Lack of a type-2 glycosyltransferase in the fish pathogen Flavobacterium psychrophilum determines pleiotropic changes and loss of virulence

Flavobacterium psychrophilum is an important fish pathogen, responsible for Cold Water Disease, with a significant economic impact on salmonid farms worldwide. In spite of this, little is known about the bacterial physiology and pathogenesis mechanisms, maybe because it is difficult to manipulate, being considered a fastidious microorganism. Mutants obtained using a Tn4351 transposon were screened in order to identify those with alteration in colony morphology, colony spreading and extracellular proteolytic activity, amongst other phenotypes. A F. psychrophilum mutant lacking gliding motility showed interruption of the FP1638 locus that encodes a putative type-2 glycosyltransferase (from here on referred to as fpgA gene, Flavobacterium psychrophilum glycosyltransferase). Additionally, the mutant also showed a decrease in the extracellular proteolytic activity as a consequence of down regulation in the fpgA mutant background of the fpp2-fpp1 operon promoter, responsible for the major extracellular proteolytic activity of the bacterium. The protein glycosylation profile of the parental strain showed the presence of a 22 kDa glycosylated protein which is lost in the mutant. Complementation with the fpgA gene led to the recovery of the wild-type phenotype. LD₅₀ experiments in the rainbow trout infection model show that the mutant was highly attenuated. The pleiotropic phenotype of the mutant demonstrated the importance of this glycosyltranferase in the physiology and virulence of the bacterium. Moreover, the fpgA mutant strain could be considered a good candidate for the design of an attenuated vaccine.     

Characterization of biological activities of feline eosinophil granule proteins

OBJECTIVE:To characterize eosinophil granule-derived proteins in cats. SAMPLE POPULATION: Eosinophils collected via peritoneal lavage from 2 cats. PROCEDURE: The cats were infested orally with Toxocara canis eggs and subsequently challenge-exposed with T. canis antigen injected IP to induce peritoneal eosinophilia; eosinophils were collected via peritoneal lavage. Eosinophil granule proteins were acid-extracted, separated by gel-filtration chromatography, and examined for their peroxidase, ribonuclease, and bactericidal activities; the N-terminal sequence of some of these proteins was determined and compared with homologue proteins from other species. RESULTS: 3 protein peaks were separated in the chromatogram. The first peak had both peroxidase and bactericidal activities. The second peak had ribonuclease and bactericidal activities, and the N-terminal sequence of the major protein was homologous with that of proteins of the ribonuclease A superfamily, including eosinophil ribonucleases from humans and other animal species. The third protein peak had bactericidal activity, and the N-terminal sequence of the major protein was homologous with that of human and murine major basic proteins. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that feline eosinophil granules contain major basic protein and eosinophil-associated ribonuclease and the granule proteins have peroxidase, ribonuclease, and bactericidal activities. In cats, characterization of eosinophil granule proteins may be useful in elucidation of the mechanism of tissue damage in eosinophil-associated diseases and development of new treatment options for those diseases. In addition, the identification of conserved structure and function of eosinophil granule proteins in cats is relevant from an evolutionary viewpoint.     

Myoplasmic calcium regulation in myotubes from horses with recurrent exertional rhabdomyolysis

OBJECTIVE: To determine whether alterations in myoplasmic calcium regulation can be identified in muscle cell cultures (myotubes) and intact muscle fiber bundles derived from Thoroughbreds affected with recurrent exertional rhabdomyolysis (RER). ANIMALS: 6 related Thoroughbreds with RER and 8 clinically normal (control) Thoroughbred or crossbred horses. PROCEDURES: Myotube cell cultures were grown from satellite cells obtained from muscle biopsy specimens of RER-affected and control horses. Fura-2 fluorescence was used to measure resting myoplasmic calcium concentration as well as caffeine- and 4-chloro-m-cresol (4-CMC)-induced increases in myoplasmic calcium. In addition, intact intercostal muscle fiber bundles were prepared from both types of horses, and their sensitivities to caffeine- and 4-CMC-induced contractures were determined. RESULTS: Myotubes of RER-affected and control horses had identical resting myoplasmic calcium concentrations. Myotubes from RER-affected horses had significantly higher myoplasmic calcium concentrations than myotubes from control horses following the addition of > or = 2mM caffeine; however, there was no difference in their response to 4-CMC (> or = 1 mM). Caffeine contracture thresholds for RER and control intact muscle cell bundles (2 vs 10mM, respectively) were significantly different, but 4-CMC contracture thresholds of muscle bundles from RER-affected and control horses (500 microM) did not differ. CONCLUSIONS AND CLINICAL RELEVANCE: An increase in caffeine sensitivity of muscle cells derived from a family of related RER-affected horses was detected in vitro by use of cell culture with calcium imaging and by use of fiber bundle contractility techniques. An alteration in muscle cell calcium regulation is a primary factor in the cause of this heritable myopathy.     

Influence of age and purpose for testing on the cut-off selection of serological methods in bovine neosporosis

The aim of this study was to investigate the need for different cut-off points, according to animal age and the purpose of testing, for two of the most widely used serological techniques in bovine neosporosis, IFAT and a crude antigen ELISA (Civtest, HIPRA). Therefore, the population reference sera used were defined using a combination of multiple criteria such as epidemiological/clinical and histopathological parameters and an immunoblot test. Firstly, foetuses and breeding cattle (heifers and cows) were considered as separate subpopulations for serological evaluation. Secondly, cut-off points for each serological technique (IFAT and ELISA) according to age group (foetuses and breeding cattle) and the different practical applications (detection of infection and abortion) were calculated following the receiver operating characteristics (ROC) analysis. Cut-off points were defined, for IFAT and ELISA for aborted breeding cattle and for IFAT alone in the case of the foetuses, assuming an equivalent cost of false positive and negative results. In infected breeding cattle, for IFAT and ELISA and in foetuses for ELISA, two possible cut-off values were obtained, one for a maximum sensitivity and one for a maximum specificity and the intervals of unclear results were defined. In this case, a cut-off value for equal sensitivity and specificity was also estimated. When cut-off points for infected breeding cattle, 1:100-1:250 for IFAT and 0.306-0.451 for ELISA were applied to a target population, optimal and similar negative and positive predictive values together with similar apparent and true prevalence results were observed suggesting the possibility of using both tests interchangeably.     

Prevalence and risk-factor analysis of Shiga toxigenic Escherichia coli in faecal samples of organically and conventionally farmed dairy cattle

Cattle are a natural reservoir for Shiga toxigenic Escherichia coli (STEC), however, no data are available on the prevalence and their possible association with organic or conventional farming practices. We have therefore studied the prevalence of STEC and specifically O157:H7 in Swiss dairy cattle by collecting faeces from approximately 500 cows from 60 farms with organic production (OP) and 60 farms with integrated (conventional) production (IP). IP farms were matched to OP farms and were comparable in terms of community, agricultural zone, and number of cows per farm. E. coli were grown overnight in an enrichment medium, followed by DNA isolation and PCR analysis using specific TaqMan assays. STEC were detected in all farms and O157:H7 were present in 25% of OP farms and 17% of IP farms. STEC were detected in 58% and O157:H7 were evidenced in 4.6% of individual faeces. Multivariate statistical analyses of over 250 parameters revealed several risk-factors for the presence of STEC and O157:H7. Risk-factors were mainly related to the potential of cross-contamination of feeds and cross-infection of cows, and age of the animals. In general, no significant differences between the two farm types concerning prevalence or risk for carrying STEC or O157:H7 were observed. Because the incidence of human disease caused by STEC in Switzerland is low, the risk that people to get infected appears to be small despite a relatively high prevalence in cattle. Nevertheless, control and prevention practices are indicated to avoid contamination of animal products.     

A description of local pig feeding systems in village smallholder farms of Western Kenya

We used face-to-face interviews to gather data on pig feeding practices in rural Busia District, Kenya. We visited 164 pig farms three times in the course of the study period. The pigs were weighed in kilograms during the visits. Feeds offered to pigs were described during the interviews. The most frequently fed feedstuffs were; ground maize or "ugali" (88%), kitchen leftovers (83%) and dried fish locally called "omena" (78%). Farmers provided pigs with water separately from the feeds. Sweet potatoes, "ugali" and cassava were available and could serve as good sources of energy for pigs in the district. Fruits and vegetables were also available and could potentially act as good sources of vitamins. Sweet potato vines, "omena" fish and slaughter blood were available and could provide pigs with proteins. The average daily gain (ADG) for pigs ≤ 5 months of age, pigs of 5.1-9.9 months of age and pigs of ≥ 10 months old was 94.5 (± 43), 127 (± 49.8) and 99 (± 92) g, respectively (p = 0.000). This study has outlined the different local pig feeds available in Busia district. We recommend two things: first, additional research on nutrient composition for the identified local feeds, and second, developing and validating simple local feed combinations that would achieve balanced local pig rations.     

Evaluation of reticulocyte hemoglobin content (RET‐He) in the diagnosis of iron‐deficient erythropoiesis in dogs

Background

Reticulocyte hemoglobin content provided by the Siemens ADVIA (CHr) is an established marker of iron deficiency. The IDEXX ProCyte Dx hematology analyzer now provides a similar variable, reticulocyte hemoglobin equivalent (RET‐He).

Objectives

The objective was to evaluate RET‐He and its diagnostic utility in dogs, and to calculate a cutoff value for diagnosing iron‐deficient erythropoiesis (IDE). Furthermore, the prevalence of RET‐He values below this cutoff value was established.

Methods

One hundred and seventy‐one CBCs of healthy dogs were used to establish a RI. Stability of RET‐He was evaluated by repeated measurements over 48 hours (n = 10). The 25‐run coefficient of variation (CV) was calculated, and correlation and bias between measurements of RET‐He and CHr were assessed (n = 190). A cutoff value for diagnosing IDE was calculated. The utility of RET‐He in the detection of IDE was evaluated in 123 dogs. The prevalence of low RET‐He values was assessed retrospectively in a multicenter study (2012–2014) under participation of 7 veterinary clinics.

Results

Reticulocyte hemoglobin equivalent with an RI of 22.2 to 28.6 pg was statistically stable over 48 hours (P = .10). The CV was 1.8%. A fair correlation (ρ = 0.74) between RET‐He and CHr with a small bias of ?0.6 pg was found. The cutoff value for diagnosing IDE was 20.9 pg (sensitivity: 85%; specificity: 99%). The prevalence of RET‐He values below 20.9 pg was 10.3% (1084/10,553 dogs).

Conclusions

RET‐He on the ProCyte Dx is a precise screening tool in dogs to detect iron‐deficient erythropoiesis.