Ticks and hemoparasitoses of livestock in Senegal. III. The Northern Sudan area

The authors describe the results of a study on ticks and hemoparasitoses of cattle and small ruminants in the Senegalese north-sudanian area. For 15 months, 40 bovine, 40 sheep and 40 goats received a routine dipping treatment, aimed at the determination of the tick population dynamics together with an accurate localization of the preferential sites for the different species. The following parasites were collected from the animals: Hyalomma marginatum rufipes, H. truncatum, Rhipicephalus lunulatus, Rh. e. evertsi, Rh. sulcatus, Rh. senegalensis, Boophilus decoloratus. Joint studies were conducted on the hemoparasites using blood smear and splenectomy. Among bovine, Anaplasma marginale, Ehrlichia bovis, Theileria mutans, Th. velifera, Trypanosoma congolense, T. brucei and microfilariae from Setaria labiatopapillosa were observed. Babesia bigemina was observed after a splenectomy. In small ruminants, the detected infections are brought about by A. ovis, Th. ovis and T. vivax. Hematocrite value of apparently healthy animals are studied as well as the seasonal variation of this hematological factor.     

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.     

Simultaneous detection of Anaplasma and Ehrlichia species in ruminants and detection of Ehrlichia ruminantium in Amblyomma variegatum ticks by reverse line blot hybridization

The detection of Anaplasma and Ehrlichia species is usually based on species-specific PCR assays, since no assay is yet available which can detect and identify these species simultaneously. To this end, we developed a reverse line blot (RLB) assay for simultaneous detection and identification of Anaplasma and Ehrlichia species in domestic ruminants and ticks. In a PCR the hypervariable V1 region of the 16S ribosomal RNA (rRNA) gene was amplified with a set of primers unique for members of the genera Anaplasma and Ehrlichia [Int. J. Syst. Evol. Microbiol. 51 (2001) 2145]. Amplified PCR products from blood of domestic ruminants or Amblyomma variegatum tick samples were hybridized onto a membrane to which eight species-specific oligonucleotide probes and one Ehrlichia and Anaplasma catch-all oligonucleotide probe were covalently linked. No DNA was amplified from uninfected blood, nor from other hemoparasites such as Theileria annulata, or Babesia bigemina. The species-specific probes did not cross-react with DNA amplified from other species. E. ruminantium, A. ovis and another Ehrlichia were identified by RLB in blood samples collected from small ruminants in Mozambique. Finally, A. variegatum ticks were tested after feeding on E. ruminantium infected sheep. E. ruminantium could be detected in adult ticks even if feeding of nymphs was carried out 3.5 years post-infection. In conclusion, the developed species-specific oligonucleotide probes used in an RLB assay can simultaneously detect and identify several Ehrlichia and Anaplasma species. However, as no quantitative data for the detection limit are available yet, only positive results are interpretable at this stage.     

Blood parasites of sheep in the Netherlands. I. Anaplasma mesaeterum sp.n. (Rickettsiales, Anaplasmataceae)

Summary On two occasions an anaplasm was isolated from sheep on the Dutch island of Ameland. The organism proved to be highly pathogenic for splenectomised sheep; a non-splenectomised animal recovered spontaneously after the packed cell volume had decreased by 40%. Treatment with oxytetracycline was effective. Its pathogenicity for goats appeared to be low, and the organism was apparently not infective to splenectomised cattle. This anaplasm differs from Anaplasma ovis in that less than 30% of the organisms are marginally situated in the red cell, as against over 70% in A. ovis; cross-immunity with A. ovis was incomplete and the latter appeared to be far more pathogenic to goats than the Dutch anaplasm, for which the name Anaplasma mesaeterum sp.n. is proposed. Its ultrastructure is similar to that of A. marginale and A. ovis. The vector is either Ixodes ricinus or Haemaphysalis punctata. Its practical importance remains to be ascertained.     

Observed prevalence of tick-borne pathogens in domestic animals in Sicily, Italy during 2003-2005

The objective of this study was to characterize the observed prevalence of tick-borne pathogens (TBP) in domestic animals in Sicily, Italy during 2003-2005. Serological (competitive ELISA and indirect immunofluorescence antibody, n = 3299) and DNA tests (polymerase chain reaction and reverse line blot, n = 2565) were conducted on horse, donkey, cattle, sheep, goat, pig and dog samples. Pathogens analysed included Anaplasma, Ehrlichia, Rickettsia, Babesia and Theileria species, and Coxiella burnetii. The most prevalent TBP were Anaplasma and Babesia species. The results reported herein suggested that cattle could serve as the major reservoir for Babesia and Theileria spp. while for Anaplasma spp. cattle, dogs, sheep and goats may be the most important reservoir species. These results expanded our knowledge about the prevalence of TBP in Sicily and provided information to understand the epidemiology of tick-borne diseases and may help to implement measures to diagnose, treat and control transmission to humans and animals in this region.     

Blood parasites of sheep in the Netherlands. I. Anaplasma mesaeterum sp.n. (Rickettsiales,Anaplasmataceae)

Summary

On two occasions an anaplasm was isolated from sheep on the Dutch island of Ameland. The organism proved to be highly pathogenic for splenectomised sheep; a non‐splenectomised animal recovered spontaneously after the packed cell volume had decreased by 40%.

Treatment with oxytetracycline was effective. Its pathogenicity for goats appeared to be low, and the organism was apparently not infective to splenectomised cattle. This anaplasm differs from Anaplasma ovis in that less than 30% of the organisms are marginally situated in the red cell, as against over 70% in A. ovis; cross‐immunity with A. ovis was incomplete and the latter appeared to be far more pathogenic to goats than the Dutch anaplasm, for which the name Anaplasma mesaeterum sp.n. is proposed. Its ultrastructure is similar to that of A. marginale and A. ovis. The vector is either Ixodes ricinus or Haemaphysalis punctata. Its practical importance remains to be ascertained.     

羊泰勒虫PCR检测方法的建立和初步应用

利用羊泰勒虫18SrRNA基因的序列特点,设计合成种特异性引物,建立羊泰勒虫PCR检测方法,该方法能特异性扩增398bp的羊泰勒虫18SrRNA基因片段,而对羊巴贝斯虫、羊无浆体、牛环形泰勒虫和牛伊氏锥虫的基因组DNA没有扩增带出现。对羊泰勒虫基因组DNA的最小检测量为0.12fgDNA。通过检测124份临床样品,24份为羊泰勒虫感染阳性,其余为阴性。结果表明,建立的PCR检测方法具有极高的敏感性和特异性,可用于羊泰勒虫病和临床健康带虫羊的诊断。     

Detection and quantification of Anaplasma marginale DNA in blood samples of cattle by real-time PCR

A TaqMan-based real-time PCR assay was developed for the diagnosis of Anaplasma marginale infection of cattle. The established assay was proven to be highly specific, since no cross-reactions were observed with other Anaplasma species of ruminants, including the closely related Anaplasma centrale, or other haemoparasites of ruminants (Anaplasma bovis, Anaplasma ovis, Anaplasma phagocytophilum, Babesia bovis, Babesia bigemina, Theileria annulata and Theileria buffeli). The detection limit was equal to that of nested (n)PCR (10(1) copies of standard DNA and 3 x 10(1) infected erythrocytes ml(-1) of blood). The assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. Fifty-four blood samples of ruminants (cattle, n = 51; sheep, n = 2; goats, n = 1), that had been tested previously by reverse line blot (RLB) hybridisation, were subjected to an nPCR assay and the newly established real-time PCR assay. By using real-time PCR, A. marginale DNA was detected in 39/51 bovine samples, with DNA titres ranging from 3.60 x 10(3) to 5.70 x 10(8) copies ml(-1) of blood, whereas sheep and goat samples tested negative. The concordance with nPCR was 100%, whereas a unique sample that had tested negative by RLB gave positive results by nPCR and real-time PCR. The established assay could overcome the limitations of existing diagnostic methods, allowing for simultaneous detection and quantification of the A. marginale DNA in bovine blood, that is essential to support the clinical diagnosis, to assess the carrier status of the animals and to evaluate the efficacy of vaccines and antirickettsial drugs.     

Detection of haemoparasites in cattle by reverse line blot hybridisation with a note on the distribution of ticks in Sicily

A reverse line blot hybridisation (RLB) of 21 oligonucleotides with polymerase chain reaction (PCR) amplified regions of 16S rRNA (Ehrlichia/Anaplasma group) or 18S rRNA (Babesia/Theileria group) genes of haemoparasites detected Theileria annulata, T. buffeli/orientalis, Babesia bovis, B. bigemina, B. divergens, Ehrlichia bovis, Anaplasma marginale, A. centrale and unknown species within the Rickettsia tribe.A very high prevalence of mixed infections was detected, which indicated that animals infected with Babesia spp. were also infected with Theileria spp. and/or Anaplasma spp.The tick distribution appeared to be seasonal with Hyalomma marginatum as the most frequently observed tick and Boophilus annulatus and Ixodes ricinus as the least frequently observed ticks. Other species identified in the 818 ticks collected during the five sampling periods between April 1998 and November 1999 included H. lusitanicum, Rhipicephalus sanguineus group, R. bursa, Dermacentor marginatus, Haemaphysalis punctata, B. annulatus and I. ricinus.     

A survey of ixodid ticks feeding on cattle and prevalence of tick-borne pathogens in the Black Sea region of Turkey

The study reports the frequency of infestation and the prevalence of tick-borne pathogens in feeding adult ticks detached from cattle in two climatic zones of the Black Sea region of Turkey. A total of 2160 adult ticks were collected during 2007-2008. Of these, 1062 were randomly selected, divided into 224 pools, and tested for the presence of bovine Theileria, Babesia, and Anaplasma species. Eleven tick species were recognized on cattle in the study. Hyalomma marginatum was widely disrubuted in the semi-arid bioclimatic zone, but few specimens were collected in the humid bioclimatic zone. The most prevalent tick species in the humid climatic zone was Ixodes ricinus. Infection rates were calculated as the maximum likelihood estimation with 95% confidence intervals (CI). Overall, 4% (CI 2.87-5.44) of 224 tick pools were found to be positive for the pathoges by Reverse line blot. Maximum likelihood estimation of the infection rate varied among tick species, ranging from 2.68% (CI 0.16-12.68) in Haemaphysalis sulcata to 10.49% (CI 4.07-23.66) in Rhipicephalus bursa. The most prevalent tick-borne pathogen was Anaplasma phagocytophilum at 6.78% (CI 3.41-12.18) followed by A. centrale (6.56%, CI 0.42-31.47), Anaplasma/Ehrlichia spp. (3.61%, CI 1.99-6.06), Babesia spp. (3.33%, CI 1.65-6.03), and T. buffeli/orientalis (2.71%, CI 0.73-7.18). Sequencing results indicated that Babesia spp. shared 99% to 100% similarity with the unnamed Babesia sp. Kashi 1 and 2, Babesia sp. Kayseri 1 and Babesia sp.CS58. Anaplasma/Ehrlichia spp. were 98% and 100% identical to Ehrlichia canis and Ehrlichia sp. Omatjenne strain, respectively.     

Phylogenetic analysis of the erythrocytic Anaplasma species based on 16S rDNA and GroEL (HSP60) sequences of A. marginale,A. centrale,and A. ovis and the specific detection of A. centrale vaccine strain

Phenotypic criteria for the identification of erythrocytic ruminant Anaplasma species has relied on subjective identification methods such as host pathogenicity (virulence for cattle or sheep) and/or the location of Anaplasma inclusion bodies within the host's red cells. Sequence comparisons of new and available GenBank Accessions were investigated to elucidate the relationships among these closely related Anaplasma species. Twenty-one 16S rDNA and GroEL (HSP60) sequences from 13 Anaplasma marginale (South Africa, Namibia, Zimbabwe, Israel, USA, Australia and Uruguay), three A. centrale (South Africa and Japan), two A. ovis (USA and South Africa), and two unknown Anaplasma species isolated from wild ruminants (South Africa), were compared. 16S rDNA maximum-likelihood and distance trees separated all A. marginale (and the two wild ruminant isolates) from the two South African A. centrale (including original vaccine strain, Theiler, 1911). The Japanese A. centrale (Aomori) demonstrated the lowest sequence identity to the remaining erythrocytic Anaplasma species. A. ovis inter-species relationships could not be resolved through the 16S rDNA analyses, whereas strong bootstrap branch support is demonstrated in the GroEL distance tree using A. ovis OVI strain. All erythrocytic Anaplasma species and isolates were confirmed to belong to the same cluster showing strong branch support to Anaplasma (Ehrlichia) phagocytophilum with Ehrlichia (Cowdria) ruminantium and Rickettsia rickettsii serving as appropriate out-groups. Based on groEL sequences, a specific PCR method was developed which amplified A. centrale vaccine (Theiler, 1911) specifically. This study confirms the suitability of 16S rDNA sequences to define genera and demonstrates the usefulness of GroEL sequences for defining species of erythrocytic Anaplasma.     

Molecular detection of Theileria sp. in ticks and naturally infected sheep

Seven healthy sheep and 10 sheep diagnosed with piroplasmosis based on clinical signs were tested for the presence of babesiae and theileriae. Using the molecular techniques, two species of theileriae were detected and characterized. Theileria ovis was present mostly in healthy sheep and in Rhipicephalus ticks collected from infected sheep. Theileria sp. OT3 parasite was detected mostly in ill animals which represent additional evidence to the possible pathogenic nature of Theileria sp. OT3. The presence of babesiae in sheep or in ticks was not determined. The results of this study showed that ovine piroplasmosis due to Theileria is present in Southern Croatia. It was concluded that clinical diagnosis of ovine piroplasmosis should be confirmed by molecular analysis in order to identify the species of piroplasm, to select the appropriate treatment and to exclude the threat for public health.     

Experiments on transmission of an unidentified Theileria sp. to small ruminants with Haemaphysalis qinghaiensis and Hyalomma anatolicum anatolicum

Experiments on the transmission of an unidentified Theileria sp. infective for small ruminants by Haemaphysalis qinghaiensis and Hyalomma anatolicum anatolicum were carried out. Three Theileria-free batches of adult, larvae, and nymphs of laboratory reared H. qinghaiensis and Hy. a. anatolicum ticks were infected by feeding them on sheep infected with Theileria sp. The Theileria sp. was originally isolated from adult ticks of H. qinghaiensis, by inoculation of blood stabilates or tick transmission. H. qinghaiensis has been shown to be capable of transmitting the Theileria sp. infective for small ruminants transstadially to sheep and goats. The nymphs developed from the larvae engorged on the sheep infected with the parasite transmitted the pathogen to splenectomized sheep with prepatent periods of 30, 31 days, respectively; but the subsequent adult ticks of H. qinghaiensis derived from the nymphs did not transmit the pathogen to sheep. However, adults developed from the nymphs engorged on the sheep infected with the parasite transmitted the pathogen to sheep with prepatent periods of 24-27 days. The larvae, nymphs and adult ticks derived from female H. qinghaiensis ticks engorged on infected sheep were not able to transmit the parasite transovarially. The same experiments were done with Hy. a. anatolicum, but examination for presence of piroplasma of Theileria sp. from all animals were negative, demonstrating that Hy. a. anatolicum could not transmit the organism to sheep or goats.     

Serologic and molecular characterization of Anaplasma species infection in farm animals and ticks from Sicily

Although Anaplasma marginale was known to be endemic in Italy, the diversity of Anaplasma spp. from this area have not been characterized. In this study, the prevalence of Anaplasma spp. antibodies in randomly selected farm animals collected on the island of Sicily was determined by use of a MSP5 cELISA for Anaplasma spp. and an immunofluorescence test specific for Anaplasma phagocytophilum. Genetic variation among strains of Anaplasma spp. from animals and ticks was characterized using the A. marginale msp1alpha and the Anaplasma spp. msp4 genes. Eight species of ticks were collected and tested by PCR. Seropositivity for Anaplasma spp. and A. phagocytophilum was detected in bovine and ovine samples. All the donkeys were seropositive for A. phagocytophilum but not for Anaplasma spp. Four A. marginale genotypes were identified by msp4 sequences from bovine and tick samples. Two new genotypes of Anaplasma ovis were characterized in sheep. The sequences of A. phagocytophilum from three donkeys proved to be identical to the sequence of the MRK equine isolate from California. Six A. marginale genotypes were found in cattle and one tick using the A. marginale msp1alpha sequences. All genotypes had four repeated sequences in the N-terminal portion of the MSP1a, except for one that had five repeats. The Italian strains of A. marginale contained three repeat sequences that were not reported previously. Definition of the diversity of Anaplasma spp. in Sicily reported, herein is fundamental to development of control strategies for A. marginale, A. ovis and A. phagocytophilum in Sicily.     

Tick infestations on livestock in the yemen arab republic and their potential as vectors of livestock diseases

A survey of ticks on cattle, camels, sheep, goats and donkeys in four different geographical locations of the Yemen Arab Republic (YAR) was carried out to provide more information on the possible risk of tick-borne diseases to imported exotic cattle included in the YAR's plans for livestock development. The most abundant ticks were Hyalomma spp. particularly on camels. Ticks found on cattle included Hyalomma spp., Amblyomma variegatum, Boophilus annulatus and Rhipicephalus spp. In general with the exception of camels tick burdens on all species of domestic livestock were very low. Two hundred and ninety eight serum samples from miscellaneous adult cattle throughout the country were negative to a test for Anaplasma marginale antibodies. It is speculated that tick burdens in the YAR are too low for significant disease transmission and the implications of the findings are discussed.     

Characterization of Anaplasma phagocytophilum and A. ovis infection in a naturally infected sheep flock with poor health condition

Anaplasma species are transmitted by ticks and cause diseases in humans and animals. These pathogens infect sheep, an economically important domestic animal worldwide. The current study was designed to characterize in 200 animals the infection with Anaplasma phagocytophilum and Anaplasma ovis and the genetic diversity of A. ovis strains collected from a naturally infected sheep flock with poor health condition. Sheep had 98% seroprevalence to Anaplasma spp. antibodies. PCR results confirmed the presence of A. phagocytophilum and A. ovis DNA in 11.5% and 37% of the sheep, respectively. Concurrent infections were detected in 6.5% of the sheep. Seventy-one adult ticks were collected from 45 sheep with infestations ranging from one to 15 ticks per animal. The analysis of A. ovis msp4 sequences demonstrated a previously unreported polymorphism for this pathogen with 17 different haplotypes in infected sheep. These results demonstrated that, although A. ovis msp4 haplotypes may be less variable when compared with Anaplasma marginale and A. phagocytophilum strains on a global scale, genetic polymorphisms occur in this locus in strains obtained from an infected sheep flock with poor health condition.     

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.     

Determination of prevalence and risk factors of infection with Babesia in small ruminants from Greece by polymerase chain reaction amplification

A total of 124 blood samples were collected from 92 sheep and 32 goats from 21 randomly selected herds located in two regions of Greece. Data on the characteristics of the animals (species, gender, age, tick burden, presence of haemoglobinuria, prior treatment for babesiosis) and the herd (location, size, species of animals, dogs associated with the herds, tick burden of dogs associated with the herds) were collected through questionnaires. Nineteen animals (15%) produced the DNA fragment specific for Babesia of which 16 were sheep and three were goats. Nucleotide sequence of PCR products revealed 100% homology with Babesia ovis 18S rRNA gene. Nine farms (43%) were found positive for B. ovis. The percentage of positive animals in each farm varied between 10 and 61%. The relative risk of the presence of ticks in sheep and goats (p<0.01) and farm dogs (p<0.01) for PCR-positive results for B. ovis in sheep and goats was found 6.63 and 4.14, respectively.     

Fatal bovine anaplasmosis in a herd with new genotypes of Anaplasma marginale, Anaplasma ovis and concurrent haemoplasmosis

Haematological and molecular analysis of blood samples was carried out during an outbreak of bovine anaplasmosis in Hungary. Acute disease was observed in five animals, two of which died. Anaplasma-carrier state was diagnosed in 69 (92%) of cattle. Further evaluation of 24 blood samples revealed concurrent infections with Mycoplasma wenyonii and 'CandidatusM. haemobos' in 22 and 21 animals, respectively. In addition, two cows were identified with rickettsaemia. Regarding molecular investigation of potential hard tick vectors, Haemaphysalis inermis and Dermacentor marginatus males collected from the animals were PCR-negative. However, in one pool (out of 18) of Ixodesricinus males, and in six pools (out of 18) of D. reticulatus males the msp4 gene of Anaplasma marginale was detected. In the same I. ricinus pool Anaplasma ovis was also identified. All ticks were negative for haemoplasmas. Anaplasma sequences yielded 97-99% homology to sequences deposited in the Genbank. This is the first report of fatal bovine anaplasmosis associated with divergent A. marginale genotypes and concurrent 'CandidatusM. haemobos' infection, as well as of an A. ovis strain in ticks collected from cattle.     

Molecular evidence of tick-borne hemoprotozoan-parasites (Theileria ovis and Babesia ovis) and bacteria in ticks and blood from small ruminants in Northern Algeria

Using qPCR, standard PCR and/or sequencing, we investigated the presence of tick-associated microorganisms in ticks and blood from sheep and goats from Souk Ahras, Algeria.Borrelia theileri, was detected in (7/120, 5.8%) blood from sheep and (13/120, 10.8%) goats. Anaplasma ovis was screened in (38/73, 52%) Rhipicephalus bursa and (5/22, 22.7%) R. turanicus and in (74/120, 61.7%), (65/120, 54.2%) blood of sheep and goats respectively.Coxiella burnetii tested positive in R. bursa (4/73, 5.5%) and (7/120, 5.8%) blood of sheep and (2/120, 1.7%) goats. Theileria ovis was detected in (50/147, 34%) R. bursa and (3/22, 13.6%) R. turanicus and in (64/120, 53.3%) blood of sheep and (25/120, 20.8%) goats. Babesia ovis was screened positive in (23/147, 15.6%) R. bursa and (7/48, 14.6%) R. turanicus.Our findings expand knowledge about the repertoire of tick-borne microorganisms present in ectoparasites and/or the blood of small ruminants in Algeria.