Comparison by experimental infections in cattle of a Dictyocaulus species occurring naturally in red deer and a dictyocaulus of bovine origin

Dictyocaulus species larvae were obtained from young red deer which had become infected on pastures considered to be carrying the Dictyocaulus species indigenous to the red deer of Scotland. These larvae were cultured to third stage and transmitted to five bovine calves. Five other bovine calves were infected with third stage Dictyocaulus viviparus larvae of bovine origin. Microscopic appearances of both groups of larvae were indistinguishable and their lengths were similar. Results indicated that the Dictyocaulus species derived from deer induced milder though similar clinical and pathological responses in cattle than did the D viviparus derived from cattle. It was concluded that there are strains of different pathogenicity within the species D viviparus, that the deer derived Dictyocaulus species was a strain of D viviparus, and that the hazards to animal health associated with infection by D viviparus in farming systems where red deer and cattle may graze alternately are likely to be acceptable.     

Experimental infections with Dictyocaulus viviparus in vaccinated and unvaccinated red deer

Four of eight red deer calves which had been artificially reared and were lungworm free were vaccinated with bovine lungworm oral vaccine when eight weeks old; the other four were not vaccinated. Three of each category were challenged daily with 500 Dictyocaulus viviparus infective stage larvae per kg liveweight for 17 days when six months old while one in each category was left as an unchallenged control. The effects of challenge were monitored and all challenged deer and one control were killed for post mortem assessment. Challenge with D viviparus was associated with reduced food intakes and weight gains but vaccinated calves were less affected than unvaccinated ones. The reaction of the alveolar tissue of red deer lung to D viviparus was mild in vaccinated and unvaccinated animals and differed from that of bovine lung in that alveolar epithelialisation was limited and hyaline membrane formation and interstitial emphysema were not seen. The disease was most evident in and around airways and was less in vaccinated calves. It was concluded that young red deer are tolerant to D viviparus but will readily acquire infection.     

Distribution of Pasteurella haemolytica in the respiratory tracts of carrier calves and those subsequently infected experimentally with Dictyocaulus viviparus

Distribution of Pasteurella haemolytica in the respiratory tracts of calves with no apparent clinical signs of illness and those infected experimentally with Dictyocaulus viviparus was determined so as to define carrier sites for this organism. The calves had been positive by nasopharyngeal swab for either P haemolytica A2 or A1 for at least two months or for over a month, respectively, before slaughter. P haemolytica A1 was acquired following horizontal spread from other infected calves. It was observed post mortem that P haemolytica A1 or A2 resided in the tonsils and retropharyngeal lymph nodes of calves of both groups. In addition to these sites, P haemolytica A1 was also isolated from the right cranial lung lobe of one of the calves from the D viviparus infected group although there was no evidence of pasteurella associated pneumonia. It was concluded that tonsil and retropharyngeal lymph nodes appear to be the most important carrier sites for P haemolytica when compared to other tissues of the bovine respiratory tract.     

Alternative pathway fof bovine complement Immunochemical studies on factor B-like serum protein and its conversion product B gamma 2.

Rabbits produced antibodies to a factor B-like serum protein (factor Bbov), its conversion product B gamma 2 and some other bovine serum proteins after repeated immunization with zymosan which previously had been incubated with fresh bovine serum. Such antisera were used to monitor purification of B gamma 2 from fresh bovine sera incubated with zxymosan. Subsequently, antisera specific for factor Bbov and B gamma 2 were produced. Antiserum produced against B gamma 2 cross-reacted with factor Bbov. Functional assays for factor Bbov were carried out in a hemolytic system with guinea pig erythrocytes in EGTA buffer. Heat inactivation (56 degrees C/5 min) of bovine serum destroyed the antigenicity of factor Bbov but not that of B gamma 2. Factor Bbov had an apparent molecular weight of 95,000 and B gamma 2 a molecular weight of 40,000 daltons. Conversion of factor Bbov to B gamma 2 was determined qualitatively by immunoelectrophoresis and quantitatively by radial immunodiffusion. Conversion of factor Bbov to B gamma 2 in bovine serum, in the presence of zymosan or cobra venom factor (CoVF) required Mg++ but not Ca++, did not occur in heat inactivated (56 degrees C/5 min) serum and was maximal, but not complete, when fresh bovine serum was incubated with zymosan (20 mg/mL) at 37 for two hours.     

Bovine eotaxin (CCL11)--an unusual member of the eotaxin group--attracts eosinophils in vitro but is not responsible for eosinophilia in the ovary

Under physiological conditions normally characterised by low tissue infiltration of eosinophils, a conspicuous number of these cells are attracted into the human and ruminant ovary. Eosinophils suddenly increase in the thecal layer of the preovulatory follicle and corpus luteum at very early development. Currently, we only have a limited understanding of the mechanism for the recruitment of the ovarian eosinophils. Eotaxin (CCL11) may be one of the chemoattractants involved in stimulating eosinophils to migrate selectively into ovary. As a prerequisite for the analysis of eotaxin expression in the bovine ovary, we determined the complete bovine eotaxin mRNA sequence since it was not available from databases. The bovine eotaxin is the first member of the monocyte chemoattractant protein (MCP)/eotaxin subfamily with two mRNA isoforms varying in length in the untranslated 3'-untranslated region. The unusual amino-acid sequence of bovine eotaxin contains structural features that are so far known to be characteristic for MCP, but not eotaxin. In our microchemotaxis assays, recombinant bovine eotaxin showed a functional pattern orthologous to known eotaxins. Thus, the chimeric structure of bovine eotaxin did not affect the favoured chemotactic activity on eosinophils. Semiquantitative RT-PCR was used to investigate the expression of eotaxin in different regions of the bovine ovary. We only detected faint eotaxin mRNA signals that did not indicate physiological significance even in stimulated granulosa cell cultures, follicle-derived macrophages or fibroblasts. Taken together, bovine eotaxin attracts eosinophils in vitro but is not responsible for eosinophilia in the ovary. Its unusual chimeric structure confirms the unity of the MCP/eotaxin subfamily of CC chemokines and distinguishes it from other CC chemokine subfamilies.     

Cleavage of bovine immunoglobulin G1 in whey by an extracellular material from Brucella abortus.

Culture extracts of in vitro grown Brucella abortus were demonstrated to cleave a part of the Fc portion of bovine immunoglobulin G1 in whey but not in serum or as a purified protein from serum. Supernates from Strains 19 and 2308 of B. abortus were both capable of this hydrolysis whereas living cells were not. The cleavage process was independent of antibody activity to B. abortus, appeared to require factor(s) found only in some whey samples and was ineffective with the other bovine immunoglobulins.     

Dictyocaulus species: cross infection between cattle and red deer

Aim: To discover whether cross infection between red deer (Cervus elaphus) and cattle is possible with either a bovine isolate of the cattle lungworm, Dictyocaulus viviparus, or with a cervine isolate of the lungworm, Dictyocaulus eckerti which is thought to be maintained primarily in deer. Method: Twelve cattle and 12 red deer were reared parasite-free from birth. At 3-4 months of age, half of each species (n=6) were experimentally infected with D. viviparus and the other half with D. eckerti. The course of infection was monitored for 34 days, after which the animals were slaughtered and the lungs removed to assess levels of infection. Results: Faecal larval counts demonstrated that patent Dictyocaulus infections occurred in all groups. At necropsy, adult worms were found in the lungs in all groups except the cattle that were infected with D. eckerti. The largest numbers of adult worms were found in the red deer infected with D. eckerti. Conclusion: It was demonstrated that both cattle and red deer could be infected with either D. viviparus or D. eckerti. However, D. eckerti larvae that originated from deer established more successfully in deer and D. viviparus larvae that originated from cattle established more successfully in cattle.     

Acute phase proteins in response to Dictyocaulus viviparus infection in calves

Three experiments were carried out to examine the acute phase response, as measured by the acute phase proteins (APP) haptoglobin, serum amyloid A (SAA) and fibrinogen, in calves infected with lungworm, Dictyocaulus vivparus. In addition, eosinophil counts were analysed. Three different dose models were used in 3 separate experiments: 1) 250 D. viviparus infective third stage larvae (L3) once daily for 2 consecutive days, II) 100 D. viviparus L3 once daily for 5 consecutive days, and III) 2000 L3 once. All 3 dose regimes induced elevated levels of haptoglobin, SAA and fibrinogen, although there was considerable variation both between and within experiments. A significant increase was observed in all 3 APP at one or several time points in experiment I and III, whereas in experiment II, the only significant elevation was observed for fibrinogen at one occasion. The eosinophil numbers were significantly elevated in all 3 experiments. The results show that lungworm infection can induce an acute phase response, which can be monitored by the selected APP. Elevated APP levels in combination with high numbers of eosinophils in an animal with respiratory disease may be used as an indicator of lung worm infection, and help the clinician to decide on treatment. However, high numbers of eosinophils and low levels of APP do not exclude a diagnosis of lungworm. Thus, lungworm infection may not be detected if measurements of APP are used to assess calf health in herds or individual animals.     

In vitro pre-exposure of Haemonchus contortus L3 to blood eosinophils reduces their establishment potential in sheep

Different authors have reported that eosinophils are capable of immobilising infective larvae of different species of nematodes in vitro. However, classifying larvae as mobile or immobile is so subjective that it does not always mean all apparently immobile larvae are dead or those that are mobile are capable of surviving further immune responses if administered to their natural hosts. The objective of this experimental study was therefore to substantiate the role of eosinophils in the killing of Haemonchus contortus infective larvae by comparing the infectivity in sheep of larvae that had been incubated with eosinophil-enriched cell suspensions with control larvae. Since it was not possible to isolate pure eosinophils from sheep blood, we were compelled to evaluate the effects of other blood cells contaminating our eosinophil-enriched suspensions. Although eosinophils and neutrophils were the only cells found adherent to H. contortus infective larvae in vitro, induced eosinophils in the presence of immune serum were primarily responsible for the drastic reduction in larval motility compared to the minor effects of neutrophils and mononuclear cells. Corresponding reductions in faecal egg count and worm numbers were observed when the incubated larvae were transferred intra-abomasally to sheep. Interestingly, the proportion of larvae that failed to establish was much higher following incubation with induced eosinophils compared with other cells or with immune serum alone. Although this study did not address the in vivo role of eosinophils in sheep, the results strongly indicate that sheep blood eosinophils have a larval killing potential in vitro, and a larval mobility test alone may not fully explain the level of damage inflicted on the larvae.     

Bovine polymorphonuclear neutrophil-mediated phagocytosis and an immunoglobulin G2 protease produced by Porphyromonas levii.

Acute interdigital phlegmon (AIP) is a commonly occurring anaerobic bacterial infection in cattle. This study examined in vitro the interaction of bovine polymorphonuclear granulocytic neutrophils (PMN) from blood with bacterial species involved in AIP. Polymorphonuclear neutrophils were purified from whole bovine blood, exposed to one of the three putative etiologic agents of AIP and comparatively assessed for phagocytosis using light microscopy. Fusobacterium necrophorum and Prevotella intermedia were effectively phagocytosed by PMN, but Porphyromonas levii was phagocytosed significantly less effectively by PMN. The effect of high titre anti-P. levii bovine serum on antibody-mediated phagocytosis by PMN was also evaluated. High titre serum increased the efficiency of phagocytosis of P. levii by bovine PMN. This was independent of heat labile complement factors. Antibodies specific for P. levii were assessed for protease activity capable of cleaving bovine immunoglobulins (IgG, IgG1, IgG2, and IgM). Partially purified supernatant from broth cultures of P. levii were incubated with biotinylated immunoglobulins (Igs). Samples were taken from times 0 to 72 h and examined using SDS-PAGE followed by Western blot analysis. Streptavidin-alkaline phosphatase and NBT-BCIP were used to visualize the Igs for heavy and light chains as well as lower molecular weight fragments of these glycoproteins. Porphyromonas levii produced an immunoglobulin protease which readily cleaved bovine IgG into fragments, but did not act against IgM. Specifically, the enzyme may be a significant virulence factor as it may act to neutralize the antibodies demonstrated necessary for effective PMN-mediated phagocytosis.     

A simple isolation procedure for functionally pure components of the bovine alternative complement pathway (ACP) C3 convertase and bovine conglutinin (K)

A simple multicomponent isolation procedure for bovine C3, factor B, factor D and conglutinin (K) from a single serum sample is described. The components of the alternative pathway C3 convertase were isolated in milligram quantities from 800 ml bovine serum and were found to be functionally pure with respect to each other and to factors H and I.     

Analysis of selective mobilization of L-selectin and Mac-1 reservoirs in bovine neutrophils and eosinophils

Following activation of granulocytes, L-selectin (CD62L) is generally shed from the cellular surface, whereas Mac-1 (CD11b/CD18) expression is well known to increase. However, a number of studies in bovines and humans show that the expression of L-selectin may increase as well. This urged us to examine the possible existence of both L-selectin and Mac-1 reservoirs in bovine neutrophil and eosinophil populations through the use of flow cytometry in combination with an optimized method for cell membrane permeabilization. Augmented L-selectin and Mac-1 expression was detected in both granulocyte populations upon saponin treatment. Confocal microscopic studies indicated that both molecules exhibit a different pattern of subcellular localization. Incubation with sialidase revealed the existence of hidden L-selectin epitopes at the cell surface, while no additional Mac-1 epitopes were exposed. Platelet-activating factor stimulation decreased surface and total expression of L-selectin to the same extent in both populations, but solely affected Mac-1 surface expression on eosinophils. Moreover, cytoskeletal actin filaments and microtubules were found to be involved in the regulation of Mac-1 surface expression on bovine neutrophils and eosinophils. In marked contrast, expression of L-selectin was minimally affected by cytoskeleton perturbing agents. The present study indicates that L-selectin and Mac-1 adhesion molecules reside in distinctly located reservoirs in bovine granulocytes and can be selectively mobilized upon in vitro stimulation.     

不同共培养体细胞和胎牛血清浓度对牛体外受精后早期胚胎的影响

利用屠宰黄牛的卵母细胞经体外成熟(IVM)、体外受精(IVF)后的早期胚胎,与单层颗粒细胞(GC)、输卵管上皮细胞(BOEC)等体细胞共培养及在胎牛血清的胚胎培养液中的后续发育进行了研究,并探讨了其影响因素,以期筛选出最佳的体外培养条件。结果表明:使用GC和BOEC体外共培养牛体外受精后胚胎,均取得了较好的囊胚发育率;且牛体外受精后早期胚胎体外培养体系中,添加10%血清能有效地促进牛体外受精后胚胎的囊胚率。     

Colostral growth factors. Possible role in bovine udder epithelial cell regeneration.

Growth of the secretory epithelium during prepartum time, and for a short period after calving, is under hormonal control by estrogen, progesterone and prolactin. The mechanism(s) by which these hormones act is not known but colostrum and milk have been shown to contain different growth promoting substances. In an attempt to unravel these relationships the effect of bovine colostrum on cellular proliferation in vitro have been characterized. Colostral thermostable factors not present in milk nor associated with fat, potently induce the proliferation associated enzyme, ornithine decarboxylase, in fibroblast cell lines. However, mammary epithelial cells appear to proliferate in response to different colostral heat sensitive factor(s) that await further characterization.     

The efficacy of two isolates of the nematode-trapping fungus Duddingtonia flagrans against Dictyocaulus viviparus larvae in faeces.

A series of experiments was carried out to examine the effects of two different isolates of the nematode-trapping fungus Duddingtonia flagrans to reduce the number of free-living larvae of the bovine lungworm, Dictyocaulus viviparus. A laboratory dose-titration assay showed that isolates CI3 and Troll A of D. flagrans significantly reduced (P < 0.05 to P < 0.001) the number of infective D. viviparus larvae in cultures at dose-levels of 6250 and 12,500 chlamydospores/g of faeces. The larval reduction capacity was significantly higher for Troll A compared to CI3 when lungworm larvae were mixed in faecal cultures with eggs of Cooperia oncophora or Ostertagia ostertagi and treated with 6250 chlamydospores/g of faeces. Both fungal isolates showed a stronger effect on gastrointestinal larvae than on lungworm larvae. Two plot trials conducted in 1996 and 1997 involved deposition of artificial faecal pats containing free-living stages of D. viviparus and C. oncophora on grass plots. Herbage around the pats was collected at regular intervals and infective larvae recovered, counted and identified. These experiments showed that both D. flagrans isolates reduced the number of gastrointestinal as well as lungworm larvae in faecal pats. During both plot trials, the transmission of C. oncophora larvae, but not D. viviparus, from faecal pats to the surrounding herbage was clearly affected by climatic conditions. After collection of faecal pats from the grass plots one month after deposition, the wet and dry weight of pats as well as organic matter content were determined. No differences were found between the fungus-treated and non-treated control pats. This indicated that the rate of degradation of faeces was not affected by the addition of the fungus.     

A review of eosinophil chemotaxis and function in Taenia taeniaeformis infections in the laboratory rat

The eosinophil has long been associated with diseases of acute hypersensitivity and with parasite infections, but its exact role in the pathogenesis of these conditions remains uncertain. Characterization of factors associated with migration of eosinophils into tissues has helped to elucidate eosinophil function. Eosinophil chemotactic factors associated with acute hypersensitivity reactions include the eosinophil chemotactic factors of anaphylaxis, histamine, and arachidonic acid metabolites, all of which are released from mast cells, and the lymphokine eosinophil stimulation promoter (ESP). Eosinophilotaxins associated with parasitic diseases include the lymphokine ESP and the low molecular weight factor ECF-G, both associated with schistosome infection in mice. In addition, in several parasite infections parasite-derived protein eosinophil chemotactic factors have been identified and characterized. The proteins associated with Ascaris, Anisakis, and Schistosoma infections appear to be distinct from one another. We have recently partially characterized a protein from Taenia taeniaeformis larvae which has marked chemotactic activity for eosinophils. In addition we have demonstrated eosinophil chemotactic activity associated with metabolism of arachidonic acid by T. taeniaeformis metacestodes. The results of studies in taeniasis and other parasite infections, therefore, indicate that parasite-derived factors may directly influence migration of eosinophils.     

Interactions between lungworms and gastrointestinal worms in calves

Interactions between gastrointestinal worms (Ostertagia ostertagi, Cooperia oncophora) and lungworms (Dictyocaulus viviparus) in calves were studied by assessing the effect of primary infections with either group of worms on the development of homologous or heterologous challenge infections. Primary infections with lungworms resulted in some degree of resistance to challenge with gastrointestinal worms, but this resistance was lower than that found after homologous infection. Primary infections with gastrointestinal worms did not confer any resistance to challenge with lungworms. On the contrary, an indication was found of some enhancing effect of previous gastrointestinal worm infection on the establishment of lungworms. The highest degree of resistance against lungworm challenge was found where calves have been primarily infected with lungworms. Lungworm infections produced some elevation of serum pepsinogen levels. Gastrointestinal worms evoked a rise in circulating eosinophils, although this rise was smaller and occurred later than in lungworm-infected calves. Under the conditions of the experiment, the effect of 6000 infective lungworm larvae on weight gain was larger than the effect of 100,000 L3 of Ostertagia ostertagi and 100,000 L3 of Cooperia oncophora.     

Identification and isolation of a specific antigen with diagnostic potential from Dictyocaulus viviparus.

The purpose of the investigation was to isolate and identify a specific antigen of Dictyocaulus viviparus that can be used to diagnose lungworm infections in cattle. Somatic, excretion and secretion antigens of adult D. viviparus and somatic antigens of L3 larvae were examined in an indirect enzyme-linked immunosorbent assay (ELISA) to determine whether they cross-reacted with sera collected from calves with mono-infections of Fasciola hepatica. Ostertagia ostertagi, Ascaris suum, or Cooperia oncophora. Serum samples containing antibodies directed against F. hepatica, A. suum, and O. ostertagi cross-reacted with somatic antigens of adult D. viviparus; these sera cross-reacted less with excretion and secretion antigens. When somatic antigens of adult D. viviparus were analysed in a Western blot, a 17-kDa protein that did not react with the heterologous sera was detected. This protein was isolated by ultrafiltration and anion chromatography. Sera collected from calves infected with D. viviparus was tested in indirect ELISAs with either somatic antigens of adult D. viviparus or with a low molecular antigen fraction of this preparation containing the 17-kDa protein. The extinction values that were measured in both assays correlated well. We conclude that the 17-kDa protein isolated from somatic antigens of adult D. viviparus may be useful in developing an improved immunoassay to diagnose lungworm infections in cattle.     

A survey on Dictyocaulus viviparus antibodies in bulk milk of dairy herds in Northern Germany

Parasitic bronchitis caused by the bovine lungworm, Dictyocaulus viviparus, occurs worldwide in temperate areas. The parasite is found predominantly in calves and heifers, but dairy cattle can suffer from lungworms when they become infected for the first time or if they have lost immunity due to lack of exposure to lungworm larvae during the grazing season. The present study was performed to determine the D. viviparus bulk milk antibody prevalence in dairy herds in the East Frisian region of northwestern Germany, Lower Saxony, by analysing bulk milk samples collected in January (860 samples), September (866 samples) and November (860 samples) 2008, thereby representing 906 dairy farms. These samples were tested for antibodies against D. viviparus by a milk ELISA. This test detects patent infections only since it is based on recombinant major sperm protein as antigen. While in January 12.8% of dairy farms were positive for D. viviparus antibodies, the bulk milk samples collected in September and November revealed 6.9% and 6.6% positive dairy herds. From the 906 dairy farms included in the study, 191 (21.1%) tested positive at least once for antibodies against lungworm. From 810 dairy farms from which bulk milk samples were obtained during all three samplings, 146 (18.0%) farms were positive at one sampling date, 27 (3.3%) at two, and 4 (0.5%) on all three sampling dates. The majority of the farms represented in the study belonged to four districts of East Frisia, which showed no significant difference in the proportion of positive dairy farms.     

Ultrastructural and cytochemical studies in normal Japanese quail (Coturnix coturnix japonica) eosinophils and in those from birds with experimentally induced eosinophilia

Normal eosinophil development in the Japanese quail (Coturnix coturnix japonica) was similar to that described in the fowl and the duck, with granulogenesis occurring in the Golgi apparatus. The characteristic lipid droplets were small in the immature eosinophils, and after staining specifically for lipid, small moieties were also traced to the Golgi apparatus. In mature eosinophils the lipid droplets measured between 1.0 and 1.5 micron in diameter and they were surrounded by profiles of smooth endoplasmic reticulum. Eosinophilia was difficult to induce in quails; injections of either horse serum or bovine serum albumin (BSA)/aluminium hydroxide produced a poor response. In some quails in which eosinophilia was produced, however, eosinophil granules showed many crescentic and vacuolated forms. The lipid droplets in the activated eosinophils were fused in many cells to form large intracellular aggregates of lipid. Quail eosinophils, which hitherto have been regarded as peroxidase-negative, had strong activity in the lipid droplets of cells from stimulated birds. It is postulated that this peroxidase-positive reaction may represent a form of ceroid or lipofuscin pigment resulting from lipid peroxidation. Acid phosphatase and trimetaphosphatase reactions were reduced in many activated cells, with a large proportion of granules being non-reactive. The results of dietary manipulations in quails appear to suggest that in stressful situations the eosinophil metabolism is altered and there is a reduction in the number of lipid droplets in the cell.