γ-干扰素释放法检测奶牛结核病的比较试验

目的：评价γ-干扰素释放试验（IGRA）牛结核病检疫过程中的效果。方法在牛结核病检疫过程中对皮试初筛为结核病和疑似结核病的89（74/15）头奶牛同时进行r干扰素试验、单纯颈部皮试变态反应（SCT）和比较皮试变态反应（CCT）检测,结果单纯颈部皮试变态反应二次检疫中89头牛均判为阳性,单纯颈部皮试变态反应与比较皮试变态反应的阳性符合数为67头。阳性符合率为75.3％;γ-干扰素释放试验和单纯颈部皮试变态反应检测两者的阳性符合数为64头,阳性符合率为71.9％;γ-干扰素释放试验与比较皮试变态反应的阳性符合数为58头,阳性符合率为79.5％。阴性符合数为16头,阴性符合率为48.4％;结论γ-干扰素释放试验在保持较高灵敏度的同时,具有比变态反应更好的特异性。     

Growth characteristics of canine distemper virus in a new cell line CCT cells originated from canine malignant histiocytosis

Canine distemper virus (CDV) growth and the morphological characterization were examined in a cell line established from a canine malignant histiocytosis (CCT cell line). The susceptibility of the CCT cells to 3 CDV strains, FXNO, YSA-TC and MD-77 was shown by detection of the antigen in the indirect fluorescent assay. After passaging 4 and 9 times through the CCT cells, only FXNO strain could produce the syncytia where demonstrated the antigens. Titers of 9 passaged viruses through the CCT cells showed slightly higher in the CCT cells than those in Vero cells. Morphological characterization of karyorrhexis and specific DNA ladder by extracted DNA electrophoresis indicated apoptosis in the CDV infected CCT cells.     

Postnatal development of central corneal thickness in chicks of Gallus gallus domesticus

Objective To investigate the changes in corneal thickness that occur during maturation of the Gallus gallus domesticus chick eye over the first 450 days of life. Animals studied Twenty‐nine chicks, of which 12 were males and 17 were females. Procedures The central corneal thickness (CCT) was measured by ultrasonic pachymetry from hatch until 450 days of age. Segmented regression was applied to capture the two phases observed in the CCT plotted against age. Eye and gender were also included in the model. Results Mean CCT values initially decreased, with the lowest point being reached at around 12 days of age. CCT then gradually increased as the chick matured. At 70 days of age the animals have completed corneal development and reached the plateau value of 0.242 ± 0.0002 mm. CCT differences between gender or between left and right eyes were not statistically significant. Prediction equations for mean CCT according to the bird's age are presented. Conclusions There is an initial decrease in corneal thickness until approximately 12 days of age, which presumably mirrors maturation of corneal endothelial cell function. The pattern of changes in corneal thickness during the first phase of development of the chick CCT was similar to the one reported for dogs and humans. However, a unique feature of the development of CCT in chicks is that after reaching a plateau at 70 days corneal thickness did not significantly change over the remainder of the study period. Additionally, unlike in humans and dogs, there is no gender difference for corneal thickness in chicks.     

牛卵形巴贝斯虫伴侣素CCT_η基因的克隆与生物信息学分析

对牛卵形巴贝斯虫延边株的伴侣素CCTη基因进行分子克隆,并利用生物信息学软件对CCTη基因进行了同源性、系统进化树、编码蛋白等电点、信号肽、跨膜区、糖基化位点及疏水性分析。结果显示,克隆的CCTη基因由1 008个核苷酸组成,其中编码335个氨基酸。与GenBank上发表的牛卵形巴贝斯虫CCTη基因序列(AB367928.1)同源性100%。CCTη基因编码蛋白等电点为7.32,不存在信号肽结构、跨膜区及糖基化位点。疏水性最大值3.38,最小值-3.79。本研究从CCTη基因方面近一步证实了该分离株的分子分类。     

Central corneal thickness in koi fish: effects of age, sex, body length, and corneal diameter

OBJECTIVE: To establish the central corneal thickness (CCT) of normal koi fish by ultrasonic pachymetry, and its relationship to age, sex, body length and corneal diameter. METHODS: Age, sex and body length of 33 koi fish (17 male and 16 female fish) were recorded. Horizontal and vertical corneal diameters of each eye were obtained using Jameson calipers. Central corneal thickness of all eyes was measured by ultrasonic pachymetry. Intraocular pressure (IOP) by rebound tonometry was obtained for a subgroup of nine koi (18 eyes). RESULTS: Mean central corneal thickness was 325.9 microm. Central corneal thickness of female koi was greater than CCT of male fish (P < 0.01). Central corneal thickness increased with increasing age overall and within both sexes (P < 0.01). Central corneal thickness increased with increasing body length (P < 0.001). For male and female fish, CCT increased with increasing horizontal and vertical corneal diameters (P < 0.01). Mean horizontal corneal diameter (HCD) was 8.05 mm, mean vertical corneal diameter (VCD) was 7.38 mm, and HCD was consistently greater than VCD. Mean IOP of a subgroup of these koi was 4.9 mmHg by rebound tonometry. CONCLUSIONS: Koi CCT increases with increasing age, body length and corneal diameter.     

Reliability of manual measurements of corneal thickness obtained from healthy canine eyes using spectral-domain optical coherence tomography (SD-OCT)

The purpose of this study was to manually measure corneal thickness in canine eyes using a spectral-domain optical coherence tomography (SD-OCT) device and to assess intra- and inter-observer reliability of this technique. Twenty healthy dogs with a mean age of 4.7 y were examined. A 6-mm corneal pachymetry protocol was carried out by 1 operator using 1 SD-OCT device in both eyes of each animal. Measurements were obtained manually and in duplicate by 2 independent investigators (> 24 h apart), using the built-in caliper function. Measurements included epithelial thickness (ET), non-epithelial thickness (NET), and central corneal thickness (CCT). The overall mean ET, NET, and CCT for all eyes examined were 72.3 ± 4.6 μm, 538.9 ± 42.5 μm, and 611.2 ± 40.3 μm, respectively. There was no significant difference in ET, NET, or CCT based on the eye examined [oculus dexter (OD) versus oculus sinister (OS)], age, or gender of the animal. There was no significant difference in replicate measurements of ET, NET, or CCT done by the same operator, although a small but significant difference was noted between operators for ET measurements only. The mean difference in ET between operators was 0.6 μm (P = 0.03). The coefficient of variation ranged from 0.5% to 9.27% and intraclass correlation coefficient ranged from 0.35 to 0.97. Based on these results, manual measurements of corneal thickness in canine eyes using a portable SD-OCT device provided ET, NET, and CCT measurements with clinically acceptable intra- and inter-observer reliability.     

Antigen beads: a novel approach for the delivery of a soluble Anaplasma immunogen

An important consideration in the development of effective immunization regimens against bovine anaplasmosis is the optimization of vaccine delivery systems. This report describes the formulation of proteinaceous antigen-containing spheres derived from supernatant fluids of Analplasma marginale cultures. The antigen beads were designed for use as potential dispersal agents for a soluble Anaplasma immunogen. Immunospecificity assays demonstrated that A marginale antigen was located on the surfaces of individual antigen beads. Use of Anaplasma antigen beads with a potent adjuvant, such as saponin, was proposed.     

In vivo measurement of central corneal thickness in normal chicks (Gallus gallus domesticus) with the optical low-coherence reflectometer

Objective To determine the practicability and accuracy of central corneal thickness (CCT) measurements in living chicks utilizing a noncontact, high‐speed optical low‐coherence reflectometer (OLCR) mounted on a slit lamp. Animals studied Twelve male chicks (Gallus gallus domesticus). Procedures Measurements of CCT were obtained in triplicate in 24 eyes of twelve 1‐day‐old anaesthetized chicks using OLCR. Every single measurement taken by OLCR consisted of the average result of 20 scans obtained within seconds. Additionally, corneal thickness was determined histologically after immersion fixation in Karnovsky’s solution alone (20 eyes) or with a previous injection of the fixative into the anterior chamber before enucleation (4 eyes). Results Central corneal thickness measurements using OLCR in 1‐day‐old living chicks provide a rapid and feasible examination technique. Mean CCT measured with OLCR (189.7 ± 3.34 μm) was significantly lower than histological measurements (242.1 ± 47.27 μm) in eyes with fixation in Karnovsky’s solution (P = 0.0005). In eyes with additional injection of Karnovsky’s fixative into the anterior chamber, mean histologically determined CCT was 195.2 ± 8.25 μm vs. 191.9 ± 8.90 μm with OLCR. A trend for a lower variance was found compared to the eyes that had only been immersion fixed. Conclusion Optical low‐coherence reflectometry is an accurate examination technique to measure in vivo CCT in the eye of newborn chicks. The knowledge of the thickness of the chick cornea and the ability to obtain noninvasive, noncontact measurements of CCT in the living animal may be of interest for research and development of eye diseases in chick models.     

Efficiency assessment of a swift method to enhance substandard viability ram ejaculates

Semen availability in ram semen processing facilities is of great importance for the genetic improvement of sheep. Accordingly, any method that would increase sperm viability in low viability ejaculates could be useful. In this study, the possibility of a glass beads filtration method by estimating the beads total surface provided (TSP) for adhesion of spermatozoa, was evaluated. Initially, two different TSP (102 and 154 cm(2)) achieved by various sizes of beads (1500, 2000 and 3000 microm) were tested and no significant difference in sperm viability improvement was noticed for the same TSP by different beads (p > 0.05). Next optimization tests were performed in which three different funnels were used for filtration at a standard TSP (154 cm(2)). The pear-shaped funnel was found to be the most appropriate for filtration, as semen volume recovery and sperm viability improvement were more pronounced (p < 0.05). Finally, filtration tests were conducted with pear-shaped funnels with different TSP (102 and 154 cm(2)) obtained by the aforementioned beads sizes (1500, 2000 and 3000 microm) in equal aliquots. Total surface provided of 102 cm(2) proved to be the more appropriate for filtration than 154 cm(2), as shown by the significant improvement of sperm viability (p < 0.01) and the significantly higher filtrate semen volume (p < 0.05). In conclusion, ram sperm viability improvement by more than 20% of its initial value and semen volume recovery by more than 60%, along with the fact that the total filtration time did not exceed 6 min in any case, suggest that through further development this method could be successfully used during ram semen processing.     

Alginate as a new biomaterial for the growth of porcine retinal pigment epithelium

Objective Determine the effect of a 3‐dimensional alginate matrix on the growth and differentiation of cells isolated from porcine retinal pigment epithelium (RPE). Procedures Porcine RPE cells were harvested from enucleated eyecups, isolated by differential gravity sedimentation and cultured in either alginate alone (Group 1) or on plastic tissue culture plates followed by alginate (Group 2). Group 1 cells were cultured in alginate to evaluate the efficacy of the matrix as a culture medium. Group 2 cells were initially cultured on plastic to induce dedifferentiation. The cells were then harvested, suspended in alginate beads, and incubated for a second culture period to determine if the induced dedifferentiation was reversible. Results The number of Group 1 cells was significantly greater (P ≤ 0.01) at the end of the culture period. The amount of pigment and cell morphology of Group 1 cells at the end of the culture period was similar to that seen at initial cell isolation. The initial culture of Group 2 cells on plastic showed characteristic features of dedifferentiation marked by the loss of pigment and alterations in microscopic appearance. Secondary culture of dedifferentiated Group 2 cells in alginate beads resulted in a return to pigmentation and characteristic morphology for a majority of the cultured cells. Conclusions Porcine RPE cells can be propagated in alginate culture with a significant increase in cell numbers while maintaining normal morphology. Under the conditions described in the present study, the dedifferentiation of porcine RPE induced by standard in vitro culture methods is reversible.     

Detection of bovine respiratory syncytial virus using a heterologous antigen-capture enzyme immunoassay

Based on the marked antigenic similarities that exist between antigens of the human and bovine strains of respiratory syncytial virus (RSV), an enzyme immunoassay (EIA) designed to detect human RSV was used to detect bovine RSV. The commercial test kit (RSV EIA) consists of a solid phase (beads) coated with a capture antiserum prepared against the Long strain of human RSV. The RSV EIA test was compared with the method of inoculation of cell cultures and fluorescent antibody (FA) staining of lung tissue for the detection of bovine RSV. Using a cell culture-propagated stock of strain 375 of bovine RSV, the threshold of sensitivity of the EIA test for the cattle strain of RSV was determined to be less than or equal to 10(2.3) CCID50/ml. In addition, RSV EIA detected the bovine RSV in nasal samples obtained from 3 experimentally inoculated cattle. The RSV EIA exhibited a sensitivity of greater than or equal to 80% during the period that shedding of infectious virus took place. All of the bovine RSV FA-positive lung samples (n = 37) were positive by the RSV EIA. Twenty-six of the remaining 214 bovine RSV FA-negative lung samples were positive by the RSV EIA. The RSV EIA was also used to test 137 nasal swabs obtained from cases of bovine respiratory disease. Of these, 38 tested positive by RSV EIA. All samples that tested positive by EIA were confirmed by blocking assays using hyperimmune serum anti-bovine RSV and a pool of monoclonal antibodies specific for that virus.     

Effect of central corneal thickness on intraocular pressure with the rebound tonometer and the applanation tonometer in normal dogs

Objective To evaluate the effect of central corneal thickness (CCT) on the measurement of intraocular pressure (IOP) with the rebound (TonoVet^®) and applanation (TonoPen XL^®) tonometers in beagle dogs. Animal studied Both eyes of 60 clinically normal dogs were used. Procedures The IOP was measured by the TonoVet^®, followed by the TonoPen XL^® in half of the dogs, while the other half was measured in the reverse order. All CCT measurements were performed 10 min after the use of the second tonometer. Results The mean IOP value measured by the TonoVet^® (16.9 ± 3.7 mmHg) was significantly higher than the TonoPen XL^® (11.6 ± 2.7 mmHg; P < 0.001). The IOP values obtained by both tonometers were correlated in the regression analysis (γ² = 0.4393, P < 0.001). Bland–Altman analysis showed that the lower and upper limits of agreement between the two devices were ?0.1 and +10.8 mmHg, respectively. The mean CCT was 549.7 ± 51.0 μm. There was a correlation between the IOP values obtained by the two tonometers and CCT readings in the regression analysis (TonoVet^® : P = 0.002, TonoPen XL^® : P = 0.035). The regression equation demonstrated that for every 100 μm increase in CCT, there was an elevation of 1 and 2 mmHg in IOP measured by the TonoPen XL^® and TonoVet^®, respectively. Conclusions The IOP obtained by the TonoVet^® and TonoPen XL^® would be affected by variations in the CCT. Therefore, the CCT should be considered when interpreting IOP values measured by tonometers in dogs.     

Immunomagnetic isolation of canine circulating endothelial and endothelial progenitor cells

Background: Increased concentrations of circulating endothelial cells (CECs) are thought to be a biomarker of vascular injury in human patients with cardiovascular disease, neoplasia, vasculitis, sickle cell anemia, shock, and sepsis. Immunomagnetic isolation is a technique currently used to enumerate human CECs and can detect low numbers of cells. Objectives: The purpose of this study was to determine whether a standard protocol for immunomagnetic isolation could be used to obtain and enumerate CECs and a subpopulation of endothelial progenitor cells (EPCs) from canine whole blood. Methods: Cultured canine aortic endothelial cells were stained immunohistochemically with von Willebrand factor to verify morphology and number. Using magnetic beads conjugated with anti‐CD146, CECs/EPCs were isolated in culture and in canine whole blood. CD146‐positive cells were stained with fluorescein‐conjugated Ulex europaeus agglutinin 1 (UEA‐1) to confirm endothelial origin and cells were counted manually using a fluorescent microscope. The method was then applied to EDTA‐anticoagulated whole blood samples from 10 healthy client‐owned dogs. Results: The anti‐CD146–coated magnetic beads (>5/cell) bound the cultured canine aortic endothelial cells. Only rare UEA‐1–positive cells were obtained from whole blood, while >85–90% of cultured canine aortic endothelial cells were UEA‐1 positive. The percentage recovery of cultured canine aortic endothelial cells was >86%. CECs in canine whole blood had >8 beads attached to the surface and were 10–40 μm in size. Using immunomagnetic isolation, 43.4 ± 15.6 CECs/mL (range 24–70/mL) were isolated from canine whole blood samples. Conclusions: Immunomagnetic isolation is an acceptable method for enumerating canine CECs/EPCs in whole blood. Further studies are warranted to evaluate the clinical significance of CEC/EPC concentration in different canine diseases.     

Prevalence of Mycobacterium bovis skin positivity and associated risk factors in cattle from Western Uganda

Purpose

A cross-sectional study was undertaken to determine the prevalence of Mycobacterium bovis skin positivity and associated risk factors in cattle in western Uganda.

Methods

Herds were selected using multi-stage cluster sampling. The comparative cervical intradermal tuberculin test (CCT) was used to determine cattle tuberculosis status using US Department of Agriculture protocols. Risk factor data were collected from cattle owners through questionnaires collected by in-person interviews. Multivariable logistic regression models were used to measure the association between risk factors and herd CCT reactor prevalence.

Results

A total of 525 cattle from 63 herds were screened for M. bovis infection. Of the 525 cattle tested, 2.1 % were CCT reactors and 15.43 % were CCT suspects. Of herds tested, 14.28 % had at least 1 CCT reactor. Using a private water source for cattle and not introducing new cattle into the farm were associated with lower prevalence of M. bovis skin positivity. The herd-level prevalence of M. bovis reactors in Kashaari County of Mbarara District was 14.5 %, and the individual cattle prevalence was low (2.1 %).

Conclusions

Using communal sources of drinking water for cattle and introducing new cattle on the farm were farm management practices associated with increased risk of M. bovis exposure in cattle. Despite the low prevalence of bovine tuberculosis (TB), there is a need to educate the populace on the possibility of human infection with zoonotic TB and for educating farmers on practices to reduce the risk of acquiring M. bovis in the Mbarara District.     

Expression of a transforming gene (E5) of bovine papillomavirus in sarcoids obtained from horses

OBJECTIVE: To determine expression of a transforming gene (E5) of bovine papillomavirus in sarcoids, other tumors, and normal skin samples collected from horses with and without sarcoids. SAMPLE POPULATION: 23 sarcoids and 6 samples of normal skin obtained from 16 horses with sarcoids, 2 samples of normal skin and 2 papillomas obtained from horses without sarcoids, and 1 papilloma obtained from a cow. PROCEDURE: Protein was extracted from tissue samples collected from horses and incubated with agarose beads covalently coupled to Staphylococcus aureus protein A and an anti-E5 polyclonal antibody. Following incubation, proteins were eluted from the beads and electrophoresed on a 14% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. The E5 protein was detected by use of western blot analysis, using a chemiluminescence detection system. RESULTS: All 23 sarcoids had positive results for expression of E5 protein. Quantity of viral protein appeared to vary among sarcoids. All other tissues examined had negative results for E5 protein. Highest expression for E5 protein was observed in biologically aggressive fibroblastic variants of sarcoids, compared with expression in quiescent tumors. CONCLUSIONS AND CLINICAL RELEVANCE: This study documented that activation and expression of the E5 gene is evident in sarcoids obtained from horses. These data support the conclusion that infection with bovine papillomavirus is important in the initiation or progression of sarcoids in horses. Treatment strategies designed to increase immune recognition of virally infected cells are warranted.     

Detection of botulinum type E toxin in channel catfish with visceral toxicosis syndrome using catfish bioassay and endopep mass spectrometry.

Visceral toxicosis of catfish (VTC) is a syndrome characterized by sudden mortality in apparently healthy market- and brooder-sized catfish (Ictalurus punctatus). This paper reports the design of a catfish neutralization assay to detect botulinum in catfish with VTC and verification by Endopep mass spectrometry (Endopep-MS). Sera from 6 affected catfish were incubated with botulinum antitoxin serotypes A, B, C, D, E, or F. For each serum sample, 3 experimental fingerlings were injected intracoelomically with each serotype-serum mixture and placed separately in an aquarium. Three fish were injected with VTC-affected serum only, and 3 fish were injected with unaffected serum only and also placed in separate aquaria. Signs of morbidity and mortality were seen in fish injected with sera combined with serotype A, B, C, or D, as well as in positive controls. No morbidity or mortality was seen in fish injected with sera combined with antitoxin serotypes E or F or negative control serum. Sera from affected and unaffected catfish were sent to Centers for Disease Control and Prevention for detection and differentiation of botulinum neurotoxin. Aliquots of 0.5 ml of sera were incubated with magnetic beads coated with antibodies to botulinum, and the beads were subjected to the Endopep-MS reaction. Sera from affected catfish tested positive for botulinum E. Sera from 34 unaffected catfish tested negative for botulinum. Although there was not enough botulinum present in affected samples to obtain exact quantification, the estimated quantity of botulinum E in these sera samples was between 0.01 and 0.5 mouse LD50/ml.