Detection of Babesia and Theileria species infection in cattle from Portugal using a reverse line blotting method

Babesiosis and Theileriosis are tick-borne diseases widespread in tropical and sub-tropical regions with high economic impact worldwide. In Portugal there are at least 4 tick vectors known to be competent for the transmission of Babesia and Theileria sp. identified: Rhipicephalus bursa, Rhipicephalus (Boophilus) annulatus, Ixodes ricinus and Haemaphysalis punctata. All these potential Babesia and Theileria tick vectors are widely distributed in Portugal, although they are predominant in the Southern region. In this study, 1104 cattle blood samples were randomly collected from Central and Southern regions of Portugal and analyzed by PCR-reverse line blotting (RLB) for the detection of Babesia and Theileria sp. Testing indicated that 74.7% of the bovines tested were positive for either Babesia and/or Theileria sp. In addition, five different apicomplexan species, namely, Theileria buffeli, Theileria annulata, Babesia divergens, Babesia bovis, and Babesia bigemina were detected by RLB among the bovines tested. T. buffeli was the most frequently found species, being present in 69.9% of the positive samples either as single infections (52.4%), or as mixed infections (17.5%). The Babesia specie most frequently found was B. divergens, detected in 4.2% of the infected bovines. Overall, infected bovines were found in all regions tested; however the highest number of infected bovines was observed in évora district (96.2%) and in cattle from Limousin breeds (81.7%). The results indicate widespread Babesia and Theileria infections in Portuguese bovines, suggesting the need for improved control of ticks and tick-borne diseases.     

High serum concentrations of iron, transferrin saturation and gamma glutamyl transferase in captive black rhinoceroses (Diceros bicornis)

Iron storage disease (haemochromatosis) is thought to be the cause of many disorders unique to captive black rhinoceroses (Diceros bicornis). To establish reliable reference ranges for iron parameters, serum samples from 27 eastern black rhinoceroses (Diceros bicornis michaeli) from a translocation programme in Kenya were analysed and compared with the samples from 17 captive individuals. The transferrin saturation, serum iron concentration and gamma glutamyl transferase were significantly higher in the captive rhinoceroses, but these elevations were not evident when the results were compared with previously published data.     

Bovine piroplasms in Minorca (Balearic Islands, Spain): a comparison of PCR-based and light microscopy detection

The present study provides the first epidemiological data regarding infection by Theileria and Babesia piroplasms in cattle in Minorca. More than 94% of the studied animals were positive for the presence of Theileria sp., and of those, 41.3% were positive for the presence of Theileria annulata. These results indicate that the prevalence of Mediterranean theileriosis caused by T. annulata is very high in Minorcan dairy farms and that other Theileria sp. are also present in the area. The prevalence of infection was similar throughout the study indicating an endemic situation in this island. The use of PCR resulted in significantly higher efficacy of detection of Theileria sp. compared to microscopical observation (MO) of blood smears and allowed the specific discrimination between pathogenic and non-pathogenic theilerias which cannot be accomplished by traditional diagnosis by MO. Babesia infection in the area was mainly due to Babesia bigemina (6.0% of the studied animals were infected), while one animal (0.75%) was found to be infected by Babesia bovis. It was observed that 31% of animals infected with B. bigemina had a concurrent infection of T. annulata. PCR also resulted in a significantly higher efficacy of detection of Babesia sp. compared to MO when infection levels were higher, towards the end of the study period. The results clearly demonstrate that parasitic infection by piroplasms, especially Theileria sp. is common and endemic in the island of Minorca and that PCR is the optimal approach for the detection and discrimination of these important parasites.     

Salmonella spp. survey of captive rhinoceroses in U.S. zoological institutions and private ranches.

A survey was mailed to 72 institutions in the USA requesting information on Salmonella spp. cultures in black rhinoceroses (Diceros bicornis), white rhinoceroses (Ceratotherium simum), and Indian rhinoceroses (Rhinoceros unicornis). Sixty-one institutions responded (85% return rate), with seven reporting positive cultures (11% prevalence rate; 10% if survey nonresponders had negative cultures). There were 17 positive cultures from 16 different animals, with nine different serotypes of Salmonella and 2 additional cultures identified to the group level.     

Incidence and prevalence of tick-borne haemoparasites in domestic ruminants in Ghana

Giemsa-stained thin blood smears prepared monthly from cattle, sheep and goats in the Greater Accra region of Ghana between May 1994 and December 1996 were examined for presence of tick-borne haemoparasites. The majority of animals were less than 2 months old at the start of the survey. Monthly and cumulative incidences are presented of Anaplasma sp., Babesia bigemina, Borrelia sp., Eperythrozoon sp., Theileria mutans and Theileria velifera in cattle, Anaplasma sp., Borrelia sp., and Theileria sp. in sheep, and Anaplasma sp. in goats. T. mutans was the commonest parasite in cattle, with 100% incidence in calves by 10 months of age, and Anaplasma was commonest in small ruminants. The relative prevalence of these haemoparasites in blood smears from cattle, sheep and goats sampled on a single occasion at sites in all 10 regions of Ghana was found to be similar, though actual infection rates were lower. Packed cell volume (PCV) measurements from the sampled animals are also presented; no seasonal trends were evident in the PCV of the cattle, sheep and goats sampled monthly. In animals sampled on a single occasion, mean PCV was significantly higher in cattle and sheep without detectable haemoparasite infection, and in cattle was lowest in animals positive for both Babesia and Anaplasma, while there was no difference in mean PCV levels between parasitised and non-parasitised goats.     

Comparison of diagnostic methods to detect piroplasms in asymptomatic cattle

This study was carried out to compare different diagnostic techniques to reveal the presence of piroplasms in asymptomatic cattle kept at pasture. Nineteen blood samples were collected from animals of two different areas of Emilia Romagna Region of Italy and processed for microscopic observation, PCR, serological test (IFAT) for Babesia bovis and Babesia bigemina antibodies and in vitro cultivation. The cultures were performed on both bovine and ovine erythrocytes. Seventeen blood smears (89%) were positive for piroplasms, while PCR was positive on 18 samples (95%). DNA sequencing of 18S rRNA identified the piroplasms as Theileria spp. In vitro cultures were successful for 6 samples (32%) cultured on bovine blood and subsequent identified these as Babesia major by PCR. On IFAT analyses of 16 samples, 36.8% resulted positive for B. bovis and 31.6% positive for B. bigemina. These results show, in the same animals, the co-infection with Babesia spp. and Theileria spp.; the detection of B. major was possible only using the in vitro cultures.     

Risk factors associated with a skewed natal sex ratio in captive black rhinoceroses (Diceros bicornis) in the United States.

The skew toward males in the sex ratio of calves is considered to be a problem for the captive population of black rhinoceroses (Diceros bicornis) in North America. This study used a retrospective case-control design to determine whether there was a predisposition toward birth of male calves over female calves and to examine risk factors associated with the occurrence of any detected skewed natal sex ratio in captive black rhinoceroses in the United States. The study population included captive female black rhinoceroses housed in the United States that had given birth to at least one calf of known sex. This study confirmed a skewing of the natal sex ratio toward male calves in the captive black rhinoceros population. The skewed ratio was found in calves born to wild-born dams, for which an increased time in captivity, irrespective of age, was associated with an increased likelihood of a male calf. Dams between 12 and 19 yr of age had a decreased likelihood of a male calf. The data also suggested a possible trend for the southern subspecies of black rhinoceros (Diceros bicornis minor) to be more likely to produce male calves than the eastern subspecies (Diceros bicornis michaeli). No associations were found with the sex of offspring in captive-born dams: however, this lack of association could be a result of low power in the study.     

Tick-borne blood parasites in nyala (Tragelaphus angasii, Gray 1849) from KwaZulu-Natal, South Africa

A total of 97 blood samples of nyala (Tragelaphus angasii, Gray 1849) from South Africa were tested for the presence of tick-borne haemoparasites by means of polymerase chain reaction (PCR) and reverse line blot (RLB) hybridization. The majority of blood samples contained several different haemoparasites, often in combination. Prevalent haemoparasites were Theileria sp. (kudu), T. buffeli, Theileria sp. (sable), T. bicornis, Ehrlichia sp. Omatjenne, Anaplasma marginale and A. bovis. This serves as the first report of Theileria sp. (kudu), T. buffeli, T. bicornis, Ehrlichia sp. Omatjenne, A. marginale and A. bovis in nyala, who seem to carry multiple haemoparasites without ill effect.     

Beta-globin chain hemoglobin polymorphism and hemoglobin stability in black rhinoceroses (Diceros bicornis)

To evaluate the syndrome of acute intravascular hemolytic anemia in black rhinoceroses (Diceros bicornis), the hemoglobin of this species was evaluated by use of isopropanol- and heat-stability tests and was further characterized by electrophoretic studies. Samples were obtained from 22 apparently healthy captive North American black rhinoceroses, though 3 of the study animals had survived previous hemolytic events, and 3 others were parents of 3 offspring that had suffered hemolysis. The eastern African (Diceros bicornis michaeli) and the southern African subspecies (D b minor) were represented. Comparative samples were also obtained from 2 white (Ceratotherium simum) and 1 Indian (Rhinoceros unicornis) rhinoceroses. The hemoglobin of all 3 species appeared stable when tested by use of the heat and isopropanol methods. Thus, an unstable hemoglobin does not appear to be involved in the hemolytic crises of captive black rhinoceroses. Black rhinoceros hemoglobin had a striking polymorphism. Thirteen of the samples from black rhinoceroses had a single hemoglobin band, based on results of alkaline electrophoresis. Nine had, in addition to this major band, a slow (more cathodic) minor band that comprised about 10% of the total hemoglobin. Further studies indicated that the major band and the slower minor band may contain globin chains analogous to human beta- and delta-chains respectively; these bands have been tentatively designated B and C. Phenotypes B and BC are common, in a ratio of 4:3. A genetic mechanism is proposed that assumes beta b and beta c gene loci and that beta c-locus-expressed (beta c+) and beta c-locus-inhibited (beta c degrees) are common alleles for the beta c-locus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of Theileria and Babesia in brown brocket deer (Mazama gouazoubira) and marsh deer (Blastocerus dichotomus) in the State of Minas Gerais, Brazil

Intraerythrocytic protozoan species of the genera Theileria and Babesia are known to infect both wild and domestic animals, and both are transmitted by hard-ticks of the family Ixodidae. The prevalences of hemoprotozoa and ectoparasites in 15 free-living Mazama gouazoubira, two captive M. gouazoubira and four captive Blastocerus dichotomus from the State of Minas Gerais, Brazil, have been determined through the examination of blood smears and the use of nested polymerase chain reaction (nPCR). The cervid population was inspected for the presence of ticks and any specimens encountered were identified alive under the stereomicroscope. Blood samples were collected from all 21 animals, following which blood smears were prepared, subjected to quick Romanowsky staining and examined under the optical microscope. DNA was extracted with the aid of commercial kits from cervid blood samples and from tick salivary glands. The nPCR assay comprised two amplification reactions: the first was conducted using primers specific for a 1700 bp segment of the 18S rRNA gene of Babesia and Theileria species, whilst the second employed primers designed to amplify a common 420 bp Babesia 18S rRNA fragment identified by aligning sequences from Babesia spp. available at GenBank. The ticks Amblyomma cajennense, Rhipicephalus microplus and Dermacentor nitens were identified in various of the cervids examined. Of the animals investigated, 71.4% (15/21) were infected with hemoprotozoa, including Theileria cervi (47.6%), Theileria sp. (14.3%), Babesia bovis (4.8%) and Babesia bigemina (4.8%). However, only one of the infected wild cervids exhibited accentuated anaemia (PCV=17%). This is first report concerning the occurrence of Theileria spp. in Brazilian cervids.     

Comparative studies of mitogen- and antigen-induced lymphocyte proliferation in four captive rhinoceros species.

Cellular immune function in four rhinoceros species was evaluated by way of in vitro lymphocyte proliferation responses to mitogenic and antigenic stimuli to establish normative data on white blood cell activity for each species and to identify species-specific differences that might help explain the predisposition of black rhinoceroses (Diceros bicornis) to disease. A cross section of the U.S. rhinoceros population encompassing all four captive species was sampled, including the Sumatran rhinoceros (Dicerorhinus sumatrensis) (n = 3); Indian rhinoceros (Rhinoceros unicornis) (n = 4); African black rhinoceros (n = 16); and African white rhinoceros (Ceratotherium simum) (n = 10). Of the four species evaluated, African black rhinoceroses exhibited the weakest (P < 0.05) lymphocyte proliferative responses to the mitogens: pokeweed (0.1 microg/ml), phytohemagglutinin (0.3 microg/ml), and concanavalin A (5.0 microg/ml). Total cell density at the end of culture was only 70% of that achieved with lymphocytes isolated from African white rhinoceroses, Indian rhinoceroses, and Sumatran rhinoceroses. However, lymphocyte response to bacterial endotoxin lipopolysaccharide was similar (P > 0.05) across species. Antigenic stimulation produced much weaker responses than mitogenic stimulation. No differences (P > 0.05) were observed among rhinoceros species in response to 1 and 10 microg/ml of Leptospira icterohemorrhagiae or Leptospira gryppotyphosa. Lymphocytes from African white rhinoceroses proliferated weakly in the presence of Aspergillus fumigatus filtrate, whereas lymphocytes from the southern black rhinoceros subspecies appeared slightly suppressed in the presence of increasing doses (0.1, 1, and 10 microg/ml) of Aspergillus filtrate. This comparative data set characterizing lymphocyte proliferation in the rhinoceros reveals several differences in immune cell responses among rhinoceros species and provides some evidence that lymphocytes of captive African black rhinoceroses are less vigorous than those of the other rhinoceros species.     

Comparison of blood smear and indirect fluorescent antibody techniques in detection of haemoparasite infections in trade cattle in Nigeria

The incidence of blood parasites in trade cattle was surveyed with emphasis on tick-borne parasites, using blood smears and immunofluorescent antibody (IFA) techniques. With the blood smear method, about 9 and 8.9% of cattle examined were found positive for Babesia bigemina and Anaplasma marginale, respectively. Percentage infections with other parasites were 3.33, 1.92, 0.75, 0.75 and 0.58, respectively, for Babesia bovis, Trypanosoma brucei, Anaplasma centrale, Eperythrozoon and Theileria species as well as Trypanosoma congolense. The incidence of A. marginale infection was at its peak during the rainy season while B. bigemina was most prevalent during the dry season. There were mixed infections of Anaplasma and Babesia (1.42%); Babesia and trypanosomes (1.00%); Babesia and Eperythrozoon (0.75%) and Babesia and Theileria (0.75%). Using the indirect fluorescent antibody test, 93, 55 and 68% of cattle sera examined were found to be positive for B. bigemina, B. bovis and A. marginale, respectively. Forty-nine percent of the positive sera of B. bigemina had highest titres. The importance of using serological means for determining the endemic levels of tick-borne diseases in cattle in Nigeria is discussed.     

Digestion coefficients achieved by the black rhinoceros (Diceros bicornis), a large browsing hindgut fermenter

In contrast to the grazing white (C. simum) and Indian (R. unicornis) rhinoceros, the black rhinoceros (D. bicornis) is an exclusive browser. Due to the particular fermentation characteristics of browse, one would expect browsers to display both shorter ingesta retention times and lower digestion coefficients on comparable diets than grazers. In order to generate a database to test this hypothesis, we performed digestibility studies in eight black rhinoceroses (D. bicornis) from three zoological institutions, using total faecal collection for the quantification of faecal output. One to three regularly fed zoo rations of roughage, concentrates and varying proportions of browse material were used per animal. Additional data was taken from three hitherto unpublished studies as well as several published sources. When compared with horses on similar rations, black rhinoceroses achieved lower digestion coefficients for organic matter and CF. In general, an increase in dietary CF content led to a steeper decrease in organic matter and GE digestibility in black rhinoceroses than in horses. When comparing available data for rhinoceroses, browsing species showed a steeper decrease in organic matter digestibility than grazing species with increasing dietary cell wall content. Endogenous losses as determined by linear regression analysis were within the range reported for horses and Indian rhinoceroses. The results suggest that the horse is not a useful model animal for evaluating diets for black rhinoceroses energetically. In general, diets fed to captive black rhinoceroses seem to include higher proportions of concentrates than diets for other rhinoceros species, and an increase in browse or roughage would reduce digestion coefficients to levels observed in animals fed natural forage.     

Molecular identification, genetic diversity and distribution of Theileria and Babesia species infecting small ruminants

Detection and identification of Theileria and Babesia species in 920 apparently healthy small ruminants in eastern Turkey, as well as parasite genetic diversity, was investigated using a specifically designed reverse line blot (RLB) assay. The hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene was amplified and hybridized to a membrane onto which catchall and species-specific oligonucleotide probes were covalently linked. Three Theileria and one Babesia genotype were identified. Comparison of the Theileria genotypes revealed 93.6-96.2% similarity among their 18S rRNA genes. Two Theileria shared 100% and 99.7% similarity with the previously described sequences of T. ovis and Theileria sp. OT3, respectively. A third Theileria genotype was found to be clearly different from previously described Theileria species. The genotype was provisionally designated as Theileria sp. MK. The Babesia genotype shared 100% similarity with Babesia ovis. The survey indicated a high prevalence of piroplasm infections in small ruminants (38.36%). Theileria spp. prevalence was 36.08%. Prevalence of B. ovis was 5.43%. The most abundant Theileria species identified was T. ovis (34.56%) followed by Theileia sp. MK (1.30%) and Theileria sp. OT3 (0.43%).     

Detection of Theileria and Babesia species in ticks collected from cattle

The present study was carried out to detect tick species that infest cattle, and Theileria and Babesia species transmitted by these ticks in Kayseri province (Turkey). A total of 300 cattle were examined for tick infestations. Of the 300 cattle, 117 (39%) were infested with ticks. A total of 1160 ticks belonging to 11 Ixodid genera were collected from the infested animals and their shelters. The most prevalent tick species was Boophilus annulatus 26.37% (306/1160) followed by Hyalomma marginatum marginatum 21.12% (245/1160) and Rhipicephalus turanicus 18.7% (217/1160). The collected ticks were separated into 43 tick pools, according to their species. These pools were examined for bovine Theileria and Babesia species (Theileria sp., Babesia sp., Theileria annulata, T. buffeli/orientalis, Babesia bigemina, B. bovis and B. divergens) by using the reverse line blotting method (RLB). Of the 43 tick pools examined, 6 (14%) were infected with B. bigemina, 4 (9.3%) with T. annulata, and 1 (2.3%) with Babesia sp., whereas 1 (2.3%) displayed mixed infection with T. annulata + B. bigemina. The sequence and phylogenetic analyses of Babesia sp., which could not be identified to the species level by RLB, were performed. In the phylogenetic tree, Babesia sp. (Kayseri 1) grouped with Babesia sp. (Kashi 2), Babesia sp. (Kashi 1), Babesia sp. (Xinjiang) and B. orientalis with 96.8-100% identity.     

Development of a polymerase chain reaction method for diagnosis of Babesia ovis infection in sheep and goats

In this study, a pair of oligonucleotide primers were designed according to the nucleotide sequence of the small subunit ribosomal RNA (ssu rRNA) gene of Babesia ovis isolated from sheep in eastern Turkey. The primers were used to detect parasite DNA from blood samples of B. ovis-infected sheep and goats by polymerase chain reaction (PCR). A 549-bp DNA fragment was specifically amplified from blood samples from sheep and goats, naturally infected with B. ovis. No PCR products resulted from Babesia motasi, T. ovis, Theileria sp. OT1, Theileria sp. OT3, T. lestoquardi, B. canis, B. microti,T. annulata or normal sheep leucocytes DNA using these specific primers. B. ovis-infected erythrocytes with 1% parasitemia were subjected to 10-fold serial dilutions (from 10(-1) to 10(-9)) using an uninfected sheep erythrocytes, and DNA was extracted from each diluted sample for testing the sensitivity of the PCR. The PCR was sensitive enough to detect parasite DNA from the dilution of 10(-5) with 0.00001% parasitemia. This is more sensitive than examining 200 fields under light microscopy. In addition, 98 field samples collected from small ruminanats in eastern Turkey were tested for B. ovis infection. Four samples were positive Babesia spp. in blood smears, 21 samples were positive for B. ovis DNA by PCR. These results indicate that the PCR provides a useful diagnostic tool for the detection of B. ovis infection in sheep and goats.     

Acute intravascular hemolytic anemia in the black rhinoceros: hematologic and immunohematologic observations

To investigate the syndrome of acute intravascular hemolytic anemia in the black rhinoceros (Diceros bicornis), laboratory techniques used in the differential diagnosis of hemolytic anemias were performed on blood samples from 6 black rhinoceroses: 3 nonrelated healthy rhinoceroses, 1 rhinoceros with iron deficiency anemia, and 2 rhinoceroses with intravascular hemolysis. Osmotic fragility, erythrocyte membrane protein composition, hemoglobin electrophoresis, and hemoglobin stability did not distinguish between healthy and affected (anemia or hemolysis) rhinoceroses. Polyclonal antiglobulin reagents were prepared in rabbits, using whole rhinoceros serum and purified rhinoceros immunoglobulin G. These reagents were nonreactive against erythrocytes of the healthy and iron-deficient rhinoceroses. Reactions with RBC from the rhinoceros with fatal hemolytic anemia indicated increased membrane coating by the third component of complement; this was not evident in a second rhinoceros that survived a hemolytic event.     

Molecular identification of tick-borne pathogens in Nigerian ticks

A molecular epidemiology investigation was undertaken in two Nigerian states (Plateau and Nassarawa) to determine the prevalence of pathogens of veterinary and public health importance associated with ticks collected from cattle and dogs using PCR, cloning and sequencing or reverse line blot techniques. A total of 218 tick samples, Amblyomma variegatum (N=153), Rhipicephalus (Boophilus) decoloratus (N=45), and Rhipicephalus sanguineus (N=20) were sampled. Pathogens identified in ticks included piroplasmids (Babesia spp., Babesia bigemina and Babesia divergens), Anaplasma marginale and Rickettsia africae. Piroplasmids were identified in A. variegatum, A. marginale was found in R. decoloratus, while R. africae was detected in all tick species examined. Ehrlichia spp. and Theileria spp. were not identified in any of the ticks examined. Of the 218 ticks examined, 33 (15.1%) contained pathogen DNA, with the presence of B. divergens and R. africae that are zoonotic pathogens of public health and veterinary importance. The variety of tick-borne pathogens identified in this study suggests a risk for the emergence of tick-borne diseases in domestic animals and humans, especially amongst the Fulani pastoralists in Plateau and Nassarawa states of Nigeria.     

Anaplasma phagocytophilum in horses and ticks: a preliminary survey of Central Italy

Anaplasma phagocytophilum, the causative agent of granulocytic ehrlichiosis, affects several species of wild and domesticated mammals, including horses. In this work we compared direct and indirect methods to evaluate A. phagocytophilum presence in Central Italy: 135 sera were screened by IFA for A. phagocytophilum and other haemopathogens (Theileria equi and Babesia caballi). Each horse was also tested for A. phagocytophilum 16S rRNA with a nested-PCR technique. In order to examine the risk of A. phagocytophilum transmission, 114 ticks were examined for the presence of A. phagocytophilum by PCR targeting the 16S rRNA. The seroprevalence against A. phagocytophilum was 17.03% and 11 horses (8.14%) showed positive PCR results. The concordance rate of A. phagocytophilum detection between IFAT and PCR had a K value of 0.34.     

A case of transplacental transmission of Theileria equi in a foal in Trinidad

Equine piroplasmosis due to Theileria equi and Babesia caballi is endemic in Trinidad. A case of equine piroplasmosis due to T. equi was diagnosed in a thoroughbred foal at 10h post-partum. A high parasitaemia (63%) of piroplasms was observed in a Wright-Giemsa(?) stained thin blood smear from the foal. In addition, the 18S rRNA gene for Babesia/Theileria was amplified from DNA extracted from the blood of the foal and the mare. Amplified products were subjected to a reverse line blot hybridization assay (RLB), which confirmed the presence of T. equi DNA in the foal. The mare was negative by RLB but was positive for T. equi using a nested PCR and sequence analysis. In areas where equine piroplasmosis is endemic, severe jaundice in a post-partum foal may be easily misdiagnosed as neonatal isoerythrolysis. Foals with post-partum jaundice should be screened for equine piroplasmosis, which may be confirmed using molecular methods if available.