Blood parasites of sheep in the Netherlands. II. Babesia motasi (Sporozoa,Babesiidae)

Summary

A large Babesia species occurs in sheep on the North Sea islands of the Netherlands. The tick Haemaphysalis punctata is a vector. Its pathogenicity appears to be low. It is morphologically similar to a Turkish strain, considered to be B. motasi, which is also transmitted by Haemaphysalis ticks. It differs from the Turkish parasite serologically as well as in cross‐immunity tests and in not being effective to goats.

There may be a group of morphologically similar parasites with serological differences and different infectivity for sheep and goats. As it is impossible to know which one is to be considered as the original B. motasi, we designate the Dutch parasite as B. motasi (Netherlands). Anaplasma mesaeterum was found to occur on the island of Texel as well as on Ameland, where it had been found initially.     

Babesia crassa n.sp. (Sporozoa,Babesiidae) of domestic sheep in Iran

Summary

A large Babesia sp., isolated from a sheep in Iran, proved to be serologically and morphologically different from B. motasi and B. ovis.

The parasite, designated B. crassa n. sp., is characterized by the frequent occurrence of four organisms in one erythrocyte, which is the result of quadruple division and, in other cases, of two successive binary divisions. Parasites resulting from the first of two successive binary divisions are exceptionally broad. B. crassa appears to be of low pathogenicity for sheep and goats, to which it is also infective. The vector is unknown.     

Serodiagnosis of Babesia motasi (Wales), Theileria recondita (Wales) and Cytoecetes phagocytophila infection in sheep

The indirect fluorescent antibody test (IFAT) was used to diagnose some tick-borne infections of sheep, Babesia motasi (Wales), Theileria recondita (Wales) and Cytoecetes phagocytophila. Antigen was prepared from blood derived from splenectomised sheep except for C phagocytophila which was derived from a normal animal. A field survey was made to assess the prevalence of B motasi and T recondita in North Wales and a comparison made between the titres using the B motasi (Wales) antigen with those previously reported. IFA titres reported in the homologous system were consistently lower than those described previously. The results of the field survey suggested that B motasi (Wales) infection is more widespread than was originally thought and more widespread than the known distribution of its vector Haemaphysalis punctata. No serological cross reactions occurred between B motasi (Wales), T recondita (Wales), C phagocytophila, B divergens, Sarcocystis ovicanis and Toxoplasma gondii.     

Isolation and identification of Babesia ovis in Extremadura (Spain).

A morphological and morphometric comparison was made of a Babesia ovis strain isolated in Extremadura (Spain) and a B. ovis strain from Turkey. Strains were morphologically similar, with a clear predominance of single sub-spherical or anaplasmoid forms. The use of an image analyser to measure B. ovis merozoites revealed significant differences in the long axis, short axis and volume between the two strains. Both strains however behaved in a similar manner in the indirect fluorescent antibody (IFA) test, which suggests a high degree of common antigenicity, despite morphometric differences. Conversely, no cross-reaction was observed between these strains of B. ovis and the other antigens used: B. motasi (The Netherlands), B. motasi (Turkey), B. crassa (Iran) and Theileria ovis (Turkey). B. ovis is therefore considered to be the aetiological agent of babesiosis in the area under study.     

An epidemiological study on ovine babesiosis in the Mashhad suburb area,province of Khorasan,Iran

The prevalence of Babesia spp. infection was studied in sheep of the Mashhad area in Iran from 1998 to 2000. A total of 677 sheep originating from 115 flocks were clinically examined and investigated for the presence of Babesia spp. in appropriate blood smears and any tick species on the body of the animals. The study revealed that the infection rate for Babesia ovis and Babesia motasi were 167 (24.6%) and 4 (0.5%), respectively. Double (mixed) infections occurred in 21 (3%) sheep. Differences in infection rates were statistically non-significant between male and female sheep and between different age groups. Seasonally, the prevalence of Babesia spp. infection started to increase in April and reached highest values in August (56%), while a decrease was observed in September, reaching the lowest levels In February and March. The study demonstrated that 1.7% of sheep infected with B. ovis and 50% of sheep infected with B. motasi exhibited clinical signs. Sheep infected with B. motasi showed the highest levels of parasitemia. We found that 550 (73%) of the animals harbored Rhipicephalus sanguineus; 166 (21%) Hyalomma marginatum; 19 (2.5%) Dermacentor daghestanicus; 14 (1.8%) Hyalomma anatolicum; 6 (0.66%) Hyalomma asiaticum; and one (0.13%) Haemaphysalis punctata. The examination of 727 tick haemolymph samples and 52 tick egg smears showed that one sample (0.2%) of haemolymph of R. sanguineus, two (1.2%) haemolymphs of H. marginatum and two (2%) eggs of R. sanguineus harbored kinetes morphologically matching the criteria described for B. ovis.     

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.     

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.     

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.     

At least two genetically distinct large Babesia species infective to sheep and goats in China

A fatal disease of sheep and goats in the northern part of China has been reported to be due to Babesia ovis. However, some characteristics of the causative agent in recent reports are not in accordance with the original attributes ascribed to this parasite. Therefore, the 18S small subunit ribosomal RNA (18S rRNA) genes of a number of Babesia isolates in China were sequenced and compared with that of other Babesia and Theileria species in an attempt to clarify their taxonomic position. In the present study, seven Babesia isolates were collected from distinct areas of northern China, and the 18S rRNA genes were amplified and sequenced. The phylogenetic trees were inferred based on 18S rRNA gene sequences of the Chinese ovine Babesia isolates and some of ovine Babesia and Theileria species available in GenBank. In the phylogenetic tree, Babesia sp. isolates from Madang, Tianzhu, Lintan, Ningxian, Hebei and Liaoning all grouped with B. motasi with 88.2-99.9% identity, while Babesia sp. Xinjiang grouped in a separate clade between B. ovis and B. crassa with 79.7-81.2% identity. The results indicated that there are at least two distinct Babesia species groups-B. motasi and Babesia sp. Xinjiang, the latter was distinctly different from other ovine Babesia isolates from China with less than 86.6% identity.     

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.     

Ixodid ticks on ruminants, with on-host initiated moulting (apolysis) of Ixodes, Haemaphysalis and Dermacentor larvae

To screen the host-dependent abundance of hard tick (Acari: Ixodidae) developmental stages on ruminants in South Hungary, red and roe deer, as well as goats and sheep were examined in a season, when larvae and nymphs are active. Altogether 2271 ticks were collected. In the relevant period the prevalence of tick-infestation was significantly higher among goats, than among sheep kept in the very same area, most likely in association with the browsing habit of the former. Roe deer and goats were found to be important hosts for Ixodes ricinus and Haemaphysalis concinna larvae, in contrast to the view that this stage does not usually feed on medium-sized mammals. Interestingly, one third of I. ricinus larvae and one larva of H. concinna and of Dermacentor reticulatus collected from goats in the same herd in August have started the moulting process (showed apolysis) on their host, despite being three-host ticks. This is the first survey involving four species of domestic and wild ruminants in Europe to compare the host-preference of ixodid ticks in the same region.     

A comparison of four serological tests for the diagnosis of caseous lymphadenitis in sheep and goats

A double antibody sandwich ELISA (ELISA A) developed for the detection of Corynebacterium pseudotuberculosis infection in sheep and goats was modified to improve its sensitivity. To establish the sensitivity and specificity of this modified ELISA (ELISA B), sera from 183 sheep and 186 goats were tested using ELISAs A and B. Comparison was also made with two further ELISAs (C and D) developed in Australia that, respectively, detect antibodies to cell wall antigens or toxin.ELISA B had the best performance of the four tests. Its specificity was 98+/-1% for goats and 99+/-1% sheep. Its sensitivity was 94+/-3% for goats and 79+/-5% for sheep. ELISA B will now be tested for use in caseous lymphadenitis eradication and control programmes in The Netherlands. It will also be used in experimental studies of CL in Scotland.     

Molecular detection of the B1 gene of Toxoplasma gondii in blood samples of female sheep and goats in Tebessa,northeastern Algeria

Toxoplasmosis, caused by Toxoplasma gondii, is a parasitic zoonosis of crucial medical and veterinary importance. It is mainly diagnosed by serological methods which are limited by insufficient sensitivity. Therefore, it is necessary to rely on direct detection of the parasite. The present study was aimed for direct detection of the parasite DNA in the blood samples of sheep and goats using PCR targeting the B1 gene. The study was carried out in 20 small ruminant farms between 2016 and 2018 in Tebessa region, part of north-eastern Algeria, and concerned 227 and 91 aborted female sheep and goats respectively. DNA of T. gondii was detected in 35.24 % and 18.68 % blood samples of sheep and goats respectively (p < 0.001). Molecular prevalence was higher in 13−24 month old female sheep (93.33 %) than 1−12 month old female sheep (14.37 %) (p < 0.0001). While, in goats no significant difference was observed in relation to age. Female sheep that aborted between 1−60 days of gestation were found to be more infected (46.41 %) compared to females that aborted between 61−120 days of gestation (12.16 %) (p < 0.001). Whereas, female goats that aborted between 61−120 days of gestation were found to be more infested (30.77 %) compared to females that aborted between 1−60 days of gestation (16.67 %) (p < 0.001). This study revealed that small ruminants are highly infected with T. gondii, which represents a major risk for the consumer in Tebessa. Further studies are needed to improve our knowledge of the different genotypes of T. gondii infecting small ruminant population.     

The incidence of Chorioptes bovis (Acarina: Psoroptidae) on the feet of horses, sheep, and goats in the Netherlands

The feet of horses, sheep, and goats of different breeds and from many different localities were examined for Chorioptes bovis. In horses, mites were mainly found in the Belgian and Frisian breeds (40% and 62% infected, respectively). In sheep and goats, respectively 63% and 86% were infected. In horses as well as in sheep and goats, mange-lesions were rarely seen. A number of sheep and goats were examined for mites and lesions quantitatively. In sheep all mites were restricted to the region close to the accessory digits and the claws. In goats the average number of mites was higher than in sheep, and mites could be found on all locations of the feet at least as far as the carpal and tarsal joint. Both in sheep and goats the biggest density of mites was found just below the accessory digits. When crusts were present, they were generally small and hidden under the coat. In sheep, which were housed for a long period, crusts were seen more often and were more distinct than in pastured animals. A negative correlation between the number of mites and the presence and extensiveness of crusts was observed. A possible explanation for this phenomenon is suggested. From the results of this study it is clear that there is no necessity to list chorioptic mange in sheep and goats as a notifiable disease.     

The incidence of Chorioptes bovis (Acarina: Psoroptidae) on the feet of horses,sheep, and goats in the Netherlands

Summary

The feel of horses, sheep, and goats of different breeds and from many different localities were examined for Chorioptes bovis. In horses, mites were mainly found in the Belgian and Frisian breeds (40% and 62% infected, respectively). In sheep and goats, respectively 63% and 86% were infected. In horses as well as in sheep and goats, mange‐lesions were rarely seen. A number of sheep and goats were examined for mites and lesions quantitatively. In sheep all mites were restricted to the region close to the accessory digits and the claws. In goats the average number of mites was higher than in sheep, and mites could be found on all locations of the feet at least as far as the carpal and tarsal joint. Both in sheep and goats the biggest density of mites was found just below the accessory digits. When crusts were present, they were generally small and hidden under the coat. In sheep, which were housed for a long period, crusts were seen more often and were more distinct than in pastured animals. A negative correlation between the number of mites and the presence and extensiveness of crusts was observed A possible explanation for this phenomenon is suggested. From the results of this study it is clear that there is no necessity to list chorioptic mange in sheep and goats as a notifiable disease.     

Q fever serologic surveillance program for sheep and goats at a research animal facility

Since Q fever is a potential risk to personnel working with small ruminants, the serologic status of sheep and goats received at a medical school animal facility for research was evaluated. A total of 104 sheep and 102 goats were subjected to blood sample-collection procedures on arrival, as well as after a 2-week quarantine period, and the sera were tested for Q fever specific antibodies by complement-fixation (CF) and microagglutination (MA) tests. The results from the 2 tests were compared and analyzed for seroconversion. On the basis of the CF test, 14 sheep and 3 goats were considered positive; these included 7 sheep and 2 goats that seroconverted during the quarantine period. In contrast, 1 sheep and 5 goats were found positive by the MA test, which also detected seroconversion of 1 sheep and 1 goat. The use of both tests for serologic surveillance of Q fever in sheep and goats increased the likelihood of detection. Management safety practices are also required to minimize the risk of disease transmission.     

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.     

Immunoprotective effect of cysteine proteinase fractions from two Haemonchus contortus strains adapted to sheep and goats

A preliminary analysis of the significance of genetic diversity in cysteine proteinase genes has been performed simultaneously in sheep and goats, with regard to the immunological control using these enzymes against haemonchosis. For this purpose, we have studied the cross-immunoprotective effect of cysteine protease-enriched protein fractions (CPFs) in adult worms of two Haemonchus contortus strains from North America and Spain that are adapted to sheep and goats, respectively. Previous genetic analysis of cysteine proteinase genes in both strains has shown that some of loci are polymorphic and these differences are translated into changes in the amino acid sequences. However, our results show that CPFs from H. contortus adult worms have a protective effect against the parasite in both sheep and goats. These results are similar regardless of whether they were obtained from sheep or goat-adapted H. contortus strains, which could be very important in case H. contortus CPFs were commercially used in different countries, as vaccines to prevent the negative effects of this parasite. Interestingly, this experimental inoculation of both species with a heterologous strain of H. contortus contributes to the idea shown in previous studies about how difficult is the interpretation and the comparison of vaccination where strains not adapted to a specific host are used. Therefore, the challenger of using heterologous strains could provide similar results to those observed in immunised animals. This study suggests the possibility of exploring the mechanisms involved in natural protection against non-adapted strains, in order to develop strategies to control haemonchosis.     

The contribution of molecular biology to the development of vaccines against nematode and trematode parasites of domestic ruminants.

Rapid developments in molecular biology have had an enormous impact on the prospects for the development of vaccines to control the major nematode and trematode infestations of livestock. Vaccine candidates are purified using conventional protein chemistry techniques but the limitations imposed by the scarcity of parasite material provide an insurmountable barrier for commercial vaccine production by this means. The ability to purify mRNA from different parasite life-cycle stages and to prepare cDNA expression libraries from it has proven central to the identification of immunogenic parasite proteins. Potentially, protective parasite antigens can now be produced in recombinant form in a variety of vectors and this represents a key breakthrough on the road to commercial vaccine production. The contribution of molecular biology to this process is discussed using several examples, particularly in vaccine development against the pathogenic abomasal nematode of sheep and goats, Haemonchus contortus, and the liver fluke of sheep and cattle, Fasciola hepatica. The difficulties of producing recombinant proteins in the correct form, with appropriate post-translational modification and conformation, are discussed as well as emerging means of antigen delivery including DNA vaccination. The opportunities offered by genome and expressed sequence tag analyses programmes for antigen targeting are discussed in association with developing microarray and proteomics technologies which offer the prospect of large scale, rapid antigen screening and identification.