Semi-nested PCR detection of Babesia orientalis in its natural hosts Rhipicephalus haemaphysaloides and buffalo

Babesiosis has recently been recognized as an emerging infectious disease of buffalo in China. In order to investigate the epidemiology and enzootic potential of this parasite in Hubei province, we sought to develop a semi-nested PCR to detect Babesia orientalis in buffalo and the potential tick vector-Rhipicephalus haemaphysaloides by amplifying a specific 257bp fragment of B. orientalis 18S rRNA gene. The practical limit of detection showed that it had high sensitivity and an approximate parasitemia of 0.00000012% was detected by the PCR system. The blood samples of 121 asymptomatic buffaloes collected from four babesia endemic counties and that of 71 asymptomatic buffaloes collected from three babesia free counties in Hubei province of China were examined for the presence of B. orientalis using both Wright-Giemsa stained blood smear and semi-nested PCR. Microscopic examination revealed that 5/121 animals were positive, whereas 24/121 animals were positive by the semi-nested PCR assay. Of 378 ticks (R. haemaphysaloides) collected from buffaloes and examined by the semi-nested PCR, 35 were positive. The results showed that the semi-nested PCR was a useful method to investigate the epidemiology of buffalo babesiosis (B. orientalis), which is widely distributed in Hubei province, China.     

Identification of ticks and detection of blood protozoa in friesian cattle by polmerase chain reacton test and estimation of blood parameters in district Kasur,Pakistan

The study was carried out to detect Theileria annulata, the causative agent of theileriosis, and Babesia bovis, the causative agent for babesiosis, in Friesian cattle by PCR and conventional blood smear examination. One hundred blood samples obtained from diseased Friesian cattle kept on private livestock farms at Pattoki, District Kasur, Pakistan were collected in addition to 20 blood samples obtained from non-diseased animals. The disease manifestations observed clinically included high fever, swelling of sub mandibular and sub scapular lymph nodes, weakness, increased respiration and pulse, anorexia, loss of condition and rough hair coat. Neurologic sign of in coordination was also seen in weak animals. Signs of lacrimation, pale conjunctiva, diarrhoea, dyspnea and frothy nasal discharge were observed in only one animal. Clinically nine animals showed signs of haemoglobinuria. Diagnosis of bovine theileria and babesia species was based on finding many intraerythrocytic piroplasms of both blood protozoa with clinical signs associated with anaemia, lymph node hyperplasia and haemoglobinuria. One hundred samples of ticks were also collected for identification of vector. Results showed that the prevalence of Hyalomma tick was highest (15%) followed by Boophilus (12%), Haemaphysalis (5%) and Rhipicephalus (3%). The blood smear examination showed 21% (21/100) samples positive for blood parasites out of which 66.6% (14/ 21) samples were positive for theileriosis while 42.8% (9/21) were positive for babesiosis. It was also recorded that 66.66% (6/9) samples were positive for B.bigemina while 33.33% (3/9) were positive for B.bovis. The results showed that 60% (60/100) samples were positive for blood parasites by PCR test. Out of these 60% (36/60) were positive for T.annulata while 33.33% (20/60) were positive for babesia. The specificity and sensitivity of PCR test was higher than blood smear examination. The blood parameters in haemoparasites infection were also analyzed and the results showed significant decrease in total erythrocyte count and haemoglobin while MCV, MCH values increased and MCHC was slightly less than normal indicating macrocytic hypochromic anaemia.     

Importance of PCR for the diagnostics of canine babesiosis

Clinical standards to confirm babesiosis in dogs include the direct identification of the infectious agent in blood smears and serological assays for Babesia canis-specific antibodies. Here, we demonstrate in seven cases (with data on anamnesis, clinics, laboratory diagnostics, and therapeutic outcomes) that a new diagnostic procedure is required. This is the molecular-genetic identification of babesia by real time PCR allowing an unequivocal identification of the infectious agents. Indeed, all seven patients presenting severe clinical symptoms were PCR-positive, but only two of them had specific antibodies and showed babesia in their bloodstream. Six of the dogs appeared to have acquired babesiosis while travelling abroad, and one in the Swiss canton of Schaffhausen.     

The indirect hemagglutination test for the detection of antibodies in cattle naturally infected mycoplasmas.

Stable mycoplasma antigens for the indirect hemagglutination test (IHA) were prepared employing glutaraldehyde treated sheep erythrocytes sensitized with Mycoplasma agalactiae subsp. bovis and Mycoplasma bovigenitalium antigens. Employing these antigens mycoplasma antibodies were detected in sera from cattle which had mastitic symptoms due to natural infection with either M. agalactiae subsp. bovis or M. bovigenitalium. A total of 200 cows from four herds were examined at varying intervals for the presence of M. agalactiae subsp. bovis and for the detection of antibody using growth inhibition and IHA tests. Mycoplasmas were isolated from 37 animals. Growth inhibiting antibody was detected from 56 of the 200 animals. In the IHA tests, antibody titer greater than or equal to 1:80 were detected in 148 animals, 76 of these having antibody titers greater than or equal to 1:160, while sera of 116 normal control animals had no growth inhibiting antibody and none had IHA antibody titers greater than 1:40. M. bovigenitalium was isolated from the milk of three of 26 animals in a fifth herd during an outbreak of mastitis. Growth inhibiting antibodies were demonstrated in the sera of ten of the 26 animals. However, the IHA test detected antibody titers of greater than or equal to 1:160 in 13 animals and of 1:80 in one of the 26 animals. To determine the specificity of the IHA tests, M. agalactiae subsp. bovis and M. bovigenitalium antigens were reacted with rabbit hyperimmune typing sera produced against 12 species of bovine mycoplasmatales. Homologous antisera showed IHA antibody titers of 1:1280 and 1:2560 against M. agalactiae subsp. bovis and M. bovigenitalium respectively, whereas heterologous antisera showed IHA antibody titers of less than or equal to 1:20. Also eight type-specific bovine antisera were reacted with M agalactiae subsp. bovis and M. bovigenitalium antigens in homologous and heterologous tests. Homoogous reactions showed IHA antibody titers greater than or equal to 1:320, whereas heterologous reactions showed IHA titers of less than or equal to 1:20. This IHA test promises to be useful for the detection of bovine mycoplasma antibodies in sera from cattle infected with M. agalactiae subsp. bovis or M. bovigenitalium. Thes test is sensitive, reproducible and specific and the technique is relatively simple and rapid. The antigens were stable for at least seven months.     

Cell-mediated immune response in hamsters infected with Babesia microti

A study was made of the cell-mediated immune (CMI) response in hamsters infected with Babesia microti isolated from a woman with clinical babesiosis. The leukocyte migration inhibition test (LMIT) using spleen leukocytes exposed to a soluble B. microti antigen was used for in vitro measurement of cellular immunity. The earliest detectable response was noted at the end of the second week after infection and persisted at relatively high levels throughout the 41-week observation period. Challenge infection caused a transitory but strong rise in the LMIT results. No hematologic manifestations of babesiosis were related to the challenge inoculation. The CMI responses were compared wiht humoral antibody measurements using the indirect fluorescent antibody (IFA) test.     

Electron microscopic study of canine Babesia gibsoni infection.

Canine babesiosis is a tick-borne parasitic disease caused by the intraerythrocytic parasites, Babesia canis and Babesia gibsoni. A lethargic, weak, American Staffordshire Terrier (pit bull) dog, which had regenerative, normocytic, normochromic anemia, was shown by polymerase chain reaction analysis to be infected with B. gibsoni. Transmission electron microscopy of ethylenediamine tetraacetic acid-treated blood disclosed many well-preserved, intraerythrocytic babesia trophozoites. Four morphologic forms of babesia trophozoites are described (small spheres, small rods, irregular forms lacking pseudoinclusions, and large spheres having pseudoinclusions) and are compared with intraerythrocytic forms of B. canis and B. gibsoni described in other light and electron microscopic studies of in vivo and in vitro Babesia infections. This is the first detailed transmission electron microscopic study of canine B. gibsoni-infected red blood cells in North America.     

An outbreak of babesiosis (B. divergens) in a dairy herd comprising different age groups of cattle

Outbreaks of acute bovine babesiosis affecting several individuals within a herd are well known among veterinary practitioners and farmers within different parts of Sweden. Although outbreaks may not occur every year, they are feared by the farmers due to the unpredictability of the disease, its suddeness of onset, its rapid course, and the high mortality unless treated. Sporadic outbreaks of acute bovine babesiosis comprising several animals within a limited time period have been previously reported in Sweden (Linnaeus 1747, Bergman & Waxberg 1915, Heijbel 1928), in Finland (Hinderson 1928), in Norway as cited by Tambs-Lyche (1943), in the United Kingdom (Donnelly et al. 1970) and elsewhere within the area where B. divergens is distributed (Tambs-Lyche 1943, Purnell 1980). In all of these cases groups of animals had been transferred onto pastures where clinical cases of bovine babesiosis were known to occur from areas where the disease was rare or had never occurred; i.e. unexposed animals were transferred onto pastures with Babesia infected ticks. The farmers first became aware of this situation after the outbreak had occurred.     

Autochthonous cases of canine babesiosis in the canton Solothurn

Starting in November 2003 a series of five clinical cases of canine babesiosis was registered in the region of Oberg?sgen (canton Solothurn). All presented dogs showed increased body temperature, thrombocytopenia and hemoglobinuria, and none of the dogs had been abroad or visited endemic regions in the southern or western part of Switzerland so far. Babesia canis was detected in the blood smears of all five patients, but only three had detectable specific antibodies against this parasite. However, seroconversion was found in a second sample collected from the negative dogs at a later timepoint, confirming the diagnosis of canine babesiosis. The blood samples of two parasitized dogs were used for DNA-isolation and were tested with a Babesia-specific PCR, detecting the 18S rRNA-gene. Sequencing of the amplified products revealed a 100% identity with the sub-species B. canis canis. The ticks Rhipicephalus sanguineus and Dermacentor marginatus are potential vectors for B. canis. In the area where the infection with B. canis was suspected a total of 152 ticks was collected and characterized; one was a female R. sanguineus.Although babesia could not be detected in the latter tick and the final prooffor the complete life cycle is still lacking, it is very probable that B. canis has become autochthonous in the canton Solothurn.     

Serological comparison of strains of Babesia bovis occurring in Australia and Mozambique

An antigen prepared in Australia and antisera collected from cattle having had field infections of babesiosis were taken to Mozambique for a serological comparison of strains of babesia bovis from the 2 countries. The reactivity of antisera collected in Mozambique against the Australian antigen was similar to that of the imported antisera. This suggested a close serological relationship between b. bovis of the 2 countries. The practical implication of this finding is that Australian vaccine should protect cattle being introduced into southern Africa from B. bovis-free environments.     

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.     

Indirect haemagglutination test for the detection and assay of antibody to bovine respiratory syncytial virus

An indirect haemagglutination (IHA) test was used for the rapid assay of antibody to bovine respiratory syncytial virus. Antigens for the sensitisation of formalised tanned erythrocytes were prepared by treatment of virus infected cells with non-ionic detergent. A close serological relationship was shown by the IHA test between the strain of bovine respiratory syncytial virus used and the A2 strain of human respiratory syncytial virus. The IHA test was sensitive and reproducible. A linear correlation was demonstrated between antibody titres obtained by the IHA test and the serum neutralisation test. Titres obtained by the IHA test were approximately 60 times greater than serum neutralisation titres. Serum samples from 803 two-year-old heifers in 48 herds in England were examined by the IHA test. Ninety-four per cent of the animals had antibody to respiratory syncytial virus. Examination of paired serum samples from outbreaks of respiratory disease by the IHA test showed that respiratory syncytial virus was associated with seven out of 15 outbreaks.     

Immunologic and pathologic responses of cows to naturally occurring Mycoplasma bovis mastitis

Systemic humoral and cell-mediated immune responses of four Holstein cows with natural Mycoplasma bovis mastitis were evaluated to determine whether a relationship exists between systemic cellular and humoral responses and the pathogenesis and resolution of infection. In vitro lymphocyte activation tests of peripheral blood lymphocytes and in vivo skin tests with M. bovis antigens provided evidence that cell-mediated immune responses against M. bovis may be involved in successful resolution or containment of infection. In several observation it appeared that viable M. bovis and their aqueous extracts are suppressive to cell-mediated responses.Humoral responses were determined by the serum indirect hemagglutination (IHA) test and the growth inhibition test. The IHA titers after approximately 2 weeks of infection were elevated; however, 75–87% of the IHA activity was in the IgM antibody class.The cell-mediated immune responses may be necessary for resolution of mycoplasmal mastitis both directly and via their helper cell function on antibody production. However, it appears that immune injury to mammary tissue results from the immunologic response to infective mycoplasma. Presence of locally secreted antibody and locally active immune cells may provide a better indication of those animals in the process of resolving the infection than was observed using systemic indicators of immune responsiveness such as indirect hemagglutination or growth inhibition tests.     

Differential Bos taurus cattle response to Babesia bovis infection

Bovine babesiosis is a tick-borne disease caused by Babesia spp. haemoprotozoans. The disease is of great importance at tick enzootic unstable areas and hampers cattle production in several developing countries. The available immunisation alternatives are pre-immunition and attenuated vaccines. Despite being efficient and protective, they are unsafe as they use cattle blood cells as inoculum and may potentially spread other diseases. Another alternative to help in babesiosis control would be the identification of genetically resistant cattle to Babesia bovis infection. The objective of this work was to phenotype cattle based on primary response against B. bovis infection. Two-hundred and forty half-sib Hereford and Aberdeen Angus heifers (120 animals from each breed), 12-18-month-old na?ve cattle, originated from a tick-free area in Southern Brazil, were used in the experiment. Animals were monitored following an inoculation with 1x10(7)B. bovis parasitised erythrocytes. Results showed three different phenotypes: 1-'susceptible', animals with babesiosis clinical signs that received treatment to avoid death; 2-'intermediate', animals with clinical signs: parasitaemia, >or=21.5% reduction in packed cell volume (PCV) and increase in body temperature when compared to their pre-challenge physiological parameters, no specific treatment was needed as animals self recovered from the disease, and 3-'resistant', animals without clinical signs that showed B. bovis presence in blood smears, <21.5% PCV reductions, with little or no increase in body temperature and no need for babesiosis treatment. The frequencies of each phenotype were: 45.4, 26.7, and 27.9%, respectively, demonstrating the existence of phenotypic variation for B. bovis in Bos taurus cattle.     

Development and evaluation of an enzyme-linked immunosorbent assay for bovine antibody (IgG) to Pasteurella haemolytica

The sensitivity of an indirect enzyme-linked immunosorbent assay (ELISA) for bovine IgG serum antibody to Pasteurella haemolytica was compared with that of an indirect hemagglutination (IHA) test. Pasteurella haemolytica serotypes were grown in a chemically defined cell culture medium, and soluble antigens released into the growth medium were used in the ELISA and IHA test. An ELISA with serotype-1 antigen consistently detected antibody in sera that were positive by IHA test (correlation, 99%). Sera reacting with serotype-1 ELISA antigens also reacted with ELISA antigens prepared from other serotypes. Although ELISA titers averaged 5 log2 units higher than IHA titers, plots of titers determined by the 2 methods were approximately linear. Titer increases detected in paired serum samples by either test were similar. The ELISA was more sensitive than was the IHA in detecting colostral IgG antibody in serum of newborn calves. The ELISA uses a simple, stable antigen preparation and detects antibody to P haemolytica serotypes that commonly infect cattle.     

Concurrent babesiosis and ehrlichiosis in the dog: blood smear examination supplemented by the indirect fluorescent antibody test, using Cowdria ruminantium as antigen

Giemsa-stained, peripheral blood smears of 67 dogs, showing clinical signs typical of babesiosis or reminiscent of concurrent babesiosis and ehrlichiosis, were examined for the presence of Babesia canis and Ehrlichia canis. Since Cowdria ruminantium cross-reacts with Ehrlichia, the sera of these dogs were also subjected to the indirect fluorescent antibody (IFA) test in which C. ruminantium was used as antigen. Fifty-five per cent of these dogs had mixed infections of B. canis and E. canis, as judged by blood smear examination and serology. The serum of 32% of these dogs with mixed infections reacted positively in the IFA test. Six out of 9 dogs, the blood smears of which were negative for both B. canis and E. canis, were serologically positive for E. canis. Furthermore, sero-conversion from a negative in the initial serum sample to titres of up to 1:160 in a subsequent sample was recorded in 9 out of 13 dogs with suspected mixed infection on blood smear.     

Epidemiological study of the prevalence of Babesia divergens in a veterinary practice in the mid-east of France

To assess the epidemiology of Babesia divergens in a veterinary practice based in the mid-east of France ("Monts du Lyonnais"), blood was collected from 254 cattle belonging to 24 herds. To assess the dynamics of the carrier state, six carriers were identified, treated with flumethrin and sampled once every 3 weeks during 6 months. Two different DNA extraction methods were compared. Each sample was tested for the presence of parasites using a PCR-RFLP test based on the 18S rRNA gene. The sensitivity of the test was equivalent to a parasitaemia as low as 10(-5)% (in "Filter Paper" samples) and 10(-6)% in 1 ml blood (extracted using "Matrix"). With the latter method, the rate of detection diminishes in the low parasitaemia range but could probably be improved. This test proved to be very useful in the detection of B. divergens carriers. Serology using IFAT showed 7% of the cattle seropositive, which is suggestive of a disease situation with a low clinical risk level. Analysis of the PCR results suggests a 20% prevalence rate of carriers in the cattle population. The use of the mean parasitaemia is proposed to serve as a babesiosis clinical risk indicator. This approach could also be used in other babesia infections provided the lowest detectable parasitaemia level (threshold level) could be resolved for each parasite species.     

Babesia ovis infections: Detailed clinical and laboratory observations in the pre- and post-treatment periods of 97 field cases

Ovine babesiosis, caused by Babesia ovis, is of major economic importance in Turkey. The changes in the blood profile of infected animals are informative about the course of infection. The aim of the present study was to evaluate the hematological and biochemical changes in the pre- and post-treatment periods of the natural B. ovis infections. The presence of the parasites was confirmed by microscopy and polymerase chain reaction (PCR) analysis. On the basis of the clinical and laboratory findings, the infections were categorized into different groups according to the degree of anemia and the level of parasitemia. All infected sheep were treated with imidocarb dipropionate (IMDP). The blood pictures in the pre- and post-treatment periods were compared.Pancytopenia occurred in animals with severe anemia and very high parasitemia, and bicytopenia in the other groups. The platelet count (PLT), plateletcrit (PCT) and mean platelet volume (MPV) returned to the normal ranges after treatment, except those in the group with severe anemia. In the biochemical profile, B. ovis infection caused an increase in blood urea nitrogen and total bilirubin, and these parameters returned to normal levels after treatment.The indirect fluorescein antibody test (IFAT) results showed that 38.1% of the cases raised specific antibodies during the period of infection, with titers ranging from 1/160 to 1/640. All of 45 animals re-examined after treatment were seropositive, with high titers that rose up to 1/5120.     

Conflicting results of serological,PCR and microscopic methods clarify the various risk levels of canine babesiosis in Slovakia: A complex approach to Babesia canis diagnostics

We have performed a survey of Babesia canis prevalence within group of dogs living in Southern and Western Slovakia. Blood samples and sera from 217 dogs, including individuals suspected of having babesiosis, were examined by nested PCR-RFLP, light microscopy and indirect fluorescence antibody test (IFAT). The detection of B. canis DNA revealed the highest number of infected dogs in the region of Nové Zámky, with 23 B. canis-positive blood samples (35.4%, n = 65), followed by an area close to Komárno (both areas of Southern Slovakia), where 1 dog out of 52 collected (1.9%) had detectible B. canis DNA in the blood stream. The serological method revealed an opposing pattern, with only 3 dogs (4.8%, n = 63) sampled at Nové Zámky presenting IgG antibodies against B. canis, while in Komárno region such antibodies were detected in 15 dogs (28.8%, n = 52). This discrepancy may be because the majority of samples from Nové Zámky were dogs suspected of an acute phase of canine babesiosis, whereas dogs at Komárno were sampled during a vaccination campaign, and thus were without any clinical signs of the disease. The latter group contains evidently recovered carriers of IgG against B. canis. Hence, the combination of PCR-based and serological methods enabled us to discover both recently infected as well as recovered dogs, thus obtaining a more realistic view on the epidemiological situation. Remarkably, we did not find any positive samples in the vicinity of Stupava (district Malacky, Western Slovakia), either by PCR-RFLP, microscopy or IFAT (n = 100). Considering the numerous falsely diagnosed cases of canine babesiosis, we suggest that light microscopy as the simplest and most accessible diagnostic test. Southern Slovakia was confirmed as an area of high risk of canine babesiosis, whereas conclusions about B. canis spreading over Western Slovakia should be considered with wariness.     

Live vaccines against bovine babesiosis

Bovine babesiosis is an important tick-borne disease caused by Babesia bovis, B. bigemina and B. divergens. The first steps taken in the development of an effective vaccination strategy against bovine babesiosis followed the observations that animals, recovered from natural infection with Babesia were strongly protected against subsequent challenge. Further investigation indicated that the use of donor blood from recovered animals to infect recipient animals did not produce the severe form of the disease. The past century has seen a refinement of this original carrier-donor system to one using attenuated less virulent strains with standardized doses of known parasite concentration to ensure reliability. With the implementation of good manufacturing practices further changes were necessary in the production of these vaccines, such as freezing for long-term storage to allow sufficient time for pre-release safety and effectivity testing. Regardless of these improvements the vaccines are not without problems and breakdowns and breakthroughs occur from time to time. Despite considerable research efforts into the development of alternative more consumer friendly vaccines, none is immediately forthcoming and the live attenuated babesiosis vaccines are still used in many countries.     

Canine babesiosis caused by Babesia canis vogeli in rural areas of the State of Minas Gerais, Brazil and factors associated with its seroprevalence

This epidemiological survey on canine babesiosis was carried out in three distinct rural regions (Lavras, Belo Horizonte and Nanuque) of the State of Minas Gerais, Brazil. Ticks and blood samples were collected during a dry season (Lavras, n = 92; Belo Horizonte, n = 50; Nanuque, n = 102) and the subsequent rainy season (Lavras, n = 71; Belo Horizonte, n = 28; Nanuque, n = 66) from dogs living on farms. Plasma samples were analyzed by the indirect fluorescent antibody test for detection of anti-Babesia canis vogeli antibodies. DNA was extracted from blood of serologically positive dogs and molecular characterization of Babesia species was performed. Rhipicephalus sanguineus, Amblyomma cajennense and Boophilus microplus were the tick species identified in all regions. In Lavras, in addition to those tick species, A. tigrinum and A. ovale were also identified. The most prevalent tick species was A. cajennense (35.3%), followed by R. sanguineus (19%) and B. microplus (4.0%). Dogs living in Nanuque region were more heavily infested with ticks than dogs living in Belo Horizonte and Lavras regions. The overall frequency of anti-B. c. vogeli antibodies in the canine population in rural areas of Minas Gerais was 28.7%, with prevalence rates of 49.0% in Nanuque, 34.0% in Belo Horizonte and 3.3% in Lavras. The age of the animals and tick infestation were associated with seroprevalence of B. c. vogeli. The sequence analysis showed that B. c. vogeli was the only Babesia species present in all three regions. This study showed different rates of prevalence and incidence of canine babesiosis among the three rural regions sampled in Minas Gerais State. The results point to the importance of canine babesiosis in rural areas and to the need for further studies related to its transmission and maintenance in nature.