Response of efferent lymph and popliteal lymph node to epidermal infection of sheep with orf virus

Functional and phenotypic changes in the cell populations were monitored in the popliteal efferent lymph of sheep following experimental epidermal infection with orf virus. In another group of sheep, cells from the popliteal lymph node draining the site of infection were similarly monitored and compared with the cells from contralateral popliteal and mesenteric lymph nodes. All sheep showed serological evidence of previous exposure to orf virus. Following infection, anti-orf antibody titres rose and efferent lymphocyte and blast cell output increased. Interferon-like activity was detected in efferent lymph early after orf virus but not mock infection. Lymphocytes from the draining popliteal lymph node showed antigen-specific lymphoproliferation on Days 3-7 while cells in the efferent lymph demonstrated proliferative activity on Days 4-6. The requirement for exogenous antigen-presenting cells in the culture of efferent lymphocytes varied between individual sheep. The culture supernatant from proliferating lymph node cells contained interferon-like activity but no anti-orf antibodies, the reverse of that from cultured efferent lymphocytes, perhaps indicating a different reactive T cell population. During the course of the experiment there was an increase in the percentage of efferent lymphocytes expressing MHC Class II antigens and surface immunoglobulins, the latter being recorded as a double peak. The short-term nature of the local T cell response may in part explain the incompleteness of immunity to orf virus in sheep.     

The cytokine response of afferent lymph following orf virus reinfection of sheep

Abstract The in vivo dynamics of differentiated cells and interleukin (IL)-lβ, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon (IFN)-γ titres in afferent lymph were compared following orf virus reinfection and inactivated virus injection of previously infected sheep. The biphasic lymphoblast and cytokine response in the lymph to virus reinfection is consistent with a response initially to orf virus as recall antigen followed by a response to viral replication. CD4 T cells increased in output over other cell types in the lymph in both groups. A rapid immune/inflammatory response was detected in lymph plasma as an increase in cytokine titres within 24 h of virus reinfection or injection. Lymph cells producing IL-1β and IL-8 appeared prior to those producing GM-CSF in both groups. In spite of variations in the concentration of individual cytokines in lymph following reinfection, both the size of the orf lesion and the time to resolve were similar in all cases. Résumé— Les dynamiques in vivo des cellules différenciées et des taux d'IL-1β, de GM-CSF et d'interferon γ dans la lymphe afférente furent comparés chez des moutons antéricurement infectés après une réinfection par le virus de l'ecthyma contagieux et après injection d'un virus inactivé. La réponse biphasique lymphoblastique et des cytokines à la réinfection virale est compatibles avec une réponse primaire au virus de l'ecthyma contagieux comme antigène mémoire suivie par une réponse secondaire à la réplication virale. Dans les 2 groupes, le nombre de TCD4 est plus élevé que les autres populations cellulaires mises en évidence dans la lymphe. Une réponse de type immune/inflammatoire est révélée dans le plasma par une élévation des taux de cytokines dans les 24 heures qui suivent la réinfection virale ou l'injection de virus inactivé. Les lymphocytes producteurs dTL-1β et d'IL-8 apparaissent avant les lymphocytes producteurs de GM-CSF dans les deux groupes. En dépit des variations de concentration des cytokines individuelles dans le lymphe après reinfection, à la fois la taille des lesions d'ecthyma et les délais de guérison sont identiques dans tous les cas. [Haig, D., Deane, D., Percival, A., Myatt, N., Thomson, J., Inglis, L., Rothel, J., Heng-Fong Seow, Wood, P., Miller, H. R. P., Reid, H. W. The cytokine response of afferent lymph following orf virus reinfection of sheep (Effet sur les cytokines de la lymphe afferente d'une reinfection par les virus de l'ecthyma contagieux chez le mouton.) Veterinary Dermatology 1996; 7 : 11–20.] Resumen Se comparó la actividad de células diferenciadas y los titulos de interleuquina (IL)-1β, IL-8, factor de estimulación de colonias de granulocitos y macrófagos (GM-CSF) e interferon (IFN)-y entre reinfecciones por el virus del ectima contagioso e inyección de virus inactivado de ovinos previamente infectados. La respuesta a la reinfección en forma bifásica linfoblástica y de citoquinas en la linfa está de acuerdo con una respuesta inicial al virus del ectima por estimulación antigénica, seguida por una respuesta a la replicación viral. Las células T CD4 aumentaron respecto a otros tipos celulares en la linfa de ambos grupos. Se detectó una respuesta inmune/inflamatoria rápida en el linfa-plasma en forma de aumento de los titulos de citoquinas dentro de las 24 h de reinfección o inyección del virus. Las células linfáticas productoras de IL-1β e IL-8 aparecicron antes que las productoras de GM-CSF en ambos grupos. A pesar de las variaciones en la concentración de citoquinas individuates en la linfa después de la reinfección, tanto el tamaño de la lesión por el virus del ectima contagioso como el tiempo de resolución fueron similares en todos los casos. [Haig, D., Deane, D., Percival, A., Myatt, N., Thomson, J., Inglis, L., Rothel, J., Heng-Fong Seow, Wood, P., Miller, H. R. P., Reid, H. W. The cytokine response of afferent lymph following orf virus reinfection of sheep (Produccion de citoquinas en el ganglio linfatico afferente tras la reinfección por el virus del ectima contagioso.) Veterinary Dermatology 1996; 7 : 11–20.] Zusammenfassung— Es wurde die in-vivo-Dynamik der Titer differenzierter Zellen und Interleukin (1L)-1β, IL-8, Granulozytenmakrophagenkolonien-stimulierender Faktor (GM-CSF) und Interferon (IFN)-γ in afferenter Lymphe nach einer Reinfektion mit ORF-Virus und einer Injektion inaktivierten Virusmaterials von früher infizierten Schafen verglichen. Die biphasische Lymphoblasten- und Zytokin-Reaktion in der Lymphe auf die Virusreinfektion stimmte mit einer initialen Reaktion gegenüber ORF-Virus überein, da der Wiederabruf von Antigen von einer Reaktion auf die Virusreplikation gefolgt wird. CD4-T-Zellen vermehrten sich im Output stärker als andere Zelltypen in der Lymphe beider Gruppen. Es wurde eine rasche immunologische/entzündliche Reaktion im Lymphplasma in Form eines Anstieges von Zytokin-Titern innerhalb von 24 Stunden nach Virusreinfektion oder -injektion festgestellt. Lymphatische Zellen, die IL-1β und IL-8 produzieren, erschienen vor dem Auftreten von solchen, die GM-CSF produzieren, in beiden Gruppen. Trotz des Schwankens der Konzentration der individuellen Zytokine in der Lymphe nach Reinfektion, waren sowohl die Größe der ORF-Veränderungen und die Zeit der Heilung ähnlich in alien Fällen. [Haig, D., Deane, D., Percival, A., Myatt, N., Thomson, J., Inglis, L., Rothel, J., Heng-Fong Seow, Wood, P., Miller, H. R. P., Reid, H. W. The cytokine response of afferent lymph following orf virus reinfection of sheep (Die ZytokinReaktion afferenter Lymphe nach einer Reinfektion mit orf-virus beim schaf.) Veterinary Dermatology 1996; 7 : 11–20.]     

A qualitative and quantitative assessment of the humoral antibody response of the sheep to orf virus infection

The serological response of naturally and experimentally infected lambs to orf virus infection was analysed using an enzyme-linked immunosorbent assay (ELISA) together with the Western blotting technique. The combination of these two methods permitted a qualitative and quantitative assessment of the response, which revealed considerable variation between animals. Despite this, all post-exposure sera reacted with a polypeptide (molecular weight 40 kilodaltons) which appears to be a component of the surface tubules that are characteristic of the virus.     

Pathogenesis of Brucella abortus infection of the mammary gland and supramammary lymph node of the goat

Goats, both in late pregnancy and soon after parturition, were inoculated intravenously with Brucella abortus, and mammary glands and supramammary lymph nodes were examined by light and electron microscopy at 2 to 55 days post-inoculation. After 7 days, lymphoplasmacytic, histiocytic interstitial mastitis with a lobular and periductal distribution were detected microscopically. Brucellae, identified in tissues with immunoperoxidase staining and antibody-coated colloidal gold stain, were first seen in macrophages and neutrophils throughout mammary parenchyma, but most commonly in mammary alveoli. In subsequent samples, infected phagocytes progressively increased in number, especially in ductal and alveolar lumina, and adjacent parenchyma. B. abortus was in phagosomes and phagolysosomes in macrophages and neutrophils; degenerate and necrotic phagocytes were often filled with brucellae. Extracellular brucellae were associated with ruptured necrotic infected phagocytes. Supramammary lymph nodes draining infected mammary glands were enlarged. Lymphofollicular hyperplasia, medullary plasmacytosis, and sinus histiocytosis were seen microscopically. Brucellae were seen exclusively in macrophages, which were most often located in subcapsular and cortical sinuses. This study suggests that phagocytic leukocytes 1) transport brucellae into mammary glands; 2) provide a site for intracellular replication in mammary secretions; and 3) transport brucellae from mammary glands to supramammary lymph nodes.     

Vaccination of sheep with cell culture grown orf virus

Orf virus, derived from contagious pustular dermatitis (scabby mouth) lesions in sheep, was adapted to cell culture and subsequently evaluated as a potential vaccine for sheep. The traditional vaccine virus, prepared from the infected scabs of orf virus lesions in sheep, was used to vaccinate sheep by scratching with an applicator (mounted pins) dipped in virus. Less than 10 TCID50 (50% tissue culture infectious doses) of virus was required to produce large lesions (greater than 5 mm diameter) which developed during a period of 10 to 14 d prior to onset of healing which was complete by 28 to 30 d. A serum neutralising antibody response was also detected and protection against challenge by application of virulent virus to abraded skin was demonstrated in that challenge lesions developed and healed more quickly (14 d against 30 d). However, cell culture-adapted virus required more than 10(5) TCID50 to induce even small lesions (less than 2 mm diameter). An antibody response could not be detected and no evidence of protection against challenge with virulent virus was demonstrated. In contrast, a recent field isolate has yielded a cell culture-adapted virus preparation that readily infects sheep, produces large lesions, detectable antibody and protects against challenge. This isolate is distinct from the traditional vaccine strain on the basis of restriction enzyme analysis but provides cross-protection in sheep inmmunisation and challenge studies. These results demonstrate that a cell culture produced scabby mouth vaccine is feasible.     

Drainage of lymph from the foreleg to the superficial cervical lymph node in sheep

The superficial cervical (prescapular) node in sheep is large and readily accessible. It lies medial to the omotransverse muscle and 40 to 60 mm cranial to the ventral end of the scapular spine. Lymph vessels reaching this node from the foreleg all enter its ventral half. These lymphatics can be approached surgically opposite the elbow joint where they lie adjacent to the cephalic vein. Carbon particles infused into a single afferent lymphatic were restricted to a small segment of the ventrocranial quadrant of the node. When Evans' Blue dye, or a suspension of carbon particles, was injected subcutaneously just above the hoof they were carried to the node in two to five afferent lymphatics and were found mainly in the ventrocranial quadrant. A knowledge of the anatomy of the superficial cervical node and its afferent lymphatics may be of value in immunological studies, especially where it is desired to introduce antigen into a known part of a node and to leave the remainder for control observations.     

Antibody response of the ovine lymph node to experimental infection with an ovine abortion strain of Chlamydia psittaci

After primary infection with Chlamydia psittaci in the draining area of the popliteal lymph node, viable organisms could be isolated from the efferent lymph only before the primary immune response developed. The lymph antibody response, as assayed by the complement fixation and immunofluorescence (IF) antibody tests, showed a rise in titre that peaked approximately 2 weeks after infection. Immunoblotting revealed that antibodies produced during this period were predominantly directed against the major outer membrane protein (MOMP). In secondary infection of convalescent sheep, an elevated IF antibody titre, already present in the lymph and blood, could not be boosted. Viable organisms could not be isolated from these sheep. Antibodies produced reacted to 12-14 bands in the immunoblot profile including the MOMP band. These potentially immunoprotective antigens, particularly MOMP, should be considered as useful candidates for an improved vaccine against ovine enzootic abortion, in further investigations.     

Cellular profiles in the abomasal mucosa and lymph node during primary infection with Haemonchus contortus in sheep

Cellular changes in the abomasal tissue and draining abomasal lymph nodes were examined after primary infection of lambs with Haemonchus contortus for 3, 5 or 27-36 days.Infection with H. contortus larvae resulted in a rapid and selective increase in the percentage of CD4(+) T-cells in the abomasal lymph node at 3 days post-infection (PI). By 5 days PI, the lymph node weight had increased two-fold; however, the percentage of lymphocyte populations in the abomasal lymph node resembled that seen in uninfected sheep. Lymph node weights remained at increased levels in the adult nematode infected sheep and down-regulation of B-cell surface markers (sIg and MHC Class II) was apparent in this group. Significant increases in the percentage of CD4(+) T-cells co-expressing MHC Class II, but not CD25, were observed in the larval infected groups except in adult nematode infected sheep. Increased numbers of eosinophils, CD4(+), gamma delta(+) T-cells and B-cells were found in the abomasal tissue by 5 days PI, but no further increases in these cell populations were observed in the adult nematode infected group. In contrast, the level of both lamina propria and intraepithelial mast cells observed in the abomasal mucosa was highest in the sheep carrying an adult nematode burden. These findings indicate that sheep are able to generate an early immune response to infection with H. contortus larvae, characterised by the activation of CD4 T-cells and B-cells in the draining lymph nodes and recruitment of eosinophils, CD4(+) and gamma delta-TCR,WC1(+) T-cells and B-cells in larval infected tissues. However, these changes do not seem to be maintained during infection with the adult parasite where increases in mast cell numbers dominate the local response, indicating that different parasite stages may induce distinct and possibly counteractive immune responses.     

Subcutaneous injection of thymopentin in the area of the supramammary lymph node to reduce milk somatic cell count in subclinically mastitic cows

The objective of this study was to evaluate the therapeutic effects of thymopentin (TP‐5) injections on subclinical intramammary infection (IMI) in lactating cows. In Experiment I, 40 cows were randomly divided into four groups. The cows in groups 1, 2, and 3 received subcutaneous injections of TP‐5 in the region of the supramammary lymph node at doses of 1, 2, and 4 mg, respectively, for 3 days. In Experiment II, 20 cows were randomly divided into two groups. The cows in group 1 were treated with injections of TP‐5 (4 mg) for 3 days in the same area as in Experiment I. Group 4 in Experiment I and group 2 in Experiment II were not treated and served as control groups. Milk samples were collected before and after treatment for bacteriological examination and analysis of the somatic cell count and level of N‐acetyl‐β‐d ‐glucosaminidase (NAGase). The results showed that treatment with TP‐5 significantly reduced the somatic cell count (SCC) and NAGase activity of the milk and numerically reduced IMI. A dose of 4 mg was found to be optimal for the reduction of SCC and NAGase in milk. Therefore, further study of the use of TP‐5 in the treatment of bovine mastitis is warranted.     

Differences in the lymphoproliferative response of cattle and sheep to bovine leucosis virus infection

Lymphoblastic leukaemia, preceded by a significantly increasing percentage of prolymphocytes in peripheral blood smears for from 12 to 68 weeks before death was a feature of sheep which developed lymphosarcoma following inoculation with the Australian strain of bovine leucosis virus (BLV). Lymphocytosis and/or the appearance of immature cells were a reliable predictor of tumour formation in sheep, but not in cattle. There was a terminal lymphoblastic leukaemia in only 43 of 84 cattle with lymphosarcoma. Differences in the morphological appearance and glycogen content of the leukaemic lymphoblasts of sheep and cattle were observed. In spite of these differences the high frequency of lymphocytosis and lymphosarcoma in experimentally infected sheep suggests that they could be a useful model for studying the pathological and immunological responses to BLV infection.     

Humoral immune response of sheep to infection with Eperythrozoon ovis

Circulating antibody was detected by an indirect fluorescent antibody test (IFAT) in the serum of sheep infected experimentally with Eperythrozoon ovis. Antibodies were first detected 15 to 32 days after infection with E ovis and titres peaked at 41 days. This antibody may be associated, at least in part, with protection against infection with E ovis since the initial increase in antibody titre coincided with a fall in the primary parasitaemia. A role for antibody is suggested further by the fact that the prepatent period of infection was prolonged by one day and the parasitaemia initially remained at low levels in infected sheep protected by passively transferred hyperimmune serum. Moreover, following primary infection, acquired immunity was manifest by a lack of parasitaemia following challenge infections while increased IFA titres were observed. No evidence of opsonic activity was observed in an in vitro erythrophagocytosis test in that neither mouse macrophages nor sheep monocytes phagocytosed E ovis infected or uninfected erythrocytes sensitised with hyperimmune serum.     

The response of Awassi and Merino sheep to primary infection with Haemonchus contortus

The possible existence of breed differences in the response of sheep to primary infection with Haemonchus contortus was examined by comparing worm establishment and pathogenic effects of the parasite in Awassi and Merino sheep of haemoglobin type B infected with 500 third stage H. contortus larvae per kg body weight. The results showed that the Merino sheep had lower faecal egg counts and worm burdens and suffered less severe clinical disturbances than sheep of the Awassi breed. This suggests that genetic resistance operates primarily at the level of worm establishment.     

Suppression of influenza virus infection by the orf virus isolated in Taiwan

Orf virus (ORFV), a member of parapoxvirus, is an enveloped virus with genome of double-stranded DNA. ORFV causes contagious pustular dermatitis or contagious ecthyma in sheep and goats worldwide. In general, detection of viral DNA and observing ORFV virion in tissues of afflicted animals are two methods commonly used for diagnosis of orf infection; however, isolation of the ORFV in cell culture using virus-containing tissue as inoculum is known to be difficult. In this work, the ORFV (Hoping strain) isolated in central Taiwan was successfully grown in cell culture. We further examined the biochemical characteristic of our isolate, including viral genotyping, viral mRNA and protein expression. By electron microscopy, one unique form of viral particle from ORFV infected cellular lysate was demonstrated in the negative-stained field. Moreover, immunomodulating and anti-influenza virus properties of this ORFV were investigated. ORFV stimulated human monocytes (THP-1) secreting proinflammatory cytokines IL-8 and TNF-α. And, pre-treatment of ORFV-infected cell medium prevents A549 cells from subsequent type A influenza virus (IAV) infection. Similarly, mice infected with ORFV via both intramuscular and subcutaneous routes at two days prior to IAV infection significantly decreased the replication of IAV. In summary, the results of a current study indicated our Hoping strain harbors the immune modulator property; with such a bio-adjuvanticity, we further proved that pre-exposure of ORFV protects animals from subsequent IAV infection.