Piroplasms of domestic animals in the Macedonia region of Greece. 1. Serological cross-reactions.

During a serological survey on haemoparasites in Macedonia, serum samples were collected from cattle, sheep and goats. All sera were tested by the indirect immunofluorescence test (IFAT); the cattle sera against Theileria orientalis, T. annulata, Babesia bigemina, B. bovis, B. divergens and B. major antigens; the sheep and goat sera against T. ovis, B. ovis, B. motasi and B. crassa antigens. Parallel tests of negative and positive control sera against all the antigens showed the existence of cross-reactions of different degrees between species of the same genus. In cattle, the most important cross-reactions were obtained against B. bigemina antigen, especially with the anti-B. bovis serum, in small ruminants against B. motasi with the anti-B. crassa serum. In the field sera, there was a high correlation between the antibody titres of B. bigemina and B. bovis, and also between the titres of these two Babesia spp. and B. divergens. A high correlation was also found between B. motasi and B. crassa, and lower ones between these two and B. ovis. The correlations of the sera titres were due to mixed infections or to cross-reactions. Therefore, the use of the IFAT is not always satisfactory for diagnosing infections in regions where animals are infected with different piroplasms.

Piroplasms of domestic animals in the Macedonia region of Greece. 2. Piroplasms of cattle.

A study was carried out on piroplasms of cattle in the Macedonia region of Greece. During 1984-1986, 602 serum samples were collected from cattle in 33 localities. Blood smears were also prepared from 50 of the animals in 13 localities. The indirect fluorescent antibody (IFA) test revealed that 41.4% of cattle sera were positive to Theileria orientalis, 2.0% to Theileria annulata, 21.6% to Babesia bovis, 15.2% to Babesia bigemina, 5.1% to Babesia major and 2.7% to Babesia divergens. Some of the positive titres were probably due to cross reactions. T. orientalis was widely distributed. T. annulata was limited to a small number of foci. B. bigemina and B. bovis, often present together, were widespread, whereas B. major was only present in a small number of localities. No convincing evidence of B. divergens infection was found. By examination of Giemsa stained blood smears T. orientalis, T. annulata and a Babesia sp. were detected.

Piroplasms of domestic animals in the Macedonia region of Greece. 3. Piroplasms of small ruminants.

A study was carried out on piroplasms of small ruminants in the Macedonia region of Greece. During 1984-1985, 721 serum samples were collected from sheep in 49 localities and 487 from goats in 43 localities. Blood smears were also prepared from 26 sheep and eight goats in 16 localities. The prevalence of positive IFA titres for sheep and goat sera, respectively, was: 24.6% and 0.6% for Theileria ovis, 52.1% and 36.4% for Babesia ovis, 10.5% and 4.2% for Babesia motasi, 12.6% and 6.6% for Babesia crassa. Many of the positive reactions, specially for the large Babesia species, B. motasi and B. crassa, probably resulted from cross-reactions and the presence of these two species is doubtful. Antibodies against B. ovis were found in animals from more than 90% of the localities. T. ovis was also common, but it was confined essentially to sheep. The examination of Giemsa stained blood smears revealed the presence of T. ovis and B. ovis in sheep, and Anaplasma ovis in a goat.

An observational study of Eimeria species in housed cattle on Dutch dairy farms.

The prevalence of oocysts of Eimeria species in calves (n = 334), yearlings (n = 254) and cows (n = 1314) was determined on 38 Dutch dairy farms. Twelve species of Eimeria were identified in faecal specimens by sucrose-flotation. The prevalences of Eimeria spp. differed markedly in the different age classes on individual farms as well as between farms. The overall prevalence of Eimeria oocysts in faecal specimens was 46% for calves, 43% for yearlings and 16% for cows. The number of oocysts excreted was generally low in cows and yearlings, whereas high numbers of oocysts per gram of faeces (OPG) were exclusively observed in calves. No cases of clinical coccidiosis were observed in this survey. Linear regression analysis showed that there is significant reduction in the OPG levels (P < 0.05) in calves infected with Eimeria, aged between 7 and 38 weeks. Finally, the data are discussed in relation to management practices and the acquisition of immunity.

The indirect fluorescent antibody test for bovine anaplasmosis.

An indirect fluorescent antibody test was used successfully for the serodiagnosis of experimental Anaplasma infections in cattle. Specific antibodies were detected three to ten days after anaplasma bodies were found in the blood, and persisted at least 15 weeks post-infection. An American and an African stock of A. marginale were used to prepare antigens, and gave comparable results when tested on sera positive to either of these stocks, as well as to an A. central-like stock from Korea. There were no cross-reactions with several Theileria, Babesia, Trypanosoma and Eperythrozoon species.

[Presence of Theileria annulata in Mauritania]. / Présence de Theileria annulata en Mauritanie.

A fatal case of bovine theileriosis by Theileria annulata is reported from Mauritania in a Friesian cow born in a dairy farm in Nouakchott. The farm was originally established with cattle imported from France. Specific diagnosis was based on piroplasm morphology and on high specific antibody titres in some of the animals. The only tick found in the herd was Hyalomma dromedarii. Four of 49 local zebus sampled at random in the south of the country had high specific antibody titres to T. annulata, while 14 had lower or doubtful titres. The infection therefore appears to be autochthonous.

Serotypes in Cowdria ruminantium and their relationship with Ehrlichia phagocytophila determined by immunofluorescence.

Two tick-borne rickettsial pathogens of ruminants, Cowdria ruminantium (causative agent of heartwater disease) and Ehrlichia phagocytophila (causative agent of tick-borne fever), were successfully cultivated in caprine or ovine neutrophilic granulocytes. Infected cultures were subsequently used as antigens in the indirect fluorescent antibody test. Low-level bilateral serological cross-reactions could be detected between Cowdria and Ehrlichia. In addition, comparison of five Cowdria stocks using immunofluorescence demonstrated the existence of distinct serotypes within the genus of Cowdria. It is concluded that the occurrence of these serotypes will considerably complicate the current serodiagnosis of heartwater.

Three groups of Babesia canis distinguished and a proposal for nomenclature.

Two stocks of large Babesia from dogs originating in France, transmitted by Dermacentor reticulatus, two from North Africa, having Rhipicephalus sanguineus as vector, and one from South Africa, transmitted by Haemaphysalis leachi, were compared in cross-immunity tests in dogs and in the indirect fluorescent antibody test (IFAT). The French and North African stocks did not immunise against the South African one, while the North African stocks did not protect against a French one. The South African stock partially protected against a French one. The three groups could be clearly distinguished in the IFAT. These differences have practical implications for existing and future vaccines against canine babesiosis and for the serological diagnosis of atypical and chronic cases. It is proposed to use a trinomial system of nomenclature for these groups: Babesia canis canis (Piana and Galli-Valerio, 1895), Babesia canis vogeli Reichenow, 1937, and Babesia canis rossi (Nuttall, 1910), having Dermacentor, Rhipicephalus and Haemaphysalis ticks as their vectors respectively.

Borrelia infections of ruminants in Europe.

The occurrence of Borrelia spirochaetes in cattle in the Netherlands is reported for the second time, and in red deer and roe-deer in Austria for the first time. It is postulated that these spirochaetes are Borrelia burgdorferi rather than B. theileri. The reservoir role of ruminants in the epidemiology of human disease caused by B. burgdorferi in Europe should be investigated.

Relationships between Cytauxzoon felis and African piroplasmids.

A cat which had recovered from Cytauxzoon felis infection following treatment with the anti-theilerial drug, parvaquone, showed an increase in piroplasm parasitemia after splenectomy and its blood was suitable for the preparation of antigen smears for the indirect fluorescent antibody test. High levels of antibodies were found in this cat after recovery, in two other cats sub-lethally infected with piroplasms and in sera of naturally infected bobcats. Cats recovered from piroplasm infection died from cytauxzoonosis when challenged with organ material containing C. felis schizonts. Tests with piroplasm antigens and positive sera of C. felis, South African Babesia felis and African Theileria taurotragi showed no significant serological relationship between C. felis and the African parasites. C. felis was not shown to be infective for splenectomized sheep.

Observations on mouse-infective stocks of Cowdria ruminantium: attempts to demonstrate persistence of the organism in mice immune to the Kwanyanga stock.

Mice immunised against the Kwanyanga stock of Cowdria ruminantium by infection and treated with oxytetracycline proved immune to challenge on day 40 and also to a second challenge on day 125 after infection. Treatment with the experimental dithiosemicarbazone gloxazone on days 59 and 73 did not abolish immunity to challenge on day 125. No persistence of the organism in immune mice that had been challenged on day 40 could be demonstrated by subinoculating blood and liver homogenate on day 126. These results are different from findings reported elsewhere with the mouse-infective Kumm stock.

Theileria orientalis occurs in Central Africa.

An outbreak of bovine theileriosis in Burundi was investigated and the occurrence of Theileria orientalis in Central Africa was confirmed for the first time. Its identity was established on morphological and serological grounds. The parasite was associated with clinical signs and further complicates the disease picture of bovine theileriosis in Africa. Amblyomma variegatum is suspected as being the local vector.

Stage-specific antigenicity in Theileria annulata: a case report.

Blood from sick cattle in Bahrain transmitted piroplasms of Theileria annulata to a splenectomized calf. Larvae of Hyalomma anatolicum anatolicum were infected on the calf and, after moulting, induced clinical theileriosis, associated with numerous schizonts, in the same calf. The animal was cured by specific treatment. Antigenic differences thus shown between piroplasms on the one hand, and sporozoites and schizonts on the other hand, were confirmed in the indirect fluorescent antibody test, as a significant titre to T. annulata piroplasm antigen developed after the inoculation of blood, but to schizont antigen only after the infective ticks had induced the appearance of schizonts.

Theileria orientalis, a cosmopolitan blood parasite of cattle: demonstration of the schizont stage.

Serological and morphological comparison of Theileria orientalis stocks from Australia, Britain, Iran, Japan and the USA with a more pathogenic stock from Korea, corresponding to T sergenti of Russian literature, showed that they all belong to one species, for which the name T orientalis is recommended. T orientalis is now known to occur on all continents. Macroschizonts and microschizonts, found in some of the calves infected with the Korean stock, are described and illustrated. Infections with the Korean stock were associated with early hyperthermia during the period schizonts are found, and commonly with high parasitaemias and anaemia even in unsplenectomised calves. The higher pathogenicity of this stock may be related to a faster rate of division. T orientalis may cause latent infection in sheep. With the exception of the stock from the USA, all stocks could be transstadially transmitted by Haemaphysalis longicornis and, or, H punctata.

Cultivation of Theileria. I. Attempts to complete the cycle of Theileria parva in vitro.

Microschizonts and free merozoites developed in bovine lymphoblastoid cell cultures containing macroschizonts of 6 different strains of Theileria parva. Clean bovine red cells were added to the cultures, which were incubated in various ways. No penetration of red cells by merozoites was observed, not even when cultures in diffusion chambers were introduced into the peritoneal cavity of non-infected cattle.

Cultivation of Theileria. II. Attempts to cultivate erythrocytic stages of Theileria mutans.

Blood from calves with a parasitaemia of Theileria mutans was cultivated in vitro in various ways. No significant increase in the percentage of infected red cells was observed, but the percentage of dividing forms of the parasite rose sharply during the first two days of incubation. The conditions of the cultures were apparently unsuitable for the invasion of erythrocytes.

Cross immunity between strains of Cowdria ruminantium.

Cross immunity tests with strains of Cowdria ruminantium from South Africa, the Sudan, São Tomé and Nigeria failed to demonstrate antigenic differences. The antibiotic ampicillin showed no activity against heartwater.

Causal agents of bovine theileriosis in southern Africa.

One pathogenic and 4 mild bovine Theileria strains from southern Africa, all transmitted by Rhipicephalus appendiculatus, were compared amongst themselves as well as to bovine and buffalo strains of the T. parva complex from eastern and southern Africa and to bovine strains of T. taurotragi from Tanzania considered to be derived from eland antelope. Criteria used were parasitological, clinical, serological and cross-immunity characters. The mild strains are similar to bovine T. taurotragi. Serological evidence suggested that T. taurotragi is also infective to sheep. The pathogenic strain belongs to the T. parva complex; the latter consists of a series of types with different behaviour ranging from the lawrencei-type (of buffalo) causing Corridor disease, through the bovis-type causing Rhodesian malignant theileriosis to the parva-type causing classical East Coat fever. Seven cattle-tick passages of a bovis-type strain did not result in transformation into a parva-type. Four species of bovine Theileriae are now known to occur in southern Africa: T. parva (lawrencei- and bovis-types) and T. taurotragi, both transmitted by R. appendiculatus, and T. mutans and T. velifera both with Amblyomma spp. as vectors.

Isoenzyme studies on Theileria (Protozoa, Sporozoa). Enzyme activity associated with the erythrocytic stage.

Bovine blood containing piroplasms of Theileria parva, as well as non-infected blood, was lysed and subjected to iso-electric focussing. Staining for 13 different enzymes revealed parasite-associated bands of glucose phosphate isomerase (GPI) activity, not of any of the other enzymes. There were no variations between individual donor animals in the host cell GPI bands and these bands did not interfere with the recognition of the parasite-associated bands, so that purification of the piroplasms was unnecessary. Blood from cattle infected with T. mutans also gave parasite-associated bands of GPI, but no such bands were seen in zymograms of blood from cattle infected with a Theileria sp. from Japan. Dependent on the level of parasitaemia, up to four parasite-associated bands were found in one strain of T. parva and up to three in two other strains. Among the disadvantages of using piroplasm material for the study of isoenzymes of T. parva is the fact that animals often die before their parasitaemia is sufficiently high, and that some strains never give rise to a high parasitaemia.

Artificial infection of Rhipicephalus appendiculatus with Theileria parva by percutaneous injection.

Rhipicephalus appendiculatus nymphs were inoculated with fresh or cryopreserved blood containing Theileria parva piroplasms, or with cell culture grown stages of T parva. The use of fresh blood was successful. Cryopreserved blood containing dimethylsulphoxide (DMSO), killed most nymphs after inoculation: DMSO could be removed by slow dialysis, without destroying the infectivity of the blood. Attempts to infect ticks by inoculating cell culture grown stages of T parva failed, even when large numbers of merozoites were present in the inoculum.