Prevalence of Toxoplasma gondii infection in Belgian house cats.

Five hundred and sixty seven sera of healthy house cats aged 3 months to 7 years, were examined for the presence of anti-toxoplasma antibodies by indirect immunofluorescence assay and compared to SAG1 and TLA enzyme linked immunosorbent assays as alternative test. Twenty-five percent of cats tested positive for IgG and/or IgM. Seroprevalence increased with age from 2% below 12 months of age up to 44% at age 7. Sensitivities of SAG1 and TLA ELISA were 84.1% and 88.6%, respectively. Peak levels in seroprevalence were correlated to increased IgG titers in TLA ELISA. Our results suggest that T. gondii infections are common in house cats and that there is a high chance for a negative cat to seroconvert in its second life-year.

Post-exposure Treatment with Anti-rabies VHH and Vaccine Significantly Improves Protection of Mice from Lethal Rabies Infection.

Post-exposure prophylaxis (PEP) against rabies infection consists of a combination of passive immunisation with plasma-derived human or equine immune globulins and active immunisation with vaccine delivered shortly after exposure. Since anti-rabies immune globulins are expensive and scarce, there is a need for cheaper alternatives that can be produced more consistently. Previously, we generated potent virus-neutralising VHH, also called Nanobodies, against the rabies glycoprotein that are effectively preventing lethal disease in an in vivo mouse model. The VHH domain is the smallest antigen-binding functional fragment of camelid heavy chain-only antibodies that can be manufactured in microbial expression systems. In the current study we evaluated the efficacy of half-life extended anti-rabies VHH in combination with vaccine for PEP in an intranasal rabies infection model in mice. The PEP combination therapy of systemic anti-rabies VHH and intramuscular vaccine significantly delayed the onset of disease compared to treatment with anti-rabies VHH alone, prolonged median survival time (35 versus 14 days) and decreased mortality (60% versus 19% survival rate), when treated 24 hours after rabies virus challenge. Vaccine alone was unable to rescue mice from lethal disease. As reported also for immune globulins, some interference of anti-rabies VHH with the antigenicity of the vaccine was observed, but this did not impede the synergistic effect. Post exposure treatment with vaccine and human anti-rabies immune globulins was unable to protect mice from lethal challenge. Anti-rabies VHH and vaccine act synergistically to protect mice after rabies virus exposure, which further validates the possible use of anti-rabies VHH for rabies PEP.

A two-step lyssavirus real-time polymerase chain reaction using degenerate primers with superior sensitivity to the fluorescent antigen test.

A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤ 1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible.

No evidence of coronavirus infection by reverse transcriptase-PCR in bats in Belgium.

No coronavirus was detected by PCR in lung and intestine samples of 100 bats, mostly common pipistrelles (Pipistrellus pipistrellus), collected dead between 2008 and 2013 for rabies surveillance in Belgium. The negative results contrast with the high prevalence of coronaviruses detected in fecal pellets from live-captured bats in some European countries.

Protective effect of different anti-rabies virus VHH constructs against rabies disease in mice.

Rabies virus causes lethal brain infection in about 61000 people per year. Each year, tens of thousands of people receive anti-rabies prophylaxis with plasma-derived immunoglobulins and vaccine soon after exposure. Anti-rabies immunoglobulins are however expensive and have limited availability. VHH are the smallest antigen-binding functional fragments of camelid heavy chain antibodies, also called Nanobodies. The therapeutic potential of anti-rabies VHH was examined in a mouse model using intranasal challenge with a lethal dose of rabies virus. Anti-rabies VHH were administered directly into the brain or systemically, by intraperitoneal injection, 24 hours after virus challenge. Anti-rabies VHH were able to significantly prolong survival or even completely rescue mice from disease. The therapeutic effect depended on the dose, affinity and brain and plasma half-life of the VHH construct. Increasing the affinity by combining two VHH with a glycine-serine linker into bivalent or biparatopic constructs, increased the neutralizing potency to the picomolar range. Upon direct intracerebral administration, a dose as low as 33 µg of the biparatopic Rab-E8/H7 was still able to establish an anti-rabies effect. The effect of systemic treatment was significantly improved by increasing the half-life of Rab-E8/H7 through linkage with a third VHH targeted against albumin. Intraperitoneal treatment with 1.5 mg (2505 IU, 1 ml) of anti-albumin Rab-E8/H7 prolonged the median survival time from 9 to 15 days and completely rescued 43% of mice. For comparison, intraperitoneal treatment with the highest available dose of human anti-rabies immunoglobulins (65 mg, 111 IU, 1 ml) only prolonged survival by 2 days, without rescue. Overall, the therapeutic benefit seemed well correlated with the time of brain exposure and the plasma half-life of the used VHH construct. These results, together with the ease-of-production and superior thermal stability, render anti-rabies VHH into valuable candidates for development of alternative post exposure treatment drugs against rabies.

Infectivity of rabies virus-exposed macrophages.

Rabies virus distributes widely in infected mice, including lymphoid tissues and spleen macrophages. The infection characteristics in murine macrophages and the infectivity of virus-exposed macrophages were examined upon inoculation in mice. In vitro, Mf4/4 spleen macrophages supported mild virus production (10(4)-fold less than neuroblastoma), with formation of typical virions. Bone marrow-derived macrophages (BMM) were most efficient to capture virus, but new virus production was not detected. Virus-induced cell death was significantly stronger in BMM, which might have eliminated BMM with productive infection. Still, viral RNA remained detectable in the remaining BMM for at least 4 weeks. Injection of in vitro-infected Mf4/4 in the nose or brain proved efficient to propagate infection in mice, even when cells were pre-incubated with neutralizing antibodies. Surprisingly, injection of ex-vivo-infected BMM in the brain also led to lethal infection in 8 out of 12 mice. Injection of infected Mf4/4 in the muscle mostly favoured a protective antibody response. Despite that macrophages are less fit to support virus production, they can still act as a source of infectious virus upon transfer in mice. This may be relevant for screening donor organs/cells, for which RT-PCR should be preferred over the traditional antigen or virus isolation assays.

A non-invasive intranasal inoculation technique using isoflurane anesthesia to infect the brain of mice with rabies virus.

Methods for intranasal inoculation of viruses are often described poorly and the effects of variations in the technique on the outcome are unknown. Standardization of protocols is key to compare studies and minimize animal use. The clinical and virological outcome of infection with rabies virus (genotypes 1 and 5) upon administration of different inoculum volumes (25, 50 and 100µl) and different anesthetic regimens were examined. Administration of 25µl of virus as a drop on both nostrils under brief superficial isoflurane anesthesia (92µl/dm(3), recovery after 85 ± 1 0s) was the most effective to infect the brain and induced 100% lethal infection 9 days later. Increasing the inoculum volume reduced infectivity significantly, with decreased viral loads in the brain and only 40% mortality. Increasing the depth of isoflurane anesthesia (230µl/dm(3)) improved the infectivity of the large-volume inoculum (90% mortality), probably because of suppression of swallow and sneeze reflexes. Compared to isoflurane anesthesia, xylazine-ketamine anesthesia reduced the infectivity of the inoculum significantly. Thus, administration of a small volume of virus on the nostrils under brief gas anesthesia is a safe and reproducible technique to induce infection of the brain. Since needles are not required, this helps to preserve the integrity of the physical barriers, animal welfare and the manipulator's safety.