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Theileria parva epidemics: a case study in eastern Zambia
Veterinary Parasitology 107 (2002) 51–63
Theileria parva epidemics: a case study in eastern Zambia M. Billiouw a , J. Vercruysse b , T. Marcotty a , N. Speybroeck a , G. Chaka c , D. Berkvens a,∗ a
b
Department of Animal Health, Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerpen, Belgium Department of Parasitology, Faculty of Veterinary Medicine, Salisburylaan 133, B-9820 Merelbeke, Belgium c Provincial Veterinary Office, P.O. Box 510155, Chipata, Zambia Received 19 October 2001; received in revised form 26 February 2002; accepted 2 April 2002
Abstract This paper presents the results of the follow-up of three sentinel herds between 1994 and 2000 during an East Coast fever (ECF) epidemic in eastern Zambia. The animals of the sentinel herds were closely monitored clinically and serologically together with detailed Rhipicephalus appendiculatus counts. Peaks of disease incidence occurred in the rainy season (December–February) and the dry months of May–July with nymph-to-adult tick transmission dominating the infection dynamics. A second wave of adult R. appendiculatus at the start of the dry season is essential for the occurrence of a full-blown epidemic while the size of the susceptible cattle population acts as a most important limiting factor. The majority of adult cattle of the sentinel herds became infected less than 2 years after the introduction of the disease. The median age at first contact for calves born towards the end of the study (1999) was about 6 months. The case-fatality ratio (including sub-clinical cases) is estimated at 60%. It is argued that part of the so-called ‘natural mortality’ is actually due to ECF and that ECF occurrence and mortality are systematically underestimated. The direct financial cost of the epidemic, based on loss of animals and cost of treatment only and calculated over 4 years running, is estimated at about US$ 6 per year per animal at risk. The value of the traditional seroprevalence survey as a tool for monitoring ECF epidemiology is put in question and the prevalence of maternal antibodies in new-born calves, reflecting the immune status of the dam population, is introduced as an alternative. It is demonstrated that an efficient immunisation campaign should concentrate its efforts in the period of low adult R. appendiculatus abundance (July–October). © 2002 Elsevier Science B.V. All rights reserved. Keywords: Theileria parva; East coast fever; Epidemiology
∗ Corresponding author. Tel.: +32-3-2476393; fax: +32-3-2476268. E-mail address: [email protected] (D. Berkvens).
0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 2 ) 0 0 0 8 9 - 4
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1. Introduction Epidemics of East Coast fever (ECF) in cattle, defined as occurrence of Theileria parva infections in previously or recently unaffected areas (Norval et al., 1992), are renowned for their devastating impact on the cattle population. These epidemics usually strike with high case fatality, causing major cattle loss and significant social and economic distress to the individual farmer. Extinction of entire herds is not uncommon especially when no measures are taken to mitigate the deadly impact of the outbreak (Nambota et al., 1994). Often, chemotherapy is the only remedy. The use of acaricides will, if anything, only delay the inevitable lethal challenge (Berkvens, 1991). Immunisation by means of the infection and treatment method creates a reservoir in both cattle and vector, Rhipicephalus appendiculatus (Bishop et al., 1992; Koch et al., 1992) and its use as a control measure is therefore, not without danger in a newly infected area with a fully susceptible cattle population if only calves are immunised, as is traditionally done. The diagnosis of an ECF epidemic is usually straightforward when the link with the increase in R. appendiculatus abundance can be made, but unawareness often leads to misdiagnosis when the disease is new. More commonly the diagnosis is missed in individual cases, which leads to an underestimation of the importance of the infection (own unpublished observations). The spread of the infection is mainly through cattle movement, e.g. oxen working in neighbouring or distant villages and feeding making migration for distances of 15 km or more not exceptional (Billiouw et al., 1999). Monitoring the evolution of the epidemic towards a state of endemicity is routinely done by listing the numbers of cases and deaths, in function of the age category and by documenting the trends in seroprevalence (Perry et al., 1992). This paper gives a detailed account of an ECF epidemic, its impact at herd level, its natural epidemiological evolution and features related to transmission dynamics in a traditional herd in the eastern province of Zambia. The study provides valuable information for decision making in terms of ECF disease control. Prevalence, incidence and age at first contact are evaluated for their use in measuring ECF frequency. Finally, the proportion of new-born calves with maternal antibodies against T. parva is introduced as a potential alternative to the traditional seroprevalence in adult cattle when monitoring the epidemiological status of theileriosis.
2. Materials and methods The study was carried out between 1994 and 2000 at Mtandaza (14◦ 25 S, 31◦ 54 E) in the Eastern Province of Zambia, involving three sentinel herds. This paper focuses on the results from the sentinel herd study at Mayela, which started before T. parva was introduced into the herd. Results obtained from the follow-up of two neighbouring sentinel herds at Lupenga and Mkambi, both of which were already infected before the start of the study, were used to provide background information where necessary. Weekly, R. appendiculatus were counted in both ears. Also weekly, all calves were examined clinically and blood sampled for an indirect fluorescence antibody test (IFAT; Burridge and Kimber, 1972) in vitro cultured T. parva (Katete) infected lymphoblasts were used as antigen to detect antibodies in diluted plasma or serum. Anti-bovine fluorescein-labelled conjugate
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was applied after the antigen–antibody binding, producing fluorescent schizonts in positive samples. The adult animals which made up the herd at the start of the study, were inspected on a weekly basis while tick counts and blood sampling for IFAT were carried out monthly. Clinical ECF was diagnosed when symptoms of the disease were confirmed by the demonstration of T. parva schizonts in lymphnode smears or when the clinical episode was followed by seroconversion. The only diagnostic criterion for sub-clinical ECF was seroconversion. Seroconversion was defined as the event of three consecutive seropositive samples. The cut-off titre for seropositivity was taken as >1/40 to allow distinction between a primary infection and maternal antibodies. Presence of maternal antibodies was defined as seropositivity at cut-off 1/40 in the first 2 months of age and is referred to as maternal immunity. For seroprevalence estimates in the adult animals the cut-off was 1/40. Statistical analysis was carried out in Stata (StataCorp, 2001). The number of calves born each year were compared using a Poisson model and equiprobability between groups evaluated with χ 2 -tests (Hardin and Hilbe, 2001). Equivalence of age at first contact functions was tested by means of the log likelihood ratio test (χ 2 ) using a Cox proportional hazard model, each time verifying the proportional hazard assumption (Hosmer and Lemeshow, 1999). Case fatality ratios were compared between groups using logistic regression and tested for equivalence by means of a χ 2 -test (Hardin and Hilbe, 2001).
3. Results 3.1. General Fig. 1 plots per month the herd size, the cumulative number of new-born calves, the cumulative off-take of animals through transfer, sale and slaughter, the cumulative number of cattle deaths due to ECF and the cumulative number of cattle deaths due to other causes. The original herd size was 39. ECF was introduced in the herd in January 1995 but few cases occurred during the first 2 years. The cases were treated and consequently the ECF mortality was minimal. The owner kept the size of the herd around 40 by taking off the surplus of cattle. During this quasi ECF-free period there were very few reports of non-ECF mortality. The maximum herd size in this period was 48 (early 1996). The ECF epidemic struck first in the cold dry season (May–July) of 1996. Three more waves of ECF challenge followed coinciding with the adult (rainy season, December–March) and the adult/nymph (cold dry season) waves of R. appendiculatus. Due to a shortage of suitable drugs a total of 16 cattle had died of ECF by the end of the rainy season in March 1998. Coinciding with the ECF epidemic, the incidence of diagnosis of non-ECF mortality peaked as well. A minimum herd size of 30 animals was recorded in February–March 1998. The herd recovered to its original size in about 4 months. Off-take was put on hold for the period of 1 year while no ECF cases were recorded throughout 1998. Thereafter, the incidence of ECF cases was moderate until a second epidemic was recorded with peaks in the rainy season of 1999–2000. The birth rate remained quite stable throughout the study period and the number of reproductive cows did not fluctuate much compared to the other categories.
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Fig. 1. Herd statistics during the ECF epidemic at Mayela (A) and Lupenga (B) (- - -), herd size; (—), cumulative ), ECF mortality; ( ), natural mortality. births; (– – –), sales, slaughters, transfers; (
3.2. Economic impact of the epidemic The size of the cow population at the end of each study year ranged from 14 to 19 (median 15). Sixty-six calves were born in this period (an average of 9.5 per year) corresponding to a calving rate of about 60%. The number of calves born per year remained constant during the study (X2 = 6.4, d.f. = 5, P = 0.3). Seasonal variation in calving was statistically
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significant (X 2 = 24, d.f. = 11, P = 0.012). About 70% of the calves were born during the dry season with a peak in July (18%). The seasonal pattern of calving remained constant throughout the study. Part of the economic impact of the epidemic can be derived directly from the herd size fluctuations demonstrated in Fig. 1. During the first (almost ECF-free) 2 years, the potential annual off-take was on average seven animals (17% of the herd). This matches the number of calves born minus the non-ECF mortality which was estimated at about 10%. After the introduction of ECF the off-take decreased to about three animals per year (8%). The average value of an animal in the area was US$ 100 (own observations and interviews of cattle owners). Taking into account a 50% salvage price the loss due to reduction in off-take amounts to US$ 200 per year. The cost of ECF treatment in the study (21 treatments over 4 years) was on average US$ 50 per year. The direct cost of the epidemic is thus estimated at US$ 250 per year for a herd of 40 animals or US$ 6.25 per year per animal at risk. 3.3. Loss to follow-up and non-ECF mortality The size of the sentinel herd at the end of the study was 46. This includes 14 animals of the original herd and 32 animals born during the study. A total of 59 animals were lost to follow up. As for the original herd, 25 animals dropped out of the study for the following reasons: ECF (5); slaughtered (5); sold (3); transferred (7); non-ECF (5, tick-borne disease, old age, poison, lion kill, complicated calving). Out of 66 calves born during the study, 34 disappeared from the herd: ECF (19); slaughtered (1); sold (7); transferred (4); non-ECF (3, malnutrition, accident, missing). The cause of death of eight animals was allegedly non-ECF. However, in three of these ECF cannot be excluded as cause or contributing factor: one cow died after aborting but was treated 1 month before for ECF; one calf of 3 months old died due to malnutrition but the dam was treated for ECF about 3 months before; one calf went missing and was found dead later, but it had seroconverted shortly before disappearing. Slaughters are another source of confounding since two of the six slaughtered animals (33%) had clinical ECF. 3.4. ECF in the original herd There was no indication that any of the 39 animals of the original herd had been infected with T. parva before the study. All were seronegative on IFAT from August to December 1994. The first animal to seroconvert was also the first confirmed clinical case diagnosed in January 95. Excluding 11 animals that were censored and two that never seroconverted, all cattle (26) had been infected by September 1997 (Kaplan–Meier plot in Fig. 2). In fact, three waves of epidemic ECF challenge infected the majority of the herd. Twenty animals showed clinical symptoms. Six were left untreated of which five died. The others survived after treatment. Six sub-clinical cases were diagnosed by means of IFAT (Table 1). 3.5. ECF in the enrolled calves A total of forty T. parva infections were diagnosed in the 66 calves enrolled during the study: 19 lethal infections, seven clinical infections that were successfully treated, two cases recovered without treatment and twelve sub-clinical infections (Table 2). Ten calves were
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Fig. 2. ECF-free survival of adult cattle present in the herd at Mayela before the introduction of the disease. Table 1 Number of ECF cases per year (adult cattle)
Sub-clinical Clinicala Lethal Censored a
1994
1995
1996
1997
1998
1999
2000
Total
0 0 0 4
0 3 1 4
4 3 3 2
2 9 1 1
0 0 0 0
0 0 0 0
0 0 0 0
6 15 5 11
Excluding lethal cases.
censored. Out of the 34 calves currently in the herd, 16 are susceptible, among them all 13 born in 2000. 3.6. Age at first contact The Kaplan–Meier age at first contact curves (or ECF-free survivor curves) which are displayed in Fig. 3, demonstrate the decreasing trend in age at first contact with T. parva Table 2 Number of ECF cases per year (calves)
Subclinicala Clinicala Lethal Censored a
1995
1996
1997
1998
1999
2000
Total
0 0 0 1
2 1 1 3
6 4 7 2
0 0 3 0
4 2 4 3
2 0 4 1
14 7 19 10
Excluding lethal cases
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Fig. 3. ECF-free survival of calves at Mayela per year of birth.
for calves born from 1994 to 1999. This trend was statistically significant (X 2 = 8.1; P = 0.004). The median was 28 months for calves born in 1994, 12 months for those born in 1996 and just over 6 months for those born in 1997. For the calves born in 1998 the median age at first contact estimate increased but it dropped again to 6 months for those born in 1999. Almost all calves born in 1997 and 1999 became infected below the age of 1 year. 3.7. Incidence The monthly ECF incidence, i.e. the risk for a susceptible calf to become infected during a particular month, is shown in Fig. 4. The resulting epidemic curve indicates that waves of ECF challenge mainly coincide with peaks of adult R. appendiculatus abundance. These peaks occur in the rainy season (December–March) and in the cold dry season (May–July). Minor peaks of transmission are observed in September. No accurate information on nymphal abundance was obtained but their presence was recorded in May–June and August–September. The incidence during the first major epidemic increased from 5% in 1996, to 20% during the first adult wave of R. appendiculatus in 1997 and to 50% during the second adult wave. In January 1998 all three remaining susceptible calves were infected. More than 10 calves were born later that year, mainly in the second half, but ECF incidence remained zero throughout. A new period of transmission started in early 1999. Seasonal incidence steadily increased with each wave of R. appendiculatus adults and reached peaks of 30% in the 2000 rainy season, infecting most susceptible calves. As in 1998, this announced a long period of zero transmission during which the susceptible population increased. A similar pattern was also seen at Mkambi and Lupenga where virtually all calves born during the cold and hot dry seasons become infected during two waves of ECF challenge (Fig. 5b and c). The incidence during the cold season is usually higher than during the rainy season.
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Fig. 4. ECF hazard in calves compared to susceptible population and vector burden at Mayela (A); Mkambi (B) and Lupenga (C). ( ), number of susceptible calves; ( ), average number of R. appendiculatus per calf; ( ), hazard of T. parva contact in calves; ( ), window for immunisation.
3.8. Lethality of T. parva infection Out of a total of 66 T. parva infections (both adults and calves) 24 were fatal. The minimum lethality estimate is thus 24 out of 66 or 36%. A total of 21 survived without treatment which yields a maximum lethality of 45 out of 66 or 68%. Since it may be safely assumed that the majority of the clinical cases would have died without treatment the lethality is estimated to be around 60%. Case-fatality ratios were equivalent in the different years (X 2 = 6.7, d.f. = 4, P = 0.15).
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Fig. 5. Seroprevalence in adult cattle at Mayela, compared to ECF incidence in calves: ( ), seroprevalence in adults animals; ( ), T. parva incidence in calves.
3.9. IFAT results The seroprevalence patterns in animals older than 2 years are plotted in Fig. 5, together with ECF incidence in calves. The seroprevalence increased together with disease incidence during the cycles of transmission. Outside transmission episodes, the seroprevalence decreased steadily (e.g. throughout 1998) and eventually drops to zero (e.g. May–December 2000). The maternal immunity prevalence in new-born calves increased from 0% for years of birth 1994–1996, 55% for 1997, 75% for 1998 and 70% for the calves born in 1999 and 2000. The sensitivity of maternal immunity (the proportion of calves, born from immune cows, that is seropositive) was 72% (28 out of 39; 95% c.i. 55–84%). The specificity (the proportion of calves, born from na¨ıve cows, that does not have maternal antibodies) was 96% (22 out of 23; 95% c.i. 77–99%). The single false positive case was a calf born from one of the two animals, from the original herd, which never seroconverted.
4. Discussion 4.1. Transmission dynamics In this study of an ECF epidemic, the incidence of T. parva infection recurs in cycles during which the intensity of transmission increases with each wave of transmitting tick instars until no susceptible cattle population is left. Thereafter, the susceptible calf population is replaced during a period of low challenge. Thus at herd level, the size of the susceptible
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population acts as the most important limiting factor for the start of an epidemic. Nymph to adult transmission appears to be the main mode of transmission in eastern Zambia and clinical cases are the main source of infection with the carrier population merely acting as a reservoir of the infectious parasite. Observations in favour of this hypothesis are the increasing trend of the incidence during cycles of transmission, the low seroprevalence observed in adult animals during periods of low disease incidence in calves and the high case-fatality ratios observed throughout the study. The fact that the nymphal wave in September was never responsible for initiating a new cycle of disease transmission, even in the presence of a substantial susceptible population, indicates that larvae picking up from the carrier population played a minor role in the transmission dynamics (Ochanda et al., 1996). The second adult wave appears then again to be crucial in the regeneration of full-blown transmission (Billiouw et al., 1999). Similar but faster transmission cycles were observed in the more established ECF areas, e.g. Mkambi and Lupenga in the later stages of the study, where the first adult wave typically infects a proportion of calves and the second wave infects the remaining larger proportion. Because of the fact that the size of susceptible calf population regulates the transmission pattern, the seasonal incidence in one herd maybe zero while it reaches high levels in neighbouring herds. It is in these situations that the age at first contact statistic proves to be a more robust epidemiological estimator than incidence. 4.2. Age at first contact and incidence Incidence and prevalence statistics have been widely used to express the frequency of clinical events. Disease prevalence has a limited value in measuring ECF abundance because of the short duration and the high case fatality of the infection. Monthly incidence estimates give snapshots of the ECF challenge but the interpretation of the statistic is often troubled by the interactions between the size of the susceptible population, the seasonality of calving and the seasonality of T. parva challenge. The age at first contact statistic is inherently less sensitive to these variables and provides information on both past and current ECF situation, but this information may be delayed, as the following examples explain. In 1998 at Mayela, the ECF incidence could not be estimated or was zero for the whole year (except January) because no or few susceptible calves were remaining when ECF challenge was high. However, the interpretation of the age at first contact curve remained straightforward, indicating that all calves born that year were ECF-free up to the age of 6 months and that 50% were infected before the age of 15 months. High incidence figures were recorded in 1997 but the high age at first contact of the calves that became infected indicates that this challenge was new (i.e. epidemic). A combination of both age at first contact and incidence provides a comprehensive picture of the ECF epidemiology. The main drawback is that both statistics can only be estimated in longitudinal studies through close monitoring of a cattle population. 4.3. Endemic stability Endemic stability in the case of T. parva may be defined as the epidemiological state where all calves become infected before the age of 6 months and clinical disease is rare (Moll et al., 1986). This implies that all adult animals are immune and are being re-infected
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at least twice per year (calves must be infected once per 6 months and adult animals carry more ticks). Among the factors contributing to the state of endemic stability are a low innate susceptibility of the cattle, a continuous exposure to all instars of R. appendiculatus and sufficient infection prevalence of T. parva in R. appendiculatus (Perry et al., 1992; Norval et al., 1992). Although a second generation of R. appendiculatus (adults and nymphs) ensures a continuous presence of tick instars transmitting T. parva to cattle in eastern Zambia, tick abundance and consequently the ECF challenge is strongly seasonal (Berkvens et al., 1998). Median age at first contact estimates may drop to levels close to those observed in endemically stable situations but case-fatality ratios remain invariably high. The low larva-to-nymph transmission from carrier animals (Norval et al., 1992) and the important highly infected second generation adults may explain the high case fatality and may therefore, be the major constraints in attaining endemic stability in the near future. 4.4. Natural mortality This study suggests that a large proportion of what is diagnosed in the field as non-ECF mortality is still due, directly or indirectly, to ECF. Also slaughter as cause of death is often related to ECF. Thus, even in our closely monitored herds, observation biases distort the estimation of ECF frequency. Retrospective data obtained from routine field reports and surveys, which depend strongly on the farmers’ recollection of events, are therefore, likely to be unreliable. ‘Broad spectrum’ surveys covering a large number of items are in this context even more vulnerable and may lead to wrong decision making. 4.5. Seroprevalence and maternal immunity Seroprevalence is currently the method of choice for monitoring ECF epidemiology (Perry et al., 1985). However, the results from the present study indicate that seasonal or annual large-scale serological surveys may face quite a number of scientific and logistical problems. The organisation of these surveys is labour intensive and expensive. The blood sampling is generally done in older animals, increasing the risk of selection bias and fraud (i.e. dividing blood from one animal over several test tubes). The large sample sizes make the processing of the samples, from storage to analysis, prone to negligence and error. Seroconversion after a booster challenge appears to be immediate and short-lived so that when estimating trends in seroprevalence, the timing of surveys becomes critical in areas with a high degree of seasonality in challenge. Furthermore, it is demonstrated in this study that seroprevalence trends merely reflect trends in disease incidence rather than the build up of immunity in the adult cattle population. On the other hand, estimation of the trend in maternal antibody prevalence gives consistent and reliable results. The collection of data can be done on a continuous basis from calves born in monitored herds. 4.6. Disease control The present study indicates that 2–3 years after the start of a major epidemic, the majority of the adult animals are immune and calf immunisations may in principle be considered. An earlier start would endanger the adult population by increasing the challenge of ECF at
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a time when the herd is particularly vulnerable, often even at risk of extinction. Possible constraints are the definition of the area and population at risk and the level of knowledge of the ECF epidemiology and vector ecology in that area. The study further demonstrates that the period June–November, when high calving rates and low T. parva transmission prevail, offers a suitable window for immunisations (Fig. 4A–C). The farmers should be recommended to keep their calves inside a pen for up to 6 months (not an uncommon practice in eastern Zambia). Temporary acaricide treatment may be an alternative. These management practices could minimise ECF mortality during the rainy season and maximise the efficiency of well-timed immunisation efforts. 4.7. Conclusions and recommendations This paper confirms that longitudinal studies are a powerful tool to evaluate the epidemiological status of ECF in a region. The combination of incidence and age at first contact provides necessary and sufficient information to allow accurate assessment of the epidemiological status of the disease, something that is not possible using either parameter on its own, nor by relying on transversal surveys. Although the economic importance of ECF in Zambia is fully acknowledged it is shown here that the frequency of occurrence of the disease may still be underestimated. The transmission dynamics in epidemic areas of ECF in eastern Zambia is dominated by nymph-to-adult transmission originating from clinical cases. The size of the susceptible population acts as the main limiting factor. Endemic stability is unlikely until larvae become more important in picking up the infection from carriers. This interaction is probably crucial to ensure a more constant and less lethal disease pressure. Infection and treatment immunisation remains the disease control method of choice, where it is applicable, but this study recommends that extra efforts should be done to immunise in the window of low adult tick abundance between July and October and to protect the calves born from November to May against ticks. The seroprevalence in adult cattle, measured to monitor ECF from its epidemic status towards endemicity, appears to be less reliable than expected. The presence of maternal antibodies in new-born calves, reflecting the immune status of the cow population, is introduced as an alternative but more studies are needed to confirm its potential value. References Berkvens, D.L., 1991. Re-assessment of tick control after immunisation against east coast fever in the eastern province of Zambia. Annales de la Société belge de la Médecine tropicale 71, 87–94. Berkvens, D.L., Geysen, D.M., Chaka, G., Madder, M., Brandt, J.R.A., 1998. A survey of the ixodid ticks (Acari: Ixodidae) parasitising cattle in the eastern province of Zambia. Med. Veterinary Entomol. 12, 234–240. Billiouw, M., Mataa, L., Marcotty, T., Chaka, G., Brandt, J., Berkvens, D., 1999. The current epidemiological status of bovine theileriosis in eastern Zambia. Trop. Med. Int. Health 4, A28–A33. Bishop, R., Sohanpal, B., Kariuki, D.P., Young, A.S., Nene, V., Baylis, H., Allsopp, B.A., Spooner, P.R., Dolan, T.T., Morzaria, S.P., 1992. Detection of a carrier state in Theileria parva-infected cattle by the polymerase chain reaction. Parasitology 104, 215–232. Burridge, M.J., Kimber, C.D., 1972. The indirect fluorescent antibody test for experimental east coast fever (Theileria parva infection of cattle): evaluation of a cell culture schizont antigen. Res. Vet. Sci. 13, 451–455. Hardin, J., Hilbe, J., 2001. Generalised Linear Models and Extensions. Stata Press, College Station TX, p. 245.
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Hosmer Jr., D.W., Lemeshow, S., 1999. Applied Survival Analysis: Regression Modelling of Time to Event Data. Wiley, New York, p. 386. Koch, H.T., Norval, R.A.I., Ocama, J.G.R., Munatswa, F.C., 1992. A study on the Theileria parva bovis carrier state. Prev. Vet. Med. 12, 197–203. Moll, G., Lohding, A., Young, A.S., Leitch, B.L., 1986. Epidemiology of theileriosis in calves in an endemic area of Kenya. Vet. Parasitol. 19, 255–273. Nambota, A., Samui, K., Sugimoto, C., Kakuta, T., Onuma, M., 1994. Theileriosis in Zambia: etiology epidemiology and control measures. Jpn. J. Vet. Res. 42, 1–18. Norval, R.A.I., Perry, B.D., Young, A.S., 1992. The Epidemiology of Theileriosis in Africa. Academic Press, London, p. 481. Ochanda, H., Young, A.S., Wells, C., Medley, G.F., Perry, B.D., 1996. Comparison of the transmission of Theileria parva between different instars of Rhipicephalus appendiculatus. Parasitology 113, 243–253. Perry, B.D., Musisi, R.G., Pegram, R.G., Schels, H.F., 1985. Assessment of Enzootic Stability to Tickborne Diseases. World animal review, October–December 1985, pp. 24–32. Perry, B.D., Deem, S.L., Medley, G.F., Morzaria, S.P., Young, A.S., 1992. The ecology of Theileria Parva infections of cattle and the development of endemic stability. In: Munderloh, U.G., Kurtii, T.J. (Eds.), Proceedings of the First International Conference on Tick-Borne Pathogens. Minnesota, USA, 15–18 September 1992, pp. 290−296. StataCorp, 2001. Stata Statistical Software: Release 7.0., Stata Corporation, College Station, TX.
Theileria parva epidemics: a case study in eastern Zambia M. Billiouw a , J. Vercruysse b , T. Marcotty a , N. Speybroeck a , G. Chaka c , D. Berkvens a,∗ a
b
Department of Animal Health, Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerpen, Belgium Department of Parasitology, Faculty of Veterinary Medicine, Salisburylaan 133, B-9820 Merelbeke, Belgium c Provincial Veterinary Office, P.O. Box 510155, Chipata, Zambia Received 19 October 2001; received in revised form 26 February 2002; accepted 2 April 2002
Abstract This paper presents the results of the follow-up of three sentinel herds between 1994 and 2000 during an East Coast fever (ECF) epidemic in eastern Zambia. The animals of the sentinel herds were closely monitored clinically and serologically together with detailed Rhipicephalus appendiculatus counts. Peaks of disease incidence occurred in the rainy season (December–February) and the dry months of May–July with nymph-to-adult tick transmission dominating the infection dynamics. A second wave of adult R. appendiculatus at the start of the dry season is essential for the occurrence of a full-blown epidemic while the size of the susceptible cattle population acts as a most important limiting factor. The majority of adult cattle of the sentinel herds became infected less than 2 years after the introduction of the disease. The median age at first contact for calves born towards the end of the study (1999) was about 6 months. The case-fatality ratio (including sub-clinical cases) is estimated at 60%. It is argued that part of the so-called ‘natural mortality’ is actually due to ECF and that ECF occurrence and mortality are systematically underestimated. The direct financial cost of the epidemic, based on loss of animals and cost of treatment only and calculated over 4 years running, is estimated at about US$ 6 per year per animal at risk. The value of the traditional seroprevalence survey as a tool for monitoring ECF epidemiology is put in question and the prevalence of maternal antibodies in new-born calves, reflecting the immune status of the dam population, is introduced as an alternative. It is demonstrated that an efficient immunisation campaign should concentrate its efforts in the period of low adult R. appendiculatus abundance (July–October). © 2002 Elsevier Science B.V. All rights reserved. Keywords: Theileria parva; East coast fever; Epidemiology
∗ Corresponding author. Tel.: +32-3-2476393; fax: +32-3-2476268. E-mail address: [email protected] (D. Berkvens).
0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 2 ) 0 0 0 8 9 - 4
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1. Introduction Epidemics of East Coast fever (ECF) in cattle, defined as occurrence of Theileria parva infections in previously or recently unaffected areas (Norval et al., 1992), are renowned for their devastating impact on the cattle population. These epidemics usually strike with high case fatality, causing major cattle loss and significant social and economic distress to the individual farmer. Extinction of entire herds is not uncommon especially when no measures are taken to mitigate the deadly impact of the outbreak (Nambota et al., 1994). Often, chemotherapy is the only remedy. The use of acaricides will, if anything, only delay the inevitable lethal challenge (Berkvens, 1991). Immunisation by means of the infection and treatment method creates a reservoir in both cattle and vector, Rhipicephalus appendiculatus (Bishop et al., 1992; Koch et al., 1992) and its use as a control measure is therefore, not without danger in a newly infected area with a fully susceptible cattle population if only calves are immunised, as is traditionally done. The diagnosis of an ECF epidemic is usually straightforward when the link with the increase in R. appendiculatus abundance can be made, but unawareness often leads to misdiagnosis when the disease is new. More commonly the diagnosis is missed in individual cases, which leads to an underestimation of the importance of the infection (own unpublished observations). The spread of the infection is mainly through cattle movement, e.g. oxen working in neighbouring or distant villages and feeding making migration for distances of 15 km or more not exceptional (Billiouw et al., 1999). Monitoring the evolution of the epidemic towards a state of endemicity is routinely done by listing the numbers of cases and deaths, in function of the age category and by documenting the trends in seroprevalence (Perry et al., 1992). This paper gives a detailed account of an ECF epidemic, its impact at herd level, its natural epidemiological evolution and features related to transmission dynamics in a traditional herd in the eastern province of Zambia. The study provides valuable information for decision making in terms of ECF disease control. Prevalence, incidence and age at first contact are evaluated for their use in measuring ECF frequency. Finally, the proportion of new-born calves with maternal antibodies against T. parva is introduced as a potential alternative to the traditional seroprevalence in adult cattle when monitoring the epidemiological status of theileriosis.
2. Materials and methods The study was carried out between 1994 and 2000 at Mtandaza (14◦ 25 S, 31◦ 54 E) in the Eastern Province of Zambia, involving three sentinel herds. This paper focuses on the results from the sentinel herd study at Mayela, which started before T. parva was introduced into the herd. Results obtained from the follow-up of two neighbouring sentinel herds at Lupenga and Mkambi, both of which were already infected before the start of the study, were used to provide background information where necessary. Weekly, R. appendiculatus were counted in both ears. Also weekly, all calves were examined clinically and blood sampled for an indirect fluorescence antibody test (IFAT; Burridge and Kimber, 1972) in vitro cultured T. parva (Katete) infected lymphoblasts were used as antigen to detect antibodies in diluted plasma or serum. Anti-bovine fluorescein-labelled conjugate
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was applied after the antigen–antibody binding, producing fluorescent schizonts in positive samples. The adult animals which made up the herd at the start of the study, were inspected on a weekly basis while tick counts and blood sampling for IFAT were carried out monthly. Clinical ECF was diagnosed when symptoms of the disease were confirmed by the demonstration of T. parva schizonts in lymphnode smears or when the clinical episode was followed by seroconversion. The only diagnostic criterion for sub-clinical ECF was seroconversion. Seroconversion was defined as the event of three consecutive seropositive samples. The cut-off titre for seropositivity was taken as >1/40 to allow distinction between a primary infection and maternal antibodies. Presence of maternal antibodies was defined as seropositivity at cut-off 1/40 in the first 2 months of age and is referred to as maternal immunity. For seroprevalence estimates in the adult animals the cut-off was 1/40. Statistical analysis was carried out in Stata (StataCorp, 2001). The number of calves born each year were compared using a Poisson model and equiprobability between groups evaluated with χ 2 -tests (Hardin and Hilbe, 2001). Equivalence of age at first contact functions was tested by means of the log likelihood ratio test (χ 2 ) using a Cox proportional hazard model, each time verifying the proportional hazard assumption (Hosmer and Lemeshow, 1999). Case fatality ratios were compared between groups using logistic regression and tested for equivalence by means of a χ 2 -test (Hardin and Hilbe, 2001).
3. Results 3.1. General Fig. 1 plots per month the herd size, the cumulative number of new-born calves, the cumulative off-take of animals through transfer, sale and slaughter, the cumulative number of cattle deaths due to ECF and the cumulative number of cattle deaths due to other causes. The original herd size was 39. ECF was introduced in the herd in January 1995 but few cases occurred during the first 2 years. The cases were treated and consequently the ECF mortality was minimal. The owner kept the size of the herd around 40 by taking off the surplus of cattle. During this quasi ECF-free period there were very few reports of non-ECF mortality. The maximum herd size in this period was 48 (early 1996). The ECF epidemic struck first in the cold dry season (May–July) of 1996. Three more waves of ECF challenge followed coinciding with the adult (rainy season, December–March) and the adult/nymph (cold dry season) waves of R. appendiculatus. Due to a shortage of suitable drugs a total of 16 cattle had died of ECF by the end of the rainy season in March 1998. Coinciding with the ECF epidemic, the incidence of diagnosis of non-ECF mortality peaked as well. A minimum herd size of 30 animals was recorded in February–March 1998. The herd recovered to its original size in about 4 months. Off-take was put on hold for the period of 1 year while no ECF cases were recorded throughout 1998. Thereafter, the incidence of ECF cases was moderate until a second epidemic was recorded with peaks in the rainy season of 1999–2000. The birth rate remained quite stable throughout the study period and the number of reproductive cows did not fluctuate much compared to the other categories.
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Fig. 1. Herd statistics during the ECF epidemic at Mayela (A) and Lupenga (B) (- - -), herd size; (—), cumulative ), ECF mortality; ( ), natural mortality. births; (– – –), sales, slaughters, transfers; (
3.2. Economic impact of the epidemic The size of the cow population at the end of each study year ranged from 14 to 19 (median 15). Sixty-six calves were born in this period (an average of 9.5 per year) corresponding to a calving rate of about 60%. The number of calves born per year remained constant during the study (X2 = 6.4, d.f. = 5, P = 0.3). Seasonal variation in calving was statistically
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significant (X 2 = 24, d.f. = 11, P = 0.012). About 70% of the calves were born during the dry season with a peak in July (18%). The seasonal pattern of calving remained constant throughout the study. Part of the economic impact of the epidemic can be derived directly from the herd size fluctuations demonstrated in Fig. 1. During the first (almost ECF-free) 2 years, the potential annual off-take was on average seven animals (17% of the herd). This matches the number of calves born minus the non-ECF mortality which was estimated at about 10%. After the introduction of ECF the off-take decreased to about three animals per year (8%). The average value of an animal in the area was US$ 100 (own observations and interviews of cattle owners). Taking into account a 50% salvage price the loss due to reduction in off-take amounts to US$ 200 per year. The cost of ECF treatment in the study (21 treatments over 4 years) was on average US$ 50 per year. The direct cost of the epidemic is thus estimated at US$ 250 per year for a herd of 40 animals or US$ 6.25 per year per animal at risk. 3.3. Loss to follow-up and non-ECF mortality The size of the sentinel herd at the end of the study was 46. This includes 14 animals of the original herd and 32 animals born during the study. A total of 59 animals were lost to follow up. As for the original herd, 25 animals dropped out of the study for the following reasons: ECF (5); slaughtered (5); sold (3); transferred (7); non-ECF (5, tick-borne disease, old age, poison, lion kill, complicated calving). Out of 66 calves born during the study, 34 disappeared from the herd: ECF (19); slaughtered (1); sold (7); transferred (4); non-ECF (3, malnutrition, accident, missing). The cause of death of eight animals was allegedly non-ECF. However, in three of these ECF cannot be excluded as cause or contributing factor: one cow died after aborting but was treated 1 month before for ECF; one calf of 3 months old died due to malnutrition but the dam was treated for ECF about 3 months before; one calf went missing and was found dead later, but it had seroconverted shortly before disappearing. Slaughters are another source of confounding since two of the six slaughtered animals (33%) had clinical ECF. 3.4. ECF in the original herd There was no indication that any of the 39 animals of the original herd had been infected with T. parva before the study. All were seronegative on IFAT from August to December 1994. The first animal to seroconvert was also the first confirmed clinical case diagnosed in January 95. Excluding 11 animals that were censored and two that never seroconverted, all cattle (26) had been infected by September 1997 (Kaplan–Meier plot in Fig. 2). In fact, three waves of epidemic ECF challenge infected the majority of the herd. Twenty animals showed clinical symptoms. Six were left untreated of which five died. The others survived after treatment. Six sub-clinical cases were diagnosed by means of IFAT (Table 1). 3.5. ECF in the enrolled calves A total of forty T. parva infections were diagnosed in the 66 calves enrolled during the study: 19 lethal infections, seven clinical infections that were successfully treated, two cases recovered without treatment and twelve sub-clinical infections (Table 2). Ten calves were
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Fig. 2. ECF-free survival of adult cattle present in the herd at Mayela before the introduction of the disease. Table 1 Number of ECF cases per year (adult cattle)
Sub-clinical Clinicala Lethal Censored a
1994
1995
1996
1997
1998
1999
2000
Total
0 0 0 4
0 3 1 4
4 3 3 2
2 9 1 1
0 0 0 0
0 0 0 0
0 0 0 0
6 15 5 11
Excluding lethal cases.
censored. Out of the 34 calves currently in the herd, 16 are susceptible, among them all 13 born in 2000. 3.6. Age at first contact The Kaplan–Meier age at first contact curves (or ECF-free survivor curves) which are displayed in Fig. 3, demonstrate the decreasing trend in age at first contact with T. parva Table 2 Number of ECF cases per year (calves)
Subclinicala Clinicala Lethal Censored a
1995
1996
1997
1998
1999
2000
Total
0 0 0 1
2 1 1 3
6 4 7 2
0 0 3 0
4 2 4 3
2 0 4 1
14 7 19 10
Excluding lethal cases
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Fig. 3. ECF-free survival of calves at Mayela per year of birth.
for calves born from 1994 to 1999. This trend was statistically significant (X 2 = 8.1; P = 0.004). The median was 28 months for calves born in 1994, 12 months for those born in 1996 and just over 6 months for those born in 1997. For the calves born in 1998 the median age at first contact estimate increased but it dropped again to 6 months for those born in 1999. Almost all calves born in 1997 and 1999 became infected below the age of 1 year. 3.7. Incidence The monthly ECF incidence, i.e. the risk for a susceptible calf to become infected during a particular month, is shown in Fig. 4. The resulting epidemic curve indicates that waves of ECF challenge mainly coincide with peaks of adult R. appendiculatus abundance. These peaks occur in the rainy season (December–March) and in the cold dry season (May–July). Minor peaks of transmission are observed in September. No accurate information on nymphal abundance was obtained but their presence was recorded in May–June and August–September. The incidence during the first major epidemic increased from 5% in 1996, to 20% during the first adult wave of R. appendiculatus in 1997 and to 50% during the second adult wave. In January 1998 all three remaining susceptible calves were infected. More than 10 calves were born later that year, mainly in the second half, but ECF incidence remained zero throughout. A new period of transmission started in early 1999. Seasonal incidence steadily increased with each wave of R. appendiculatus adults and reached peaks of 30% in the 2000 rainy season, infecting most susceptible calves. As in 1998, this announced a long period of zero transmission during which the susceptible population increased. A similar pattern was also seen at Mkambi and Lupenga where virtually all calves born during the cold and hot dry seasons become infected during two waves of ECF challenge (Fig. 5b and c). The incidence during the cold season is usually higher than during the rainy season.
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Fig. 4. ECF hazard in calves compared to susceptible population and vector burden at Mayela (A); Mkambi (B) and Lupenga (C). ( ), number of susceptible calves; ( ), average number of R. appendiculatus per calf; ( ), hazard of T. parva contact in calves; ( ), window for immunisation.
3.8. Lethality of T. parva infection Out of a total of 66 T. parva infections (both adults and calves) 24 were fatal. The minimum lethality estimate is thus 24 out of 66 or 36%. A total of 21 survived without treatment which yields a maximum lethality of 45 out of 66 or 68%. Since it may be safely assumed that the majority of the clinical cases would have died without treatment the lethality is estimated to be around 60%. Case-fatality ratios were equivalent in the different years (X 2 = 6.7, d.f. = 4, P = 0.15).
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Fig. 5. Seroprevalence in adult cattle at Mayela, compared to ECF incidence in calves: ( ), seroprevalence in adults animals; ( ), T. parva incidence in calves.
3.9. IFAT results The seroprevalence patterns in animals older than 2 years are plotted in Fig. 5, together with ECF incidence in calves. The seroprevalence increased together with disease incidence during the cycles of transmission. Outside transmission episodes, the seroprevalence decreased steadily (e.g. throughout 1998) and eventually drops to zero (e.g. May–December 2000). The maternal immunity prevalence in new-born calves increased from 0% for years of birth 1994–1996, 55% for 1997, 75% for 1998 and 70% for the calves born in 1999 and 2000. The sensitivity of maternal immunity (the proportion of calves, born from immune cows, that is seropositive) was 72% (28 out of 39; 95% c.i. 55–84%). The specificity (the proportion of calves, born from na¨ıve cows, that does not have maternal antibodies) was 96% (22 out of 23; 95% c.i. 77–99%). The single false positive case was a calf born from one of the two animals, from the original herd, which never seroconverted.
4. Discussion 4.1. Transmission dynamics In this study of an ECF epidemic, the incidence of T. parva infection recurs in cycles during which the intensity of transmission increases with each wave of transmitting tick instars until no susceptible cattle population is left. Thereafter, the susceptible calf population is replaced during a period of low challenge. Thus at herd level, the size of the susceptible
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population acts as the most important limiting factor for the start of an epidemic. Nymph to adult transmission appears to be the main mode of transmission in eastern Zambia and clinical cases are the main source of infection with the carrier population merely acting as a reservoir of the infectious parasite. Observations in favour of this hypothesis are the increasing trend of the incidence during cycles of transmission, the low seroprevalence observed in adult animals during periods of low disease incidence in calves and the high case-fatality ratios observed throughout the study. The fact that the nymphal wave in September was never responsible for initiating a new cycle of disease transmission, even in the presence of a substantial susceptible population, indicates that larvae picking up from the carrier population played a minor role in the transmission dynamics (Ochanda et al., 1996). The second adult wave appears then again to be crucial in the regeneration of full-blown transmission (Billiouw et al., 1999). Similar but faster transmission cycles were observed in the more established ECF areas, e.g. Mkambi and Lupenga in the later stages of the study, where the first adult wave typically infects a proportion of calves and the second wave infects the remaining larger proportion. Because of the fact that the size of susceptible calf population regulates the transmission pattern, the seasonal incidence in one herd maybe zero while it reaches high levels in neighbouring herds. It is in these situations that the age at first contact statistic proves to be a more robust epidemiological estimator than incidence. 4.2. Age at first contact and incidence Incidence and prevalence statistics have been widely used to express the frequency of clinical events. Disease prevalence has a limited value in measuring ECF abundance because of the short duration and the high case fatality of the infection. Monthly incidence estimates give snapshots of the ECF challenge but the interpretation of the statistic is often troubled by the interactions between the size of the susceptible population, the seasonality of calving and the seasonality of T. parva challenge. The age at first contact statistic is inherently less sensitive to these variables and provides information on both past and current ECF situation, but this information may be delayed, as the following examples explain. In 1998 at Mayela, the ECF incidence could not be estimated or was zero for the whole year (except January) because no or few susceptible calves were remaining when ECF challenge was high. However, the interpretation of the age at first contact curve remained straightforward, indicating that all calves born that year were ECF-free up to the age of 6 months and that 50% were infected before the age of 15 months. High incidence figures were recorded in 1997 but the high age at first contact of the calves that became infected indicates that this challenge was new (i.e. epidemic). A combination of both age at first contact and incidence provides a comprehensive picture of the ECF epidemiology. The main drawback is that both statistics can only be estimated in longitudinal studies through close monitoring of a cattle population. 4.3. Endemic stability Endemic stability in the case of T. parva may be defined as the epidemiological state where all calves become infected before the age of 6 months and clinical disease is rare (Moll et al., 1986). This implies that all adult animals are immune and are being re-infected
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at least twice per year (calves must be infected once per 6 months and adult animals carry more ticks). Among the factors contributing to the state of endemic stability are a low innate susceptibility of the cattle, a continuous exposure to all instars of R. appendiculatus and sufficient infection prevalence of T. parva in R. appendiculatus (Perry et al., 1992; Norval et al., 1992). Although a second generation of R. appendiculatus (adults and nymphs) ensures a continuous presence of tick instars transmitting T. parva to cattle in eastern Zambia, tick abundance and consequently the ECF challenge is strongly seasonal (Berkvens et al., 1998). Median age at first contact estimates may drop to levels close to those observed in endemically stable situations but case-fatality ratios remain invariably high. The low larva-to-nymph transmission from carrier animals (Norval et al., 1992) and the important highly infected second generation adults may explain the high case fatality and may therefore, be the major constraints in attaining endemic stability in the near future. 4.4. Natural mortality This study suggests that a large proportion of what is diagnosed in the field as non-ECF mortality is still due, directly or indirectly, to ECF. Also slaughter as cause of death is often related to ECF. Thus, even in our closely monitored herds, observation biases distort the estimation of ECF frequency. Retrospective data obtained from routine field reports and surveys, which depend strongly on the farmers’ recollection of events, are therefore, likely to be unreliable. ‘Broad spectrum’ surveys covering a large number of items are in this context even more vulnerable and may lead to wrong decision making. 4.5. Seroprevalence and maternal immunity Seroprevalence is currently the method of choice for monitoring ECF epidemiology (Perry et al., 1985). However, the results from the present study indicate that seasonal or annual large-scale serological surveys may face quite a number of scientific and logistical problems. The organisation of these surveys is labour intensive and expensive. The blood sampling is generally done in older animals, increasing the risk of selection bias and fraud (i.e. dividing blood from one animal over several test tubes). The large sample sizes make the processing of the samples, from storage to analysis, prone to negligence and error. Seroconversion after a booster challenge appears to be immediate and short-lived so that when estimating trends in seroprevalence, the timing of surveys becomes critical in areas with a high degree of seasonality in challenge. Furthermore, it is demonstrated in this study that seroprevalence trends merely reflect trends in disease incidence rather than the build up of immunity in the adult cattle population. On the other hand, estimation of the trend in maternal antibody prevalence gives consistent and reliable results. The collection of data can be done on a continuous basis from calves born in monitored herds. 4.6. Disease control The present study indicates that 2–3 years after the start of a major epidemic, the majority of the adult animals are immune and calf immunisations may in principle be considered. An earlier start would endanger the adult population by increasing the challenge of ECF at
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a time when the herd is particularly vulnerable, often even at risk of extinction. Possible constraints are the definition of the area and population at risk and the level of knowledge of the ECF epidemiology and vector ecology in that area. The study further demonstrates that the period June–November, when high calving rates and low T. parva transmission prevail, offers a suitable window for immunisations (Fig. 4A–C). The farmers should be recommended to keep their calves inside a pen for up to 6 months (not an uncommon practice in eastern Zambia). Temporary acaricide treatment may be an alternative. These management practices could minimise ECF mortality during the rainy season and maximise the efficiency of well-timed immunisation efforts. 4.7. Conclusions and recommendations This paper confirms that longitudinal studies are a powerful tool to evaluate the epidemiological status of ECF in a region. The combination of incidence and age at first contact provides necessary and sufficient information to allow accurate assessment of the epidemiological status of the disease, something that is not possible using either parameter on its own, nor by relying on transversal surveys. Although the economic importance of ECF in Zambia is fully acknowledged it is shown here that the frequency of occurrence of the disease may still be underestimated. The transmission dynamics in epidemic areas of ECF in eastern Zambia is dominated by nymph-to-adult transmission originating from clinical cases. The size of the susceptible population acts as the main limiting factor. Endemic stability is unlikely until larvae become more important in picking up the infection from carriers. This interaction is probably crucial to ensure a more constant and less lethal disease pressure. Infection and treatment immunisation remains the disease control method of choice, where it is applicable, but this study recommends that extra efforts should be done to immunise in the window of low adult tick abundance between July and October and to protect the calves born from November to May against ticks. The seroprevalence in adult cattle, measured to monitor ECF from its epidemic status towards endemicity, appears to be less reliable than expected. The presence of maternal antibodies in new-born calves, reflecting the immune status of the cow population, is introduced as an alternative but more studies are needed to confirm its potential value. References Berkvens, D.L., 1991. Re-assessment of tick control after immunisation against east coast fever in the eastern province of Zambia. Annales de la Société belge de la Médecine tropicale 71, 87–94. Berkvens, D.L., Geysen, D.M., Chaka, G., Madder, M., Brandt, J.R.A., 1998. A survey of the ixodid ticks (Acari: Ixodidae) parasitising cattle in the eastern province of Zambia. Med. Veterinary Entomol. 12, 234–240. Billiouw, M., Mataa, L., Marcotty, T., Chaka, G., Brandt, J., Berkvens, D., 1999. The current epidemiological status of bovine theileriosis in eastern Zambia. Trop. Med. Int. Health 4, A28–A33. Bishop, R., Sohanpal, B., Kariuki, D.P., Young, A.S., Nene, V., Baylis, H., Allsopp, B.A., Spooner, P.R., Dolan, T.T., Morzaria, S.P., 1992. Detection of a carrier state in Theileria parva-infected cattle by the polymerase chain reaction. Parasitology 104, 215–232. Burridge, M.J., Kimber, C.D., 1972. The indirect fluorescent antibody test for experimental east coast fever (Theileria parva infection of cattle): evaluation of a cell culture schizont antigen. Res. Vet. Sci. 13, 451–455. Hardin, J., Hilbe, J., 2001. Generalised Linear Models and Extensions. Stata Press, College Station TX, p. 245.
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Hosmer Jr., D.W., Lemeshow, S., 1999. Applied Survival Analysis: Regression Modelling of Time to Event Data. Wiley, New York, p. 386. Koch, H.T., Norval, R.A.I., Ocama, J.G.R., Munatswa, F.C., 1992. A study on the Theileria parva bovis carrier state. Prev. Vet. Med. 12, 197–203. Moll, G., Lohding, A., Young, A.S., Leitch, B.L., 1986. Epidemiology of theileriosis in calves in an endemic area of Kenya. Vet. Parasitol. 19, 255–273. Nambota, A., Samui, K., Sugimoto, C., Kakuta, T., Onuma, M., 1994. Theileriosis in Zambia: etiology epidemiology and control measures. Jpn. J. Vet. Res. 42, 1–18. Norval, R.A.I., Perry, B.D., Young, A.S., 1992. The Epidemiology of Theileriosis in Africa. Academic Press, London, p. 481. Ochanda, H., Young, A.S., Wells, C., Medley, G.F., Perry, B.D., 1996. Comparison of the transmission of Theileria parva between different instars of Rhipicephalus appendiculatus. Parasitology 113, 243–253. Perry, B.D., Musisi, R.G., Pegram, R.G., Schels, H.F., 1985. Assessment of Enzootic Stability to Tickborne Diseases. World animal review, October–December 1985, pp. 24–32. Perry, B.D., Deem, S.L., Medley, G.F., Morzaria, S.P., Young, A.S., 1992. The ecology of Theileria Parva infections of cattle and the development of endemic stability. In: Munderloh, U.G., Kurtii, T.J. (Eds.), Proceedings of the First International Conference on Tick-Borne Pathogens. Minnesota, USA, 15–18 September 1992, pp. 290−296. StataCorp, 2001. Stata Statistical Software: Release 7.0., Stata Corporation, College Station, TX.