Differentiation Among Strains of Goat Mycoplasma by Incident Light Immunofluorescence

Antigenic differentiation between strains of goat mycoplasma was studied by direct fluorescent antibody reactions employing incident (vertical) ultraviolet light. Agar colonies of the mycoplasma grown in petri dishes were fixed by alcohol in situ, and stained with conjugated globulin before examination with ultraviolet light.

The fluorescent antibody (FA) conjugate against Vom strain of Mycoplasma mycoides var. capri was Vom strain-specific, no cross reaction with Mexico, Connecticut, or Maryland strains. Similarly, the Mexico strain conjugate was specific for colonies of Mexico, and did not cross with the Vom, strain. Additionally, the conjugate of the PG-2 strain of Mycoplasma agalactiae, which was specific for the colonies of PG-2 was refractory for the strain #99 of M. agalactiae.

It was therefore possible to utilize an immunofluorescent technique (incident ultraviolet light) to demonstrate differences among strains of M. mycoides var. capri and M. agalactiae.

     

Comparative Study of Agar Mediums for Propagation of Ruminant Mycoplasma

A brain heart infusion agar supplemented with 16.7% rabbit serum (BHIR) was found the most suitable for the culturing of ruminant mycoplasma. Gourlay medium and Perreau medium (4, 5) were not suitable for growth of Mycoplasma mycoides var. mycoides or M. agalactiae, but were satisfactory for M. mycoides var. capri.

Four strains of M. mycoides var. mycoides, three strains of M. agalactiae and three strains of M. mycoides var. capri were grown in our laboratory.

     

Enzootic Pneumonia of Pigs: Complement-Fixation Tests for the Detection of Mycoplasma Antibodies in the Serum of Immunized Rabbits and Infected Swine

The direct, the modified direct and the indirect complement-fixation tests were investigated as methods for the detection of antibodies for the enzootic pneumonia mycoplasma and for Mycoplasma hyorhinis in the serum of infected pigs and of immunized rabbits.

Only the modified direct complement-fixation test in which the guinea-pig complement is supplemented with fresh, normal unheated calf serum was suitable for the detection of mycoplasma antibodies in sera of infected swine. Based on the close correlation between the production of typical lung lesions in experimentally infected pigs and the appearance of significant serum antibody titres, the modified direct complement-fixation test provides for the first time a sensitive, specific in vitro method for the detection of enzootic pneumonia in the live pig. This test also permitted the in vitro differentiation of the mycoplasma causing enzootic pneumonia from M. hyorhinis which causes polyserositis.

Antibodies in the sera of rabbits were demonstrable by the ordinary direct complement-fixation test. However, in contast to the observation made with swine sera, only a slight quantitative antigenic difference between the enzootic pneumonia mycoplasma and M. hyorhinis was seen when the tests were performed with rabbit serum antibodiies.

     

Characterisation of protein and antigen variability among Mycoplasma mycoides subsp. mycoides (LC) and Mycoplasma agalactiae field strains by SDS-PAGE and immunoblotting

Mycoplasma mycoides subsp. mycoides (LC) (Mmm LC) and Mycoplasma agalactiae are the most important mycoplasma species involved in the contagious agalactia syndrome. A total of 25 field strains from Spain and the two type strains were analysed by SDS-PAGE and immunoblotting. Two polyclonal antisera (PAbs) raised against a pool of strains of each mycoplasma species were used. The results revealed a high degree of protein variability among the field strains. The type strain of Mmm LC appeared to be representative of the field strains of this species, whereas this was not the case with the M. agalactiae type strain. Whereas M. agalactiae is known to possess a gene family regulating surface antigen diversity, there is a need to study the mechanisms used byMmm LC to generate antigenic variability in more detail.     

Enzootic Pneumonia of Pigs: Identification of a Causative Mycoplasma in Infected Pigs and in Cultures by Immunofluorescent Staining

Immunofluorescent staining has been used to identify Mycoplasma hyopneumoniae in smears of broth cultures, in infected pig testicle cell cultures, and in frozen cut sections of pneumonic lungs from field and experimentally produced cases of enzootic pneumonia. In the pneumonic pig lung, fluorescent staining was limited to the surface of the bronchial and bronchiolar epithelium and to the contained exudate. In a series of trials using experimentally infected pigs fluorescence was not detected until 25 days post-infection and was regularly seen in pigs killed thereafter. Porcine immune globulin precipitated from the serum of experimentally infected pigs and conjugated with fluorescein isothiocyanate was reactive and specific for the detection of M. hyopneumoniae. Immune globulin conjugates prepared from the serum of hyperimmunized rabbits were reactive but in some cases produced a faint non-specific staining of frozen tissue sections. No such non-specific reactions were noted on stained culture smears or cell cultures.

Fluorescence was not seen in known positive preparations stained with non-immune pig globulin conjugates or in preparations from uninoculated cell cultures or pigs, stained with non-immune or immune globulin conjugates.

Mycoplasma hyorhinis was detected by immunofluorescent staining with homologous conjugates, in smears of broth cultures and in tissue sections from pigs with polyserositis.

Immunofluorescent staining was found to be species specific and useful for the early species identification of mycoplasma isolated from pigs.

     

Vibriosis: Demonstration of Vibrio fetus and Vibrio bubulus Organisms in Preputial Fluid by Immunofluorescence and Cultural Techniques

Fluorescent conjugates were prepared from the sera of calves immunized with four Vibrio fetus strains and one Vibrio bubulus strain. The fluorescent antibody technique (FAT) was then used to detect vibrio organisms in preputial fluid collected from 67 bulls belonging to a Canadian artificial insemination (AI) unit. The V. fetus conjugates reacted with both V. fetus var venerealis and V. fetus var intestinalis. V. fetus was found in 20 animals (29.9%), 13 of which also harboured V. bubulus. In two cases, the FAT failed to detect V. fetus which was isolated by concurrent bacteriological examinations.

It was concluded that the FAT can be a rapid method of detecting some carrier bulls but more reliable results are obtained when a combination of FAT and bacteriological methods is employed. It was found that a single sample giving negative results is inconclusive and additional tests are required before making a final diagnosis. The FAT can also be used to differentiate V. fetus isolates from V. bubulus.

     

A Mycoplasma Isolated from Cattle with Infectious Bovine Keratoconjunctivitis

A mycoplasma has been recovered from the eyes of calves in two naturally-occurring outbreaks of infectious bovine keratoconjunctivitis; also from a third group of calves accidentally exposed to an animal which had ocular exudates from one of the outbreaks instilled into its eyes.

The severity of the ocular lesions in infectious bovine keratoconjunctivis outbreaks may be related to a mixed infection with the mycoplasma and Moraxella bovis.

Preliminary typing studies indicate the mycoplasma is not serologically related to any known bovine mycoplasma.

     

Bovine Infectious Keratoconjunctivitis: Serological Aspects of Moraxella bovis Infection

A modification of a gel diffusion precipitin test (GDPT) was used to detect antibodies for Moraxella bovis (M. bovis) in the sera of cattle affected with bovine infectious keratoconjunctivitis (BIK). The test was also used for the detection of sequential antibody development in cattle vaccinated with cultures of M. bovis. Also, strains of M. bovis isolated from cattle herds affected with BIK were characterized serologically as a part of an identification scheme using the test.

A comparison of the antigenic properties of various strains of M. bovis and M. bovis-like organisms was conducted using the test. The results indicated that there might be antigenic relationships between M. bovisand M. bovis-like organisms such as Moraxella liquefaciens, Moraxella nonliquefaciens, an unidentified hemolytic diplococcus, Mima polymorpha, Mima polymorpha var. oxidans and Herellea vaginicola

The authors suggest that the GDPT can be used for serological studies of BIK, and the identification and antigenic analysis of M. bovis. They indicate, however, that a more definitive study is needed to evaluate the reliability of the test for quantitative work.

     

Investigations of Outbreaks of Contagious Caprine Pleuropneumonia in Eritrea

Mycoplasmas were isolated from freeze-dried lung samples from goats from the western lowlands of Eritrea suspected of being affected by contagious caprine pleuropneumonia. The goats belonged to two herds in which mortality and morbidity rates were high. Mycoplasma capricolum subsp. capripneumoniae was identified in most samples by the polymerase chain reaction and by conventional serological tests. The latex agglutination test detected more positive serum samples in both herds than did the complement fixation test. Following cloning, the isolates of M. capricolum subsp. capripneumoniae were analysed biochemically and shown to be metabolically similar. They oxidized glucose, N-acetylglucosamine, pyruvate and L-lactate with high affinity and mannose, glucosamine and 2-oxobutyrate with low affinity; they were unable to utilize maltose, trehalose, fructose or ethanol. Major improvements were seen in the growth yield of the Eritrean strains with the addition of pyruvate to the medium. Thus, it may be that organic acids are important energy sources for these strains and may be used in addition to or in place of glucose. In contrast to most other strains of the M. mycoides cluster, the Eritrean strains produced large amounts of hydrogen peroxide during the oxidation of NADH by lysed cells. This characteristic had previously been reported for strain M. F38, the type strain of M. capricolum subsp. capripneumoniae, although strain F38 did not metabolize sugars. Hydrogen peroxide has long been considered a pathogenicity factor in mycoplasma infections. This is the first isolation of M. capricolum subsp. capripneumoniae from Eritrea.     

Mycoplasma detection and isolation from one-humped camels (Camelus dromedarius)

In scientific literature, a small amount of information is found concerning mycoplasmosis in camel species. Mycoplasma (M.) arginini, Acholeplasma (A.) laidlawii, and Acholeplasma oculi have been reported to be isolated from these host species. Serologically positive results have been reported for Mycoplasma mycoides subsp. mycoides SC type, Mycoplasma capricolum subsp. capripneumoniae, and M. mycoides subsp. capri. The aims of this study were to detect, isolate, and identify mycoplasmas from camels (Camelus dromedarius). Initially, saliva and ear smears plus conjunctival and vaginal secretions were taken from five female animals, but only conjunctival secretions in three male animals, all belonging to the same farm. An unknown mycoplasma was isolated from one of the vagina samples. Additionally, another unknown and uncultured mycoplasma was detected with molecular biology in the same sample. In the second stage, 23 vaginal secretions were taken from the same farm plus another secretion from a different one. Ten isolates of the same unknown and previously isolated mycoplasma were detected, nine of them recovered from the vagina of female camels. Some mycoplasmas have been related to reproductive disorders; however, there is no evidence that the isolated mycoplasmas are related to such disorders.     

Haemadsorption inhibition test for the identification of Mycoplasma gallisepticum and Mycoplasma synoviae

Summary

Colonies of the avian mycoplasma strains Mycoplasma gallisepticum S6 and Mycoplasma synoviae WVU 1853 and two Mycoplasma synoviae isolates from this laboratory were shown to be haemadsorption positive for chicken erythrocytes. Three Mycoplasma synoviae isolates from this laboratory proved to be haemadsorption and haemagglutination negative. The haemadsorption of the mycoplasma colonies mentioned above was inhibited with specific antisera of either high or low titre. No cross‐inhibition was observed. It is suggested that this test could be used for a quick tentative identification of the two avian mycoplasmas on primary solid‐medium cultures.     

Minimal Inhibitory Concentrations of Twenty Antimicrobial Agents to Animal Pathogens

Minimal inhibitory concentration (M.I.C.) values as determined by an agar-plate-dilution method for 60 bacterial isolates, consisting of Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus of animal origin against 20 antimicrobial drugs are presented. Of all the drugs, gentamicin had the best in vitro antibacterial activity in terms of M.I.C. when considering all the species of organisms together, while spectinomycin had the least activity.

An inoculum replicator was a convenient tool in carrying out the agar-plate-dilution method.

A comparison of the M.I.C. values of 42 isolates of S. typhimurium with the results obtained by low level method and the Bauer-Kir-by method showed that with few exceptions, there is a general agreement.

     

Field trial of a combined vaccine against caprine contagious agalactia: humoral immune response in lactating goats

Two vaccines against Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (large colony type) were developed using inactivated strains selected in previous characterization studies. Formaldehyde and phenol were used as the inactivating agents for vaccines A and B, respectively. Aluminium hydroxide plus purified saponin (Quil-A) were added to both vaccines as adjuvant. The field trial was designed to evaluate the specific humoral immune response to the two mycoplasma species in lactating goats over a period of 7 months. The vaccines were tested on 120 goats randomly assigned to three groups of 40 animals each. Two groups received two injections of vaccine A or B respectively, and a third group remained in the herd as control. Antibody titres determined by ELISA indicated a significant difference between both vaccines and the control group over a 6-month period. Immunoblotting assays also revealed the production of antibodies against the two mycoplasma species. Further field trials are underway to evaluate the efficacy and protection conferred to the animals by these specific antibodies.     

Response of Gnotobiotic Pigs to Escherichia coli

In a study of the response of gnotobiotic pigs to coliform infections, 45 one-week-old germfree pigs were divided into five groups and each group was inoculated orally with a different strain of Escherichia coli. Three of these were enteropathogenic swine strains, P307[08:K87(B), K88 a,b (L):H19]; P570 [0138:K81]; P568[0141:K85a,b(B), K88a,b(L):H4], one was a virulent human strain, H224, [026:K60(B6)], and one was a non-enteropathogenic swine strain, P581[OX13:K68]. It was attempted to protect a portion of the pigs with orally administered specific antisera and sera from non-immunized specific pathogenfree (SPF) pigs. Observations were made on the clinical response, bacterial counts of feces and intestinal contents, gross pathological changes, distribution of the organisms in organs and serum hemagglutinin titers.

Infection with E. coli P307 resulted in diarrhea, dehydration and death, unless the pig was protected with specific antiserum. The pigs infected with E. coli P570 had a transient diarrhea but retained their appetites and recovered. Those infected with the other three strains remained healthy throughout. No circulating hemagglutinating antibody against the test strains of E. coli could be detected in any of the pigs seven days or earlier post-inoculation.

Relationship could not be established between the numbers of viable E. coli in the feces and the presence of clinical colibacillosis. Orally administered specific antiserum afforded protection against strain P307, but did not reduce the number of E. coli in the gut or alter their distribution in the internal organs. This suggested that the protective effect of specific antibody in the intestine was due to its action on a metabolite (enterotoxin) produced by E. coli P307 rather than the organism itself.

     

Heterogeneity of Mycoplasma dispar.

Seventy bovine mycoplasma strains recovered from cases of calf pneumonia, and all displaying the cultural characteristics of Mycoplasma dispar, were compared to the type strain of this species by the disc growth inhibition test, the metabolism inhibition test and indirect epi-immunofluorescence test applied to colonies on agar. Sixty-seven strains were found to be identical with M. dispar. The remaining three strains formed a distinct serogroup partially separate from the type strain of M. dispar, but the difference from the type strain was not considered great enough to warrant the establishment of a subspecies.     

Experimences with a lyophilised contagious bovine pleuropneumonia vaccine in the ivory coast

Summary Trials on a lyophilised vaccine prepared fromM. mycoides var.mycoides strain T1, were carried out on humpless N'dama and Baoulé cattle and on zebu cattle. Post-vaccinal complications in some animals made the vaccine unsuitable under prevailing conditions for widespread field use on the humpless cattle in Ivory Coast. However the post-vaccinal lesions can be controlled and the proven immunological effectiveness of this lyophilised product have convinced the author that its use should be gradually extended as staff training and improved facilities permit. The product was acceptable for field vaccination of the zebu herds. The number and extent of local lesions were much reduced in herds revaccinated after one year. Serological findings based on the complement fixation test are discussed.

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Sumario Se realizaron vacunaciónes con una vacuna preparada deM. mycoides var.mycoides cepa T1, en ganado bovino N'dama sin joroba, ganado Baoulé y en ganado cebú. Las complicaciones post-vaccinales en algunos animales hizo que la vacuna no sea la adecuada considerando las condiciones prevalentes para su uso en el campo en el ganado sin joroba ne la Costa de Marfil. Sin embargo, las lesions post-vacunales pueden ser controladas y la probada efectividad inmunológica de este producto liofilizado han convencido al autor de que su uso debería de extenderse gradualmente tanto como el adiestramiento del personal y la mejora de facilidales lo permitan. El producto fue aceptable para la vacunación de campo del ganado cebú. El número y extensión de las lesions locales fue mucho mas reducido en hatos revacunados despues de un a?o. Se discuten hallazgos serológicos basados en la prueba de fijación de complemento.

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Résumé Des essais avec un vaccin lyophilisé préparé avec la souche T1 deM. myco?des var.myco?des ont été effectués sur du bétail zébu et sur du bétail sans bosse des races N'dama et Baoulé. Des réactions post-vaccinales observées chez certains animaux font que ce vaccin n'est pas recommandable pour son emploi à large échelle chez le bétail taurin de la C?te d'Ivoire dans les circonstances actuelles. Cependant ces complications vaccinales peuvent être combattues et l'efficacité immunologique indubitable de ce vaccin lyophilisé a convaincu l'auteur que son emploi devrait être progressivement étendu dans la mesure oú l'entra?nement du personnel et des facilités améliorées le permettraient. Ce vaccin est satisfaisant pour la vaccination de masse dans les troupeaux de zébus. Le nombre et l'importance des réactions locales sont réduites de beaucoup chez les animaux revaccinés après un an. Des données sérologiques obtenues par la méthode de fixation du complément sont discutées.

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Serving on a United Nations Development Programme/Technical Assistance assignment on the control of contagious bovine pleuropneumonia with the Ministry of Animal Production, Republic of the Ivory Coast     

Toxicité et effet immunosuppresseur de la sulfaquinoxaline chez la poule

Secondary effects of sulfaquinoxaline have been studied by comparing specific antibody titres of treated birds with the titres of those birds which were not treated.

Previously these birds had been immunized by intramuscular injections of a killed antigen of S. pullorum, standard strain, and S. pullorum, variant strain. This experiment demonstrated that this drug possesses an immuno-suppressing action. This secondary effect was confirmed by an experimental infection with the GB strain of Newcastle disease virus.

     

Bovine Viral Diarrhea Virus and Escherichia Coli in Neonatal Calf Enteritis

This study was initiated to determine the etiologic and pathogenic significance of an American strain of bovine viral diarrhea (BVD) virus (strain NADL-MD) in enteritis of neonatal calves (calf scours).

Three colostrum-fed calves from dams exposed intravenously to BVD virus at 6, 16 and 25 days prepartum, respectively, had moderate diarrhea persisting until the eighth day of life. The BVD virus was isolated from all 3 calves and persisted up to 93 days in 1 calf, indicating either that BVD was transmitted in utero or via the dam's milk.

Three specific pathogen free (SPF) calves permitted dams' colostrum for the first 4 feedings and then given milk replacer were exposed orally on the day of birth to BVD virus. One calf died of neonatal enteritis 28 hours post-exposure and at necropsy the BVD virus was isolated from several of its organs. The remaining 2 calves had a mild diarrhea persisting to the eighth day of age.

Two calves permitted dams' colostrum ad lib. for 72 hours, and then weaned, were exposed orally to BVD virus. Both calves had a mild persistent diarrhea and BVD virus was isolated from their blood for 56 days post-exposure.

Of 13 SPF, colostrum-deprived calves exposed orally or intranasally at birth to the BVD virus, 4 had severe diarrhea and died of neonatal enteritis from 38 hours to 13 days postexposure. Isolations of BVD virus were made from several of the organs of the calves at necropsy. All of the 9 surviving calves had a moderate to severe diarrhea frequently persisting for 7 to 10 days, and BVD virus was isolated from the survivors up to 103 days postexposure.

Several strains of Escherichia coli were isolated from calves after the second day of life, but were neither pathogenic for mice, nor serologically related to strains of E. coli usually associated with outbreaks of calf scours. Four colostrum-deprived SPF calves were exposed orally at birth to a strain of E. coli isolated from the intestine of the calf with the most acute symptoms and fatal neonatal enteritis. None of the four calves receiving the E. coli had diarrhea. One calf, however, had respiratory distress and died on day 5.

Two SPF colostrum-deprived control calves had neither diarrhea nor respiratory distress.

The above findings support the conclusion that BVD virus should not be overlooked as a primary cause of the neonatal calf enteritis complex.

     

Detection of Mycoplasma mycoides sub-species mycoides small colony by a specific capture/enrichment monoclonal antibody-based sandwich ELISA

The present study describes the development of a specific Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC) monoclonal antibody (MAb), 6E3, and its application in a sandwich ELISA (sELISA) format. Mab 6E3 reacted only to the 12 MmmSC within the 32 M. mycoides cluster strains and 12 representative strains of other bovine, ovine and caprine associated mycoplasmas examined. A capture/enrichment format of the sELISA that combined MAb 6E3 with a previously developed MAb 3H12 that cross reacted with Mmm Large Colony [Rodriguez, F., Ball, H.J., Finlay, D., Campbell, D., Mackie, D.P., 1996. Detection of Mycoplasma mycoides sub-species mycoides by monoclonal antibody-based sandwich ELISA. Veterinary Microbiology 51, 69–76], retained MmmSC specificity and improved the sensitivity from the 1.2 × 10⁷ cfu/ml for a standard 2 h capture stage sELISA down to as low as 2 cfu/ml for a 72 h capture. A low level of false positives (1%) was observed when this assay was applied to 200 bovine respiratory and milk samples submitted for diagnostic investigation. This simple and specific sELISA provides a suitable assay for screening large numbers of samples for CBPP.     

Enzootic Pneumonia in Pigs: Propagation of a Causative Mycoplasma in Cell Cultures and in Artificial Medium

Three strains of a new species of mycoplasma were recovered from pneumonic pig lungs, known free of Mycoplasma hyorhinis, by prolonged incubation in pig testicle cell cultures. The three strains produced a characteristic cytopathic effect in the cell cultures. A highly enriched meat-infusion-broth medium was evolved and permitted regular propagation of these organisms. Pneumonia could consistently be produced by intratracheal inoculation of pigs with the mycoplasma propagated in the enriched broth medium or in cell cultures. The mycoplasma were recovered from the lungs of experimentally infected pigs by inoculation into the broth medium. Comparative studies of the pneumonia producing mycoplasma and of M. hyorhinis were carried out in cell cultures, broth media, and in pigs infected experimentally by different routes. The morphological characteristics of the mycoplasma, grown in the different media, are described and illustrated.