Isolates of Theileria annulata collected from different parts of India show phenotypic and genetic diversity.

Phenotypic and genetic polymorphism was studied amongst four Theileria annulata isolates collected from three different parts of India. Amongst various markers studied for the comparison of growth characteristics of schizont cell lines established from these isolates, viability, non-viability counts and nitric oxide (NO) production showed significant variation. A negative correlation was observed between NO production and mRNA expression for TNF-alpha, a potent proinflammatory cytokine related to the pathogenesis of the disease. Phenotypic polymorphism was also revealed by T. annulata schizont-specific monoclonal antibodies (Mabs), viz. 1C7, 1E11, 2G2 and EU-106, which recognized variable number of cells in indirect fluorescent antibody and indirect immunoperoxidase tests, when tested against the four T. annulata isolates collected from India. Genetic polymorphism was recognized amongst the four isolates by restriction digestion analysis of Tams-1 gene PCR products. These observations revealed that the four isolates of T. annulata are different from each other and might be expressing different antigenic determinants on their cell surface.

In vivo immunostimulatory effects of CpG oligodeoxynucleotide in cattle and sheep.

Oligodeoxynucleotides (ODN) containing cytosine-phosphate-guanosine (CpG) motifs have been shown to activate the innate immune system and protect mice and chicken from bacterial and viral infections. Unfortunately, similar studies in other veterinary species are lacking. In this study we assessed the in vivo immunostimulatory effects of CpG ODN 2007, an ODN with previously demonstrated in vitro biological activity. The in vivo effects of ODN 2007 were compared in two closely related outbred species, sheep and cattle, to determine if there were common biological responses. We demonstrated that subcutaneous (s.c.) injection of the CpG ODN induces an acute phase response in the form of a transient fever, a mild transient increase in circulating neutrophils and elevated serum haptoglobin in both sheep and cattle. Sheep injected with CpG ODN also exhibited increased serum 2'5'-oligoadenylate (2'5'-A) synthetase activity, but no increase in serum 2'5'-A synthetase was detected in cattle. The ODN-induced responses were stronger in animals injected with CpG ODN formulated in 30% emulsigen than phosphate buffer saline (PBS) alone. These in vivo data demonstrate for the first time that a CpG ODN induces acute phase immunostimulatory responses in sheep and cattle. However, CpG ODN-induced antiviral effector molecule 2'5'-A synthetase was detected only in sheep but not in cattle.

Characterization of efferent lymph cells and their function following immunization of cattle with an allogenic Theileria annulata infected cell line.

Immunization of cattle with in vitro propagated bovine mononuclear cells infected with Theileria annulata induces a protective immune response. Activation and effector function of T cells exiting the lymph node draining the site of cell line immunization were investigated to understand the mechanisms involved in the generation of immunity. Immunized animals exhibited a biphasic immune response in efferent lymph as well as peripheral blood. The first phase corresponded to allogenic responses against MHC antigens of the immunizing cell line and the second was associated with parasite specific responses. An increase in the output of CD2(+) cells and MHC class II(+) cells in efferent lymph was observed after cell line immunization with a corresponding decrease in WC1(+) cells. Although the percentage of CD4(+) T cells did not change significantly over the course of the experiment, they became activated. Both CD25 and MHC class II expressing CD4(+) T cells were detected from day 7 onwards, peaking around day 13. Efferent lymph leukocytes (ELL) exhibited sustained responses to IL-2 in vitro following cell line immunization. Antigen specific proliferation was also detected first to the immunizing cell line and then to parasite antigens. The two peaks of CD2(+) cells were observed, which corresponded to similar peaks of CD8(+) cells. The increase in CD8(+) cells was more pronounced during the second parasite specific phase than the first allogenic phase. Activated CD8(+) T cells mainly expressed MHC class II and some expressed CD25. Significantly the peak of activated CD4(+) T cells preceded the peak of activated CD8(+) T cells, highlighting the role of T. annulata specific CD4(+) T cells in inducing parasite specific CD8(+) cytotoxic responses. A biphasic cytotoxic response also appeared in efferent lymph and peripheral blood, the first directed against MHC antigens of the immunizing cell line followed by MHC class I restricted parasite specific cytotoxicity. The cytotoxic responses in efferent lymph appeared earlier than peripheral blood, suggesting that activated CD8(+) cells exiting the draining lymph node following immunization with T. annulata infected schizonts play an important role in the development of protective immune responses.

Identification of immunoreactive polypeptides of Babesia equi parasite during immunization.

This study was carried out to identify immunoreactive polypeptides in Babesia equi merozoite antigen. Three fractions of killed B. equi merozoite antigen viz.; whole merozoite (WM), cell membrane (CM) and high speed supernatant (HSS) antigens were prepared from the parasite infected erythrocytes. These antigenic preparations along with ghost antigen from non-infected erythrocytes were fractionated on 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with sera showing high antibody titres. On SDS-PAGE, 16 polypeptides with molecular weight (Mr) in the range of 112-17kDa were obtained from the WM and CM antigens. But only six polypeptides were detected (96.5-28kDa) in the HSS antigen. On immunoblotting with high titred serum collected from donkeys following two immunizations with a killed B. equi merozoite immunogen, 11 polypeptides were observed in the WM and CM antigens (Mr 112-18kDa). Of these, four polypeptides (Mr 112, 45, 33 and 18kDa) were identified as most immunoreactive. Besides these, a 28kDa was observed as strong immunoreactive protein in WM and CM antigens. The HSS antigen showed only six polypeptides and one peptide (28kDa) was identified as immunoreactive. When high titred serum collected from immunized donkeys following challenge with B. equi infected blood and was used for immunoblotting, the protein profile of WM and CM antigens remained the same. However, three additional polypeptides (Mr 81, 54.5 and 39kDa) were detected in HSS antigen.

Vaccination of donkeys against Babesia equi using killed merozoite immunogen.

Protective efficacy of a killed Babesia equi immunogen was assessed in donkeys. The immunogen was prepared from B. equi infected blood so as to contain lysate of 2 x 10(10) parasitised erythrocytes per dose. The immunogen was mixed with an adjuvant Quil A (3mg) and inoculated into four susceptible donkeys (group I). A booster inoculation was given after 21 days of first inoculation followed by challenge with fresh infected blood containing 1x10(11) parasitised erythrocytes 14 days later. Two groups of two donkey each were included as adjuvant only control (group II) and uninoculated control (group III), respectively. After challenge, donkeys were observed for a period of 4 weeks. The immunised donkeys (group I) showed significantly high (P<0.05%) enzyme linked immunosorbant assay (ELISA) antibody titres and significantly high (P<0.05%) stimulation indices (SI) in lymphocyte proliferation assay (LPA) than that of groups II and III donkeys from day 14 PI and day 7 PI onwards, respectively. All the immunised donkeys withstood lethal challenge, whereas, control donkeys died within 10 days post-challenge (PC). Parasitaemia rose to mean maximum 8.0+/-6.0% for 5-7 days in group I donkeys after challenge, whereas, it rose to 55.5% in control groups. The percent rise in rectal temperature, total leucocyte count (TLC), fall in haemoglobin (Hb) was less severe in immunised group as compared to the control groups. Two immunised-challenged donkeys were splenectomised recovery. No parasites appeared in the blood during the observation period following splenectomy 4-week. Three times increase in skin-fold thickness at 24h of intradermal inoculation prior to challenge in group I donkeys was observed, thus, indicating a good in vivo cell mediated immunity. It can be concluded that the B. equi immunogen along with adjuvant Quil A, used in the present study, was optimum to elicit a strong immune response against B. equi in experimental donkeys.

Impact of field vaccination with a Theileria annulata schizont cell culture vaccine on the epidemiology of tropical theileriosis.

Tropical theileriosis, caused by Theileria annulata, is an important tick-borne disease of cattle. A cell culture attenuated vaccine has been developed in our laboratory by long-term in vitro propagation of the schizont stage of the parasite. A longitudinal study was conducted at selected farms housing indigenous, cross-bred and exotic animals to investigate the effect of vaccination on the epidemiology of the disease. A total of 120 animals in 4 age groups were vaccinated with the vaccine before the onset of disease season. An equal number of age-matched animals were kept as controls at the same sites. Animals were monitored for 14 months at monthly intervals. The 97.5% vaccinated animals showed a rise in antibody titres 1 month post-vaccination, as determined by single dilution ELISA. The 78.3% of non-vaccinated animals became sero-positive over the period of observation. Mean antibody titres were significantly higher in vaccinated than non-vaccinated animals. Cross-bred animals showed higher antibody titres followed by exotic and indigenous animals in both the vaccinated and non-vaccinated groups. However, the antibody titres in animals of different ages were similar. The 36.7% vaccinated and 64.2% non-vaccinated animals became carriers (<0.5% piroplasms in erythrocytes) during the observation period. Clinical cases of theileriosis were recorded only in the non-vaccinated group suggesting that vaccinated animals were sufficiently immune to withstand field tick challenge for at least 14 months.

Effect of vaccination in the field with the Theileria annulata (Hisar) cell culture vaccine on young calves born during the winter season.

The responses were monitored of young crossbred calves vaccinated against tropical theileriosis during the winter against a field tick challenge in the disease season. Thirty-eight calves below 2 months of age, born after the end of the disease season, were selected at an organized farm. Twenty-five animals were vaccinated with Theileria annulata (Hisar) cell culture vaccine (developed at CCS HAU Hisar laboratory) after the end of the disease season and 13 calves were kept as non-vaccinated controls. These calves were observed for their susceptibility to theileriosis in the new disease season. There was an increase in antibody titre in 18 of the 25 vaccinated animals one month after vaccination. The antibody titre then declined gradually, but remained higher than those of the non-vaccinated animals at month 0. No fever or other clinical signs of tropical theileriosis were observed in any of the vaccinated animals. Nine out of 25 (36%) vaccinated calves showed occasional piroplasms (<0.5%) in blood smears. All the vaccinated animals withstood the field tick challenge. On the other hand, 9 of the 13 (69%) unvaccinated calves exhibited occasional piroplasms, and included three clinical cases of tropical theileriosis. These observations suggest that young crossbred calves vaccinated with the T. annulata (Hisar) cell culture vaccine at the end of the disease season were relatively resistant during the next disease season.

Single dilution ELISAs using soluble piroplasm, cellular schizont and soluble schizont antigens for the detection of antibodies against Theileria annulata.

Single dilution ELISAs were standardised for the determination of antibody titres against Theileria annulata using three antigens namely soluble piroplasm, cellular schizont or soluble schizont antigens. Antibody titres of 20 cattle serum samples of known identity were determined by multi-dilution ELISA using the three antigens. The ratio of the optical density (OD) of known positive and known negative sera at different serum dilutions were calculated and termed as positive/negative (P/N) ratios. Coefficients of correlation (r) were calculated between the P/N ratios at different dilutions of known sera and their log10 antibody titres by multi-dilution ELISA. The value of "r" was the highest at the dilution of 1:400. From the log10 antibody titres of known sera and their P/N ratios at the dilution of 1:400, regression equations (Y = a + bX, where Y = predicted log10 titre, X = the P/N ratio at 1:400 dilution) were calculated separately for the three antigens. Thus, the equations Y = 1.63 + 1.35X for soluble piroplasm, Y = 2.67 + 0.547X for cellular schizont and Y = 1.817 + 0.663X for soluble schizont antigens were derived. Test sera were diluted to 1:400 and their OD were read in duplicate wells and converted to P/N ratios. The antibody titres were predicted from the P/N ratios using the above mentioned regression equations. Twenty randomly selected sera tested by single and multidilution ELISAs showed non-significant differences (P < 0.01) between antibody titres. Antibody titres of 90 unknown field sera of cattle were determined by single dilution ELISA. The piroplasm antigen detected higher antibody titres followed by cellular schizont and soluble schizont antigens. The study revealed that a single dilution ELISA could be successfully used for field epidemiological studies of tropical theileriosis.

Proinflammatory cytokine expression by Theileria annulata infected cell lines correlates with the pathology they cause in vivo.

Control of Theileria annulata is currently best achieved by the use of live attenuated cell line vaccines. However, the mechanisms underlying attenuation are unclear and there is a need to rapidly produce new cell line vaccines, which could safely and effectively vaccinate cattle against tropical theileriosis. There is increasing evidence to suggest that proinflammatory cytokines produced by T. annulata infected cells play a central role in both pathology and immune evasion. This study aimed to test this hypothesis and to evaluate cytokine expression as a marker of virulence. The pathogenicity and protective efficacy of cloned T. annulata cell lines that expressed different levels of proinflammatory cytokines were compared. In two independent trials using different stocks of T. annulata, cell lines that expressed higher levels of proinflammatory cytokines induced severe reactions, and in some cases death, when used to vaccinate groups of cattle. In contrast, low cytokine expressing lines induced low post-vaccinal reactions. The results clearly demonstrated that cytokine expression by T. annulata infected cells could be used as a marker of virulence and provided strong evidence to support a role for cytokines in the induction of pathology. Both high and low cytokine expressing cell lines protected cattle against heterologous challenge infection, offering the possibility of using cytokine expression to rapidly select new safe, potent vaccines against tropical theileriosis without the need for culture attenuation.

Determination of duration of immunity of calves vaccinated with the Theileria annulata schizont cell culture vaccine.

Bovine tropical theileriosis caused by Theileria annulata is a serious haemoprotozoan disease of cattle affecting exotic cattle, their crossbreeds and young indigenous calves. Cell culture vaccines have been developed and used effectively in various countries for the control of this disease. However, the duration of immunity provided by these vaccines is poorly understood. The present experiments were planned to study the duration of immunity in animals after vaccination with the T. annulata (Hisar) schizont cell culture vaccine. Two groups of calves were vaccinated and challenged after a period of 3 and 6 months, respectively. There was no fever in any of the vaccinated calves after challenge. However, the vaccinated animals exhibited mild to moderate enlargement of lymph nodes and parasitological reactions. The parasitological reactions were very mild in calves challenged after 3 months and moderate in calves challenged after 6 months. There was a mild but significant decrease in the haematological values of calves after challenge. A significant rise in the anti-theilerial antibody titres was observed in all calves after vaccination, which increased further, by many folds after challenge. On the other hand, all the challenge control calves showed symptoms of acute theileriosis and died. The observations suggested that the T. annulata (Hisar) schizont cell culture vaccine provided immunity in vaccinated animals for at least 6 months in the absence of field tick challenge. However, there was some decline in immunity after 6 months, if the animals are not exposed to ticks during this period.

Comparison of cellular schizont, soluble schizont and soluble piroplasm antigens in ELISA for detecting antibodies against Theileria annulata.

The efficacy and suitability of cellular schizont, soluble schizont and soluble piroplasm antigens was compared for detecting antibodies against Theileria annulata. Fifty bovine sera of known identity were evaluated in ELISA using the above mentioned antigens. Antibody titres of 1:100 to 1:51,200 were detected while using soluble piroplasm and cellular schizont antigen in ELISA. The titres ranged between 1:100 to 1:25,600 with the soluble schizont antigen. Soluble piroplasm antigen exhibited the highest antibody titres followed by cellular schizont and soluble schizont antigens. Cellular schizont antigen proved to be better than soluble schizont antigen for detecting anti-schizontal antibodies. Antibody titres obtained by the three antigens exhibited a good linear correlation amongst each other. The study showed that soluble piroplasm and cellular schizont antigens can be used successfully for detecting antibodies against piroplasm and schizont stages of T. annulata, respectively in bovine sera.

In vivo development of Theileria annulata: major changes in efferent lymph following infection with sporozoites or allogeneic schizont-infected mononuclear cells.

The object of these experiments was to study the pathogenesis and kinetics of Theileria annulata infection in the efferent lymph of the draining lymph nodes of calves. Efferent lymphatics of calves were cannulated prior to infection with T. annulata sporozoite or an allogeneic schizont cell line. Potentially lethal sporozoite challenge induced cell shut-down from days 4-6 and then a massive increase in output of blasting cells (both infected and non-infected) in the efferent lymph. The rate of lymph flow and total cell output increased to 5 to 10-fold from day 6 onwards. Sporozoites were never isolated from the efferent lymph. However, large numbers of parasite-infected cells were seen in efferent lymph from the sixth day of infection. The animals inoculated with an allogeneic T. annulata-infected cell line exhibited only a small increase in flow rate and cell output. Parasite-infected cells of recipient origin were seen in efferent lymph from day 11 onwards. However, cells of donor origin were never isolated either from efferent lymph or peripheral blood. Thus the parasite transferred from the inoculated donor cell line to the cells of the recipient before schizonts appeared in efferent lymph.

Diminished IL-2 responses and alteration of CD2 expression on CD8+ T cells are associated with a lack of cytotoxic T cell responses during Theileria annulata infection.

Theileria annulata is a tick-borne protozoan parasite which causes the disease bovine tropical theileriosis. In immunized or drug-treated animals, the pathogenic macroschizont stage of the parasite is destroyed by MHC class I-restricted cytotoxic T lymphocytes (CTL). Here we show that although CD8+ T cells increase greatly in number and display activation markers during an acute infection, they exhibit no killing of infected cells. During the ineffectual response, efferent lymph cells' ability to proliferate to IL-2 drops, coinciding with loss of MoAb binding to CD2 by CD8+ cells. When animals were treated with the anti-parasite drug 'Butalex', IL-2 responses, anti-CD2 antibody binding by CD8+ cells and strong CTL activity were restored within 24 h. The initial activation of CD4+ T cells by parasite-infected cells altering the IL-2 production in the draining lymph node is the likely cause of the failure of CTL responses.

Successful long-term in vitro cultivation of Theileria annulata schizonts in media supplemented with homologous and heterologous sera.

The efficacy of medium RPMI-1640 supplemented with either foetal bovine, normal bovine, goat or sheep sera was compared for prolonged in vitro propagation of Theileria annulata (Hisar) schizonts. Medium RPMI-1640 supplemented with 20% foetal bovine serum (standard growth medium) resulted in optimum growth of T. annulata (Hisar) schizonts in vitro. Comparable viability and non-viability counts were observed in growth media supplemented with normal bovine or goat sera. However, viability counts in medium supplemented with sheep serum were significantly lower than that of the standard medium. Mitotic indices of cultures of T. annulata (Hisar) schizonts were directly related to the extent of cell growth and were lower in various growth media supplemented with normal bovine, goat or sheep sera than in that of the standard medium. The results suggested that normal bovine and goat sera could be successfully used in place of foetal bovine serum in the growth medium for long-term in vitro propagation of T. annulata schizonts. The study will help in reducing the cost of large-scale in vitro propagation of T. annulata aimed at mass production of the cell culture vaccine.

Evidence for strain specificity in cytotoxic T-lymphocyte-mediated, major histocompatibility complex class I-dependent killing of Theileria annulata-infected cells.

Cattle immunised against Theileria annulata with one parasite strain have been found to be immune to re-challenge with different strains of the parasite. However, recent evidence of apparent strain specificity has been documented in cattle immunised with attenuated parasite-infected cells. In this study the strain specificity of major histocompatibility complex class I-restricted cytotoxic T-lymphocytes (CTL), a major anti-parasite effector mechanism, was examined. CTL generated following challenge with the Hissar (Indian) strain effectively lysed autologous cells infected with this strain of the parasite. However, CTL were less effective against cells infected with the Gharb (Moroccan) strain and showed virtually no reactivity against the Ankara (Turkish) strain, providing the first direct evidence for strain specificity in immune responses against T. annulata.

A stage-specific, parasite-induced, "window" of in vivo interferon-gamma production is associated with pathogenesis in Theileria annulata infection.

The tick-borne protozoan parasite Theileria annulata causes tropical theileriosis, a severe leukoproliferative disease of cattle, which naive susceptible animals fail to control. The parasite infects and transforms macrophages, developing in the local draining lymph node. IFN gamma has been shown to block parasite development in newly infected cells, and inhibits the growth of fully differentiated macroschizont stage-infected cells in vitro. However, the parasite has been found to specifically induce IFN gamma production by T cells and appears to flourish in the face of this T cell-derived response in vivo. Here we show that the production of IFN gamma in vivo is tightly controlled by the parasite. Induction of cytokine production by T cells is not initiated until the parasite has developed beyond the IFN gamma sensitive trophozoite stage. Cytokine production is kept high as infected macrophages develop, and IFN gamma appears to play an active role in maintaining the growth of these cells. Once the infection is fully established, IFN gamma is down regulated, avoiding potential inhibitory effects. Thus by controlling T cell IFN gamma production, the parasite induces a "window" of cytokine expression which promotes its own growth, but avoids potential inhibitory effects of the cytokine.

A novel cell surface proliferation-associated marker expressed on T cells and up-regulated on germinal center B cells.

In this study we present data on a novel cell surface antigen recognized by monoclonal antibody (mAb) VPM30, originally thought to recognize only bovine and ovine sIg+ B cells from peripheral blood. Here we show that the antigen, molecular mass 28 kDa, is not only found in B cell follicles in frozen sections, but when used on paraffin sections VPM30 specifically stains B cells in the light zone of germinal centers but not in the mantle or dark zones. In addition we show that the antigen is also expressed by 90% of T cells after activation, with kinetics of antigen expression mirroring those of proliferation. By both size and distribution, the antigen appears to be novel, corresponding to no known cluster of differentiation, and will be of great use in the study of ruminant cellular immune responses.

Susceptibility to tropical theileriosis of calves born to dams immunized with Theileria annulata (Hisar) cell culture vaccine.

The susceptibility/immune status to tropical theileriosis of calves born of immunized dams was evaluated. Six cows were vaccinated with the Theileria annulata cell culture vaccine in the eighth month of pregnancy. Sera from the immunized dams exhibited very high post-vaccination antibody titres as determined by the indirect fluorescent antibody (IFA) test. The calves born to these dams did not show antibodies against T. annulata at the time of birth (IFA titres of < 1:20). The new-born calves were fed colostrum from their mothers and were challenged with T. annulata-infected ground tick supernate at 5-7 days of age. All the calves developed fever (from day 5-6 onwards) and parasitological reactions (from day 8-9 onwards) after challenge. There was a significant decrease in the haemoglobin and packed cell volume of the calves after challenge. All the calves showed signs of acute theileriosis by day 9-10 after challenge and had to be treated with buparvaquone in order to save their lives. The study indicated that detectable levels of anti-theilerial antibodies were not transferred from immune dams to their offspring. All the calves born to immunized dams were fully susceptible to theileriosis and thus themselves needed vaccination.

A non-protective T helper 1 response against the intra-macrophage protozoan Theileria annulata.

Theileria annulata is a protozoan parasite which infects and transforms bovine macrophages. Infected macrophages possess augmented antigen presentation capabilities, as they are able to activate the majority of T cells from unexposed animals. In vivo, T cells in the draining lymph node (principal site of parasite development) are activated 'non-specifically' by the parasite. This event is followed by failure of the immune response to control the infection. Protective immune responses against intra-macrophage protozoa are usually mediated by T helper 1 (Th1) T cell responses. Here we examine the cytokine responses made by T. annulata-activated T cells. We show that the outcome of in vitro activation of T cells by parasitized macrophages is a skewing of their cytokine responses towards preferential expression of interferon-gamma (IFN-gamma) mRNA. The in vitro response is mirrored during in vivo infection, as greatly elevated amounts of IFN-gamma protein are found in lymph efferent from infected lymph nodes, while expression of IL-4 mRNA within the node stops. IFN-gamma production does not correlate with protection against the parasite, as infected cells flourish during peak IFN-gamma production, and only very small amounts of IFN-gamma are produced during the effective immune response of an immunized animal. Overproduction of IFN-gamma and loss of IL-4 expression are also likely to account for the failure of B cells to reach the light zone of germinal centres, a developmental step which is tightly regulated by cytokines.

Allograft responses can interfere with the development of immunity against Theileria annulata following vaccination with parasite infected cell lines.

Theileria annulata macroschizont-infected cell lines are successfully used as vaccines in several countries. The inoculated animals produce a strong allogeneic response against the MHC antigens of the immunizing cell line followed by an anti-parasite response. Immunity against the parasite wanes in the absence of challenge and re-immunization is sometimes recommended. However, it is not known if allogeneic responses generated by the first immunization with a T. annulata infected cell line will interfere with the boosting of immunity against the parasite at the time of re-immunization with the same cell line. Animals were primed against MHC antigens by skin grafting, followed by immunization with a T. annulata infected cell line prepared from the skin donor. A strong anti-MHC response was produced. This interfered with parasite transfer and the development of an anti-parasite immune response; the effect was more marked when a low vaccine cell dose was used. There was a negative correlation between the ease of isolating infected cells from the animals after cell line immunization, and the subsequent response to challenge. Where no cell lines could be isolated, the animals were fully susceptible to sporozoite challenge. These observations are of immediate importance in endemic areas where cell lines of T. annulata schizonts are being used as vaccines to control the disease.