Functional Diversification, Redundancy, and Epistasis among Paralogs of the Drosophila melanogaster Obp50a-d Gene Cluster.

Large multigene families, such as the insect odorant-binding proteins (OBPs), are thought to arise through functional diversification after repeated gene duplications. Whereas many OBPs function in chemoreception, members of this family are also expressed in tissues outside chemosensory organs. Paralogs of the Obp50 gene cluster are expressed in metabolic and male reproductive tissues, but their functions and interrelationships remain unknown. Here, we report the genetic dissection of four members of the Obp50 cluster, which are in close physical proximity without intervening genes. We used CRISPR technology to excise the entire cluster while introducing a PhiC31 reintegration site to reinsert constructs in which different combinations of the constituent Obp genes were either intact or rendered inactive. We performed whole transcriptome sequencing and assessed sexually dimorphic changes in transcript abundances (transcriptional niches) associated with each gene-edited genotype. Using this approach, we were able to estimate redundancy, additivity, diversification, and epistasis among Obp50 paralogs. We analyzed the effects of gene editing of this cluster on organismal phenotypes and found a significant skewing of sex ratios attributable to Obp50a, and sex-specific effects on starvation stress resistance attributable to Obp50d. Thus, there is functional diversification within the Obp50 cluster with Obp50a contributing to development and Obp50d to stress resistance. The deletion-reinsertion approach we applied to the Obp50 cluster provides a general paradigm for the genetic dissection of paralogs of multigene families.

Using milk leukocyte differentials for diagnosis of subclinical bovine mastitis.

This research study aimed to evaluate the use of the milk leukocyte differential (MLD) to: (a) identify quarter milks that are culture-positive; and (b) characterize the milk leukocyte responses to specific groups of pathogens causing subclinical mastitis. The MLD measures the absolute number and relative percentage of inflammatory cells in milk samples. Using the MLD in two dairy herds (170 and 172 lactating cows, respectively), we studied all lactating cows with a most recent monthly Dairy Herd Improvement Association somatic cell count (SCC) >200 × 103 cells/ml. Quarter milk samples from 78 cows meeting study criteria were analysed by MLD and aseptically collected milk samples were subjected to microbiological culture (MC). Based upon automated instrument evaluation of the number and percentage of inflammatory cells in milk, samples were designated as either MLD-positive or - negative for subclinicial mastitis. Positive MC were obtained from 102/156 (65·4%) of MLD-positive milk samples, and 28/135 (20·7%) of MLD-negative milk samples were MC-positive. When MC was considered the gold standard for mastitis diagnosis, the calculated diagnostic Se of the MLD was 78·5% (IC(95%) = 70·4 to 85·2%) and the Sp was 66·5% (IC(95%) = 58·6 to 73·7%). [corrected]. Quarter milks positive on MC had higher absolute numbers of neutrophils, lymphocytes and macrophages, with higher neutrophils% and lymphocytes% but lower macrophages%. The Log10 (N/L) ratios were the most useful ratio to differentiate specific subclinical mastitis quarters from healthy quarters. Use of the MLD on cows with monthly composite SCC > 200 × 103 cells/ml for screening at quarter level identified quarters more likely to be culture-positive. In conclusion, the MLD can provide an analysis of mammary quarter status more detailed than provided by SCC alone; however, the MLD response to subclinical mastitis was not found useful to specifically identify the causative pathogen.

Molecular epidemiology of Enterococcus cecorum isolates recovered from enterococcal spondylitis outbreaks in the southeastern United States.

Enterococcus cecorum, a normal intestinal inhabitant, is increasingly responsible for outbreaks of arthritis and osteomyelitis in chickens worldwide. Enterococcal spondylitis (ES) is a specific manifestation of E. cecorum-associated disease in which increased flock morbidity and mortality result from chronic infection involving the free thoracic vertebra. In this study the genetic relatedness and antimicrobial resistance of isolates recovered from ES-affected flocks in the southeastern United States were determined. ES outbreaks from 2007 to 2011 were investigated in North Carolina (15 flocks, 13 farms, four integrators), South Carolina (one flock, one farm, one integrator) and Alabama (six flocks, six farms, one integrator). From these 22 epidemiologically distinct outbreaks, 326 isolates of E. cecorum were recovered. Isolates from spinal lesions and caeca of affected birds (cases) and caeca of unaffected birds (controls) were genotyped using pulsed-field gel electrophoresis; phenotyped using both GenIII MicroPlate™ (Biolog; Hayward, CA, USA) microbial identification plates and antimicrobial sensitivity testing; and compared with each other. Isolates from spinal lesions were incapable of mannitol metabolism and the majority of these isolates were genetically clonal. In contrast, caecal isolates from control birds varied in their ability to metabolize mannitol and were genetically diverse. Isolates from both case and control birds had high levels of antimicrobial resistance. These findings indicate that the increase in E. cecorum-associated disease in the southeast United States is due to the emergence of new clones with increased pathogenicity and multidrug resistance.

An outbreak and source investigation of enterococcal spondylitis in broilers caused by Enterococcus cecorum.

Enterococcus cecorum was isolated from spondylitis lesions in broilers from two flocks in North Carolina that were experiencing increased mortality. Affected birds showed paresis and paralysis, clinical signs characteristic of enterococcal spondylitis (ES). Affected birds rested on their hocks and caudal abdomens with legs extended forward and were unable to stand or walk. Necropsy examination of affected birds revealed firm to hard inflammatory masses involving the vertebral bodies at the level of the free thoracic vertebra that bulged dorsally and compressed the spinal cord. When opened, lesions contained pale, tan to yellow caseonecrotic material. Microscopically, necrosis and fibrinoheterophilic spondylitis with intralesional gram-positive bacteria were seen. Heavy growth of E. cecorum recovered from vertebral lesions confirmed the diagnosis of ES. To investigate possible sources of the organism for one of the flocks bacterial cultures were made from the environment, water lines, mice trapped on the farm, cecal/cloacal swabs from one of the parent broiler breeder flocks, egg residue, hatching eggs, and the hatchery environment. Except for cecal/cloacal swabs from the breeders, E. cecorum was not isolated from any of these samples. When compared phenotypically and genotypically, cecal/cloacal isolates of E. cecorum from the breeders differed from isolates from spondylitis lesions in the broilers. The source of E. cecorum for the broiler flocks was not determined, but vertical transmission appears unlikely.

Genetic diversity and antimicrobial susceptibility profiles among mastitis-causing Staphylococcus aureus isolated from bovine milk samples.

OBJECTIVE: To determine whether particular antimicrobial susceptibility profiles of bovine mastitis-causing Staphylococcus aureus isolates were associated with specific S aureus genotypes. SAMPLE POPULATION: 357 S aureus isolates recovered from milk samples submitted for diagnostic bacteriologic testing from 24 dairy herds. PROCEDURES: Antimicrobial susceptibility of S aureus isolates was assessed by determining minimum inhibitory concentrations (MICs) to 14 antimicrobial agents. After digestion of S aureus genomic DNA by SmaI, electrophoretic patterns were obtained via pulsed-field gel electrophoresis (PFGE) and used to classify isolates into types. Gels were analyzed, and data were used to prepare dendrograms. RESULTS: 308 of 357 (86%) S aureus isolates were susceptible to all antimicrobials evaluated. Forty-nine S aureus isolates were resistant to 1 or more antimicrobials; of these isolates, 37 were resistant only to penicillin, 9 were resistant to penicillin and erythromycin, 2 were resistant to tetracycline, and 1 was resistant to erythromycin. Isolates were assigned to 7 PFGE types. An association was found between PFGE type and antimicrobial susceptibility profile. Organisms with resistance to at least one of the tested antimicrobial agents were identified in only 4 of the 7 types of S aureus. CONCLUSIONS AND CLINICAL RELEVANCE: Antimicrobial resistance was uncommon among the mastitis-causing S aureus isolates identified in the milk samples. A limited number of genotypes were associated with mastitis in these herds. Antimicrobial resistance phenotypes were associated with particular S aureus PFGE types; this association may have implications for future treatment and control of S aureus-associated mastitis in cattle.

Efficacy of vaccination and antimicrobial treatment to eliminate chronic intramammary Staphylococcus aureus infections in dairy cattle.

OBJECTIVE: To determine whether a combination of vaccination and extended intramammary antimicrobial treatment would eliminate chronic intramammary Staphylococcus aureus infections in lactating dairy cows. DESIGN: Randomized controlled clinical trial. ANIMALS: 50 dairy cows with chronic mastitis caused by S aureus. PROCEDURE: Cows were identified and paired within herd on the basis of days in milk, lactation number, milk production, and numbers of quarters infected. Treated cows (n=20) received 3 doses of a polyvalent S aureus bacterin on days 1, 15, and 21 of the study along with intramammary administration of pirlimycin in all 4 quarters once daily for 5 treatments (days 16 to 20). Control cows (n=23) received no treatment. Follow-up samples for bacteriologic culture were collected for at least 3 months after treatment to determine treatment success rates. RESULTS: Significantly more S aureus infections were eliminated from treated cows (8/20 [40%]), compared with control cows (2/23 [9%]). The proportion of infected quarters that yielded negative results throughout the follow-up period was also significantly higher in treated cows (13/28 [46%]) than in control cows (2/41 [5%]). CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that a combination of vaccination and antimicrobial treatment can be successful in eliminating some cases of chronic intramammary S aureus infections in dairy cattle. However, it is important to consider extended treatment protocols carefully because many cows are likely to remain infected with S aureus despite treatment and vaccination.

Comparison of microbial receptor assay and liquid chromatography for determination of penicillin G and amoxicillin in milk powder.

A microbial receptor assay (Charm II Tablet Beta-Lactam Test) and liquid chromatography (LC) were compared for determination of penicillin G (PG) and amoxicillin (AMOX) in reconstituted milk powder. Nonfat dry milk and whole dry milk were reconstituted (10%, w/v) to concentrations of 0, 2.5, 5, 7.5, and 10 ppb PG; nonfat dry milk was reconstituted (10%, w/v) to 0, 7.5, 10, and 15 ppb AMOX. Reconstituted samples were analyzed blindly by each method. Concentrations determined by both methods demonstrated good agreement. A significant difference between methods (p < or = 0.05) was observed only for 7.5 ppb PG in defatted dry milk. Significant differences were not observed between known concentrations and concentrations determined by the Charm II assay for PG or AMOX in defatted dry milk and PG in whole dry milk. Results by LC showed significant differences (p < 0.05) between known and measured concentrations at 10 ppb PG in both milks and 0 ppb AMOX in defatted dry milk. These results suggest that both the microbial receptor assay and LC may be useful for determination of PG and AMOX near safe level and tolerance, respectively, in reconstituted milk powder.