The Mondrian matrix: Culicoides biting midge abundance and seasonal incidence during the 2006-2008 epidemic of bluetongue in the Netherlands.

During the northern Europe epidemic of bluetongue (BT), Onderstepoort-type blacklight traps were used to capture Culicoides Latreille (Diptera: Ceratopogonidae) biting midges weekly between November 2006 and December 2008 on 21 livestock farms in the Netherlands. Proven and potential vectors for the bluetongue virus (BTV) comprised almost 80% of the midges collected: the Obsoletus complex, constituting C. obsoletus (Meigen) and C. scoticus Downes & Kettle (44.2%), C. dewulfi Goetghebuer (16.4%), C. chiopterus (Meigen) (16.3%) and C. pulicaris (Linnaeus) (0.1%). Half of the 24 commonest species of Culicoides captured completed only one (univoltine) or two (bivoltine) generations annually, whereas multivoltine species (including all BTV vectors) cycled through five to six generations (exceeding the one to four generations calculated in earlier decades). Whether this increment signals a change in the phenology of northern Europe Culicoides or simply is an adaptive response that manifests during warmer episodes, thus heightening periodically the incursive potential of midge-borne arboviruses, remains to be clarified. Culicoides duddingstoni Kettle & Lawson, C. grisescens Edwards, C. maritimus Kieffer, C. pallidicornis Kieffer and C. riethi Kieffer are new records for the biting midge fauna of the Netherlands. It is suggested that C. punctatus (Meigen) be added to the European list of vector Culicoides.

Endophily in Culicoides associated with BTV-infected cattle in the province of Limburg, south-eastern Netherlands, 2006.

Culicoides were captured at a BTV-infected dairy near Gulpen in the province of Limburg (south-east Netherlands) between 14 September and 4 October 2006. Onderstepoort-type blacklight traps were used to sample Culicoides both inside and outside a partially open shed housing 11 cattle. A total of 28 light trap collections were made at the shed and yielded: 9371 Culicoides representing 11 species; >90% comprised five potential vectors of BTV and in order of abundance were Culicoides obsoletus and Culicoides scoticus (of the Obsoletus Complex), Culicoides dewulfi, Culicoides pulicaris and Culicoides chiopterus; Culicoides imicola, the principal Mediterranean (and African) vector of BTV, was absent. 2339 Culicoides representing seven species were captured inside (endophily) the cattle shed; >95% comprised the Obsoletus Complex and C. dewulfi. Conversely, the Pulicaris Complex, represented by five species and including C. pulicaris, showed strong exophily with >97% captured outside the shed. 7032 Culicoides were captured outside the shed, approximately threefold more than inside. This trend was reversed on an overcast day, when eightfold more Culicoides were captured inside; this indicates that when the light intensity outdoors is low Culicoides will attack (i) earlier in the day while cattle are still at pasture, and (ii) might follow cattle into the sheds in the late afternoon leading to elevated numbers of biting midges being trapped inside the shed during the subsequent hours of darkness. Culicoides were captured inside the shed on all 14 sampling nights. On occasion up to 33% were freshly blood fed indicating they had avidly attacked the cattle inside (endophagy); because half the cattle had seroconverted to BTV, and because no cattle were left outdoors at night, the data indicate that (i) the housing of animals in partially open buildings does not interrupt the transmission of BTV, and/or (ii) BTV is being transmitted while cattle are grazing outdoors during the day. The capture of partially engorged midges inside the shed shows they are being disturbed while feeding; this may lead to cattle being attacked repeatedly, and if these attacks include older parous BTV-infected Culicoides, may enhance virus dissemination (particularly in sheds where cattle stand close together). Endo- and exophagy by potential vector Culicoides--coupled to increased adult longevity and multiple feeding events in single (potentially) infected midges--would ensure an R0 of >1, resulting in the continued maintenance and spread of BTV within local vertebrate populations. Four light trap collections made additionally in a mature deciduous forest 70 m from the shed yielded a high proportion (48%) of gravid females amongst which 10% had incompletely digested blackened blood meals in their abdomens; the absence of this age category in Culicoides captured at the sheds indicates that all Culicoides, after engorgement, exit the buildings to undergo oogenesis elsewhere. In Europe, the blacklight trap is used widely for the nocturnal monitoring of Culicoides; a drawback to this approach is that this trap cannot be used to sample midges that are active during the day. Because diurnal biting in vector Culicoides may constitute a significant and underestimated component of BTV transmission a novel capture methodology will be required in future and is discussed briefly.

The development and structure of mouse nasal-associated lymphoid tissue: an immuno- and enzyme-histochemical study.

This study deals with the structure and development of Nasal-Associated Lymphoid Tissue (NALT) in the mouse. NALT is present in the nasal cavity on both sides at the entrance of the pharyngeal duct. The lymphocytes are organized in B- and T-cell areas covered by an epithelium in which M-cells are present. Immuno- and enzyme-histochemistry showed that NALT is already present at birth. Before birth, accumulations of Ia-positive dendritic cells and a few B lymphocytes were the first signs of the formation of NALT. These cell accumulations occurred just under the epithelium in the nasal floor. At birth, NALT could be distinguished as an accumulation of mainly B lymphocytes and Ia positive dendritic cells in a network of reticulum cells. The covering simple epithelium showed crypt-like invaginations. B- and T-cell areas appeared at seven days after birth. High endothelial venules (HEV) were observed from day 7 onward; from day 14 they could be stained with the HEV-specific monoclonal antibody MECA-325. Ia-positive dendritic cells increased in number during ontogeny. They occurred both in NALT and between the covering epithelial cells. Also, some epithelial cells expressed the Ia-antigen. The number of acid phosphatase positive macrophages increased steadily during ontogeny. The cells were mainly observed in the connective tissue surrounding NALT and just under the epithelium. They displayed antigenic determinants characteristic of macrophages (Moma-1, Moma-2). The structure and development of NALT are compared with those of other parts of the mucosa-associated lymphoid tissue.

Mucosal and systemic antibody formation in the rat after intranasal administration of three different antigens.

In order to study the role of nasal-associated lymphoid tissue (NALT) in the local nasal immune response, rats were immunized intra-nasally with either of the following trinitrophenylated (TNP) antigens; the thymus-dependent keyhole limpet haemocyanin (KLH), or the thymus-independent lipopolysaccharide (LPS), or with the particulate (thymus-dependent) sheep red blood cells (SRBC). Primary responses hardly occurred, while only TNP-KLH elicited a considerable secondary response. The major responding organ was the posterior cervical lymph node. Specific antibody-forming cells (AFC) occurred in the medulla and were mainly of the IgA or IgG isotype. Hardly any specific AFC were found in NALT or the surrounding mucosa. Intranasal immunization evoked no antibody response in the lung. Ample anti-TNP antibodies could be detected in the sera of animals, primed and boosted with TNP-KLH or TNP-LPS. No specific serum antibodies occurred after immunization with TNP-SRBC. The results are discussed in view of the immunological defence in the upper respiratory tract.

Anti-TNP-forming cells in rats after different routes of priming with TNP-LPS followed by intranasal boosting with the same antigen.

To study the reactivity of nasal-associated lymphoid tissue (NALT) and its position in the mucosal immune system, rats were intranasally challenged with 200 micrograms TNP-LPS. Priming had occurred 15 days previous to the challenge with the same antigen and dose, either intranasally, intratracheally, subcutaneously in the cheek or intraperitoneally. The number of anti-TNP antibody-forming cells (AFC) was determined in various tissues using the conjugate TNP/alkaline phosphatase. Generally, anti-TNP AFC were predominantly found in the posterior cervical lymph nodes, while NALT contained hardly any such AFC. The highest response in the posterior cervical lymph nodes occurred on day 5, after subcutaneous priming and intranasal boosting. This also evoked peak responses in several other tissues. The highest response in spleen and lung occurred on day 7 after intraperitoneal priming and intranasal boosting. Irrespective of the immunization route, IgA was the least produced isotype in the spleen as compared to antigen-specific IgG and IgM. In the posterior cervical lymph nodes, besides specific IgG and IgM, a considerable proportion of specific IgA was produced. All four immunization routes yielded anti-TNP antibodies in serum. As for the non-lymphoid cells, the intratracheal-intranasal immunization protocol induced an increase in pulmonary macrophages on days 3 and 5. The immunological role of lung macrophages is discussed.

Lymphoid and non-lymphoid cells in nasal-associated lymphoid tissue (NALT) in the rat. An immuno- and enzyme-histochemical study.

Lymphocyte and macrophage subpopulations and the stroma of mucosa-associated lymphoid tissue in the nasal cavity of the rat were examined by application of immunohistochemical and enzyme histochemical methods to cryostat sections. Nasal-associated lymphoid tissue was composed of a loose reticular network with lymphocytes and macrophages, covered by epithelium. The epithelium was infiltrated with B cells. T helper (W3/13-positive) and T suppressor/cytotoxic or large granular cells (OX8-positive), ED1-positive macrophages and Ia-positive cells. The B cell areas were populated by B cells, immunopositive for surface IgM or IgG. B cells with surface IgA or IgE were rare. Germinal centres were found infrequently. T helper cells were scattered throughout the B cell area. A few ED1-positive macrophages and ED5-positive follicular dendritic cells were observed. Strong Ia staining (mostly of B cells) was found in this area. The T cell areas contained T helper and T suppressor/cytotoxic cells in about equal amounts, and numerous ED1-positive macrophages. ED1 staining was also found in the subepithelial area. Numerous ED1-, ED2- and ED3-positive macrophages were found in the border between the lymphoid mass and the surrounding connective tissue. A few non-lymphoid cells showed weak acid phosphatase or non-specific esterase activity. The morphological observations suggest that nasal-associated lymphoid tissue plays an important role in the first contact with inhaled antigens.

Intra-epithelial lymphocytes and non-lymphoid cells in the human nasal mucosa.

Immunohistochemical staining of biopsy specimens was used to investigate the occurrence of lymphocyte subsets and non-lymphoid cells within the epithelial layer of the human nasal mucosa. The CD19 (B cell) marker was not expressed on the intra-epithelial lymphocytes, whereas the pan T cell marker CD2 was varyingly detected. The HLA-Dr antigen was abundantly present on epithelial cells, lymphocytes, and non-lymphoid cells. The latter are probably dendritic or Langerhans' cells. The findings stated above were the same in patient and control samples. In biopsy sections of 9 ear, nose, and throat patients, many CD8-positive (T suppressor/cytotoxic) cells and very few weakly stained CD4-expressing (T helper/inducer) cells were present. Quantification on single-cell preparations showed an average of 67% of the lymphocytes to be CD2 positive, 73% to be CD8 positive, while only 12% of the lymphocytes expressed the CD4 antigen. In control sections CD8 was similarly present as in patient sections, and, in addition, some clearly stained CD4-positive cells were seen.