Ehrlichia ruminantium is a tick-borne pathogen transmitted by ticks in the genus Amblyomma. This bacterial pathogen causes heartwater, a fatal disease affecting domestic and wild ruminants in sub-Saharan Africa. The prevalence of heartwater in western Kenya is not well documented. In this study, reverse line blot (RLB) hybridization and quantitative real-time PCR (qPCR) assays were used to detect E. ruminantium DNA in 545 blood samples collected from calves from twenty sublocations distributed across five agro-ecological zones of western Kenya. Ehrlichia ruminantium DNA was detected in 1.10% and 0.92% of the samples using RLB and qPCR, respectively. There were discrepancies in the detection of E. ruminantium by the RLB and the qPCR. Five samples were positive with the qPCR while six were positive with the RLB, but only three of the samples were positive by both tests. The occurrence of E. ruminantium in western Kenya appears to be low, but this might be attributed to the inability of the tests used to detect E. ruminantium carriers. The most prevalent haemoparasites detected by the RLB in the Ehrlichia/Anaplasma group were Anaplasma (formerly Ehrlichia) sp. Omatjenne and Anaplasma bovis at 37.98% each, while Theileria mutans (66.61%) was the most prevalent in the Theileria/Babesia group. In addition, a nested p104 PCR was used to detect Theileria parva in a subset of 86 of the samples; T. parva was detected in 32.56% (28/86) of these samples. The RLB detected T. parva in 27.91% (24/86) of the same sample subset, but only 17 were positive by both tests. The molecular tests used in this study suggest that, of the pathogenic haemoparasites known to cause disease in Kenya, T. parva occurs the most commonly in western Kenya, while E. ruminantium, A. marginale, B. bigemina and B. bovis are less frequently detected. / Dissertation (MSc)--University of Pretoria, 2012. / Veterinary Tropical Diseases / unrestricted

The most important tick-borne diseases (TBDs) occurring in Zambia that affect domestic animals, particularly cattle and small ruminants, are theileriosis (East Coast fever and Corridor disease), anaplasmosis (gall sickness), babesiosis (red water), and heartwater (cowdriosis). Of these, theileriosis is the most important, causing significantly more deaths than the other tick-borne diseases combined. Despite their importance, little is known about the occurrence and prevalence of haemoparasites in cattle in the communal areas of Zambia. Clinical signs and post mortem lesions are pathognomonic of mixed tick-borne infections especially babesiosis, anaplasmosis and East Coast fever (ECF). The main objective of this study was, therefore, to screen selected communal herds of cattle for tick-borne haemoparasites and identify the tick vectors associated with the high cattle mortalities due to suspected TBDs in the local breeds of cattle grazing along the banks of the Chambeshi River in Mungwi, Zambia. East Coast fever is endemic to the district of Mungwi, Northern Province, Zambia and vector control using acaricides has proved to be very costly for the small scale farmers. Also, Mungwi experiences increased cattle mortalities between December to March and May to July. All age groups of cattle are affected. A total of 299 cattle blood samples were collected from July to September 2010 from Kapamba (n=50), Chifulo (n=102), Chisanga (n=38), Kowa (n=95) and Mungwi central (n=14) in the Mungwi District, Northern Province, Zambia. Ticks were also collected from the sampled cattle from April to July 2011. DNA was extracted and the parasite hypervariable region of the small subunit rRNA gene was amplified and subjected to the reverse line blot (RLB) hybridization assay. The results of the RLB assay revealed the presence of tick-borne haemoparasites in 259 samples occurring either as single or mixed infections. The most prevalent species present were the benign Theileria mutans (54.5%) and T. velifera (51.5%). Anaplasma marginale (25.7%), Babesia bovis (7.7%) and B. bigemina (3.3%) were also detected in the samples. Nine percent of the samples tested negative for the presence of haemoparasites. In a number of samples (4%) the PCR products failed to hybridize with any species-specific probes but hybridized only with the genus-specific probes which could suggest the presence of a novel species or variant of a species. Of the four Theileria species known to occur in Zambia (T. parva, T. mutans, T. velifera and T. taurotragi), T. parva is the most economically important, causing Corridor disease in the Southern, Central, Lusaka and the Copper-belt provinces, while causing ECF in the Northern and Eastern provinces of Zambia. In our study, only one sample (from Kapamba) tested positive for the presence of T. parva. This was an unexpected finding; also because the tick vector, Rhipicephalus appendiculatus, was identified on animals from Kowa (14%), Chisanga (8.5%), Chifulo (6%) and Kapamba (1.4%). We can only speculate that the RLB hybridization assay may not have been able to detect the parasite in the animals sampled due to a too low parasitaemia. The samples should also be subjected to the T. parva specific real-time PCR assay to determine a more accurate T. parva prevalence in cattle in the Mungwi district, Northern Province. In Zambia, Babesia bovis and B. bigemina are recognized as being of economic importance in cattle. In our study, B. bovis was present in 7.7% of the sampled animals and B. bigemina in 3.3% of the animals. We detected B. bovis in all of the five sampled areas with the highest detection in Mungwi central (14.3%) and Kowa (10.5%). As expected, the tick vector Rhipicephalus (Boophilus) microplus was identified from animals from all of these areas. Babesia bigemina was only reported from Kowa (10.5%). The most abundant ticks identified from the sampled animals from Kowa were Rhipicephalus (Boophilus) decoloratus (36.3%) and Rhipicephalus (Boophilus) microplus (8.8%). These tick vectors have been implicated in the transmission of B. bigemina. Our findings are in concordance with results obtained by other authors who have speculated that an increase in the detection of B. bovis may indicate that B. bovis is becoming endemic in this part of the country. This could be due to uncontrolled movement of cattle that frequently occurs within Zambia. Heartwater (cowdriosis) is caused by Ehrlichia ruminantium, a rickettsial disease that affects domestic and wild ruminants. In Zambia, heartwater is mainly a disease of cattle, although outbreaks in sheep and goats have been reported and recorded. In our study, only one sample (from Kapamba) tested positive for the presence of E. ruminantium even though Amblyomma variegatum ticks were identified from 52.9% of the sampled animals from all study areas. The cattle sampled in our study are not regularly dipped and no game has been spotted in cattle grazing areas. It is possible that these cattle may have attained a state of endemic stability to heartwater. It is also possible that the RLB hybridization assay may not have been sensitive enough to detect E. ruminantium infections if the parasitaemia was very low. Samples should also be subjected to the E. ruminantium-specific pCS20 real-time PCR assay to determine more accurately the E. ruminantium prevalence in cattle in the Mungwi district, Northern Province. Anaplasma marginale (the causative agent of bovine anaplasmosis) has previously been shown to be present in all the provinces of Zambia and is the only Anaplasma species of importance to cattle in Zambia. In our study, 25.7% of the sampled cattle tested positive for A. marginale; it was detected in all areas except Chisanga. Amblyomma variegatum was identified from 52.9% of the sampled cattle, and Rhipicephalus (Boophilus) microplus from 12.1% of the cattle. Rhipicephalus (Boophilus) microplus has been incriminated as being a vector of A. marginale. Furthermore, three samples (from Kowa) tested positive for the presence of Anaplasma centrale. To our knowledge, no vaccination regime using A. centrale is being conducted in the Mungwi district of Zambia. The presence of A. centrale is, therefore, an interesting finding. The results of our study suggest that the cause of cattle mortalities in Mungwi during the winter outbreaks is mainly due to A. marginale, B. bovis and B. bigemina infections. This was confirmed by the results of the RLB hybridization assay, clinical manifestation of the disease in the affected cattle (own observation) and the tick species identified on the animals. It appears that in Mungwi, babesiosis due to B. bovis mostly infects cattle above one year of age. Calves appear to be less affected by B. bovis infection. There is need for further epidemiological surveys in Mungwi district, Northern Province, Zambia to get a better understanding of the epidemiology of these tick-borne haemoparasites affecting cattle. We conclude that integrated control policies should be developed to take account of multi-species pathogen communities that are commonly associated with clinical and sub-clinical TBD infections in Zambia. / Dissertation (MSc)--University of Pretoria, 2012. / Veterinary Tropical Diseases / unrestricted

A total of 143 blood samples of nyala (Tragelaphus angasii) from two regions in South Africa were tested for the presence of tick-borne haemoparasites by means of polymerase chain reaction (PCR) and reverse line blot (RLB) hybridisation. While most blood samples taken in EDTA blood turned out negative for the presence of haemoparasites, the majority of blood samples collected on Whatman® filter paper contained several different haemoparasites, often in combination. Samples from the Eastern Cape Province as well as from KwaZulu- Natal turned out positive. Prevalent haemoparasites were Theileria sp. (kudu), T. buffeli, T. bicornis, Theileria sp. (sable), T. taurotragi, Ehrlichia sp. Omatjenne, Anaplasma bovis and A. marginale. This serves as the first report of T. buffeli, T. sp. (kudu), T. bicornis, T. taurotragi, Ehrlichia sp. Omatjenne, A. marginale and A. bovis in nyala. Copyright / Dissertation (MSc (Veterinary Tropical Diseases))--University of Pretoria, 2009. / Veterinary Tropical Diseases / unrestricted

Hemoparasitos do filo Apicomplexa merecem destaque devido ao impacto que podem ter na conservação da biodiversidade dos mamíferos. O presente estudo objetivou avaliar a ocorrência de agentes Piroplasmida (Cytauxzoon, Theileria e Babesia) e Hepatozoon spp., utilizando o Exame Microscópico Convencional (EMC) e a Reação em Cadeia da Polimerase (PCR), e identificar os carrapatos presentes em mamíferos silvestres destinados ao LAPAS-UFU, CETAS-GO/IBAMA e CETAS-BH/IBAMA. Obteve-se 152 amostras de sangue total e realizou-se a PCR para Hepatozoon spp., com a utilização dos primers HepF300/Hep900, Hep2F/Hep2R e HAM-1/HPFR2, e para a ordem Piroplasmida, realizou-se a PCR com auxílio dos primers BAB- 33-47/ BAB432-439, PIRO 1F/PIRO 5R. Com punção de sangue periférico, confeccionou-se 352 extensões de 123 animais. Pelo EMC, não foram observados gamontes de Hepatozoon spp., mas pela PCR obteve-se 5,2% de positividade (oito positivos). Foram obtidos quatro novos haplótipos de Hepatozoon canis e Hepatozoon felis nos mamíferos de espécies lobo-guará (Chrysocyon brachyurus), raposa-do-campo (Lycalopex vetulus), irara (Eira barbara) e onça-parda (Puma concolor). Para os piroplasmas, pelo EMC e PCR, obteve-se 34,9% (43 animais positivos) e 26,3% (40 animais positivos), respectivamente. Foram obtidos três novos haplótipos de espécies de piroplasmas, sendo sequências similares à Theileria cervi em veado-catingueiro, Theileria equi em anta e Cytauxzoon felis em jaguatirica. Foram identificadas 432 espécimes, de 30 animais. Os carrapatos identificados foram: Amblyomma nodosum, A. parvum, A. ovale, A. sculptum, A. longirostre, A. dubidatum, Rhipicephalus microplus e R. sanguineus. Além disso, outros hemoparasitos foram observados em microscopia óptica, como Ehrlichia spp. em 23 animais (18,6%) e microfilárias em dois indivíduos de espécie Coendou prehensilis (ouriço-cacheiro). O conjunto de informações trazidas por este trabalho comprova a abundância de hemoparasitos que mamíferos brasileiros podem albergar. Além disso, algumas espécies de carrapatos identificados foram determinantes para inferir possíveis modos de transmissão de hemoparasitos observados nos animais. Sobre o resultado obtido pelo uso das técnicas de diagnóstico, a PCR, aliada ao sequenciamento, mostrou-se mais precisa que o EMC para identificação de espécies de hemoparasitos-alvo deste trabalho. / Haemoparasites belonging to filum Apicomplexa deserve attention because can affect the conservation of mammals’s biodiversity. This work aimed to avalue the occurencce of Piroplasmida agents (Cytauxzoon, Theileria e Babesia) e Hepatozoon spp. using Convencional Microscopic Exam (CME) and Polymerase Chain Reaction (PCR) and indetify ticks on wild mammals belonging to LAPAS-UFU, CETAS-GO/IBAMA e CETAS-BH/IBAMA. On total, this work obtained 152 total blood samples that were submited to PCR for Hepatozoon spp. with primers HepF300/Hep900, Hep2F/Hep2R e HAM-1/HPFR2, and for Piroplasmida agents, PCR was done using primers BAB- 33-47/ BAB432-439, PIRO 1F/PIRO 5R. To exam slides on optic microscope, was used peripheral samples of 123 mammals. There wasn’t observed gamonts of Hepatozoon spp. on blood slides, but on PCR was observed prevalence of 5,2% (eight positive mammals). From these, was obtained four new haplotypes of Hepatozoon spp. like similar sequence of Hepatozoon canis in maned-wolf (Chrysocyon brachyurus) and hoary fox (Lycalopex vetulus) and similar sequence of Hepatozoon felis in tayra (Eira barbara) and puma (Puma concolor). For Piroplasmida agents, the CME and PCR results were 34,9% (43 positive mammlas) and 26,3% (40 positive mammals), respectively. There was obtained three new haplotypes of Piroplasmida agents, being similar sequences of Theileria cervi in brown brocket deer (Mazama gouazoubira), similar sequence of Theileria equi in brazilian tapir (Tapirus terrestris) and similar sequence of Cytauxzoon felis in ocelot (Leopardus pardalis). There was identified 432 specimnes of ticks on 30 mammals. The species were Amblyomma nodosum, A. parvum, A. ovale, A. sculptum, A. longirostre, A. dubidatum, Rhipicephalus microplus e R. sanguineus. Besides this results, other haemoparasites were observed on optic microscope, like Ehrlichia spp. in 23 mammals (18,6%) and microfilaria in two mammals of the species Coendou prehensilis (brazilian porcupine). The information gathered by this work confirms the abundance of haemoparasites that the Brazilian mammals can harbor. In addition, some species of ticks identified were determinant to infer possible ways of transmitting hemoparasites identified in mammals. The result obtained by the use of molecular diagnostic techniques (PCR and sequencement) was more accurate than the CME for identification of hemoparasites on wild mammals of this work. / Dissertação (Mestrado)