Master of Science / Department of Animal Science / R. Scott Beyer / Daniel Y.C. Fung / Clostridium perfringens causes necrotic enteritis (NE), resulting in decreased feed efficiency and increased mortality, costing the poultry industry USD 2 billion a year worldwide. The objective of the first trial was to validate the Fung Double Tube (FDT) to detect and enumerate C. perfringens in chicken intestines. Two methods (FDT and petri plates) and three media (Shahidi Ferguson Perfringens [SFP] with egg supplement, polymyxin B [p], and kanamycin [k; E]; SFP with p and k [P]; and SFP with cycloserine [C]) were arranged in a 2 x 3 factorial, resulting in six treatments. The FDT with medium C (5.35 log CFU/g) had significantly (P<0.05) higher C. perfringens counts than any other media/method combination. The objective of the second and third trials was to determine the effect of diet type on the population of C. perfringens in broiler intestines using the FDT. Trial 2 tested: corn-soybean meal (SBM), low-crude protein (19.8%)/high synthetic amino acids (SAA), and barley (56%)-fishmeal (4%; BF). Diets in Trial 3 included: corn-SBM, barley (7.46%), fishmeal (4%), and BF. Diets in Trial 2 contained an antibiotic and a coccidiostat; diets in Trial 3 did not. After 21 days, birds in Trial 2 fed BF had significantly higher (P<0.05) counts (5.96 log CFU/g) of C. perfringens, as compared to all other diets. Both, corn-SBM and SAA diets resulted in 3.89 log CFU/g. In Trial 3, birds fed the corn-SBM diet (2.7 log CFU/g) had significantly lower (P<0.05) counts than broilers fed BF (4.15 log CFU/g). When broilers were fed fishmeal (3.583 log CFU/g) and barley (3.577 log CFU/g) separately, C. perfringens counts were numerically higher compared to the corn-SBM diet, but numerically lower than birds fed BF. Barley and fishmeal inclusion increased the incidence of C. perfringens, and their combination resulted in a cumulative effect. The FDT method is able to detect C. perfringens at higher levels than the conventional petri plate method (P<0.001) and it also proved to be an effective method to detect differences in C. perfringens counts from the intestines of chickens fed different diet.

Chikungunya is a mosquito-transmitted disease caused by Chikungunya virus (CHIKV). Symptoms of Chikungunya include debilitating joint pain and swelling, fever and rash. CHIKV was first discovered in 1953 in Tanzania, and has since caused periodic outbreaks of disease. The virus reemerged recently in 2004 and has since spread around the world affecting more than 3 million people. The different strains of CHIKV have been grouped into three phylogenetic clades: West African, Asian and East/Central/South African (ECSA). There are no FDA approved medicines or vaccines used to treat or prevent CHIKV infection. The antiviral drug, T-705 (commercially known as Favipiravir), has recently been shown to have activity against CHIKV. T-705 has already been approved in Japan for the treatment of influenza and is currently going through clinical trials in the US. Since there may be phenotypic differences between the clades of CHIKV, it is important to first characterize distinctions between the strains and determine the susceptibility of these strains to treatment. To do this, we obtained two different CHIKV strains from each of the three phylogenetic groups. These CHIKV strains displayed differences in their ability to replicate in cell culture and exhibited only slight differences in susceptibility to T-705 treatment. However, more profound differences were observed in mouse models where differences in disease severity and response to T-705 treatment were observed.

Thesis (M. Sc.) Uppsala : Sveriges lantbruksuniv., 2007.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2001. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniviversitet, 2006. / Härtill 7 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2006. / Härtill 5 uppsatser.

The fusion protein of the Newcastle Disease Virus (NDV) contains three hydrophobic domains. To explore the topogenic signals of these domains, mutants were constructed in which each of the hydrophobic domains was deleted. The membrane insertion and topology of these proteins was characterized in a wheat germ cell-free translation system supplemented with canine microsomal membranes. The results indicated that the first 13 amino acids of the fusion protein are necessary to confer translation inhibition by SRP. Translocation of the nascent chains containing all or part of the first hydrophobic sequence resulted in the appearance of a species of higher molecular weight consistent with glycosylation of at least four of the five potential N-linked glycosylation sites. When glycosylation was inhibited with a glycosylation competitor peptide, signal sequence cleavage was detected. Protease digestion of mutants missing the C-terminal hydrophobic domain indicated that the C-terminus has stop transfer activity. A comparison of membrane insertion of the wild-type fusion protein to that of a mutant missing the second hydrophobic domain, the fusion sequence, indicated that the fusion domain has stop-transfer activity when synthesized in vitro. Furthermore, the fusion domain shows little signal sequence activity when positioned near the amino terminus of the fusion protein. The fusion protein has a highly conserved leucine zipper motif immediately upstream from the transmembrane domain of the F1 subunit. In order to determine the role that the conserved leucines have for the oligomeric structure and biological activity of the NDV fusion protein, the heptadic leucines at positions 481,488, and 495 were changed individually and in combination to an alanine residue. Whereas single amino acid changes had little effect on fusion, substitution of two or three leucine residues abolished the fusogenic activity of the protein although cell surface expression of the mutants and sedimentation in sucrose gradients was similar to that of the wild type. Furthermore, deletion of the C-terminal 91 amino acids, including the leucine zipper motif and transmembrane domain resulted in secretion of an oligomeric structure. These results indicate that the conserved leucines do not play a role in oligomer formation but are required for the fusogenic ability of the protein. When the polar face of the potential alpha helix was altered by nonconservative substitutions of a serine-to-alanine (position 473), glutamic acid-to-lysine (position 482) or an asparagine-to-lysine (position 485), the fusogenic ability of the protein was not significantly disrupted. A phenylalanine residue is at the amino terminus of the F1 protein of all paramyxovirus fusion proteins with the exception of the avirulent strains which have a leucine residue in this position. To explore the role of this phenylalanine in the fusion activity of the protein, this residue was changed to leucine (F117L) or to glycine (F117G) by site-specific mutagenesis while maintaining the cleavage site sequence of virulent strains of NDV. Whereas both the wild-type and the F117G proteins were proteolytically cleaved and F1 was detected, the leucine subsitution abolished cleavage. When co-expressed with the HN protein, the fusion protein with either a phenylalanine and glycine residue at position 117, but not a leucine, was shown to stimulate membrane fusion. However, incubation in trypsin activated the fusion activity of the F117L protein. Thus the presence of a leucine at position 117 of the precursor sequence blocks cleavage, but not fusion acitivity, and indicated that the phenylalanine at the amino terminus of the F1 subunit is not conserved for the fusion activity of the protein.