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.     

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.     

Effects of rapid passage in the gerbil (Meriones unguiculatus) on the course of infection of the bovine piroplasm Babesia divergens in splenectomised calves

The antigenicity and virulence of the cattle piroplasm Babesia divergens was compared in splenectomised calves before and after adaptation to the gerbil. It was apparent that adaptation of B divergens to the gerbil did not result in attenuation of the parasite in cattle. The gerbil-adapted parasite had a shorter prepatent period, produced higher parasitaemias, and more severe haematological and biochemical changes than the initial bovine strain. The antigenicity of the parasite was unchanged by gerbil passage. The changes in the virulence of the gerbil-adapted parasite were attributed to selection of a rapidly replicating parasite.     

Immunological changes in Babesia rodhaini infected BALB/c mice after treated with anti-babesial drug; diminazene diaceturate.

A model system capable of investigating immunological changes was first established in Babesia rodhaini infected mice with an aid of a drug, diminazene diaceturate (DD). Intraperitoneal (ip) inoculation with B. rodhaini resulted in acute death in euthymic (nu/+) and athymic (nu/nu) BALB/c mice. Treatment with DD at an early stage of infection saved both mice from acute death. Parasitemia recurred in some of them but resulted in death only in nu/nu mice. A re-challenge with 10(5) parasitized erythrocytes (PE) on the surviving mice on day 28 post infection revealed resistance in nu/+ but not in nu/nu mice. The results suggested a participation of the thymus in the protective mechanisms. Immunological changes were then observed on nu/+ and nu/nu mice which were inoculated ip with 10(4)PE and treated with the drug, and then challenged with 10(5)PE ip on day 28. An antibody response was measured with immediate reaction by footpad injection of a soluble antigen of B. rodhaini and by ELISA of serum antibody using the antigen and protein A, on day 10 and later, and further a pronounced response was detected after re-challenge in nu/+ mice. No response was detected by ELISA in nu/nu mice. Delayed footpad reaction was seen in nu/+ mice by day 14 and later but it was suppressed after the re-challenge.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Inhibition of Babesia divergens in cattle by oxytetracycline

The effects of continuous oxytetracycline administration on the development of parasitaemia of Babesia divergens during both natural and artificial infections were studied. During natural exposure on grazing heavily infested with Ixodes ricinus, seven out of 42 cattle with no previous exposure to tick-borne diseases were injected every four days with a long acting preparation of oxytetracycline at a dose rate of 20 mg/kg. During the six week grazing period 21 untreated cattle developed a patent parasitaemia of B divergens and all became seropositive by the fluorescent antibody test. In contrast, no parasites were observed in treated cattle and antibody titres remained low. Artificial infections were studied with different dose levels of oxytetracycline and their effects on antibody stimulation noted. First, four groups of cows were infected with 10(8) erythrocytes infected with B divergens, three groups being injected every four days with the long acting oxytetracycline formulation at dose levels of 20, 10 and 5 mg/kg, respectively. The highest level completely inhibited parasite replication and antibody formation; the same was observed in one animal dosed at 10 mg/kg but the remainder, plus those treated at 5 mg/kg, developed both low parasitaemia and high antibody titres. The untreated cows developed severe babesiosis. A further untreated control group was added and three weeks after cessation of oxytetracycline treatment all were infected with 10(9) erythrocytes infected with a homologous isolate of B divergens. The controls, plus those in which the previous infection had been completely inhibited, developed severe clinical babesiosis but the remainder were refractory to parasite development.     

THE DURATION OF LATENT INFECTION AND FUNCTIONAL IMMUNITY IN DROUGHTMASTER AND HEREFORD CATTLE FOLLOWING NATURAL INFECTION WITH BABESIA ARGENTINA AND BABESIA BIGEMINA

Tne Droughtmaster and 9 Hereford cattle were born in an enzootic babesiasis area and became naturally infected with Babesia argentina and B.bigemina during a 3 year period. They were then kept free of cattle ticks (Boophilus microplus) for the remainder of the experiment. Annually for the next 3 years their individual infection status with Babesia was determined by sub-inoculation of blood into splenectomised calves. At the end of this period the functional immunity of all cattle was challenged by blood inoculation of heterologous strains of B. argentina and B. bigemina. Infection with B. argentina persisted in all Herefords for 2 years and in 7 for 3 years after they had been freed of B. microplus. The number of Droughtmasters with detectable B. argentina infection progressively declined, and at the end of 3 years only 2 of 10 were still infected. No Herefords were shown to be infected with B. bigemina following 1 year's freedom from B. microplus but latent B. bigemina infection of at least 2 year's duration was demonstrated in one of the Droughtmasters. A marked degree of resistance was apparent in all cattle when they were challenged with an heterologous strain of B. argentina. There were no differences between the response to challenge of the Herefords and Droughtmasters nor between the reactions of cattle which had apparently naturally sterilised B. argentina infection and those which were still infected. The heterologous strain of B. bigemina produced parasitaemia in the majority of animals but only minimal fever and anaemia resulted with no significant differences between the breeds.     

Immune responses of immunocompetent and immunocompromised mice experimentally infected with Bartonella henselae

The aim of this study is to understand host immune responses in immunocompetent and immunocompromised mice against Bartonella henselae infection. BALB/c and nude (BALB/c nu/nu) mice were inoculated intraperitoneally with 10(8) colony forming units of B. henselae (Houston-1 strain). Blood, brain, liver, spleen, kidney and bone marrow samples were collected 0, 3, 7, 14, 21 and 28 days after infection and submitted to bacteriological, serological and genetical examinations. B. henselae was isolated only from the liver 3 days after infection. DNA of the inoculums was detected by polymerase chain reaction from blood, liver, and spleen samples collected from BALB/c and blood from nude mice 3 and 7 days after infection. No bacterial DNA was detected from both BALB/c and nude mice thereafter during 4 weeks observation periods. These results indicate that the T-cell may not participate in the effective elimination of the organisms from mice. In addition, western blot analysis revealed that the antigens of 27.3- and 31.5-kDa reacted with IgM antibodies from the blood of BALB/c and nude mice after 3 days of infection, suggesting that these antigens were recognized by thymus-independent mechanism. Furthermore the antigens were detected from the culture-supernatants of B. henselae, indicating that these antigens were secreted from the organisms.     

The pathogenesis of Babesia motasi (Wales) infection in sheep

Studies on the pathogenesis of Babesia motasi (Wales) infection following blood transfusion of infected blood to normal or splenectomised recipients showed that the intact animal is refractory to infection but that infection in splenectomised animals caused weight loss, fever, anorexia, lassitude and a macrocytic hypochromic anaemia which coincided with the peak of parasitaemia. There was an initial leucocytosis, largely due to a neutrophilia. The prepatent period following blood transfusion was 2-3 days. Unconjugated and conjugated (direct) bilirubin levels increased from pre-infection levels to peaks of 1.43 and 0.70 mg/100 ml of blood, respectively. Serum glutamic pyruvic acid transaminases (SGPT) increased slightly but serum glutamic-oxaloacetic acid transaminases (SGOT) and blood sugar (glucose) levels did not show significant changes after infection. Total serum protein levels increased temporarily and then returned to normal. Blood urea nitrogen levels increased, with biphasic peaks (76.32 and 86.29 mg/100 ml) on Days 2 and 8 post-patency. Clinical infections even in splenectomised sheep, were mild and of short duration, although recovered sheep remained carriers.     

Establishment of Theileria parva-infected bovine tissue culture in Swiss and athymic (nude) mice

Theileria parva-infected bovine lymphoid cells, grown in culture, were inoculated by different routes into neonatal and adult Swiss mice immunosuppressed by irradiation, thymectomy or inoculation of anti-lymphocyte serum. Tumour-like masses, composed of parasitized bovine lymphoid cells, formed at the site of subcutaneous inoculation in immunosuppressed neonatal and adult mice, but consistent establishment of cells following intra-peritoneal inoculation occurred only in neonatal mice. In all cases the degree of cellular establishment was proportional to the degree of immunosuppression. The best “take” was in irradiated neonatally thymectomized mice.Cells underwent short-term multiplication in mice but, as immune competence returned, the cells were rejected. There was no evidence that cells, on passage, became more adapted to grow in mice, nor that mouse cells became parasitized.Culture-derived cells were also inoculated subcutaneously into irradiated and non-irradiated nu/nu, nu/+ and Swiss mice. Tumour-like masses, composed of parasitized bovine lymphoid cells, developed at the site of inoculation in all irradiated mice. In nu/+ and Swiss mice these masses regressed after 2–3 weeks, but in the athymic nu/nu mice there was generally no rejection or cellular degeneration and parasitized cells became widely disseminated in the host's tissues and organs, in some cases causing death.T. parva-infected cells could not be established in non-irradiated nu/nu mice, nor when irradiated nu/nu mice were inoculated by the intra-peritoneal route. “Take” in irradiated neonatal nu/nu mice was also poor.Cells were passaged three times in irradiated nu/nu mice inoculated subcutaneously and it seems probable that indefinite passage of T. parva in mice can now be achieved.     

Piroplasms of ruminants in Switzerland and zoonotic significance of Babesia

Piroplasms are tick-transmitted blood parasites belonging to the genera Babesia and Theileria. In western and southern Switzerland, B. divergens, a small Babesia species, has been known for a long time as a parasite of cattle. Recent investigations have revealed the autochthonous occurrence of this parasite also in central and eastern Switzerland. On the occasion of an outbreak of anaplasmosis in the canton of Grisons, however, B. bigemina, a large Babesia species, and Theileria of the buffeli/sergenti/orientalis species complex were for the first time identified; the epidemiology of these two piroplasms in Switzerland remains unknown until now. The recent identification by genetic analyses of B. divergens in wild ruminants contradicts the hitherto postulated strict host specificity of this Babesia species for cattle. B. divergens as well as the closely related Babesia spp. genotype EU1 have in single cases also been identified in splenectomized humans.The rodent babesia B. microti which causes a human infection that is considered an "emerging tick-borne disease" in the U.S.A., is widespread in rodent populations in Switzerland, but seems to be of minor relevance as zoonotic pathogen here. Reasons for this could be differences in virulence of the parasites or in the transmission by the respective tick-vectors on the two continents.     

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.     

Immune response to foot-and-mouth disease virus in an experimental murine model. II. Basis of persistent antibody reaction

A murine model was used to study the mechanisms involved in the prolonged immune response to live and inactivated foot-and-mouth disease virus (FMDV). The antibody response elicited by the infection persisted throughout the entire life of the animal, while immunization with inactivated virus induced a transient response. The administration of inactivated virus in a water-in-oil emulsion increased antibody titres to values as high as those obtained by infection. There was a high correlation between neutralizing antibody titre and transfer of immunity with primed cells, and the protection afforded against challenge with infectious virus. It appears that the mechanism involved in the induction of prolonged immune memory in infected animals is not due to viral persistence. Nude mice infected with FMDV also evidenced a prolonged immune response, showing marked differences in antibody levels but equal effectiveness against challenge when nu/nu and nu/+ animals were compared. Furthermore, athymic and euthymic littermates were efficient in conferring protection when cells were transferred to irradiated animals. It is concluded that there is an effective, T-cell-independent, prolonged immune memory against FMDV in this murine model, and that the difference in the immune responses to live and inactivated virus is due mainly to differential antigenic processing rather than to a difference in the degree of sensitization of effector cells.     

Concurrent infection with Theileria buffeli caused depression of parasitaemia in Babesia bovis and Anaplasma centrale infections in splenectomised calves but not in B bigemina infections

In five experiments undertaken in splenectomised calves it was found that Theileria buffeli infections depressed the parasitaemias of superimposed Babesia bovis and Anaplasma centrale infections, but not B bigemina infections. The course of A centrale infections did not appear to be affected by a concurrent Eperythrozoon teganodes infection.     

Evaluation of an enzyme immunoassay for serodiagnosis of infections with Theileria parva and T annulata

An enzyme linked immunosorbent assay (ELISA) was used to determine antibody levels in cattle infected with Theileria parva and T annulata, using antigens prepared from the intra-erythrocytic piroplasm stage of the parasites. Antibody levels in calves infected with T parva increased from the 16th day after infection to reach peak values at days 28 to 35 and then declined rapidly, but in calves infected with T annulata antibody levels rose steadily up to day 40. Similar patterns of antibody production were shown by indirect fluorescent antibody tests. Sera from animals infected with T parva gave higher ELISA values with the antigen prepared from the homologous parasite species than with the antigen prepared from T annulata, but sera from cattle infected with T annulata gave similar high ELISA values with antigens prepared from both T parva and T annulata. Sera from animals infected with T mutans, T sergenti, T velifera, Babesia divergens, B major and B bovis gave only slight or no cross reactions with the piroplasm antigens, but serum from a calf infected with B bigemina cross reacted at a significant level with both piroplasm antigens.     

Acquired immunity to Fasciola hepatica in splenectomised rats.

Significant resistance to a second infection with Fasciola hepatica was obtained in splenectomised and sham operated rats (P less than 0.001, 78 and 76 per cent respectively) when compared with singly infected splenectomised or sham operated control groups. For both the stimulating and challenge infections, there were no significant differences in the number of flukes recovered from rats that had been splenectomised as compared to those receiving the sham operation. Thus, splenectomy did not significantly affect the ability of rats to develop an acquired immunity to F hepatica, nor were fluke recoveries from a particular schedule of infection significantly affected by the presence of absence of a spleen. It is concluded that the presence of a spleen is not necessary for the development of acquired immunity to F hepatica in the rat.     

Comparison of early splenic changes associated with replication of viruses in murine monocytic macrophages

An effect of replication of certain viruses in murine monocytic macrophages was manifested by depletion of cells through degenerative and necrotizing changes in thymus-dependent areas of lymphoid structures. In mice infected with murine hepatitis virus (MHV-3) or lactate dehydrogenase virus, these changes were transient in mice killed on postinoculation day (PID) 2. To study these morphologic changes due to viral replication, adult Swiss specific-pathogen-free homozygous nude mice (nu/nu) and their heterozygous haired littermates (nu/+) were inoculated with 10(5) LD50 of MHV-3, euthanatized, and necropsied on PID 1, 2, 4, 6, 8, and 10 along with noninoculated controls. The nu/+ and nu/nu mice killed on PID 2 had lymphocytic karyorrhexis and depletion of cells in the thymus-dependent area. In the heterozygote, these characteristic lesions were transient; whereas in the homozygote, lesions persisted and were present in survivors euthanatized and necropsied on PID 16. Although the intensity of lesions due to MHV-3 varied between nu/+ and nu/nu mice, virus titers determined on liver homogenates were similar for the homozygote and heterozygote during acute disease. Nude and nonnude mice given lactate dehydrogenase virus and killed on PID 2 had a transient depletion of lymphocytes; whereas mice given lymphocytic choriomeningitis virus and killed on PID 4 had a similar lesion. Lesions neither occurred when mice were treated with silica before inoculation, indicating that functional monocytic macrophages were required, nor occurred when another virus, herpes simplex virus type 1, was given.     

Pathogenesis of infection with Theileria recondita (Wales) isolated from Haemaphysalis punctata from north Wales

The pathogenicity of Theileria recondita infection in both splenectomised and non-splenectomised sheep was investigated. Following transmission of T. recondita to splenectomised animals both by adult ticks and blood transfusion a fever and severe macrocytic hypochromic anaemia developed which lasted the 9 days or more of patent infection. The prepatent period following tick attachment was about 8 days, but was variable following blood transfusion. Infection of normal animals caused a transient macrocytic hypochromic anaemia and slight fever which lasted throughout the period of low parasitaemia (7 days). The increase in mean cell volume was due to an increase in numbers of juvenile red cells in the peripheral blood. There was a transient neutrophilia and lymphocytopenia but no significant changes in thrombocyte or leucocyte numbers.     

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.