Mutants in the lipopolysaccharide of Brucella ovis are attenuated and protect against B. ovis infection in mice

Brucella spp. are Gram-negative bacteria that behave as facultative intracellular parasites of a variety of mammals. This genus includes smooth (S) and rough (R) species that carry S and R lipopolysaccharides (LPS), respectively. S-LPS is a virulence factor, and mutants affected in the S-LPS O-polysaccharide (R mutants), core oligosaccharide or both show attenuation. However, B. ovis is naturally R and is virulent in sheep. We studied the role of B. ovis LPS in virulence by mutating the orthologues of wadA, wadB and wadC, three genes known to encode LPS core glycosyltransferases in S brucellae. When mapped with antibodies to outer membrane proteins (Omps) and R-LPS, wadB and wadC mutants displayed defects in LPS structure and outer membrane topology but inactivation of wadA had little or no effect. Consistent with these observations, the wadB and wadC but not the wadA mutants were attenuated in mice. When tested as vaccines, the wadB and wadC mutants protected mice against B. ovis challenge. The results demonstrate that the LPS core is a structure essential for survival in vivo not only of S brucellae but also of a naturally R Brucella pathogenic species, and they confirm our previous hypothesis that the Brucella LPS core is a target for vaccine development. Since vaccine B. melitensis Rev 1 is S and thus interferes in serological testing for S brucellae, wadB mutant represents a candidate vaccine to be evaluated against B. ovis infection of sheep suitable for areas free of B. melitensis.     

Antibacterial susceptibility patterns of Pseudomonas strains isolated from chronic canine otitis externa

Development of antibiotic resistance in bacteria is a problem of great concern. It is important to establish the convenience of antimicrobial susceptibility tests in animal infections. The aim of this study was to test the susceptibility to antibiotics of Pseudomonas strains isolated from chronic canine otitis externa. We tested 23 strains of Pseudomonas: 19 Ps. aeruginosa, three Ps. fluorescens and one Pseudomonas spp. The most effective antibiotics were tobramycin (100% susceptible), marbofloxacin (91.3%) and ceftazidime (91.3%). Ticarcillin and gentamicin, commonly used for the treatment of otitis externa also showed good results (susceptibility of strains was 86 and 65.2% respectively). Lower susceptibility was found using enrofloxacin (52.1%) probably due to its indiscriminate use. We emphasize the need for a rational policy of antibiotic prescribing in order to prevent the selection of resistant strains.     

Migration of Strongyloides venezuelensis in rats after oral inoculation of free-living infective larvae

Strongyloides venezuelensis (SVZ) infection was chronologically monitored in 85 Sprague-Dawley rats (SDR), which were orally inoculated with approximately 1,000 infective larvae. In order to describe the characteristics of migrating larvae (MLS) in various visceral organs (the liver, lung, cardiac blood, and small intestine), 5 SDR were sacrificed at 20 min, 45 min, 1 hr, 2 hr, 3 hr, 4 hr, 8 hr, 12 hr, 16 hr, 48 hr, 72 hr, 96 hr, 120 hr, 144 hr, 168 hr and 192 hr post inoculation (PI). MLS were recovered from the liver and blood 20 and 45 min PI and measured 788 +/- 26 microm and 846 +/- 40 microm in length, respectively. MLS were first observed in the lung tissue 45 min PI and measured 925 +/- 38 microm on the average. In the trachea, MLS measuring 849 +/- 75 microm appeared 3 to 96 hrs PI. Adult worms (AWS) measuring 1,926 +/- 521 microm to 2,956 +/- 159 microm in length were observed in the small intestine from 120 hr PI. The worms appeared to mature more than 168 hr PI and attained the average maximum length of 2,420 +/- 532 microm. At 3 hr PI focal hyperemic and necrotic lesions were evidently observed in the liver and lung, together with eosinophilic infiltration in the stomach, liver, and lung. The parasites were histologically detectable in the lung tissues but were very difficult to find in the liver and the epithelial layer of small intestine. These data demonstrate that SVZ parasites take 20 min to reach the liver via the stomach and only three hours to reach the trachea through the same route. The development from eggs to adults takes 168 hr in the SDR model.     

Serum α-globulin fraction in horses is related to changes in the acute phase proteins

Serum electrophoresis is a useful technique to diagnose some diseases characterized by quantitative and qualitative changes in the serum proteins, such as the acute phase proteins. An increased synthesis of acute phase proteins has been associated with acute infection and inflammation. We wanted to study whether gastrointestinal diseases and disorders of the locomotor system in horses produce enough inflammatory reaction so the acute phase proteins can be detected by electrophoresis in serum. Serum total proteins were calculated, and the serum electrophoresis from 63 horses was performed. The horses were classified in 3 groups: Group I (19 healthy horses), Group II (20 horses suffering from inflammatory disorders of the gastrointestinal tract), and Group III (24 horses with inflammatory disorders of the locomotor system). Results indicated an increase of the acute phase proteins only in patent inflammatory disorders that showed a rapid progression. A global increase in those proteins was observed only for α₁-globulins from Group II.     

Protection of olive planting stocks against parasitism of root-knot nematodes by arbuscular mycorrhizal fungi

The effects were investigated, under controlled conditions, of single and joint inoculation of olive planting stocks cvs Arbequina and Picual with the arbuscular mycorrhizal fungi (AMF) Glomus intraradices , Glomus mosseae or Glomus viscosum , and the root-knot nematodes Meloidogyne incognita and Meloidogyne javanica , on plant performance and nematode infection. Establishment of the fungal symbiosis significantly increased growth of olive plants by 88·9% within a range of 11·9–214·0%, irrespective of olive cultivar, plant age and infection by M. incognita or M. javanica . In plants free from AMF, infection by Meloidogyne spp. significantly reduced the plant main stem diameter by 22·8–38·6%, irrespective of cultivar and plant age. Establishment of AMF in olive plants significantly reduced severity of root galling by 6·3–36·8% as well as reproduction of both Meloidogyne spp. by 11·8–35·7%, indicating a protective effect against parasitism by root-knot nematodes. Infection by the nematodes influenced root colonization by AMF, but the net effect depended on the AMF isolate–olive cultivar combination. It is concluded that prior inoculation of olive plants with AMF may contribute to improving the health status and vigour of cvs Arbequina and Picual planting stocks during nursery propagation.     

Intravaginal progesterone devices in synchronization protocols for artificial insemination in beef heifers

Two experiments were designed to investigate the administration of intravaginal progesterone in protocols for oestrus and ovulation synchronization in beef heifers. In Experiment 1, cyclic Black Angus heifers (n = 20) received an Ovsynch protocol and were randomly assigned to receive (CIDR‐Ovsynch) or not (Ovsynch) a progesterone device between Days 0 and 7. Treatment with a controlled internal drug release (CIDR) device significantly increased the size of the dominant follicle prior to ovulation (12.8 ± 0.4 CIDR‐Ovsynch vs 11.4 ± 0.4 Ovsynch) (p < 0.02). Plasma progesterone concentrations throughout the experiment were affected by the interaction between group and day effects (p < 0.004). In Experiment 2, cyclic Polled Hereford heifers (n = 382) were randomly assigned to one of the six treatment groups (3 × 2 factorial design) to receive a CIDR, a used bovine intravaginal device (DIB), or a medroxiprogesterone acetate (MAP) sponge and GnRH analogues (lecirelin or buserelin). All heifers received oestradiol benzoate plus one of the devices on Day 0 and PGF on Day 7 pm (device withdrawal). Heifers were detected in oestrus 36 h after PGF and inseminated 8–12 h later, while the remainder received GnRH 48 h after PGF and were inseminated on Day 10 (60 h). The number of heifers detected in oestrus on Day 8 and conception rate to AI on Day 9 were higher (p < 0.01) in the used‐DIB than in the CIDR or MAP groups, while the opposite occurred with the pregnancy rate to FTAI on Day 10 (p < 0.01). There was no effect of progesterone source, GnRH analogue or their interaction on overall pregnancy rates (64.9%). Progesterone treatment of heifers during an Ovsynch protocol resulted in a larger pre‐ovulatory follicle in beef heifers. Progesterone content of intravaginal devices in synchronization protocols is important for the timing of AI, as the use of low‐progesterone devices can shorten the interval to oestrus.     

Upregulation of LENK and VIP in paracervical ganglion neurons supplying the urinary bladder of tetrodotoxin- and resiniferatoxin-treated female pigs

Both resiniferatoxin (RTX) and tetrodotoxin (TTX) have been reported to be effective in several clinical trials aiming to cure urinary bladder dysfunction. The goal of this experiment was to study the effect of intravesical administration of RTX and TTX on the chemical coding of paracervical ganglion (PCG) neurons that supply the urinary bladder in pigs. The vasoactive intestinal peptide (VIP) and the opioid family member Leu5-enkephalin (LENK) are both known for their regulatory effects in the function of the porcine genitourinary tract. The PCG neurons innervating the urinary bladder were identified by application of the retrograde tracer Fast Blue (FB), injected into the bladder wall prior to intravesical RTX or TTX administration. Immunocytochemical detection of LENK and VIP expression in the FB-labelled perikarya revealed that in the control group 25.15% of the FB-positive PCG neurons contained LENK, and 9.22% of them expressed VIP. Intravesical infusion of RTX resulted in an increase in the number of LENKIR neurons to 48.19% and VIP-IR perikarya to 11.25%. Optional treatment with TTX induced increase of LENK-IR neurons up to 81.67% and VIP-IR population to 16.46% of the FB-positive PCG cells. The present results show that both neurotoxins affect the chemical coding of PCG nervous cells supplying the porcine urinary bladder and that they stimulate both LENK and VIP expression. Furthermore, the results indicate a possible involvement of LENK and VIP neurons in the mechanisms of action of RTX and TTX in the therapy of overactive bladder disorder.     

Phenolic composition in grape (Vitis vinifera L. cv. Malbec) ripened with different solar UV-B radiation levels by capillary zone electrophoresis

The responses of Vitis vinifera L. cv. Malbec to different solar ultraviolet-B radiation (UV-B) levels were assessed in two contrasting situations, under sunlight with full UV-B (+UV-B) and filtered UV-B (-UV-B), in three different locations at 500, 1000, and 1500 m above sea level (asl). To evaluate the effects of radiation, a simple, accurate, and rapid method for the separation and simultaneous determination of representative phenolic compounds in grape berry skins by capillary zone electrophoresis was developed. Separation was carried out in less than 20 min with 20 mM sodium tetraborate buffer containing 30% methanol, pH 9.00. The procedure is fast and reliable, and extracted grape berry skins can be directly analyzed without prior sample cleanup procedure. Berry skins from the +UV-B treatment at 1500 m asl showed the highest levels of total polyphenols anthocyanins, and resveratrol, compared with the -UV-B treatment at this altitude.     

Do pine plantations provide mycorrhizal inocula for seedlings establishment in grasslands from Patagonia,Argentina?

We investigated if Pinus ponderosa plantations in Patagonia are able to produce viable mycorrhizal inocula towards adjacent grasslands, which only harbor endomycorrhizal vegetation. We hypothesized that these inocula have the potential to contribute to the establishment of naturally disseminated seedlings. Also, we determined the main fungal taxa involved in this process. Seven plantations in the onset of their reproductive phase and located in the Patagonian native forest/steppe ecotone (Argentina) were selected. Soil samplings were obtained at nine points along a 450 m long, W-E transect established in each plantation. Soil bioassays were performed in a greenhouse, with P. ponderosa seedlings acting as hosts for mycorrhizal inocula present in soil samples, during 12 months. Mycorrhization percentage, morphotype richness and morphotype composition was determined through morphological evaluation. Viable ecto- and ectendomycorrhizal inocula were found disseminated outside plantations. The amount of mycorrhizal inoculum followed a decreasing function with distance to plantation edges. Mycorrhizal fungal genus Rhizopogon and “E-strain” mycorrhizal types appeared as pioneering taxa regarding seedlings colonization, being the most persistent and frequent symbionts found. Plantations, thus, facilitate the surrounding terrain for newcoming seedlings through the dispersion of mycorrhizal fungal inocula.