Sub-cycle pulse revealed with carrier-envelope phase control of soliton self-compression in anti-resonant hollow-core fiber.

The influence of the carrier-envelope phase (CEP) of a pump pulse on the multioctave supercontinuum (SC) generation in a gas-filled anti-resonant hollow-core fiber (AR HCF) by soliton self-compression (SSC) has been explored. We have shown an octave-wide third harmonic generation (THG) in the visible-to-near-infrared range during the pulse compression down to a sub-cycle duration. The CEP of a multi-cycle pump pulse provides control of interference between the third harmonic (TH) and the SC that indicates the coherent synthesis of a sub-cycle pulse with a duration of about 0.4 optical cycles and a peak power of more than 2 GW at the fiber output.

Neospora caninum in crows from Israel.

A cross-sectional Neospora caninum seroprevalence study was performed on free ranging crows (Corvus cornix, Corvus monedula and Corvus splendens) from Israel in order to assess their exposure to this pathogen and evaluate their role as potential hosts or as sentinels of infection. Using the modified agglutination test (MAT) with a cutoff titer of 1:100, 30 out of 183 crows (16.4%) were found to be N. caninum seropositive. Positive results were validated and confirmed by the indirect fluorescent antibody test (IFAT). There was 100% agreement between tests when cut-off titers of 1:50 and 1:100 were applied for the IFAT and MAT, respectively. PCR analysis of brain extracts from all crows resulted in the detection of N. caninum DNA for the first time in crows belonging to two species, C. cornix and C. monedula. The high N. caninum seroprevalence in crows suggests that widespread exposure to infection with N. caninum exists especially in central and northern Israel and that crows may act as suitable markers for disease prevalence in the areas in which they are found.

The effect of a live Neospora caninum tachyzoite vaccine in naturally infected pregnant dairy cows.

Neosporosis, caused by the intracellular protozoan Neospora caninum, is a major cause of abortion and reproductive failure in cattle worldwide. The principal route of transmission of neosporosis is via in utero infection of the offspring. There is no effective prophylactic treatment or vaccine available against bovine neosporosis. A N. caninum NcIs491 isolate was examined for its ability to immunize and reduce abortions in naturally infected dairy cows under field conditions. N. caninum-seropositive pregnant dams were inoculated with 10(8) live tachyzoites during mid-term pregnancy. A total of 520 N. caninum seropositive dams were included in this study, of these, 146 were immunized and 374 cows served as a non-vaccinated control group. A significantly lower incidence of abortion was observed in vaccinated compared to non-vaccinated cows, 16 and 26% respectively (P=0.01), with a vaccine efficacy of 39%. However, the number of seropositive offspring remained similar in both groups. Overall, this field trial suggests that vaccination with live N. caninum tachyzoites should be considered as an effective measure to reduce abortions caused by neosporosis in naturally infected cows.

Concomitant infection of cattle with the vaccine strain Anaplasma marginale ss centrale and field strains of A. marginale.

Bovine anaplasmosis, caused by Anaplasma marginale, the intraerythrocytic rickettsia, is controlled by vaccination with live Anaplasma marginale ss centrale (A. centrale), a subspecies of relatively low pathogenicity. We have experimentally demonstrated that an animal primarily infected with A. marginale, or with the related vaccine subspecies A. centrale can be infected with the heterologous subspecies, and carries both bacteria. The co-infection was detected in experimentally cross-infected calves for up to 3 months after the last inoculation with the heterologous subspecies. The occurrence of characteristic cyclic rickettsemia of A. centrale and A. marginale was observed by examination of Giemsa-stained blood smears, or by the presence of specific rickettsial DNA confirmed in PCR assays based on specific msp1a and msp4 for A. marginale, and on specifically designed msp3 and msp4 primers for A. centrale. Sequence analysis of msp4-specific fragments for each subspecies revealed the presence of dual infection in both calves on days 30 and 60 after cross-inoculation with the heterologous Anaplasma subspecies. The experimental cross-infection of calves clearly demonstrated that the concept of "infection exclusion" does not apply to Anaplasma infection in cattle; as there was no infection exclusion of A. marginale in A. centrale-infected cattle, and vice versa. The present results confirmed our previous findings that cattle grazing in an anaplasmosis-endemic field were subject to concomitant infection with both the vaccine A. centrale and the field A. marginale strains.

Isolation of Neospora caninum from dairy zero grazing cattle in Israel.

First Israeli Neospora caninum isolates were obtained from brain tissues of aborted fetuses (NcIs491 and NcIs580) from dairy farms endemic for neosporosis and maintaining cattle on zero grazing. Tissues from different parts of the fetus brains were used to infect Vero cells. Tachyzoites of N. caninum were first observed in cultures from days 30 and 32 after infection. To confirm the identity of the isolated parasites, DNA extracts from brains and cultures were tested by PCR with specific primers based on the Nc5 gene. Specific fragments were amplified by PCR from infected cultures of both fetuses on day 25. Susceptible seronegative gerbils (Meriones tristrami) were inoculated intraperitoneally with 10(3) to 10(5) tenfold dilutions of subculture tachyzoites. The inoculated gerbils developed specific antibodies to N. caninum, with end-point serum dilution of 1:4096 in the IFA assay, whereas no neurological signs or deaths were seen during 4 months of observation.

Babesia bigemina: attenuation of an Uzbek isolate for immunization of cattle with live calf- or culture-derived parasites.

The virulence of an Uzbek isolate of Babesia bigemina, obtained from infected Boophilus annulatus ticks from an endemic area in Uzbekistan, was attenuated for immunization of cattle with autochthonous calf- or culture-derived parasites in Uzbekistan. After four "slow passages" in vivo the virulence was reduced, as evidenced by the response of calves inoculated with an experimental live frozen vaccine produced from the following passage. The vaccine was safe and protective against homologous virulent challenge under laboratory conditions. The culture-derived experimental vaccine was produced from cultures initiated after 3 passages in vivo followed by 22 passages in vitro. The cultured parasites did not elicit any clinical sign, but inoculated calves seroconverted following vaccination and were protected against the virulent homologous challenge. Both calf- and culture-derived vaccines were safe for cattle grazing in an endemic area in Uzbekistan. Despite the high polymorphism of B. bigemina, as reported from various geographical regions, the Central Asian strain was attenuated similarly to those that form the basis of the existing live B. bigemina vaccines in other parts of the world.

Low seroprevalence of antibodies to Neospora caninum in wild canids in Israel.

The role of domestic dogs in the epidemiology of Neospora caninum as well as the relationship between N. caninum infection of farm dogs and cattle were demonstrated, however, evidence is scarce regarding the role of wild canids in domestic animal neosporosis. The present study was undertaken to evaluate the role of wild canids in the epidemiology of bovine neosporosis in Israel by analyzing the prevalence of antibodies to N. caninum in wild canids. Sera samples were collected from 114 free ranging wild golden jackals (Canis aureus), 24 red foxes (Vulpes vulpes) and nine wolves (Canis lupus), which were collected in Israel during the years 1999-2004. Of a total of 147 wild canids tested antibodies to N. caninum were only found in two golden jackals with IFAT titers of 1:50, and in one red fox and one wolf with IFAT titer of 1:400. The low seroprevalence found in this study (2.7%) indicated that wild canids probably do not have an important role in the epidemiology of N. caninum in Israel. However, since the diet of different species of wild canids and even diverse populations of the same canid species vary, it is possible that other results might be obtained from specific wild canids populations, which scavenge in the vicinity of infected bovines.

Molecular and serological detection of A. centrale- and A. marginale-infected cattle grazing within an endemic area.

A reverse line blot hybridization (RLB) one-stage nested PCR (nPCR) for Anaplasma centrale and a nested PCR for Anaplasma marginale were used to detect infected cattle grazing within an endemic region in Israel. A novel set of PCR primers and oligonucleotide probes based on a 16S ribosomal RNA gene was designed for RLB detection of both Anaplasma species, and the performance of the molecular assays compared. The immunofluorescent antibody test (IFA) was used to detect antibodies to both Anaplasma species, whereas, a highly sensitive and specific competitive enzyme-linked immunosorbent assay (cELISA) was used to detect antibodies in A. centrale-vaccinated cattle. The RLB and the nested PCR procedures showed bacteremia with sensitivity of 50 infected erythrocytes per milliliter. Up to 93% of the A. centrale vaccinates carried specific antibodies that were detected by cELISA, and up to 71% of the vaccinated cattle were found to be naturally infected with A. marginale according to the PCR and the RLB assays. Nevertheless, no severe outbreaks of A. marginale infection occurred among vaccinated herds in this endemic region. It appears that both, molecular tools and serology are useful for evaluation of the vaccine efficacy. In the light of wide natural field infection with A. marginale, strong recommendations to continue the A. centrale vaccination program regime will continue until a new generation of non-blood-based vaccine will be developed.

Vaccination of older Bos taurus bulls against bovine babesiosis.

Two separate groups of Bos taurus bulls, one of 106 and the second of 27 animals, imported to Israel from areas free of Babesia bovis and Babesia bigemina, were vaccinated against babesiosis with a bivalent live attenuated vaccine. In light of the fact that routine vaccination is recommended at the weaning age, these bulls--of highly susceptible breeds--were kept under close surveillance to prevent losses that might be caused by severe clinical reactions to their vaccination at the age of 16-18 months. Seven days after vaccination, about one-third of the 106 bulls in the first group developed clinical signs of B. bigemina infection, which peaked at day 9, and then diminished from day 11, when the patent period known for B. bovis infection was observed. Because of the severe clinical responses a total of 36% of the bulls required babesicidal treatment. Despite the treatment Babesia were not sterilized: 33 and 68% of the animals remained PCR positive for B. bigemina and B. bovis, respectively. To mitigate the severe responses to vaccination, the 27 bulls of the second group were vaccinated in two-steps: they were inoculated initially with avirulent culture-derived parasites and then vaccinated with the conventional donor-derived vaccine a month later. None of the bulls in the latter group developed clinical babesiosis, all were serologically positive to B. bigemina, and 67% showed seroconversion to B. bovis. In light of the experience described here, it is suggested that sensitive older cattle be vaccinated against babesiosis by priming them with avirulent in vitro-cultured parasites and then inoculating them with the conventional donor-derived vaccines.

Proteolytic enzyme activity and attenuation of virulence in Theileria annulata schizont-infected cells.

A field isolate of Theileria annulata (Uzbek strain) was obtained from calves infected by Hyalomma anatolicum ticks collected from an endemic region in Uzbekistan. Schizont-infected bovine cells that had been established and propagated in cell culture were examined for attenuation both in vivo, by inoculating cells from various passages into calves, and in vitro for metalloproteinase activity. During serial subcultivation a gradual reduction in virulence and in enzyme activity in cells infected with the Uzbek strain were observed. Complete attenuation of the Uzbek isolate was obtained at about passage 80, and only traces of proteolysis were detected in gelatin substrate gels. In contrast, there was no direct correlation between virulence and enzyme levels in an Israeli strain. While schizonts of the Israeli strain were completely attenuated at passage 80, proteolysis in the substrate gels was detected up to passage 197. Solid immunity was observed in calves immunized with attenuated T. annulata schizonts of the Uzbek strain upon challenge with the homologous H. excavatum sporozoites. For a strain to be used for vaccine production, it appears that animal inoculation still remains the most reliable method to assess the degree of attenuation and protection.

Babesia bovis and B. bigemina: Persistence of infection in friesian cows following vaccination with live antibabesial vaccines.

The persistence of Babesia bovis and B. bigemina infection in Friesian cows, following vaccination with attenuated live vaccines, was assessed by subinoculation of blood into splenectomized calves. Subinoculation tests showed that B. bigemina persisted in two out of 20 cows vaccinated 10 and 46 months previously, and that B. bovis persisted in 11 out of 22 cows vaccinated 10 to 47 months previously. Antibody was detected in five B. bigemina - and 15B. bovis -vaccinated cows. Parasites of both species persisted among the serologically negative cows, whereas blood obtained from serologically positive cows failed to transmit infection. It is concluded that in the absence of reinfection Friesian cattle may spontaneously eliminate B. bigemina and B. bovis infection after various periods following vaccination.

Susceptibility of Psammomys obesus and Meriones tristrami to tachyzoites of Neospora caninum.

The susceptibility of Psammomys obesus (sand rat) and Meriones tristrami (Tristram's jird) to Neospora caninum was investigated by subcutaneous (s.c.) and intraperitoneal (i.p.) inoculation of 10-fold doses of culture-derived tachyzoites. Groups of 5 animals were inoculated with doses of 10-10(7) parasites via each route of inoculation. All but 2 of the sand rats inoculated with doses of 10-10(4) parasites succumbed to the infection by 7-18 days postinfection. All jirds inoculated with 10(7) tachyzoites succumbed by 5-16 days postinfection and those inoculated with 10(6) tachyzoites by 9-25 days. A considerable proportion of the jirds inoculated with 10-10(5) tachyzoites survived. Fibrinous peritonitis with ascites containing numerous tachyzoites was observed in the i.p.-inoculated sand rats and jirds that succumbed to the infection. In the jirds, tachyzoites were also found in pleural exudate. A considerable number (42.8%) of the jirds inoculated s.c. or i.p. exhibited neuromuscular symptoms, expressed in ataxia, head tilt, circling movement, and posterior paralysis. Seven successive passage of tachyzoites were achieved in sand rats with doses of 10(5) parasites and in jirds with doses of 10(7) parasites. All surviving jirds became seroconverted and were immune to lethal challenge.

Antibody response to Hepatozoon canis in experimentally infected dogs.

Canine hepatozoonosis is a disease caused by the tick-borne protozoan Hepatozoon canis. Five puppies were inoculated by ingestion of Rhipicephalus sanguineus ticks experimentally infected with H. canis, and all became infected with H. canis: gametocytes were detected in blood smears from four dogs and schizonts were observed in the spleen and bone marrow of the fifth. Antibodies reactive with H. canis gametocytes were detected by the indirect fluorescent antibody test (IFA), with IgM detected initially in all dogs 16 to 39 days post infection (PI) and IgG 22 to 43 days PI. The presence of gametocytes was first observed within peripheral blood neutrophils in Giemsa-stained blood smears between days 28 and 43 PI. Gametocyte-reactive antibodies were detected before the appearance of blood gametocytes in three of the four parasitemic dogs and also in a dog with no observed parasitemia. The detection of serum antibodies prior to the detection of blood gametocytes, or without apparent parasitemia, suggests that antibodies reactive with gametocytes may be formed against earlier forms of the parasite developing in the parenchymal tissues. Sera of dogs experimentally infected with Babesia canis, Babesia gibsoni and Ehrlichia canis exhibited no reactivity when tested with H. canis antigen. Additionally, sera positive for H. canis were not reactive with antigens of Toxoplasma gondii, Neospora caninum, Leishmania donovani and E. canis. In conclusion, incoculation of dogs with ticks infected with H. canis results in production of antibodies reactive with peripheral blood gametocytes. Detection of IgG titres would be beneficial for the diagnosis of progressive infections with undetectable parasitemia, for seroprevalence studies, and as an adjunct to IgM titres in early infections.