H Post Inoculation Research Articles

Anti-TNF-α therapy does not ameliorate disease in a model of acute virus-endotoxin mediated respiratory disease in pigs

Tumour necrosis factor-α (TNF-α) has been shown to play a role in many inflammatory conditions. Currently anti-TNF-α drugs (e.g. etanercept) are used in humans for treatment of autoimmune diseases. In this study we aimed to elucidate the role of TNF-α in the development of virus-endotoxin-induced respiratory disease. Twenty-two caesarean derived colostrum deprived pigs were used. Initially, the availability in the lungs and circulation, and possible clinical and inflammatory effects of etanercept alone were assessed in 4 pigs after intratracheal and intraperitoneal administration of 0.5 mg/per route/per pig. High anti-TNF-α activity was detected in bronchoalveolar lavage (BAL) fluids, peritoneal lavage fluids and serum of all animals for at least 8 h post-inoculation (HPI). No clinical symptoms, lung lesions, lung cell infiltration or induction of IFN-α, IL-1, IL-6, IL-12 and TNF-α in BAL were detected. Subsequently, the ability of etanercept to block porcine TNF-α and its effect on the above mentioned parameters and on lung virus titres were assessed in 8 pigs. They were inoculated intratracheally with porcine respiratory coronavirus (PRCV) followed by lipopolysaccharide (LPS) 24 h later. Etanercept was administered at the time of LPS inoculation via the same routes and dose as in the initial experiment. The parameters were compared with a control group ( n = 8), receiving only PRCV-LPS. Half of the animals from each group were euthanized at 4 and the rest at 8 h after LPS inoculation. TNF-α was completely neutralized in 3 of the 4 animals euthanized at 4 HPI and significantly lower than in the PRCV-LPS group at all times. No significant differences in disease severity, lung lesions, virus replication, lung cell infiltration or levels of IFN-α, IL-1, IL-6 and IL-12/IL-23 were observed between the two groups. Blocking of TNF-α alone was not sufficient to ameliorate disease in the PRCV-LPS model of respiratory disease, possibly due to the redundancy in the proinflammatory cytokine cascade, or the involvement of other unidentified disease mediators.

A Novel Gene Involved in Lesion Formation in Magnaporthe grisea

AbstractTo date, a number of genes that are expressed in the early stages of infection have been reported, while few genes that are expressed during the course of colonization, after the initiation of plant infection, have been studied. Plant inoculations, real‐time polymerase chain reaction (PCR), gene cloning, protein expression and bioinformatics analysis were combined to identify a novel gene, MgNIP04, in the rice blast fungus, Magnaporthe grisea. Bioinformatics analysis showed that the amino acid sequence contained a signal peptide. The wounded inoculation resulted in necrosis specks when the total expressed proteins including MBP‐MgNIP04 was inoculated on the wounded rice leaves, which demonstrated the protein directly interacted with rice. The real‐time PCR result of infected leaves showed that it is upregulated during late stages of infection of rice. Additionally, the copies of the gene expression absolute quantity of the gene were 7.61 × 102, 1.06 × 103, 1.31 × 103, 4.13 × 103, and 4.00 × 103, at 24, 48, 72, 96 and 168 h postinoculation (HPI), respectively. The higher copies of MgNIP04 were found at 96 and 168 HPI. The copies of MgNIP04 in mycelia of Y98‐63C, Y99‐16, 94‐64‐1b and 95‐23‐4a were significantly lower than those of infected leaves. Through the above bioinformatics and experimental analysis, our result suggested that the MgNIP04 was novel pathogenicity‐related protein in rice blast fungus.

Role of two cysteine proteinases in the susceptible response of Nicotiana benthamiana to Colletotrichum destructivum and the hypersensitive response to Pseudomonas syringae pv. tomato

Two cysteine proteinase genes of the papain family, NbCYP1 and NbCYP2, were amplified from cDNA of Nicotiana benthamiana leaves infected with the hemibiotrophic fungus Colletotrichum destructivum. Both genes showed peak expression corresponding with the switch from biotrophic to necrotrophic growth by C. destructivum at 72 h post-inoculation (HPI). For N. benthamiana inoculated with the incompatible bacterium, Pseudomonas syringae pv. tomato, expression of NbCYP1 significantly decreased at 12 HPI, whereas NbCYP2 expression increased at 3 HPI. Expression of both genes then returned to near pre-inoculation levels and remained constant as necrosis later appeared due to a non-host hypersensitive response (HR). Virus-induced gene silencing of NbCYP1 and NbCYP2 resulted in increased susceptibility of N. benthamiana to C. destructivum but did not affect the HR necrosis or population levels of P. syringae pv. tomato. These two cysteine proteinase genes do not appear to be involved in the programmed cell death of the HR resistance to P. syringae pv. tomato, but they are involved in limiting the host's susceptibility to C. destructivum.

Reaction of Rice (Oryza sativa) Cultivars to Penetration and Infection by Curvularia tuberculata and C. oryzae.

Isolates of Curvularia species were collected from weedy Cyperaceae species and are being evaluated as possible biocontrol agents of sedge weeds in rice (Oryza sativa). Curvularia species have been reported from rice; thus cultivars of rice were tested to determine rice seedling responses to these potential biocontrol agents. All 13 rice cultivars were resistant to Curvularia tuberculata isolate 93-022, 12 were resistant to C. tuberculata isolate 93-020, and 7 were resistant to C. oryzae isolate 93-061. In the resistant cultivars, lesions on the leaf laminae were small, light to dark brown, with a dry appearance. Spots on the leaf margins and leaf tips were light brown to cream and dry. In the susceptible cultivars, the brown lesions coalesced with necrotic centers. Sporulation was observed in the lesions on susceptible cultivars but not on the resistant cultivars. The histopathology of C. tuberculata and C. oryzae was studied in two resistant rice cultivars, IR 64 (IRRI Acc. no. 66970) and Norin 21 (IRRI Acc. no. 7686), by light microscopy. C. tuberculata exhibited polar germination beginning at 4 h postinoculation (HPI); whereas C. oryzae was characterized by bipolar germination starting at 2 HPI. Simple terminal or intercalary appressoria were initiated at 24 HPI over stomatal apertures, or rarely, on the epidermal cell walls and bulliform cells. No infection cushions were formed. Penetration occurred by the formation of a fine penetration peg beneath the appressorium. A chlorotic reaction was observed in areas beneath and adjacent to the appressoria and germ tubes and in the infected cells. Resistance of IR 64 and Norin 21 to C. tuberculata and C. oryzae infection was mainly expressed after penetration as a slow and restricted mycelial growth and no sporulation. C. tuberculata isolate 93-022 is the preferred isolate for further study as a biological control agent against Cyperus difformis, C. iria, and Fimbristylis miliacea.

Variability of phenylalanine ammonia-lyase and peroxidase activities in leaves of subterranean clover is determined by their susceptibility to Kabatiella caulivora

Kabatiella caulivora is the causal organism of northern anthracnose or clover (Trifolium spp.) scorch disease. The activities of phenylalanine ammonia-lyase (PAL) and soluble peroxidase were determined in seedling leaves of two cultivars of subterranean clover (T. subterraneum) inoculated with race 1 or race 2 of K. caulivora. A small increase in activity of PAL was recorded in both cultivars 2–4 h post inoculation with either race. A second, large increase in PAL activity was observed only in the incompatible interaction (cv. Daliak inoculated with race 1), increasing 18-fold between 8 and 48 h post inoculation. Peroxidase activity in cv. Daliak increased rapidly within 2 h post inoculation with either race but was significantly higher in the incompatible interaction. Peroxidase activity in cv. Woogenellup increased by 4 h post inoculation with either race, but was significantly lower than that in cv. Daliak. Subsequent increases in peroxidase activity were recorded in both cultivars, however the levels remained constant in cv. Daliak infected with race 1, while activities in the other race-cultivar combinations decreased to control levels. It is hypothesised that the peak of activity of PAL at 48 h, and the rapid increase in peroxidase at 2 h are related to the race-specific resistance response of cv. Daliak to race 1 of K. caulivora, and that minor peaks of activity in the compatible interactions are general defence responses.

Early Development of Eimeria Papillata (Apicomplexa: Eimeriidae) In the Mouse

Early development of Eimeria papillata (Apicomplexa) in the mouse was evaluated using Nomarski interference-contrast and brightfield microscopy. Sporozoite-shaped meronts, which were motile and contained a large posterior refractile body and a smaller anterior refractile body, were observed entering and leaving host cells in the jejunum of an experimentally infected mouse at 26 h post inoculation (HPI). However, early developmental stages were not observed in tissue of the duodenum, ileum, cecum and colon. The mean length and width of these meronts (n = 20) were 12.0 microns and 3.7 microns, respectively. Spherical or subspherical meronts containing crescent-shaped merozoites were observed at 36 HPI.

Life cycle of Isospora rivolta (Grassi, 1879) in cats and mice.

The endogenous development of Isospora rivolta (Grassi) was studied in cats fed oocysts, and was compared with the endogenous cycle after feeding them mice infected with I. rivolta. For the mouse-induced cycle, 14 newborn cats were killed 12 to 240 h after having been fed mesenteric lymph nodes and spleens ofmice. Asexual and sexual development occurred throughout the small intestine, in epithelial cells of the villi and glands of Lieberkühn. The number of asexual generations was not determined with certainty, but there were at least 3 structurally different meronts. Type I meronts appeared at 12-48 h postinoculation (HPT). They were 8.5(6-13) x 5.1(3-6) micrometer, contained 2-8 merozoites, and divide by binary division or endodyogeny. Type II meronts were multinucleate merozoite-shaped meronts within a single parasitophorous vacuole. They were found at 48-172 HPI and measured 12.6(9-18) x 9.8(9-13) micrometer. Individual multinucleate merozoite-shaped meronts were 7-13 x 3-5 micrometer in sections and contained 2-30 slender (5.5 x 1.0 micrometer) merozoites. Type III meronts occurred at 72-192 HPI and gamonts at 72-96 HPI. Mature microgamonts measured 11.3(9-15) x 8.0(6-9) micrometer in sections and up to 21.5 x 14 micrometer in smears, and contained up to 70 microgametes. Macrogamonts measured 13.3(11-18) x 9.0(5-13) micrometer in sections and 18 x 16 micrometer in smears, and contained up to 70 microgametes. Macrogamonts measured 13.3(11-18) x 9.0(5-13) micrometer. Sporulation was completed within 24 h at 22-26 C. For the study of the oocyst-induced cycle in cats, 18 newborn cats were killed between 6 and 192 HPI. The endogenous development was essentially similar to the mouse-induced cycle, but merogony and gametogony occurred 12-48 h later than in the latter cycle. Isospora rivolta was pathogenic for newborn but not for weaned cats. Newborn cats fed 10(6) sporocysts or infected mice usually developed diarrhea 3-4 days after inoculation. Microscopically, desquamation of the tips of the villi and cryptitis were seen in the ilium and cecum in association with meronts and gamonts. For the study of the development of I. rivolta in mice, mice were killed from day 1 to 23 months after having been fed 10(5)-10(6) sporocysts, and their tissues were examined for the parasites microscopically, and by feeding to cats. The following conclusions were drawn. (A) Isospora rivolta most freqeuntly invaded the mesenteric lymph nodes ofmice and remained there for 23 months at least. Ii also invaded the spleen, liver, and skeletal muscles of mice. This species could not be passed from mouse to mouse. Sporozoites increased in size from approximately 6.8 x 4.9 micrometer on day 1 to approximately 13.4 x 6.9 micrometer on day 31 postinoculation. Division was not seen. Prepatent period was 4-7 days and patent periods ranged from 2 to several weeks.

Pathogenesis of Infectious Pancreatic Necrosis in Atlantic Salmon (Salmo salar)

Atlantic salmon (Salmo salar) fry and yearling were found to undergo an active but subclinical infection following exposure to infectious pancreatic necrosis (IPN) virus. Eight-week-old Atlantic salmon fry were fed live virus and the mortality and virus concentration in these fry were recorded over a period of 78 d following exposure. Yearling Atlantic salmon were inoculated intraperitoneally with IPN virus and the concentration of virus in the pancreas–intestine, kidneys, spleen, liver, and gonads of inoculated fish was determined over a period of 74 d following inoculation. Virus first appeared in the fry 3 d postinoculation (DPI) and virus titers reached a peak by 8 DPI. Low levels of virus persisted in fry sampled 78 DPI. Virus first appeared in the pancreas–intestine of Atlantic salmon 12 h postinoculation (HPI) and was detected in the kidneys, liver, and spleen by 72 HPI. Peak virus titers were reached in the pancreas–intestine by 72 HPI. Both the pancreas–intestine and kidneys continued to support viral growth frequently and appeared to be important reservoirs of virus. At no time was virus isolated from the gonads of inoculated yearling. Histological examination of yearlings revealed degenerative changes in the pancreatic acinar tissue and in some cases focal necrosis in the liver. No histological abnormalities were found in IPN-infected fry. Key words: infectious pancreatic necrosis, Atlantic salmon, virus, histology