Cloning,expression, purification,and biological activity of five feline type I interferons

Type I interferons (IFN) are important mediators of the host defense against viral infections in mammals. In humans multiple subtypes of IFN-alpha exist, most of which possess antiviral activity. Little is known about the type I IFN genes in cats and the role they may play in feline immunological responses to viruses. We have isolated cDNAs encoding five feline IFN-alpha (feIFN) subtypes that share from 95 to 99% amino acid sequence identity. FeIFN-alpha5 has five additional amino acids inserted at position 139, which are not present in the other four subtypes. Sequence identity of the feIFN proteins encoded by the five clones compared to human IFN-alpha2 is approximately 60%. Unlike most of the human subtypes, each of the five feline IFN sequences has an N-glycosylation recognition site. Expression of all five feIFN-alpha subtypes in Chinese hamster ovary (CHO) cells was confirmed by Western blot analysis, and all resulting proteins were glycosylated. The antiviral activity of each feIFN-alpha subtype produced in transiently transfected CHO cell cultures was tested in vitro. In addition, subtype feIFN-alpha6 was expressed in the yeast, Pichia pastoris. The resulting secreted mature recombinant protein was purified and demonstrated significant antiviral activity and induction of 2',5'-oligoadenylate synthetase activity in vitro.     

The effect of diet fed to lambs on subsequent development of Trichostrongylus colubriformis larvae in vitro and on pasture

Contrasting herbage diets were fed to lambs to evaluate their effect on subsequent development of Trichostrongylus colubriformis larvae in faeces and on pasture. The diets had either no condensed tannin (CT), lucerne (Medicago sativa cv. Otaio), white clover (Trifolium repens cv. Tahora), or had moderate to high concentrations of CT, sulla (Hedysarum coronarium cv. Grassland Aokau), Lotus corniculatus (cv. Grasslands Goldie), L. pedunculatus (cv. Grassland Maku), Dorycnium pentophyllum, and Dorycnium rectum. Trials were carried out in summer (warm) and in autumn (cool and moist). In summer, egg viability was evaluated in vitro with egg hatch and larval development assays. In both seasons faeces were placed on pasture to compare recovery of eggs and larvae from faeces and larvae from herbage on the high and low fertility farmlets on the AgResearch Ballantrae Hill Country Research Station. D. rectum and D. pentophyllum diets decreased (P<0.01) egg hatching and larval development in laboratory assays relative to other diets. In summer, the number of larvae recovered from faeces placed on pasture was far greater (P<0.001) if the lambs had been fed lucerne than any other diet, whereas recovery was always lowest from faeces of sheep fed D. rectum and D. pentophyllum. Although dietary differences were lower in autumn than in summer, larval recoveries were lower (P<0.05) from faeces of lambs fed D. rectum and L. corniculatus than from white clover, lucerne and sulla diets. This study indicates that the diet of the host can have a significant impact on egg hatching and the subsequent development of T. colubriformis larvae in the laboratory and in the field. In particular, D. rectum consistently reduced T. colubriformis development. Effects measured in vitro generally under-estimated effects measured under field conditions.     

Evaluation of cystatin C as an endogenous marker of glomerular filtration rate in dogs

Cystatin C is a cysteine protease inhibitor produced by all nucleated cells. It is freely filtered by the glomerulus and is unaffected by nonrenal factors such as inflammation and gender. Because of greater sensitivity and specificity, cystatin C has been proposed to replace creatinine as a marker of glomerular filtration rate (GFR) in humans. The aims of this study were to validate an automated assay in canine plasma and to evaluate the usefulness of cystatin C as a marker of GFR in dogs. Western blotting was used to demonstrate cross-reactivity of an anti-human cystatin C antibody. An immunoturbidimetric assay was used to detect cystatin C in 25 clinically healthy dogs and 25 dogs with renal failure. Mean cystatin C concentration in the healthy dogs and the dogs with renal failure was 1.08 +/- 0.16 mg/L and 4.37 +/- 1.79 mg/L respectively. Intra- and interassay variability was <5%. The assay was linear (r = .974) between 0.14 and 7.53 mg/L. Both cystatin C and creatinine concentrations were measured in banked, frozen serum from 20 remnant kidney model dogs and 10 volume-depleted dogs for which GFR measurements by exogenous creatinine clearance had been determined previously. In the remnant kidney model, cystatin C was better correlated with GFR than creatinine (r = .79 versus .54) but was less well correlated with GFR in volume-depleted dogs (r = .54 versus .95). GFR measurements were repeated in the remnant kidney model dogs 60 days after initial GFR measurements. At this time, cystatin C and creatinine concentrations correlated equally well with GFR (r = .891 versus .894, respectively). Cystatin C concentration is a reasonable alternative to creatinine for screening dogs with decreased GFR due to chronic renal failure.     

Geo-referenced spatiotemporal analysis of the urban citrus canker epidemic in Florida

ABSTRACT Five areas in urban Miami were identified to study the spread of Xanthomonas axonopodis pv. citri to determine if the practice of removing exposed citrus trees within 38.1 m of trees affected by citrus canker was adequate to curtail further bacterial spread. To accomplish this, 18,769 trees in dooryards were surveyed, geo-referenced by differential global positioning systems (GPS), and assayed for disease severity, age of infection, citrus cultivar, location of infection in tree, and canopy size. For each tree, the date the tree became infected was estimated and used to separate trees into contiguous 30-day categories. For each area studied, distance measurements between focal trees and newly infected trees were calculated for various temporal periods of 30, 60, 90, and 120 days in duration, corresponding to intervals of inspection survey. A visual basic application was used to calculate the distances between each newly diseased tree and all prior focal trees. The nearest distance was used because it was considered the most conservative estimate possible. It is therefore likely to be an underestimate of spread but is a good estimate of the minimum possible distances of spread. For the first four 30-day periods among the five study areas, calculated maximum distances of spread ranged from 12 to 3,474 m, indicating a broad continuum of distance for bacterial spread was possible. Disease increased during the first two-thirds of the time studied and reached an asymptote due to dry conditions in the final one-third of the duration of the study. Cross correlation analysis indicated that disease was best visualized 107 days following rainstorms with wind. Analysis of regional spatial point patterns was performed temporally for each 30-day period via a modified Ripley's K-function. Spatiotemporal analyses between periods over areas larger than previously examined were accomplished via spatiotemporal semivariogram analysis. These methods in combination demonstrated rapid increases in range of spatial dependency and range of spatiotemporal dependency for all study sites. This corresponded to rapid spread of disease across the regions studied in response to rainstorms with wind followed by a "filling in" of disease on remaining noninfected susceptible trees through time by less intense rain events. A stochastic quadratization technique demonstrated that disease incidence and disease severity were not greatly affected by urban host density but were positively correlated to host susceptibility within local 0.25-km(2) quadrats.     

Reference values for activated coagulation time in cats

OBJECTIVE: To establish reference values for activated coagulation time (ACT) in cats by use of jugular venipuncture and direct collection of blood into ACT vacuum tubes. ANIMALS: 100 clinically normal cats that were to have elective surgery performed at a private practice. PROCEDURE: Collection of 3 blood samples for ACT measurement was attempted for each cat at the time of elective surgery: sample 1, obtained before sedation; sample 2, tube 1 of 2 consecutive samples obtained from a single venipuncture of the contralateral jugular vein after sedation with acepromazine and ketamine hydrochloride; and sample 3, tube 2 collected immediately following collection of sample 2 without removing the needle from the vein. Venipuncture quality was rated subjectively on a 3-point scale. RESULTS: Median ACT were 95 seconds for each sample group. The middle 95% of values ranged inclusively from 55 to 185 seconds (sample 1), 65 to 135 seconds (sample 2), 45 to 145 seconds (sample 3), and 55 to 165 seconds overall (samples 1, 2, and 3). Significant differences in ACT values were not detected between sample groups. Significant relationships between ACT and venipuncture quality or sex of cat were not detected. CONCLUSIONS AND CLINICAL RELEVANCE: With the ACT protocols used, clinically normal cats had ACT of < 165 seconds. The ACT in cats does not appear to be significantly affected by sex, sedation with acepromazine and ketamine, or by moderately traumatic venipunctures. These results refute widespread statements that ACT should be < 65 seconds in healthy cats. Cats with ACT repeatedly > 165 seconds should be further evaluated for hemostatic disorders.     

Theileriosis in a Missouri beef herd caused by Theileria buffeli: case report, herd investigation, ultrastructure, phylogenetic analysis, and experimental transmission

A 6-year-old Simmental cow infected with Theileria buffeli had a clinical disease characterized by theilerial parasitemia, macrocytic normochromic anemia with acanthocytosis and spherocytosis, lymphoid hyperplasia (lymphocytosis, edematous lymphadenomegaly), dysproteinemia, evidence of liver disease, and a low serum antibody titer against T. buffeli. The cow was in a herd in which all cattle originated in Missouri; 22/75 (29%) of cattle had a theilerial parasitemia and 26/75 (35%) had titers to T. buffeli of > or =1:160. Classification of the Missouri bovine organism as T. buffeli was based on DNA sequencing and comparison to sequences for T. buffeli and Theileria sp. type A obtained from GenBank. Intraerythrocytic veils and piroplasms were seen during transmission electron microscopy. The organism was successfully transmitted to two splenectomized calves, which developed mild anemias while parasitemic. Blood from the second calf was used as the source of T. buffeli antigen for an indirect immunofluorescence antibody test. Theilerial isolates from a Missouri white-tailed deer were also sequenced and resembled Theileria sp. types F and G and were not consistent with the bovine organism.     

A sequential study of the pathology associated with the infection of sheep with adult and larval Ostertagia circumcincta

Disturbances in the physiology of the abomasa of sheep infected with either adult Ostertagia circumcincta given via abomasal cannulae, or larvae (L3) given intraruminally were matched by pathological changes in tissues collected by repeated mucosal biopsy. Within 2-3 days of the transplant of adult worms, abomasal pH had increased markedly in five out of six animals, and there also had been rapid increases in serum gastrin and pepsinogen concentrations in all animals. Reductions in parietal cell number were recorded as early as 1 day after the transplant of adults and were associated with the rapid accumulation of many neutrophils and eosinophils. Mucosal hyperplasia, with increased numbers of cells closer in appearance to mucous/mucous neck cells, was a relatively late development, being most pronounced in the latter part of the infection. In sheep given larvae, changes in secretory physiology were again matched by a concurrent fall in parietal cell number and by the accumulation of inflammatory cells. Changes became maximal when most worms could be expected to be present as adults, confirming the role of adults in the natural disease. Some abnormalities were detected in biopsies collected from animals maintained free of parasites and, although milder in degree, there were similarities to those observed in parasitised tissues, there being fewer parietal cells, a modest degree of mucous cell hyperplasia and inflammatory infiltrates of predominantly neutrophils. These changes were the likely result of trauma to the tissues in the immediate vicinity of the cannula, due either to the presence of the cannula itself or to the frequent collection of biopsy material from areas close to it.     

Patterns of antimicrobial susceptibility in Michigan wildlife and bovine isolates of Mycobacterium bovis.

The state of Michigan has recognized the presence of Mycobacterium bovis in its free-ranging white-tailed deer population since 1994. This endemic infection is primarily located in a 12-county area in the northeastern lower peninsula of Michigan. A statewide surveillance and eradication program of the disease has been in effect since 1994. Worldwide, Mycobacterium tuberculosis complex organisms have a known predilection toward development of antimicrobial resistance. The objective of this study was to investigate the antimicrobial susceptibility of M. bovis isolates from white-tailed deer in Michigan and detect any changes in susceptibility over time. M. bovis isolates from 2 fall hunting seasons (1999 and 2004) were used in this study. The fall season of 2004 marked the first documented case of direct transmission of M. bovis from a wild deer to a human in Michigan. Since M. bovis is a zoonotic disease, knowledge of susceptibility can expedite treatment options in humans. M. bovis isolates were obtained from 58 deer, 4 coyotes, 3 cattle, 2 raccoons, and 1 human case from the 2 years combined. Methods of susceptibility testing included 1% proportion agar plates and Bactec radiometric broth testing. M. bovis was found to be uniformly resistant to the antibiotic pyrazinamide; this resistance is common to all M. bovis isolates. No other antimicrobial resistance was found in any of the tested M. bovis isolates, which may be, in part, attributed to the lack of any significant treatment pressure in wildlife.