Serological relationship of a Japanese Babesia species and Babesia bigemina by the complement fixation and capillary-tube agglutination tests

The complement fixation (CF) test and the capillary-tube agglutination (CA) test were used to study the antigenic relationship between Babesia bigemina and the large Babesia species frequently infecting cattle in Japan. The CF antigen was prepared from parasitized erythrocytes by extraction with distilled water. The CA antigen was prepared from parasitized erythrocytes by mild sonification of mixtures of Babesia and erythrocyte stroma, following lysis of the erythrocytes with hypotonic saline solution. All the sera used were collected from experimentally-infected cattle. Cross reaction was demonstrated between the Japanese Babesia species and B. bigemina. There was, however, a difference of two dilutions in titer between homologous and heterologous antibody in the CF test, and a difference of more than three tubes in titer between both antibodies in the CA test. It was possible, therefore, to distinguish the Japanese Babesia species from B. bigemina by the CF and CA tests.     

Characterization of the in vitro core surface proteome of Mycoplasma mycoides subsp. mycoides, the causative agent of contagious bovine pleuropneumonia

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides (Mmm) is a severe cattle disease, present in many countries in sub-Saharan Africa. The development of improved diagnostic tests and vaccines for CBPP control remains a research priority. Polyacrylamide gel electrophoresis and mass spectrometry were used to characterize the Triton X-114 soluble proteome of nine Mmm strains isolated from Europe or Africa. Of a total of 250 proteins detected, 67 were present in all strains investigated. Of these, 44 were predicted to be lipoproteins or cytoplasmic membrane-associated proteins and are thus likely to be members of the core in vitro surface membrane-associated proteome of Mmm. Moreover, the presence of all identified proteins in other ruminant Mycoplasma pathogens were investigated. Two proteins of the core proteome were identified only in other cattle pathogens of the genus Mycoplasma pointing towards a role in host–pathogen interactions. The data generated will facilitate the identification and prioritization of candidate Mycoplasma antigens for improved control measures, as it is likely that surface-exposed membrane proteins will include those that are involved in host–pathogen interactions.     

Isolation of Mycoplasma mycoides subspecies mycoides from Polyarthritis and Mastitis of Goats in Canada

The clinical signs, pathomorphological changes, and microbiological findings in Canadian goats infected with Mycoplasma mycoides subspecies mycoides are discussed. The disease affected mainly young goats and was characterized by septicemia and polyarthritis. Mastitis followed by septicemia was seen in two mature goats. The diagnosis was made by culture and identification of the mycoplasma. Infected goats without clinical signs were identified by cultural and serological (complement fixation) techniques. Healthy carriers are presumably able to transmit the infection and may have brought the disease to Canada.     

A radial growth precipitation test and its comparison with certain other serological tests for the detection of antibody in bovine sera to Mycoplasma agalactiae subsp. bovis

A radial growth precipitation test is described for measuring antibody to Mycoplasma agalactiae subsp. bovis. The test is quantitative and appears to depend on the production of soluble antigen by growing organisms. When compared with indirect haemagglutination, complement fixation and inhibition of film production, for measuring antibody to M. agalactiae subsp. bovis in bovine sera, it was found to have a sensitivity comparable to that of the complement fixation test.     

Kinetics of substrate oxidation and hydrogen peroxide production by Mycoplasma mycoides subsp. mycoides Large Colony (LC) type and Mycoplasma mycoides subsp. capri

Mycoplasma mycoides subsp. mycoides Large Colony (LC) type is a pathogen of goats causing contagious agalactia and respiratory disease, found on all continents where small ruminants are kept. It shares close genetic characteristics with M. mycoides subsp. capri. Substrate oxidation by 22 strains of M. mycoides subsp. mycoides LC from nine countries was compared with that of eight strains of M. mycoides subsp. capri from five countries. There was considerable similarity in the substrates used, but substrate saturation coefficients (K_s) varied for different substrates. Substrate utilization patterns and K_s values did not (1) significantly differentiate the LC strains from each other, (2) show any correlation with geographical origin, or (3) distinguish the LC strains from the capri strains. These results support previous studies justifying the reclassification of these subspecies as a single species.     

Experimental immunization of calves against Anaplasma marginale infection: observations on the use of living A. centrale and A. marginale

Groups of Bos indicus cross calves aged 6 months were immunized with Anaplasma centrale or A. marginale. All animals were challenged with 10¹⁰A. marginale 7 months later. Groups immunized with A. marginale were refractory to challenge, whereas only a portion of the animals vaccinated with A. centrale were immune. Following immunization, animals that had experienced a good primary antibody response as measured by a complement fixation test, a marked reduction in packed cell volume, and a high parasitaemia, resisted a subsequent challenge with A. marginale. Resistance was characterised by a weak secondary antibody response and the absence of A. marginale in blood films     

Anthelmintic activity of Cocos nucifera L. against sheep gastrointestinal nematodes

The development of anthelmintic resistance has made the search for alternatives to control gastrointestinal nematodes of small ruminants imperative. Among these alternatives are several medicinal plants traditionally used as anthelmintics. This work evaluated the efficacy of Cocos nucifera fruit on sheep gastrointestinal parasites. The ethyl acetate extract obtained from the liquid of green coconut husk fiber (LGCHF) was submitted to in vitro and in vivo tests. The in vitro assay was based on egg hatching (EHT) and larval development tests (LDT) with Haemonchus contortus. The concentrations tested in the EHT were 0.31, 0.62, 1.25, 2.5 and 5 mg ml⁻¹, while in the LDT they were 5, 10, 20, 40 and 80 mg ml⁻¹. The in vivo assay was a controlled test. In this experiment, 18 sheep infected with gastrointestinal nematodes were divided into three groups (n = 6), with the following doses administered: G1—400 mg kg⁻¹ LGCHF ethyl acetate extract, G2—0.2 mg kg⁻¹ moxidectin (Cydectin^®) and G3—3% DMSO. The worm burden was analyzed. The results of the in vitro and in vivo tests were submitted to ANOVA and analyzed by the Tukey and Kruskal–Wallis tests, respectively. The extract efficacy in the EHT and LDT, at the highest concentrations tested, was 100% on egg hatching and 99.77% on larval development. The parameters evaluated in the controlled test were not statistically different, showing that despite the significant results of the in vitro tests, the LGCHF ethyl acetate extract showed no activity against sheep gastrointestinal nematodes.     

Contagious bovine pleuropneumonia in northern tanzania,culture confirmation and serological studies

Summary After an absence of about 25 years contagious bovine pleuropneumonia (CBPP) appeared again in 1990 in Tanzania. It was preceded by a spread in Kenya to an area bordering Tanzania. Due to the frequent cattle movements across the border it was soon introduced into Loliondo in northern Tanzania. One month after the first cases, CBPP was suspected in a total of 9 herds comprising 1,500 cattle. However, few animals showed clear clinical signs and frequent antibiotic treatment at an early stage further obscured the clinical picture. In one herd with acute cases, the diagnosis was confirmed by autopsy andMycoplasma mycoides subsp.mycoides, SC type, was isolated. From this herd several serum samples were positive in the complement fixation test and gave high absorbance values in an ELISA withM. mycoides subsp.mycoides antigen. From 5 other herds with suspected cases blood samples were negative by the complement fixation test but in the enzyme-linked immunosorbent assay at least one in each herd was positive.

---

Pleuroneumonia Contagiosa Bovina En Tanzania Septentrional, Confirmacion Mediante Cultivo Y Estudios Serologicos

Resumen Después de un período de 25 años sin ningún brote, la pleuroneumonía contagiosa bovina apareció de nuevo en Tanzania en 1990, después de que la enfermedad se extendiera a una zona de Kenya fronteriza con Tanzania. Debido a los frecuentes movimientos de ganado vacuno a través de la frontera, la enfermedad se declaró pronto en Loliondo, en el norte de Tanzania. Un mes después de los primeros casos, 9 rebaños, que contabilizaban en total 1500 animales, eran sospechosos de estar infectados. Sin embargo, pocos animales mostraron síntomas clínicos claros y et tratamiento precoz con antibióticos contribuyó a disminuir la intensidad de las manifestaciones clinicas. En un rebaño en el que hubo casos agudos el diagnóstico se confirmó mediante necropsia y se aislóMycoplasma mycoides subsp.mycodies del tipo SC. Varias muestras de sangre de este rebaño dieron resultado positivo en el test de fijación del complemento y dieron valores de absorbancia altos en un test de ELISA conM. mycoides subsp.mycoides. Las muestras de sangre provenientes de otros 5 rebaños en los que se sospechó la existencia de la enfermedad dieron resultado negativo en el test de fijación de complemento mientras que al menos un animal de cada rebaño dio resultado positivo en el test ELISA.

---

Peripneumonie Contagieuse Bovine Dans Le Nord De La Tanzanie, Confirmation Par Culture Et Etudes Serologiques

Résumé Après une absence d'environ 25 ans la péripneumonie contagieuse bovine est de nouveau apparue en 1990 en Tanzanie. Elle a été prècedée d'une progression au Kenya vers une zone bordant la Tanzanie. Par suite de mouvements frontaliers fréquents du bétail, elle a été beintôt introduite dans la région de Loliondo, dans le nord de la Tanzanie. Un mois après les premiers cas, 9 troupeaux totalisant 1500 têtes ont été supectés. Cependant, peu d'animaux ont présenté des signes cliniques nets et, de surcroît, les traitements antibiotiques entrepris en début de maladie obscurissent le tableau clinique. Le diagnostic a été confirmé à l'autopsie dans un troupeau présentant des cas cliniques etMycoplasma mycoides subsp.mycoides, type SC, a été isolé. Plusieurs échantillons de sérum de ce troupeau ont été positifs en fixation du complément et ont donné de grandes valeurs d'absorption dans un test ELISA avec un antigèneM. mycoides subsp.mycoides. Pour 5 autres troupeax avec des cas suspects, les échantillons de sang ont été négatifs en fixation du complément mais un au moins pour chaque troupeaux a été positif dans un test ELISA.

     

Serological response of cattle after vaccination and challenge with Brucella abortus

New and currently used serological procedures were evaluated using sera from cattle that were challenged with B. abortus S544 (S544) after vaccination with either B. abortus S19 (S19) or B. abortus 45/20 (S45/20) as calves or adults. In animals vaccinated with S19, titres to the indirect haemolysis test (IHLT) rose more slowly, declined more rapidly and involved fewer animals than did titres to the complement fixation test (CFT). In animals vaccinated with S45/20 the rough antigen complement fixation test (RCFT) showed persistent titres. At slaughter the IHLT and CFT were found to be more specific and more sensitive than the Rose Bengal Plate Test (RBPT) and Serum Agglutination Test (SAT) in the detection of cattle infected with B. abortus.     

Kinetics of substrate oxidation and hydrogen peroxide production by Mycoplasma mycoides subsp. mycoides Large Colony (LC) type and Mycoplasma mycoides subsp. capri

Mycoplasma mycoides subsp. mycoides Large Colony (LC) type is a pathogen of goats causing contagious agalactia and respiratory disease, found on all continents where small ruminants are kept. It shares close genetic characteristics with M. mycoides subsp. capri. Substrate oxidation by 22 strains of M. mycoides subsp. mycoides LC from nine countries was compared with that of eight strains of M. mycoides subsp. capri from five countries. There was considerable similarity in the substrates used, but substrate saturation coefficients (K_s) varied for different substrates. Substrate utilization patterns and K_s values did not (1) significantly differentiate the LC strains from each other, (2) show any correlation with geographical origin, or (3) distinguish the LC strains from the capri strains. These results support previous studies justifying the reclassification of these subspecies as a single species.     

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.     

Performance of skin tests with allergens from B. melitensis B115 and rough B. abortus mutants for diagnosing swine brucellosis

Swine brucellosis by Brucella suis biovar 2 is an emerging disease whose control is based on serological testing and culling. However, current serological tests detect antibodies to the O-polysaccharide (O/PS) moiety of Brucella smooth lipopolysaccharide (S-LPS), and thus lack specificity when infections by Yersinia enterocolitica O:9 and other gram-negative bacteria carrying cross-reacting O/PS occur. The skin test with the protein-rich brucellin extract obtained from rough B. melitensis B115 is assumed to be specific for discriminating these false positive serological reactions (FPSR). However, B115 strain, although unable to synthesize S-LPS, accumulates O/PS internally, which could cause diagnostic problems. Since the brucellin skin test has been seldom used in pigs and FPSR are common in these animals, we assessed its performance using cytosoluble protein extracts obtained from B. abortus rough mutants in manBcore or per genes (critical for O/PS biosynthesis) and B. melitensis B115. The diagnostic sensitivity and specificity were determined in B. suis biovar 2 culture positive and brucellosis free sows, and apparent prevalence in sows of unknown individual bacteriological and serological status belonging to B. suis biovar 2 naturally infected herds. Moreover, the specificity in discriminating brucellosis from FPSR was assessed in brucellosis free boars showing FPSR. The skin test with B. abortus ΔmanBcore and B. melitensis B115 allergens performed similarly, and the former one resulted in 100% specificity when testing animals showing FPSR in indirect ELISA, Rose Bengal and complement fixation serological tests. We conclude that O/PS-free genetically defined mutants represent an appropriate alternative to obtain Brucella protein extracts for diagnosing swine brucellosis.     

Effect of Eperythrozoon ovis infection on the reductive potential of sheep erythrocytes

Reduced glutathione (GSH) and methaemoglobin were measured in sheep blood during an infection cycle of Eperythrozoon ovis and in uninfected control blood. The GSH levels in infected erythrocytes were significantly lower in the latter half of the infection cycle. Incubation with acetylphenylhydrazine (APH) resulted in negligible leves in infected cells whereas control cells were approximately 45% normal. Infection with E. ovis had little effect on methaemoglobin levels. However, incubation with APH caused a marked increased of the methaemoglobin levels in both infected and control blood; the control methaemoglobin levels were significantly higher.It was concluded from these observations that E. ovis interfered with maintenance of GSH within erythrocytes to an extent that when challenged with an oxidizing chemical erythrocyte membrane integrity could be lost. In addition E. ovis may bave provided some protection against oxidative damage for the heme portion of the haemoglobin molecule or the amount of oxidative damage to haemoglobin was further advanced in the infected cells.     

Rapid latex agglutination (RLA) test for the diagnosis of Babesia Argentina

A rapid test, utilizing latex particles (0.81-μm diameter), sensitized with Babesia argentina antigens, proved to be effective in the diagnosis of B. argentina in natural and experimental infections. Two drops of plasma or serum and one drop of B. argentina antigen placed on a glass plate were used in the test. Reaction was observed after 3—10 min rotation. The positive agglutination reaction was characterized by the formation of fine latex particle clumss. In experimental infections with B. argentina, the first detectable positive agglutination reactions coincided with the appearance of parasitemia in thin blood films. Plasma from animals with natural infections of B. argentina, proven by blood smears and indirect fluorescent antibody and complement fixation tests, also showed a reaction to the latex agglutination test.     

Effect of aqueous extracts of Baccharis trimera on development and hatching of Rhipicephalus microplus (Acaridae) eggs

This study evaluated the effects of aqueous extracts of Baccharis trimera (Less.) DC (Asteraceae), colloquially known as carqueja, on egg production, and hatching rate of larvae of Rhipicephalus microplus. Plant samples were collected in Montes Claros, north of Minas Gerais, Brazil. Adult female ticks were distributed into 24 homogeneous groups of 10. The in vitro test was performed by immersing each group in 10 ml solutions of aqueous extracts at 50, 100, 150, or 200 mg of fresh leaves ml⁻¹. These concentrations were compared with distilled water as negative control and a commercial product as positive control and the tests were repeated four times. The carqueja extract at concentrations of 150 and 200 mg of fresh leaves ml⁻¹ showed 100% efficacy in inhibiting egg hatching and therefore could have potential as an acaricide.     

Functional and antigenic properties of GlpO from Mycoplasma mycoides subsp. mycoides SC: characterization of a flavin adenine dinucleotide-binding site deletion mutant

L-α-glycerophosphate oxidase (GlpO) plays a central role in virulence of Mycoplasma mycoides subsp. mycoides SC, a severe bacterial pathogen causing contagious bovine pleuropneumonia (CBPP). It is involved in production and translocation of toxic H₂O₂ into the host cell, causing inflammation and cell death. The binding site on GlpO for the cofactor flavin adenine dinucleotide (FAD) has been identified as Gly ₁₂−Gly₁₃−Gly ₁₄−Ile₁₅−Ile₁₆−Gly ₁₇. Recombinant GlpO lacking these six amino acids (GlpOΔFAD) was unable to bind FAD and was also devoid of glycerophosphate oxidase activity, in contrast to non-modified recombinant GlpO that binds FAD and is enzymatically active. Polyclonal monospecific antibodies directed against GlpOΔFAD, similarly to anti-GlpO antibodies, neutralised H₂O₂ production of M. mycoides subsp. mycoides SC grown in the presence of glycerol, as well as cytotoxicity towards embryonic calf nasal epithelial (ECaNEp) cells. The FAD-binding site of GlpO is therefore suggested as a valuable target site for the future construction of deletion mutants to yield attenuated live vaccines of M. mycoides subsp. mycoides SC necessary to efficiently combat CBPP.     

An immunofluorescence technique for the detection of Toxocara canis antibodies

An immunofluorescent (IF) test for the serodiagnosis of Toxocara canis infections in puppies is described. Frozen sections of male adult T. canis worms were used as antigen.A group of seven puppies, 6 weeks of age, was infected orally with 10 000 embryonated T. canis eggs each. In the sera of all animals IF antibodies could be detected from approximately 4 weeks after infection onwards. Titers were detectable until the end of the observation period (22 weeks).Two puppies of the same age were infected with 30 000 or 50 000 embryonated T. canis eggs respectively. Positive IF results were also obtained in the sera of these pups from week 4 post infection (p.i.) onwards. No correlation between titer and initial number of egges administered was observed. Furthermore, no correlation was noticed between titer and number of adult worms recovered from the dogs. For comparison all sera were tested with the complement fixation (CF) test, using cuticle material of adult worms as antigen. Complement fixing antibodies could be detected in none of the serum samples.     

Evaluation of the in vitro growth-inhibitory effect of epoxomicin on Babesia parasites

Epoxomicin potently and irreversibly inhibits the catalytic activity of proteasomal subunits. Treatment of proliferating cells with epoxomicin results in cell death through accumulation of ubiquinated proteins. Thus, epoxomicin has been proposed as a potential anti-cancer drug. In the present study, the inhibitory effects of epoxomicin on the in vitro growth of bovine and equine Babesia parasites were evaluated. The inhibitory effect of epoxomicin on the in vivo growth of Babesia microti was also assessed. The in vitro growth of five Babesia species that were tested was significantly inhibited (P < 0.05) by nanomolar concentrations of epoxomicin (IC₅₀ values = 21.4 ± 0.2, 4 ± 0.1, 39.5 ± 0.1, 9.7 ± 0.3, and 21.1 ± 0.1 nM for Babesia bovis, Babesia bigemina, Babesia ovata, Babesia caballi, and Babesia equi, respectively). Epoxomicin IC₅₀ values for Babesia parasites were low when compared with diminazene aceturate and tetracycline hydrochloride. Combinations of epoxomicin with diminazene aceturate synergistically potentiated its inhibitory effects in vitro on B. bovis, B. bigemina, and B. caballi. In B. microti-infected mice, epoxomicin caused significant (P < 0.05) inhibition of the growth of B. microti at the non-toxic doses of 0.05 and 0.5 mg/kg BW relative to control groups. Therefore, epoxomicin might be used for treatment of babesiosis.     

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.

     

In vitro effects of Tabernaemontana citrifolia extracts on Haemonchus contortus

Tabernaemontana citrifolia (Apocynaceae) is traditionally used as an anthelmintic preparation for ruminants in Guadeloupe (French West Indies). This study was carried out to evaluate the in vitro effect of this plant against the parasitic nematode of small ruminants Haemonchus contortus. Three extracts (aqueous, methanolic and dichloromethane) of T. citrifolia fruit, leaf and root were tested on four developmental stages of the parasite, using egg hatch assay (EHA), larval development assay (LDA), L3 migration inhibition assay (LMI), and adult worm motility assay (AWM). Compared to the negative control, significant effects were observed for the different parts of T. citrifolia but with differences depending on the parasitic stage; efficacies on the larval development of H. contortus from 88.9% to 99.8% for fruit, from 72.1% to 83.8% for root and from 33.5% to 85% for leaf with dose-dependent effect for the methanolic extract. The root gave the best result on EHA (22.7% efficacy for dichloromethane extract) and AWM (56% efficacy, with dose-dependent effect for dichloromethane extract) and the leaf on LMI (49.4% efficacy). These results suggest that T. citrifolia possess anthelmintic activity against H. contortus. The active ingredients responsible for the activity could be the alkaloid compounds present in the plant parts of the plant.