobwe cooperative society. The choice of animal sampled depended on results of automatic random number generator version 4. Data on every sampled farm was collected using a questionnaire, and individual sample data was collected, this included the, sex, breed and farm location. Every individual farm was given a unique code that differentiates it from others. Each blood sample was given a unique code and the same code was written on the questionnaires to link them with the blood samples. The GPS locations of the sampled farms were collected that were later used to map the location of the sampled farms with intention to show the distribution of the brucellosis and leptospirosis. At herd level, data on herd size, grazing type, biosecurity and disease control was also collected. The study was challenged by poor restraint structures of farmers and impassable roads to some farms.
3.4: Blood sample collection
Blood samples were collected into sterile plain vacutainers from either jugular vein or coccygeal vein and maintained at sample transportation temperature to the National Veterinary Diagnostics and epidemiology Centre (NADDEC). At NADDEC samples were kept in a refrigerator until the time of analysis.
Figure 1: Blood collection from coccygeal vein
3.5 Serological investigation
Serum samples were analyzed for presence of B. abortus antibodies in series by using the Rose Bengal Plate Test (RBPT) as described by Alton et al (1988) where equal volumes (20 ??ls of each) of the test serum and RBPT antigen on a white tile and swirled for 1 minute and Dot ELISA where Immunocomb?? Bovine Brucella antibody test kit was used according to manufacturer’s instructions. Rose Bengal plate test was used to act as an internal control test and compare with Dot ELISA. The kit is designed to determine cow serum IgG antibody titers for Brucella. This was done to compare the agreement of the results between the two tests. The same samples were run with Bovine Leptospira?? antibody test kit in a bid identify the presence of Leptospira antibodies and consequently the prevalence.
3.6 Risk factor questionnaire
A detailed brucellosis and leptospirosis risk factor questionnaire was prepared, pre tested and finally administered to all the participating farms purposely to find out the farmers’ understanding of the diseases in relation to abortion, risk factors, management practices as well as control and preventive measures used by farmers in Zirobwe.
3.7 Data analysis
Quantitative row data was entered in Microsoft excel?? and later by WINPEPI statistical package, qualitative data was analyzed using excel mainly while ESRI-ArcGIS 9.1 was used to draw the map to show the distribution of the sampled farms.
3.8 Ethical clearance
Ethical clearance was obtained from the Ethical Review Committee of the College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University. The farmers were informed of the study and their verbal consent was sought prior to commencement of data collection and sampling. Securing consent was easy since the farmers were also eager to know the sero-status of their herds following the explanation that was given.
Chapter 4
Results
To determine the seroprevalence of Brucella, Leptospira antibodies and associated risk factors in Zirobwe meat producer’s cooperative society in Zirobwe Sub County, Luwero district in Central Uganda, a cross sectional study was done where a total of 140 blood samples from 28 farms out of 30 member farms to the society were collected. Sample information including the GPS, the breed, breeding type, management was collected in a questionnaire. ESRI-ArcGIS 9.1 software was used to locate distribution of the farms on a map and data analyzed using Microsoft excel?? and Winipep statistical software. Antibodies for Brucella and Leptospira were detected using Rose Bengal plate test and Dot ELISA partly as internal control test and compare the results of the two tests for brucellosis and only Dot ELISA for leptospirosis.
4.0 Serological results
Rose Bengal plate test: This was performed according to the described methodology and a total of 140 serum samples were screened using this test and observed for agglutination. Out of the 140 samples, 45 samples tested positive for the Rose Bengal plate test giving a sero prevalence of 32 %. Typical agglutination reaction for a strong positive reaction appeared as in figure 2.
Figure 2: Typical agglutination reaction between Rose Bengal antigen and brucella antibodies
Dot ELISA (Brucella immunocomb test kit??). A total of 140 serum samples were analyzed using Brucella immunocomb kit, a total of 32 samples tested positive for Brucella antibodies equivalent to a sero prevalence of 23 %. Positive samples were read as in the figure below.
Figure 3: Reading of Immunocomb ELISA results
A kappa tests statistic was done using Winipep to compare the results of Rose Bengal plate test and DOT Elisa of kappa index 0.0.042 (SE Zero).
Dot ELISA (Leptospira immunocomb test kit?? ) for detection of leptospira antibodies: A total of 140 serum samples were tested using Leptospira immunocomb kit for presence of antibodies as per the described methodology, a total of 68 samples out of 140 tested positive for Leptospira antibodies equivalent to seroprevalence of 49%.
Table1: Summary of results by the three serological tests
Serological test method Number of samples (N) Number of positive samples (n) Prevalence (%)
Rose Bengal Plate test 140 45 32
Dot ELISA-Brucella 140 32 23
Dot ELISA-Leptospira 140 68 49
Prevalence by farms
Out of the 28 sampled farms, 17 farms (60.7%) tested positive for the presence of Brucella antibodies using both the Rose Bengal plate test and Dot ELISA (kappa index ‘..), although rose Bengal plate test had more positive samples than Brucella immunocomb test kit??, 13 farms (46.4 %) had mixed infections while 20(71.4 %) farms had Leptospira antibodies as in the summary below. The individual farm seroprevalence for both Brucella and Leptospira was between 0 to 100%
Table2: Summary of seropositivity by farms
Status of farms No of farms sampled (N) Number positive farms (n) Seropositivity by farm (%)
Brucella infection 28 17 60.7
Leptospira infection 28 20 71.4
Mixed infections 28 13 46.4
Table 3: Summary of individual farm seroprevalence for Brucella, Leptospira and mixed infections.
Farm code (F) Brucella antibodies (%) Leptospira antibodies (%) Mixed infections (%)
1 75 25 25
2 25 50 13
3 25 92 8.3
4 7 50 7.1
5 7 36 0
6 36 50 33.3
7 0 0 0
8 50 0 0
9 25 50 25
10 17 67 17
11 0 0 0
12 0 100 0
13 0 100 0
14 100 50 50
15 20 20 0
16 35 41 25
17 100 100 0
18 50 100 0
19 0 50 0
20 40 100 20
21 0 0 0
22 0 0 0
23 40 60 20
24 0 0 0
25 0 0 0
26 25 0 0
27 0 100 0
28 50 100 50
Foot Notes: Yellow signifies presence of Brucella antibodies on a farm , blue indicates presence of Leptospira antibodies on a farm , red indicates presence of both infections on a farm whether in same animals or different. Brucella positive farms are those that had positive samples by both Rose Bengal plate test and Dot ELISA while a true positive Brucella infected animal is the one that tests positive by both tests.
Figure 4: Map showing the distribution of Leptospira and Brucella positive farms in Zirobwe
4.2: Questionnaire results
A total of twenty eight (28) individual farm questionnaires were filled together with one hundred and forty (140) individual sample data sheets. Out of all the 28 farms sampled, none of them had a history of vaccination against brucellosis and or leptospirosis, one farm was exclusively fenced with no possibility of accessing common pastures, A total of 26 farms out of 28 farms admitted to have brought in new animals in the last 2 years of the study and all the farms that reported to have had previous abortions occurred during the 2nd and 3rd trimester as described by the farmers that aborted fetuses had hairs and some had the primary teeth. Aborted fetuses on farms were disposed differently either by throwing in the bush or being fed to dogs.
A total of 27 farms admitted to have received training either from the cooperative or the other government extension system and had heard about brucellosis but not about leptospirosis and all the interviewed farmers knew that all the abortions on the farms were due to brucellosis.
Table 4: Summary of risk factors for Brucella and Leptospira infections
Risk factor Brucella Leptospira
Positive farms (n) Negative farms (n) Positive farms(n) Negative farms (n) Odds Brucella Odds Leptospira
Grazing type Tethered/ communal grazing 8 15 15 8 0.13 1.25
Fenced farm 4 1 3 2
Breeding type Communal bull /borrows bull 8 7 12 3 3.4 12
Artificial insemination 1 3 1 3
Training of farmers Had trainings 12 6 14 5
No trainings 3 7 8 1 4.7 0.4
New animals to the farm New animals 12 14 21 5
No new animals 2 0 1 1 0.0 4.2
Previous abortions Had previous abortions 6 4 8 2
No previous abortions 7 11 13 5 2.4 1.54
Disposal of aborted fetuses Given to dogs 5 4 5 4 1.25 0.42
Thrown in bush 2 2 3 1
Chapter 5
Discussion
The study confirmed the presence of Leptospira and Brucella antibodies in cattle farms of Zirobwe meat producers’ cooperative society. The seroprevalence of Brucella antibodies was 23% by Brucella immunocomb test kit?? and 49 % for leptospira by leptospira immune comb test kit??, the seroprevalence of brucella antibodies was higher than the one determined by Nizeyimana et al, 2012 of 5%, in the same area probably due to the limited sample size and scope in the former study and ‘also decline in animal husbandry practices as well as government disease control programs clearly shown by the increasing levels of all other livestock diseases’. The study findings are however consistent with those reported by Kungu et al 2010 48.9% and 46.8% in Amuru and Gulu districts respectively justifying the overall decline in brucellosis control measures in the country. The findings again contrast the findings by Mwebe et al, 2010), of 11 and 17% in Luwero and Nakasongola districts respectively and other surveys in various parts of Uganda by researchers who reported varying seroprevalence: Nakavuma (1994) reported 14.7% in Central Region, Kagumba & Nandoka (1978) reported 5% in North Eastern Uganda and Ndyabahinduka (1978) that reported 18.1% in East Ankole and Central Region. The general explanation for the differences in the reported seroprevalence could be due to temporal, spatial, sampling and assay differences , highlighting the fact that there is a problem of brucellosis distribution in many parts of Uganda possibly due to variation in time to time control measures that are not coordinated, animal husbandry differences and beef sector developments as was in Uganda meat export development program in Uganda and Zirobwe meat producers cooperative society in particular(UMED ,2008).
The seroprevalence of Leptospira antibodies was higher than that of Brucella antibodies at 49 % by the leptospira immunocomb test kit??, a finding that is consistent with the study by Artherstone et al, 2014 in Uganda that indicated a high sero-positivity against Leptospira serovars in buffalos (42%) and cattle (29%). The status quo is worrying given the fact that this is the second study that too does not comprehensively study leptospirosis including its zoonotic effects. The seroprevalence of leptospira antibodies in the cattle farms poses a threat to animal health workers including farmers, abattoir workers and veterinarians because it’s a zoonotic and that There is a likelihood that the sero prevalence of leptospirosis in humans could be alarming partly because farmers have not been aware of leptospirosis as per the questionnaire findings that revealed that none of the farmers interviewed had heard about it, all that was known was brucellosis as the only cause of abortion in cattle. There could be a similar situation as in New Zealand where the seroprevalence was up to 17-19 % in abattoir workers ((Dreyfus et al, 2015) The implication for this in Uganda where the disease level in humans is not known even in people at high risk leaves room for urgent studies’. This could be explained by the fact that brucellosis in humans has ably been identified, diagnosed and awareness created amongst people on how it spreads and thus ‘the publicity could be due to the effects in humans not the animals’. The high level of leptospira antibodies in cattle could be explained by the ubiquitous nature and wide distribution of leptospira organisms coupled with the cattle grazing behavior (grazers) that led to constant exposure to the organisms from pastures contaminated by urine of infected anima and the environment (Justine et al, 2015)
Brucella immunocomb test kit??(Dot ELISA) (23%) detected lower number of Brucella positive samples rose Bengal plate test (32% ) that is in agreement with previous findings by Nizeyimana et al , 2012 of higher sensitivity than ELISA possibly due to cross reaction from other bacterial infections like Yersinia enterocolitica and Salmonella whose smooth lipopolysaccharides, thereby confounding the interpretation of RBPT results in addition to the failure to distinguish between vaccinated and infected animals ( Poester et al, 20111, Radostits et al. 2000). However the results by both tests had aslight agreement as indicated by a kappa index of 0.042, 95 % Confidence interval (50% level of agreement).
Risk factors:
The key risk factors observed included the method of breeding, cattle grazing systems, farm biosecurity systems , history of abortions at farms and disposal of aborted materials as potential contributors for the observed seroprevalence of both Brucella and Leptospira antibodies in Zirobwe meat producers cooperative union.
All the sampled farms in Zirobwe had no history of vaccination against leptospira and or Brucella done in the 28 farms studied and as such the antibodies detected could be due to natural infections as opposed to vaccination because neither the vaccination nor the source of leptospira vaccine has been done and is available in Uganda and so is Brucella vaccines save for the cost.
Disposal of aborted fetuses by farmers was a big challenge as was found out in the questionnaire where a total of 5 farms that reported abortions some were feeding the fetuses to dogs while others were throwing them in bushes, the reason for this was that farmers did not know why and how to dispose the fetuses and as such did as they wished’. All the abortions observed occurred during the 2nd and 3rd trimester as described by the farmers that aborted fetuses had hairs and some had the primary teeth.
It is important to note that throwing the fetuses could be a potential risk factor to occurrence of both leptospirosis and brucellosis in cattle herds (Odds ratio, 0.42, 1.25 respectively) while feeding the fetuses to dogs could equally be a danger to them as found out in a study in Nigeria where up to 30.6 % seroprevalence for Brucella species (abortus and canis) in dogs was recorded (Cadmus et al, 2011). Unfortunately no parallel study is being done in Uganda in dogs from the farming communities to determine the seroprevalence including studying any possible zoonotic effects. Feeding dogs and throwing in the bush all did not have a big contribution to the disease (OR 0.42) probably because the organisms are very sensitive to ultra violet rays from the sun (Stamm and Charon, 1988).
As briefly described in the methodology, the Uganda meat export development program that led to the creation of Uganda meat producers’ cooperative union where Zirobwe meat producers’ cooperative society a cattle producers group was formed with an aim of improving the quality of breeds of cattle kept to improve the productivity of farms changed the breeding system from the traditional breeds to exotic crosses both the beef and dairy because they had become aware of the attributes, farmers started buying bulls and females animals from all over the region for breeding but all this did not take into consideration the animal health status save for the inefficient biosecurity systems at farms to isolate new animals before admission to the herds partly because the farmers were not aware and the government capacity to do this was limited that could be responsible for the current status quo. This was evidenced by the questionnaire findings where up to 93% (26 farmers) confessed that they had admitted new animals to the farms at least for the last two years without bothering about screening and or isolation. The reason was that farmers were not so sure of the likely impacts of this.
Bringing new animals to the farms contributed significantly to the seroprevalence of leptospirosis as opposed to brucellosis (Odds ratio, 4.2, 0.0 respectively). Farms that were using artificial insemination as a method of breeding were less likely to have brucellosis and leptospirosis compared to those that were using a communal bull/borrow a bull (Odds ratio for brucellosis 3.4, and 12 for leptospirosis). Communal and tethered animals were at higher risk of having leptospirosis (Odds ratio 1.25) than brucellosis (Odds ratio, 0.35) than animals in fenced farms. This was possibly because there was always contact between animals of different health status increasing exposure.
In general; farms with brucellosis were at high risk of having leptospirosis which indicated a general problem with farm management and animal husbandry Brucella infected farms. The fact fenced farms that were using artificial insemination were at low risk of both Brucella and Leptospira infection suggest that controlled production with use of artificial insemination and strict biosecurity systems and methods could control both brucellosis and leptospirosis infections at farms.
Chapter 6:
Conclusion
1. The study findings of high seroprevalence of leptospira antibodies present a serious implication on the public health of the cattle keeping communities and animal health workers given its zoonotic potential and significance calling for the urgent surveillance studies in both cattle nationwide and humans to determine the prevalence.
2. The infectious causes of cattle abortions in Uganda have been confounded by brucellosis and the study has found out that other infectious causes are higher than brucellosis sand are possibly responsible for the ever increasing complaints of abortions.
3. The sero prevalence of brucellosis in cattle has continued to rise, increased awareness notwithstanding
4. The seroprevalence of leptospirosis in cattle presents an eminent danger to cattle farmers, workers and herdsmen including abattoir workers.
5. There is limited awareness on leptospirosis in cattle, effects in cattle and its zoonotic effects.
Chapter Seven
Recommendations
1. Based on the study findings, a comprehensive study on the infectious causes of abortion in cattle in Uganda is recommended
2. A study on the prevalence of brucellosis in canines in cattle keeping communities with elucidation on the epidemiology and possible zoonotic consequences in Uganda.
3. A study in human beings in cattle keeping communities, abattoir workers to determine the level of leptospirosis in people.
4. A national awareness program on the brucellosis and leptospirosis control program with emphasis on biosecurity, disposal of aborted fetuses and proper breeding methods.
5. A Comprehensive control program by vaccination of both brucellosis and leptospirosis following a national diseases survey and mapping.
6. As a starting point, the Uganda meat producers cooperative union and Uganda meat export development program conducts similar studies in other cooperatives since it is easy to do these studies in organized groups and cheaper . These diseases are serious setbacks to meat sector improvement.
7. Increase availability of leptospirosis testing kits in both humans and animals for continued research.
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Appendix 1: QUESTIONAIRE
Introduction:
This to introduce Dr. Gerald Nizeyimana who is a student of International Animal Health at Edinburgh University. He is undertaking a research study on Value chain study of the risks, causes and control of Bovine brucellosis in Zirobwe Beef Producers Cooperative Society in Uganda. This questionnaire therefore, is designed purely to generate academic information although the work coming from this research may be published, all names will be anonymized and names kept confidential. Any assistance rendered to him will be highly appreciated.
Bovine Brucellosis Investigation
a) Contact details of the personnel collecting the sample
Name Address
Phone number Email
b) Location of the epidemiological unit
District Name of Unit , Name of the farmer and contact details
Sub county Unique unit ID
Village Latitude / longitude
Type of unit Date of investigation
c) Demographics of the epidemiological unit
Number of Local cattle Number of dairy animals Number of beef animals Number of Goats
Cattle less than 6 months
>6 months old)
d) Sample Information: Please fill all the bullets.
Unique unit ID
Date Sample Collected
Unique sample ID
Animal ID
Unit /Farm Name
Species
Breed
Sex
Age
e) History and details of the epidemiological unit ( Please Tick and fill in as appropriate)
1.
Have you ever had abortions in cattle on this Farm? YES
NO
2. If yes, what stage? ( Stage will depend on the description of the abortus) 1st Trimester
2nd Trimester
3rd Trimester
3.
How many have you had since the year begun?
4.
How is the number of abortions this year compared to previous years?
5. In what breeds are the abortions common?
6. What do you think are the causes of this abortion?
7. Have you heard about brucellosis and
Leptospirosis? YES
NO
8.
Have you confirmed these diseases at your Farm? YES
NO
9. If yes, how have you controlled them?
10.
Do you have ready access to both
Leptospirosis and brucellosis vaccines? YES
NO
11.
If yes, have you vaccinated the animals
YES
NO
12. If yes to 11, what vaccine did you use
13. When did you vaccinate your cattle Last 6 months
Last 1 year
2 years ago
14.
Did any vaccinated animals abort? YES
NO
15. Have you brought in new animals in the last 6 months Yes
No
Do you isolate new animals to the farm Yes
No
16. If yes to 15, How many and after how long?
17 Have you had any training in animal husbandry? Yes
No
18 If yes to 17, how many and mention at least one
19 Who provided the training?
20
How do you breed your animals
By Artificial Insemination
Natural mating by a bull at the farm
Natural Mating by neighbor’s bull
21 Have grazed in common pastures since the
year begun Yes
No
22 If yes, why and when?
23 How do you dispose of the aborted material
Given to dogs
Thrown in bush
None
21.
Do you keep records at the farm Yes
No
22.
If yes, what records are kept?
t in here…