RESUMEN

INTRODUCTION: Tick-borne pathogens (TBPs) constitute an emerging threat to public and animal health especially in the African continent, where land-use change, and wildlife loss are creating new opportunities for disease transmission. A review of TBPs with a focus on ticks determined the epidemiology of Rhipicephalus ticks in heartwater and the affinity of each Rickettsia species for different tick genera. We conducted a systematic review and meta-analysis to collect, map and estimate the molecular prevalence of Anaplasmataceae, Rickettsiaceae and Coxiellaceae in African wildlife. MATERIALS AND METHODS: Relevant scientific articles were retrieved from five databases: PubMed, ScienceDirect, Scopus, Ovid and OAIster. Publications were selected according to pre-determined exclusion criteria and evaluated for risk of bias using the appraisal tool for cross-sectional studies (AXIS). We conducted an initial descriptive analysis followed by a meta-analysis to estimate the molecular prevalence of each pathogen. Subgroup analysis and meta-regression models were employed to unravel associations with disease determinants. Finally, the quality of evidence of every estimate was finally assessed. RESULTS: Out of 577 retrieved papers, a total of 41 papers were included in the qualitative analysis and 27 in the meta-analysis. We retrieved 21 Anaplasmataceae species, six Rickettsiaceae species and Coxiella burnetii. Meta-analysis was performed for a total of 11 target pathogens. Anaplasma marginale, Ehrlichia ruminantium and Anaplasma centrale were the most prevalent in African bovids (13.9 %, CI: 0-52.4 %; 20.9 %, CI: 4.1-46.2 %; 13.9 %, CI: 0-68.7 %, respectively). Estimated TBPs prevalences were further stratified per animal order, family, species and sampling country. DISCUSSION: We discussed the presence of a sylvatic cycle for A. marginale and E. ruminantium in wild African bovids, the need to investigate A. phagocytophilum in African rodents and non-human primates as well as E. canis in the tissues of wild carnivores, and a lack of data and characterization of Rickettsia species and C. burnetii. CONCLUSION: Given the lack of epidemiological data on wildlife diseases, the current work can serve as a starting point for future epidemiological and/or experimental studies.

Asunto(s)

Anaplasmataceae , Animales Salvajes , Animales , Prevalencia , Animales Salvajes/microbiología , Anaplasmataceae/aislamiento & purificación , África/epidemiología , Rickettsiaceae , Enfermedades por Picaduras de Garrapatas/veterinaria , Enfermedades por Picaduras de Garrapatas/epidemiología , Enfermedades por Picaduras de Garrapatas/microbiología , Infecciones por Anaplasmataceae/veterinaria , Infecciones por Anaplasmataceae/epidemiología , Infecciones por Anaplasmataceae/microbiología

RESUMEN

A participatory epidemiological (PE) study was conducted with livestock keepers in Moroto and Kotido districts, Karamoja Region, Uganda, between October and December 2013 to determine the management options and relative importance of tick-borne diseases (TBDs) amongst transhumant zebu cattle. Data collection involved 24 focus group discussions (each comprising 8-12 people) in 24 settlement areas (manyattas), key informant interviews (30), direct observation, a review of surveillance data, clinical examination, and laboratory confirmation of cases of TBDs. Methods used in group discussions included semi-structured interviews, simple ranking, pairwise ranking, matrix scoring, proportional piling and participatory mapping. The results of pairwise comparison showed the Ngakarimojong-named diseases, lokit (East Coast fever, ECF), lopid (anaplasmosis), loukoi (contagious bovine pleuropneumonia, CBPP), lokou (heartwater) and lokulam (babesiosis), were considered the most important cattle diseases in Moroto in that order, while ECF, anaplasmosis, trypanosomosis (ediit), CBPP and nonspecific diarrhoea (loleo) were most important in Kotido. Strong agreement between informant groups (Kendall's coefficient of concordance W=0.568 and 0.682; p<0.001) in pairwise ranking indicated that the diseases were a common problem in selected districts. East Coast fever had the highest median score for incidence (18% [range: 2, 33]) in Moroto, followed by anaplasmosis (17.5% [8,32]) and CBPP (9% [1,21]). Most animals that suffered from ECF, anaplasmosis, heartwater and babesiosis died, as the respective median scores for case fatality rates (CFR) were 89.5% (42, 100), 82.8% (63, 100), 66.7% (20, 100) and 85.7% (0, 100). In Kotido, diseases with high incidence scores were ECF (21% [6,32]), anaplasmosis (17% [10,33]) and trypanosomosis (8% [2,18]). The CFRs for ECF and anaplasmosis were 81.7% (44, 100) and 70.7% (48, 100), respectively. Matrix scoring revealed that disease indicators showed strong agreement (W=0.382-0.659, p<0.05-p<0.001) between informant groups. Inadequate knowledge, poor veterinary services and limited availability of drugs were the main constraints that hindered the control of TBDs. Hand picking of ticks was done by all pastoralists while hand spraying with acaricides was irregular, often determined by availability of drug supplies and money. It was concluded that TBDs, particularly ECF and anaplasmosis were important diseases in this pastoral region. Results from this study may assist in the design of feasible control strategies.

Asunto(s)

Crianza de Animales Domésticos/métodos , Enfermedades de los Bovinos/epidemiología , Enfermedades de los Bovinos/prevención & control , Enfermedades por Picaduras de Garrapatas/epidemiología , Enfermedades por Picaduras de Garrapatas/prevención & control , Animales , Bovinos , Enfermedades de los Bovinos/microbiología , Enfermedades de los Bovinos/parasitología , Enfermedades por Picaduras de Garrapatas/microbiología , Enfermedades por Picaduras de Garrapatas/parasitología , Garrapatas/fisiología , Uganda/epidemiología

RESUMEN

We conducted a study to investigate tick species diversity, seroprevalence of antibodies to Anaplasma marginale and Theileria parva, and the risk factors for these infections among cattle under a transhumant production system in Karamoja Region, Uganda, from November 2013 through January 2014. Twenty herds were randomly selected from 20 purposively-selected superherds. Semi-structured interviews and piling for annual proportion of tick-borne disease (TBD) cases in different age groups, with pastoralist groups, clinical examinations and field observations were employed to obtain information related to the epidemiology of TBDs. Ticks were collected and identified from whole body inspections of at least seven systematically selected cattle in each herd. Concurrently, serum was collected from 397 cattle. Antibodies to A. marginale were detected by MSP-5 competitive inhibition enzyme-linked immunosorbent assay, and to T. parva by indirect fluorescent antibody test. Clinical examinations and informant interviews showed that TBDs affected all age groups of cattle. Tick species that have not been reported in recent studies from other parts of Uganda were collected, namely Amblyomma lepidum, Hyalomma truncatum, Amblyomma gemma, and Rhipicephalus pulchellus. Out of the 10,923 ticks collected, Rhipicephalus appendiculatus (54.4%) was the most abundant species followed by Rhipicephalus (Boophilus) decoloratus (17.7%), Amblyomma variegatum (12%) and A. lepidum (11.6%). Two-thirds of the sampled cattle had moderate (37.3%, 11-50 ticks) to abundant (28.6%, >50 ticks) numbers of ticks. Seroprevalence of A. marginale was high (86.6%, 95% confidence interval [CI] 80.8%-91.8%), while that of T. parva was low (14.6%, 95% CI 7.1%-22.4%). Cattle of 5-12months (18.3%, Odds ratio [OR]=4.1) and 13-24months (30.3%, OR=8.0) were more likely to be seropositive for T. parva than those >24months. For A. marginale, cattle of 13-24months (92.4%, OR=2.7) and >24months (89.7%, OR=2.0) were more likely to be seropositive than those 5-12months. There was a significant difference (p<0.001, OR=6.5) in the proportion of T. parva seropositive animals between Moroto (24.5%) and Kotido districts (4.8%), but not for A. marginale. In conclusion, the low seroprevalence for T. parva, possibly due to limited exposure in calves, may suggest a high likelihood of ECF in cattle. High seroprevalence for A. marginale suggests that a high proportion of cattle were exposed to infection. The findings provide knowledge of epidemiology of TBDs in Karamoja cattle and support for strategic control and improvement of cattle productivity.

RESUMEN

Ehrlichia ruminantium is an obligate intracellular bacterial pathogen which causes heartwater, a serious tick-borne disease of ruminants throughout sub-Saharan Africa. The development of promising recombinant vaccines has been reported previously, but none has been as effective as immunisation with live organisms. In this study we have used reverse vaccinology to identify proteins that elicit an in vitro cellular immune response similar to that induced by intact E. ruminantium. The experimental strategy involved four successive steps: (i) in silico selection of the most likely vaccine candidate genes from the annotated genome; (ii) cloning and expression of the selected genes; (iii) in vitro screening of the expressed proteins for their ability to induce interferon-gamma (IFN-γ) production in E. ruminantium-immune lymphocytes; and (iv) further examination of the cytokine response profiles of those lymphocytes which tested positive for IFN-γ induction. Based on their overall cytokine induction profiles the recombinant proteins were divided into four distinct groups. Eleven recombinant proteins induced a cytokine profile that was similar to the recall immune response induced by immune peripheral blood mononuclear cells (PBMC) stimulated with intact E. ruminantium. This response comprised the upregulation of cytokines associated with adaptive cellular immune responses as well as innate immunity. A successful vaccine may therefore need to contain a combination of recombinant proteins which induce both immune pathways to ensure protection against heartwater.

Asunto(s)

Proteínas Bacterianas/inmunología , Ehrlichia ruminantium/inmunología , Animales , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/fisiología , Proteínas Bacterianas/farmacología , Vacunas Bacterianas/inmunología , Bovinos/inmunología , Bovinos/microbiología , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/microbiología , Hidropericardio/inmunología , Inmunidad Celular/efectos de los fármacos , Inmunidad Celular/inmunología , Interferón gamma/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Ovinos/inmunología , Ovinos/microbiología , Enfermedades de las Ovejas/inmunología , Enfermedades de las Ovejas/microbiología

RESUMEN

Blood samples were collected from 71 tsessebes relocated from the deproclaimed Vaalbos National Park to Mokala National Park, South Africa. DNA was extracted from the samples and the reverse line blot (RLB) hybridization technique was used to detect and identify any haemoparasites present. Six samples hybridized to the Theileria/Babesia genus-specific probe, the Theileria genus-specific probe and the Theileria sp. (sable) probe, while 3/6 also hybridized to the Theileria separata probe. Full-length 18S rRNA genes of the Theileria spp. detected were amplified, cloned and sequenced. Two novel Theileria 18S rRNA gene sequences were identified which are phylogenetically very closely related to both Theileria sp. (sable) and T. separata. All animals appeared to be in good health. It seems likely, therefore, that these Theileria spp. do not cause disease under normal circumstances. Nevertheless, care should be taken when translocating wild animals, as introduction of novel piroplasm parasites into new areas could cause clinical disease and losses in naïve wildlife and domestic animals, and new parasite species could become established in areas in which they previously did not occur.

Asunto(s)

Antílopes/parasitología , Vectores Arácnidos/parasitología , Rhipicephalus/parasitología , Theileria/aislamiento & purificación , Theileriosis/parasitología , Animales , Secuencia de Bases , ADN Protozoario/sangre , ADN Protozoario/química , ADN Protozoario/genética , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Filogenia , ARN Protozoario/genética , ARN Ribosómico 18S/genética , Análisis de Secuencia de ADN/veterinaria , Sudáfrica/epidemiología , Theileria/genética , Theileriosis/epidemiología , Theileriosis/transmisión , Enfermedades por Picaduras de Garrapatas/epidemiología , Enfermedades por Picaduras de Garrapatas/parasitología , Enfermedades por Picaduras de Garrapatas/transmisión

RESUMEN

Theileria parva, the most important bovine theilerial species in sub-Saharan Africa, causes widespread mortality and morbidity in endemic areas. A survey was conducted using buffy-coat specimens from 60 apparently healthy adult communally herded Nguni-type cattle at the northeastern edge of the Hluhluwe-iMfolozi Park to determine, by means of PCR and Reverse Line Blot (RLB) hybridisation, the occurrence of Theileria and Babesia species. The presence of Trypanosoma species was determined using PCR-RFLP. Results showed that 6.7 % of the specimens were positive for Theileria parva. This significant finding suggests that cattle in South Africa, and not only African buffaloes (Syncerus caffer), may be subclinical carriers of T. parva. Other species identified were T. mutans (83.3%), T. velifera (70.0%), Theileria sp. (sable) (46.8%) and T taurotragi (1.7%). Two specimens (3.3%) were positive for Babesia bovis and single specimens (1.7%) positive for B. bigemina and B. rossi, respectively. Mixed infections, of up to 4 species, were common (65.0%). Only 1 specimen was found to be positive for Trypanosoma vivax, and 2 for T theileri, of which only the first species is pathogenic.

Asunto(s)

Babesiosis/veterinaria , Enfermedades de los Bovinos/epidemiología , Theileria parva/aislamiento & purificación , Theileriosis/epidemiología , Animales , Babesia/aislamiento & purificación , Babesiosis/epidemiología , Búfalos/parasitología , Bovinos , Enfermedades de los Bovinos/parasitología , Reservorios de Enfermedades/parasitología , Reservorios de Enfermedades/veterinaria , Femenino , Incidencia , Masculino , Reacción en Cadena de la Polimerasa/veterinaria , Sudáfrica/epidemiología

RESUMEN

A previously identified polymorphic Ehrlichia ruminantium gene, Erum2510, was investigated to determine its ability to induce protective immunity in ruminants following two different DNA immunisation strategies; DNA-only and a DNA prime/recombinant protein (rprotein) boost immunisation. The DNA-only vaccine was also compared to a cocktail of three polymorphic E. ruminantium (Welgevonden) open reading frames (ORFs) adjacent to Erum2510 in the genome. Weak protection was observed in animals immunised with the pCMViUBs_Erum2510 construct alone, while none of the animals immunised with the DNA cocktail were protected. In contrast, all five animals immunised using a DNA prime/rprotein boost strategy survived challenge, thereby indicating that Erum2510 is a good candidate for inclusion in a recombinant vaccine against heartwater. One drawback of using polymorphic genes is a possible lack of cross-protection between genotypes, therefore the genetic diversity of Erum2510 was investigated to establish the degree of polymorphism among different E. ruminantium stocks. Three distinct genotypes were identified indicating that if this gene is used as a vaccine (prime/boost strategy) the vaccine should include a representative Erum2510 gene from each genotype.

Asunto(s)

Vacunas Bacterianas/inmunología , Ehrlichia ruminantium/genética , Hidropericardio/prevención & control , Animales , Clonación Molecular , Ehrlichia ruminantium/inmunología , Genotipo , Hidropericardio/inmunología , Inmunización Secundaria , Sistemas de Lectura Abierta , Filogenia , Polimorfismo Genético , Ovinos , Vacunas Sintéticas/inmunología

RESUMEN

A 10-year-old tame zebra gelding was presented after suffering from lethargy, nervousness, reported anaemia and icterus as well as a decreased appetite. These symptoms were seen over some months, with changing severity. The animal was immobilised, treated, and blood specimens were submitted for haematology and biochemistry. This report describes molecular characterisation of Theileria equi recovered from this animal, as well as the clinical findings, treatment and historical relevance of piroplasmosis in zebra in southern Africa.

Asunto(s)

Antiprotozoarios/uso terapéutico , Equidae , Imidocarbo/uso terapéutico , Theileria/aislamiento & purificación , Theileriosis/diagnóstico , Animales , ADN Protozoario/análisis , Diagnóstico Diferencial , Masculino , Recurrencia , Theileriosis/tratamiento farmacológico , Resultado del Tratamiento

RESUMEN

Heterologous prime/boost immunisation strategies using the Ehrlichia ruminantium 1H12 pCMViUBs_ORFs [Pretorius A, Collins NE, Steyn HC, Van Strijp F, Van Kleef M, Allsopp BA. Protection against heartwater by DNA immunisation with four Ehrlichia ruminantium open reading frames. Vaccine 2007;25(12):2316-24] were investigated in this study. All the animals immunised twice with a recombinant (r) DNA cocktail of four 1H12 pCMViUBs_ORFs followed by a r1H12 protein and those immunised 3x with 1H12 plasmid rDNA showed 100% protection against a virulent E. ruminantium Welgevonden needle challenge. In addition, 90% of the sheep immunised twice with rDNA and boosted with r1H12 lumpy skin disease virus (LSDV) survived. Only the lymphocytes isolated from the r1H12 protein boost group showed specific proliferation and increased interferon (IFN)-gamma expression. In contrast, only 20% protection was obtained in animals immunised with the rDNA prime/r1H12 protein boost when subjected to natural tick challenge in the field. Thus this heterologous prime/boost immunisation strategy had not conferred any significant protection against a field challenge.

Asunto(s)

Transmisión de Enfermedad Infecciosa/prevención & control , Ehrlichia ruminantium/inmunología , Hidropericardio/prevención & control , Inmunización Secundaria/métodos , Enfermedades de las Ovejas/prevención & control , Vacunas de ADN/inmunología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proliferación Celular , Vectores de Enfermedades , Fiebre/etiología , Vectores Genéticos , Hidropericardio/inmunología , Interferón gamma/biosíntesis , Virus de la Dermatosis Nodular Contagiosa/genética , Linfocitos/inmunología , Ovinos , Enfermedades de las Ovejas/inmunología , Análisis de Supervivencia , Garrapatas/microbiología , Factores de Tiempo , Vacunas de Subunidad/inmunología

RESUMEN

We have reported previously that a recombinant DNA vaccine consisting of four Ehrlichia ruminantium (Welgevonden) open reading frames (ORFs) known as the 1H12 cocktail provided protection against a virulent E. ruminantium (Welgevonden) needle challenge in sheep. In this study, we have investigated the vaccine effectiveness of two other cocktails of E. ruminantium (Welgevonden) ORFs, as well as single ORFs from the 1H12 cocktail, to protect sheep against a virulent needle challenge with the homologous strain. Each individual 1H12 ORF provided protection, but all the animals vaccinated with the other cocktails succumbed to the challenge.

Asunto(s)

Ehrlichia ruminantium/inmunología , Hidropericardio/inmunología , Inmunización/métodos , Sistemas de Lectura Abierta/genética , Vacunas de ADN/inmunología , Animales , Islas de CpG/genética , Ehrlichia ruminantium/genética , Hidropericardio/prevención & control , Linfocitos/inmunología , Ovinos , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética

RESUMEN

Heartwater, an economically important tickborne disease of wild and domestic ruminants, is caused by the intracellular rickettsia Ehrlichia (formerly Cowdria) ruminantium. The only commercially available immunization procedure is more than 50 years old and uses an infection and treatment regimen using a preparation of virulent organisms in cryopreserved sheep blood. Much research has been conducted into the development of attenuated, inactivated, and nucleic acid vaccines over the last half-century, with relatively little success until recently. We describe here the development of two new experimental vaccines, a live attenuated vaccine and a nucleic acid vaccine. The attenuation of virulent E. ruminantium was achieved by growing the organisms in a continuous canine macrophage-monocyte cell line. After more than 125 passages the cultures produced no disease when inoculated into mice or sheep, and the animals were completely protected against a subsequent lethal homologous needle challenge. The nucleic acid vaccine consists of a cocktail of four E. ruminantium genes, from a genetic locus involved in nutrient transport, cloned in a DNA vaccine vector. Sheep immunized with this cocktail were completely protected against a subsequent lethal needle challenge, either with the homologous isolate or with any one of five different virulent heterologous isolates. Protection against a field challenge in a heartwater endemic area, however, was relatively poor. Genetic characterization of the E. ruminantium genotypes in the challenge area did not identify any having major differences from those used in the heterologous needle challenge experiments, so lack of cross-immunity between the vaccine genotype and those in the field was unlikely to be the main reason for the lack of protection. We believe that a needle challenge is far less severe than a tick challenge, and that the immunity engendered by the DNA vaccine alone was not sufficient to protect against the natural route of infection. Boosting with live organisms after DNA vaccination results in much higher levels of protection against tick challenge than DNA vaccination alone, suggesting that improved methods of boosting could lead to more effective immunization.

Asunto(s)

Vacunas Bacterianas/inmunología , Ehrlichia ruminantium/inmunología , Hidropericardio/inmunología , Vacunas de ADN/inmunología , Animales , Animales Domésticos , Animales Salvajes , Línea Celular , Perros , Ehrlichia ruminantium/genética , Ehrlichia ruminantium/patogenicidad , Geografía , Hidropericardio/prevención & control , Sistemas de Lectura Abierta , Ovinos , Sudáfrica , Vacunas Atenuadas/inmunología , Virulencia

RESUMEN

Immune responses of infected animals and humans have been reported to be directed against variable outer membrane proteins of Ehrlichia species that are encoded by polymorphic multigene families. In Ehrlichia (= Cowdria) ruminantium, two immunodominant proteins have been identified, namely major antigenic protein 1 (MAP1) and open reading frame 2 (ORF2). The aim of the present study was to identify additional map1-like genes in the E. ruminantium genome. A 12 kb clone that hybridized with the map1 probe was amplified using long template PCR. The PCR product was partially digested, cloned, and sequenced. Four map1-like genes are located in tandem, namely map1-1 (orf2) and map1-2 upstream of map1 as well as map1+1 downstream of map1. A large ORF (2.4 kb) at the 3' end is homologous to secA genes of other organisms. The sequence data in this study support other findings that outer membrane proteins are located in tandem and are encoded by a polymorphic multigene family.

Asunto(s)

Antígenos Bacterianos , Proteínas de la Membrana Bacteriana Externa/genética , Ehrlichia ruminantium/genética , Variación Genética , Familia de Multigenes , Secuencia de Aminoácidos , Animales , Proteínas de la Membrana Bacteriana Externa/inmunología , Secuencia Conservada , Ehrlichia ruminantium/inmunología , Genoma Bacteriano , Humanos , Epítopos Inmunodominantes/genética , Epítopos Inmunodominantes/inmunología , Sistemas de Lectura Abierta/genética , Sistemas de Lectura Abierta/inmunología , Reacción en Cadena de la Polimerasa/veterinaria , Polimorfismo Genético

RESUMEN

A 1.2 kb polymorphic fragment from the Gardel isolate of Ehrlichia (formerly Cowdria) ruminantium was used to isolate a 15kb clone from the E. ruminantium Welgevonden LambdaGEM-11 library. This clone, WL2EL1, was subcloned and sequenced. Eight open reading frames (ORFs) were identified. The ORF in WL2EL1 which contained the Welgevonden homologue of the 1.2 kb polymorphic fragment was designated Cowdria polymorphic gene 1 (cpg1). The cpg1 ORF was cloned into pCMViUB, a genetic vaccine vector. Mice and sheep were immunized with pCMViUB/cpg1 by intramuscular injection and gene gun inoculation. Although all of the immunized mice died, there was a trend for mice that received larger amounts of pCMViUB/cpg1 DNA to survive longer. Four out of five sheep immunized with the construct survived lethal challenge.

Asunto(s)

Vacunas Bacterianas , Ehrlichia ruminantium/genética , Ehrlichia ruminantium/inmunología , Hidropericardio/prevención & control , Sistemas de Lectura Abierta/inmunología , Enfermedades de las Ovejas/prevención & control , Animales , Clonación Molecular , Relación Dosis-Respuesta Inmunológica , Genes Bacterianos , Vectores Genéticos , Hidropericardio/inmunología , Ratones , Ratones Endogámicos C57BL , Sistemas de Lectura Abierta/genética , Polimorfismo Genético , Homología de Secuencia , Ovinos , Enfermedades de las Ovejas/inmunología , Vacunación/veterinaria

RESUMEN

Ehrlichia ruminantium GroEL and GroES genes were amplified from E. ruminantium Welgevonden genomic DNA and were cloned into genetic vaccine and Salmonella expression vectors. These constructs were used to inoculate Balb/c and C57BL/6J mice. Both GroEL and GroES induced low levels of protection in Balb/c and C57BL/6J mice immunized with the Salmonella expression vectors. None of the mice inoculated with the genetic vaccine survived. Immunological memory was also tested in these mice and a correlation between splenocyte proliferation and the survival rate was observed.

Asunto(s)

Vacunas Bacterianas , Chaperonina 10/inmunología , Chaperonina 60/inmunología , Ehrlichia ruminantium/inmunología , Hidropericardio/prevención & control , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Vacunas Bacterianas/genética , Vacunas Bacterianas/inmunología , Chaperonina 10/genética , Chaperonina 60/genética , ADN Bacteriano/inmunología , Ehrlichia ruminantium/genética , Amplificación de Genes , Vectores Genéticos , Inmunización , Memoria Inmunológica , Dosificación Letal Mediana , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Salmonella , Bazo/citología , Vacunas de ADN , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología

RESUMEN

Three Lambda GEM11 clones were isolated from a large-insert Ehrlichia ruminantium Welgevonden library. The inserts were amplified, sequenced, and analyzed. A total of 39 827 bp was obtained, and 18 different open reading frames (ORFs) were identified. Long repeats (100-200 kbp) were found in all three sequences. These repeats may play a role in the induction of antigenic variation. Along with a 20-kbp sequence of a clone from the E. ruminantium cosmid library, these sequences are the first large sequences to be yielded by the E. ruminantium genome sequencing project.

Asunto(s)

Proteínas Bacterianas/genética , ADN Bacteriano/química , Ehrlichia ruminantium/genética , Sistemas de Lectura Abierta/genética , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Clonación Molecular , Cósmidos , Biblioteca de Genes , Genoma Bacteriano , Sistemas de Lectura Abierta/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Homología de Secuencia

RESUMEN

A Cowdria ruminantium genomic library was constructed in a cosmid vector to serve as a source of easily accessible and pure C. ruminantium DNA for molecular genetic studies. The cosmid library contained 846 clones which were arrayed into microtitre plates. Restriction enzyme digestion patterns indicated that these clones had an average insert size of 35 kb. Probing of the arrays did not detect any bovine clones and only one of the known C. ruminantium genes, pCS20, was detected. Due to the high AT content and the fact that C. ruminantium genes are active in the Escherichia coli host, the C. ruminantium clones were unstable in the SuperCos1 vector and most clones did not grow reproducibly. The library was contaminated with E. coli clones and these clones were maintained with greater fidelity than the C. ruminantium clones, resulting in a skewed representation over time. We have isolated seven C. ruminantium clones which we were able to serially culture reproducibly; two of these clones overlap. These clones constitute the first large regions of C. ruminantium DNA to be cloned and represent almost 10% of the C. ruminantium genome.

Asunto(s)

Cósmidos/genética , Ehrlichia ruminantium/genética , Animales , Bovinos , Clonación Molecular/métodos , Técnicas Genéticas/normas , Pruebas Genéticas , Genoma Bacteriano , Biblioteca Genómica , Hidropericardio/etiología , Hibridación de Ácido Nucleico

RESUMEN

We sequenced the rRNA genes and internal transcribed spacers (ITS) of several Theileria parva isolates in an attempt to distinguish between the causative agents of East coast fever and Corridor disease. The small subunit (SSU) and large subunit (LSU) rRNA genes from a cloned T. p. lawrencei parasite were sequenced; the former was identical to that of T. p. parva Muguga, and there were minor heterogeneities in the latter. The 5.8S gene sequences of 11 T. parva isolates were identical, but major differences were found in the ITS. Six characterization oligonucleotides were designed to hybridize within the variable ITS1 region; 93.5% of T. p. parva isolates examined were detected by probe TPP1 and 81.8% of T. p. lawrencei isolates were detected by TPL2 and/or TPL3a. There was no absolute distinction between T. p. parva and T. p. lawrencei and the former hybridized with fewer of the probes than did the latter. It therefore seems that a relatively homogenous subpopulation of T. parva has been selected in cattle from a more diverse gene pool in buffalo. The ITSs of both T. p. parva and T. p. lawrencei contained different combinations of identifiable sequence segments, resulting in a mosaic of segments in any one isolate, suggesting that the two populations undergo genetic recombination and that their gene pools are not completely separate.

Asunto(s)

ADN Ribosómico/genética , Genes Protozoarios , ARN Protozoario/genética , ARN Ribosómico/genética , Theileria parva/genética , Animales , Secuencia de Bases , Evolución Biológica , Búfalos , Bovinos , Variación Genética , Datos de Secuencia Molecular , Sondas de Oligonucleótidos/genética , Polimorfismo Genético , Alineación de Secuencia , Theileria parva/clasificación , Theileriosis/parasitología

Asunto(s)

ADN Bacteriano/química , Ehrlichia ruminantium/genética , Animales , Secuencia de Bases , Cósmidos , Genoma Bacteriano , Sistemas de Lectura Abierta , Mapeo Restrictivo , Rumiantes

RESUMEN

Bacteriophage clones containing ribosomal RNA genes of Theileria parva were isolated from genomic DNA libraries. Physical mapping studies revealed 2 ribosomal DNA units, which were distinguishable by restriction enzyme site polymorphisms in flanking sequences. The cloned ribosomal DNA units were mapped to 2 separate T. parva chromosomes. Analysis of sequences contained in lambda EMBL3 recombinants, together with Southern blot analysis of genomic DNA and data on the copy number of the rRNA genes, suggested that the rDNA units were not tandemly repeated. This organisation of ribosomal transcription units is similar to that described for other genera of apicomplexan protozoa, but 2 rDNA units, each containing single copies of the rRNA coding genes, would be the lowest copy number described for any eukaryote in which amplification of rRNA genes is not known to occur. EcoRI restriction fragment length polymorphisms, which were revealed using rRNA gene probes, separated T. parva stocks into 2 categories. Nucleotide sequence analysis of polymerase chain reaction-amplified internal transcribed spacer DNA revealed 2 different ITS sequences derived from rDNA transcription units within the genome of a cloned T. parva parasite. Polymorphism was also observed between ITS sequences amplified from the DNA of different T. parva stocks. A synthetic oligonucleotide derived from T. parva Uganda ribosomal ITS DNA sequences hybridised to DNA from the T. parva Uganda stock, but not to the DNA of the T. parva Muguga stock. This oligonucleotide is potentially useful as a marker for the T. parva Uganda stock.

Asunto(s)

Genes Protozoarios , ARN Protozoario/genética , ARN Ribosómico/genética , Theileria parva/genética , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Cartilla de ADN/genética , ADN Protozoario/genética , ADN Ribosómico/genética , Datos de Secuencia Molecular , Polimorfismo Genético , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido Nucleico