Determination of the protease activity in a Cuban strain of Babesia bovis]

The attenuation of a Babesia bovis strain depends on its protease content. The present work evaluates this parameter on a virulent strain, before and after attenuation by quick passages on splenectomized calves. The protease activity at different pH values was determined in protein fractions from the blood of calves. The enzymatic test showed marked differences between the protease content of both substrains.     

Babesia, Theileria and Anaplasma spp. infecting sable antelope, Hippotragus niger (Harris, 1838), in southern Africa

Some observations are recorded on blood parasites of sable antelopes. Blood smears of 124 of these antelopes from South Africa and Zimbabwe were examined and 7 were found to be positive for a Babesia sp., identified as Babesia irvinesmithi Martinaglia 1936. A total of 70 of the smears were positive for theilerial piroplasms, while 1 smear had macroschizonts (with cytomeres) and microschizonts of a Theileria (= Cytauxzoon) sp. One blood smear was positive for an Anaplasma sp. Attempts to isolate the Babesia sp. by subinoculating blood from sable to splenectomized and intact sable and splenectomized cattle were unsuccessful. Attempts to infect sable with Babesia bovis and Babesia bigemina were likewise unsuccessful. Theilerial piroplasms reached high levels in a splenectomized sable but could not be transmitted with blood to cattle. The Anaplasma sp. was found to be infective for sheep but not for cattle.     

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.     

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%).     

Antibody response and duration of latent infection in sheep following experimental infection with Babesia ovis

Babesia ovis isolated in Extremadura (Spain) was the subject of a serological study in experimentally inoculated sheep. The first antibody titres, determined by the indirect fluorescent antibody (IFA) test, were observed 7-8 days post infection (p.i.) in all animals except the splenectomized group, in which the only animal that survived showed antibody response 10 days p.i. A faster response following challenge was observed in sheep which were seropositive before inoculation, which suggests the existence of an antigen memory. The highest titres were reached 16-25 days p.i., and subsequently began to fall, reaching minima at the end of the experimental period (330 days p.i.). The chronic carrier state in experimental B. ovis infection had a duration of at least 2 years. Passive transmission of antibodies from experimentally infected mothers to newborn lambs was also detected. Antibody levels were observed for a period not longer than 2 months after birth.     

The effect of the ovine host parasitaemia on the development of Babesia ovis (Babes, 1892) in the tick Rhipicephalus bursa (Canestrini and Fanzago, 1877)

Batches of Rhipicephalus bursa adult ticks were fed on two lambs with 10.0% (batch 1) and 0.3% (batch 2) Babesia ovis parasitaemia, respectively. Haemolymph and eggs were checked for parasites daily after detachment, before and after appearance of B. ovis in the lamb's blood.B. ovis kinetes were found in the haemolymph and eggs earlier in the engorged ticks detached before appearance of the parasite in the host blood. Rates of haemolymph and egg infection with B. ovis as well as the percentage of infected eggs were much higher in batch 1 (10% lamb parasitaemia) than in batch 2 ticks (0.3% lamb parasitaemia). In eggs incubated at 28 degrees C the optimal period to look for kinetes seems to be days 4-9. Heavily infected ticks laid fewer less eggs within a shorter oviposition period. Pre-oviposition, pre-hatching periods and egg hatchability were not affected. Various parasitic forms are described in the haemolymph and the eggs.     

Histopathological changes in sheep experimentally infected with Babesia ovis

Histopathological study was made of 12 Merino sheep - five splenectomized and seven intact - experimentally infected with Babesia ovis. Non-purulent encephalitis; initially exudative and subsequently interstitial pneumonia; pericarditis, myocarditis and haemorrhagic endocarditis; centrilobular necrotic hepatitis; hyperplasia of the lymphoreticular system; necrosis and vascular changes in adrenal glands were observed. The kidney was the most severely affected organ, exhibiting acute tubular necrosis typical of kidney shock syndrome. The lesions observed were suggestive of hypovolemic shock culminating in haemorrhagic diathesis owing to consumptive coagulopathy. Additionally, the massive release of catabolites from lysis and necrosis apparently produced endotoxic shock.     

The inability of a South African Babesia bovis vaccine strain to infect Boophilus microplus

A strain of Babesia bovis that had been attenuated by rapid syringe passage through a series of 23 splenectomized calves was unable to infect its vector Boophilus microplus. An attempt to transmit the attenuated Australian Babesia bigemina G strain with a South African strain of B. microplus was likewise unsuccessful. The epidemiological implication of these observations in terms of babesiosis control is discussed.     

The Effect of Diminazene Aceturate on Spienectomized Dogs with Babesia gibsoni Infection

The effect of diminazene aceturate on splenectomized and nonspienectomized dogs with Babesia gibsoni (B. gibsoni) infection was investigated. In splenectomized dogs, the fissional and multiplicational stages of B. gibsoni were observed in peripheral blood films, and hemoglobinuria was frequently observed. These findings were different from previous reports and were not changed by administration of diminazene aceturate. It is clear that the intramuscular administration of diminazene aceturate at the dose of 3 mg/kg body weight for 3 days is not effective against B. gibsoni infection in splenectomized dogs.     

Experimental Babesia bovis infection in Holstein calves

One intact and two splenectomized Holstein calves were infected intravenously with a Mexican strain of Babesia bovis and killed following the onset of severe clinical disease. A light and electron microscopic study was conducted on selected tissues to examine the relationship between parasitized erythrocytes and microvascular endothelial cells. The pattern and degree of specific organ sequestration of parasitized erythrocytes was assessed and correlated to lesions. Red blood cells infected with Babesia bovis exhibited stellate membrane protrusions. This morphological change appeared to mediate erythrocyte sequestration in the microvascular and capillary beds of the brain, kidney, and adrenal gland by an as yet unknown mechanism(s).     

Therapeutic and prophylactic efficacy of imidocarb dipropionate on experimental Babesia ovis infection of lambs

The objective of this study was to evaluate the therapeutic and prophylactic efficacy of imidocarb dipropionate (IMDP) against babesiosis and to determine specific antibodies against Babesia ovis in experimentally infected lambs. Thirty-six 6-month-old splenectomized lambs were used. The lambs were randomly divided into six groups with six animals each, and were intravenously inoculated with 50 mL B. ovis-infected erythrocytes as follows: group I (therapy group) was treated with IMDP (1.2 mg/kg body weight) starting on the day of onset of clinical signs of babesiosis after the inoculation; group II (untreated control animals) was not treated with any therapeutic treatment after the inoculation; groups III, IV, V and VI (prophylaxis groups) were administered IMDP (2.4 mg/kg body weight) 1, 2, 3 and 4 weeks before the inoculation, respectively. The animals were housed in a tick-proof room with water and food ad libitum up to the 30th day post-inoculation (PI). The lambs were monitored from the first day PI by recording the manifestation of clinical disease, rectal temperature, and the degree of parasitaemia. All the lambs became infected with B. ovis, except five animals from group III, which were treated 1 week prior to experimental infection. Other animals showed signs of acute clinical babesiosis. The animals treated with IMDP (group I) were able to clear the parasite from the blood circulation after 48 h post-treatment. The recrudescence of B. ovis was observed in two lambs 7 days after treatment, and they were treated with the second similar dose of the drug. Six lambs (1, 1, 2 and 2 lambs in group III, IV, V and VI, respectively) from the prophylaxis groups died within 7-17 days after showing high parasitaemia and clinical symptoms of the disease. Regardless of the clinical symptoms, 83.30% and 66.66% of the lambs which were administered IMDP 1-2 and 3-4 weeks before, were determined to be protected against the virulent field strain of B. ovis.     

Molecular studies on Babesia,Theileria and Hepatozoon in southern Europe. Part I. Epizootiological aspects

Molecular epizootiology of piroplasmids (Babesia spp., Theileria spp.) and Hepatozoon canis was studied in mammals from southern Europe (mainly from Spain, but also from Portugal and France). Partial amplification and sequencing of the 18s rRNA gene was used for molecular diagnosis. In some particular cases (B. ovis and B. bovis) the complete 18s rRNA gene was sequenced. Blood samples were taken from domestic animals showing clinical symptoms: 10 dogs, 10 horses, 10 cows, 9 sheep and 1 goat. In addition, DNA samples were isolated from blood of 12 healthy dogs and from spleen of 10 wild red foxes (Vulpes vulpes). The results of the survey were the following: Piroplasmid infections: Approximately from 50 to 70% of wild or domestic mammals (symptomatic) were infected.Piroplasmids detected in ruminants were:COW: B. bovis, T. annulata and Theileria sp. (type C). Sheep and goat: B. ovis. Piroplasmids present in canids were: Babesia canis vogeli, Babesia canis canis, Theileria annae and B. equi. The only piroplasmid found in asymptomatic dogs was B. equi. Piroplasmids found in horse were: B. equi and B. canis canis.H. canis infections in canids: H. canis was absent of domestic dog samples, whereas all foxes studied were infected by this protozoa.Genetic analysis showed that most of piroplasmid and Hepatozoon isolates from southern Europe matched unambigously with previously described species, as demonstrated by the high level sequence identity between them, usually between 99 and 100%. Minor differences, usually detected in hypervariable regions of 18s rRNA gene are probably due to strain variations or rare genetic polymorphisms. A possible exception was B. bovis, which shows a relatively lower degree of homology (94%) with regard to other B. bovis isolates from several countries. The same is true for B. ovis, that showed a 94% identity with regard to Babesia sp. from South African cow and a 92% with rapport to B. bovis from Portugal.     

The isolation,separation and preservation ofBabesia bigemina

Summary Experiments were performed in Colombia to separateBabesia bigemina from contaminating organisms.Babesia bigemina was passaged serially through five splenectomized calves. The first calf was inoculated with blood carrying several different organisms, and subsequent subinoculations were done soon after blood smears from each calf were found to be positive forB. bigemina. Five blood passages were carried out in 6.5 days.Babesia argentina, B. major andA. marginale were eliminated as contaminants of theB. bigemina isolated after four passages. A stabilate of the isolatedB. bigemina was established.

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El aislamiento, separación y preservacion de babesia bigemina

Sumario Se realizaron experimentos en Colombia para separarBabesia bigemina de organismos contaminantes.Babesia bigemina fue pasada en forma seriada en cinco terneros esplenectomizados. El primer ternero fue inoculado con sangre conteniendo algunos organismos diferentes, y las siguientes subinoculaciones fueron hechas tan pronto como los frotices de sangre de cada ternero fueron encontrados ser positivos conB. bigemina. Se llevaron a cabo cinco pases de sangre en 6.5 dias.Babesia argentina, B. major, yA. marginale fueron eliminados como contaminantes del aislamiento deB. bigemina despues del cuarto pase. Una forma estable del aislamiento deB. bigemina ha sido obtenida.

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Isolement, séparation et conservation debabesia bigemina

Résumé Des expériences ont été effectuées en Colombie pour séparerBabesia bigemina des organismes infectés.Babesia bigemina fut passé en série sur cinq veaux splénectomisés. Le premier veau a été inoculé avec du sang contenant plusieurs organismes différents. Les subinoculatinos suivantes ne furent faites que lorsque les étalements de sang de chaque veau furent positif avec.B. bigemina. Les cinq passages furent effectués en 6,5 jours.Babesia argentina, B. major etAnaplasma marginale ont été élimines, en tant qu'éléments contaminants, après quatre passages. Un stabilat desB. bigemina isolés a été effectué.

     

利用套式PCR检测牛羊乏质体

为同时检测和鉴定牛边缘乏质体、中央乏质体及绵羊乏质体,根据这3种病原体的msp4基因核苷酸序列,自行设计、合成了针对3种乏质体的2对通用引物,及分别针对三者的特异引物,通过PCR条件优化,建立了检测乏质体及分别鉴定3种乏质体的套式PCR方法,并与OIE推荐的msp5半套式PCR比较.结果显示:该方法对牛巴贝斯虫、双芽巴贝斯虫、羊莫氏巴贝斯虫、山羊泰勒虫、温氏附红细胞体、东方巴贝斯虫、刚地弓形虫、伊氏锥虫均未扩增出特异性片段.套式PCR检测乏质体DNA量为0.2 pg(相当于6个感染红细胞).检测l 119份来自6个不同地区的奶牛、肉牛、水牛及羊的临床样品,阳性106份,经鉴定边缘乏质体46份,中央乏质体15份,绵羊乏质体35份,混合感染中央乏质体和绵羊乏质体4份,混合感染边缘乏质体和绵羊乏质体3份,混合感染边缘乏质体和中央乏质体3份.首次在分子生物学水平证明中央乏质体存在于中国.同时,证明牛可以混合感染边缘乏质体和中央乏质体或绵羊乏质体,以及混合感染中央乏质体和绵羊乏质体.上述848份样品用OIE推荐的msp5半套式PCR同时检测,两者符合率为98.5％(835/848).检测结果表明,msp4套式PCR特异、敏感,可用于边缘乏质体、中央乏质体、绵羊乏质体的检测和鉴定.     

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.     

Detection of Theileria ovis in naturally infected sheep by nested PCR

A nested polymerase chain reaction (PCR) for the detection of Theileria ovis in sheep using oligonucleotide primers designed from the small subunit ribosomal RNA (SSU rRNA) gene sequence of T. ovis from sheep in eastern Turkey is described. A 398-bp DNA fragment was specifically amplified from blood samples from sheep, naturally infected with T. ovis. No PCR products resulted from T. lestoquardi, T. annulata, T. parva, T. buffeli and Babesia spp. DNA using these specific primers. The sensitivity of the nested PCR for T. ovis, which was assessed showed that one infected cell in 10(7) sheep erythrocytes, equivalent to a blood parasitemia of 0.00001%, could be detected. This is more sensitive than examining 200 fields under light microscopy. In addition, of the 124 field samples obtained from sheep in eastern Turkey tested, 19.35% (24/124) were positive for the presence of Theileria spp. by microscopic examination compared to 54.03% (67/124) positive for T. ovis by nested PCR. The primer pairs described in this study will be useful for epidemiological studies on ovine theileriosis and for discrimination between T. lestoquardi and T. ovis infections in sheep.     

Efficacy, toxicity and metabolism of imidocarb dipropionate in the treatment of Babesia ovis infection in sheep

An intramuscular dose of 1.2 mg kg-1 of imidocarb dipropionate (IMDP) was effective in controlling fatal infections with Babesia ovis in sheep. The sheep were infected by the intravenous injection of sheep blood containing B ovis. A severe recrudescence of infection occurred in most sheep 10 to 14 days after therapy but this could be controlled by a second dose of 1.2 mg kg-1 IMDP. Studies on the toxicology, residues and metabolism of IMDP showed this to be a safe dosage regimen. Transient or mild signs of toxicity were seen at 2.4 and 4.8 mg kg-1 IMDP. Severe toxicity was observed in sheep treated with 9.6 mg kg-1.     

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.     

Clinical and hematologic effects of experimental infection of dogs with recently identified Babesia gibsoni-like isolates from Oklahoma

OBJECTIVE: To characterize clinical and hematologic responses in dogs following experimental inoculation with Babesia gibsoni-like isolates from infected dogs in Oklahoma. DESIGN: Prospective study. ANIMALS: 6 mixed-breed dogs. PROCEDURE: 2 dogs were inoculated with organisms from a naturally infected dog, and 3 were inoculated with organisms from a second naturally infected dog (1 of these 3 dogs was splenectomized 1 week prior to inoculation). One dog was not inoculated. Complete blood counts were performed weekly. RESULTS: In the 5 dogs inoculated with organisms, parasites were initially detected 1 to 5 weeks after inoculation, and severity of parasitemia peaked with 1.9 to 6.0% of RBC infected by 4 to 6 weeks after inoculation. Parasitemia was easily detectable (> 0.1% of RBC infected) for 3 to 4 weeks. Clinical abnormalities included lethargy, fever, and pale mucous membranes but were mild to nearly inapparent in 2 dogs. All dogs developed regenerative anemia and marked thrombocytopenia. Thrombocytopenia developed before and lasted longer than the parasitemia. Profound but transient neutropenia was detected in some dogs. The splenectomized dog developed more severe parasitemia and anemia and more pronounced clinical abnormalities. Three dogs with intact spleens recovered without treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that 2 or more genotypically distinct, but morphologically identical, small Babesia parasites can infect dogs in the United States. Compared with infection with small Babesia parasites from California, infection with these isolates resulted in less severe parasitemia and clinical abnormalities. Parasitemia was transient, indicating that identification of organisms in blood smears may be difficult in some dogs.     

Evaluation of antioxidant status and oxidative stress in sheep naturally infected with Babesia ovis

The present study aimed to assess antioxidant status and oxidative stress in sheep naturally infected with Babesia ovis. Red blood cell (RBC) count, hemoglobin (Hb) concentration, packed cell volume (PCV), piroplasm parasitemia percentage, malondialdehyde (MDA) concentration, erythrocyte superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) activities and total antioxidant capacity (TAC) were determined in 52 sheep naturally infected with B. ovis as well as same number of healthy sheep in West-Azerbaijan province, Iran. Microscopic examination of Giemsa-stained peripheral blood smears revealed B. ovis infection. The parasitological diagnosis was confirmed using polymerase chain reaction (PCR) analysis by amplifying a partial small subunit ribosomal RNA (ssu rRNA) gene sequence of B. ovis of 52 diseased sheep, 18 (34.61%), 11 (21.15%), 16 (30.76%) and 7 (13.48%) had <1%, 1-2%, 2-3% and >3% parasitemia, respectively. Compared to controls, the activities of erythrocyte GSH-Px, SOD, TAC and CAT showed a significant decrease, whereas the concentration of MDA in erythrocytes of infected sheep increased significantly. Parasitemia rate was positively correlated with MDA and negatively correlated with PCV, SOD, CAT, GSH-Px and TAC. Also, MDA was negatively correlated with PCV, SOD, catalase, GSH-Px and TAC. The study demonstrated that B. ovis plays an important role in the occurrence of oxidative damage to RBCs and anemia in ovine babesiosis.