Applications of a cell culture system for studying the interaction of Anaplasma marginale with tick cells

A cell culture system for the tick-borne rickettsia Anaplasma marginale offers new opportunities for research on this economically important pathogen of cattle. A. marginale multiplies in membrane-bound inclusions in host cells. Whereas erythrocytes appear to be the only site of infection in cattle, A. marginale undergoes a complex developmental cycle in ticks and transmission occurs via the salivary glands during feeding. We recently developed a cell culture system for A. marginale using a cell line derived from embryos of Ixodes scapularis. Here we review the use of this cell culture system for studying the interaction of A. marginale with tick cells. Several assays were developed using the A. marginale/tick cell system. An adhesion assay was developed for the identification of proteins required by A. marginale for adhesion to tick cells. The effect of antibodies against selected major surface proteins in inhibiting A. marginale infection was tested in an assay that allowed further confirmation of the role of surface proteins in the infection of tick cells. A drug screening assay for A. marginale was developed and provides a method of initial drug selection without the use of cattle. The culture system was used to test for enhancing effects of tick saliva and saliva components on A. marginale infection. The tick cell culture system has proved to be a good model for studying A. marginale-tick interactions. Information gained from these studies may be applicable to other closely related tick-borne pathogens that have been propagated in the same tick cell line.     

A DNA-Based Procedure for In Planta Detection of Fusarium oxysporum f. sp. phaseoli

ABSTRACT We have characterized strains of Fusarium oxysporum from common bean fields in Spain that were nonpathogenic on common bean, as well as F. oxysporum strains (F. oxysporum f. sp. phaseoli) pathogenic to common bean by random amplified polymorphic DNA (RAPD) analysis. We identified a RAPD marker (RAPD 4.12) specific for the highly virulent pathogenic strains of the seven races of F. oxysporum f. sp. phaseoli. Sequence analysis of RAPD 4.12 allowed the design of oligonucleotides that amplify a 609-bp sequence characterized amplified region (SCAR) marker (SCAR-B310A280). Under controlled environmental and greenhouse conditions, detection of the pathogen by polymerase chain reaction was 100% successful in root samples of infected but still symptomless plants and in stem samples of plants with disease severity of >/=4 in the Centro Internacional de Agricultura Tropical (CIAT; Cali, Colombia) scale. The diagnostic procedure can be completed in 5 h and allows the detection of all known races of the pathogen in plant samples at early stages of the disease with no visible symptoms.     

Rapid Identification of Clavibacter michiganensis Subspecies sepedonicus Using Two Primers Random Amplified Polymorphic DNA (TP-RAPD) Fingerprints

Twenty strains of Clavibacter michiganensis subsp. sepedonicus from different geographic origins and other reference strains of the same and different species, including other potato pathogens, were analysed with a new procedure named TP-RAPD that originates fingerprints of bacterial species. This procedure uses two primers to amplify the 16S rDNA gene. At 45 °C of annealing, the PCR product electrophoresed in agarose gels produced a band pattern that was different in all bacterial species studied as well as in the subspecies of C. michiganensis. All strains of C. michiganensis subsp. sepedonicus displayed the same TP-RAPD number of pattern. Unlike Gram negative bacteria, Gram positives of high G + C content, such as Clavibacter, produced low bands in TP-RAPD. By using a different set of two primers also based in the 16S rDNA sequence from Escherichia coli a more adequate amplification of Gram positives of high G + C including a greater number of bands was obtained. TP-RAPD patterns using the new set of primers described in this work is a reliable and fast method to identify C. michiganensis subsp. sepedonicus.     

Genetics of tomato spotted wilt virus resistance coming from Lycopersicon peruvianum

New resistance sources coming from Lycopersicon peruvianum, especially those introgressed in UPV 32 line, are studied. UPV 32 resistance is controlled by a single gene. Resistance and dominance levels of this gene are conditioned by thrips transmission and isolate aggressiveness. A partial overcoming of resistance occurs due to the incomplete penetrance of the gene. Incomplete dominance of resistance also happens, which suggests gene dosage dependence. UPV 32 gene segregates independently of both Sw-5 gene and UPV 1 resistance gene, also coming from Lycopersicon peruvianum. It is proposed to name Sw-6 this new locus from UPV 32. Sw-5 gene and UPV 1 resistance gene show higher resistance than Sw-6. Heterozygotes for UPV 1 resistance gene were more resistant than heterozygotes for Sw-5. The lower dependence of UPV 1 resistance gene on the gene dosage effect makes it very useful for the development of commercial hybrids.     

Evaluation of a laparoscopic technique for collection of serial full-thickness small intestinal biopsy specimens in standing sedated horses

OBJECTIVE: To assess a technique for laparoscopic collection of serial full-thickness small intestinal biopsy specimens in horses. ANIMALS:13 healthy adult horses. PROCEDURES: In the ex vivo portion of the study, sections of duodenum and jejunum obtained from 6 horses immediately after euthanasia were divided into 3 segments. Each segment was randomly assigned to the control group, the double-layer hand-sewn closure group, or the endoscopic linear stapler (ELS) group. Bursting strength and bursting wall tension were measured and compared among groups; luminal diameter reduction at the biopsy site was compared between the biopsy groups. In the in vivo portion of the study, serial full-thickness small intestinal biopsy specimens were laparoscopically collected with an ELS from the descending duodenum and distal portion of the jejunum at monthly intervals in 7 sedated, standing horses. Biopsy specimens were evaluated for suitability for histologic examination. RESULTS: Mean bursting strength and bursting wall tension were significantly lower in the ELS group than in the hand-sewn and control groups in both the duodenal and jejunal segments. Use of the hand-sewn closure technique at the biopsy site reduced luminal diameter significantly more than use of the stapling technique. In the in vivo part of the study, all 52 biopsy specimens collected during 26 laparoscopic procedures were suitable for histologic examination and no clinically important perioperative complications developed. CONCLUSIONS AND CLINICAL RELEVANCE: Laparoscopic collection of serial full-thickness small intestinal biopsy specimens with a 45-mm ELS may be an effective and safe technique for use in healthy adult experimental horses.     

Evaluation of inciting causes, alternative targets, and risk factors associated with redirected aggression in cats

OBJECTIVE: To identify inciting causes, alternative targets, and risk factors associated with redirected aggression in cats. DESIGN: Case-control study. ANIMALS: 19 cats with a history of redirected aggression and 64 cats with no such history. PROCEDURES: Medical records were reviewed to identify cats evaluated for problems with redirected aggression (case cats), in which the primary inciting stimulus and alternative target of aggression were clearly identifiable. Data obtained from the records and from follow-up interviews included details about the cats and incidents of redirected aggression. Owners of control cats were interviewed via telephone to obtain similar information on their cats. RESULTS: 22 incidents of redirected aggression were reported for the 19 case cats. In 95% of those incidents, loud noises or interactions with other cats were identified as the inciting stimuli. Case cats most commonly redirected their aggression toward the owner, followed by another cat living in the same household. Compared with control cats, case cats were more likely to have a sound phobia but were less likely to be outdoor cats. In addition, case cats were more likely to be from small households (相似文献   

Prolactin stimulates the internalization of Staphylococcus aureus and modulates the expression of inflammatory response genes in bovine mammary epithelial cells

The incidence of mastitis in dairy cattle is highest at the drying off period and parturition, which are characterized by high levels of the lactogenic hormone prolactin (PRL). One of the most frequently isolated contagious pathogens causing mastitis is Staphylococcus aureus. However, the role of PRL on S. aureus infection in mammary epithelium has not been studied. In this work we evaluated the effect of bovine PRL (bPRL) on S. aureus internalization in a primary culture of bovine mammary epithelial cells (bMEC) and on the expression of cytokine and innate immune response genes. Our data show that 5ng/mL bPRL enhances approximately 3-fold the internalization of S. aureus (ATCC 27543) into bMEC. By RT-PCR analysis, we showed that bPRL is able to up-regulate the expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and inducible nitric oxide synthase (iNOS) mRNAs. However, bPRL together with S. aureus did not modify the expression of TNF-alpha and iNOS mRNAs, while it down-regulated the expression of beta-defensin and IL-1beta mRNAs, as well as nitric oxide production, suggesting that infection and bPRL together can inhibit elements of the host immune response. To our knowledge, this is the first report that shows a role of bPRL during the internalization of S. aureus into bMEC.     

Salmonella in Belgian laying hens: an identification of risk factors

Since the 1980s, the prevalence of Salmonella in Belgian poultry layers and broilers has greatly fluctuated with a rise observed in 2003 and a significant decrease in 2005. In order to alleviate the risk at egg consumer level, it is crucial to understand the factors which influence the contamination and the spread of Salmonella in laying hens. To study such determinants we explored the Belgian data from the 2005 baseline study on the prevalence of Salmonella in laying flocks of Gallus gallus in the European Union. The response variables corresponded to presence or absence of Salmonella from dust and faecal samples taken from the environment of a Belgian layer flock. The explanatory variables included: region of Belgium, sampling time (month the flock was sampled), production type (cage or barn and free range), Salmonella vaccination status, flock age and flock size. Analyses of these data were performed using a bivariate logistic regression model assuming independence between the two responses and bivariate generalized estimating equations model, which incorporates the correlation between the two responses on the same flock. The main risk factor that was identified was rearing flocks in cages compared to barns and free-range systems. The results also showed a significant higher risk for Salmonella for a 1 week increase in flocks’ age as well as with a unit increase in the size of the flock.     

Occurrence of antibodies to Neospora caninum and Toxoplasma gondii in dogs from Pernambuco, Northeast Brazil

A serological survey was carried out to assess the occurrence of anti-Neospora caninum antibodies in dogs from the State of Pernambuco. A total of 625 serum samples of dogs (289 from Paulista, 168 from Amaraji and 168 from Garanhuns) were tested by an immunofluorescence antibody assay for the detection of anti-N. caninum antibodies. A total of 177 (28.3%; IC 95%, 24.9-32.1) samples were positive. The seropositivity rates found in Paulista, Amaraji and Garanhuns were 26% (IC 95%, 21-31.4), 26.2% (IC 95%, 19.7-33.5) and 34.5% (IC 95%, 27.4-42.2), respectively. Of the 177 serum samples positive to anti-N. caninum antibodies, 170 were additionally tested for the presence of anti-Toxoplasma gondii antibodies and out of these 57.6% (IC 95%, 49.8-65.2) were positive. The results indicate that dogs from Amaraji, Paulista and Garanhuns are exposed to both N. caninum and T. gondii infections. The presence of dogs infected by N. caninum in Pernambuco represents a potential risk factor for the occurrence of outbreaks of abortion in cattle and small ruminants in this state. This study is the largest serological survey on the presence of anti-N. caninum antibodies in dogs carried out in Brazil and reports for the first time the exposure to N. caninum and T. gondii in dogs from Pernambuco.     

Assessment of primers designed for the subspecies-specific discrimination among Babesia canis canis, Babesia canis vogeli and Babesia canis rossi by PCR assay

Canine babesiosis is an infectious disease caused by either Babesia gibsoni or Babesia canis protozoans. The latter is also classified under three different phylogenetic groups, referred to as subspecies B. canis canis, B. canis vogeli and B. canis rossi. The objective of the present study was to validate and standardize a PCR assay to discriminate the organisms at the subspecies level. First, the reference sequences of the 18S rRNA, 5.8S rRNA and 28S rRNA genes, including the internal transcribed spacer 1 (ITS1) and 2 (ITS2) of the most common species and subspecies of the genus Babesia were retrieved from the GenBank database. Subspecies-specific primers (BAB3, BAB4 and BAB5) and one genus-specific primer were designed from the alignment of the sequences. The PCR assays were evaluated in three different combinations of primer pairs in order to assure complete specificity for each reaction. The results of the tests had demonstrated effectiveness of the novel primer pairs BAB1/BAB3, BAB1/BAB4 and BAB1/BAB5 for the amplification of the subspecies-specific target fragments of 746 bp (B. c. canis), 546 bp (B. c. vogeli) and 342 bp (B. c. rossi) by PCR. The original enzymatic amplification assays with novel primers reported in this paper were confirmed to be a reliable tool for the specific discrimination among B. canis subspecies by single-step PCR assays.