The dynamics of cooperative bacterial virulence in the field

Laboratory experiments have shown that the fitness of microorganisms can depend on cooperation between cells. Although this insight has revolutionized our understanding of microbial life, results from artificial microcosms have not been validated in complex natural populations. We investigated the sociality of essential virulence factors (crystal toxins) in the pathogen Bacillus thuringiensis using diamondback moth larvae (Plutella xylostella) as hosts. We show that toxin production is cooperative, and in a manipulative field experiment, we observed persistent high relatedness and frequency- and density-dependent selection, which favor stable cooperation. Conditions favoring social virulence can therefore persist in the face of natural population processes, and social interactions (rapid cheat invasion) may account for the rarity of natural disease outbreaks caused by B. thuringiensis.     

Interaction of proteinase inhibitors with Cry1Ac toxicity and the presence of 15 chymotrypsin cDNAs in the midgut of the tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae)

BACKGROUND: The potential development of resistance to Bacillus thuringiensis (Bt) cotton and surging of non‐targeted insects is a major risk in the durability of Bt plant technology. Midgut proteinases are involved in Bt activation and degradation. Proteinase inhibitors may be used to control a wide range of insects and delay Bt resistance development. Proactive action to examine proteinase inhibitors for synergistic interaction with Bt toxin and cloning of proteinase cDNAs for RNAi is necessary to make transgenic cotton more versatile and durable. RESULTS: A sublethal dose (15 ppb) of Cry1Ac, 0.5% benzamidine and 0.02% phenylmethylsulfonyl fluoride significantly suppressed midgut azocaseinase, tryptic and chymotryptic activities, and resulted in reductions in larval and pupal length and mass of Heliothis virescens. The combination of proteinase inhibitor and Bt suppressed 20–37% more larval body mass and 26–80% more enzymatic activities than the inhibitor only or Bt only. To facilitate knockdown‐resistance‐related proteinase genes, 15 midgut chymotrypsin cDNAs were sequenced. Most predicted chymotrypsins contained the conserved N‐termini IVGG, three catalytic center residues (His, Asp and Ser), substrate specificity determinant (Ser or Gly) and cysteines for disulfide bridges. These putative chymotrypsins were separated into three distinct groups, indicating the diverse proteinases evolved in this polyphagous insect. CONCLUSION: H. virescens has evolved diverse midgut proteinase genes. Proteinase inhibitors have potential insecticidal activity, and the interaction of Bt with proteinase inhibitors is desirable for enhancing Bt toxicity and delaying resistance development. Intensive sequencing of chymotrypsin cDNAs will facilitate future functional examinations of individual roles in Bt toxicity and resistance development and facilitate targeted control using RNAi and/or proteinase inhibitors. Copyright © 2012 Society of Chemical Industry     

The effect of individual liveweight and use of teaser rams prior to mating on the reproductive performance of ewe hoggets

AIM: To determine the effects of liveweight (LW) and use of teaser rams prior to mating on both the mating performance and pregnancy rate of ewe hoggets. METHODS: Romney hoggets (n=841) were weighed and randomly assigned to one of two treatment groups; either exposed to seven harnessed, vasectomised (teaser) rams for 17 days prior to the breeding period (n=283); or not exposed to teaser rams (unteased, n=558). Harnessed Perendale rams were then introduced for two oestrous cycles. Using crayon marks, hoggets were identified as having been mated during the first 17 days only, during the second 17 days only, during both periods, or not mated. All mated hoggets were then scanned for pregnancy using ultrasound, 48 days after the end of the breeding period, and identified as being either non-pregnant, or as single-, twin or triplet-bearing. RESULTS: Hoggets mated in either the first 17 days only or in both the first and second 17-day periods were heavier than those mated in the second period only (p<0.001). Those mated in the first or second 17-day period only, and those mated in both periods were all heavier prior to mating than those not mated at all (p<0.001). Twin-bearing hoggets were heavier than both their single-bearing and non-pregnant counterparts (p<0.001). A greater proportion of the teased hoggets were mated in the first 17 days only compared with unteased hoggets (62.6 vs 32.1%; p<0.001). In contrast, more of the unteased hoggets were marked in the second 17-day period only (p<0.001). A greater proportion of mated, unteased hoggets were found to be non-pregnant in comparison to their teased counterparts (17.8 vs 11.2%; p=0.02). CONCLUSIONS: Heavier hoggets at breeding were more likely to be mated in the first 17 days of the breeding period and more likely to be twin-bearing. Use of teaser rams for 17 days prior to the breeding period increased the proportion of hoggets mated in the first 17 days, increased conception rates, and thus increased the proportion of pregnant hoggets. These results indicate that farmers should utilise teasers for 17 days prior to commencement of the breeding period and avoid mating hoggets that weigh below 36 kg.     

Epidemiologic study of results of pulsed-field gel electrophoresis of isolates of Rhodococcus equi obtained from horses and horse farms

OBJECTIVE: To compare isolates of Rhodococcus equi on the basis of geographic source and virulence status by use of pulsed-field gel electrophoresis (PFGE). SAMPLE POPULATION: 290 isolates of R equi (218 virulent isolates from foals and 72 avirulent isolates from feces, soil, and respiratory tract samples) obtained between 1985 and 2000 from horses and horse farms from 4 countries. PROCEDURE: DNA from isolates was digested with the restriction enzyme Asel and tested by use of PFGE. Products were analyzed for similarities in banding patterns by use of dendrograms. A similarity matrix was constructed for isolates, and the matrix was tested for nonrandom distributions of similarity values with respect to groupings of interest. RESULTS: There was little grouping of isolates on the basis of country, virulence status, or region within Texas. Isolates of R equi were generally < 80% similar, as determined by use of PFGE. Isolates from the same farm generally were rarely of the same strain. CONCLUSIONS AND CLINICAL RELEVANCE: Considerable chromosomal variability exists among isolates of R equiobtained from the same farm, sites withinTexas, or among countries from various continents. Only rarely will it be possible to link infections to a given site or region on the basis of analysis of isolates by use of PFGE of chromosomal DNA.     

Improved Resistance Management for Durable Disease Control: A Case Study of Phoma Stem Canker of Oilseed Rape (Brassica napus)

Specific resistance loci in plants are generally very efficient in controlling development of pathogen populations. However, because of the strong selection pressure exerted, these resistances are often not durable. The probability of a resistance breakdown in a pathosystem depends on the evolutionary potential of the pathogen which is affected by: (i) the type of resistance (monogenic and/or polygenic), (ii) the type of reproduction of the pathogen (sexual and/or asexual), (iii) the capacity of the pathogen for dispersal, (iv) the resistance deployment strategy (pyramiding of specific resistances, mixture of cultivars, spatio-temporal alternation), (v) the size of the pathogen population, which is affected by control methods and environmental conditions. We propose the concept of Integrated Avirulence Management (IAM) to enhance the durability of specific resistances. IAM involves a strategy to limit the selection pressure exerted on pathogen populations and, at the same time, reduce the size of pathogen populations by combining cultural, physical, biological or chemical methods of control. Several breakdowns of resistance specific to Leptosphaeria maculans, the causal agent of phoma stem canker have occurred in Europe and in Australia. This review paper examines control methods to limit the size of L. maculans populations and discusses how this limitation of population size can enhance the durability of specific resistances. It proposes pathways for the development of a spatially explicit model to define IAM strategies. Simulation results are presented to demonstrate the potential uses of such a model for the oilseed rape/L. maculans pathosystem.     

Babesia gibsoni infection among dogs in the southeastern United States

OBJECTIVE: To identify subclinical Babesia gibsoni infection in American Pit Bull Terriers from the southeastern United States and to determine the genetic sequence of parasite DNA isolated from these dogs. DESIGN: Case series. ANIMALS: 33 American Pit Bull Terriers and 87 dogs of various other breeds. PROCEDURE: Blood smears were examined for microscopic evidence of the parasite, and DNA was extracted from blood samples and used in a polymerase chain reaction (PCR) assay designed to amplify the small subunit ribosomal RNA gene sequence of B. gibsoni. Amplification products of the expected size were sequenced, and sequences were compared with published sequences for B. gibsoni isolates. Hematocrit, platelet count, mean platelet volume, WBC count, and eosinophil count were compared between dogs with positive PCR assay results and dogs with negative results. RESULTS: Results of the PCR assay were positive for 18 of the 33 (55%) American Pit Bull Terriers, including all 10 dogs with microscopic evidence of parasitemia. Only 1 of these dogs was clinically ill at the time blood samples were collected. Results of microscopic evaluation of blood smears and of the PCR assay were negative for the 87 other dogs. Hematocrit and platelet count were significantly lower in dogs with positive PCR assay results than in dogs with negative results. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that American Pit Bull Terriers in the southeastern United States may be subclinically infected with B. gibsoni. However, subclinical infection was not identified in dogs of other breeds from the same geographic area.     

The efficacy of a subcutaneous injection of soluble Vitamin B12 in lambs

AIMS: To assess the efficacy of a soluble Vitamin B12 injection in lambs by measuring changes in the serum and liver Vitamin B12 concentrations. METHODS: Thirty-six lambs were injected subcutaneously with 2 mg of soluble Vitamin B12 while another group of 36 served as untreated controls. Blood and liver biopsy samples for Vitamin B12 determinations were collected just before the injection and at days 1, 2, 5, 8, 16, 24, 30 and 45. RESULTS: The serum Vitamin B12 concentrations of the Vitamin B12 treated lambs increased rapidly compared to the untreated lambs. Concentrations peaked at day 2, decreased rapidly to day 8, and then decreased more slowly until day 24 when there were no longer differences between the groups. Liver Vitamin B12 concentrations of the Vitamin B12 treated lambs were significantly greater over days 8-24. CONCLUSION: A subcutaneous injection of 2 mg of soluble Vitamin B12 was effective in increasing and maintaining the Vitamin B12 status of lambs for about 24 days. CLINICAL SIGNIFICANCE: This Vitamin B12 product is only effective for preventing cobalt deficiency in lambs for about 4 weeks.     

The effects of age, weaning, drench volume and yarding on ruminoreticulum bypass in sheep, with reference to the anthelmintic efficacy of benzimidazole drenches

AIMS: To determine the incidence of ruminoreticulum bypass in lambs before and after weaning, following drenching with anthelmintic-equivalent volumes of fluid; to investigate the influence of withholding feed for 24 hours before drenching on the incidence of ruminoreticulum bypass; and to investigate the hypothesis that ruminoreticulum bypass occurs more frequently following drenching with large than with low volumes. The relationship between these factors and the efficacy of benzimidazole anthelmintic drenches has been widely promoted in New Zealand and Australia. METHODS: Video-taped fluoroscopy, using barium sulphate suspension with a specific gravity of 1.38 as a contrast medium, was used to study closure of the reticular groove and ruminoreticulum bypass in sheep. RESULTS: When lambs were drenched with 3 ml of barium sulphate suspension/10 kg liveweight soon after removal from pasture, ruminoreticulum bypass only occurred before weaning and during the first 8 weeks after weaning. However, when a low volume drench (1 ml/10 kg) was used in lambs soon after removal from pasture during the same period, ruminoreticulum bypass was not observed. When recently weaned lambs were yarded for 24 hours before drenching with barium sulphate suspension, substantial (> or = 20%) ruminoreticulum bypass occurred in 25-58% of the animals. When 8-9 month-old hoggets were yarded for 24 hours before drenching, substantial ruminoreticulum bypass occurred in 35% of the animals. Ruminoreticulum bypass was not observed in the same group of hoggets when drenched soon after removal from pasture, in a reversed crossover experiment. This effect of withholding feed for 24 hours on ruminoreticulum bypass was significant (p < 0.0001). In hoggets which had been yarded for 24 hours before drenching with barium sulphate suspension, the incidence of ruminoreticulum bypass was significantly lower (p = 0.0026) following the use of a low volume (1 ml/10 kg) when compared to high volume (3 ml/ 10 kg) of drench. Thus, the effect of withholding feed for 24 hours on ruminoreticulum bypass was largely overcome when the low volume drench was used. CONCLUSIONS: These results support the use of low volume formulations of benzimidazole drenches for New Zealand pasture-fed lambs less than 6 months old. The results indicate that, in order to obtain the optimum efficacy of benzimidazole anthelmintics in New Zealand pasture-fed sheep, which have been yarded for 24 hours, low volume drench formulations should be used.     

Acaricidal 1,2,4,5-tetrazines

A series of 1,2,4,5-tetrazines has been synthesised and screened for acaricidal activity. Laboratory tests have shown several of the compounds to be highly active mite ovicides. Further examination in the field has identified a highly active compound, which gives prolonged control of mites on top fruit. The selected compound (clofentezine) is shown to be safe to mite predators and beneficial species and has been developed commercially for use on top fruit. (Clofentezine is the B.S.I. approved common name of 3,6-bis-(2-chlorophenyl)-1,2,4,5-tetrazine).