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Induction of Acquired Immunity in Pastoral Zebu Cattle Against East Coast Fever After Natural Infection by Early Diagnosis and Early Treatment
J. A. Matovelo†
P. S. Gwakisa‡
R. S. Silayo‡
D. M. Kambarage§
†Department of Veterinary Pathology, Faculty of Veterinary Medicine, Sokoine University of Agriculture, Morogoro, Tanzania
‡Department of Veterinary Microbiology and Parasitology, Faculty of Veterinary Medicine, Sokoine University of Agriculture, Morogoro, Tanzania
*Department of Education and Extension, Faculty of Agriculture, Sokoine University of Agriculture, Morogoro, Tanzania
§Department of Veterinary Medicine and Public Health, Faculty of Veterinary Medicine, Sokoine University of Agriculture, Morogoro, Tanzania.
This study was facilitated by a research grant by the TARP II-SUA project.
KEY WORDS: East Coast fever (ECF), chemotherapy, immunity, pastoral, zebu
This study was conducted to optimize farm regimens for control of East Coast fever (ECF). Particular interest was paid to the opportunities that early diagnosis by farmer-recognizable signs and early treatment could offer in the management of ECF. The performance of early diagnosis and early treatment (EDET) as a method of reducing calf mortality and enhancing conditions for the development of endemic stability was investigated. A group of 280 Maasai-owned zebu cattle were screened serologically and parasitologically for Theileria parva infection once every month and daily by the farmers themselves for clinical signs over a period of 6 months. A total of 100 calves were tentatively diagnosed as ECF-positive based on clinical symptoms. The clinically sick animals were treated with Butalex (buparvaquone 2.5mg/kg) and monitored over 24 days to evaluate treatment outcome. Recovered animals were also monitored for 6 months to show the patterns of specific anti-T. parva antibodies in blood and incidences of recurrent ECF. The percentages of cases that could be detected by each test were LS, 67%, and BS, 41%, whereas 27% of cases were detected using the two tests together. In aggregate terms, 81 (81%) animals were confirmed as positive for ECF, and 19 animals with clear ECF clinical signs were negative using both laboratory screening methods. There was an overall recovery rate of 98%; only two calves died at variable time points after treatment. Of the recovered animals, 91.84% showed prompt recovery as manifested by the disappearance of clinical signs. Parasite recrudescence was 10 (10.2%) and 4 (4.08%) as detected by BS and LS, respectively. There was a significant increase (P < .05) in levels of specific antibodies in the blood of treated animals from 21.23% to 51.13% 6 months after treatment, and disease recurrence was recorded in only five animals at different time points. Therefore, we concluded that with pastoral zebu cattle kept in ECF endemic areas, EDET for the management of ECF offers not only the opportunity for prompt recovery but also an alternative and pastoral-friendly method of immunizing cattle against ECF.
East Coast fever (ECF), a lymphoproliferative disorder of cattle caused by protozoan parasite Theileria parva, is one of the most important diseases in the Central Eastern and Southern African regions.1 Control of this disease has, for many years, relied on control of the vector tick by regular application of acaricides and treatment of clinical cases. Acaricides are, however, expensive to apply. Additionally, they may cause environmental pollution, and with time ticks develop resistance to acaricides.2 Recently, immunization through the infection and treatment method (immunoprophylaxis) has been introduced and proved efficacious both at the laboratory and field levels.3,4 The resulting solid immunity protects cattle against homologous challenge for up to 3.5 years.
However, this method also has several disadvantages, including the creation of carriers in immunized herds, which may act as sources of infection for other nonimmune animals in the same herd and in ECF-free areas where the vector tick is present.5 This may also constitute a risk of introducing new strains in endemically stable areas, which may consequently lead to endemic instability. Furthermore, the failure of consistent cross immunity among cattle immunized with different stabilate stocks is a major drawback to the reputation of the method in the field.6 The collective limitations of the current immunization method prompt research for a cheaper, safe, convenient, and practical method to control ECF in the field. It is known that cattle that recover from ECF, either naturally or after treatment, acquire resistance against subsequent homologous challenge. The immunity engendered is life long provided that a constant parasite challenge occurs through repeated tick infestations. The goal of this study was to explore the efficiency of chemotherapy of natural ECF cases as a way of conferring protective immunity against ECF in cattle.
It is a common understanding that effective and rational treatment requires early and accurate diagnosis. However, diagnosis of ECF in the field to date depends mainly on the detection of parasites in Giemsa-stained blood and lymph node needle biopsy smear,1 which may not be a true indicator of clinical ECF. This is particularly common in ECF-endemic areas in which the detection of parasites not only indicates an active infection but may also indicate either a slow proliferation of macroschizont-infected lymphocyte or a carrier state of the animal.7 As a result, diagnosing ECF cases in such areas may be confusing, unrealistic, and even impractical. Therefore, this study also attempted to design farmer-manageable regimens to minimize losses from ECF in pastoral zebu cattle through the establishment of accurate early disease diagnosis and early treatment.
MATERIALS AND METHODS
Sites and Animals
This study was conducted in two villages (Melela and Wami–Sokoine) in Morogoro Rural District in a period that includes a wet and dry season between April and November 2001. Animals used were the local zebu cattle kept under a pastoral management system. Twenty-four Maasai herds were purposively selected based on the animal management practices. At the beginning of the study, a total 280 calves were ear-tagged for identification purposes. Farmers were assigned to monitor the animals for clinical symptoms of ECF throughout the study.
Treatment of ECF cases
Each ECF case was qualified with the following clinical symptoms: enlargement of superficial lymph nodes, increase in body temperature, dullness, and respiratory distress. Participating farmers were trained to identify and record these signs as soon as they appeared and to convey messages on the same to researchers at the earliest opportunity. Animals that were diagnosed with ECF were subject to treatment using buparvaquone (Butalexâ, Pitman-Moore). The drug was administered intramuscularly at a dose rate of 2.5 mg/kg body weight and repeated at the same dosage after 48 hours. Treated animals were monitored for 24 days and samples were taken on days 10 and 24 after treatment. The severity of the disease at the time of treatment was categorized based on the criteria set for ECF clinical reactions8 with minor modifications as shown in Table 1. Responses to treatment were recorded as prompt/rapid, slow, or died. A “prompt” response was recorded when animal showed marked clinical improvement within 48 hours after the first dose of drug. A “slow” or “sluggish” response was recorded when treated animals did not show a marked clinical improvement during the follow-up period. The category “dead” was used if the animal died within the follow-up period and if necropsy findings were suggestive of ECF with detection of T. parva parasites in either lymphocytes or erythrocytes.
Detection of serum antibodies to T. parva
Antibody levels to T. parva were determined twice: at the onset of the study (April) and at the end of the study (November). Serum samples were assayed for antibodies to T. parva by ELISA using recombinant polymorphic immunodominant molecule (PIM) as the antigen.9 Optical density (OD) readings for test sera were expressed as percentage positivity (PP), and were calculated by OD (test serum)/OD (+ve control serum) ¥ 100.9
Microscopic Examination of Blood and Lymph Node Smears for T. parva Parasites
Smears of blood and lymph node biopsies were prepared each time an animal was suspected of having ECF and thereafter were taken at days 10 and 24 after treatment. For the establishment of carrier or immunity status in the treated population, blood and lymph node smears were taken at the end of the study. Smears were fixed in absolute methanol for 5 minutes and then placed in 10% Giemsa’s stain for 30 minutes. In total, 200 fields of each smear were examined at x1000 magnification. Blood smears were assessed for piroplasm parasitaemia and lymph node smears for schizont parasitosis. These were scored as either negative or positive and expressed in percentages for expression of disease prevalence.
Data Handling and Statistical Analysis
Data were entered in a record form that included clinical observations, interpretation of blood and lymph node smears, antibody titers, treatment regimens, and information on response to treatment. Data were analyzed by STATISTIX 7 computer software using descriptive statistics, one-way analysis of variance (ANOVA), whereas comparisons of any differences of means of all parameters were made by the LSD method, at a rejection level of P < .05.
Clinical and Laboratory Diagnosis
One hundred sick animals were diagnosed with ECF based on farmer-recognizable clinical signs. The clinical signs varied from one animal to another, ranging from mild and moderate to a severe reaction. Febrile reaction and lymph node enlargements were the most prominent clinical signs in early ECF cases, and dullness and respiratory distresses were recorded in advanced cases. Table 2 shows the number of cases, categorized clinical findings, and general laboratory findings. Laboratory evaluation showed that lymph node smear (LS) examination detected 67 (67.68%) cases and blood smear (BS) examination detected 41 (41.84%) cases. The sensitivity difference between the two diagnostic means was statistically significant (P < .05). We also noted that 19 animals (19%) that displayed clinical signs of ECF were negative on both (LS and BS) laboratory screening methods. We found that 81% of the animals were positive on at least one of the screening methods. As shown in Table 3, responses to treatment based on clinical reaction at treatment showed that there was an overall recovery rate of 98%. Of the animals that recovered from the clinical disease, 90 (91.84%) showed rapid or prompt response to treatment, and 8 (8.16%) showed slow recovery. All animals (100%) with mild clinical reactions recovered promptly; the majority (95.45%) of moderate clinical reactors also recovered promptly, and of the animals that were severely affected 76.67% recovered promptly, 20% recovered slowly, and one (3.33%) died. Post-treatment follow-up revealed the presence of parasites in 14 animals. Blood smear examination detected parasites in 10 animals, whereas lymph node smear examination showed 4 animals to be ECF positive. However, animals that showed parasite recrudescence were clinically normal.
Reaction and Response in Relation to Prevalence of T.
parva in Lymph, Blood Smears, and Specific
Laboratory findings showed a significantly (P < .05) higher schizont parasitosis in severely affected animals than in the other two categories (mild and moderate). Piroplasm parasitemia was extremely low in all categories, and there was no significant difference among them. Severely affected cattle had slightly lower levels of specific antibodies to T. parva than mildly and moderately affected animals (Fig. 1). Furthermore, a retrospective analysis of antibody prevalence revealed an interesting pattern in which dead animals had low values as opposed to high values in promptly recovered animals. High schizont parasitosis was observed in cattle that had a prolonged convalescence (slow response category), but the difference among the groups was not significant (Fig. 2).
Prevalence of Parasites in Lymph Node and Blood Smears and Specific Anti-T. parva Antibodies
Mean percentage positivity (PP) of specific antibodies of all tagged animals at the beginning of the study was 27.13 ± 1.75 (n = 280); this titer increased significantly to 43.58 ± 2.34 in a 6-month period. Meanwhile, a retrospective analysis of antibodies levels in all treated animals revealed a mean value of 21.41 ± 2.6 (n=100) at the beginning of study, which increased significantly (P < .05) to 51.23 ± 3.69 (Fig. 3) 6 months later. Percentage piroplasm parasitemia was 0.034% ± 0.18% during treatment, which increased significantly (P < .05) to 0.32% ± 0.75% at the end of study, whereas percentage schizont parasitosis was significantly high during treatment 1.63% ± 2.73% as compared with 0.53% ± 2.44% at the end of study (Fig. 3). The screening results for parasite prevalence in the recovered population (n = 98) revealed 10.2% and 9.2% of clinically normal cattle were harboring piroplasms and schizonts, respectively. The overall parasite prevalence at the end of the study was 14.06% and 9.84% as detected in blood and lymph node smears, respectively.
ECF Recurrence (Morbidity) and Mortality
Cattle that recovered from ECF showed a degree of resistance to subsequent field disease challenge. Only five (5.1%) animals had a second disease episode at different time points during a follow-up period, and no fatal cases were recorded during these attacks. A positive correlation was obtained between the level of specific antibodies to T. parva and resistance to subsequent infection (r = 0.95, P < .05).
In the present study, we have shown that a majority of cattle (98%) that were symptomatically diagnosed as ECF cases recovered after treatment with the antitheilerial drug. The proportion of recovered animals suggests that, in endemic areas, the diagnosis of ECF based on clinical signs can be a reliable diagnostic means. We have seen that a febrile reaction and enlargement of lymph nodes, especially those draining the head, were the most prominent early clinical symptoms of the disease; therefore, it would seem practical to depend on their first appearance for early diagnosis of ECF. These findings are in agreement with the observations by other researchers10 who found that systematic monitoring of body temperature is a good method for early detection of T. parva infection. The symptomatic diagnostic approach is also preferable because it does not necessarily require highly trained personnel to perform the diagnosis. That is seen in this study, in which farmers themselves made most of the diagnoses. Furthermore, this study emphasizes the need for early diagnosis and early treatment (EDET) for prompt recovery from ECF; we noted that the majority of animals displaying mild and moderate clinical symptoms recovered rapidly after treatment as compared with the severely affected animals.
Results from this study have also revealed that chemotherapy using the present approach (EDET) not only reduces mortalities against ECF but also contributes to the acquisition of immunity to subsequent parasite challenges in pastoral indigenous cattle. This observation is clearly reflected in the significantly low number of recurrent ECF cases and remarkable increase in the mean values of specific anti-T. parva antibodies in the treated animal group 6 months later. Several authors have shown a correlation between level of specific antibodies to T. parva and degree of resistance to ECF.11 Studies on the immunology of T. parva infection and the application of molecular tools have led to the conclusion that sera of recovered animals neutralize sporozoites of various isolates.6 The absence of disease recurrences and elimination of ECF mortalities in calves during the follow-up period is clearly indicative of such a correlation. We, therefore, assert that EDET as an approach for the management of ECF presents a feasible alternative to the currently used infection and treatment method (ITM) for immunization against ECF. EDET is particularly relevant for pastoral herds where ITM has not made many inroads. EDET is far more advantageous than ITM if financial aspects are to be taken into account, since in this approach the targeted animal population involves young calves that require a small amount (dose) of curative drugs and the costs attributable to delivery of the ITM are obviously reduced. Also EDET stands to offer the benefits of use of local strains and thereby eliminates the risk of introducing new strains. The fact that unlike ITM, it eliminates the need for cold chains facilities, means that EDET presents better potential for adaptability in pastoral areas.
Despite diagnostic problems that are usually encountered on discriminating Theileria spp based on the detection of schizonts in Theileria endemic areas, LS emerged as a more effective laboratory diagnostic means for ECF than BS did. This method is the most valuable and straightforward means of diagnosing ECF, although regrettably it is not always performed.1 Through LS examination, we were able to detect the disease at early stages even before the clinical signs were conspicuous. Failure to detect parasites in 32% of the cases, which had apparent clinical signs of ECF, may be viewed as a source of error in diagnosing ECF, especially in early stages of the disease, when only a few lymphocytes are parasitized. In this respect, use of alternative methods with higher sensitivity, like PCR, may improve the detection of such parasites, especially when screening a high number of samples.12
Parasite prevalence results suggest that schizont parasitosis could play a prognostic role in predicting the outcome of ECF cases, because this parameter correlated well with disease severity and response to treatment. Thus, we have shown that highly parasitized animals displayed severe clinical reaction and underwent a long convalescence period (Figs. 1 and 2). As a result, we suggest that supportive treatment would have been a necessity for rapid recovery.
This study has also revealed that BS examination is not useful in the diagnosis of ECF at early stages and should not be used as a sole means of defining ECF case in the absence of clinical data. The increasing trend of piroplasms parasitemia at the end of study period would instead indicate the role of BS on defining carrier status of animals. This is particularly common in endemically stable areas, where most recovered animals become carriers of parasites and show these parasites in blood even without clinical disease.1,13 These observations further reinforce the diagnostic value of a clinically made diagnosis. We therefore conclude that, with pastoral zebu cattle kept in ECF endemic areas, early diagnosis and early treatment (EDET) as an approach to management of ECF not only offers good opportunities for prompt recovery but also an alternative and pastoral friendly method of immunizing cattle against ECF.
The expert technical assistance of Mr. Edson Rugaimukamu, Mr. Geoffrey Mulungu, and Mr. W. Hozza is gratefully acknowledged. The authors also extend their appreciation to the collaborating pastoral farmers in Melela and Wami Sokoine villages.
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Table 1. Categories of Clinical Status of ECF Cases Before Treatment
variable Rectal Lymph node Respiratory
Mild Above 39.5 ˚C Swollen parotid Normal Normal
Moderate Above 39.5˚C Swollen parotid Slight labored Normal
Severe Above 39.5˚C Swelling of all Respiratory Reluctant to move
Table 2. Cases Attended and Laboratory Findings
Clinical signs of ECF 100 0 100 (100%) 4 66 30
Lymph node smears (LS) 99 1 67 (67.68%) 3 42 22
Blood smears (BS) 98 2 41 (41.84%) 1 27 13
No test detected – – 19(19%) 1 13 5
Table 3. Responses to Treatment in Relation to Clinical Status Before Treatment
Responses to treatment
Clinical Status Prompt Slow Dead % Total
Mild 4 0 0 4
Moderate 63 2 1 66
Severe 23 6 1 30
% Total 90 8 2 100
Figure 1. Clinical reaction and prevalence of T. parva in lymph, blood smears, and specific anti-T. parva antibodies.
Figure 2. Responses to treatment and prevalence of T. parva in lymph, blood smears, and specific anti-T. parva antibodies.
Figure 3. Percentage schizont parasitosis, piroplasm parasitemia, and anti-T. parva antibody prevalence in treated animals at different time points of study.
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