Transport of Babesia venatorum-infected Ixodes ricinus to Norway by northward migrating passerine birds

Background

Bovine babesiosis is regarded as a limited health problem for Norwegian cows, and the incidence has decreased markedly since the 1930s. Rare cases of babesiosis in splenectomised humans from infection with Babesia divergens and B.venatorum have been described. The objective of this study was to determine whether birds can introduce Babesia-infected ticks. There are between 30 and 85 million passerine birds that migrate to Norway every spring.

Methods

Passerine birds were examined for ticks at four bird observatories along the southern Norwegian coast during the spring migrations of 2003, 2004 and 2005. The presence of Babesia was detected in the nymphs of Ixodes ricinus by real-time PCR. Positive samples were confirmed using PCR, cloning and phylogenetic analyses.

Results

Of 512 ticks examined, real-time PCR revealed five to be positive (1.0%). Of these, four generated products that indicated the presence of Babesia spp.; each of these were confirmed to be from Babesia venatorum (EU1). Two of the four B. venatorum-positive ticks were caught from birds having an eastern migratory route (P< 0.001).

Conclusions

Birds transport millions of ticks across the North Sea, the Skagerrak and the Kattegat every year. Thus, even with the low prevalence of Babesia-infected ticks, a substantial number of infected ticks will be transported into Norway each year. Therefore, there is a continuous risk for introduction of new Babesia spp. into areas where I. ricinus can survive.     

Experimental in vitro transmission of Babesia sp. (EU1) by Ixodes ricinus

Babesia sp. (EU1), first characterized in 2003, has been implicated in human cases of babesiosis in Italy, Austria and Germany. It has been identified in roe deer and in its suspected tick vector, Ixodes ricinus, in several European countries. The aim of the present study was to validate the competence of I. ricinus as a vector of Babesia sp. (EU1) via experimental infections. For this purpose, a parasite strain isolated from roe deer was cloned in sheep erythrocytes. After experimental infections, parasite DNA was successfully amplified by PCR in both eggs and larvae originating from infected I. ricinus females and in the salivary glands of females exposed to Babesia sp. (EU1) as nymphs. We also demonstrate that infected females were able to transmit parasite DNA during a new blood meal. Together with previous epidemiological studies, these results validate I. ricinus as a competent vector for Babesia sp. (EU1).     

A note on the transmission of Babesia bovis (syn B argentina) by the one-host tick, Boophilus microplus.

Boophilus microplus infected with Babesia bovis were transferred artificially from one splenectomised calf to another during each moult in the parasitic life cycle of the tick. Eggs from the engorged female ticks recovered at the end of the cycle were incubated and the resulting larvae used to infest more splenectomised calves. Babesia bovis was transmitted only by the original larvae used at the commencement of the experiment and it was concluded that the protozoan parasite did not persist in an infective form in the ticks beyond the larval stage.     

PCR-based detection of the transovarial transmission of Uruguayan Babesia bovis and Babesia bigemina vaccine strains

Bovine babesiosis is responsible for serious economic losses in Uruguay. Haemovaccines play an important role in disease prevention, but concern has been raised about their use. It is feared that the attenuated Babesia bovis and Babesia bigemina vaccine strains may be transmitted by the local tick vector Boophilus microplus, and that reversion to virulence could occur. We therefore investigated the possibility that these strains could be transmitted via the transovarial route in ticks using a Babesia species-specific polymerase chain reaction (PCR) assay. DNA was extracted from the developmental stages of the tick vector that had fed on calves immunized with the haemovaccine. It was possible to detect Babesia DNA not only in adult ticks, but also in their eggs and larvae. In addition, it was shown that calves infested with larvae derived from eggs laid by ticks fed on acutely infected calves, were positive for Babesia using PCR. Caution should therefore be shown with the distribution of the haemovaccine in marginal areas. It is still advisable that suitable tick control measures be used to prevent transovarial transmission and the potential risk of attenuated Babesia reverting to virulence.     

Bovine babesiosis (Babesia bovis infection) in Zambia

Two cases of Babesia bovis, a parasite associated with the tick Boophilus microplus, are reported for the first time from the central part of Zambia. It is concluded that infected B. microplus ticks are occasionally introduced into central Zambia by tick-infested cattle from the north-eastern part of the country where B. bovis is endemic. The spread of B. microplus in Southern Africa in a westward direction is discussed and related to the epidemiology of bovine babesiosis in Zambia.     

Bovine babesiosis (Babesia bovis infection) in Zambia

Summary

Two cases of Babesia bovis, a parasite associated with the tick Boophilus microplus, are reported for the first time from the central part of Zambia. It is concluded that infected B. microplus ticks are occasionally introduced into central Zambia by tick‐infested cattle from the north‐eastern part of the country where B. bovis is endemic. The spread of B. microplus in Southern Africa in a westward direction is discussed and related to the epidemiology of bovine babesiosis in Zambia.     

A morphological and molecular study of Anaplasma phagocytophilum transmission events at the time of Ixodes ricinus tick bite

Background

Anaplasma phagocytophilum is the causative agent of human granulocytic anaplasmosis (HGA) in humans and tick-borne fever (TBF) in ruminants. The bacterium invades and replicates in phagocytes, especially in polymorphonuclear granulocytes.

Methods

In the present study, skin biopsies and ticks (Ixodes ricinus) were collected from tick feeding lesions on 38 grazing lambs between two and three weeks after access to pastures. The histopathological changes associated with tick bites and A. phagocytophilum infection, were described. In addition the skin biopsies were examined by immunohistochemistry. Furthermore, samples from blood, skin biopsies and ticks were examined by serology, PCR amplification of msp2 (p44), genotyping of rrs (16S rRNA) variants, and compared with the results obtained from histological and immunohistochemical investigations.

Results

Tick bites were associated with chronic and hyperplastic inflammatory skin lesions in this study. A. phagocytophilum present in skin lesions were mainly associated with neutrophils and macrophages. Bacteria were occasionally observed in the Tunica media and Tunica adventitia of small vessels, but were rarely found in association with endothelial cells. PCR and genotyping of organisms present in blood, ticks and skin biopsies suggested a haematogenous and a local spread of organisms at the tick attachment sites.

Conclusions

The present study describes different aspects of A. phagocytophilum infection at the site of tick bite, and indicates that A. phagocytophilum rarely associates with endothelium during the early pathogenesis of infection.     

Babesia microti-like rodent parasites isolated from Ixodes persulcatus (Acari: Ixodidae) in Heilongjiang Province, China

A Babesia microti-like rodent parasite was isolated from the tick, Ixodes persulcatus, collected from the northern forest area of Heilongjiang province, China. The collected I. persulcatus were allowed to feed on specific pathogen-free SCID mice and red blood cells from the mice were used to isolate Babesia spp. with the microareophilous stationary-phase culture technique. Paired and tetrad forms of merozoites were observed by light microscope in red blood cells of SCID mice. In vitro growth of the parasites was also achieved in mice erythrocytes, which indicated the presence of Babesia spp. in I. persulactus. To further identify the Babesia species, polymerase chain reaction screening and subsequent sequencing of nuclear small subunit ribosomal RNA (nss-rRNA) was employed. The results indicate that the observed parasites might be an isolate strain responsible for human babesiosis -B. microti - which has 99.3% identity with that of B. microti isolate RcM5201 (AB112050) from Mishan in Heilongjiang and Kobe isolates from Japan. In addition, the infection rate of B. microti in I. persulcatus ticks in the region was 3.6-4.0% in adult females and no infection in males. Though the infection rate is low, the high attack frequency of tick species on local residents indicates the risk of human babesiosis in the region and the necessity of precautionary measures.     

Use of cerebellar brain smears in the diagnosis of babesiosis (Babesia bovis) in cattle

Summary Cerebral and cerebellar smears were made from 4 animals acutely reacting toBabesia bovis and 94 animals free from clinical babesiosis. The brain smears were stained by the Giemsa method and examined for the presence ofB. bovis parasites. In animals showing clinical babesiosis capillaries congested with parasitised erythrocytes were abundant in cerebral and cerebellar smears. Results obtained from both types of brain smears in animals free from clinical babesiosis agreed closely (83% conformity) as to the presence or absence of parasites. A third group of 39 animals from which cerebral and cerebellar smears were taken was also examined serologically by the indirect immunofluorescent antibody test (IFAT); about 69% of the IFAT positive and doubtful animals showed parasites in the cerebellar brain smears. The existence of false negatives in the IFAT test has been shown and discussed. It has been concluded that cerebellar samples obtained through the foramen occipitale can be used for the microscopic detection ofB. bovis parasites in latently infected bovines. This method can also be used in field cases suspected of cerebral babesiosis permitting brain sampling without resorting to the opening of the skull. Such an approach might prove particularly useful in areas where rabies occurs and the animal's head has to be sent to a diagnostic centre.

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El Empleo De Frotis De Cerebelo En El Diagnostico De Babesiosis (Babesia Bovis) En Bovinos

Resumen Se hicieron frotis de cerebro y cerebelo de 4 animales con síntomas agudos de babesiosis porBabesia bovis y de 94 animales clínicamente sanos. Los frotis se tiñeron con Giemsa y se examinaron por la presencia deB. bovis. En animales enfermos, los capilares del cerebro y cerebelo se encontraron congestionados y repletos de eritrocitos parasitados. Los resultados obtenidos en ambos tipos de frotis en animales libres de la enfermedad clínica, estuvieron de acuerdo (83% de conformidad) en cuanto a la presencia o ausencia de parásitos. Un tercer grupo de 39 animales de los cuales se tomaron frotis de cerebro y cerebelo, se examinaron también serológicamente mediante la técnica indirecta de anticuerpos fluorescentes (TIAF); cerca del 69% de animales positivos y dudosos por immunofluorescencia revelaron parásitos en los frotis de cerebelo. La existencia de falsos positivos se demostró mediante TIAF. Se concluye que los frotis de cerebelo tomados a través del agujero occipital pueden usarse para detectarB. bovis microscópicamente en animales con infecciones latentes. Este método se puede utilizar en el campo para no tener que abrir el craneo, sobre todo en áreas en donde existe rabia y las cabezas se envían al laboratorio.

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Examen De Frottis De Matière Cervelette Pour Le Diagnostic De La Babésiose (babesia bovis) Du Bétail

Résumé Des frottis de cerveau et de cervelet ont été effectués à partir de 4 animaux atteints de babésiose aiguë et de 94 autres n'ayant présenté aucun symptôme de la maladie. Les frottis ont été colorés au Giemsa et examinés pour mettre en évidence la présence deB. bovis. Chez les animaux atteints de babésiose clinique, les capillaires étaient congestionnés avec présence abondante d'érythrocytes parasités. Il existe une excellente concordance (83 p.100) entre les résultats des examens des deux types de frottis, qu'il s'agisse d'absence ou de présence des parasites. Un troisième groupe de 39 animaux dont les frottis de cerveau et de cervelet ont été faits ont également fait l'objet d'une étude sérologique pour déceler des anticorps par le test de l'immunofluorescence indirecte (I.F.A.T.); environ 69 p.100 des animaux reconnus positifs et douteux par l'I.F.A.T. ont montré des parasites dans les frottis du cervelet. L'existence de cas négatifs faux dans le test IFAT a été montrée et discutée. Il en a été conclu que les échantillons de cervelet obtenus à travers le foramen occipital peuvent être utilisés pour la détection microscopique deB. bovis chez des animaux en état d'infection latente. Cette méthode peut être également utilisée en brousse lors de suspicion de babésiose cérébrale car elle permet d'obtenir les prélèvements de tissus cérébraux nécessaires sans ouverture de la boite cranienne. Une telle approche peut être particulièrement utile dans les régions où sévit la rage et où il est nécessaire d'expédier la tête des animaux suspects à un centre de diagnostic.

     

Proteomic profiling of Rhipicephalus (Boophilus) microplus midgut responses to infection with Babesia bovis

Differences in protein expression in midgut tissue of uninfected and Babesia bovis-infected southern cattle ticks, Rhipicephalus (Boophilus) microplus, were investigated in an effort to establish a proteome database containing proteins involved in successful pathogen transmission. The electrophoretic separation of midgut membrane proteins was greatly improved by using liquid-phase isoelectric focusing combined with one-dimensional or two-dimensional (2-D) gel electrophoresis. A selection of differentially expressed proteins were subjected to analysis by capillary-HPLC-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). Among the identified Babesia-affected tick midgut proteins were six proteins that are implicated in signaling processes, including three Ca(2+)-binding proteins, a guanine nucleotide-binding protein, a protein with signal peptide activity and a translocon-associated receptor protein. Up-regulation of five metabolic enzymes indicated parasite-induced changes in electron and proton transport, protein processing and retinoic acid metabolism. Among the down-regulated proteins were a molecular chaperone, a cytoskeletal protein and a multifunctional protein of the prohibitin family. Identification of these proteins may provide new insights into the molecular interactions between B. bovis and its tick vector, and could lead to identification of anti-tick and transmission-blocking vaccine candidates.     

Borrelia burgdorferi sensu lato and Rickettsia spp. infections in hard ticks (Ixodes ricinus) in the region of Hanover (Germany)

In a total of 605 Ixodes (I.) ricinus ticks collected in the spring-months March, April and May 2005, quantitative real time PCR (qPCR) revealed 26.6% Borrelia (B.) burgdorferi sensu lato (sl)-positive ticks, i. e. divided by sex and stage into 31.9% positive adults (34.8% females and 29.0% males) and 18.5% positive nymphs. Mono-infections with genospecies from the B. burgdorferi sl-complex were found in over two thirds of the positive individuals, whereas almost one third showed double- or even triple-infections. Genospecies-specific conventional PCR determined B. afzelii as the most frequent genospecies followed by B. garinii, B. spielmanii, B. valaisiana and B. burgdorferi sensu stricto (ss). Rickettsia spp. were found in 34.2% of the collected ticks, divided into 37.6% adults (42.5% females and 32.8% males) and 29.0% nymphs. Co-infections of Rickettsia-positive ticks with B. burgdorferi sl spirochaetes were present in 10.1% of the ticks. Thereby, adult ticks exhibited a co-infection rate of 13.4% (15.5% females and 11.3% males) and nymphs of 5.0%. Independently of the above mentioned study, 3939 Ixodes ticks, sent in between 2006 and 2010 for B. burgdorferi sl-diagnostic, were examined by qPCR exclusively for B. burgdorferi sl. The resulting B. burgdorferi sl prevalence was 23.1% and 24.4% in 2006 and 2007, respectively, followed by a continuous decrease to 12.8% in 2010. To analyse whether this observed decrease in infection frequency is due to sampling bias, in a current study randomly sampled ticks collected from defined sites equally distributed over the city of Hanover are investigated in a statistically relevant sample size.     

Molecular detection of Babesia bovis and Babesia bigemina in white-tailed deer (Odocoileus virginianus) from Tom Green County in central Texas

Serologic and molecular evidence suggest that white-tailed deer in South Texas and North Mexico carry the agents of bovine babesiosis, Babesia bovis and Babesia bigemina. To determine if white-tailed deer in central Texas, which is outside the known occurrence of the vector tick at this time, harbor these parasites, blood samples from free-ranging and captive white-tailed deer (Odocoileus virginianus) in Tom Green County were tested by polymerase chain reaction (PCR) assays for B. bovis and B. bigemina 18S rDNA. Of the 25 samples tested, three (12%) were positive by nested PCR for B. bovis. This identity was confirmed by sequence analysis of the cloned 18S rDNA PCR product. Further confirmation was made by sequence analysis of the rRNA internal transcribed spacer (ITS) 1, 5.8S rRNA gene, and ITS 2 genomic region in two (representing samples from two different ranches) of the B. bovis positive samples. Three samples were positive by B. bigemina nested PCR, but sequencing of the cloned products confirmed only one animal positive for B. bigemina; Theileria spp. DNA was amplified from the other two animal samples. In addition to Theileria spp., two genotypically unique Babesia species sequences were identified among the cloned sequences produced by the B. bigemina primers in one sample. Phylogenetic analysis showed no separation of the deer B. bovis or B. bigemina 18S rDNA, or deer B. bovis ITS region sequences from those of bovine origin. Clarification of the possible role of white-tailed deer as reservoir hosts in maintaining these important pathogens of cattle is critical to understanding whether or not deer contribute to the epidemiology of bovine babesiosis.     

Effect of breed of cattle on transmission rate and innate resistance to infection with Babesia bovis and B bigemina transmitted by Boophilus microplus

OBJECTIVE: To assess the effect of breed of cattle on the transmission rates of and innate resistance to Babesia bovis and B bigemina parasites transmitted by Boophilus microplus ticks. DESIGN: Groups of 56 purebred B indicus and 52 B indicus cross B taurus (50%, F1 generation) steers were placed in a paddock seeded with and also naturally infested with B microplus which were the progeny of females ticks fed on B taurus cattle specifically infected with a virulent isolate of B bovis. The cattle were placed in the infested paddock 50 days after seeding had started. PROCEDURE: Cattle were inspected from horseback daily for 50 days. Clinically ill cattle were brought to yards and assessed by monitoring fever, depression of packed-cell volume, parasitaemia and severity of clinical signs. Any animals that met preset criteria were treated for babesiosis. Blood samples were collected from all cattle on day 28, 35 and 42 after exposure and antibodies to Babesia spp and packed cell volume measured. RESULTS: All steers, except for one crossbred, seroconverted to B bovis and B bigemina by day 35 and 75% of the crossbred steers showed a maximum depression in packed cell volume of more than 15% due to infection with Babesia spp compared with only 36% of the B indicus group. Ten of the 52 crossbreds and 1 of the 56 B indicus steers showed severe clinical signs. Two of the crossbreds required treatment of which one died 2 weeks after initial treatment. CONCLUSIONS: Pure-bred B indicus cattle have a high degree of resistance to babesiosis, but crossbred cattle are sufficiently susceptible to warrant the use of preventive measures such as vaccination. Transmission rates of B bovis and B bigemina to B indicus and crossbred cattle previously unexposed to B microplus were the same.     

Acaricidal properties of the essential oil of Melaleuca alternifolia Cheel (tea tree oil) against nymphs of Ixodes ricinus

The aim of the study was to examine the acaricidal effect of essential oil of Melaleuca alternifolia (tea tree oil, TTO) at different doses (4, 6, 8 and 10 microl) and for different exposure times (30, 60, 90 and 120 min) on nymphs of Ixodes ricinus. A dose of 8 microl TTO was lethal for more than 70% of ticks when inhaled and this effect was enhanced when the dose was increased to 10 microl (> 80%). The effect was correlated with the duration of exposure of ticks to TTO, with a significant effect being observed after 90 min exposure. The findings show that TTO has acaricidal properties and could be extremely useful in controlling ticks that are efficient vectors of pathogens.