The genome of Brucella melitensis

The genome of Brucella melitensis strain 16M was sequenced and contained 3,294,931 bp distributed over two circular chromosomes. Chromosome I was composed of 2,117,144 bp and chromosome II has 1,177,787 bp. A total of 3198 ORFs were predicted. The origins of replication of the chromosomes are similar to each other and to those of other α-proteobacteria. Housekeeping genes such as those that encode for DNA replication, protein synthesis, core metabolism, and cell-wall biosynthesis were found on both chromosomes. Genes encoding adhesins, invasins, and hemolysins were also identified.     

Evaluation of a rough mutant of Brucella melitensis in pregnant goats

Brucella melitensis strain VTRM1, a rough derivative of B melitensis strain 16M, is able to colonise the lymph nodes of goats, does not induce abortion in pregnant goats when used at doses leading to abortions with virulent strain 16M, and does not induce anti-O chain antibodies. However, strain VTRM1 as a single dose vaccine induces only partial protection against both infection and abortion following challenge.     

Discrimination between sheep antibodies to Brucella melitensis and to Brucella ovis

When preparations containing smooth Brucella abortus lipopolysaccharide (LPS) were used as antigens in an ELISA, strong positive reactions were obtained with sera from sheep infected with Brucella melitensis or with Brucella ovis. Oxidation of the LPS with sodium metaperiodate greatly reduced the extent of the cross-reactions with antisera to B. ovis, with little effect on the reactions with antisera to smooth B. melitensis. Periodate oxidation of hot saline extract (HSX) antigen of B. ovis markedly reduced its reactivity in ELISA with anti-B. ovis sera and eliminated cross-reactivity with anti-B. melitensis sera. The reactivity of HSX was maintained after treatment with proteinase K.

A simple ELISA system, in which replicate samples from a single serum dilution were tested in parallel against both B. ovis HSX antigen and periodate-oxidised smooth phase B. abortus LPS, was evaluated. It was found to discriminate well between antibodies induced by vaccination or virulent infection with B. melitensis strains and those induced by infection with B. ovis.     

Identification of Brucella abortus, B. canis, B. melitensis, and B. suis by carbon substrate assimilation tests

By using the results of seven carbon substrate assimilation tests from the Biotype 100 system (bioMérieux, Marcy-l’Etoile, France), we correctly identified 79 (85.9%) of 92 Brucella strains tested. The specificity of the method varied from 97.4 to 100% depending on the species. Although a biological safety cabinet must be used, this method represents an easy and fast alternative for the identification of Brucella species.     

Seroprevalences and lack of abortions after vaccination of Churra sheep with reduced doses of Rev. 1 Brucella melitensis vaccine by subcutaneous or conjunctival routes

A field study to evaluate the serological response and the safety of different doses and administration routes of the Rev. 1 vaccine was carried out on two Churra breed flocks. Reduced doses of 2.3 × 10⁶ and 3 × 10⁷ live organisms were administered by the subcutaneous or the conjunctival route, respectively. In those animals which were seropositive before vaccination, the percentage of positive sera declined progressively in a similar way in all groups over the 36 weeks that the study lasted; the antibody titers also dropped continuously in the group vaccinated by the conjunctival route with the lower dose, while in the remaining three groups there was a transitory increase in the 4th week after vaccination. In those animals which were serologically negative prior to vaccination, the percentage of positive sera and the antibody titers generally reached their peak in the 4th week after vaccination, followed by a progressive decline in succeeding weeks. Similarly, titers were higher in animals vaccinated subcutaneously than in those vaccinated by the conjunctival route. The differences between the frequencies of positive sera and the levels of antibodies were important when routes were compared. Animals receiving a dose of 2.3 × 10⁶ CFU subcutaneously had a satisfactory serological response, with a more rapid decline in their level of antibodies than in the animals which were vaccinated with 3 × 10⁷ CFU by the same route. No cases of abortion were reported in the 461 vaccinated ewes.     

Phylogentic analysis of Anaplasma ovis strains isolated from sheep and goats using groEL and mps4 genes

Evidence of Anaplasma spp. in goats and sheep in Cyprus has been demonstrated by previous research. Herein, further research was performed for the identification of the exact Anaplasma spp. resulting in the identification of Anaplasma ovis strains in all samples examined. We used a bioinformatics as well as a molecular approach (study of groEl and mps4 genes) in order to verify the validity of the results. All samples depicted the presence of A. ovis regardless of the host (goat or sheep).     

A review of the use of B. melitensis Rev 1 vaccine in adult sheep and goats

The live Brucella melitensis Rev 1 strain is considered the best vaccine available for the prophylaxis of brucellosis in small ruminants. The classically recommended exclusive vaccination of young replacement animals has failed to control brucellosis in some developed countries and is frequently inapplicable in the developing world. Accordingly, whole-flock vaccination is the only feasible alternative to control B. melitensis infection in small ruminants under the extensive management conditions characteristic of these countries. This review describes the practical problems encountered and the experience acquired over the past decade (particularly in Spain) using the Rev 1 based control strategy. The vaccination of pregnant animals with full standard doses of Rev 1 administered subcutaneously is followed by abortion in most vaccinated animals. Reducing the dose of vaccine has been suggested as a method of avoiding this problem and, accordingly, a reduced-dose vaccination strategy has been widely used and has been reported as a safe and effective method of controlling small ruminant brucellosis. However, we reviewed field and experimental results supporting the fact that as a result of the induction of abortion in pregnant animals and the low degree of immunity conferred, reduced doses of Rev 1 should not be recommended as an alternative to the full standard doses.

When tested in a mouse model, differences in residual virulence and immunogenicity have been demonstrated between the different Rev 1 vaccines produced world-wide. These differences could account for the discrepancies in safety results obtained in mass vaccination trials in different countries. The induction of abortions when vaccinating pregnant animals means that there is no entirely safe strategy for Rev 1 vaccination. Conjunctival vaccination is safer than subcutaneous vaccination but is not safe enough to be applied regardless of the pregnancy status of the animals, and should be used only under restricted conditions. For sheep, conjunctival administration of standard doses of Rev 1 during the late lambing season or during lactation is recommended as a whole-flock vaccination strategy.     

Epidemiology of Oestrus ovis in southwest France

From July 1989 to June 1990, 555 heads of adult sheep obtained from Pamiers slaughterhouse (southwest France) were examined for infestation by Oestrus ovis. Infestation was present in 65% of the heads and the mean larval burden per positive case over the year was 24.8. The monthly prevalence rate varied from 44% in April to 88.2% in November. There are usually three generations of Ovis each year: the first March–April, the second in June–July and the last in September–October. There was no fly activity in winter and during the hottest months of summer. On the other hand, nearly all the larvae overwintered as the firs stage.This study emphasizes the seriousness of the problem in the region and the authors recommend three strategic treatments per year during periods of high fly activity.     

A prediction model for strike in the sheep nasal fly, Oestrus ovis, in Namibia

A model was developed for predicting outbreaks of Oestrus ovis throughout the main sheep farming areas of Namibia. Pupal developmental periods were studied, concomitant air and soil temperatures enabling degree-day calculations to be made for prediction of adult fly strike. Monitoring of larval infection established seasonal incidence of O. ovis infestation and acted as verification of predictions. The establishment of relevant isothermal maps for Namibia made possible extrapolation from the several study sites to the entire sheep farming area. Retrospective and actual predictions of the important first peak after winter were considered accurate enough to recommend timing of control measures. No evidence of overwintering of first stage instars was found, the strategy used instead being extended pupation. Adult fly energy reserves were determined.     

Using remote sensing to predict outbreaks of Oestrus ovis in Namibia

The Directorate of Veterinary Services of the Ministry of Agriculture, Water and Rural Development of Namibia issues warnings to farmers in the south of the country about the likelihood of infestation of small-stock by the nasal bot fly, Oestrus ovis. Farmers can then treat their stock at the most appropriate time. The O. ovis puparia develop at shallow depths in the soil and the timing of emergence is directly dependent on climatic conditions, specifically the number of degree-days above a particular threshold soil temperature. Based on temperature measurements from only a few stations scattered throughout the country, the veterinary department warnings lack precision in space and time. This paper presents an attempt to support the programme of warnings with accumulated temperature information from Meteosat satellite images, in order to strengthen predictions of the time of emergence in specific places, and to improve the precision and reliability of warnings given to farmers.     

A study of an enzootic focus of sheep babesiosis (Babesia ovis, Babes, 1892)

Morbidity and mortality due to Babesia ovis in sheep flocks grazing in an enzootic area of Israel occur yearly, about 2 weeks after detection of adult Rhipicephalus bursa ticks on the animals. Disease incidence peaks in May, but lasts throughout the active period of the adult ticks in the spring-summer months of April–July. No clinical cases of babesiosis have been registered during the active period of the preimaginal stages of R. bursa, from October to February. Incidence of parasitaemia during the spring-summer months was variable, ranging between 2 and 25%. However, in the winter months the incidence of parasitaemia in hoggets increased considerably, reaching 4–60% of the animals.

A positive serological response to B. ovis was found in 84.5% of the hoggets and 88.9% of the ewes. In ewes, the prevalence of the serological response showed no marked seasonal variations. Colostral sera of 67.5% and 75% of the ewes and hoggets, respectively, were serologically positive for B. ovis. No antibodies were detected in the sera of lambs less than 3–4 months of age. The epizootiology of sheep babesiosis appears to differ from that of bovine babesiosis.     

The intramacrophagic environment of Brucella suis and bacterial response

Phagocytes have developed various antimicrobial defense mechanisms to eliminate pathogens. They comprise the oxidative burst, acidification of phagosomes, or fusion of phagosomes with lysosomes. Facultative intracellular bacteria, in return, have developed strategies counteracting the host cell defense, resulting in intramacrophagic survival.Until lately, only very little was known about the phagosomal compartment containing Brucella spp., the environmental conditions the bacteria encounter, and the pathogen’s stress response. Recently, we have determined that the phagosomes acidify rapidly to a pH of 4.0–4.5 following infection, but this early acidification is crucial for intracellular replication as neutralization results in bacterial elimination. A vacuolar proton-ATPase is responsible for this phenomenon that is not linked to phagosome–lysosome fusion. On the contrary, in vitro reconstitution assays revealed association only between phagosomes containing killed B. suis and lysosomes, describing the absence of phagolysosome fusion due to specific recognition inhibition for live bacteria. Further evidence for the necessity of an intact, acidic phagosome as a predominant niche of brucellae in macrophages was obtained with a strain of B. suis secreting listeriolysin. It partially disrupts the phagosomal membranes and fails to multiply intracellularly.How does B. suis adapt to this environment? We have identified and studied a series of genes that are involved in this process of adaptation. The bacterial heat shock protein and chaperone DnaK is induced in phagocytes and it is essential for intracellular multiplication. A low-level, constitutive expression of dnaK following promoter exchange does not restore intramacrophagic survival. Another chaperone and heat shock protein, ClpB, belonging to the family of ClpATPases, is important for the resistance of B. suis to several in vitro stresses, but does not contribute to intramacrophagic survival of the pathogen. Additional bacterial genes specifically induced within the phagocyte were identified by an intramacrophagic screen of random promoter fusions to the reporter gene gfp. A large majority of these genes are encoding proteins involved in transport of nutrients (sugars, amino acids), or cofactors, such as nickel. Analysis of the intracellular gene activation reveals that low oxygen tension is encountered by B. suis.Altogether, these results suggest three major stress conditions encountered by brucellae in the phagosome: acid stress, starvation and low oxygen tension.     

Larval salivary gland proteins of the sheep nasal bot fly, (Oestrus ovis L.), are major immunogens in infested sheep

Tissue extracts from larval instars of the sheep nasal bot, Oestrus ovis, were resolved by gel electrophoresis under both native and denaturing conditions. Polypeptides resolved under these conditions were tested by immunoblotting against sera of infested sheep. Of all tissues examined in this study, salivary glands proved to be major immunogens in infested sheep. Salivary gland polypeptides were also detected in the washing solution as larval secretory products (LSP). To a minor extent, a few polypeptides from the larval cuticle were also found to be immunogenic, but they did not contribute to LSP. These results were further corroborated by nasal infestation of rabbits that also developed specific antibodies against larval salivary gland polypeptides from Oestrus ovis.     

Salmonella abortus ovis (Etude de 112 souches)

The bacteriological examination of 112 strains of Salmonella abortus ovis shows some particular properties of this serotype, and the existence of two different types according to their geographical source.     

Culex annulirostris as a vector of Eperythrozoon ovis infection in sheep

Eperythrozoon ovis, a blood parasite of sheep, was transmitted from carrier lambs to susceptible lambs by the interrupted feeding of the mosquito Culex annulirostris. The circumstances of some of the transmissions were similar to those encountered in the field, in that carrier and susceptible lambs were exposed to a natural population of mosquitoes, while in another transmission an artificial means of containment of the mosquitoes was used. Infective E. ovis organisms were shown to be present in and/or on Cx. annulirostris for at least 14 hours by the inoculation of physiological saline-extracted material from macerated mosquitoes fed on E. ovis carrier lambs.     

Duration of immunity, efficacy and safety in sheep of a recombinant Taenia ovis vaccine formulated with saponin or selected adjuvants

The efficacy and safety of a recombinant Taenia ovis protein was tested in sheep using 13 different adjuvant formulations, including oil adjuvants, aluminium salts, saponin, Iscoms and DEAE-dextran. The oil adjuvants, saponin and DEAE-dextran gave the highest antibody responses and greatest degree of protection against challenge infection with T. ovis eggs. Duration of immunity studies with a saponin based vaccine showed that highly significant protection (>90% reduction of cyst numbers) was achieved when sheep were challenge infected one month after immunisation. Significant protection (79%) was still present when sheep were challenged 6 months after immunisation. The optimum dose for this batch of saponin was 10 mg, which stimulated a peak antibody titre of 38,400, 4 weeks after immunisation and did not cause injection site reactions. Dialysed saponin was shown to retain its adjuvant properties and allowed an increase in dose to 30 mg without site reaction, resulting in a peak antibody titre of 51,200.     

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.     

Validation of an ELISA method for the serological diagnosis of canine brucellosis due to Brucella canis

In the present study, the validation of an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of canine brucellosis is described. Two different antigenic extracts, obtained by heat or ultrasonic homogenization of microbial antigens from a wild isolate of Brucella canis bacteria, were compared by ELISA and Western blot (WB). A total of 145 canine sera were used to define sensitivity, specificity and accuracy of the ELISA as follows: (1) sera from 34 animals with natural B. canis infection, confirmed by blood culture and PCR, as well as 51 sera samples from healthy dogs with negative results by the agar–gel immunodiffusion (AGID) test for canine brucellosis, were used as the control panel for B. canis infection; and (2) to scrutinize the possibility of cross reactions with other common dog infections in the same geographical area in Brazil, 60 sera samples from dogs harboring known infections by Leptospira sp., Ehrlichia canis, canine distemper virus (CDV), Neospora caninum, Babesia canis and Leishmania chagasi (10 in each group) were included in the study. The ELISA using heat soluble bacterial extract (HE-antigen) as antigen showed the best values of sensitivity (91.18%), specificity (100%) and accuracy (96.47%). In the WB analyses, the HE-antigen showed no cross-reactivity with sera from dogs with different infections, while the B. canis sonicate had various protein bands identified by those sera. The performance of the ELISA standardized with the heat soluble B. canis antigen indicates that this assay can be used as a reliable and practical method to confirm infection by this microorganism, as well as a tool for seroepidemiological studies.     

Seroprevalence of antibodies to Encephalitozoon cuniculi and Encephalitozoon intestinalis in humans and animals

The presence of antibodies against Encephalitozoon cuniculi (E. cuniculi) and Encephalitozoon intestinalis (E. intestinalis) was examined in 215 samples from humans and in 488 samples from five different species of domestic and companion animals in Slovakia. The 215 human samples and samples from 90 swine, 123 non-infected cattle (cattle), 24 cattle infected with bovine leukosis virus (BLV-positive cattle), 140 sheep and 111 dogs were examined by the enzyme-linked immunosorbent assay (ELISA). Samples with serum titres 1:200 or higher were considered as positive. Specific anti-E. cuniculi antibodies were found in humans (0.9%), swine (52%), cattle (2%), sheep (9%) and dogs (15%) except for the BLV-positive cattle at the titre of 1:200. The titre of 1:400 was detected only in humans (0.5%). The presence of specific anti-E. intestinalis antibodies at the titre of 1:200 was confirmed in humans (6%), swine (51%), cattle (11%), BLV-positive cattle (13%) and dogs (6%) but not in sheep. The anti-E. intestinalis antibodies reached the 1:400 in humans (1%), swine (4%) and BLV-positive cattle (17%). The presence of specific anti-E. intestinalis antibodies at the titre of 1:600 was observed only in one swine (1%). Significant differences were observed in animals at titres 1:200 and 1:400 (chi-squared test: p < 0.0001) for both pathogens and in humans only for E. cuniculi at the titre of 1:400 (chi-squared test: p < 0.0075).