A genetically engineered H5 protein expressed in insect cells confers protection against different clades of H5N1 highly pathogenic avian influenza viruses in chickens.

The evolution of highly pathogenic H5N1 avian influenza viruses (HPAI-H5N1) has resulted in the appearance of a number of diverse groups of HPAI-H5N1 based on the presence of genetically similar clusters of their haemagglutinin sequences (clades). An H5 antigen encoded by a recombinant baculovirus and expressed in insect cells was used for oil-emulsion-based vaccine prototypes. In several experiments, vaccination was performed at 10 days of age, followed by challenge infection on day 21 post vaccination (PV) with HPAI-H5N1 clades 2.2, 2.2.1, and 2.3.2. A further challenge infection with HPAI-H5N1 clade 2.2.1 was performed at day 42 PV. High haemagglutination inhibition titres were observed for the recH5 vaccine antigen, and lower haemagglutination inhibition titres for the challenge virus antigens. Nevertheless, the rate of protection from mortality and clinical signs was 100% when challenged at 21 days PV and 42 days PV, indicating protection over the entire broiler chicken rearing period without a second vaccination. The unvaccinated control chickens mostly died between two and five days after challenge infection. A low level of viral RNA was detected by reverse transcription followed by a quantitative polymerase chain reaction in a limited number of birds for a short period after challenge infection, indicating a limited spread of HPAI-H5N1 at flock level. Furthermore, it was observed that the vaccine can be used in a differentiation infected from vaccinated animals (DIVA) approach, based on the detection of nucleoprotein antibodies in vaccinated/challenged chickens. The vaccine fulfilled all expectations of an inactivated vaccine after one vaccination against challenge with different clades of H5N1-HPAI and is suitable for a DIVA approach.

A new animal model for implant-related infected non-unions after intramedullary fixation of the tibia in rats with fluorescent in situ hybridization of bacteria in bone infection.

There is no adequate animal model to mimic the difficult clinical situation of infected non-union of the tibia after intramedullary stabilization. The purpose was to establish an animal model of implant-related infected non-unions of the tibia in rats. Furthermore, it was evaluated if detection of bacteria by fluorescent in situ hybridisation (FISH) technique is possible in bone infection. 17 rats were used in which osteotomy of the midshaft tibia was performed and stabilized with an intramedullary device. Two groups were tested: group 1: contamination of the osteotomy site with 10(4) colony forming units (CFUs) of Staphylococcus aureus (11 animals), group 2: no bacterial contamination (6 animals). The animals were sacrificed after 42 days and bone healing and infection were assessed clinically, by X-ray, micro-CT, and microbiological methods including FISH technique using EUB and STAPHY probes. Histology and scanning electron microscopy (SEM) for biofilm formation were performed. All animals of the control group showed uneventful bone healing after 6 weeks without any signs of local infections. 10 of 11 (90.9%) animals of group 1 with bacterial contamination exhibited infected non-union formation with positive clinical, radiological and microbiological infection signs of the tibia but without any systemic infection signs. FISH technique was able to identify bacteria in the infected bone. All intramedullary implants from the infected animals showed positive biofilm formation in SEM. This work presents the first animal model for the induction of intramedullary device-related infected non-union in the tibia and detection of bacteria by FISH technique in infected bone.

Teste de avaliação in vitro e criopreservação do sêmen de cão utilizando diferentes diluidores

Visando verificar o tempo de viabilidade das células espermáticas do cão, na temperatura de 38C, e comparar a eficiência dediferentes diluentes na sua criopreservação, foram realizadas 40 colheitas através da manipulação peniana para, no primeiroexperimento, realizar o teste de termorresistência (TIR) do sêmen fresco, onde se observou motilidade e vigor espermático,a cada 15 minutos, durante duas horas. No segundo experimento, as amostras obtidas foram diluídas em leite desnatado(LO), Tris+gema de ovo de galinha (TGG) e Tris+gema de ovo de codorna (TGC), refrigeradas a 5C e, em seguida, adicionadasde glicerol (7%), envasadas e congeladas (-196 ac). O TIR demonstrou que a motilidade e o vigor decaíram gradativamentee, após 45 minutos, observou-se uma redução mais acentuada destes parâmetros espermáticos. Em contrapartida, após ocongelamento, verificou-se motilidade de 11 ,92 %, 41 ,54 % e 50,00 % e 1 ,23, 2, 77 e 3,00 de vigor espermático, demonstrandodiferença significativa (P ~ 0,05) entre o primeiro grupo e os outros dois, que, por sua vez, não diferiram entre si, além de 17,00%, 11,35% e 10,95% de patologias do acrossoma, para os grupos LO, TGG e TGC, respectivamente. Conclui-se, portanto, queo sêmen da espécie canina até 45 minutos a 38C pode ser utilizado na inseminação artificial ou criopreservação e que odiluente Tris, acrescido de gema de ovo (galinha ou codorna), de