Laboratory Studies on the Transmission of Western Equine Encephalitis Virus by Saskatchewan Mosquitoes I. Culex tarsalis

A Saskatchewan strain of the mosquito Culex tarsalis, transmitted a local strain of western equine encephalitis virus from chick to chick, between four and 44 days after an infective blood meal. At incubation temperatures of 69 and 75°F, 120 transmissions occurred out of a possible 141, and all but seven of these were by single infected mosquitoes. At 75°F virus titers in individual mosquitoes were more uniform and transmission was more efficient, than at 69°F, although infection rates were similar at both temperatures. The minimum concentration of virus required to infect 50% of C.tarsalis was 10^2.5 intracerebral three-week old mouse LD₅₀ per 0.03 ml of donor blood. These findings provide direct evidence that C. tarsalis of Saskatchewan is a highly efficient vector of western equine encephalitis virus.     

Establishing conditions for the storage and elution of rabies virus RNA using FTA? cards

The Flinders Technology Associates filter paper cards (FTA^® cards) can be used to store nucleic acid from various samples and are easily portable. However, RNA is physicochemically unstable compared with DNA, and appropriate methods have not been established for storage and extraction of RNA from FTA^® cards. The present study investigated the optimum conditions for storage and elution of viral RNA (vRNA) using rabies virus (RABV) applied to FTA^® cards. When TE buffer was used, the elution rates of vRNA increased with the length of the elution time. When the cards were stored at −80°C or −20°C, vRNA was stable over 3 months. Degradation of vRNAs occurred following storage at 4°C and room temperature, suggesting that RNA should be extracted from cards as soon as possible if no freezer is available. When we tried to amplify vRNA from RABV-infected animal brains applied to FTA^® cards and stored at −80°C for 6 months, we did not detect any amplified products with the primer set for 964 bp of RABV N gene. However, we were able to detect amplified products by increasing the elution time of vRNA from FTA^® cards from 30 min to 24 hr or by changing the primer sets to amplify 290 bp of N gene. Thus, we recommend extending the elution time for damaged or low concentration samples in FTA^® cards.     

Studies on Aspergillus Fumigatus; Toxin Production Ry Different Strains and Serological Comparison of the Strains

Saline extracts of mycelial material from 14 different strains of Aspergillus fumigatus showed haemolytic and toxic activities. These activities were present in filtrates obtained from mycelial material incubated at 20°C for 6, 15 and 30 days and at 37°C for 6 days. The haemolysin and toxin titers decreased with an increase in incubation time and at higher incubation temperatures.The haemolytic and toxic activities could be inactivated by heating the filtrates at 70°C for 10 min.The results of serological tests demonstrated immunological uniformity for the haemolytic and toxic factors.     

Immunofluorescence Plaque Assay for African Swine Fever Virus

Suitably diluted cell culture adapted African swine fever virus preparations were inoculated on VERO cell monolayers and grown on coverslips. Gum tragacanth was used as an overlay. After three days incubation at 37°C the infected cultures were fixed with acetone and stained with fluorescent antibody conjugate. Fluorescing plaques consisted of 20-30 infected cells.

Three statistical criteria for a quantitatively reliable assay were met: the Poisson distribution for plaque counts, linearity of the relationship between the concentration of virus and the plaque count and reproducibility of replicate titrations. The method is suitable for counts up to at least 70 plaques per 5 cm² coverslip and computed titers are reproducible within 0.16 log units with a total of 300 plaques enumerated.

     

Association between in-transit loss, internal trailer temperature, and distance traveled by Ontario market hogs

An observational study was conducted from July to October 2004 to determine the association between in-transit losses of swine and internal trailer temperature after controlling for loading density, trip distance, herd size, and random trip effect. A convenience sample of 3 trucking companies was used to collect temperature, relative humidity, and global positioning data for 104 trips that delivered 21 834 pigs from 371 producers to Ontario abattoirs. The association between in-transit loss and trailer temperature was determined using the 90th percentiles of internal temperature for each trip. Average loading density was 0.36 m²/100 kg pig (range 0.28 to 0.50 m²/100 kg pig). Average in-transit loss was 0.12%; however, 94% of producers experienced no losses. As the 90th percentile of internal trailer temperature increased from a range of 8.6°C to 23.3°C to a range of 23.4°C to 26.1°C, average in-transit loss ratio increased approximately 3-fold, with an additional 2-fold increase as the range increased from 26.2°C to 28.9°C to 29.0°C to 30.5°C. As the 90th percentile of temperature increased by 1°C over the full range of temperatures in this study, in-transit loss was expected to increase 1.26 times. The in-transit loss was expected to decrease 0.81 times for each 50-km increase in distance traveled between the farm and the abattoir.     

Bovine respiratory syncytial virus infection in an ontario cattle herd

Bovine respiratory syncytial virus was recovered from the lung of a six month old calf that died during an outbreak of respiratory disease in a cattle herd in Ontario. Lung tissue removed from the calf at necropsy, performed within two hours of death, was frozen at -70°C prior to virus isolation attempts. Syncytia and intracytoplasmic inclusions were demonstrated both in histological sections of the calf's lung and in stained cell culture preparations infected with the bovine respiratory syncytial virus isolate. Direct fluorescent antibody and virus neutralization tests serologically confirmed the identity of the isolate.     

Generation of a cold-adapted PRRSV with a nucleotide substitution in the ORF5 and numerous mutations in the hypervariable region of NSP2

A cold-adapted porcine reproductive and respiratory syndrome virus (CA-VR2332) was generated from the modified live virus strain VR2332. CA-VR2332 showed impaired growth when cultured at 37°C with numerous mutations (^S731^F, ^E819^D, ^G975^E, and ^D1014^N) in the hypervariable region of the NSP2, in which the mutation ^S731^F might play a vital role in viral replication at 30°C. Conserved amino acid sequences of the GP5 protein suggests that CA-VR2332 is a promising candidate for producing an effective vaccine against PRRSV infection. Further studies on replication and immunogenicity in vivo are required to evaluate the properties of CA-VR2332.     

Comparison between core temperatures measured telemetrically using the CorTemp? ingestible temperature sensor and rectal temperature in healthy Labrador retrievers

This study evaluated the CorTemp^® ingestible telemetric core body temperature sensor in dogs, to establish the relationship between rectal temperature and telemetrically measured core body temperature at rest and during exercise, and to examine the effect of sensor location in the gastrointestinal (GI) tract on measured core temperature. CorTemp^® sensors were administered orally to fasted Labrador retriever dogs and radiographs were taken to document sensor location. Core and rectal temperatures were monitored throughout the day in 6 resting dogs and during a 10-minute strenuous retrieving exercise in 6 dogs. Time required for the sensor to leave the stomach (120 to 610 min) was variable. Measured core temperature was consistently higher than rectal temperature across all GI locations but temperature differences based on GI location were not significant (P = 0.5218). Resting dogs had a core temperature that was on average 0.4°C above their rectal temperature with 95% limits of agreement (LoA) between 1.2°C and −0.5°C. Core temperature in exercising dogs was on average 0.3°C higher than their concurrent rectal temperature, with LoA of +1.6°C and −1.1°C.     

Temperature Related Absorption and Excretion of Sulphadimidine in Rainbow Trout,Salmo Gairdneri

The main purpose of the present study was to see if a kinetic model and a computer program for characterizing the rate of uptake and clearance of chemicals in aquatic organisms (Biofac, OECD’s Chemicals Testing Programme) was applicable to pharmacokinetic studies in fish. The program seems suitable for such studies.The effect of temperature upon rate of absorption of sulphadimidine from medicated water and elimination of the same drug was investigated in rainbow trout. A rise in the temperature from 7° C to 14° C more than doubled the calculated rate constants of absorption and elimination. The biological half life (t_½ β) and the time to reach 90 % of steady state (t_{90% ss}) at 14° C were approximately half the values at 7° C.     

Concentration and heritability of immunoglobulin G and natural antibody immunoglobulin M in dairy and beef colostrum along with serum total protein in their calves

Immunoglobulin (Ig) G and natural antibody (NAb) IgM are passively transferred to the neonatal calf through bovine colostrum. Maternal IgG provides pathogen- or vaccine-specific protection and comprises about 85% of colostral Ig. NAb-IgM is less abundant but provides broad and nonspecific reactivity, potentially contributing to protection against the dissemination of pathogens in the blood (septicemia) in a calf’s first days of life. In the dairy and beef industries, failure of passive transfer (FPT) of colostral Ig (serum total protein [STP] <5.2 g/dL) is still a common concern. The objectives of this study were to: (1) compare colostral IgG concentrations and NAb-IgM titers between dairy and beef cows; (2) assess the effect of beef breed on colostral IgG; (3) compare passive transfer of colostral Ig in dairy and beef calves; and (4) estimate the heritability of colostral IgG and NAb-IgM. Colostrum was collected from Holstein dairy (n = 282) and crossbred beef (n = 168) cows at the University of Guelph dairy and beef research centers. Colostral IgG was quantified by radial immunodiffusion and NAb-IgM was quantified by an enzyme-linked immunosorbent assay. In dairy (n = 308) and beef (n = 169) calves, STP was estimated by digital refractometry. Beef cows had significantly greater colostral IgG (146.5 ± 9.5 standard error of the mean [SEM] g/L) than dairy cows (92.4 ± 5.2 g/L, P <0.01). Beef cows with a higher proportion of Angus ancestry had significantly lower colostral IgG (125.5 ± 5.8 g/L) than cows grouped as “Other” (142.5 ± 4.9 g/L, P = 0.02). Using the FPT cutoff, 13% of dairy and 16% of beef calves had FPT; still, beef calves had a significantly larger proportion with excellent passive transfer (STP ≥6.2 g/dL, P <0.01). The heritability of colostral IgG was 0.04 (±0.14) in dairy and 0.14 (±0.32) in beef. Colostral NAb-IgM titers in dairy (12.12 ± 0.22, log₂ [reciprocal of titer]) and beef cows (12.03 ± 0.19) did not differ significantly (P = 0.71). The range of NAb-IgM titers was 9.18–14.60, equivalent to a 42-fold range in antibody concentration. The heritability of colostral NAb was 0.24 (±0.16) in dairy and 0.11 (±0.19) in beef cows. This study is the first to compare colostral NAb-IgM between dairy and beef cows. Based on the range in NAb-IgM titers and the heritability, selective breeding may improve colostrum quality and protection for neonatal calves in the early days of life.     

Simplified isolation and enrichment of spermatogonial stem-like cells from pubertal domestic cats (Felis catus)

The efficiency of spermatogonial stem cell (SSC) isolation and culture from pubertal donors is currently poor primarily, because of contamination with other testicular cells. This study aimed to purify SSC-like cells using different extracellular matrixes and a discontinuous gradient density. In experiment 1, testes (n=6) were analyzed for histology and SSC-related protein expressions (laminin, SSEA-4, DDX-4 and GFRα-1). After enzymatic digestion, the cell suspension was plated onto either a laminin- or gelatin-coated dish. The number of SSC-like cells was determined at 15, 30 and 60 min of culture (experiment 2). Experiment 3 was performed to test whether or not the additional step of Percoll gradient density centrifugation could really improve purification of SSC-like cells. Testicular histology revealed complete spermatogenesis with laminin expression essentially at the basal lamina of the seminiferous tubules. SSEA-4 and GFRα-1 co-localized with DDX-4 in the spermatogonia. The relative percentage of SSC-like cells, as determined by cells expressing SSEA-4 (59.42 ± 2.18%) and GFRα-1 (42.70 ± 1.28%), revealed that the highest SSC-like cell purity was obtained with the 15-min laminin-coated dish compared with other incubation times and gelatin treatment (P<0.05). Percoll treatment prior to laminin selection (15 min) significantly improved SSC-like cell recovery (91.33 ± 0.14%, P<0.001) and purity (83.82 ± 2.05% for SSEA-4 and 64.39 ± 1.51% for GFRα-1, P<0.05). These attached cells demonstrated a typical SSC-like cell morphology and also expressed POU5F1, RET and ZBTB16 mRNA. In conclusion, double enrichment with Percoll gradient density centrifugation and laminin plating highly enriched the SSC-like cells population.     

The Effects of Cesarean Section Anesthesia on Heat Loss and Heat Production in the Newborn Rabbit

The newborn of some smaller animals rely upon heat produced by nonshivering thermogenesis in the brown fat to prevent a fall in body temperature after birth. Because of their pharmacological properties, some drugs may affect nonshivering thermogenesis. Therefore, in this study, the ability of newborn rabbits delivered under Innovar-Vet, ketamine hydrochloride, methoxyflurane and epidural anesthesia to maintain the rectal, subcutaneous interscapular and lumbar temperature was investigated at an ambient temperature of 35°C or 22°C and the results compared with control newborns delivered without anesthesia. When the newborns were exposed to 35°C, the anesthetics studied had no effect on the ability of the newborn to maintain the rectal temperature and the subcutaneous temperature over the interscapular fat pad was similar to the lumbar subcutaneous temperature thereby indicating that nonshivering thermogenesis was not activated. However, at an ambient temperature of 22°C Innovar-Vet or methoxyflurane reduced the temperature difference between interscapular and lumbar temperatures to 1.6°C compared to 2.5°C in controls and the difference between core temperature and ambient temperature to 3.5°C greater compared to 7.5°C in controls. Ketamine hydrochloride or lidocaine hydrochloride plus meperidine has less effect because these compounds lack adrenergic blocking properties. These data suggest that newborns delivered under anesthetics or tranquillizers that have adrenergic blocking properties require a warm (35°C) environment to prevent a fall in core temperature.     

Urinary albumin and transferrin as early diagnostic markers of chronic kidney disease

Feline renal diseases are increasingly noted in veterinary practice. It is important to diagnose and identify the pathological basis of renal dysfunction accurately at an early stage, but there are only a few reports on this area in clinical veterinary medicine. We investigated the efficacy of measurement of urinary albumin (u-Alb) and urinary transferrin (u-Tf) for early diagnosis using 5-µl urine samples collected noninvasively by catheterization from normal (IRIS stage I) cats and cats with stage I chronic kidney disease (CKD). The u-Alb levels in normal and stage I CKD cats were 6.0 ± 4.5 and 11.2 ± 8.4 mg/dl, respectively, and the u-Tf levels were 0.09 ± 0.42 and 0.52 ± 0.79 mg/dl, respectively. Based on ROC curve analysis, the sensitivity and specificity of u-Alb and u-Tf were higher than those of the currently used biomarker, the plasma creatinine level. The sensitivity of u-Alb was higher than that of u-Tf, whereas the specificity of u-Tf was higher than that of u-Alb. The validity of the threshold albumin level (20 mg/dl) was confirmed by measurements using SDS-PAGE. Since leakage of u-Tf in urine precedes leakage of u-Alb, inclusion of u-Tf in biochemistry tests may be appropriate for IRIS staging as a diagnostic marker of early diagnosis of renal disorder in cats.     

Studies with Bovine Parainfluenza — 3 Virus in U.A.R. (Egypt)

A bovine strain of myxovirus parainfluenza-3 (MP3) virus, designated S virus, was isolated from lung tissue collected from cattle with respiratory illness in 1963. The virus agglutinates mammalian and avian erythrocytes, and is sensitive to ether, sodium desoxycholate and trypsin. It grows in primary calf kidney, buffalo kidney, dog kidney, camel kidney and MS cell cultures. The S virus forms well-defined plaques in buffalo and calf kidney cells on the 5th or 6th day after inoculation. Examination of cell cultures following inoculation with S virus revealed giant cell formation, and introcytoplasmic and intranuclear inclusions. At 37°C the virus titer dropped from 10^10.4 to 10^2.6 in 3 days. Virus was completely inactivated at 56°C within 15 minutes. Growth-curve studies in tissue culture monolayer cells revealed a latent period of 10 hours. The intracellular virus titer was slightly lower than that of extracellular virus. The isolate was identified as MP3 virus by serum neutralization and hemagglutination-inhibition tests. Antibodies (HI) to S virus were shown to be present in a significant proportion of Egyptian cattle. The epidemiological significance of MP3 (bovine strain) virus in U.A.R. is discussed.     

Synergistic effects of quaternary ammonium compounds and food additive grade calcium hydroxide on microbicidal activities at low temperatures

The microbicidal activities of mixtures of quaternary ammonium compounds (QACs) and food additive grade calcium hydroxide (FdCa(OH)₂) were evaluated in a suspension test at −20°C using an anti-freeze agent (AFA) containing methanol, or at 1°C, with varying contact time, toward avian influenza virus (AIV), Newcastle disease virus (NDV), fowl adenovirus (FAdV), avian reovirus (ARV), Salmonella Infantis (SI) and Escherichia coli (EC). At −20°C, the mixtures could inactivate AIV and NDV within 30 min, FAdV and ARV within 5 sec, and SI and EC within 3 min, respectively. AFA did not inactivate viruses and bacteria within 30 min and 10 min, respectively. At 1°C, the mixtures inactivated FAdV and ARV within 30 sec, AIV within 10 min, and NDV within 30 min. A mixture of slaked lime (SL) and QAC could inactivate FAdV and ARV within 30 sec, but could not inactivate AIV and NDV even after 60 min at 1°C. SL could not substitute FdCa(OH)₂ in order to exert the synergistic effects with QAC. Thus, QACs microbicidal activities were maintained or enhanced by adding FdCa(OH)₂. It is hence recommended to use QACs with FdCa(OH)₂, especially in the winter season.     

Physicochemical Characterization of a Neonatal Calf Diarrhea Virus

Physicochemical characteristics of two isolates of a neonatal calf diarrhea virus were investigated. Neither isolate was sensitive to ether or chloroform, both were stable at pH 3.0, were relatively heat resistant, but were thermolabile when heated to 50°C for one hour in the presence of 1.0 M MgCl₂. Multiplication of virus was not inhibited by concentrations of 5-iodo-2'-deoxyuridine (IDUR) up to 500 µg/ml, which indicated that the nucleic acid was ribonucleic acid (RNA). Also, multiplication was not inhibited by concentrations of actinomycin D (AD) up to 0.5 µg/ml. Thermal denaturation studies demonstrated that the nucleic acid had a high melting temperature.

Resistance to lipid solvents, stability at an acid pH, relatively high thermostability, type of nucleic acid, plus previous reports from this laboratory on general morphology and cytopathogenicity suggest that the virus may belong to the diplornavirus (reovirus) group. However, thermolability in the presence of 1.0 M MgCl₂ is not consistent with characteristics of this group.