Actions of BW540C in an equine model of acute inflammation: A preliminary study

Summary

An equine model of acute non‐immune inflammation has been developed to facilitate studies of the inflammatory process and the actions of novel anti‐inflammatory drugs. Five polyester sponge strips soaked in sterile 2% carrageenin solution were placed in subcutaneous pouches prepared under local anaesthesia in the necks of conscious ponies. Serial removal of the strips and harvesting of the exudate enabled studies to be made of the cellular, biochemical and mediator aspects of the localised, acute inflammation, and the heat generated by the lesion was monitored by infra‐red thermometry. Maximal concentrations of the eicosanoids 6‐keto‐prostaglandin F_1α, thromboxane B₂ and leukotriene B, occurred at 9 h, whereas leukocyte numbers, lactate dehydrogenase (LDH) and total protein concentrations were greatest at 24 h. Lesional skin temperature was increased by approximately 4°C throughout the 24 h period. The novel anti‐inflammatory agent BW540C, administered orally at a dose‐rate of 20 mg/kg, did not affect leukocyte infiltration or the concentrations of protein, LDH and eicosanoids in exudate but serum thromboxane B₂ levels were reduced. Skin temperature rises were greater in drug‐treated animals.

It is concluded that higher doses of BW540C will be required for a clinically useful anti‐inflammatory action in horses.     

Interferon induction in porcine leukocytes with transmissible gastroenteritis virus

Leukocytes were harvested from the peripheral blood, mesenteric lymph node and small intestinal lamina propria from groups of three piglets before, and 1,2 and 3 weeks after infection with virulent transmissible gastroenteritis virus (TGEV) at 2 weeks of age. The donor piglets developed clinical signs of transmissible gastroenteritis which persisted for up to 3 days, and they developed peak serum titres of TGEV-neutralizing antibodies 2 weeks post-infection. The leukocytes were cultured in the presence of pokeweed mitogen (PWM), various dilutions of purified TGEV, or control media for 3 or 5 days, and the culture supernatants were tested for antiviral activity in MDBK cells challenged with vesicular stomatitis virus. The antiviral activity was characterized as porcine interferon (IFN)- or porcine IFN-τ on the basis of its stability at pH 2.0 and neutralization by anti-human IFN- antibodies. Viability of the leukocytes in culture, determined by trypan blue exclusion, was highest for the peripheral blood leukocytes and lowest for the mesenteric lymph node leukocytes. There were no consistent differences in antiviral activity between cultures incubated for 3 or 5 days. Porcine IFN- was found in the supernatants of the leukocyte cultures stimulated with TGEV antigen, harvested before or after infection of the donor piglets with TGEV. Porcine IFN-τ was demonstrated in the supernatants of the leukocyte cultures stimulated with PWM, more frequently when the leukocytes were harvested post-infection. This was the first demonstration of IFN induction in vitro in leukocytes from porcine gut-associated lymphoid tissue.     

The Leukocyte Culture Method in the Diagnosis of Free-martinism

The clinical application and reliability of the leukocyte culture method for the diagnosis of freemartinism were examined and the length of time that blood samples could be held at room temperature and in the refrigerator prior to culturing, was investigated.

The chromosome findings by the leukocyte culture method in 14 freemartins and 9 non-freemartin females belonging to heterosexual twins or triplets revealed that XX-XY cell chimerism exists only in the former, whereas the latter were exclusively of normal female complement.

The mitotic index in bovine blood after preservation for varying periods was studied on samples from two animals. Blood samples from these two animals stored at 5°C for 6 hours in a refrigerator showed the mitotic index to be 3.8 and 5.3 per cent which gradually decreased in samples stored for longer than 12 hours. After 72 hours, a very rapid decrease in mitotic index occurred in both cases, reaching zero in samples stored for 96 and 108 hours. Samples kept at room temperature followed a similar pattern as under refrigeration but with slightly lower values throughout.

     

Glasser's Disease of Swine Produced by the Intratracheal Inoculation of Haemophilus suis

The intracheal inoculation of pigs with Haemophilus suis led to the production of Glasser's disease at every attempt without significant pulmonary involvement. Isolation of this organism from the experimental animals was possible only in the acute phase of the disease.

The indirect fluorescent antibody technique when applied to frozen sections of tissues obtained from the experimentally infected pigs at autopsy, revealed a few rod forms but mostly “round bodies” of H. suis in animals from which the organism was isolated, and “round bodies” only in the pigs from which the organism was not isolated.

Attention is drawn to the similarities between the lesions caused by H. suis and Mycoplasma hyorhinis, and to the confusion which may result therefrom. It is stressed that the laboratory diagnosis of these two diseases is complicated by the fact that both agents may not be isolated on the media commonly used in diagnostic laboratories. Both organisms necessitate the use of special media where the clinical and autopsy results indicate polyserositis and arthritis.

     

Observations on Trypanosoma theileri Infection in Cattle

Naturally occurring Trypanosoma theileri infection was studied in two cattle herds. Herd A was a dairy herd of approximately 250. Herd B was an isolated herd of 32 and contained both dairy and beef breeds. Blood samples were collected from all animals in Herd A during July and August on two successive years. Samples were collected from Herd B at monthly intervals. Total leukocyte and differential counts packed cell volume determinations, and trypanosome cultures were made on each sample.

Infection was detected in all age groups between seven months and fifteen years but it was rare in calves. Infected animals were not consistently positive for trypanosomes on consecutive blood cultures and there was considerable variation between infected individuals. Positive cultures were usually obtained from some animals while others were positive intermittently. No correlation was found between trypanosome isolations and the season of the year.

A correlation was found between trypanosome isolation and lymphocytosis. Of the 920 blood samples examined, approximately one in every five trypanosome positive samples had lymphocyte levels in the Bendixen positive range. Approximately one in every twenty trypanosome negative samples had lymphocyte numbers in the Bendixen positive range. Evidence indicated that trypanosome isolation from animals with lymphocytosis was not caused by increased numbers of infected buffy coat cells in the inoculum cultured.

Eight calves were inoculated intravenously with trypanosome-infected blood. Lymphocyte numbers increased an average of 3549 per cumm above pre-inoculation levels in seven and remained essentially unchanged in one. Prior to inoculation with infective blood, two of the calves were intravenously inoculated with trypanosome-infected blood that had been frozen and thawed to kill the trypanosomes contained in it. Neither developed lymphocytosis following this inoculation.

No clinical disease problems which could be attributed to trypanosome infection were found.

     

Guide for Authors

Abstract

The etiological agent of bacterial cold-water disease, Flavobacterium psychrophilum, can cause significant losses of salmonid fishes in aquaculture facilities. Few studies describing the value of media components on the growth of F. psychrophilum are available in the literature. We therefore conducted a study that began with the standard enriched Anacker-Ordal broth (EAO) and over the course of multiple iterations evaluated the effects of various media supplements by adding or subtracting them from the base EAO medium. Different media formulations were made, and samples were removed from each broth formulation every 24 h for 72 h. From those samples we determined bacterial density by measuring absorbance values with a spectrophotometer. The medium with the highest absorbance value from one iteration was used as the base medium in the next iteration. Using this iterative approach, we determined that sodium acetate, calcium chloride, and magnesium sulfate inhibit growth and that maltose has no effect on the proliferation of the bacterium. The addition of skimmed milk (0.2%) and horse serum (1%) appears to provide a slight improvement in bacterial proliferation. Variations in agar concentration had no effect on the growth of the bacterium. Even though the addition and removal of some ingredients increased the mean absorbance values, the benefit of these substitutions was not significant. Even so, we found that the growth of F. psychrophilum in EAO was better than that in two other widely used media: tryptone-yeast extract salts and maltose infused tryptone-yeast extract salts.

Received August 1, 2011; accepted December 6, 2011     

Histopathology of Hybrid Tilapias Infected witha Biotype of Streptococcus iniae

Abstract

A systemic streptococcal disease caused by a biotype of Streptococcus iniae affecting hybrids of Nile tilapia Tilapia nilotica × blue tilapia T. aurea at a commercial aquaculture facility in central Texas was investigated. Histological analysis revealed a general septicemia with the occurrence of cellular infiltration and numerous cocci in most organ systems. Observations included cellular infiltration of the eye, meningitis, meningeal granuloma, numerous foci of infection, and massive cellular infiltration in the kidneys. Bacteria were seen in the subcapsular capillaries of the liver, and some liver tissue was granulomatous. Splenic sinuses contained numerous cocci, and both epicarditis and myocarditis was observed in the heart tissue of some specimens. Cocci were free in the plasma of infected fish and often phagocytized by macrophages.     

Experimental infection of the possum (Trichosurus vulpecula) with Leptospira interrogans serovar balcanica II. A comparison of laboratory techniques for the detection of leptospiraemia and leptospiruria

Leptospiruria in possums (Trichosurus vulpeculu) experimentally infected with balcanica was monitored for 13 months by dark-field microscopy, inoculation of hamsters and culture. Leptospiraemia was monitored by inoculation of hamsters and culture for the first 28 days post-inoculation.

Inoculation of hamsters for the detection of leptospiraemia had a sensitivity of 63.2%, whereas culture had a sensitivity of 36.8%. The use of hamsters enabled leptospires to be demonstrated in blood up to 15 days after inoculation by which time serum agglutination titres had reached peak levels.

Dark-field microscopy was the least sensitive method for the detection of leptospiruria (37.7%). Inoculation of hamsters and direct inoculation of media containing 400𝛍g 5-fluorouracil (5FU) had sensitivities higher than 70%. The incorporation of a high level of 5FU in media significantly improved isolation rates.

Differences were seen in the sensitivity of different methods at three different stages of leptospiruria. Inoculation of hamsters was the most sensitive method during the intial stage of leptospiruria, while direct inoculation of media containing 400𝛍g 5FU/ml was the most sensitive method during the chronic stage.     

Interaction of transport distance and body weight on preslaughter stress and breast meat quality of broilers

1. The objective of the study was to investigate the effect of transport distance on blood metabolites and breast meat quality of broilers slaughtered at different weights.

2. The study was conducted on Ross 308 broilers from 27 different flocks reared under similar conditions. Slaughter weight was classified as <2·0?kg, 2·0–2·4?kg, and >2·4?kg. Transport distance was categorised as short (65?km), medium (115?km) and long (165?km) distance representing 90, 155 and 220?minutes at an average 45?km/h speed, for each slaughter weight.

3. Higher heterophils and heterophil:lymphocyte (H/L) ratios were obtained for broilers transported over a long distance. Long distance transport increased blood albumin, glucose, and triglycerides levels for <2·0?kg broilers, which did not differ from broilers slaughtered at >2·4?kg after long-distance transport.

4. Broilers slaughtered at >2·4?kg after long-distance transport had lower pH_u, and paler and tougher breast meat, than those broilers slaughtered at <2·0?kg after long-distance transport.

5. A negative correlation was obtained between pH_u and L*, thawing loss and texture. The L* value was negatively correlated with a*; and positively correlated with b*, thawing and cooking losses.

6. It was concluded that the effect of transport distance could not be evaluated independently of slaughter weight. The interaction between transport distance and slaughter weight contributes to preslaughter stress and meat quality.     

Some Clinical and Hematological Features of Virus Enteritis of Mink

Twenty-six, ten-week-old mink were infected by force feeding by pipette 2 ml of a tissue suspension containing a Wisconsin strain of mink enteritis virus. Four days later, diarrhea and partial or complete loss of appetite developed simultaneously in all of the animals. Squinting and occasional vomiting were also observed. By the sixth day after inoculation, all of the mink were anorectic and weak. Anorexia persisted for 48 to 96 hours. Diarrhea and vomiting continued until the eighth to ninth day after exposure. For the first two days after the appearance of diarrhea, the feces contained large quantities of mucus and intestinal casts were seen frequently in the droppings. Thereafter, the feces consisted mostly of yellowish green, watery fluid and contained no casts. Some of the animals died on the eighth day after infection. Those which survived were severely dehydrated and debilitated, but resumed eating and achieved complete clinical recovery within the next five to six days.

Leukopenia, i.e., total leukocyte count of less than 5,000 cells per mm³ of blood, was found in seven of nine mink examined during the height of the disease. Leukopenic animals were deficient in both lymphocytes and neutrophils.

     

Epizootiological Study of Bacterial Cold-Water Disease in Pacific Salmon and Further Characterization of the Etiologic Agent,Flexibacter psychrophila

Abstract

Isolates of Flexibacter psychrophila were obtained from chinook salmon Oncorhynchus tshawytscha and coho salmon O. kisutch that had previously sustained epizootics of coldwater disease. The pathogen was readily isolated from kidney and mucus of convalescent fish. The organisms were relatively inert in most standard microbiological media but were structurally and serologically homogenous by examination of whole cell protein lysates by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In contrast to the homogeneity observed in phenotypic and serologic assays, the isolates studied elaborated varied ribotypes. All isolates produced a single rDNA spacer amplification product of about 240 base pairs.     

An aroA Mutant of Edwardsiella ictaluri Is Safe and Efficacious as a Live,Attenuated Vaccine

Abstract

The aroA gene of Edwardsiella ictaluri was cloned and sequenced, and the sequence data were used to construct a deletion–insertion mutation in the aroA gene. The mutated gene was transferred into a virulent, wild-type E. ictaluri strain by conjugation and allelic exchange. Putative aroA mutants were confirmed phenotypically by demonstrating a need for supplementation with aromatic metabolites to support growth in minimal media. The genetic construction was evaluated by using the polymerase chain reaction to amplify appropriate regions of the aroA deletion–insertion, and DNA sequencing of the amplified products confirmed the predicted construction. A selected mutant, LSU-E1, was passed 30 times in nonselective media with no reversion to the wild-type following screening of 1.6 × 10¹¹ colony-forming units. The mutant was demonstrated via injection to be attenuated more than 5 logs₁₀ compared with the wild-type E. ictaluri strain, and it was avirulent by immersion and oral routes. Tissue persistence studies indicated that the mutant maintained the ability to invade following immersion exposure, but no viable cells were detected after 48–72 h. Significant levels of protection from disease were demonstrated following immersion vaccination of channel catfish Ictalurus punctatus.     

The effect of transportation on the immune status of Bos indicus steers

This study investigated the effect of 72 h of road transport on the immune status of Bos indicus steers (n = 10; age = 15 to 18 mo). Total and differential leukocyte numbers and lymphocyte function were determined at 2 d before transport (-48 h), immediately after 72 h of transport (72 h), and 6 d after transport (216 h). Phytohemagglutinin (PHA)-stimulated lymphocyte proliferation, interferon-gamma production, and tetanus-toxoid specific antibody levels were determined. Total leukocyte and eosinophil numbers showed a transient decrease at 72 h (immediately after transport; P < 0.05) and returned to baseline values by 6 d after transport. Lymphocyte numbers and antibody titers were unaffected by transportation. The PHA-stimulated lymphocyte proliferation decreased (P < 0.05) at 72 h and returned to baseline levels 6 d after transport. This study demonstrated that transportation of mature Bos indicus steers caused transient decreases in leukocyte numbers and lymphocyte function, although all measures recovered by 6 d after transport. Therefore, Bos indicus cattle may be vulnerable to infection during this period.     

Lung and pleural lesions of veal calves at slaughter and their relationship with carcass weight

Summary

At slaughter the lungs of 2138 veal calves (13 transport groups, mainly from different farms) were examined. Lung and pleural lesions were classified by degree of extension and pathomorphological features, and the accompanying carcass weights were measured. Seventeen percent of the calves had extensive lung lesions, extensive pleural lesions or both, and their mean carcass weight was reduced by 4.3 kg (p<0.05). The percentage of calves with extensive lesions varied between transport groups from 4% to 33%. All pleural lesions were adhesive. Most lung lesions (93%) were classified as cuffing/exudative pneumonia. Slaughterhouse inspection of lungs can be used as a non‐clinical parameter of respiratory disease in veal calves.     

Glycogen in Leukocytes from Bovine Blood and Milk

Glycogen content was determined quantitatively by the Anthrone reagent method in leukocytes obtained from blood and milk of five cows. Distribution of glycogen in leukocytes was studied by microscopic examination of slides stained by Periodic acid-Schiff (PAS) reaction. Blood glucose concentrations were investigated in these animals by standard procedures. In two of five cows both blood glucose levels and blood leukocyte glycogen levels on the same day were determined for six consecutive days. One hundred and two blood leukocyte samples from five cows had a mean glycogen content of 1.32 ± 0.04 (S.E.) mg/10⁹ WBC, and 6.11 ± 0.17 (S.E.) mg/10⁹ PMNs. Leukocyte preparations from 80 samples of milk comprising 97 to 98% PMNs contained 3.81 ± 0.18 (S.E.) mg glycogen/10⁹ milk leukocytes. In PAS preparations of blood and milk leukocytes glycogen was found almost exclusively in PMNs. Glycogen granules, present frequently in PMNs and occasionally in monocytes and large lymphocytes from blood, were not observed in those from milk. The glycogen level in milk leukocytes was significantly lower (P = <0.01) than that of the blood PMNs in every cow, and the overall mean difference between levels for milk leukocytes and blood PMNs was highly significant (P = <0.001). Mean blood glucose concentration in the five cows was 44.46 ± 0.66 (S.E.) mg%. There was no significant relationship between blood glucose and blood leukocyte glycogen levels in the five corresponding cows; nor between blood glucose and blood PMN glycogen levels on the same day in either of two cows investigated. Leukocyte preparations from milk samples obtained on the second day following intramammary infusion of endotoxin consistently contained markedly less glycogen than the leukocyte preparations from first day post-infusion samples.

These tended to level off and became intermediate between first and second day levels. It is postulated that the poor phagocytic competence of leukocytes from bovine mammary glands compared to their counterparts in blood observed by various workers may be due partially to low energy reserves in these cells.

     

Interactive effects of α-tocopheryl acetate and zinc supplementation on the antioxidant and immune systems of broilers

ABSTRACT

1. A total of 1440 one-day-old Ross 308 male broilers were allocated to 12 dietary treatments to evaluate dose-dependent effects of α-tocopheryl acetate (α-TOA) combined with zinc (Zn) supplementation on humoral and cellular immune responses, antioxidant enzymes, serum and hepatic contents of vitamins and minerals in broilers.

2. Three levels of supplemental α-TOA (0, 150 and 300 mg/kg) and 4 levels of Zn (0, 100, 200 and 400 mg/kg) were combined as a completely randomised design as a 3 × 4 factorial arrangement.

3. Concentrations of serum α-tocopherol and selenium were influenced by the interaction of α-TOA and Zn. The interaction of α-TOA and Zn affected malondialdehyde (MDA) concentration in serum and liver (p < 0.05). Incremental amounts of supplemental Zn augmented the effects of α-TOA in reducing serum and hepatic MDA concentrations.

4. The interaction of α-TOA and Zn on antibody titres (p < 0.05) was such that increasing level of Zn at each α-TOA level led to a linear enhancement in antibody titre. Moreover, dietary supplementation with α-TOA and Zn resulted in an increase in relative weight of lymphoid organs (thymus, bursa, spleen; p < 0.05) along with an increase in humoral and cellular immune responses (p < 0.05).

5. In conclusion, α-TOA with Zn showed interactive effects in improving oxidative stability and humoral immune responses, which could result from their impact on the concentrations of antioxidant vitamins and minerals in tissues.     

Cultivation and Maintenance of Sphaerophorus necrophorus Part I: An Anaerobic Medium for Aerobic Incubation

Out of 185 media formulated and studied for the growth of Sphaerophorus necrophorus, ten were selected for intensive investigation. Their ability to promote growth was compared photometrically. Medium 156 made up of Brewer's thioglycollate broth, yeast extract, L-cystine, and ascorbic acid, was found to yield a very heavy growth. The viability of S. necrophorus was maintained indefinitely by weekly subcultures. When not subcultured, all isolates remained viable for fifteen weeks and some isolates for 30 weeks at 37°C.

Twenty-nine isolates were tested. No variation in maximum growth was noticed.

Medium 156 is an optimum medium for S. necrophorus. It can be incubated aerobically without interfering with maximum growth. The medium does not deteriorate when stored for at least 12 weeks at room temperature.

     

Effects of dietary yeast extract on turkey stress response and heterophil oxidative burst activity

1. Effective nutritional approaches to counteract the negative effects of stress may provide food animal producers with useful alternatives to antibiotics. In this study, turkeys were fed on a standard diet, or the same diet supplemented with yeast extract (YE), to determine if YE would improve disease resistance in a stress model.

2. At 16 weeks of age, half of the birds were exposed to a bacterial challenge using a coarse spray of the pen environment. A subset of control and challenged birds was also treated with dexamethasone (Dex) prior to challenge (Dex/challenge). At 18 weeks, another subset was subjected to a 12?h transport stress protocol (Challenge/transport). All birds were bled and necropsied the morning after transport. The numbers and proportions of blood cells and the heterophil oxidative burst activity (OBA) were determined. Serum corticosterone (Cort) levels of male birds were measured using a commercial ELISA kit. Body weight and gain were increased by YE during week 1.

3. YE decreased mortality and bacterial isolation following Dex/challenge only in females. Cort levels in male turkeys were decreased by YE and Dex treatment. OBA was higher in males and in birds given YE and was reduced by challenge and transport.

4. These results suggest there may be gender differences in the turkey stress response and that dietary YE has potential for modulating the impact of stress on innate immunity of turkeys.     

The Impact of Mitochondrial and Thermal Stress on the Bioenergetics and Reserve Respiratory Capacity of Fish Cell Lines

Abstract

Various stressors affect the health of wild and cultured fish and can cause metabolic disturbances that first manifest at the cellular level. Here, we sought to further our understanding of cellular metabolism in fish by examining the metabolic responses of cell lines derived from channel catfish Ictalurus puntatus (CCO), white bass Morone chrysops (WBE), and fathead minnow Pimephales promelas (EPC) to both mitochondrial and thermal stressors. Using extracellular flux (EF) technology, we simultaneously measured the oxygen consumption rate (OCR; a measure of mitochondrial function) and extracellular acidification rate (ECAR; a surrogate of glycolysis) in each cell type. We performed a mitochondrial function protocol whereby compounds modulating different components of mitochondrial respiration were sequentially exposed to cells. This provided us with basal and maximal OCR, OCR linked to ATP production, OCR from ion movement across the mitochondrial inner membrane, the reserve capacity, and OCR independent of the electron transport chain. After heat shock, EPC and CCO significantly decreased OCR and all three cell lines modestly increased ECAR. After heat shock, the reserve capacity, the mitochondrial energetic reserve used to cope with stress and increased bioenergetic demand, was unaffected in EPC and CCO and completely abrogated in WBE. These findings provide proof-of-concept experimental data that further highlight the utility of fish cell lines as tools for modeling bioenergetics.

Received April 12, 2012; accepted August 5, 2012     

Reference parameters of peripheral immune cell populations in broiler chickens using a large database

ABSTRACT

1. Peripheral blood immune evaluation of broilers by flow cytometry is commonly used in avian research. However, indicative immune parameters in healthy animals have not yet been determined. In this trial, confidence intervals were determined for several circulating immune cell subsets.

2. The intervals were determined from over 1,000 healthy broilers from commercial lines. To verify the usefulness of the data, they were compared to animals under salmonellosis or mycotoxin challenges.

3. Confidence intervals were determined for several immune cells in the peripheral blood of broilers. The importance of some immune cells in discriminating diseased from healthy controls was demonstrated.

4. This work initiated the construction of reference values for cellular parameters in broilers as a tool for the avian immunology community. Accordingly, the entire dataset of healthy animals was made available for use with other analyses and for improvement by others.