The natural resistance of cattle to artificial infection with Cowdria ruminantium: the role played by conglutinin

The conglutinin titres of year-old Bonsmara-cross cattle infected with Cowdria ruminantium were inversely proportional to the severity of the reactions elicited by the infection. There was no correlation, however, between conglutinin levels of 8-month-old calves of the same breed, sex and origin and their susceptibility to heartwater. The role possibly played by conglutinin in the non-specific resistance of cattle to heartwater and in the epidemiology of the disease is discussed.     

Consequences of eicosapentaenoic acid (n-3) and arachidonic acid (n-6) supplementation on mast cell mediators

Mast cells are important players in the pathogenesis of atopic diseases. These cells release immediate-phase and late-phase mediators of inflammation. Fatty acids are incorporated in cellular membranes and therefore seem to influence mediator production and release. A study was conducted to assess the effects of eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (AA, 20:4n-6) on mast cell mediators in a canine mastocytoma cell line (C2). Cells were cultured in a basic medium (Dulbecco's modified Eagle's medium/HAM's F12 1 : 1, DEH), DEH supplemented with 14.0 microm EPA (DEH-EPA) or 14 microm AA (DEH-AA). The DEH-AA cultured cells had increased spontaneous and mastoparan-stimulated PGE2 production and histamine release. Furthermore, the tryptase activity was increased. The DEH-EPA cultured cells rendered elevated levels of PGE2 and histamine release compared with DEH only after stimulation. These levels were significantly lower in comparison to DEH-AA. The increased PGE2 production of C2 cultured in DEH-AA is the consequence of the AA enrichment, because AA is the precursor of PGE2. However, the different effects by AA and EPA on mast cell mediators possibly reflect the higher susceptibility of long chain polyunsaturated fatty acids (PUFA) to undergo lipid peroxidation, because it is known that altered cellular redox state influences mediator production and release.     

Mast cells and canine mast cell tumours. A review

This article reviews the literature on mast cells and tumours derived from mast cells in the dog. Mast cells play a central role in inflammatory and immune reactions. Mast cells, normal and neoplastic, contain and release important biologically active substances: heparin, histamine, eosinophilic chemotactic factor and proteolytic enzymes. Mast cell tumours occur in the dog, particularly in the boxer and related breeds, in the skin and less frequently in the intestines. Cytology usually provides an accurate diagnosis, but histological examination adds further information concerning the histologic grade and the completeness of surgical therapy. Cutaneous mast cell tumours should be regarded as potentially malignant and therefore be removed widely (3 cm. margin). Local recurrence, regional and distant metastases together with paraneoplastic disorders may cause the death of the pet. Histologic grading (2 or 3 grades) and clinical staging together with kinetic parameters and breed (boxers have relatively benign tumours) are important prognostic parameters. Based on prognostic criteria, surgical treatment should be completed with adjuvant radiotherapy, corticosteroids and eventually with combined chemotherapy. A novel, promising therapy is the application of the receptor kinase inhibitor. The study of the pathogenesis of mast cell tumours received new impetus by the finding of mutations, deletions and duplications, in exons 11 and 12 of the C-kit oncogene. Further study of physiological and oncological aspects of mast cells are favoured by the availability of mast cells isolated from spontaneous mast cell tumours and of cultured cell lines.     

Mast cells and canine mast cell tumours. A review

Summary

This article reviews the literature on mast cells and tumours derived from mast cells in the dog. Mast cells play a central role in inflammatory and inunune reactions. Mast cells, normal and neoplastic, contain and release important biologically active substances: heparin, histamine, eosinophilic chemotactic factor and proteolytic enzymes.

Mast cell tumours occur in the dog, particularly in the boxer and related breeds, in the skin and less frequently in the intestines. Cytology usually provides an accurate diagnosis, but histological examination adds further information concerning the histologic grade and the completeness of surgical therapy. Cutaneous mast cell tumours should be regarded as potentially malignant and therefore be removed widely (3 cm. margin). Local recurrence, regional and distant metastases together with paraneoplastic disorders may cause the death of the pet. Histologic grading (2 or 3 grades) and clinical staging together with kinetic parameters and breed (boxers have relatively benign tumours) are important prognostic parameters. Based on prognostic criteria, surgical treatment should be completed with adjuvant radiotherapy, corticosteroids and eventually with combined chemotherapy. A novel, promising therapy is the application of the receptor kinase inhibitor. The study of the pathogenesis of mast cell tumours received new impetus by the finding of mutations, deletions and duplications, in exons 11 and 12 of the C‐kit oncogene. Further study of physiological and oncological aspects of mast cells are favoured by the availability of mast cells isolated from spontaneous mast cell tumours and of cultured cell lines.     

Pasteurella haemolytica leukotoxin induces histamine release from bovine pulmonary mast cells.

Pasteurella haemolytica A1 leukotoxic culture supernatant was evaluated for its ability to induce histamine release from bovine pulmonary mast cells isolated by enzymatic dispersion of lung tissue. Histamine was measured by a radioimmunoassay technique. Leukotoxic culture supernatant of P. haemolytica significantly released histamine in a time and concentration-related manner. This effect was lost when culture supernatant was heat-inactivated or preincubated with leukotoxin neutralizing rabbit serum. Preincubation of the mast cells with propranolol or p-bromophenacyl bromide reduced the histamine-releasing effect of leukotoxin, while verapamil enhanced release. Experimental infection of calves with P. haemolytica A1 reduced the total histamine content of pulmonary mast cells recovered at postmortem. Histamine release induced by P. haemolytica leukotoxin is likely an important factor in the pathogenesis of bovine pneumonic pasteurellosis.     

Colonic mast cell infiltration in rats with TNBS-induced visceral hypersensitivity

Colonic mucosal mast cells are implicated in the pathogenesis of visceral hypersensitivity associated with irritable bowel syndromes. This study was designed to investigate the roles of mucosal mast cells in development of an experimental visceral hypersensitivity induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) in rats. TNBS, when injected into the proximal colon through laparotomy, produced a significant decrease in pain threshold of the distal colon to mechanical distention, indicating a visceral hypersensitivity. In the proximal colon that was directly insulted by TNBS, mucosal necrosis and extensive inflammatory cell infiltration were observed with concomitant increase in tissue myeloperoxide (MPO) activity. In the distal colon where distention stimuli were applied, the number of mucosal mast cells significantly increased following TNBS treatment, although neither mucosal injury nor increase in tissue MPO activity was observed. In an organ culture, spontaneous release of a mucosal mast cell-specific protease (RMCP-2) from the distal colon tissue of TNBS-treated rats was significantly larger than that of sham animals. Furthermore, TNBS-induced visceral hypersensitivity was significantly suppressed by subcutaneous pretreatment with a mast cell stabilizer doxantrazole in a dose-dependent manner. These findings suggest that prominent colonic mast cell infiltration associated with an enhanced spontaneous mediator release is responsible, at least partly, for development of visceral hypersensitivity induced by TNBS in rats.     

The use of electro-encephalography and brain biopsy in the clinical diagnosis of heartwater

Both electro-encephalography and brain biopsy were examined as possible aids in the confirmation of a clinical diagnosis of heartwater in field cases. Results showed that electro-encephalographic changes associated with heartwater were non-specific and probably caused by the associated brain oedema. Brain biopsy on the other hand appears to be a reliable technique for the confirmation of heartwater, and the technique if properly performed holds little risk for the animal.     

Problems encountered in the control of heartwater in Angora goats

This preliminary investigation confirmed that when Angora goats were immunized against heartwater, either when kids or young goats, they still had an immunity to heartwater a year later when they were challenged with a vaccine containing Cowdria ruminantium. An overwhelming majority of the uninoculated animals which were challenged at the same time were susceptible to clinical heartwater. Strategic immunization to obtain enzootic stability to heartwater is suggested. In these experiments the immunization of Angora goats was not accompanied by severe losses.     

Mortality percentages related to heartwater and the economic impact of heartwater disease on large-scale commercial farms in Zimbabwe

A postal questionnaire asking for data concerning heartwater deaths was sent to all large-scale commercial farmers in Zimbabwe's lowveld, where heartwater is endemic. One hundred and fifty returns (24% return rate) provided usable data, with 85 farms (57% of sample) reporting heartwater deaths in 1990/91. The median heartwater mortality percentage for calves (0–12 months) was 2.3%. This was statistically significantly different (Wilcoxon signed rank test, P < 0.005) from the median mortality percentage of 0.6% for cattle 13 months and older. Heartwater accounted for 51% of all mortalities on farms reporting heartwater deaths. There were no meaningful correlations between farm or herd size and heartwater mortality percentages. A plot of the farms reporting heartwater deaths found that the heartwater endemic area in 1991 is essentially unchanged from 1979. Inspection of the geographic distribution of farms reporting heartwater showed no obvious patterns, and distances from communal land farms were not significantly correlated to heartwater mortality percentages. An economic model found that the annual financial impact of heartwater on large-scale commercial farms in Zimbabwe to be some Z$19.4 million (1991 prices). The upper bound, that allowed for inflation since 1991, was Z$33.4 million. The lower bound estimate was Z$8.1 million (Z$3 = US$1, 1991 rates). Eighty-nine percent of these costs can be attributed to the cost of dipping. Since it has been shown experimentally that the carrier state of heartwater may not affect the productivity of cattle, intensive dipping may not be the best means of controlling heartwater.     

Problems with the interpretation of epidemiological data in heartwater: a study on 23 farms

In an epidemiological study undertaken on 23 farms where heartwater occurs endemically, it was found that on an overall average, antibodies to Cowdria ruminantium were detected in 64.3% of the cattle, 6 adult Amblyomma hebraeum ticks were counted per animal and 7.0% of ticks were infected with the heartwater agent. It was found that the seropositivity of the animals was determined largely by the tick loads to which they were subjected and that the influence of the tick C. ruminantium infection rate was less evident. There was no parallel between the prevalence of heartwater on the farms and the immune status of the animals. In general, higher tick counts were recorded in herds where strategic tick control is practised than on farms with a total tick control programme. The method of tick control did not, however, appear to influence the immune status of the cattle, the tick infection rate, or the prevalence of heartwater.     

Production of monoclonal antibodies specific for native equine IgE and their application to monitor total serum IgE responses in Icelandic and non-Icelandic horses with insect bite dermal hypersensitivity

Immunoglobulin E forms a minor component of serum antibody in mammals. In tissues IgE is bound by FcvarepsilonRI receptors on the surface of mast cells and mediates their release of inflammatory substances in response to antigen. IgE and mast cells have a central role in immunity to parasites and the pathogenesis of allergic diseases in horses and other mammals. This paper describes the production of several novel monoclonal antibodies that detect native equine IgE in immunohistology, ELISA and Western blotting. An antigen capture ELISA to quantify equine IgE in serum has been developed using two of these antibodies. The mean serum IgE concentration of a group of 122 adult horses was 23,523ng/ml with a range of 425-82,610ng/ml. Total serum IgE of healthy horses was compared with that of horses with insect bite dermal hypersensitivity (IBDH) an allergic reaction to the bites of blood feeding insects of Culicoides or Simulium spp. IBDH does not occur in Iceland where Culicoides spp. are absent, but following importation into mainland Europe native Icelandic horses have an exceptionally high incidence of this condition. In the present study Icelandic horses with IBDH had significantly higher total IgE than healthy Icelandic horse controls (P<0.05). By contrast in horses of other breeds the difference in total serum IgE between those affected with IBDH and healthy controls was not statistically significant. Total serum IgE was also monitored in a cohort of Icelandic horses prior to import into Switzerland and for a period of 3 years thereafter. High levels of serum IgE were present in all horses at the start of the study but dropped in the first year after import. Thereafter the total serum IgE remained low in Icelandic horses that remained healthy but rose significantly (P<0.05) in those that developed IBDH. These results support the conclusion that IBDH is a type I hypersensitivity response to insect allergens but indicate that IBDH in Icelandic horses may have a different pathogenesis from the same condition in other breeds.     

Immunoglobulin E-bearing cells and mast cells in skin biopsies of horses with urticaria

The pathogenesis of equine urticaria is not well understood. In man, urticaria has been associated with immunological and nonimmunological mechanisms leading to the release of various mediators by mast cells. Skin biopsies of 32 horses with a history of urticaria were stained with toluidine blue, a double-labelling method for chymase and tryptase, and immunohistochemistry for immunoglobulin (Ig)E. These horses were compared with horses with pemphigus foliaceus, insect bite hypersensitivity and control horses with healthy skin. Neither formalin fixation time nor biopsy site influenced the staining methods. No chymase-positive cells were found. In all groups of horses, cells staining with toluidine blue and for tryptase and IgE were found in the epidermis and hair follicle papilla and significantly more positively staining cells were observed in the subepidermal dermis compared with the deep dermis. Horses with urticaria had significantly more IgE-bearing cells in the subepidermal dermis than control horses. However, horses with urticaria had significantly fewer toluidine-blue-stained mast cells in both subepidermal and deep dermis compared with the insect bite hypersensitivity and pemphigus foliaceus groups. This study suggests that IgE-mediated reactions play a role in the pathogenesis of urticaria.     

Protection of cattle against heartwater in Botswana: comparative efficacy of different methods against natural and blood-derived challenges

Five groups of Tswana-cross castrated male cattle between 20 and 30 months of age (a total of 158 animals) were transported from a ranch in a heartwater-free area of south Botswana to a feedlot near Gaborone in the east of Botswana where heartwater is endemic. On arrival, one group was vaccinated intravenously with the Onderstepoort sheep blood heartwater vaccine, one group was vaccinated intravenously with the new Onderstepoort tick-derived heartwater vaccine and a third group was vaccinated subcutaneously with this tick-derived vaccine. Vaccine reactions were blocked with long acting oxytetracycline on the first day of fever. A fourth group had a series of injections of long acting oxytetracycline on days 0, 7, 14 and 21 after arrival, and a fifth served as untreated controls. The animals remained at the feedlot for 65 days during which time they faced a low level of challenge by Amblyomma hebraeum ticks. None contracted heartwater and so they were then challenged, together with a further group of control cattle, with a dose of the sheep blood vaccine. Some animals in all groups had severe heartwater reactions and died despite therapy, but 76.7 per cent, 64.5 per cent and 74.3 per cent of the cattle in the blood vaccine, intravenous tick vaccine and long acting oxytetracycline groups respectively were resistant to challenge, compared with 48.3 per cent of the subcutaneous tick vaccine group and 36.4 per cent of the controls. It was concluded that intravenous vaccination of susceptible adult cattle with either the blood or the tick-derived vaccine needs careful monitoring in the month after vaccination and does not necessarily result in immune animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Parainfluenza-3 virus-induced enhancement of histamine release from calf lung mast cells – effect of levamisole

Clinically healthy calves were divided into five groups. Group 1 served as control; Group 2 received levamisole (LEV), 3 mg/kg, s.c.; Group 3 was aerosolized with parainfluenza-3 virus (PI-3); Group 4 received LEV and PI-3 and Group 5 was inoculated with Pasteurella haemolytica. They were killed 6 days after virus exposure or 5-6 days after bacterial inoculation. Lung mast cells were prepared by enzymatic treatment. Mast cell histamine (HIST) release was assayed spectrofluorometrically. Total HIST (micrograms/g) in mast cells was as follows (means +/- SEM): control (5.30 +/- 0.26); LEV (5.27 +/- 0.31); PI-3 (6.37 +/- 0.65); LEV + PI-3 (6.21 +/- 0.51); P. haemolytica (7.06 +/- 0.85). Spontaneous HIST release was as follows (% total, means +/- SEM): control (10.38 +/- 1.09), LEV (11.95 +/- 2.13), PI-3v (73.57 +/- 11.97), PI-3v + LEV (19.50 +/- 3.03), and P. haemolytica (70.59 +/- 5.94). Calcium ionophore A23187 (5 X 10(-6) M)-induced release (% total, means +/- SEM) was: 51.53 +/- 3.05, 50.02 +/- 2.70, 83.91 +/- 4.09, 75.21 +/- 4.51 and 70.59 +/- 6.91 for control, LEV, PI-3, LEV + PI-3 and P. haemolytica groups, respectively. Both virus and bacteria increased HIST content of lung mast cells and enhanced ionophore-induced release. Levamisole significantly reduced spontaneous HIST release in virus-infected calves but had no effect on ionophore-induced release. Results suggest a significant role for HIST in pathogenesis of bovine microbial pneumonia and that LEV probably does not modulate non-immunologic release of HIST from bovine lungs.     

Veterinary extension on sampling techniques related to heartwater research

Heartwater, a tick-borne disease caused by Ehrlichia ruminantium, is considered to be a significant cause of mortality amongst domestic and wild ruminants in South Africa. The main vector is Amblyomma hebraeum and although previous epidemiological studies have outlined endemic areas based on mortalities, these have been limited by diagnostic methods which relied mainly on positive brain smears. The indirect fluorescent antibody test (IFA) has a low specificity for heartwater organisms as it cross-reacts with some other species. Since the advent of biotechnology and genomics, molecular epidemiology has evolved using the methodology of traditional epidemiology coupled with the new molecular techniques. A new quantitative real-time polymerase chain reaction (qPCR) test has been developed for rapid and accurate diagnosis of heartwater in the live animal. This method can also be used to survey populations of A. hebraeum ticks for heartwater. Sampling whole blood and ticks for this qPCR differs from routine serum sampling, which is used for many serological tests. Veterinary field staff, particularly animal health technicians, are involved in surveillance and monitoring of controlled and other diseases of animals in South Africa. However, it was found that the sampling of whole blood was not done correctly, probably because it is a new sampling technique specific for new technology, where the heartwater organism is much more labile than the serum antibodies required for other tests. This qPCR technique is highly sensitive and can diagnose heartwater in the living animal within 2 hours, in time to treat it. Poor sampling techniques that decrease the sensitivity of the test will, however, result in a false negative diagnosis. This paper describes the development of a skills training programme for para-veterinary field staff, to facilitate research into the molecular epidemiology of heartwater in ruminants and eliminate any sampling bias due to collection errors. Humane handling techniques were also included in the training, in line with the current focus on improved livestock welfare.     

The control of heartwater on large-scale commercial and smallholder farms in Zimbabwe.

As part of a series of studies associated with the development of improved vaccines for heartwater (a tick-borne disease of ruminant livestock caused by Cowdria ruminantium), field surveys were carried out to assess losses associated with the disease and the costs associated with controlling it in the two main agro-ecological zones of Zimbabwe (lowveld and highveld) where heartwater is believed to be endemic and epidemic, respectively. In each zone, a cross-sectional study was performed in the main farming systems (smallholder (SH) and large-scale commercial (LSC) beef and dairy), followed by longitudinal studies in the same sectors to improve data accuracy for some parameters. Suspected heartwater-specific mortality in cattle was similar in all LSC sectors (p = 0.72) accounting for a median 1% mortality risk. Heartwater-specific mortality in SH areas was not assessed due to poor diagnostic ability of the farmers. Few LSC farms and SH households kept sheep; suspected heartwater-specific mortality in LSC sheep was 0.8% in the lowveld and 2.4% in the highveld. Goats were a major enterprise in SH areas but not on LSC farms. Suspected heartwater mortality in LSC goats was 0.8% at one site in the highveld and 17.5% on a farm in the lowveld. Application of acaricides was the major control method for heartwater and other tick-borne diseases on both SH and LSC farms. On LSC farms, plunge dipping was used most frequently and the number of acaricide applications ranged widely between 3 and 52 per year. The total cost of acaricides per head per annum was higher in highveld dairies than in highveld and lowveld beef enterprises (p = 0.03). In SH areas, cattle plunge dipping was conducted by the government with an average frequency of 8 +/- 2 (sd) immersions per annum in both the lowveld and highveld. The type of tick control on sheep and goats in all production systems was highly variable (ranging from none to hand removal or intensive acaricide treatment). Suspected heartwater cases on LSC farms were treated with tetracyclines; treatment was not reported in SH areas. Reported treatment costs were high (median Z$ 120) and highly variable (range Z$-833). Vaccination against heartwater with the live, blood-based vaccine was reported on only one LSC farm. LSC farms applying acaricide 30 or more times per year reported higher morbidity (p < 0.0001) and mortality (p < 0.0001) than farms applying acaricides less than 30 times a year. This finding supports the use of reduced tick control in the management of heartwater in Zimbabwe.     

T cell-mediated immunity to Cowdria ruminantium in mice: the protective role of Lyt-2+ T cells

The inability of athymic nude mice to make a drug-aided recovery from infection with either the Kümm or the Welgevonden stocks of Cowdria ruminantium and their inability to mount an immune response, suggest that immunity in heartwater is cell-mediated. The adoptive transfer of immunity with the spleen cells of mice immune to the Welgevonden stock is supportive evidence. Immune spleen cells depleted of Lyt-2+ T cells are unable to confer resistance to challenge to recipient mice, whereas the depletion of L3T4+ T cells had no effect on the protection conferred by immune spleen cells. This is conclusive evidence that immunity in heartwater is largely cell-mediated. Immune serum, C. ruminantium and complement incubated in the presence of mouse peritoneal macrophages, inhibits the infectivity of the heartwater agent, but not in the absence of macrophages. The decreased resistance to challenge of immune mice treated with gloxazone adds further support to the concept that in heartwater persistence of C. ruminantium in the host is associated with immunity.     

Endocytosis of erythrocytes in vivo and particulate substances in vitro by feline neoplastic mast cells.

Clinical evidence for the phagocytic capability of neoplastic feline mast cells was provided by recognition of endocytosed erythrocytes in seven of 12 cytological smears of mast cell neoplasms, particularly in those cells collected from splenic tumors. The capability of these neoplastic mast cells to endocytose particulate substances was also studied in vitro. Evidence is presented that under cultural conditions, feline neoplastic mast cells are capable of endocytosing a variety of substances including polystyrene latex microspheres, zymosan particles, horse spleen ferritin, salmon sperm nuclei, horseradish peroxidase, and carbon particles.     

Electron microscopy of mast cells in the venous wall of canine liver

Although many mast cells locate under the endothelial layer along the sublobular veins in canine liver, the cell function remains to be fully defined. To establish the nature of the canine mast cell, the mast cells were examined by electron microscopy. A few monocytes contacted with luminal surface of endothelial cells under which mast cells situated. To confirm the chemotaxis of monocyte by hepatic mast cells, the hepatic venous vessels were treated with a histamine releaser (compound 48/80). The monocytes invaded into the subendothelial layer and extended their pseudopodium to the degranulated mast cells. It presumes that some mediators within the mast cell granules might act as a chemotactic substance to the monocyte. On the contrary, mast cells were migrating from subendothelial layer to venous lumen under normal condition. The migrating mast cell showed strong acid phosphatase reaction in their granules. It suggests that the granules of migrating mast cell became visible to acid phosphatase activity by a physical force such as contact stimulation, and that a part of mast cells remigrate from the venous wall to other places by the blood flow. Furthermore, hepatic mast cells were revealed to contain both endothelin-1 and histamine in their granules by immunocytochemistry. As these substances have an activity of stronger venous constriction, it seems that the mast cells play an important role in the blood flow regulation of the canine liver, mast cell, monocyte.     

Diagnosis of heartwater in the live animal: experiences with goats in Guadeloupe

A presumptive diagnosis of heartwater in the living animal can be based on clinical and epidemiologic observations. In Guadeloupe, heartwater can be confused with haemonchosis in goats or cerebral babesiosis in cattle. Confirmation of the clinical diagnosis by brain biopsy is useful in experimental infections but is hardly applicable in the field. Positive results were obtained from 92% of animals 16 to 18 days after experimental infection. In febrile animals the best results were obtained between the 3rd and 6th days of the thermal response. Diagnosis can also be supported by serological tests. These are useful for monitoring experimental infections and for checking recovered animals in the field. Nineteen goats out of 27 were negative on Day 1 of the febrile reaction but positive a week later. The remaining 8 goats were positive on Day 1 and had greatly increased antibody titres a week later. Confirmation of a diagnosis can also be achieved by subinoculating into susceptible animals either blood or suspensions of ticks collected from suspect animals and then homogenized. Ticks that have engorged on a suspect animal can be allowed to moult and then fed on a susceptible animal to test their infectivity. These methods are time consuming but useful for heartwater surveys.