Histologic evaluation of lung and bronchus-associated lymphoid tissue in young turkeys infected with Bordetella avium

One-day-old turkeys were inoculated intranasally with Bordetella avium. Noninoculated hatchmates were housed separately. At postinoculation weeks 1, 2, 3, 4, and 5, B avium-infected (BA+) and B avium-free (BA-) turkeys were necropsied; specimens of tracheas, intrapulmonary primary bronchi, and lung adjacent to primary bronchi were bacteriologically cultured. Lung tissue was collected for histologic examination. Lungs perfused with acetic acid were collected for evaluation to determine the size, number, and distribution of lymphoid nodules associated with primary bronchi. Bordetella avium was isolated from trachea and primary bronchi of all BA+ turkeys, but was never isolated from lung parenchyma. Acute purulent bronchitis was associated with colonization of the primary bronchi by B avium from postinoculation weeks 1 to 3. Macrophages and lymphocytes persisted in the peribronchial connective tissue for 5 weeks after inoculation. Bronchus-associated lymphoid tissue consisted of discrete lymphoid nodules protruding into the lumens of primary bronchi. Lymphoid nodules, morphologically similar in BA+ and BA- turkeys, were composed of nonciliated, cuboidal epithelium covering a zone of loosely arranged lymphocytes and macrophages and a deeper, sharply demarcated lymphoid follicle. Compared with bronchus-associated lymphoid tissue of BA- turkeys, lymphoid nodules of bronchus-associated lymphoid tissue in BA+ turkeys were more numerous and widely distributed along primary bronchi. In both BA- and BA+ turkeys, the mean diameter of lymphoid nodules doubled between 1 and 5 weeks of age.     

Particle uptake by conjunctiva-associated lymphoid tissue (CALT) in turkeys

Uptake of tracer particles was assessed in lower eyelid conjunctiva-associated lymphoid tissue (CALT) of 3-week-old turkeys. Tracer particle suspensions, including carbon, iron oxide, or three sizes of latex beads (0.81 micron, 1.7 microns, and 2.9 microns), were placed into the experimentally sealed conjunctival space. After 5, 15, or 30 minutes, eyelids were removed and CALT was examined by light microscopy. Uptake was confirmed for all tracers and occurred within the lymphoepithelium of CALT. The uptake of latex beads was not as frequent as for carbon and iron. Tracers were increasingly evident in lymphoepithelium and subepithelial macrophage clusters as contact time increased. These findings provide additional evidence that CALT is capable of antigen uptake and may play a role in paraocular and upper respiratory immunity in turkeys.     

Conjunctiva-associated lymphoid tissue (CALT) in normal and Bordetella avium-infected turkeys

Conjunctiva-associated lymphoid tissue (CALT) was characterized in normal and Bordetella avium-infected turkey poults during the first 5 weeks of life. At 1, 5, 12, 19, 25, and 33 days post-hatching (DPH), upper and lower eyelids were examined by gross, histologic, and electron microscopic techniques. CALT was confined to the proximal part of the lower eyelid near the conjunctival fornix; it appeared by 5 DPH as individual lymphoid nodules and as dense masses by 19 DPH. In the upper eyelid, CALT was present only as isolated nodules. Histologically, CALT was composed of dense lymphocyte infiltrates within subepithelial connective tissue, intraepithelial lymphocytes, and flattened lymphoid-associated epithelium that lacked goblet cells. Germinal centers were in CALT by 19 DPH. By scanning electron microscopy, epithelial cells over lymphoid areas were flat and had short, irregular microvilli; non-lymphoid areas were covered by cells with tall, regular microvilli. Transmission electron microscopy revealed that with increasing age of birds, the epithelium over conjunctival lymphoid infiltrates became progressively flattened and infiltrated by lymphocytes. Some blood vessels in CALT had high endothelial cells; lymphocytes were in the lumen and between or beneath endothelial cells. In B. avium-infected poults, CALT was increased, developed earlier, and contained more germinal centers than in normal poults. We conclude that CALT of turkeys closely resembles other mucosal lymphoid tissues and may serve as a site for local antigen uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in lymphocyte subsets in the bronchus-associated lymphoid tissue of goats naturally infected with different Mycoplasma species

The distribution of cells containing lysozyme, S-100 protein, CD3, CD4, CD8, major histocompatibility complex class II antigen and immunoglobulin G (IgG) was analysed in the bronchus-associated lymphoid tissue (BALT) of goats naturally infected with three Mycoplasma species. This study included the immunohistochemical characterization of the pneumonic lesions of 18 goats (3-5 months old) infected with one of the following Mycoplasma species: M. mycoides ssp. mycoides, Large Colony type (goats no. 1-6), M. mycoides ssp. capri (goats no. 7-12) and M. capricolum ssp. capricolum (goats no. 13-18). Microscopically, infected animals showed a moderate broncho-interstitial pneumonia, characterized by lymphoid hyperplasia of the BALT and infiltration of mononuclear cells in the alveolar walls and airways. The main cellular type in the BALT was represented by CD3+ T lymphocytes, and the ratio of CD4+:CD8+ cells was > 2. The BALT showed large germinal centres mainly composed of IgG+ B lymphocytes, with numerous S-100+ follicular dendritic cells. The presence of follicular dendritic cells confirmed the high degree of organization of this lymphoid tissue. The immunohistochemical results showed that activated T lymphocytes, particularly in the CD4 subset, and IgG+ B cells, play a major role in the immune response of the caprine lung infected with these species of mycoplasmas.     

Stimulation of the bronchus-associated lymphoid tissue of goats and its effect on in vitro colonization by Pasteurella haemolytica

Twenty goats of about 7 months of age were divided into five groups. The goats in groups 1 and 2 were exposed once, using an intranasal spray to 2 ml of an inoculum containing 10⁶ colony-forming units/ml of living or dead Pasteurella haemolytica A2, respectively. The goats in groups 3 and 4 were similarly exposed twice at a 2-week interval. Group 5 was the untreated control. The number and size of the bronchus-associated lymphoid tissue (BALT) in goats exposed twice to either living or dead organisms were significantly (p>0.05) increased compared with those exposed once and with the unexposed control. In vitro colonization by living P. haemolytica A2 onto the lung tissue in which the BALT had been stimulated by two exposures of either living or dead organisms was significantly (p>0.05) reduced. The study indicates that stimulation of the respiratory mucosal immunity may prevent P. haemolytica A2 infection.     

A morphologic and immunohistochemical study of the bronchus-associated lymphoid tissue of pigs naturally infected with Mycoplasma hyopneumoniae

Porcine enzootic pneumonia (PEN), caused by Mycoplasma hyopneumoniae (Mh), has been described in pigs in all geographic areas. The disease is characterized by high morbidity and low mortality rates in intensive swine production systems. A morphologic and immunohistochemical study was done to determine the cellular populations present in lung parenchyma of infected pigs, with special attention to the bronchus-associated lymphoid tissue (BALT). Polyclonal and monoclonal antibodies were used for the detection of antigens of Mh, T lymphocytes (CD3+, CD4+, and CD8+), IgG+ or IgA+ lymphocytes, and cells containing lysozyme, S-100 protein, major histocompatibility complex class II antigen or myeloid-histiocyte antigen. Findings in lung tissues associated with Mh infection were catarrhal bronchointerstitial pneumonia, with infiltration of inflammatory cells in the lamina propria of bronchi and bronchioles and alveolar septa. Hyperplasia of mononuclear cells in the BALT areas was the most significant histologic change. The BALT showed a high morphologic and cellular organization. Macrophages and B lymphocytes were the main cellular components of germinal centers. T lymphocytes were primarily located in perifollicular areas of the BALT, lamina propria and within the airway epithelium, and plasma cells containing IgG or IgA at the periphery of the BALT, in the lamina propria of bronchi and bronchioles, in alveolar septa, and around bronchial submucosal glands. The hyperplastic BALT in PEN cases consisted of macrophages, dendritic cells, T and B lymphocytes, and IgG+ and IgA+ plasma cells. CD4+ cells predominated over CD8+ cells. Local humoral immunity appears to play an important role in the infection.     

Unique genotypes of Mycobacterium avium subsp. paratuberculosis strains of Type III

Mycobacterium avium subsp. paratuberculosis (MAP) strains with two new IS900 restriction fragment length polymorphism (RFLP) BstEII types intermediate suspected to belong to the MAP Type III group were isolated from migrating sheep in Germany. Such strains have only been sporadically identified in a few studies. For a better understanding of the genomic diversity of MAP with regard to specific host associations, geographic origin, and the discussed classification into Type I, Type II and Type III, these isolates were further characterized.Using IS900-RFLP, the isolates showed unique fingerprint patterns after BstEII-, PstI-, PvuII- and BamHI-digestion which had not been published before. Additionally, using gyrB-PCR-restriction endonuclease analysis (PCR/REA) and mycobacterial interspersed repetitive unit (MIRU)-PCR, the two strains showed differences to known patterns of the Type I as well as the Type II group. Unique genotypes were also obtained with multilocus short sequence repeat (MLSSR) sequencing and MIRU-variable-number tandem-repeat (VNTR) typing.As expected, genomic profiles identical to the Type I and different from the Type II group were detected by IS1311-PCR/REA, IS1311 sequencing as well as by Large Sequence Polymorphism analysis (LSP^A 8, 17, 20, 4-II, and 18).In addition to distinct growth characteristics, the unique genotypes of the studied sheep strains support their affiliation to the assumed third group within the MAP subspecies and suggest the existence of different genotypes within this Type III group. The results could serve as further evidence that Type I and Type III groups are more closely related to each other than to the bovine Type II group.     

Antibody recognition to secreted proteins of Mycobacterium avium subsp. paratuberculosis in sera from infected ruminants

Two liquid culture media to obtain secreted proteins of Mycobacterium avium subsp. paratuberculosis at different incubation periods were evaluated. Middlebrook 7H9-OADC (7H9) and Watson-Reid (WR) broths were inoculated with a field strain of M. paratuberculosis and growth curves determined using nonlinear regression analysis. Most culture filtrate (CF) proteins were of low molecular weight and reacted strongly against sera from cultured-positive cases of paratuberculosis. CF proteins obtained in WR yielded a higher number of bands and were detected earlier than those obtained from 7H9. A high degree of variability in CF protein immunoreactivity was seen among infected animals. Sera from cattle with clinical paratuberculosis or heavy fecal shedders of M. paratuberculosis reacted more intensively and to more CF proteins than did sera from other infected cattle. Immunoblots showed differences in antibody binding to CF proteins when sera were absorbed with M. avium but not with others environmental mycobacteria. Immunoblots with sera from infected goats and a sheep showed reactivity with proteins of 32, 33 and 46 kDa both before and after the sera were absorbed with M. phlei. Antibodies found in serum of infected deer reacted with CF proteins in a similar way as did for cattle. These results suggest that a pool of CF proteins of M. paratuberculosis could be good candidates as antigens for serodiagnosis of paratuberculosis.     

Role of nitric oxide production in dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis

Nitric oxide (NO) is a crucial mediator in host defense and is one of the major killing mechanisms within macrophages. Its induction is highly affected by the types of cytokines and the infectious agents present. In the current study, NO production was evaluated after in vitro infection of unfractionated peripheral blood mononuclear cells (PBMCs) with Mycobacterium avium subsp. paratuberculosis (MAP) after 8 h, 3 and 6 days of culture for cows in different stages of disease. In addition, the effects of in vitro exposure to inhibitory cytokines such as interleukin-10 (IL-10) and transforming growth factor β (TGF-β) as well as the pro-inflammatory cytokine IFN-γ were correlated with the level of NO production. Nitric oxide production was consistently higher in cell cultures from subclinically infected animals at all time points. An upregulation of NO production was demonstrated in unfractionated cell cultures from healthy control cows after exposure to MAP infection as compared to noninfected cell cultures. A similar increase in NO due to the addition of MAP to cell cultures was also noted for clinically infected cows. NO level among subclinically infected cattle was greater at all time points tested and was further boosted with the combination of both in vitro MAP infection and IFN-γ stimulation. Alternatively, nonspecific stimulation with LPS from Escherichia coli O111:B4-W resulted in an upregulation of NO production in all infected groups at 3 and 6 days after in vitro infection. Finally, the in vitro exposure to inhibitory cytokines such as IL-10 and TGF-β prior to MAP infection or LPS stimulation resulted in the downregulation of this inflammatory mediator (NO) in all experimental groups at all time points. In summary, a higher level of NO production was associated with cows in the subclinical stage of MAP infection. As well, the results demonstrated an increase in NO production upon infection with MAP and in the presence of exogenous IFN-γ. Finally, the results suggest an important role of IL-10 and TGF-β on the profile of NO production which may explain the low NO production in MAP clinically infected cows.     

Suppression of immunologic function and degeneration of lymphoid organs in cyclophosphamide-treated turkeys

Neonatal administration of 8 to 20 mg of cyclophosphamide (CY) in turkey poults resulted in a marked alteration in the morphologic features of all major lymphoid organs. Cyclophosphamide induced severe deficiency in antibody response to multiple injections of Brucella abortus and sheep RBC and in vitro proliferative response of peripheral blood leukocytes to concanavalin A. The morphologic regeneration of thymus and of thymus-dependent areas was marked in all major lymphoid organs by posttreatment day 15. This regeneration preceded functional recovery of the mitogenic response of peripheral blood leukocytes. The regeneration of the bursa and bursa-dependent areas in other lymphoid organs was minimal through posttreatment day 27. Thus, CY may be used effectively in turkeys as an immunosuppressive reagent.     

A comparison of the gland of Harder response and head-associated lymphoid tissue (HALT) morphology in chickens and turkeys.

The immunofunctional response of the gland of Harder (GH) was compared in chickens and turkeys using an in vivo assay previously developed for use in chickens. The GH were surgically removed (GHx) from leghorn chicks at 1 day of age and from poults at 2 days of age. Intact birds of each species served as controls. During the fourth week of age, both GH-intact and GHx chicks were exposed to killed Brucella abortus antigen by the ocular or intraperitoneal route. One week later, serum and tears were collected and assayed for antibodies to B. abortus. In addition, all birds were killed at the end of the trial period, and the heads were fixed and processed for histologic examination. Various components of the head-associated lymphoid tissue (HALT) including the GH, nasal glands, lacrimal glands, lacrimal ducts, eyelid conjunctiva, and nasal cavity mucosa/submucosa, were evaluated microscopically using a scoring system to estimate quantity and degree of development of immune tissue in those sites. Results of all analyses indicate that functional response and morphology of the HALT are comparable in turkeys and chickens.     

Mycobacterium avium subsp. paratuberculosis in cow bulk tank milk in Cyprus detected by culture and quantitative IS900 and F57 real-time PCR

The possibility that Mycobacterium avium subsp. paratuberculosis (MAP) plays some role in the development of Crohn's disease in humans is attracting attention to milk and milk products originating from infected animals. In this study, we focused on the detection of MAP in 220 bulk tank milk (BTM) samples from all dairy cattle herds in Cyprus. In total, 63 (28.6%) BTM milk samples were found to be positive for MAP using quantitative real-time PCR assays for IS900 and F57. The presence of MAP in BTM was low, and was assessed to be several tens of MAP cells per one ml of BTM. Milk samples examined by cultivation were found to be negative for MAP in all 220 BTM. In two BTM samples cultivation and subsequent sequencing of 16S rRNA revealed two isolates of M. fortuitum.     

Prevalence of Michigan dairy herds infected with Mycobacterium avium subspecies paratuberculosis as determined by environmental sampling

A cross-sectional, stratified random survey of Michigan dairy herds was conducted to estimate the prevalence of herds infected with Mycobacterium avium paratuberculosis (MAP), the causative agent of Johne's disease, in Michigan using targeted environmental sampling. One pooled sample each from the primary manure storage area and a high-traffic common cow area from each herd was collected and cultured for MAP using the ESP^® culture system II. A herd was classified as positive if at least one sample was culture positive for MAP. State, agricultural district, and herd size stratum prevalence were calculated. Information on past MAP testing and cattle purchase history was collected, and logistic regression was performed to determine their importance to the MAP status of the herd. One hundred twenty-seven herds were contacted, and 94 agreed to participate in the study. The environment of 38 (40.4%) herds cultured positive for MAP. MAP was found in all herds (n = 15) with greater than 200 lactating cows. Herds that had tested for MAP or purchased cattle in the previous 5 years were 4.6 and 3.1 times, respectively, more likely to be infected than herds that had not. MAP continues to be prevalent on Michigan dairy farms, especially those with greater than 200 lactating cows. The environmental sampling protocol used in this study is an economically attractive alternative for monitoring herd level prevalence and the progress of Johne's disease control programs at the state or national level. Implementation of such a program would aid states in monitoring Johne's control program progress, and guide changes over time.     

Searching for proteins of Mycobacterium avium subspecies paratuberculosis with diagnostic potential by comparative qualitative proteomic analysis of mycobacterial tuberculins

Accurate immunodiagnosis of bovine paratuberculosis is among others hampered by the lack of specific antigens. One of the most frequently used antigen preparations is purified protein derivative (PPD), also known as tuberculin. This crude extract has limitations when used in diagnostic assays due to the presence of cross-reactive antigens. The aim of the current study was to systematically analyze the qualitative protein composition of PPD of the major mycobacterial pathogens.One-dimensional gel electrophoresis followed by tandem mass spectrometry analysis of PPD from Mycobacterium avium subspecies paratuberculosis (MAP), Mycobacterium avium subspecies avium (MAA) and Mycobacterium bovis (MB) identified 156, 95 and 132 proteins, respectively. Comparative sequence analysis led to the selection of a MAP-specific protein (MAP1718c), and finally heterologous expression in Escherichia coli of this and other diagnostic candidate proteins (MAP3515c and MAP1138c (LprG)) enabled evaluation of their immunogenicity. Lymphocyte proliferation responses did not indicate substantial diagnostic potential of the antigens tested. In contrast serum antibody levels for MAP1138c in paratuberculosis infected cows (N = 20) were significantly higher (p < 0.01) than in control animals (N = 20), despite the conserved nature of this protein.In conclusion, this study showed that a combination of proteomics and genomics, starting from complex protein mixtures, present in tuberculins, can reveal novel proteins aiding the development of immunodiagnostics for mycobacterial diseases.     

Distribution of IS900 restriction fragment length polymorphism types among animal Mycobacterium avium subsp. paratuberculosis isolates from Argentina and Europe

Sixty-one Mycobacterium avium subsp. paratuberculosis isolates from cattle and deer from the Buenos Aires province, an important livestock region in Argentina, were typed by restriction fragment length polymorphisms (RFLP) analysis based on IS900. Four different RFLP patterns (designated ‘A’, ‘B’, ‘C’ and ‘E’) were identified in BstEII digests of genomic DNA. The most frequently observed type, pattern ‘A’, was found in 46 isolates (75%). The second, pattern ‘E’, included 8 isolates (13%), while the third, pattern ‘B’, included 6 isolates (10%). Pattern ‘C’ was found for only one isolate. All of the deer isolates were classified as pattern ‘A’, while cattle isolates represented all four RFLP patterns. Twenty-one isolates representing the four different BstEII-RFLP patterns were digested with PstI. Twenty isolates showed identical PstI-RFLP pattern. BstEII-RFLP patterns from Argentine cattle and deer were compared with patterns found in cattle, goat, deer, rabbit, and human isolates from Europe. The most common pattern in Argentina, pattern ‘A’, was identical to a less frequently occurring pattern R9 (C17) from Europe. The other Argentine patterns ‘B’, ‘C’ and ‘E’, were not found in the Europe. These results indicate that the distribution of M. avium subsp. paratuberculosis genotypes in the Buenos Aires province of Argentina is different from that found in Europe.     

The recovery of Bordetella bronchiseptica strains by means of smears from the nasal cavities of pigs

The paper presents results obtained from a study on the recovery of Bordetella bronchiseptica strains from the nasal cavities of pigs by means of tampon smears. There are several important factors influencing the reliability of recovery: restraining of animals, thorough disinfection of the snout about the nostrils prior to the taking of samples, correct method of sample collection using suitable tampons, suitable culture media for cultivation, correct cultivation method and diagnostic procedure for the isolation and identification of grown cultures.