Immunomorphologic and morphometric changes in pulmonary lymph nodes of sheep with progressive pneumonia

Morphologic, immunohistochemical, and morphometric studies were conducted on the posterior mediastinal lymph nodes of eleven sheep with naturally occurring ovine progressive pneumonia and four apparently healthy sheep with no pulmonary lesions (three seropositive, one seronegative for antibody to ovine progressive pneumonia virus). Compared with lesion-free sheep, sheep with ovine progressive pneumonia had a seven-fold increase in B lymphocyte areas and a 21/2-fold increase in T lymphocyte areas of these lymph nodes. Immunochemistry revealed cytoplasmic immunoglobulin G in scattered cells of germinal centers, medullary cords and interfollicular areas and membrane-associated immunoglobulin G in dendritic cells of germinal centers. Immunoglobulin M staining cells were widely scattered in germinal centers and medullary cords. Although B cell hyperplasia seemed to be the predominant process in lymph nodes of sheep with ovine progressive pneumonia, this was not accompanied by the expected degree of plasmacytosis, morphologically and immunohistochemically. These findings may represent an aberrancy of immunoregulation in ovine progressive pneumonia.     

Vasculitis associated with ovine progressive pneumonia virus infection in sheep

Vasculitis, involving small muscular arteries and arterioles, was found in 5 of 18 sheep naturally infected with ovine progressive pneumonia (OPP) virus and in 5 of 11 sheep experimentally infected with OPP virus. In order of frequency, arterial lesions were seen in carpal joint capsules, kidneys, meninges, brains, lungs, and tracheas. The lesions were intramural edema and hemorrhage, mononuclear cell infiltration, fibrinoid necrosis of media, and thrombosis. The vascular lesions were frequently associated with interstitial pneumonitis, arthritis, and encephalitis also induced by OPP virus.     

Mastitis associated with ovine progressive pneumonia virus infection in sheep

Eighteen ewes were experimentally infected with ovine progressive pneumonia virus and their mammary glands were examined for lesions and virus at 2.5 to 10 years postinoculation. Lesions were seen in 14 of 18 sheep; virus was isolated from 4 of 8 sheep. Lesion consisted of an interstitial accumulation of lymphocytes with periductal lymphoid nodules, and epithelial vacuolation and necrosis at the site of the lymphoid nodules.     

Production and assay of ovine T cell growth factor by concanavalin A stimulated peripheral blood leukocytes

Conditions for the production and assay of ovine T cell growth factor (TCGF) activity were evaluated. Peripheral blood leukocytes were stimulated with concanavalin A (Con A) in the presence of 2% autologous serum or serum-free media. Supernatants were harvested after 30 hr and concentrated for further characterization. A 28 hr proliferation assay with 2.5 X 10(4) 24 hr Con A blasts per well was optimal for detection of TCGF. Peak TCGF activity occurred with a 30-37 KD molecular weight fraction. Production and assay of TCGF were performed under autologous conditions to reduce background stimulation which occurred when fetal bovine serum was present. This methodology required no cell lines or inbred animals and should be adaptable to the study of immunostimulatory molecules of other outbred species.     

Ovine interleukin-2: partial purification and assay in normal sheep and sheep with ovine progressive pneumonia

Interleukin-2 (IL-2), a T cell derived lymphokine, acts in nonspecific hormone-like fashion to maintain proliferation of activated lymphocytes in vitro and is believed to play a key role in cell-mediated immune function in vivo. The parameters of induction and assay of factors with IL-2 activity were examined in a group of clinically normal sheep seronegative for antibodies to ovine progressive pneumonia virus (OPPV-). Supernatants from cultures of Concanavalin A (Con A) stimulated mononuclear leukocytes (ML) derived from peripheral blood and lymph nodes contained factors with the capacity to maintain continued proliferation in Con A stimulated lymphoblasts. This activity was localized by gel chromatography to fractions containing proteins of 17,000-20,000 daltons. In a group of sheep seropositive for antibodies to OPPV (OPPV+), decreased levels of IL-2 activity were found in ML culture supernatants derived from the posterior mediastinal lymph nodes of sheep with clinical and pathological evidence of OPP, when compared to OPPV+ sheep with no lesions and sheep with visceral caseous lymphadenitis. This decrease in IL-2 activity appeared not to be associated directly with levels of prostaglandin E2 in these supernatants. These findings may correlate with virus induced alterations in cell mediated immune function in lymphoproliferative lesions of OPP.     

Serologic survey of prevalence of ovine progressive pneumonia in Idaho range sheep.

Blood samples from 2,310 mature sheep in 3 Idaho range flocks were examined by agar gel immunodiffusion to determine the prevalence of ovine progressive pneumonia. The prevalence ranged from 58% for all ages combined in one flock to 90% of cull ewes in another flock. Age-specific prevalence rates increased from 16% in yearlings to 83% in ewes greater than or equal to 7 years old. Rambouillet sheep had a significantly (P less than 0.01) lower prevalence than sheep of 5 other breeds, whereas one-half Finnsheep crosses had a significantly (P less than 0.01) higher prevalence than sheep of other breeds. Within breed and age, there was no significant difference in reproductive performance between seropositive and seronegative ewes.     

Ultrastructure and frequency of mastitis caused by ovine progressive pneumonia virus infection in sheep

Twenty-five sheep, experimentally (n = 15) or naturally (n = 6) infected with ovine progressive pneumonia virus and noninfected controls (n = 4), were evaluated for histological and ultrastructural lesions of mastitis. Histologically, nine of 15 experimentally infected sheep and all six naturally infected sheep had lympho-plasmacytic mastitis. Severity of the lesion increased with length of time after infection. Periductal lymphatic nodules were seen in five sheep experimentally infected for 2.8 years or longer and in five naturally infected sheep that were 3.7 years old or older. Ultrastructurally, responses to ovine progressive pneumonia virus were diffuse lympho-plasmacytic infiltrates in glandular interstitium, lymphocytic and occasional plasmacytic infiltrates in ductal walls and lumens, lymphoblasts surrounded by small lymphocytes in glandular interstitium, and degeneration of epithelium releasing cells and cellular debris into the lumen. Based on the prevalence of lesions, the mammary tissue was more susceptible to ovine progressive pneumonia virus than other target organs: lung, brain, and synovium. Lesions did not differ between breeds of sheep. Ovine progressive pneumonia virus was not seen in the mammary tissue but was isolated from 15 of 17 mammary glands.     

Early detection of maedi-visna (ovine progressive pneumonia) virus seroconversion in field sheep samples.

The aim of this work was to investigate whether an enzyme-linked immunosorbent assay (ELISA) was useful for early detection of maedi-visna virus (MVV) infection in sheep under field conditions. An ELISA based on p25 recombinant protein and a gp46 synthetic peptide was used. Sequentially obtained serum samples (n = 1,941) were studied for 4 years. ELISA results were compared with those of the agar gel immunodiffusion (AGID) test, and results of both tests were compared with a reference result established using consensus scores for at least 2 of 3 serologic techniques (AGID, ELISA, and western blotting, which was used to resolve result discrepancies between the other 2 techniques). A total of 247 discrepancies were observed between ELISA and AGID. Of these, 131 were due to an earlier detection of 120 sera by the ELISA and 11 sera by AGID. The remaining discrepancies (116) were due to the presence of false reactions in both tests. Fewer false-negative results were found by ELISA than with AGID (6 vs. 69 sera, respectively), whereas the number of false-positive results was virtually the same for ELISA and AGID (21 vs. 20, respectively). In relation to the reference result, ELISA sensitivity and specificity were 97.8% and 98.2%, respectively, whereas values for AGID were 76.3% and 98.3%, respectively. The agreement between ELISA and the reference result was higher than that between AGID and the reference result (K value: 0.96 and 0.77, respectively). A variation in the ELISA signal (based on optical density) was observed during the study period, suggesting different antibody levels throughout the animal's life. The ELISA was useful for detecting MVV-infected sheep in field conditions and has potential for use in control and eradication programs.     

Prevalence of ovine progressive pneumonia in a sampling of cull sheep from western and midwestern United States.

Ovine progressive pneumonia was found to be prevalent in many major sheep-producing areas in the United States. The incidence was determined in a sampling of cull sheep (5 to 10 years of age) at slaughter. Diagnostic criteria were based on finding viral-specific immunoglobulins, virus, or lesions. Each method indicated a high incidence in samplings from midwestern and northwestern states and a relatively low incidence in the sampling from southwestern states. Of those sheep examined, precipitating immuno-globulin to the causal virus was seen in 1.0% to 67.5%, virus was isolated from the lungs of 0% to 46.2%, and lymphoid hyperplasia was seen in the lungs of 0% to 38.5%. The most common lesions in the lungs (from which virus was isolated) were multiple lymphoid nodules and increased fibromuscular tissue.     

Trypanosomes in the lymph nodes of cattle and sheep infected with Trypanosoma congolense

The prefemoral lymph nodes of two calves and a sheep infected with a stock of Trypanosoma congolense transmitted by Glossina morsitans were examined histologically for the presence of trypanosomes. Ten days after infection trypanosomes were found in the subcapsular sinuses of the nodes of a calf and the sheep but parasites were absent from the blood at this time. Trypanosomes were also detected in the prefemeral lymph node of the other calf on examination 30 days after infection, when parasites were also present in the blood. These observations provide further evidence that extravascular foci of trypanosomes develop in infections with T congolense and indicate that it should not be regarded as a strict plasma parasite.     

The immune reactivity of the regional and distant lymph nodes to autochthonous ovine squamous cell carcinomata

Anti-tumor immune reactivity of lymphocytes derived from lymph nodes regional to and distant from tumor growth, as well as that of peripheral blood leucocytes, against autochthonous tumor cells, was investigated. Experiments were carried out in vitro using a 51Cr cytotoxic assay and in vivo by cannulating the afferent and efferent lymphatics of regional and distant lymph nodes and challenging via the afferent lymphatics with 10(7) cultivated autochthonous tumor cells. No anti-tumor cytotoxic reactivity was detected in vitro using lymphocytes derived from any of the sources studied. In vivo, while challenge with autochthonous tumor cells produced no response in the regional lymph node, significant blast cell response was obtained in the distant node. The response at the distant node was associated with the production of antibodies that could bind to tumor cells without causing their demise. The anergy observed at the regional lymph node, and the possibility of a relation between the events occurring at that node and those observed at the distant node, are discussed.     

Seroprevalence of ovine progressive pneumonia virus in sheep in the United States as assessed by analyses of voluntarily submitted samples.

Ovine progressive pneumonia (OPP) is a lentivirus-induced disease of sheep in the United States that is similar, if not identical, to maedi/visna in many other countries. Prevalence estimates of seropositivity to this virus in sheep in the United States have been confined to limited groups or flocks of sheep and have varied from 1 to 90%. In this study of detection of antibodies against OPP virus, we found a lower general prevalence of antibodies to OPP virus in sheep than was previously reported. Of 16,827 sheep from 29 states in the United States, 26% were seropositive and 48% of 164 flocks that were tested had 1 or more seropositive sheep. Seropositivity to OPP virus for sheep within special categories was determined, although nonrandom samples that were available may have biased the results. Within regions of the United States, prevalence was highest in the Rocky Mountain region at 49% and lowest in the northern Atlantic region at 9%. Seropositive sheep were not evenly distributed among flocks, but were clustered in a few flocks of sheep. A high number of flocks had no or few seropositive sheep. Prevalence increased with age from 4% at less than 1 year to a plateau of 34% at 4 years. Seropositivity was variable among breeds and was not associated with sex, wool class, or place of origin of ancestors.     

Effects of Pasteurella haemolytica cytotoxin on ovine peripheral blood leucocytes and lymphocytes obtained from gastric lymph

Ovine peripheral blood leucocytes were separated on discontinuous Percoll density gradients into fractions rich in lymphocytes (PBLy) and polymorphonuclear leucocytes. Supernatant fluid from a dialysis sac culture of Pasteurella haemolytica biotype A serotype 1 (A1) was cytotoxic to these leucocytes in vitro. PBLy retained viability after storage in liquid nitrogen and could be employed in cytotoxicity assays. However, sheep cannulated via the common gastric lymph duct were an excellent source of large numbers of homogeneous lymphoid cells (GLy) which also stored well in liquid nitrogen. As both freshly collected and stored GLy were killed by culture supernatant fluid GLy offer advantages as target cells for further characterisation of the extracellular cytotoxin produced by P. haemolytica. From the results obtained, it is considered that all ovine leucocytes are susceptible to P. haemolytica cytotoxin.     

Precipitating antibodies in experimental visna and natural progressive pneumonia of sheep

Serological responses of Icelandic sheep experimentally infected with visna virus (vv) were contrasted with responses in American Targhee sheep naturally infected with progressive pneumonia virus (PPV). Precipitating antibodies assayed by immunodiffusion were compared with the neutralising and complementing fixing antibody response. In experimental infections with vv, complement fixing and neutralising antibodies appeared early after infection and rose to high levels in all sheep, while precipitating antibodies were detected only at minimal titre. In natural infections with PPV, immune responses were less consistent and precipitating antibodies were detected more frequently than complement fixing or neutralising antibodies against PPV. These results may suggest important biological differences between the lytic fibroblast-tropic virus strains used for experimental infection of Icelandic sheep and the nonlytic macrophage-tropic strains of PPV circulating in nature. Lytic strains evoke a brisk response against the viral glycoprotein with high titre neutralising antibody while nonlytic strains induce a less consistent response to the glycoprotein.     

Antigen-induced production of interferon-gamma in samples of peripheral lymph nodes from sheep experimentally inoculated with Mycobacterium avium subsp. paratuberculosis.

The production of interferon-gamma (IFN-gamma) in response to Johnin purified protein derivate was measured in samples of the prescapular lymph node (PLN) from 10 sheep, aged 2 years, and nine sheep, aged 1 year that had been inoculated orally with Mycobacterium avium subsp. paratuberculosis within their first month of life. Ten non-inoculated sheep, aged 1 year, constituted the negative control group. The results obtained in the PLN IFN-gamma assay were compared with those derived from serological tests: a complement fixation test (CFT), agar gel diffusion test (AGID) and enzyme-linked immunosorbent assay (ELISA), as well as an IFN-gamma test on samples of blood. Among the 19 inoculated sheep, 16 gave positive reactions in the PLN IFN-gamma assay on samples incubated overnight, and 18 tested positive when the assay was applied to PLN samples incubated for 48h. In comparison, three, four and seven inoculated sheep gave positive reactions in the ELISA, CFT and in the blood IFN-gamma assay on samples incubated overnight, respectively. The AGID and IFN-gamma assay on blood samples incubated for 48h detected eight inoculated animals. Twelve inoculated sheep, that tested positive in the PLN IFN-gamma assay were clinically normal, gave negative results in an IS900-based polymerase chain reaction (PCR) assay on samples of ileum and ileocaecal lymph node and had no histological evidence of paratuberculosis, but tested positive on more than two occasions in sequential serological testing before necropsy. None of the 10 non-inoculated sheep tested positive in the AGID, CFT, ELISA, blood IFN-gamma assay on samples incubated overnight and for 48h or the PLN IFN-gamma assay on samples incubated overnight, but one gave a positive result in the PLN IFN-gamma assay on samples stimulated for 48h. It is likely that the positive reactions obtained by the PLN IFN-gamma assay in the 12 inoculated sheep that tested negative in the PCR assay and histopathological examination represents immunological evidence of latent infection or previous exposure to M. paratuberculosis rather than active infection.     

Innate immune responses induced by classes of CpG oligodeoxynucleotides in ovine lymph node and blood mononuclear cells

CpG ODN signal through Toll-like receptor 9 (TLR9) and trigger a cascade of events that lead to activation of innate and adaptive immune responses. Our current understanding of the immunobiology of host responses to CpG is based largely on studies on peripheral blood mononuclear cells (PBMC) and splenocytes. Little is known regarding CpG-induced responses in other lymphoid tissues. In the present study, we investigated responses induced by CpG in both PBMC and lymph nodes. Cells were isolated from the superficial cervical lymph node (LNC) and blood and then stimulated with CpG ODN (either A-, or B- or C-class ODN). Cytokine production was assayed by ELISA, and lymphocyte proliferation was determined by (3)H-thymidine incorporation. NK-like cytotoxicity was analyzed by lysis of (51)Cr-labelled target cells. All three classes of CpG induced IFNalpha and IFNgamma in LNC. In contrast, only A and C-class ODN induced IFNalpha and IFNgamma in PBMC. Moreover, the IFN levels in LNC were 20-40-fold higher than in PBMC. Furthermore, all classes of ODN induced higher IL-12 levels in LNC (five- to six-fold) than in PBMC. Both B and C-class ODN induced good proliferative responses in PBMC and LNC, but the A-class ODN did not induce proliferation of PBMC and only induced moderate proliferation of LNC. A-class ODN induced significant NK-like activity in LNC. Thus, all three classes of CpG ODN induced similar responses in LNC, and these responses were consistently higher than in PBMC. These observations indicate that CpG ODN-induced responses differ between blood and lymph nodes, and suggest that the functional classification of CpG ODN based on PBMC responses may not be directly applicable to cells from other immune tissues.     

Changes in subpopulations of lymphocytes in peripheral blood, and supramammary and prescapular lymph nodes of cows with mastitis and normal cows

Direct immunofluorescence (IF) and indirect IF techniques were employed to analyze the distribution of B and T lymphocyte populations in peripheral blood, and in supramammary (draining), and prescapular (non-draining) lymph nodes of cows with mastitis and normal cows. In the peripheral blood there was a significant decrease in the percent and absolute number of B lymphocytes in mastitic cows (n = 29; 17.1 +/- 10.2%; 3.4 +/- 2.7 X 10(5) cells/ml) as compared to normal cows (n = 38; 25.2 +/- 7.8%; 9.3 +/- 5.4 X 10(5) cells/ml). The percent T lymphocyte count in mastitic cows (71.2 +/- 7.1%) was slightly increased over that of normals (65.8 +/- 7.2%), although the absolute number of T lymphocytes was decreased in mastitic cows (1.49 +/- 0.91 X 10(6) cells/ml vs. 2.47 +/- 1.28 X 10(6) cells/ml). In the prescapular lymph node the percent of B lymphocytes, but not T or "null lymphocytes", decreased significantly in mastitic cows as compared to that of normals. The decrease, i.e. 32%, paralleled the 32.1% decrease found in peripheral blood B lymphocytes. In contrast, in the supramammary lymph node of mastitic cows, the percent B lymphocytes increased over that of normals (35.1 +/- 2.0% vs. 20.4 +/- 9.4%), whereas the percent T lymphocytes decreased to 54.5 +/- 2.8% compared to 70.7 +/- 3.5% in normal cows. There was no significant change in percent "null lymphocytes". The weight of prescapular lymph nodes did not change in mastitic cows when compared to that of normals. As a result, the estimated number of B lymphocytes, but not of T and "null lymphocytes", decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative histopathology of the lymph nodes, spleen, liver and kidney in experimental ovine trypanosomosis

The infection of Yankassa rams with three important trypanosome species affecting livestock, namely, Trypanosoma congolense, T. vivax and T. bruceiproduced both acute and chronic fatal conditions. Chronic infections were induced in the three infections by the application of subcurative doses of diaminazene aceturate (Berenil). Pathological changes in the infected animals included splenomegaly and hepatomegaly which were more pronounced in acute than in chronic T. congolense infection. However, these changes were more severe in chronic than in acute T. vivax infection. While splenomegaly was more pronounced in chronic T. bruceiinfection than in acute, hepatomegaly and lymphadenopathy were more severe in acute than in the chronic condition. The increases in size of the spleen, lymph nodes and liver were associated with congestion, increases in cell density related to increased immunological reactions in the spleen and lymph nodes as well as increase in numbers, size and activity of the phagocytic cells in these organs.     

A PCR technique for the detection of Jaagsiekte sheep retrovirus in the blood suitable for the screening of ovine pulmonary adenocarcinoma in field conditions

Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring contagious lung neoplasia caused by jaagsiekte sheep retrovirus (JSRV). Although no specific circulating antibodies against the virus can be detected in infected sheep, JSRV proviral DNA sequences can be found in peripheral blood leukocytes (PBLs) in clinically affected and in a proportion of in contact animals. In this study, existing hemi-nested PCR procedure is compared with a new one-step PCR technique that was developed to minimise potential DNA contamination and reduce sample and reagent handling. Different blood preparations were assessed and the best results were achieved on DNA prepared from buffy coat. The sensitivity of this PCR was lower in JSRV infected sheep without lesions of OPA than in clinically affected sheep, which indicate that this PCR may not be not fully appropriate for screening of individual sheep, but rather to provide results at flock level. This PCR is the only currently available blood test for detection of JSRV infected sheep and may be useful in epidemiological studies and in control programmes of OPA.