The transstadial transmission of Babesia caballi by Rhipicephalus evertsi evertsi

Rhipicephalus evertsi evertsi larvae were fed on the ears of rabbits. Seven days after larval infestation, unfed, newly moulted nymphae were manually removed to infest a splenectomized donkey showing a patent Babesia caballi infection. Engorged nymphae were collected from the donkey and the ensuing adult ticks were placed on a susceptible horse. The horse contracted a B. caballi infection showing a prepatent period of 19 days after tick infestation. A very low parasitaemia, (highest score 2), which was patent for only 10 days, was recorded. The lowest packed cell volume recorded was 16%.     

Infectivity rate and transmission potential of Hyalomma anatolicum anatolicum ticks for Babesia equi infection

The infectivity rate of Babesia equi in the salivary glands of Hyalomma anatolicum anatolicum was assessed. The hungry nymphs were fed on a donkey experimentally infected with B. equi. The engorged dropped-off nymphs were collected at different levels of parasitaemia and kept in BOD incubator. After ecdysis, the hungry adults were prefed on rabbits for different time intervals, thereafter the salivary glands were dissected out and acini were examined after methyl green pyronin (MGP) staining. A total of 134 male and 139 female ticks were dissected out. Average infected acini per tick were found to be significantly higher (p<0.05) in male as compared to the female ticks. Further, maximum infected acini in both male and female ticks were found at 24h of prefeeding on rabbits and overall infected acini per tick increased with rise in parasitaemia. The release of infected ticks on susceptible donkeys resulted in development of clinical babesiosis.     

Immunization of cattle with nymphal Hyalomma anatolicum anatolicum extracts: effects on tick biology

Antigens derived from partially engorged nymphs of Hyalomma anatolicum anatolicum were used in immunizing crossbred (Bos indicus×Bos taurus) cattle against larval, nymphal and adult H. a. anatolicum and H. dromedarii. The cattle were either infected with Theileria annulata at low parasitaemia or were uninfected. Whole nymphal extract (WNE), nymphal membrane antigens (NMA) and nymphal soluble antigens (NSA) were used for immunization. The group immunized with WNE showed significant and better rejection of H. a. anatolicum ticks as compared to calves immunized with either NMA or NSA. The moulting rates of both engorged larvae and nymphs remained unaffected. Nymphs which engorged on the immunized calves were fully susceptible to infection by T. annulata as indicated by the intensity and abundance of Theileria infections in the resulting adult ticks from immunized and unimmunized Theileria infected cattle. These ticks also transmitted fatal theileriosis to susceptible calves.     

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.     

Natural infection rates of Hyalomma anatolicum anatolicum with theileria in Sudan

Hyalomma anatolicum anatolicum ticks were collected around cattle pens at two locations close to Khartoum. They were assessed for theileria infection by four methods. Salivary glands were stained whole with methyl green pyronin and examined for parasite masses. Adult ticks were partially fed on rabbits, ground up in medium, and the suspensions were examined in Giemsa stained smears and by inoculation into bovine lymphocyte cultures. Ticks were fully fed on calves which were monitored for developing theileriosis. H a anatolicum were found infected with parasite masses similar to those seen in experimental infections with T annulata. At one site 38 per cent of 102 ticks were infected and the mean number of parasite masses per tick for the whole sample was 37. At the other site 86 per cent of 156 ticks were similarly infected and the mean parasite masses per tick was 19.5. Suspensions of sporozoites contained sporozoites typical of those found in experimental preparations of T annulata. Sporozoites harvested from ticks from both locations infected and transformed normal bovine lymphocyte cultures. H a anatolicum ticks from both locations produced fatal theileria infection in susceptible calves.     

Seroprevalence of equine piroplasms in the Republic of Korea

Equine piroplasms include two tick-borne protozoan parasites, Babesia caballi and Theileria equi. Although no clinical equine piroplasmosis has been reported in the Republic of Korea, the possible existence of the disease has been proposed due to a nationwide distribution of the vector ticks. To determine if the antibodies against B. caballi and T. equi were present, 184 sera of horses (Equus caballus) raised in the Republic of Korea from 2007 to 2010 were assessed using cELISA kits. Two (1.1%) out of 184 sera were positive for T. equi, but none were seropositive for B. caballi. Both samples tested positive came from one region (Gyeonggi province). The accuracy of the cELISA was confirmed by PCR using primers specific to the 18S rRNA of T. equi. This study presents for the first time horses infected by T. equi in the Republic of Korea. Since the infection of T. equi occurred in horses raised in the Republic of Korea, further studies with continuous monitoring of the vector ticks for equine piroplasms and appropriate control programs need to be established.     

The use of spleen antigen in the tube agglutination test for diagnosis of anaplasmosis in cattle

An antigen was prepared from the spleen of an infected bovine calf and was used in the tube agglutination test. Cases having rare and very low parasitaemia as well as the carrier cases were detected by this procedure and no non-specific reaction was observed with other haemoprotozoan infections. The maximum titre obtained with the Anplasma positive sera was 1:20, about 60 per cent showed a titre of 1:10 and 90 per cent showed a titre of 1:5 or more. The test provided satisfactory results in detecting the cases even at the early phase of the infection. The test also proved useful in judging the efficacy of a drug employed to destroy the carrier status.     

An investigation into the clinical pathological changes and serological response in horses experimentally infected with Babesia equi and Babesia caballi

Serologically negative horses, as determined with the indirect fluorescent antibody test (IFA), were infected with Babesia equi and 60 days later with Babesia caballi. The only clinical signs of disease observed in these animals were a febrile reaction and slight icterus. Haematological changes included a drop in haematocrit and haemoglobin concentration, as well as lowered platelet counts. The serum concentrations of albumin, iron and phosphorus were lowered. Mildly elevated serum bilirubin and fibrinogen concentrations were observed. Antibody titres were determined with the IFA and complement fixation (CF) tests. Antibodies to B. equi were first detected between Days 10-19 and 12-38 with the IFA and CF test, respectively, while the corresponding IFA periods for B. caballi were 6-8 days after infection. The parasitaemia of both B. equi and B. caballi infections never reached the 1% level.     

Distribution and biology of Ornithodoros erraticus in parts of Spain affected by African swine fever

Ornithodoros erraticus was found in 30.7 per cent, 35.0 per cent and 71.0 per cent of the pig-pens sampled in the provinces of Salamanca, Badajoz and Huelva in which African swine fever is a problem in the rearing of Iberian pigs. Between 38 and 65 per cent of the pig-pens in these areas are now abandoned and their populations of O erraticus are extinct or becoming so because they can no longer feed on pigs, which in Spain are their main hosts. The abandonment of pig-pens has resulted in the elimination of most soft ticks infected with the virus of African swine fever, and means that the distribution of ticks is now irregular and focal. Another factor affecting their distribution is the kind of soil on which the pig-pens are located. In abandoned pig-pens, the adults and large nymphs survive for about five years or longer when animals occasionally enter them. Hungry tick populations may transmit African swine fever when feeding in winter, whereas the populations that have continuous access to pigs do not feed until the pig-pens reach a temperature of 13 to 15 degrees C. In the latter populations, each stage exhibits a single annual peak of activity, which implies that the development from larva to adult takes two to three years. Pigs may die as a result of the bites, but on no occasion were 100 per cent of the fasting ticks seen to feed, even though they had the opportunity of doing so. This may hinder the eradication of this soft tick from infested pig-pens.     

The transovarial transmission of Babesia trautmanni by Rhipicephalus simus to domestic pigs.

Rhipicephalus simus was, for the first time, experimentally proven to be a transovarial vector of Babesia trautmanni of domestic pigs. The nymphal and adult progeny of experimentally infected female ticks transmitted the infection to 2 susceptible splenectomized pigs. Features of the infection included a prepatent period of 6-8 days post-tick infestation, a febrile reaction for 3 days and a maximum parasitaemia score of 15 (more than 6 parasites per 300 red blood cells). Other clinical signs in both pigs were mild inappetence and listlessness. Both pigs recovered without any antibabesial therapy.     

Hepatozoon canis infection in dogs: clinical and haematological findings; treatment

Hepatozoon canis gametocytes were identified in the circulating neutrophils of six dogs from different parts of southern Israel. Clinical signs included fever, anaemia, anorexia, weakness of the hind legs and numerous brown dog ticks (Rhipicephalus sanguineus) on the skin surface. Parasitaemia varied from 6 per cent to 43 per cent of the circulating neutrophils. Two of these six cases also showed Ehrlichia morulae in the cytoplasm of leucocytes. Drug therapy with tetracycline hydrochloride 22 mg/kg, three times daily, and imidocarb dipropionate, 6 mg/kg, by subcutaneous injection, repeated after 14 days, was accompanied by clinical improvement and the depression of gametocyte parasitaemia.     

High infection rates of the tick Hyalomma anatolicum anatolicum with Trypanosoma theileri

A crossbred calf (3 months old) obtained from a farm where regular control of ticks was practised and found to be free of blood parasites was inoculated with 20 ml pooled blood collected from four field cattle which had very low Trypanosoma theileri parasitaemias (one parasite per 70 microl blood as determined by the haematocrit centrifugation technique). Trypanosoma theileri was present in the blood 6 days after injection and a peak parasitaemia of 42 parasites per 70 microl blood was recorded by day 12. Hyalomma anatolicum anatolicum nymphs were applied on the ears of the calf on day 8 and they dropped engorged by days 13 and 14. The resulting adult ticks were examined for the presence of T. theileri by severing a leg and making a smear of the clear haemolymph which exuded from the wound. The smear was fixed in methanol and stained with Giemsa stain. The infection rate with T. theileri in the ticks was 43.3% (26 out of 60). The intensity of infection was very high and various developmental stages of the flagellates were observed (epimastigotes, sphaeromastigotes, trypomastigotes and other intermediate stages). The haemolymph from 12 ticks was also collected in tissue culture medium and the trypanosomes survived for 25 weeks before eventually dying. The results demonstrated unequivocally the high vectorial capacity of the tick H. a. anatolicum for T. theileri.     

Molecular detection of Babesia equi and Babesia caballi in horse blood by PCR amplification of part of the 16S rRNA gene.

Babesia equi and Babesia caballi are tick-borne haemoparasites that may cause babesiosis of Equidae. In southern Europe B. equi is enzootic and infections may occur asymptomatically and more frequently than those due to B. caballi. Complement fixation test (CFT) is the official serological test for the diagnosis of equine babesiosis, but it has low sensitivity during early and latent stages of the disease. With the aim of developing more sensitive and rapid direct diagnostic alternatives, PCR systems that amplified DNA targets of 664 or 659 bp regions of the 16S rRNA genes were designed and demonstrated to specifically detect the genomes of B. equi and B. caballi, respectively. An approximated parasitaemia of 0.000083% was detected by the PCR system for B. equi compared with reported limits of 0.001% for microscopic examination of stained blood smears, and up to 0.00025% for DNA probes. Although the sensitivity of the PCR system for B. caballi could not be estimated, samples with microscopically undetectable parasitaemia as well as those with 0.017% parasitised red blood cells were detected. DNA extracts of blood collected with EDTA as an anticoagulant from 23 horses from Portugal were tested with both PCR systems. Of these samples, 22 were positive for B. equi and 8 were positive for B. caballi with PCR tests and intraerythrocytic parasites were seen in all samples. Antibodies against both parasites were not detected by CFT in several cases, but in these cases the presence of either or both parasites was apparent by PCR tests. The PCR systems may be useful in the diagnosis of equine babesiosis covering a wider range of clinical disease, as useful adjuncts to serological, microscopic, and cultural methods, especially for the import and export testing of horses.     

Prevalence of Theileria equi and Babesia caballi infections in horses belonging to resource-poor farmers in the north-eastern Free State Province, South Africa

The prevalence of Theileria equi and Babesia caballi infections in the north-eastern Free State Province of South Africa was determined by examination of thin and thick Giemsa-stained blood smears, IFAT and PCR. No parasites were detected by microscopy from any blood samples collected at five study sites, Qwaqwa, Kestell, Harrismith, Vrede and Warden. Of the tested serum samples, 28/29 (96.5%), 20/21 (95.2%) and 42/42 (100%) were positive by IFAT for T. equi infections in Harrismith, Kestell and Qwaqwa, respectively, and 5/29 (17.2%), 13/21 (61.9%) and 30/42 (71.4%) were sero-positive for B. caballi infections in Harrismith, Kestell and Qwaqwa, respectively. All DNA samples from the study sites were negative for B. caballi infections by PCR, but five samples, two from each of Kestell and Warden and one from Vrede, were PCR positive for T. equi infections. The high prevalence of antibodies against T. equi and B. caballi in the sampled horses indicates that the animals had been exposed to T. equi and B. caballi infections but the absence of parasitaemia and very low number of positive PCR samples, however, imply that T. equi and B. caballi are endemically stable in the north-eastern Free State Province.     

Hyalomma anatolicum anatolicum (Koch, 1844) as a possible vector for transmission of Trypanosoma theileri,Laveran, 1902 in cattle

Amastigotes, sphaeromastigotes, epimastigotes and trypomastigotes of Trypanosoma theileri, Laveran, 1902 have been observed in the nymphs and adults of Hyalomma anatolicum anatolicum (Koch, 1844). These ticks were obtained from engorged larvae and nymphs collected from a cross-bred bull with detectable parasitaemia. Infection with T. theileri was established inoculation of Trypanosomatid flagellates collected from ticks and by feeding of the infected ticks on the ears of these calves.     

An electron microscopic study of Cytoecetes phagocytophila infection in Ixodes ricinus

Tick-borne fever, caused by a rickettsia-like organism, Cytoecetes phagocytophila, is transmitted by the sheep tick Ixodes ricinus. An electron microscopic technique was developed to examine I ricinus for C phagocytophila infection. Infected and uninfected ticks were obtained from a laboratory maintained tick culture. All stages of I ricinus collected from one field site were examined; 44 per cent of nymphae and 32 per cent of adults were infected with C phagocytophila, but larvae were uninfected. This supports the previously held theory of transtadial, but not transovarial transmission of tick-borne fever.     

Transmission of Babesia spp by the cattle tick (Boophilus microplus) to cattle treated with injectable or pour-on formulations of ivermectin and moxidectin

OBJECTIVE: To assess the efficacy of ivermectin and moxidectin to prevent transmission of Babesia bovis and Babesia bigemina by Boophilus microplus to cattle under conditions of relatively intense experimental challenge. DESIGN: Naive Bos taurus calves were treated with either pour-on or injectable formulations of either ivermectin or moxidectin and then exposed to larvae of B microplus infected with B bovis or larvae or adults of B microplus infected with B bigemina. One calf was used for each combination of haemoparasite, B microplus life stage, drug and application route. PROCEDURE: Groups of calves were treated with the test drugs in either pour-on or injectable formulation and then infested with B microplus larvae infected with B bovis or B bigemina. B bigemina infected adult male ticks grown on an untreated calf were later transferred to a fourth group of animals. Infections were monitored via peripheral blood smears to determine haemoparasite transmission. RESULTS: Cattle treated with either pour-on or injectable formulations of ivermectin and moxidectin became infected with B bovis after infestation with infected larvae. Similarly, larvae infected with B bigemina survived to the nymphal stage to transmit the haemoparasite to animals treated with each drug preparation. Cattle treated with pour-on formulations of ivermectin and moxidectin then infested with adult male ticks infected with B bigemina did not become infected with B bigemina whereas those treated with the injectable formulations of ivermectin and moxidectin did show a parasitaemia. CONCLUSIONS: Injectable or pour-on formulations of ivermectin and moxidectin do not prevent transmission of Babesia to cattle by B microplus. Use of these drugs can therefore not be recommended as a primary means of protecting susceptible cattle from the risk of Babesia infection.     

Transformation of Theileria parva derived from African buffalo (Syncerus caffer) by tick passage in cattle and its use in infection and treatment immunization.

A sporozoite stabilate (St. 199) of Theileria parva was obtained by feeding nymphal Rhipicephalus appendiculatus on an African buffalo (Syncerus caffer) and was used to immunize cattle by the infection and treatment method. Nymphal ticks were applied to one of the steers 90 days later and it was shown that the resultant adult tick had become infected. Using tick/cattle passage, two passage lines of T. parva were established. By the fifth tick/cattle passage, the parasite stocks had changed their behaviour to that of T. parva derived from cattle as the parasite produced relatively high schizont parasitosis and piroplasm parasitaemia in cattle, and had become highly infective to ticks. At various passage levels the parasite populations were characterized by behaviour and by monoclonal antibodies against T. parva schizonts using infected cell culture isolates from cattle during acute infections. The monoclonal antibody profile showed little evidence of antigen change of the parasite during passage through cattle, which was confirmed in a two-way cross-immunity experiment using sporozoite stabilate derived from ticks obtained from the buffalo and fourth passage in cattle. The implication of these results, particularly in relationship to immunization of cattle against T. parva derived from buffalo, is discussed.     

Effect of host resistance on the feeding and reproductive performance of Haemaphysalis punctata and Ixodes ricinus ticks

A study was made to assess the influence of host response on Haemaphysalis punctata and Ixodes ricinus biology by comparing the effects of feeding ticks on previously uninfested hosts with those fed on previously sensitised hosts. Adult H punctata were fed on sheep; larvae and nymphs of H punctata and I ricinus were fed on rabbits. The engorgement weights of all instars studied that fed on sensitised hosts fell very gradually with successive infestations. These weight reductions were greater at high than at low levels of infestation. In previously sensitised hosts, the engorgement period of all instars was prolonged: the percentage of instars which completed engorgement and engorgement weights were reduced. The host's clinical and immune response markedly affected the fecundity of adult female ticks and the viability and hatchability of the laid eggs. Eighty per cent of female H punctata successfully laid eggs following the first infestation and the hatchability of eggs was 85 per cent. The numbers successfully laying eggs decreased to 60 per cent during the fifth and sixth infestations and was less than 20 per cent following the seventh infestation; the hatchability decreased similarly. There was no marked effect on the development of immature instars when fed on sensitised rabbits.