Trichinella spiralis newborn larvae: characterization of a stage specific serine proteinase expression, NBL1, using monoclonal antibodies.

Trichinella spiralis is an intracellular parasitic nematode of mammalian skeletal muscle, causing a serious zoonotic disease in humans and showing a high economic impact mainly in pig breeding. Serine proteinases of T. spiralis play important roles in the host-parasite interactions mediating host invasion. In this study, we have focused on newborn larvae (NBL-1), the first identified serine proteinase from the NBL stage of T. spiralis. Five monoclonal antibodies (mAbs) directed against the C-terminal part of NBL1, were produced. These mAbs were IgG1κ isotype and specifically recognized as a common motif of 10 amino acids (PSSGSRPTYP). Selected mAbs were further characterized using antigens from various developmental stages of T. spiralis. Western blot revealed that selected mAbs reacted with the native NBL1 at Mr 50 kDa in the adult and NBL mixed antigens and NBL stage alone. Indirect immunofluorescence analysis revealed that selected mAbs intensely stained only the embryos within the gravid females and the NBL. Thus, the produced mAbs are useful tools for the characterization of NBL1 as a major antigen of Trichinella involved in the invasion of the host but also for the development of new serological tests with an early detection of T. spiralis infection.

Evaluation of immunogenicity and protection of the Mic1-3 knockout Toxoplasma gondii live attenuated strain in the feline host.

Toxoplasmosis is a zoonotic disease caused by the parasite Toxoplasma gondii. Up to a third of the global human population is estimated to carry a T. gondii infection, which can result in severe complications in immunocompromised individuals and pregnant women. Humans and animals can become infected by ingesting either tissue cysts containing T. gondii bradyzoites, from raw or undercooked meat, or sporulated oocysts from environmental sources. T. gondii oocysts are released in the faeces of cats and other felids, which are the parasite's definitive hosts, leading to environmental contamination. Therefore, vaccination of the feline host against T. gondii is an interesting strategy to interrupt the parasitic life cycle and subsequently limit contamination of intermediate hosts. With this goal in mind, we tested in cats, an attenuated live strain of T. gondii deleted for the Mic1 and Mic3 genes (Mic1-3KO) that was previously shown to be an efficient vaccine candidate in mouse and sheep models. Subcutaneous or oral vaccination routes induced a high specific antibody titer in the cat sera, indicating that the Mic1-3KO strain is immunogenic for cats. To assess protection induced by the vaccine candidate strain, we followed oocysts shedding by vaccinated cats, after oral challenge with a T. gondii wild-type strain. Surprisingly, a high antibody titer did not prevent cats from shedding oocysts from the challenge strain, regardless of the vaccination route. Our results show that the Mic1-3KO vaccine candidate is immunogenic in the feline host, is well tolerated and safe, but does not confer protection against oocysts shedding after natural infection with wild type T. gondii. This result highlights the particular relationship between T. gondii and its unique definitive host, which indicates the need for further investigations to improve vaccination strategies to limit environmental and livestock contaminations.

Primary characterization and assessment of a T. spiralis antigen for the detection of Trichinella infection in pigs.

A clone, designated L20h-Ts3, was selected by immunoscreening of cDNA libraries of Trichinella spiralis worms collected 14h, 20h and 48h post-infection (p.i.) from mice intestines. L20h-Ts3 encodes the full-length of a conserved hypothetical protein of 13.1kDa involving putative interaction with the immune system. PCR analysis showed that L20h-Ts3 mRNA is constitutively expressed throughout T. spiralis life cycle and not restricted to intestinal stages. The L20h-Ts3 fusion protein was obtained in an Escherichia coli expression system and purified by Ni-affinity chromatography before inoculation into mice in order to produce polyclonal antibodies. Then, immunohistochemical study and Western blot analysis revealed its presence within the stichosome of T. spiralis and in excretory/secretory products strengthening a putative fundamental role for the parasite's survival such as host tissue invasion or modification of the host muscular cell phenotype. L20h-Ts3 fusion protein was recognized in Western blot as soon as 15-20 days p.i. by sera from pigs experimentally infected with 20,000 muscle larvae (ML) of T. spiralis. Thus, an indirect L20h-Ts3 ELISA was designed and evaluated using sera from experimentally infected pigs by comparison with the only ELISA currently available for trichinellosis purposes. A gain of precocity from 7 up to 14 days and detection up to 25 weeks p.i. was possible with the L20h-Ts3 ELISA offering a large window for trichinellosis detection. The L20h-Ts3 ELISA was less effective in the case of low infections in pigs. Nevertheless, these results show that the L20h-Ts3 ELISA has a real interest due to its precocity and stability of detection in time. The association of the L20h-Ts3 fusion protein with other antigenic proteins identified previously could appreciably improve the serological test and facilitate its standardization.

Freeze-tolerance of Trichinella muscle larvae in experimentally infected wild boars.

Freeze-tolerance of encapsulated Trichinella muscle larvae (ML) is mainly determined by Trichinella species, but is also influenced by host species, the age of the infection and the storage time and temperature of the infected meat. Moreover, the freeze-tolerance of the encapsulated species appears to be correlated to the development of thick capsule walls which increases with age. An extended infection period and the muscle composition in some hosts (e.g. herbivores) may provide freeze-avoiding matrices due to high carbohydrate contents. The present experiment compares freeze-tolerance of Trichinella spiralis and Trichinella britovi ML in wild boar meat 24 weeks post inoculation (wpi). Three groups of four wild boars were infected with 200, 2000 or 20,000 ML of T. britovi (ISS 1575), respectively. Additionally, three wild boars were inoculated with 20,000 ML of T. spiralis (ISS 004) and two animals served as negative controls. All wild boars were sacrificed 24 wpi. Muscle samples of 70 g were stored at -21°C for 19, 30 and 56 h, and for 1-8 weeks. Larvae were recovered by artificial digestion. Their mobilities were recorded using Saisam(®) image analysis software and their infectivities were evaluated using mouse bioassays. Samples frozen for 19, 30 and 56 h allowed recovery of mobile ML, but samples frozen for 1 or 2 weeks did not. Correspondingly, only T. spiralis and T. britovi larvae isolated from wild boar meat frozen for 19, 30 and 56 h established in mice. This study showed that freezing at -21°C for 1 week inactivated T. spiralis and T. britovi ML encapsulated in wild boar meat for 24 weeks.