Inverse age resistance to experimental Babesia divergens infection in cattle

Two groups of calves, 1.5-2 and 7-11 months old respectively, and dairy cows were inoculated i.v. with 3 x 10(7) erythrocytes infected with Babesia divergens. High parasitaemia, fever and other clinical signs of babesiosis occurred among adult animals. A very low parasitaemia and a slightly increased body temperature but no other symptoms occurred in calves. these findings substantiate the conclusion that there exists an inverse age resistance against Babesia divergens. The kinetics of B. divergens IgG antibody formation were similar in all age groups. Consequently this antibody response was not the factor determining the development of the primary parasitaemia and thus the inverse age resistance phenomenon. However, age is not necessarily the only factor involved in the clinical expression of babesiosis. The kinetics of antibody formation was not associated with the intensity of the parasitaemia. In fact only about half the animals had a demonstrable parasitaemia although the antibody responses were similar in all age groups.     

Annual evolution of cattle immunity against Babesia divergens in Northern Switzerland

A seroepidemiological survey of Babesia divergens babesiosis was conducted on 9 farms in a focus of northern Switzerland (Clos-du-Doubs, Jura). During 1981, a total of > 300 cattle were examined for B. divergens antibodies with an indirect immunofluorescence test. Annual evolution of immunity was estimated 3 times during the year (April, July and December). Cattle were progressively sensitized against B. divergens during the grazing period. Considering all cattle, 33.9% possessed anti-B. divergens antibodies in spring, before the grazing period. This percentage increased in the summer (59.1%), reaching 90.6% in December.In the studied area, the most important calving period occurs in autumn and young calves do not leave the farm before they are 2 months old. Calves generally lacked maternal antibodies when they were moved to pastures for the first time. Although infested with B. divergens-infected ticks, they never presented acute symptoms of babesiosis. They appeared to be naturally resistant to the disease. Cattle from 9 months to 3 years old were more susceptible. In April, 73.4% did not show antibodies. During our study, 12 of the 15 cattle of that age which had babesiosis did not possess antibodies. Cattle > 3 years old were more protected. In April, only 51% lacked antibodies and 1 case of babesiosis was observed in this group.     

Indirect fluorescent antibody levels in experimental Babesia divergens infections of cattle.

In splenectomised calves infected with Babesia divergens IFA titres appeared after peak parasitaemia and maximum titres were observed 35 days after infection: the IFA response of adult non-splenectomised cattle was similar. It was concluded that the absence of a spleen did not markedly inhibit the IFA response of cattle to B divergens infection.     

Isolation of Babesia divergens from carrier cattle blood using in vitro culture

Babesia divergens, the main causative agent of bovine babesiosis in Western Europe, was isolated from naturally infected cattle. Ninety-six blood samples were examined by means of an in vitro culture technique in sheep erythrocytes: 19 of them were collected from animals in the acute phase of the disease with visible parasitemia on blood smears, while the 77 remaining animals showed no microscopically detectable parasites. B. divergens was cultured from the 19 first blood samples as well as from 31 samples collected from asymptomatic animals. The time period before parasites could be detected in the culture varied in the latter samples from 6 to 20 days. The effects of sampling condition (anticoagulant used) and storage length were tested. A good correlation was obtained between immunofluorescent antibody test and culture, with identical results (positive or negative) for 89.6% of the samples collected from asymptomatic animals. The sensitivity of the in vitro culture method was determined and was about 10 parasites/mL of whole blood from three independent experiments performed with three different isolates, confirming its suitability to detect and culture diverse B. divergens isolates from carrier cattle. The parasites could indeed be isolated 9 months after the acute babesiosis phase in the blood of naturally infected animals. The 50 isolates collected in this study were successfully subcultured, cryopreserved and resuscitated using the same culture medium. The in vitro isolation of B. divergens from asymptomatic carrier cattle was achieved and will allow the analysis of parasite diversity within cattle herds.     

The immunization of cattle against Babesia divergens with fractions of parasitized erythrocytes

A lysate of erythrocytes infected with Babesia divergens was subjected to isoelectric focusing within a pH range of 3.5-9.5. The focused proteins were split into 2 fractions depending on pH (1) those from 3.5-6.5 and (2) those from 6.5-9.5. The acidic fraction was used as an immunogen for groups of 5 adult cows using 2 different adjuvants, saponin and a mixture of muramyldipeptide (MDP) and incomplete Freund's adjuvant (IFA). Two other groups of 5 cows were simultaneously treated with the alkaline fraction and a sample of the complete lysate with the MDP/IFA adjuvant. The treated cows plus a control group were infected with 2.5 X 10(8) erythrocytes infected with a homologous strain of B. divergens. When compared with the controls all pre-treated groups were protected to some degree. There were significant differences between the treated groups with the group which received MDP/IFA combined with the acidic fraction showing most resistance to the infection. The experiment also showed that the MDP/IFA adjuvant produced more protection than the saponin.     

Tick infestation and the prevalence of Borrelia burgdorferi and Babesia divergens in cattle in Bavaria

During the grazing period 2002 319 cattle from 31 farms located in 6 districts of southern Bavaria were examined for the presence of ticks in 4- to 5-week intervals, and 287 serum samples were tested for the presence of antibodies against Borrelia burgdorferi and Babesia divergens. Ticks were detected in all 31 farms with a mean prevalence of 69%. 3218 out of 3453 collected ticks were Ixodes ricinus; 139 nymphs, 19 larvae and 77 damaged adult specimens could only be determined to the Genus level (Ixodes).The seasonal pattern revealed the highest frequencies of ticks in May/June and September.The intensity of tick infestation of positive animals was generally low. 76.5% of parasitized cattle had 1-6 ticks per day of investigation. Individual cattle showed up to 250 ticks per day. The percentage of infested animals in each herd varied within the period between 0-100%. The examination of serum samples by immunofluorescence technique (IFAT) revealed positive anti-Borrelia antibody titers (> or = 1:64) for 45.6% of the animals. The within-farm seroprevalence of borreliosis ranged from 20 to 100% in 27 of the 31 farms. A significant correlation could be detected between the number of ticks/cattle and the anti-Borrelia burgdorferi IgG-titer. By contrast, there was no significant correlation between the age of the animals and anti-Borrelia serum titers. For comparative reasons, 64 IFAT-positive serum samples were tested by Western blot techniques for the presence of antibodies cross-reacting with Borrelia garinii antigen. These analyses revealed that 69% of the samples reacted positively, 28% were unclear and 3% were negative. Examinations of the 287 serum samples for the presence of anti-Babesia divergens antibodies revealed one positive animal with a titer of 1:16.     

Use of long-acting oxytetracycline in the immunisation of cattle against Babesia bovis and B bigemina

Forty Friesian one-year-old calves were vaccinated simultaneously with live Babesia bovis and B bigemina vaccines. Three groups of 10 calves each were treated with two, three or four doses of 20 mg kg-1 long-acting oxytetracycline (OTC/LA) at six- to seven-day intervals starting from day 6 after vaccination. Ten animals remained untreated. The treated calves showed considerably fewer days of patency and higher packed cell volumes than the vaccinated untreated calves. All calves developed serum antibodies to both parasites following vaccination. Five months later the 40 vaccinated and 30 new calves were challenged with syringe-transferred virulent parasites of both species. The vaccinated calves showed no parasites or clinical manifestations while calves of the new group exhibited severe clinical babesiosis. These results show that when OTC/LA is administered following anti-babesial vaccination, parasitaemia and red blood cell destruction are significantly reduced without, however, inhibiting the development of immunity.     

Babesia divergens becoming extinct in cattle of Northeast Hungary: new data on the past and present situation

Previously unpublished data from 1958 to 1967 attest the occurrence of Babesia divergens in cattle in several endemic foci of Northeast Hungary. During that period the number of clinical cases showed fluctuation with intervals of 4-5 years and monophasic seasonality (peaking in June). In order to assess the current status of bovine babesiosis in that region, blood samples were collected from 654 cattle on 44 farms of 36 settlements in or near the endemic area during 2005, and serum levels of IgG antibodies to B. divergens were measured by indirect fluorescent antibody test (IFAT). Only 2 samples (0.3%) showed positivity. In one village clinical babesiosis was observed over the past few years. Animals brought into the endemic area during the spring developed haemoglobinuria in the summer of the same year, but those introduced during the summer or autumn showed clinical signs only after two years. Sampled animals born and raised locally had neither haemoglobinuria nor seroconversion. Reduction in the number of cases during the past decades may have been influenced by the availability of hosts (i.e. decrease of cattle breeding) and the activity of vectors associated with climate-related changes (e.g. increase of annual sunlight hours in the endemic area). This is the first report on the prevalence of antibodies to B. divergens in cattle in Hungary.     

Babesia major in Britain: cross-immunity trials with Babesia divergens in splenectomised calves.

Splenectomised calves infected with Babesia major were shown to have no resistance to challenge with Babesia divergens; however, initial infection with B divergens provided a good protection against subsequent challenge with B major. It is suggested that this might mean that B divergens would be the dominant and most commonly encountered species in areas where both occur.     

Chemotherapy of Babesia divergens in the gerbil, Meriones unguiculatus

It was found that surprisingly low doses of four babesicides were effective against Babesia divergens in gerbils and it was concluded that this was due to the involvement of host resistance, which may be of a non-specific nature. The efficacy of the drugs relative to each other was the same in gerbils as in cattle and this host-parasite system is evidently more suitable for the screening of babesicides than are other rodent babesia systems. The prophylactic dose of imidocarb dipropionate required to provide a similar degree of protection in gerbils as in cattle was found to be much higher and was very close to toxic levels. Challenge infections resulted in sterile immunity. Acute babesiosis in gerbils could be cured with all four drugs if parasitaemias were below approximately 45 per cent and packed cell volumes above 18 per cent at treatment.     

Sheep as a new experimental host for Babesia divergens

Babesia divergens was cultivated in sheep erythrocytes in RPMI 1640 supplemented with 10% Fetal Calf Serum (FCS) or sheep serum. In vitro cultures in sheep red blood cells were initiated with human erythrocytes infected in vitro with B. divergens Rouen 1987 or with gerbil blood infected with several isolates from bovine origin. After the first subcultures on sheep erythrocytes, a ten-fold multiplication of the parasites was obtained within 48 h. Erythrocytes from three splenectomized sheep were infected in vitro with B. divergens; when parasitaemia reached 10%, the animals were inoculated with homologous parasitized erythrocytes. All sheep expressed hyperthermia with a peak between the 6th and the 9th day post-infection (p-i) and a transitory parasitaemia 10 days p-i. In vitro primary cultures were performed on two of these sheep, demonstrating the parasite persistence at very low parasitaemia in the infected animals. Splenectomized sheep can be used as a new model for B. divergens chronic infection.     

Detection of Babesia divergens in southern Norway by using an immunofluorescence antibody test in cow sera

Background

The incidence of bovine babesiosis, caused by Babesia divergens (Apicomplexa: Piroplasmida) has decreased markedly since the 1930 s, but may re-emerge as a consequence of climate change and changes in legislation and pasturing practices. This is a potentially serious disease, with both economical and animal welfare consequences. Therefore, there is a need to survey the distribution of B. divergens.

Methods

We tested sera from 306 healthy pastured cows from 24 farms along the southern Norwegian coast by using an indirect immunofluorescence IgG antibody test (IFAT). Fractions of seropositive cows were compared by calculating 95% CI.

Results

The results of this test showed that 27% of the sera were positive for B. divergens antibodies. The fraction of antibody-positive sera that we detected showed a two-humped distribution, with a high fraction of positives being found in municipalities in the western and eastern parts of the study area, while the municipalities between these areas had few or no positive serum samples.

Conclusions

Neither the farmers'' observations nor the Norwegian Dairy Herd Recording System give an adequate picture of the distribution of bovine babesiosis. Serological testing of cows by using IFAT is a convenient way of screening for the presence of B. divergens in an area.     

Babesia divergens: cloning of a Ran binding protein 1 homologue

Babesia divergens is an Apicomplexa transmitted to bovines by its acarian vector, the tick I. ricinus. Babesia divergens merozoites have an intraerythrocytic development in the blood of infected mammals. The nucleocytoplasmic transport system in this parasite is not yet characterized and no protein involvement in such transport has been described. In this report, we describe the cloning of a protein that shares important homologies with Ran binding protein 1. This protein in Eukaryote belongs to the nucleocytoplasmic transport system.     

A Vegetation Index qualifying pasture edges is related to Ixodes ricinus density and to Babesia divergens seroprevalence in dairy cattle herds

Babesia divergens, transmitted by the tick Ixodes ricinus, is the main agent of bovine piroplasmosis in France. This Apicomplexa often is present in asymptomatic carriers; however, clinical cases are rare. While numerous factors are known to influence tick density, no risk factor of contact with B. divergens has been identified for cattle. Our study aimed to explore whether a Vegetation Index could serve as an indirect indicator of within-herd B. divergens seroprevalence. In February 2007, blood samples were taken from all of the cows in 19 dairy cattle herds in Western France and IFAT serology was performed individually to measure B. divergens seroprevalence. The following spring, I. ricinus nymphs were collected by drag sampling along transects on the vegetation of each farm's pasture perimeters. Tick density was related significantly to a Vegetation Index (V.I., ranging from 1 to 5) that took into account the abundance of trees and bushes on the edge of pastures: most ticks (57%) were found in transects with the highest V.I. (covering 15% of the explored surface in the study area). At the farm level, the proportion of transects presenting I. ricinus nymphs was significantly related to B. divergens seroprevalence: the farms with more than 15% of transects with I. ricinus had a significantly higher risk of high seroprevalence. The proportion of pasture perimeters where the V.I.=5 also was significantly related to B. divergens seroprevalence: the farms where more than 20% of transects had a V.I.=5 had a significantly higher risk of high seroprevalence. Given that the Vegetation Index is a steady indicator of the potential I. ricinus density in the biotope, we recommend that the risk of high B. divergens seroprevalence in cows be evaluated using this tool rather than drag samplings.