Association of bovine respiratory disease with clinical status and acute phase proteins in calves.

Eighty-four calves with respiratory disease from 18 herds in different parts of Finland were chosen for a study evaluating the capacity of different respiratory pathogens to cause changes in different acute phase protein concentrations, white blood cell (WBC) count and clinical signs. The selected acute phase proteins were fibrinogen, haptoglobin, serum amyloid-A, lipopolysaccharide binding protein and alpha1-acid glycoprotein. From each calf, a paired blood sample was obtained for serological studies of bovine parainfluenza virus-3, bovine respiratory syncytial virus, bovine coronavirus, bovine adenovirus-3 and bovine adenovirus-7. Tracheobronchial lavage was performed to detect bacteria and mycoplasma. Isolation of Pasteurella multocida was associated with increased concentrations of all tested acute phase proteins. For other pathogens, no significant relationships were observed. No association was present between viral or bacterial findings and WBC count.

Viral and bacterial pathogens in bovine respiratory disease in Finland.

Pathogens causing bovine respiratory tract disease in Finland were investigated. Eighteen cattle herds with bovine respiratory disease were included. Five diseased calves from each farm were chosen for closer examination and tracheobronchial lavage. Blood samples were taken from the calves at the time of the investigation and from 86 calves 3-4 weeks later. In addition, 6-10 blood samples from animals of different ages were collected from each herd, resulting in 169 samples. Serum samples were tested for antibodies to bovine parainfluenza virus-3 (PIV-3), bovine respiratory syncytial virus (BRSV), bovine coronavirus (BCV), bovine adenovirus-3 (BAV-3) and bovine adenovirus-7 (BAV-7). About one third of the samples were also tested for antibodies to bovine virus diarrhoea virus (BVDV) with negative results. Bacteria were cultured from lavage fluid and in vitro susceptibility to selected antimicrobials was tested. According to serological findings, PIV-3, BAV-7, BAV-3, BCV and BRSV are common pathogens in Finnish cattle with respiratory problems. A titre rise especially for BAV-7 and BAV-3, the dual growth of Mycoplasma dispar and Pasteurella multocida, were typical findings in diseased calves. Pasteurella sp. strains showed no resistance to tested antimicrobials. Mycoplasma bovis and Mannheimia haemolytica were not found.

Experimental infection of laboratory mice and rabbits with several isolates of Borrelia burgdorferi sensu lato; comparison of antigens from different genospecies in serological measurement of immune responses.

Infectivity of and immune responses to 28 Finnish Borrelia burgdorferi sensu lato isolates was studied in 3-4-week-old outbred NMRI and inbred BALB/c/Hy laboratory mice; rabbits were also inoculated. Twenty-one isolates were found to detectably infect mice. A variation among isolates in degree of infectivity was observed. Higher infection rate and higher average ELISA readings were recorded for intradermal than intraperitoneal inoculations. The results suggest differences between Borrelia genospecies in organotropism. The ear was frequently infected by representatives of all genospecies; among high infectivity experiments, this rate was highest, 100%, in infections by Borrelia afzelii. Further differences between genospecies specific organ distributions: B. burgdorferi sensu stricto and Borrelia garinii isolates seemed to infect the bladder relatively more frequently than B. afzelii did; B. afzelii isolates infected heart relatively more frequently than others did. Genospecies specific differences were demonstrated between antigens in reactivity, i.e. in their 'sensitivity' as reagents of ELISA and IFA methods to measure isolate specific immune responses. Antigens from two B. afzelii isolates differed clearly in sensitivity.

Disease patterns in field and bank vole populations during a cyclic decline in central Finland.

Declining field vole (Microtus agrestis) and bank vole (Clethrionomys glareolus) populations were sampled (117 field voles and 34 bank voles) in south-central Finland during the winter of 1988-89. The last surviving field voles were caught in April and bank voles in February. A subsample (16) of the April field voles were taken live to the laboratory for immunosuppression. The histopathology of the main internal organs and the presence of aerobic bacteria and certain parasites were studied. In the lungs, an increase in lymphoid tissue, probably caused by infections, was the most common finding (52% of all individuals). The prevalences in the voles, in the whole material, of Chrysosporium sp. and Pneumocystis carinii in lungs were 13 and 10% in field voles, and 9 and 0% in bank voles, respectively. Cysts of Taenia mustelae (9 and 27%) were the most common pathological changes in the liver. Enteritis was also rather common (14 and 34%). In field voles the prevalences of Frenkelia sp. in the brain and Sarcocystis sp. in leg muscles were low (both 6%). Bordetella bronchiseptica was commonly (31%) isolated from field vole lungs and Listeria monocytogenes from the intestines (34%). Salmonella spp. could not be found. The dynamics and abundance of inflammations in the lungs and intestines, as well as B. bronchiseptica isolations from the lungs, indicate that obvious epidemics took place in declining vole populations. Of the Luhanka subsample of 16 field voles brought to the laboratory in April, one died of listeriosis, two of Bordetella, and five died for unknown reasons. Even if small mustelids are the driving force in microtine cycles, it is possible that diseases also contribute to the decline.

Experimental infection of cattle with several Borrelia burgdorferi sensu lato strains; immunological heterogeneity of strains as revealed in serological tests.

Twenty-three experimental cattle, mainly calves, were each inoculated 1-3 times with one of ten Finnish Borrelia burgdorferi sensu lato strains. All three genospecies were represented. Borreliae were administered mainly by both intravenous (about 10(6) to 10(9) spirochaetes) and intradermal (10(4)) routes, and on six occasions subcutaneously (10(3)) only. For infectivity control and comparison purposes mice and rabbits were inoculated simultaneously. Immune responses in cattle were monitored both with whole-cell sonicate enzyme-linked immunosorbent assay (IgG-ELISA) and indirect immunofluorescent assay (IgM-IgG-IFA). Five Finnish strains and the American strain B31 were used as antigens. No clinical signs of borreliosis were observed. Of the strains, 7/10 were interpreted by the immune responses to have caused relatively short-term subclinical infections of varying intensity. Borreliae could not be isolated from blood or other organ specimens of cattle. A rough estimate of the mean infectious dose in the conditions of experiments is 10(6) to 10(7) organisms. In conclusion, the overall result appears to argue a low susceptibility of cattle to clinical borreliosis, at least when infected by Finnish strains of the agent. Significant antigen-specific differences were observed both by ELISA and IFA in detection and quantification of immune responses. As a rule, the homologous antigen was found to be the most sensitive. Genospecies differences were mostly distinct. Antigens of two Borrelia garinii isolates proved practically equal in sensitivity, whereas major differences were displayed between two Borrelia afzelii antigens. In an IFA study, an American (B31) and a Finnish B. burgdorferi sensu stricto strain proved equally sensitive as antigens. In two relatively strong primary immune responses the antigen-specific measurement differences were such that diagnostically in a cross-sectional study only the homologous antigen or an antigen of the same genospecies would have been sufficiently sensitive to show a positive result.

Mycoplasma contamination of canine gastric biopsy samples and cultures of gastric Helicobacter spp.

Helicobacter felis and H. bizzozeronii are canine gastric helicobacters related to H. pylori. The growth of gastric helicobacters requires a complex medium including blood and serum. We found that some of our blood agar cultures were contaminated with mycoplasmas, which were isolated from one biopsy sample and several blood agar cultures of canine gastric helicobacters by PCR or culture method. However, none of our other 18 Helicobacter strains, subcultured 10-15 times since 1990, were found to be contaminated when studied in spring 1996. Nor was horse serum used as a growth supporter found to be contaminated with mycoplasmas. All our mycoplasma isolates grew as pure cultures and as cocultures with H. bizzozeronii on a selective medium containing vancomycin, polymyxin B and trimethoprim used in cultivation of helicobacters. Our data suggest that mycoplasmas occurring on biopsy samples can grow as contaminants on Helicobacter cultures.

Characterization of Finnish Borrelia burgdorferi sensu lato isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and with monoclonal antibodies.

Thirty-seven Borrelia burgdorferi strains, isolated in 1992 from Ixodes ricinus in Finland, were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting and indirect immunofluorescence assay (IFA) with five to nine monoclonal antibodies (MAbs). By SDS-PAGE results and reactivities to MAbs H3TS, J 8.3, I 17.3, and D6, the 37 isolates were assigned to the species B. burgdorferi sensu stricto (n = 7), Borrelia afzelii (n = 17), or Borrelia garinii (n = 13). Twenty more isolates examined only by IFA and with part of the MAbs were distributed as follows: 9 B. burgdorferi sensu stricto and 11 other species. Among 16 of 37 isolates displaying a SDS-PAGE patterns considered typical of that of B. garinii, 3 were negative by the test with MAb D6; the rest were positive. The three MAb D6-negative isolates reacted with MAb J 8.3 but not with MAb I 17.3. It is suggested that these isolates of a previously undescribed type represent atypical B. afzelii strains deficient in the expression of OspB proteins. The misleading species designation by the SDS-PAGE result is described. The IFA results were generally consistent with those obtained by immunoblotting. The exception was for 3 of 29 isolates that were positive with MAb H5332 by immunoblotting but that were IFA negative. In the present material of 57 strains, all 16 B. burgdorferi sensu stricto isolates originated from the Aland Islands. B. afzelii and B. garinii were isolated from all three regions where ticks were collected. The distributive difference seems to offer a basis for comparative clinico-epidemiological studies of Lyme borreliosis.

Prevalence of Borrelia burgdorferi in selected tick populations in Finland.

The purpose of the studies was to determine the prevalence of Borrelia burgdorferi sensu lato in selected populations of Ixodes ricinus in Finland and to secure strains of the spirochete for further characterization. 1,210 Ixodes ricinus ticks (399 females, 419 males and 392 nymphs) were collected during June to August 1992 by flagging from 8 sites in 3 regions. The frequency of B. burgdorferi infection was determined by isolation in BSK II medium. The species identity of most of the isolates was confirmed with the immunofluorescence method. 67 B. burgdorferi strains were isolated from 8 sites; the prevalence ranged from 2.8% to 7.9%. The overall isolation percentage for adult male ticks was 6.4% (7.5% for females, 5.3% for males); for nymphs, 3.8%. No statistically significant association of the prevalence was observed with either pasture or off-pasture habitats nor with specific geographic region/regions. Prevalence figures were roughly of the same magnitude in areas and parts of the country known to differ in their incidence of human borreliosis. An additional 294 ticks, mainly engorged females, were collected from places outside the main study sites. Of the 7 positive ticks 3 were engorged females, originating from a cow, a dog and a cat, respectively. The results in general demonstrate that tick populations in various parts of Finland quite commonly harbour B. burgdorferi.

Growth and interaction of Mycoplasma bovirhinis and Mycoplasma dispar in vitro.

Two mycoplasmas were grown in pure and mixed cultures in glucose calf-serum broth with initial pH 7.8. Sensitivity to pH was also tested. The main data for Mycoplasma bovirhinis and Mycoplasma dispar, respectively, in pure cultures were as follows: lag phase, days: less than 1, 1-2; log phase: 1-2, 1-4; relatively stationary phase: 0-1, 2-4; decline phase (to the extent roughly logarithmic): 2-6, 5-10; maximal titers: 5 x 10(7) to 10(9), 5 x 10(6) to 10(8) color changing units per 0.2 ml; highest pH, approximately, at which decline started: 7.7 and 7.0; definitely toxic initial pH: 5.6, 6.8; relative production of acidity; less, more. Decline either shortly ended in loss of viability or, correlated with higher pH levels, led to a prolonged maintenance in lower numbers. The decline of M. bovirhinis was postulated to be essentially caused by an autotoxic product/products other than H+. In mixed cultures an antagonistic effect due to the faster growing M. bovirhinis against M. dispar was recorded. The effect varied according to the initial numbers of organisms and their ratio. Two mechanisms seemed to be active: 1. decrease of pH somewhat below neutrality led to the death of the sensitive M. dispar; 2. M. dispar, when present in relatively high initial numbers, was inhibited by M. bovirhinis during the latter's logarithmic growth at pH-levels above 7.0, the inhibition ending shortly afterwards. A rapidly inactivated product/products of M. bovirhinis metabolism, inhibitory to M. dispar was posited. The results offer an improved insight into diagnostic practices for M. dispar.

Inhibition of growth of Mycoplasma dispar by DL-alpha-difluoromethyllsine, a selective irreversible inhibitor of lysine decarboxylase, and reversal by cadaverine (1,5-diaminopentane).

DL-alpha-Difluoromethyllysine was shown to be a potent inhibitor of lysine decarboxylase from Mycoplasma dispar. The inhibition appeared to be specific since neither difluoromethylornithine nor difluoromethylarginine, known to inhibit other decarboxylases, inhibited the reaction catalyzed by lysine decarboxylase in extracts of M. dispar. Inhibition was irreversible since extensive dialysis could not overcome the inhibitory effect exerted by difluoromethyllysine. Difluoromethyllysine (1 mM) also totally blocked the growth of M. dispar when added at the beginning of the growth period, while 1 mM cadaverine, the product of the reaction, reversed this inhibitory effect when added to the culture medium. When difluoromethyllysine was added during the logarithmic growth phase of Mycoplasma, it inhibited the increase of the growth; 1 mM cadaverine again partially reversed this inhibitory action.