Subhash Morzaria - Academia.edu (original) (raw)

Papers by Subhash Morzaria

Research paper thumbnail of Evidence for two single copy units in Theileria parva ribosomal RNA genes

Molecular and Biochemical Parasitology, 1994

Research paper thumbnail of Agarose entrapment method for the production of SfiI linking library for Theileria parva

Genetic Analysis Biomolecular Engineering, 1991

We have developed a simple method for isolation of SfiI linking clones from a eukaryotic genomic ... more We have developed a simple method for isolation of SfiI linking clones from a eukaryotic genomic DNA. The method involves the physical separation of the small proportion of plasmids in a plasmid genomic library that are linearized by SfiI digestion, from the bulk of molecules that remain circular, by ordinary electrophoresis through high-percentage gels of SeaPlaque agarose. Following the isolation of linearized molecules, their recircularization, and introduction into Escherichia coli, 55% of recovered plasmids contained inserts of the expected size, and 73% of these had SfiI sites. This represented a 25-fold enrichment of linking clones expected to be present at a frequency of 1/60 in the original library of 4- 6-kb fragments of genomic DNA of the protozoan parasite Theileria parva. This approach is rapid and obviates the need for introduction of a selectable marker. It is uniquely appropriate for linking clones spanning SfiI sites as this enzyme leaves degenerate 3' overhanging ends that preclude the direct ligation into vector sites required by most alternative strategies, but that favor the recircularization reactions used here.

Research paper thumbnail of Bovine theileriosis: progress in immunisation methods

International Journal of Animal Sciences, 1990

Research paper thumbnail of Isolation and Transmission of Cowdria Ruminantium (Causal Agent of Heartwateb Oisease) in Blue Nile Province, Sudan

Vet Res Commun, 1984

Heartwater was diagnosed in sheep at the Umm Banein Livestock Research Station, Blue Nile Provinc... more Heartwater was diagnosed in sheep at the Umm Banein Livestock Research Station, Blue Nile Province, Sudan.Cowdriaruminantium was isolated from sheep by subinoculation of blood into a goat. AdultAmblyommalepidum ticks fed as nymphs on this goat transmitted heartwater to a Friesian (Bostaurus) calf. Brain stabilates from this calf, either snapfrozen into liquid nitrogen or frozen slowly in a deepfreezer at −70°C,

Research paper thumbnail of Production and dose determination of the Infection and Treatment Method (ITM) Muguga cocktail vaccine used to control East Coast fever in cattle

Ticks and tick-borne diseases, 2016

The Infection and Treatment Method (ITM) of vaccination against the apicomplexan parasite Theiler... more The Infection and Treatment Method (ITM) of vaccination against the apicomplexan parasite Theileria parva has been used since the early 1970s and is still the only commercially available vaccine to combat the fatal bovine disease, East Coast fever (ECF). The disease is tick-transmitted and results in annual economic losses of at least $300 million per year. While this vaccine technology has been available for over 40 years, few attempts have been made to standardize the production process and characterize the vaccine. The latest batch was produced in early 2008 at the International Livestock Research Institute (ILRI). The vaccine production involves the use of cattle free from parasites routinely monitored throughout the production process, and a pathogen-free tick colony. This paper describes the protocol used in the recent production, and the process improvements, including improved quality control tools, that had not been employed in previous ITM productions. The paper also descr...

Research paper thumbnail of Isolation and transmission ofCowdria ruminantium (causal agent of heartwater disease) in Blue Nile Province, Sudan

Vet Res Commun, 1984

Heartwater was diagnosed in sheep at the Umm Banein Livestock Research Station, Blue Nile Provinc... more Heartwater was diagnosed in sheep at the Umm Banein Livestock Research Station, Blue Nile Province, Sudan.Cowdriaruminantium was isolated from sheep by subinoculation of blood into a goat. AdultAmblyommalepidum ticks fed as nymphs on this goat transmitted heartwater to a Friesian (Bostaurus) calf. Brain stabilates from this calf, either snapfrozen into liquid nitrogen or frozen slowly in a deepfreezer at −70°C,

Research paper thumbnail of A Differential Diagnostic Criterion for Babesia major and Babesia bigemina Vermicules from Tiek Haemolymlah

Parasitol Res, 1977

Babesia major mature and immature vermicules in the haemolymph of Haemaphysalis punctata were mea... more Babesia major mature and immature vermicules in the haemolymph of Haemaphysalis punctata were measured and found to be significantly larger than vermicules of Babesia bigemina. Mature B. major vermicules had a mean length of 15.53 micrometer and mature B. bigemina vermicules had a mean length of 11.79 micrometer. This difference provides a new criterion for the differentiation of the two species.

Research paper thumbnail of Ultrastructure of Babesia major vermicules from the tick Haemaphysalis punctata as demonstrated by negative staining

Zeitschrift Fur Parasitenkunde, May 1, 1978

The ultrastructure of Babesia major vermicules was studied in samples derived from the haemolymph... more The ultrastructure of Babesia major vermicules was studied in samples derived from the haemolymph of Haemaphysalis punctata adults and negatively stained with phosphotungstic acid. Most of the organelles observed were typical of those found in apicomplexan parasites. These were the apical complex with the polar ring and the ribs, micronemes and subpellicular microtubules. The number of ribs was 27 or 28. The outer membrane of the pellicle was composed of a large number of fibrils running along the length of the parasite. The inner membrane had large numbers of irregularly scattered holes. A cytoplasmic organelle similar to the granular body described in Theileria annulata ookinetes was seen for the first time in a B. major vermicule.

Research paper thumbnail of Cloning, characterization, and expression of a 200-kilodalton diagnostic antigen of Babesia bigemina

Journal of clinical microbiology, 2000

Current serological tests for Babesia bigemina use semipurified merozoite antigens derived from i... more Current serological tests for Babesia bigemina use semipurified merozoite antigens derived from infected erythrocytes. One of the major drawbacks of these tests is that antigen quality can vary from batch to batch. Since the quality of the antigen contributes to the sensitivity and specificity of serological tests, the use of standardized recombinant antigens should ensure consistency in assay quality. Previously, a 200-kDa merozoite antigen (p200) was identified as a candidate diagnostic antigen for use in a serological assay for the detection of B. bigemina antibodies in infected cattle. In this study, we have cloned, characterized, and expressed p200. A 3.5-kbp cDNA clone encoding p200 was isolated and shown to be almost full length, lacking approximately 300 bp at the 5' end. The predicted amino acid sequence shows that p200 consists of a long, highly charged central repeat region of an uninterrupted alpha helix, indicative of a fibrous protein. Immunoelectron microscopy loc...

Research paper thumbnail of Production and delivery costs of the infection-and-treatment vaccine against East Coast Fever

Research paper thumbnail of Immunization against East Coast fever (Theileria parva infection of cattle) by infection and treatment: chemoprophylaxis with N-pyrrolidinomethyl tetracycline

Tropenmedizin und Parasitologie

ABSTRACT

Research paper thumbnail of Characterization of an insect cell-derived Theileria parva sporozoite vaccine antigen and immunogenicity in cattle

Research paper thumbnail of Lessons learned from HPAI” FAO Animal Production and Health Paper 176

Over the last 3-4 years FAO’s role and priority has evolved from a predominantly emergency respon... more Over the last 3-4 years FAO’s role and priority has evolved from a predominantly emergency response to long term capacity building to improve surveillance, early detection and response in HPAI-infected and at-risk countries. FAO has also broadened its HPAI programme to include other EIDs and adopted a One Health approach to promote greater multisectoral and multidisciplinary participation. This transition provides an opportunity to reflect on the work done so far in HPAI control in the Asia region, and identify achievements, success stories, challenges, lessons learned and impact. This document represents the outcome of this exercise and provides in one place the knowledge, insights and recommendations of experts with first-hand knowledge and over eight years of experience in dealing with H5N1 HPAI in Asia.

Research paper thumbnail of East coast fever: 4. Further studies on the protection of cattle immunized with a combination of theilerial strains

Veterinary Parasitology, 1980

ABSTRACT

Research paper thumbnail of Animal Disease and Livestock-Based Livelihoods in Southern Cambodia

Research paper thumbnail of Ultrastructure of Babesia major vermicules from the tick Haemaphysalis punctata as demonstrated by negative staining

Zeitschrift für Parasitenkunde (Berlin, Germany), Jan 20, 1978

The ultrastructure of Babesia major vermicules was studied in samples derived from the haemolymph... more The ultrastructure of Babesia major vermicules was studied in samples derived from the haemolymph of Haemaphysalis punctata adults and negatively stained with phosphotungstic acid. Most of the organelles observed were typical of those found in apicomplexan parasites. These were the apical complex with the polar ring and the ribs, micronemes and subpellicular microtubules. The number of ribs was 27 or 28. The outer membrane of the pellicle was composed of a large number of fibrils running along the length of the parasite. The inner membrane had large numbers of irregularly scattered holes. A cytoplasmic organelle similar to the granular body described in Theileria annulata ookinetes was seen for the first time in a B. major vermicule.

Research paper thumbnail of Animal disease and livestock-keeper livelihoods in Southern Cambodia

International Development Planning Review, Feb 6, 2015

Research paper thumbnail of Development of sero-diagnostic and molecular tools for the control of important tick-borne pathogens of cattle in Africa

Parassitologia, 1999

Tick-borne diseases (TBDs) are a major economic constraint to livestock production in sub-Saharan... more Tick-borne diseases (TBDs) are a major economic constraint to livestock production in sub-Saharan Africa. ILRI is focussing on developing a range of products, such as vaccines, diagnostics and decision support services to underpin improved control programmes against these diseases. We have developed three highly sensitive and specific enzyme linked immuno-assays (ELISAs), which allow precise diagnosis of Theileria parva, Babesia bigemina and Anaplasma marginale. These tests have been standardised and validated using defined experimental and field infection sera. Parasite specific recombinant antigens and monoclonal antibodies against bovine immunoglobulins as secondary antibodies have played an important role in in enhancing the sensitivity and specificity of the assays. They have been further evaluated in on-farm longitudinal sero-epidemiological studies to define infection dynamics and disease risks in various farming systems in Kenya and Uganda. In addition, DNA-based tests for d...

Research paper thumbnail of Agarose entrapment method for the production of SfiI linking library for Theileria parva

Genetic analysis, techniques and applications, 1991

We have developed a simple method for isolation of SfiI linking clones from a eukaryotic genomic ... more We have developed a simple method for isolation of SfiI linking clones from a eukaryotic genomic DNA. The method involves the physical separation of the small proportion of plasmids in a plasmid genomic library that are linearized by SfiI digestion, from the bulk of molecules that remain circular, by ordinary electrophoresis through high-percentage gels of SeaPlaque agarose. Following the isolation of linearized molecules, their recircularization, and introduction into Escherichia coli, 55% of recovered plasmids contained inserts of the expected size, and 73% of these had SfiI sites. This represented a 25-fold enrichment of linking clones expected to be present at a frequency of 1/60 in the original library of 4- 6-kb fragments of genomic DNA of the protozoan parasite Theileria parva. This approach is rapid and obviates the need for introduction of a selectable marker. It is uniquely appropriate for linking clones spanning SfiI sites as this enzyme leaves degenerate 3' overhangi...

Research paper thumbnail of Effect of timing and intensity of challenge following immunization against East Coast fever

Veterinary Parasitology, 1987

When groups of Theileria parva parva Muguga-immunized cattle were given a homologous lethal chall... more When groups of Theileria parva parva Muguga-immunized cattle were given a homologous lethal challenge at different times after immunization, it was found that 4/6, 5/6, 6/6 and 6/6 animals survived when challenged on Days 5, 10, 20 and 30, respectively, post-immunization. With a heterologous challenge (T.p.parva Marikebuni), 2/6, 5/6, 4/6, 4/6 and 5/6 cattle survived when challenged on Days 5, 10, 20 and 30, respectively, after immunization. All controls, except one, died of East Coast fever (ECF). The survivor underwent severe ECF and recovered after a prolonged convalescence. When two T.p.parva Muguga-immunized animals were each given homologous challenge by application of 1000 infected ticks (infection rate of 20 infected acini (i.a.) per tick), both survived a mild ECF reaction. When groups of T.p.parva Muguga- or T.p.parva Muguga/Marikebuni-immunized cattle were challenged with different doses of T.p.parva Muguga sporozoites (equivalent of 140, 1400 and 14,000 i.a. per animal), 28/29 cattle survived. All controls died of ECF. It was concluded that cattle could be safely exposed to tick challenge 1 week after immunization by infection and treatment using appropriate immunizing stock(s). Massive homologous challenge did not break through the immunity induced by the immunization procedure.

Research paper thumbnail of Evidence for two single copy units in Theileria parva ribosomal RNA genes

Molecular and Biochemical Parasitology, 1994

Research paper thumbnail of Agarose entrapment method for the production of SfiI linking library for Theileria parva

Genetic Analysis Biomolecular Engineering, 1991

We have developed a simple method for isolation of SfiI linking clones from a eukaryotic genomic ... more We have developed a simple method for isolation of SfiI linking clones from a eukaryotic genomic DNA. The method involves the physical separation of the small proportion of plasmids in a plasmid genomic library that are linearized by SfiI digestion, from the bulk of molecules that remain circular, by ordinary electrophoresis through high-percentage gels of SeaPlaque agarose. Following the isolation of linearized molecules, their recircularization, and introduction into Escherichia coli, 55% of recovered plasmids contained inserts of the expected size, and 73% of these had SfiI sites. This represented a 25-fold enrichment of linking clones expected to be present at a frequency of 1/60 in the original library of 4- 6-kb fragments of genomic DNA of the protozoan parasite Theileria parva. This approach is rapid and obviates the need for introduction of a selectable marker. It is uniquely appropriate for linking clones spanning SfiI sites as this enzyme leaves degenerate 3' overhanging ends that preclude the direct ligation into vector sites required by most alternative strategies, but that favor the recircularization reactions used here.

Research paper thumbnail of Bovine theileriosis: progress in immunisation methods

International Journal of Animal Sciences, 1990

Research paper thumbnail of Isolation and Transmission of Cowdria Ruminantium (Causal Agent of Heartwateb Oisease) in Blue Nile Province, Sudan

Vet Res Commun, 1984

Heartwater was diagnosed in sheep at the Umm Banein Livestock Research Station, Blue Nile Provinc... more Heartwater was diagnosed in sheep at the Umm Banein Livestock Research Station, Blue Nile Province, Sudan.Cowdriaruminantium was isolated from sheep by subinoculation of blood into a goat. AdultAmblyommalepidum ticks fed as nymphs on this goat transmitted heartwater to a Friesian (Bostaurus) calf. Brain stabilates from this calf, either snapfrozen into liquid nitrogen or frozen slowly in a deepfreezer at −70°C,

Research paper thumbnail of Production and dose determination of the Infection and Treatment Method (ITM) Muguga cocktail vaccine used to control East Coast fever in cattle

Ticks and tick-borne diseases, 2016

The Infection and Treatment Method (ITM) of vaccination against the apicomplexan parasite Theiler... more The Infection and Treatment Method (ITM) of vaccination against the apicomplexan parasite Theileria parva has been used since the early 1970s and is still the only commercially available vaccine to combat the fatal bovine disease, East Coast fever (ECF). The disease is tick-transmitted and results in annual economic losses of at least $300 million per year. While this vaccine technology has been available for over 40 years, few attempts have been made to standardize the production process and characterize the vaccine. The latest batch was produced in early 2008 at the International Livestock Research Institute (ILRI). The vaccine production involves the use of cattle free from parasites routinely monitored throughout the production process, and a pathogen-free tick colony. This paper describes the protocol used in the recent production, and the process improvements, including improved quality control tools, that had not been employed in previous ITM productions. The paper also descr...

Research paper thumbnail of Isolation and transmission ofCowdria ruminantium (causal agent of heartwater disease) in Blue Nile Province, Sudan

Vet Res Commun, 1984

Heartwater was diagnosed in sheep at the Umm Banein Livestock Research Station, Blue Nile Provinc... more Heartwater was diagnosed in sheep at the Umm Banein Livestock Research Station, Blue Nile Province, Sudan.Cowdriaruminantium was isolated from sheep by subinoculation of blood into a goat. AdultAmblyommalepidum ticks fed as nymphs on this goat transmitted heartwater to a Friesian (Bostaurus) calf. Brain stabilates from this calf, either snapfrozen into liquid nitrogen or frozen slowly in a deepfreezer at −70°C,

Research paper thumbnail of A Differential Diagnostic Criterion for Babesia major and Babesia bigemina Vermicules from Tiek Haemolymlah

Parasitol Res, 1977

Babesia major mature and immature vermicules in the haemolymph of Haemaphysalis punctata were mea... more Babesia major mature and immature vermicules in the haemolymph of Haemaphysalis punctata were measured and found to be significantly larger than vermicules of Babesia bigemina. Mature B. major vermicules had a mean length of 15.53 micrometer and mature B. bigemina vermicules had a mean length of 11.79 micrometer. This difference provides a new criterion for the differentiation of the two species.

Research paper thumbnail of Ultrastructure of Babesia major vermicules from the tick Haemaphysalis punctata as demonstrated by negative staining

Zeitschrift Fur Parasitenkunde, May 1, 1978

The ultrastructure of Babesia major vermicules was studied in samples derived from the haemolymph... more The ultrastructure of Babesia major vermicules was studied in samples derived from the haemolymph of Haemaphysalis punctata adults and negatively stained with phosphotungstic acid. Most of the organelles observed were typical of those found in apicomplexan parasites. These were the apical complex with the polar ring and the ribs, micronemes and subpellicular microtubules. The number of ribs was 27 or 28. The outer membrane of the pellicle was composed of a large number of fibrils running along the length of the parasite. The inner membrane had large numbers of irregularly scattered holes. A cytoplasmic organelle similar to the granular body described in Theileria annulata ookinetes was seen for the first time in a B. major vermicule.

Research paper thumbnail of Cloning, characterization, and expression of a 200-kilodalton diagnostic antigen of Babesia bigemina

Journal of clinical microbiology, 2000

Current serological tests for Babesia bigemina use semipurified merozoite antigens derived from i... more Current serological tests for Babesia bigemina use semipurified merozoite antigens derived from infected erythrocytes. One of the major drawbacks of these tests is that antigen quality can vary from batch to batch. Since the quality of the antigen contributes to the sensitivity and specificity of serological tests, the use of standardized recombinant antigens should ensure consistency in assay quality. Previously, a 200-kDa merozoite antigen (p200) was identified as a candidate diagnostic antigen for use in a serological assay for the detection of B. bigemina antibodies in infected cattle. In this study, we have cloned, characterized, and expressed p200. A 3.5-kbp cDNA clone encoding p200 was isolated and shown to be almost full length, lacking approximately 300 bp at the 5' end. The predicted amino acid sequence shows that p200 consists of a long, highly charged central repeat region of an uninterrupted alpha helix, indicative of a fibrous protein. Immunoelectron microscopy loc...

Research paper thumbnail of Production and delivery costs of the infection-and-treatment vaccine against East Coast Fever

Research paper thumbnail of Immunization against East Coast fever (Theileria parva infection of cattle) by infection and treatment: chemoprophylaxis with N-pyrrolidinomethyl tetracycline

Tropenmedizin und Parasitologie

ABSTRACT

Research paper thumbnail of Characterization of an insect cell-derived Theileria parva sporozoite vaccine antigen and immunogenicity in cattle

Research paper thumbnail of Lessons learned from HPAI” FAO Animal Production and Health Paper 176

Over the last 3-4 years FAO’s role and priority has evolved from a predominantly emergency respon... more Over the last 3-4 years FAO’s role and priority has evolved from a predominantly emergency response to long term capacity building to improve surveillance, early detection and response in HPAI-infected and at-risk countries. FAO has also broadened its HPAI programme to include other EIDs and adopted a One Health approach to promote greater multisectoral and multidisciplinary participation. This transition provides an opportunity to reflect on the work done so far in HPAI control in the Asia region, and identify achievements, success stories, challenges, lessons learned and impact. This document represents the outcome of this exercise and provides in one place the knowledge, insights and recommendations of experts with first-hand knowledge and over eight years of experience in dealing with H5N1 HPAI in Asia.

Research paper thumbnail of East coast fever: 4. Further studies on the protection of cattle immunized with a combination of theilerial strains

Veterinary Parasitology, 1980

ABSTRACT

Research paper thumbnail of Animal Disease and Livestock-Based Livelihoods in Southern Cambodia

Research paper thumbnail of Ultrastructure of Babesia major vermicules from the tick Haemaphysalis punctata as demonstrated by negative staining

Zeitschrift für Parasitenkunde (Berlin, Germany), Jan 20, 1978

The ultrastructure of Babesia major vermicules was studied in samples derived from the haemolymph... more The ultrastructure of Babesia major vermicules was studied in samples derived from the haemolymph of Haemaphysalis punctata adults and negatively stained with phosphotungstic acid. Most of the organelles observed were typical of those found in apicomplexan parasites. These were the apical complex with the polar ring and the ribs, micronemes and subpellicular microtubules. The number of ribs was 27 or 28. The outer membrane of the pellicle was composed of a large number of fibrils running along the length of the parasite. The inner membrane had large numbers of irregularly scattered holes. A cytoplasmic organelle similar to the granular body described in Theileria annulata ookinetes was seen for the first time in a B. major vermicule.

Research paper thumbnail of Animal disease and livestock-keeper livelihoods in Southern Cambodia

International Development Planning Review, Feb 6, 2015

Research paper thumbnail of Development of sero-diagnostic and molecular tools for the control of important tick-borne pathogens of cattle in Africa

Parassitologia, 1999

Tick-borne diseases (TBDs) are a major economic constraint to livestock production in sub-Saharan... more Tick-borne diseases (TBDs) are a major economic constraint to livestock production in sub-Saharan Africa. ILRI is focussing on developing a range of products, such as vaccines, diagnostics and decision support services to underpin improved control programmes against these diseases. We have developed three highly sensitive and specific enzyme linked immuno-assays (ELISAs), which allow precise diagnosis of Theileria parva, Babesia bigemina and Anaplasma marginale. These tests have been standardised and validated using defined experimental and field infection sera. Parasite specific recombinant antigens and monoclonal antibodies against bovine immunoglobulins as secondary antibodies have played an important role in in enhancing the sensitivity and specificity of the assays. They have been further evaluated in on-farm longitudinal sero-epidemiological studies to define infection dynamics and disease risks in various farming systems in Kenya and Uganda. In addition, DNA-based tests for d...

Research paper thumbnail of Agarose entrapment method for the production of SfiI linking library for Theileria parva

Genetic analysis, techniques and applications, 1991

We have developed a simple method for isolation of SfiI linking clones from a eukaryotic genomic ... more We have developed a simple method for isolation of SfiI linking clones from a eukaryotic genomic DNA. The method involves the physical separation of the small proportion of plasmids in a plasmid genomic library that are linearized by SfiI digestion, from the bulk of molecules that remain circular, by ordinary electrophoresis through high-percentage gels of SeaPlaque agarose. Following the isolation of linearized molecules, their recircularization, and introduction into Escherichia coli, 55% of recovered plasmids contained inserts of the expected size, and 73% of these had SfiI sites. This represented a 25-fold enrichment of linking clones expected to be present at a frequency of 1/60 in the original library of 4- 6-kb fragments of genomic DNA of the protozoan parasite Theileria parva. This approach is rapid and obviates the need for introduction of a selectable marker. It is uniquely appropriate for linking clones spanning SfiI sites as this enzyme leaves degenerate 3' overhangi...

Research paper thumbnail of Effect of timing and intensity of challenge following immunization against East Coast fever

Veterinary Parasitology, 1987

When groups of Theileria parva parva Muguga-immunized cattle were given a homologous lethal chall... more When groups of Theileria parva parva Muguga-immunized cattle were given a homologous lethal challenge at different times after immunization, it was found that 4/6, 5/6, 6/6 and 6/6 animals survived when challenged on Days 5, 10, 20 and 30, respectively, post-immunization. With a heterologous challenge (T.p.parva Marikebuni), 2/6, 5/6, 4/6, 4/6 and 5/6 cattle survived when challenged on Days 5, 10, 20 and 30, respectively, after immunization. All controls, except one, died of East Coast fever (ECF). The survivor underwent severe ECF and recovered after a prolonged convalescence. When two T.p.parva Muguga-immunized animals were each given homologous challenge by application of 1000 infected ticks (infection rate of 20 infected acini (i.a.) per tick), both survived a mild ECF reaction. When groups of T.p.parva Muguga- or T.p.parva Muguga/Marikebuni-immunized cattle were challenged with different doses of T.p.parva Muguga sporozoites (equivalent of 140, 1400 and 14,000 i.a. per animal), 28/29 cattle survived. All controls died of ECF. It was concluded that cattle could be safely exposed to tick challenge 1 week after immunization by infection and treatment using appropriate immunizing stock(s). Massive homologous challenge did not break through the immunity induced by the immunization procedure.