Studies on the infection of sandflies and biting midges with arboviruses

The research for this thesis was carried out in the Entomology department of the Animal Virus Research Institute, Pirbright. Membrane feeding and intrathoracic inoculation techniques were developed and used successfully for the first time to infect sandflies with viruses. These infection techniques were used to investigate the susceptibility of a laboratory colony of the sandfly Lutzomyia longipalpis to infection with three viruses of the Phlebotomus fever group, Sicilian and Naples sandfly fever, and Pacui. The suitability of the colony for use as a laboratory model for studies on the development of such viruses in an insect host was then assessed. Sicilian and Naples sandfly fever viruses are Old World viruses transmitted in nature by the Old World sandfly Phlebotomus papatasi. Pacui virus is a New World virus isolated from the New World sandfly L.flaviscutellata. A limited number of P.papatasi were obtained and both infection techniques were applied equally successfully to this species. The development of the New and Old World viruses in New and Old World sandflies was then compared. The susceptibility of two Culicoides species to the Phlebotomus fever group viruses was tested as part of a general programme of research into the specificity of arboviruses with respect to the insect host. Several Culicoides species are important as vectors of the orbivirus Bluetongue. The vector potential of sandflies for this virus was assessed and the development of Bluetongue virus in Culicoides and sandflies was compared. The distribution of virus in infected insects on successive days after infection was determined by infectivity titrations of dissected heads, thoraces and abdomens. A fluorescent antibody test was developed and used to detect viral antigens in tissue smears from infected Culicoides and sandflies. The possibility of using such a test to detect viral antigens in paraffin wax sections of infected insect was investigated

Immunopathological studies in the ovine lung during the course of natural and experimental parainfluenza type 3 virus infection

I lie pulmonary imnuinopalliology of parainfluenza type 3 (I'l V-3) infection in sheep was investigated firstly by isolating ttic virus from field cases of sliccp pneumonia, secondly by experimentally reproducing the disease with the isolated vims and finally try studying changes in lymphocytes subsets and alveolar macrophages, induced by HI V-3 in vivo and in vitro.Three ovine virus isolates (270-7, 390-10 and 430-7) were obtained and characterised, as l'IV-3. according to virus morphology, transmission electron microscopy (I HM); cylopalhic cll'ccl (CI'H); hacmagglutination, of guinea pig erythrocytes; physicochcmical properties; serological crossrcaclivity with anliscra raised against l'IV-3; and reactivity with monoclonal antibodies to l'IV-3 structural proteins, that crossrcact with human and bovine strains.flic ability of the virus to induce respiratory disease was investigated by experimental inoculation of ovine l'IV-3 isolate, 270-7 in colostrum deprived lambs Clinical, pathological, bacteriological and virological studies were carried out on days 2. 3, 3 and 7 post infection (p i ), l itis I'l V-3 ovine strain was able to induce clinical disease, llistopathological findings were interstitial pneumonia with hyperplasia of bronchiolar associated lymphoid tissue (HALT), degenerative bronchiolar epithelium with lymphocyte inlillration, areas of atelectasis and increased alveolar septa thickness due to proliferation of type II pncmnocytcs, lymphocytes, macrophages and later to Itbrosis. lire large number of lymphocytes, particularly on days 5 and 7 pi, combined with the minimal to moderate cytolvsis of antigen bearing cells suggests that l'IV-3-induced pulmonary disease has an inuuunopathological component. l'IV-3 particles were detected more frequently in bronchiolar epithelium cells than in alveolar septal cell This was correlated with changes of these cell populations in lung lesions, delected by iminunohistochemislry.Alter 7 days p i . virus-induced changes in the leukocyte composition of the lungs were detected using a panel of rnAbs (o ovine lymphocytes and macrophages. Changes in lung tissue were detected by inununohistology and changes in lungwash llnid (LWH) bv llowcylomctry. Association lieI ween lung cells and virus particles was investigated by double immunoslaining. The differential cell count of LWIf from l'IV-3 infected animals was characterised by a significant increase (pet).03) in lymphocytes and neutrophils. Lymphocy te phcnolyping showed a significant decrease (pet).05) of C'1711 cells, a significant increase (p<0.05) oT CDK1 cells and a significant inversion (pcO.OO I) or the (171 '/COS1 ratio. Immunoslaining of I'l V-3 infected lung sections showed a remarkable increase of lymphocy tes, particularly in HALT, and most cells were CD81. flic number of macrophages increased in peribronchial and alveolar septa and some were positive lor I'l V-3 particles.Cultures of peripheral blood monocylc-dcrivcd macrophages (MUMf) and alveolar macrophages (AMf) were established, file ability of l'IV-3 lo inlccl these cells was studied by CI'H, and virus particle immtmoslaining. Viral replication was detected by TliM and scanning electron microscopy (SBM). l'IV-3 induced lytic CI'H'w ith inlracyloplastnic eosinophilic inclusion Ixidics and syncy tia formation. I'HM revealed virus budding al the cell membrane, lilamcntous cytoplasmic inclusions and clusters of pleomorphic viral particles in the extracellular space.es in the extracellular space. flic expression of MIIC class I and MIIC class II molecules, which is associated willt antigen presenting function, was studied alter in vitro inlccl ion of MUM and AM with I'l V-3. UK and IX) Ml It.' class II expression was moderately high (60-80%) on noninl'cclcd fresh monocytes and AM but, allcr 3 days in culture the expression of this molecules was dramatically reduced lo 5%. Stimulation with y-llrN was able to promptly restore MIIC class II expression in cultured noninfcclcd MUM arid AM. This did not occur alter I'l V3 infection. The expression of MIIC class I molecules was not significantly all'cctcd (p>0,05) by culture or I 'I V-3 infection.flic phagocytic activity of macrophages lor ITfC-lnbcllcd/nnlibody-coalcd sheep red blood cclls(SRUC) alter I'l V-3 infection decreased significantly (p< ().()3) alter .3 days p i

Studies on the role of parainfluenzavirus type 3 and adenovirus in respiratory disease of sheep

Investigations were undertaken to isolate viruses from sheep, and to assess their role in respiratory diseases by means of epidemiological observations and experimental studies of their pathogenesis.Viruses were isolated from 0*7 per cent, of samples taken at necropsies and from 16 per cent, of sheep in 3 of 4 flocks currently experiencing outbreaks of respiratory disease, but not from 7 flocks with few or no signs of clinical illness.An adenovirus (strain 7769) was isolated from rectal swabs, but not nasal swabs, from 3 of 15 lambs during an outbreak of pneumonia in a group of 4 to 10-week-old lambs. This adenovirus differed antigenically from other ovine, bovine and human adenoviruses, and the species of erythro¬ cytes that were agglutinated by strain 7769 differed from those agglutinated by porcine, canine, equine and murine adenoviruses. It was concluded that strain 7769 was a previously unreported type of adenovirus and was designated ovine adenovirus type 4 (0A4).Although antibodies to the adenovirus group specific antigen were detected in only 6 per cent, of 661 sheep sera, neutralizing antibodies to 4 serotypes of ovine adenovirus were more common. Neutralizing antibodies to ovine adeno¬ virus types 1-4 were more prevalent in animals over 12 months of age.Following exposure of specific pathogen-free (SPF) - 2 - lambs to an aerosol of 0A4 virus, replication of this virus occurred in the respiratory and alimentary tracts and liver, and neutralizing antibodies could be detected in the serum and nasal secretions as early as 8 days after inoculation. Infection was associated with a mild clinical illness, detectable by auscultation only, and accompanied by lesions in the lungs and liver. The lesions found in the lungs of infected lambs were pulmonary oedema and peribronchiolar accumulations of mononuclear cells and in the livers were focal necrosis, lymphangitis and occlusive cholangitis.The clinical disease and pneumonic lesions observed in SPF lambs infected with both ovine adenovirus type 4 and Pasteurella haemolytica were no more severe than those in lambs infected with P.haemolytica alone.Enzootic pneumonia was induced consistently in SPF lambs inoculated with parainfluenza virus type 3 (PI3) followed by P.haemolytica 4 or 7 days later. Seventyeight per cent, of lambs developed severe respiratory disease by this method, 54 per cent, died and 95 per cent, had macroscopic lung lesions. The illness and lesions were more marked in lambs inoculated with both PI3 virus and P.haemolytica than in lambs inoculated with either agent alone and were associated with rapid multiplication of P.haemolytica within the lung

Einsatz eines gegen die leichte Kette von Pekingenten-Immunglobulin gerichteten monoklonalen Antikörpers für die Etablierung eines Newcastle Disease-ELISA zur Überprüfung der humoralen Impfreaktion beim Wassergeflügel

Enzymverstärkte Testsysteme wie der ELISA finden in der serologischen Diagnostik bei Hühnervögeln bereits breite Anwendung. Beim Wassergeflügel gilt dagegen immer noch der Hämagglutinationshemmungstest (HAH) als Test der ersten Wahl. Für große Probenmengen ist er jedoch ungeeignet und insbesondere bei Seren mangelhafter Qualität mit dem Problem unspezifischer Reaktionen behaftet. Daraus ergibt sich die Notwendigkeit der Etablierung alternativer serologischer Testverfahren auch für das Wassergeflügel. Basierend auf einem monoklonalen, gegen die leichte Kette von Pekingenten-Immunglobulin (Ig) gerichteten Antikörper (mAk 14A3) (Kothlow et al., 2005) wurden im Rahmen dieser Arbeit indirekte Testsysteme für das Wassergeflügel etabliert. Die Kreuzreaktivität des mAk 14A3 mit verschiedenen Entenspezies (Flugente = Cairina moschata, Stockente = Anas platyrhynchos, Weißflügelente = Asarcornis scutulatus, Spießente = Dafila acuta) sowie zwei Schwanenspezies (Höckerschwan = Cygnus olor, Schwarzhalsschwan = Sthenelides melanocoryphus) und zwei Gänsespezies (Hausgans = Anser anser var. domestica, Rothalsgans = Rufibrenta ruficollis) wurde mittels Western Blot (WB) überprüft. Der Antikörper wies Reaktivität gegenüber der leichten Kette des Serum-Ig aller getesteten Wassergeflügelspezies auf. Mit Hilfe seropositiver Seren von gegen aviäre Paramyxoviren des Serotyps 1 (APMV-1) geimpften Hausgänsen und Moschusenten konnte gezeigt werden, dass der Antispezies-Antikörper fähig ist die spezifische Färbung APMV-1-infizierter Zellen im Immunfluoreszenztest zu vermitteln. Darüber hinaus bewährte er sich auch im indirekten ELISA und im WB (Kothlow et al., 2008). Mit dem etablierten indirekten Newcastle Disease (ND)-ELISA ließ sich in Seren adulter Moschusenten und Hausgänse drei Wochen nach Vakzination mit einem inaktivierten ND-Impfstoff für Hühner eine Serokonversion nachweisen, die bis zum Versuchsende (10 Wochen p.v.) anhielt. Zwischen den ELISA-Werten und den parallel ermittelten HAH-Titern war eine positive lineare Korrelation feststellbar (Pearson’s product moment correlation; r = 0.652; P < 0.001). In Woche 7 und 10 nach der Impfung erkannte der ELISA jedoch zehn Seren geimpfter Tiere mehr positiv als der HAH, wobei für acht dieser Seren mittels WB eine spezifisch gegen aviäre Paramyxoviren gerichtete Reaktivität bestätigt werden konnte. Die sich aus dem Vergleich der Tests ergebende relative diagnostische Sensitivität (rDSe) des ELISA (100.0%) war höher als die des HAH (91.1%), seine relative diagnostische Spezifität (rDSp) etwas niedriger (ELISA: 91.7%; HAH: 97.2%). Die zweimalige Testung aller Seren unter den gleichen Versuchsbedingungen ergab eine sehr gute Wiederholbarkeit des etablierten ELISA mit einer positiven linearen Korrelation der Ergebnisse (Pearson’s product-moment correlation; r = 0.982; P < 0.001; ls = 0.05) (Häuslaigner et al., 2009). Der untersuchte monoklonale Antispezies-Antikörper stellt somit ein attraktives und vielseitig einsetzbares Reagenz dar, das die Möglichkeit eröffnet, serologische Testverfahren für das Wassergeflügel, zum Einsatz in Diagnostik und Forschung zu entwickeln. Der etablierte indirekte ELISA erwies sich als gut geeignet, die Immunantwort beim Wassergeflügel nach ND-Impfung zu detektieren. Die gute Reproduzierbarkeit seiner Ergebnisse spricht für eine hohe Aussagesicherheit. Basierend auf der sich aus dem Vakzinationsstatus ergebenden diagnostischen Sensitivität (DSe) (92.2%) und Spezifität (DSp) (96.2%), kann der etablierte ND-ELISA als geeignete Alternativ- und Ergänzungsmethode zum HAH (DSe 83.0%, DSp 100.0%) angesehen werden. Vor allem zu späteren Zeitpunkten nach Viruskontakt scheint er mehr seropositive Reagenten als der HAH zu erkennen, wohingegen sich letzterer in der DSp überlegen zeigte. Die Ergebnisse der serologischen Untersuchungen unterstreichen zudem die Immunogenität des inaktivierten ND-Impfstoffes bei domestizierten Hausgänsen und Moschusenten, wobei die doppelte Hühnerdosis eine effizientere Antikörper-Antwort zu induzieren scheint