Epidemiological studies on tick-borne diseases of cattle in Central Equatoria State,Southern Sudan

A herd-based study was carried out in Central Equatoria State, Southern Sudan, to study epidemiological aspects of tick-borne diseases. Six herds of cattle situated in three different locations were selected and investigated every 3 months during the year 2005. Blood smears for Giemsa staining and blood spots on filter paper for deoxyribonucleic acid extraction were collected from 600 apparently healthy indigenous cattle. A total of 69 (11.5%) samples showed the presence of piroplasms in Giemsa-stained blood smears, and polymerase chain reaction increased the detection limit to 297 (49.5%). Using reverse line blot, it was possible to detect and differentiate eight different piroplasms namely, Theileria parva (71.2%), Theileria mutans (73%), Theileria velifera (45.3%), Theileria taurotragi (2.7%), Theileria buffeli (0.5%), Theileria annulata (0.2%), Babesia bovis (1.7%), and Babesia bigemina (0.3%). Mixed infections were detected in 406 samples (67.7%) accounting for 17 different combinations. High infection of Theileria parva was reported among young calves compared to older cattle. The highest prevalence of Theileria parva was reported in the rainy season (October). The implications of these results on the epidemiology of tick-borne diseases are discussed with emphasis on East Coast fever. Nucleotide sequences data reported in this paper are available in GenBank™ database under the accession numbers EF469603, EF469604, EF469605, EF469606, and EF469607.     

Theileria parva genetic diversity and haemoparasite prevalence in cattle and wildlife in and around Lake Mburo National Park in Uganda

Wildlife, especially Cape buffalo (Syncerus caffer), are thought to act as a reservoir for many of the important tick-borne pathogens of cattle. In this study, we have determined the prevalence of the most significant tick-borne haemoparasites in wildlife (buffalo, impala, eland and bushbuck) as well as in cattle grazing inside and neighbouring Lake Mburo National Park (LMNP) in Uganda. A high percentage of buffalo were carriers of Theileria parva, Theileria mutans, Theileria velifera, Theileria buffeli and Theileria sp. (buffalo) as well as Anaplasma marginale and Anaplasma centrale. The majority of impala sampled were carriers of A. centrale, and all were carriers of an unidentified Babesia/Theileria species. The eland and bushbuck sampled were all carriers of Theileria taurotragi and Theileria buffeli, and the majority were carriers of T. mutans. The bushbuck sampled were also carriers for Erhlichia bovis. There were some differences in the prevalence of haemoparasites between the calves sampled inside and neighbouring LMNP. In order to address the question of whether there is evidence for interbreeding between buffalo-associated and cattle-associated T. parva populations, multi-locus genotypes (MLGs) of T. parva (based on micro-satellite markers) from buffalo and from calves grazing inside and outside LMNP were compared, and the results revealed that buffalo and cattle gene pools were distinct, showing no evidence for transmission of buffalo-derived T. parva genotypes to the cattle population.     

Changes in serum iron concentration and hepatic enzyme activities in cattle infected with Theileria annulata and Babesia bigemina

Analyses for alanine aminotransferase, aspartate aminotransferase (AST), alkaline phosphatase (ALP) activities, and iron concentration were carried out in infected and uninfected cattle in order to determine the degree of hepatic damage and blood iron status caused by Theileria annulata and Babesia bigemina. Blood samples from 30 infected and 20 uninfected cattle with T. annulata and also from 40 infected and 20 uninfected cattle with B. bigemina were taken. The biochemical analyses revealed increased serum AST and ALP activities in infected cattle with T. annulata and B. bigemina compared with uninfected cattle (P?<?0.05). Significant decreases in iron concentration were observed in cattle infected with T. annulata and B. bigemina when compared with uninfected cattle (P?<?0.05) although the level of serum iron concentration in infected cattle was within the normal reference values for cattle. It was concluded that iron deficiency anemia is not an important factor in T. annulata- and even in B. bigemina-infected cattle with severe intravascular hemolysis. The increased activities of serum enzymes indicated the hepatic injuries associated with infection with T. annulata and B. bigemina.     

Development and laboratory evaluation of a lateral flow device (LFD) for the serodiagnosis of Theileria annulata infection

Several DNA-based and serological tests have been established for the detection of Theileria annulata infection, including polymerase chain reaction, reverse line blot and loop-mediated isothermal amplification, indirect enzyme-linked immunosorbent assay (ELISA), and competitive ELISA. In this study, we have applied knowledge from the development and application of a recombinant protein-based indirect ELISA and competitive ELISA to establish a rapid test for point-of-care diagnosis of T. annulata infection in the field to be used by the veterinarian. For the development of a lateral flow test, the recombinantly expressed T. annulata surface protein (TaSP) was applied as the test antigen and anti-TaSP antiserum as the control line. TaSP antigen conjugated to colloidal gold particles was used as the detection system for visualization at the test line for the binding of anti-TaSP antibody present in the serum of infected animals. The developed test specifically detected antibodies in the serum of animals experimentally infected with T. annulata and showed no cross-reactivity with serum from animals infected with other tested bovine pathogens (Trypanosoma brucei, Anaplasma marginale, Babesia bigemina, Babesia bovis, and Theileria parva). Testing of field samples was compared to results obtained by other serological tests, resulting in a sensitivity and specificity of 96.3% and 87.5% compared to indirect fluorescence antibody test, 98.7% and 81.8% compared to indirect ELISA, and 100% and 47.6% compared to competitive ELISA. In conclusion, a rapid test for the detection of T. annulata infection (T. annulata lateral flow device, Ta-LFD) has been developed, which is easy to perform, delivers results to be read by the naked eye within 10 min, and is suitable for the detection of infection in field samples.     

Determination of potential risk factors associated with <Emphasis Type="Italic">Theileria annulata</Emphasis> and <Emphasis Type="Italic">Theileria parva</Emphasis> infections of cattle in the Sudan

A multi-variate logistic regression analysis was performed on two sets of data on the prevalence of Theileria annulata in Northern Sudan and Theileria parva in Southern Sudan, to determine the potential risk factors that might affect the distribution of the infections in those regions. The logistic regression model was fit with the tested risk factors for each disease, separately. The results indicated that locations, management systems and age could be held as risk factors for T. annulata infection in Northern Sudan, while for T. parva locations and seasons could be held as risk factors in Southern Sudan. The results of this study will assist in the development of more effective control strategies for smallholder dairy farms in the country.     

Detection of tick blood parasites in Egypt using PCR assay I—Babesia bovis and Babesia bigemina

Babesia bovis and Babesia bigemina are distributed all over the world; the etiologic agents of the animal babesiosis are considered the most important tick-borne disease. The present research work was the first attempt to determine the prevalence of B. bovis and B. bigemina infection in ticks, in Egypt, by using polymerase chain reaction (PCR). Questing 5,243 hard and soft ticks were collected from different localities throughout the Giza Governorate. Furthermore, DNA from 500 different individual tick species was extracted and PCR was performed. Primers verified from the sequence of Mexico strain of both species were used. Two fragments of 275 and 175 bp of B. bovis and B. bigemina, respectively, were generated. Fragments of the pathogens were recovered with PCR and sequenced. The prevalence of B. bovis and B. bigemina in Boophilus annulatus ticks were 55% and 66%, respectively. Also, presence of 12% dual infection with B. bovis and B. bigemina was observed. Sequence analysis of PCR product of these pathogens shares a high degree of similarity in sequence compared to similar species found in GenBank.     

Development of an enzyme-linked immunosorbent assay for detection of antibodies to Babesia bigemina in cattle

Monoclonal antibodies, directed against a 58-kDa Babesia bigemina merozoite antigen that reacted strongly with immune sera from experimentally and naturally infected cattle in Western blots, were used to develop a competitive-inhibition enzyme-linked immunosorbent assay (ELISA). As based on the testing of 70 antibody-positive sera from experimentally infected cattle and 166 antibody-negative sera collected in non-endemic areas of Australia, the sensitivity and specificity of the ELISA were 95.7% and 97.0%, respectively. In sequential sera collected from six calves during the course of experimental B. bigemina infections the ELISA detected seroconversion at about 10 days post-inoculation. The specificity of the ELISA was not affected by the presence of antibodies to B. bovis, Anaplasma marginale or Theileria buffeli. In 42 sera from cattle experimentally infected with B. bovis but negative for B. bigemina the specificity of the ELISA was 95.2%. The use of a competitive-inhibition ELISA format detecting only antibody directed against a single epitope on the 58-kDa antigen appears to have overcome many of the specificity problems that have plagued serological tests for B. bigemina in the past. The test should be useful for epidemiology studies, particularly in areas where B. bovis and B. bigemina have overlapping distributions. Received: 28 December 1997 / Accepted: 10 February 1998     

Evaluation of the resistance of indigenous Iranian cattle to Theileria annulata compared with Holstein cattle by measurement of acute phase proteins

This study was conducted to evaluate the resistance of indigenous cattle against Theileria annulata in comparison with that of Holsteins, through assessment of changes in acute phase proteins. Blood samples were collected from 24 indigenous and 26 Holstein dairy cattle, 2–3 years old, which had become naturally infected with T. annulata. Twenty-five healthy cattle, ten indigenous and 15 Holsteins were selected as a control group. The Theileria-infected group were divided into four subgroups according to their parasitemia rates (<1%, 1–3%, 3–5% and >5%). Measurement of red blood cells (RBCs), packed cell volume (PCV), haemoglobin (Hb), haptoglobin (Hp), serum amyloid A (SAA), ceruloplasmin and fibrinogen were done for all animals using validated methods. Results showed significant differences in RBCs, PCV, Hb and concentrations of Hp, SAA, ceruloplasmin and fibrinogen between healthy cattle and those infected with T. annulata with different parasitemia rates in both breeds (P < 0.05). In both breeds, there was significant negative correlation between parasitemia and RBCs, PCV and Hb (P < 0.05). In contrast, with increasing parasitemia rate, a significant increase in MCV, Hp, SAA, ceruloplasmin and fibrinogen was evident. Iranian indigenous cattle in comparison with Holsteins showed lower parasitemia rate, milder clinical manifestations and significantly lower levels of acute phase proteins including Hp, SAA, ceruloplasmin and fibrinogen (P < 0.05).     

Report of Theileria annulata and Babesia canis infections in dogs

Piroplasmosis is a zoonotic protozoan disease transmitted by ticks. The full geographical range of canine piroplasms has been found in dogs in the Middle East, parts of Africa, North America, and Europe. Following our studies on molecular detection of piroplasmosis in the south of Iran, we found Theileria annulata in two herd dogs, as well as information on their 18S rRNA gene sequences. Piroplasmosis agents were detected by PCR of 280 blood samples collected from dogs in seven regions of the Shiraz suburbia in southern Iran, between November 2009 and June 2011. Two positive samples from Shiraz were infected with T. annulata, and one sample was infected with Babesia canis. PCR positive samples were further analyzed by sequence analysis. The results of this study reconfirmed that T. annulata are not always as host specific as accepted. This is the first report of T. annulata in herd dogs in southern Iran and the second report of T. annulata in dogs worldwide.     

Differentiation of Babesia bigemina, B. bovis, B. divergens and B. major by Western blotting—first report of B. bovis in Austrian cattle

To establish an assay for the serological differentiation of bovine Babesia species (B. bigemina, B. bovis, B. divergens and B. major), antigens from experimentally infected cattle were Western blotted and probed with homologous and heterologous sera. Varying antigen patterns for each species allowed the determination of species-specific diagnostic antigens. Blood samples from 36 naturally infected cattle from the province of Styria were tested by indirect immunofluorescence antibody test (IFAT) against B. divergens, as well as by Western blotting against B. bigemina, B. bovis, B. divergens and B. major, 3 weeks after clinical babesiosis was diagnosed by blood smears. All 36 cattle were B. divergens-positive when tested by IFAT. In four cases (11%), an infection with both B. bovis and B. divergens and in two cases a single infection with B. bovis were diagnosed when tested by Western blot. B. bigemina and B. major infections were not detected. These are the first serologically confirmed cases of B. bovis in Austrian cattle.     

Concurrent infections with vector-borne pathogens associated with fatal anaemia in cattle: haematology and blood chemistry

An outbreak of a fatal haemolytic anaemia in a dairy herd of cattle in Switzerland was shown to be associated with infections with five vector-borne pathogens, namely Anaplasma marginale, A. phagocytophilum, Babesia bigemina, a Theileria spp belonging to the buffeli/sergenti/orientalis complex and haemotrophic Mycoplasma spp. The latter three had not been documented before this outbreak in Switzerland. To characterise the haematological and blood chemical changes in these unique cows, packed cell volume was determined in all 286 blood samples, blood smears, and complete haematology were performed from 285 and 173 blood samples, respectively, and biochemical parameters were assayed in 105 serum samples. Regenerative anaemia was the key sign of illness. Red blood cells of anaemic cattle were hypochromic and macrocytic. Anaemic animals had reduced platelet cell counts and increased total white cell counts. In addition, increased serum bilirubin, blood aspartate aminotransferase, gamma glutamyltransferase, glutamic dehydrogenase and blood urea nitrogen and decreased magnesium, calcium and albumin levels were found in anaemic cattle when compared to animals with normal packed cell volume. Most changes could not be attributed to a single infection. A. marginale seemed to be important in causing the outbreak, but co-infections may have aggravated the disease development and clinical signs. Thus, when encountering cattle with haemolytic anaemia, all of the mentioned pathogens should be included as differential diagnosis.     

Detection of <Emphasis Type="Italic">Theileria annulata</Emphasis> by the PCR-RFLP in ticks (Acari,Ixodidae) collected from cattle in West and North-West Iran

The presence of potential vectors, ticks, and susceptible hosts of bovine malignant theileriosis in all parts of Iran pose a real threat to food animal industry. The present study was conducted to determine the infection rate of ticks collected from naturally occurring bovine theileriosis in West and North-West Iran. Two hundred and thirty seven cattle suspected of suffering from theileriosis were investigated for the presence of Theileria annulata in the blood smears and any tick species on their body. In this study, 402 ticks were obtained from 99 cattle. The examination of 402 ticks by polymerase chain reaction (PCR) using primers derived from the gene encoding heat shock protein70 (Hsp70) revealed that 39.9% of Hyalomma anatolicum anatolicum, 3.5% of H. asiaticum asiaticum, and 18.2% H. anatolicum excavatum, were infected with T. annulata. The results suggest that H. a. anatolicum may play a major role in transmission of T. annulata infection in Iran. Finally, digestion of the PCR products of T. annulata with two different restriction enzymes produced only a single pattern.     

Prevalence of Theileria equi, Babesia caballi, and Anaplasma phagocytophilum in horses from the north of Portugal

Piroplasmid protozoa Theileria equi and Babesia caballi and zoonotic rickettsial bacterium Anaplasma phagocytophilum are important agents of equine vector-borne diseases (EVBD). This study aimed at investigating the prevalence of infections with or exposure to these pathogens in horses from the north of Portugal. Blood was randomly collected from 162 horses, living in 72 different stables, to prepare Giemsa-stained slide smears. Additionally, plasma samples were tested for antibodies to T. equi and B. caballi by two competitive enzyme-linked immunosorbent assays and to A. phagocytophilum by an indirect fluorescence antibody test. Five horses were positive to T. equi by microscopy (3.1 %), three to B. caballi (1.9 %), and none to A. phagocytophilum with no horse simultaneously positive for the two piroplasms. Clinically suspect animals had a significantly higher positivity to T. equi by microscopy in comparison with the nonsuspect ones (21.4 vs. 1.4 %). Twenty-nine horses were seropositive to T. equi (17.9 %), 18 to B. caballi (11.1 %), and 21 to A. phagocytophilum (13.0 %). Combined serology and microscopy positive results to T. equi and B. caballi were 19.1 and 11.7 %, respectively, with 33.3 % of the horses found positive to at least one agent. Forty horses were positive to single agents and 14 to more than one agent. An outdoor or mixed outdoor/indoor type of housing was found to be a risk factor for the combined positivity to T. equi. Infections with T. equi, B. caballi, and A. phagocytophilum are endemic in the north of Portugal. In addition to the treatment of positive horses, preventive measures should be put in practice to reduce exposure to and infection with agents of EVBD.     

Development of a loop-mediated isothermal amplification method for detection of Theileria lestoquardi

A loop-mediated isothermal amplification (LAMP) assay was developed for the diagnosis of Theileria lestoquardi infection. The primers were designed based on the clone-5 sequence of T. lestoquardi. The specificity and sensitivity of the assay were established. Analysis of the specificity showed that the selected LAMP primers amplified the target sequence from T. lestoquardi DNA successfully, while no amplification was seen with DNA from Theileria annulata, Theileria ovis, Babesia ovis, Anaplasma ovis, or ovine genomic DNA. The specificity of the LAMP product was further confirmed by restriction digestion and sequencing. The sensitivity of the LAMP assay was analyzed in comparison to PCR resulting in a detection limit of 10 fg/μl of plasmid DNA containing the clone-5 sequence. The suitability for utilizing the LAMP assay in the field for the diagnosis of T. lestoquardi infection was tested on 100 field samples collected in Sudan and compared with results obtained by PCR. The relative specificity and sensitivity of the established LAMP assay was determined to be 92.1% and 87.5%, respectively, indicating that it may be regarded as an alternative molecular diagnostic tool to PCR which could be used for epidemiological surveys on T. lestoquardi infection.     

Outer membrane protein sequence variation in lambs experimentally infected with Anaplasma phagocytophilum

Anaplasma phagocytophilum has long been known to cause tick-borne fever in ruminants and has been identified more recently as the causative agent of the emerging disease human granulocytic anaplasmosis. The related organism Anaplasma marginale uses gene conversion of the expression site for two major outer membrane proteins (OMPs) to generate extensive sequence and antigenic variation in these OMPs. This is thought to present a continuously varying repertoire of epitopes to the mammalian host and allow disease persistence. Recent genomic and structural data on human strains of A. phagocytophilum, together with animal studies in model systems, have implicated an orthologous OMP of A. phagocytophilum in a similar mechanism of variation. However, to date there has been little investigation of the mechanisms of antigenic variation or disease persistence in hosts naturally infected with field strains of A. phagocytophilum. Approximately 300,000 lambs in Norway suffer severe disease caused by A. phagocytophilum annually. We show here the persistent and cyclic nature of infection in these animals that is accompanied by loosely programmed sequence variation of the major OMP expression site in each rickettsemic peak. These data will allow analysis of interactions between A. phagocytophilum and the host immune system in naturally occurring persistent infections and provide an important comparison with enduring infections of cattle caused by A. marginale.     

A study on the prevalence of a tick-transmitted pathogen, Theileria annulata, and hematological profile of cattle from Southern Punjab (Pakistan)

The present study was carried out to determine the prevalence of Theileria annulata in large ruminants in Southern Punjab (Pakistan). Blood samples were collected from 144 large ruminants, consisting of 105 cattle and 39 buffaloes, from six districts of Southern Punjab including Multan, Layyah, Muzaffar Garh, Bhakar, Bahawalnagar, and Vehari. Data on the characteristics of the animals and herds were collected through questionnaires. The age of animals (P = 0.02), presence of ticks on animals (P = 0.02), and presence of ticks on dogs associated with herds (P = 0.05) were among the major risk factors involved in the spread of tropical theileriosis in the study area. Two different parasite detection techniques, PCR amplification and screening of Giemsa-stained slides, were compared, and it was found that PCR amplification is a more sensitive tool (19% parasite detection) as compared to smear scanning (3% parasite detection) for the detection of T. annulata. Twenty eight out of 144 animals produced the 721-bp fragment specific for T. annulata from five out of six sampling districts. Different blood (hemoglobin, glucose) and serum (ALT, AST, LDH, cholesterol) parameters of calves and cattle were measured and compared between parasite-positive and parasite-negative samples to assess the effect of T. annulata on the blood and serological profile of infected animals.     

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. Received: 18 August 1997 / Accepted: 5 December 1997     

<Emphasis Type="Italic">Anaplasma marginale</Emphasis> and <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis>: Rickettsiales pathogens of veterinary and public health significance

Anaplasma marginale and Anaplasma phagocytophilum are the most important tick-borne bacteria of veterinary and public health significance in the family Anaplasmataceae. The objective of current review is to provide knowledge on ecology and epidemiology of A. phagocytophilum and compare major similarities and differences of A. marginale and A. phagocytophilum. Bovine anaplasmosis is globally distributed tick-borne disease of livestock with great economic importance in cattle industry. A. phagocytophilum, a cosmopolitan zoonotic tick transmitted pathogen of wide mammalian hosts. The infection in domestic animals is generally referred as tick-borne fever. Concurrent infections exist in ticks, domestic and wild animals in same geographic area. All age groups are susceptible, but the prevalence increases with age. Movement of susceptible domestic animals from tick free non-endemic regions to disease endemic regions is the major risk factor of bovine anaplasmosis and tick-borne fever. Recreational activities or any other high-risk tick exposure habits as well as blood transfusion are important risk factors of human granulocytic anaplasmosis. After infection, individuals remain life-long carriers. Clinical anaplasmosis is usually diagnosed upon examination of stained blood smears. Generally, detection of serum antibodies followed by molecular diagnosis is usually recommended. There are problems of sensitivity and cross-reactivity with both the Anaplasma species during serological tests. Tetracyclines are the drugs of choice for treatment and elimination of anaplasmosis in animals and humans. Universal vaccine is not available for either A. marginale or A. phagocytophilum, effective against geographically diverse strains. Major control measures for bovine anaplasmosis and tick-borne fever include rearing of tick-resistant breeds, endemic stability, breeding Anaplasma-free herds, identification of regional vectors, domestic/wild reservoirs and control, habitat modification, biological control, chemotherapy, and vaccinations (anaplasmosis and/or tick vaccination). Minimizing the tick exposure activities, identification and control of reservoirs are important control measures for human granulocytic anaplasmosis.     

A PCR method targeting internal transcribed spacers: the simultaneous detection of Babesia bigemina and Babesia bovis in cattle

In this study, two pairs of oligonucleotide primers were designed according to the nucleotide sequence of the internal transcribed spacers (ITSs) of Babesia bigemina and B. bovis isolates from China. The primers were used in a multiplex PCR to detect parasite DNA in blood samples from cattle. There was no cross reactions with B. ovata, B. major, B. sp. Kashi, Theileria annulata, T. sergenti, T. sinensis or normal bovine DNA. The sensitivity of multiplex PCR assay was 1 pg and 10 pg DNA for B. bigemina and B. bovis, respectively. A total of 260 field blood samples collected from cattle in five provinces of China were analyzed by multiplex PCR and light microscopy. PCR testing revealed that 7.3% (19/260) and 5.8% (15/260) of cattle were positive for B. bigemina and B. bovis and 1.2% (3/260) of cattle were co-infected with B. bigemina and B. bovis. Using light microscopy, 2.3% (6/260) and 1.5% (4/260) of cattle were infected by B. bigemina and B. bovis, respectively, and no co-infection was found. The results showed that the multiplex PCR developed in the present study could be an alternative diagnostic tool for the detection of B. bovis and B. bigemina infection in cattle.     

Evaluation of Babesia bigemina 200?kDa recombinant antigen in enzyme-linked immunosorbent assay

A truncated fragment of the gene encoding the 200-kDa protein (P200) of Babesia bigemina was cloned into a plasmid vector, pGEX-4 T-1 and expressed in Escherichia coli as a glutathione-S-transferase fused protein. An indirect enzyme-linked immunosorbent assay (ELISA) using the rp200/CT detected specific antibodies in cattle experimentally infected with B. bigemina. Furthermore, the antigen did not cross-react with antibodies to Babesia bovis, a closely related Babesia parasite indicating that rp200/CT is a specific antigen for the diagnosis of B. bigemina infection. Additionally, ELISA using p200/CT and polymerase chain reaction were conducted on serum and corresponding DNA samples obtained from field cattle to evaluate the diagnostic utility of the p200/CT antigen. Results from the current study suggest that p200/CT ELISA is a sensitive and specific method for improved serodiagnosis of B. bigemina infection.