Identification of N-acyl homoserine lactone-degrading bacteria isolated from rainbow trout (Oncorhynchus mykiss).

AIMS: A variety of pathogens use quorum sensing (QS) to control the expression of their virulence factors. QS interference has hence been proposed as a promising antivirulence strategy. The specific aim of this study was to isolate bacteria from trout tissue able to degrade N-acyl homoserine lactones (AHL), a QS molecule family. METHODS AND RESULTS: In total 132 isolates were screened for AHL degradation using Chromobacterium violaceum CV026 as a biosensor. Twenty-four quorum-quenching (QQ) isolates were identified biochemically and characterized using 16S rDNA sequencing. They belong to Bacillus, Enterobacter, Citrobacter, Acinetobacter, Agrobacterium, Pseudomonas and Stentrophomonas genera. Four Bacillus spp. showed the highest and fastest QQ activity. AHL degradation proved to be enzymatic in most isolates (except for Stentrophomonas spp. and Pseudomonas sp.) as QQ activity could be destroyed by heat and/or proteinase K treatments. All QQ activity proved to be cell-bound except for Pseudomonas sp., where it could be detected in the supernatant. The results of aiiA gene homology analysis revealed the presence of aiiA gene encoding AHL lactonase in all examined isolates except Pseudomonas syringae and Enterobacter cloacae. The HXHXDH motif conserved in all AHL lactonases and considered to be essential for AHL degradation was detected in all AiiAs after sequence alignment. CONCLUSIONS: Some known and novel QQ bacteria were isolated from trouts and characterized in terms of enzymatic or nonenzymatic AHL degradation activity and their extracellular or intracellular location. In addition, an aiiA gene and its HXHXDH motif were detected in most isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: We could isolate and identify some novel QQ bacteria including Enterobacter hormaechei, Acinetobacter radioresistens and Citrobacter gillenii. The aiiA gene was detected for the first time in these strains as well as in Stenotrophomonas maltophilia. Our QQ isolates could be used for biocontrol of bacterial infections in aquaculture.

Pathological, bacteriological, and molecular characteristics of natural outbreaks of Johne's disease in goats of Fars Province, Iran.

OBJECTIVE/BACKGROUND: Johne's disease, also called paratuberculosis, is a very important chronic infectious disease of ruminants worldwide. The causative agent is an acid-fast bacterium, Mycobacterium avium paratuberculosis (MAP). Finding a precise method for detecting MAP is essential for the control and eradication of this disease in small ruminant herds. METHODS: In this study, appropriate samples were obtained from the ileum, cecum, colon, and mesenteric lymph nodes of 10 suspected naturally infected goats. Each sample was divided to two parts: the first part was stored at -20°C for bacteriologic culture and the second part was placed in 10% neutral formalin for molecular and histopathologic examination. To isolate MAP, the double incubation method was used for decontaminating the tissues and Middlebrook 7H9 broth-based culture associated with OADC (oleic acid, albumin, dextrose, and catalase) supplement with/without mycobactin J were used. Polymerase chain reaction (PCR; IS 900) was performed for media with positive acid-fast staining. RESULTS: Acid-fast staining was positive in 40% of ileum samples, 50% of cecum samples, 40% of colon samples, and 50% of lymph node samples with mycobactin J and in 60% of ileum samples, 60% of cecum samples, 40% of colon samples, and 40% of lymph node samples without mycobactin J. All samples were confirmed by IS 900 PCR assay. Diffuse granulomatous enteritis with multibacillary lesions and paucibacillary multifocal lymphadenitis associated with calcification were diagnosed histopathologically. CONCLUSION: MAP detection in intestinal content and in tissues is quite necessary for the diagnosis, control, and eradication of this disease in small ruminant herds.

Development of a multiplex PCR for the identification of major pathogenic bacteria of post-partum endometritis in dairy cows.

Post-partum period has an important role in cows' breeding due to its effects on reproductive efficiency and subsequent pregnancy. Escherichia coli, Trueperella pyogenes (Arcanobacterium pyogenes), Fusobacterium necrophorum and Prevotella melaninogenicus are recognized as major pathogens associated with uterine endometrial lesions. The objective of this study was to identify these pathogens using the polymerase chain reaction (PCR) as a culture-independent sensitive method. A total of 172 cows were examined 25-35 days post-partum, and 128 cows were examined at 2 weeks later (39-49 days post-partum). Uterine discharges were collected by covered plastic infusion pipettes. The prevalence of endometritis was greater in the first examination than the second (35.5% vs. 16%). E. coli was detected in eight of the samples, T. pyogenes was detected in 13 of the samples and F. necrophorum was detected in 11 of the samples. There was no positive sample of P. melaninogenicus. Uterine contamination by T. pyogenes and F. necrophorum in the first examination was higher than the second examination. T. pyogenes affected as a tendency the prevalence of clinical endometritis in first examination. Primiparous cows showed 4.02 times higher odds of clinical endometritis compared with second-parity cows in first examination. A multiplex PCR protocol as a simple, less expensive, fast assay was introduced to identify E. coli, T. pyogenes and F. necrophorum.

Acute phase response in lame cattle with interdigital dermatitis.

This study was undertaken to evaluate acute phase response via assessing the concentration of serum sialic acids (total, lipid-bound and protein-bound), inflammatory mediators (IFN-γ and TNF-α) and acute phase proteins [haptoglobin (Hp) and serum amyloid A (SAA)] in lame cattle with interdigital dermatitis. Fifteen hoof scrapings from lame cows were collected from eight commercial dairy farms. As a consequence of the difficulty in culturing and isolation, a PCR technique was used to detect the organism. None of the colonies on enriched blood agar was identified as Fusobacterium necrophorum. Four (26.6%) out of the 15 hoof scrapings examined tested positive for the presence of the lktA gene (402 bp) of F. necrophorum. It seems that culture cannot be considered as the gold standard method for F. necrophorum isolation. Molecular detection is suggested as an alternative method. In the blood serum of different groups of animals (control, lameness and F. necrophorum-positive lameness) Hp, SAA, total sialic acid, lipid-bound sialic acid, and protein-bound sialic acid, and IFN-γ and TNF-α were measured using validated standard procedures. All parameters were significantly higher in the lameness group and the F. necrophorum-positive lameness group compared with the healthy group (P < 0.01 in all cases). Mean SAA concentrations in the lameness group and the F. necrophorum-positive lameness group was relatively 4.6 and 8.0 times higher than the control group. Corresponding measures for Hp indicate a 3.3 times increase in the lameness group compared to the control. In the lameness group, significant associations were observed for Hp with PBSA, SAA with TSA, TSA with PBSA, TSA with LBSA, PBSA with LBSA, and SAA with IFN-γ.