Effects of storage on serum ionized calcium and pH from horses with normal and abnormal ionized calcium concentrations

It has been previously shown that Ca(I) concentration is stable in serum collected from healthy horses for 10 days if stored at 40 degrees C. This may not be true for horses with abnormal Ca(I) concentrations. Thus the stability of ionized calcium (Ca(I)) concentration and pH measurement in serum from horses with both normal and abnormal Ca(I) concentrations stored for various times at 40 degrees C and -10 degrees C was evaluated. Our results indicated that serum Ca(I) concentration was stable throughout 7 days of cold or frozen storage, after being received by the Clinical Chemistry Laboratory. Serum Ca(I) concentration showed a significant decrease by 14 days of frozen storage (-10 degrees C). Serum pH showed a statistically significant increase by 7 days of cold storage, and within 3 days of frozen storage. If equine serum is collected, handled and stored anaerobically, and kept cold or frozen, Ca(I) concentration can be accurately measured for approximately 7 days after collection, regardless of the health status of the animal. An accurate measurement of pH may be made within 3 days of cold or 1 day of frozen storage.     

Engraftment of severe combined immune deficient/beige mice with bovine foetal lymphoid tissues.

To develop a model of bovine thymus and lymph node growth in vivo, we have implanted bovine foetal tissues (16-23 weeks gestation) under the renal capsule of severe combined immune deficient (SCID)/beige (BG) mice and assayed for graft growth and characteristics 2-18 weeks after engraftment. Bovine foetal thymus and lymph node grew considerably following engraftment of SCID/BG mice. Growth was optimal if bovine foetal tissues were used before gestation Week 17. Bovine-mouse chimerism was confirmed using glucose phosphate isomerase analysis. Bovine thymus grew during the entire 18 weeks of study. Growth of bovine lymph node was initially rapid, reaching a maximum at 2 weeks after transplantation followed by a progressive decrease in size. Transplanted bovine lymph node and thymus were morphologically similar to age-matched bovine foetal tissue for a limited time period. Fibrosis, degeneration and depletion of lymphocytes were evident 6 weeks after engraftment; changes were more severe in lymph node than in thymus whereas increases in lymphocytes, lymphopoiesis and follicle formation were evident in age-matched bovine foetal tissue. Despite growth and morphological similarities of the transplanted tissue, blood counts suggested there was no peripheralization of bovine leucocytes. Bovine immunoglobulins (IgG1 and IgG2) were detected in serum of some SCID/BG chimeric mice for a limited time. The appearance of bovine immunoglobulins at 2 weeks in SCID/BG chimeric mice depended on the age of the foetal donor (> 18 weeks) and coincided with the appearance of morphologically mature lymphocytes in the donor foetus lymph nodes. The ability to produce bovine immunoglobulins decreased 8 weeks after engraftment, coinciding with the depletion of lymphocytes in the engrafted lymph node. Lymphocyte depletion and loss of function of engrafted tissues appear the result of a lack of lymphoid progenitors normally derived from hematopoietic stem cells in the bone marrow.     

Pathogenicity for horses of original Sagiyama virus, a member of the Getah virus group

Sagiyama virus is a member of the Getah virus group. Its pathogenicity for horses was examined. All the horses infected with the original 4 strains of Sagiyama virus (M6/Mag 33, Mag 121, Mag 132 and Mag 258) developed pyrexia ranging from 39.0 to 40.0 degrees C. Other clinical signs, characterized by eruptions, edema in the hind legs, enlargement of the submandibular lymph node and mild leukopenia, were also manifested. Viremia occurred 1-4 days post-inoculation (p.i.). Virus was recovered from spleen, liver, lung and various lymph nodes of a horse autopsied on Day 4 p.i. The maximum titer of virus (10(6.0) TCID50 g-1) was detected in the inguinal lymph node. Seroconversion was demonstrated in all the infected horses on Day 5 p.i. These clinical signs and virological findings were similar to those of horses infected naturally. The results indicate that Sagiyama virus has pathogenicity for horses and is similar to that of Getah virus.     

Stability of sorbitol dehydrogenase activity in bovine and equine sera

Serum sorbitol dehydrogenase (SDH) activities in 10 cows and nine horses were measured using an automated clinical analyzer. The serum samples were divided into aliquots that were stored at room temperature (21 degrees C), refrigerated (0-5 degrees C), or frozen (-30 degrees C). The stability of the SDH activity was monitored at various intervals. SDH activity in bovine sera remained stable for at least 5 hours at room temperature, 24 hours refrigerated, and 72 hours frozen without any significant (p < 0.05) differences from the initial serum values. In equine sera, SDH activity remained stable for at least 5 hours at room temperature and 48 hours frozen. The activity of the refrigerated equine sera was stable for at least 5 hours but less than 24 hours. An evaluation of fresh bovine serum and heparinized plasma samples indicated that there was no significant difference (p < 0.05) between the two sampling methods and that either may be employed for automated measurement of SDH activity following the established protocol. Sample type comparison indicated that there was a small but statistically significant (p < 0.05) difference between the results obtained comparing fresh serum and heparinized plasma samples for the horse. A reference range for Holstein cows was established using sera from 71 clinically healthy cattle (mean -/+ 2 SD = 32 -/+ 26 U/L).     

Effect of freezing goat milk samples on recovery of intramammary bacterial pathogens

With the aim of evaluating the effect of freezing goat milk samples on recovery of intramammary pathogens, 1200 milk samples from udder halves with subclinical intramammary infection were studied. Samples (20 ml) were frozen at -20 and at -80 degrees C. Thawing was carried out at room temperature at 7, 14, 21, 28, 58, 118, 178, 236 and 730 days after collection and bacteriological analyses were carried out to determine the number of colony forming units/ml (CFU/ml). Mixed model statistical analysis showed that bacterial group, temperature of storage, interaction of bacterial group and temperature of storage and the interaction of bacterial group, time and temperature of storage were statistically significant effects. For coagulase negative staphylococci (CNS), least squares means of log CFU/ml recovered at -20 and -80 degrees C were not different. Nevertheless, for Gram negative bacilli (GNB) a significant decrease was detected in samples frozen at -20 vs. -80 degrees C. At both temperatures and at different times of storage, significant increases were detected between log CFU/ml of CNS and values on day zero. At -20 degrees C, a significant decrease in GNB recovery was detected between freezing days zero and 730. This difference was not detected when goat milk samples infected by GNB were frozen at -80 degrees C. The results show that frozen milk samples can be useful in goat subclinical mastitis control programs.     

Aerosol stability of bovine parainfluenza type 3 virus.

Aerosols of bovine parainfluenza type 3 virus were generated with a Devilbiss 40 nebulizer from Eagle's minimum essential medium and nasal secretion from a non-infected calf and stored in a rotating drum at temperatures of 6 degrees C or 32 degrees C and relative humidities of 30% or 90%. The aerosols were sampled at seven minutes, one, two and three hours after the start of generation with an all glass impinger (AGI-30) and titrated for infectivity in cell cultures. Physical decay was determined by a rhodamine tracer technique. Media, temperature or relative humidity had little effect on the survival of parainfluenza type 3 virus during spraying (zero to seven minutes). During aging of aerosols at 32 degrees C and 30% relative humidity, parainfluenza type 3 virus was less stable in Eagle's minimum essential medium than in nasal secretion from a noninfected calf, but at 6 degrees C and 30% relative humidity, the virus was more stable in Eagle's minimum essential medium. At 32 degrees C, the virus was less stable during aging at 90% relative humidity than at 30% relative humidity. The virus was consistently more stable during aging of aerosols at 6 degrees C than at 32 degrees C.     

Pregnancy rates of mares inseminated with semen cooled for 18 hours and then frozen

The ability to ship cooled stallion sperm for subsequent freezing at a facility specializing in cryopreservation would be beneficial to the equine industry. Stallion sperm has been centrifuged, cooled to 5 degrees C for 12 h, and frozen without a detrimental effect on motility in a previous study; however, no fertility data were available. Experiment 1 compared the post-thaw motility of sperm cooled for 18 h at 15 or 5 degrees C at either 400 or 200 x 10(6) sperm/mL and then frozen. Storage temperature, sperm concentration, or the interaction of temperature and concentration had no effect on total (TM) and progressive motility (PM) after cooling. Post-thaw TM and PM were higher for control than (P < 0.05) for treated samples. There was no difference in post-thaw TM and PM due to temperature or concentration. Experiment 2 further evaluated procedures for cooling before freezing. Ejaculates were either cooled to 5 degrees C for 18 h and centrifuged, centrifuged at room temperature and then cooled to 5 degrees C for 18 h before freezing, or centrifuged and frozen immediately (control). There was no difference among treatments on post-thaw TM or PM. In Exp. 3, mares were inseminated with semen that had been extended in skim milk-egg yolk without glycerol, centrifuged, resuspended at 200 x 10(6) sperm/mL, cooled to 5 degrees C for 18 h, and then frozen or not cooled for 18 h before freezing (control). Pregnancy rates did not differ for mares receiving semen cooled and then frozen (21 of 30, 70%) or semen frozen directly without prior cooling (16 of 30, 53%). In summary, a procedure was developed for cooling stallion sperm for 18 h before freezing without a resultant decrease in fertility.     

Freezing of sheep faeces invalidates Haemonchus contortus faecal egg counts by the McMaster technique

Faecal pellets from a sheep that was artificially infected with a monoculture of Haemonchus contortus were collected over a 2-h period in the morning. In the laboratory the faeces were thoroughly mixed by hand and 48 by 1 g aliquots of the pellets were sealed in plastic bags, from which the air had gently been expressed. The faecal worm egg count of the sheep was about 14,000 g(-1). Varying numbers of the bags were either processed for faecal worm egg counting (FEC) by the McMaster technique on day 0, or were stored at one of the following temperatures: about 4 degrees C, -10 degrees C or -170 degrees C before processing. The faecal aliquots that were frozen were thawed at room temperature after having been frozen for either 2 h or 7 days, and processing of aliquots maintained at 4 degrees C proceeded shortly after the samples had been removed from the refrigerator. A dramatic reduction in egg numbers was found in all the aliquots that were frozen at -170 degrees C before faecal worm egg counts were done, as well as in those frozen for 7 days at about -10 degrees C. Numerous empty, or partially empty, egg shells were observed when performing the counts in faeces that had been frozen. In contrast, there was no significant reduction in the numbers of eggs in aliquots maintained for 7 days in a refrigerator at +/- 4 degrees C before examination, when compared with others examined shortly after collection of the faeces. Since H. contortus eggs in faeces are damaged by freezing, some methods that can be used for short term preservation are outlined. It is concluded that all nematode egg counts from cryopreserved faeces (whether in a freezer at -10 degrees C or in liquid nitrogen) should possibly be regarded as being inaccurate, unless the contrary can be demonstrated for different worm genera. However, exceptions are expected for the more rugged ova, such as those of the ascarids and Trichuris spp.     

Viability and infectivity of Trichinella spiralis muscle larvae in frozen horse tissue

Many aspects of the biology and epidemiology of Trichinella infection in the horse are poorly understood, including survival of Trichinella spp in horse muscle. In this study, we have assessed the freeze tolerance of T. spiralis in horse meat stored at 5, -5, and -18 degrees C for 1 day to 24 weeks. Results demonstrate a steady reduction in the number of live ML recovered from the cold stored meat samples. On Day 1, recovery of live larvae had been reduced by 18.6%, 50.1%, and 37.2%, and by 4 weeks, recovery of larvae had been reduced by 65.4%, 66.5%, and 96.2% in samples stored at 5, -5, and -18 degrees C, respectively. Infectivity results (measured as reproductive capacity index (RCI)) from mice inoculated with larvae recovered from non-frozen meat samples at day 0 was 23.5. Following storage at -18 degrees C for one and two days, the RCIs were 2.09 and 0.99, respectively. Small numbers of infective larvae were still present in meat samples stored at -18 degrees C for 4 weeks. The RCI of ML recovered from meat samples stored at -5 degrees C was 14.99 and 6.36 at 2 weeks and 4 weeks respectively; the RCI of samples stored at 5 degrees C was 23.1 at 8 weeks, and fell rapidly thereafter (12 week RCI 1.33; 0 at 24 weeks). These data demonstrate that infective T. spiralis, a non-freeze tolerant species, can survive for at least 4 weeks in horse tissue frozen at -5 or -18 degrees C, and that the numbers of infective larvae decrease substantially by day 2 at -18 degrees C and by week 4 at -5 degrees C.     

Infection of pigs with transmissible gastroenteritis virus from contaminated carcases

Sixteen 6-month-old pigs were exposed to transmissible gastroenteritis (TGE) virus by placing them in close contact with piglets infected at 1 week of age. Fourteen of the older pigs were slaughtered between 1 and 5 d after exposure to infection and their carcases dressed in simulated abattoir conditions. Samples of muscle, bone marrow and carcase lymph nodes were stored at -25 degrees C for at least 30 d and then homogenised and fed to groups of 1-week-old and 3-week-old pigs. Four of 12 one-week-old pigs died and TGE virus was isolated from intestinal contents of one of these. All pigs of both age groups developed neutralising antibody to TGE virus over the ensuing 4 w. The results indicate that carcases from pigs infected with TGE virus can represent a source of infection for susceptible pigs given access to them.     

Response of efferent lymph and popliteal lymph node to epidermal infection of sheep with orf virus

Functional and phenotypic changes in the cell populations were monitored in the popliteal efferent lymph of sheep following experimental epidermal infection with orf virus. In another group of sheep, cells from the popliteal lymph node draining the site of infection were similarly monitored and compared with the cells from contralateral popliteal and mesenteric lymph nodes. All sheep showed serological evidence of previous exposure to orf virus. Following infection, anti-orf antibody titres rose and efferent lymphocyte and blast cell output increased. Interferon-like activity was detected in efferent lymph early after orf virus but not mock infection. Lymphocytes from the draining popliteal lymph node showed antigen-specific lymphoproliferation on Days 3-7 while cells in the efferent lymph demonstrated proliferative activity on Days 4-6. The requirement for exogenous antigen-presenting cells in the culture of efferent lymphocytes varied between individual sheep. The culture supernatant from proliferating lymph node cells contained interferon-like activity but no anti-orf antibodies, the reverse of that from cultured efferent lymphocytes, perhaps indicating a different reactive T cell population. During the course of the experiment there was an increase in the percentage of efferent lymphocytes expressing MHC Class II antigens and surface immunoglobulins, the latter being recorded as a double peak. The short-term nature of the local T cell response may in part explain the incompleteness of immunity to orf virus in sheep.     

The isolation and preliminary characterization of a rhabdovirus in Australia related to bovine ephemeral fever virus

CSIRO 368 virus was isolated from blood collected in the Northern Territory from a healthy cow and electron microscope studies showed that the isolate had rhabdovirus morphology. Fluorescent antibody studies and complement fixation tests related the virus to bovine ephemeral fever (BEF) virus. Neutralization tests in both suckling mice and Vero cells showed that the virus was not BEF virus. Antibodies to CSIRO 368 virus were found in cattle sera from northern and eastern Australia and Papua New Guinea. Antibodies were found in 16 out of 45 buffalo, some of which also had antibodies to BEF virus. In contrast, none of the 419 deer tested had antibodies to CSIRO 368 virus, although 142 of the same deer had antibodies to BEF virus. No antibodies to CSIRO 368 virus were detected in 16 goats, 54 horses, 10 pigs, 31 sheep, 25 kangaroos, or 14 human beings. Both CSIRO 368 and BEF viruses were found to be sensitive to ether and chloroform, but were not affected by the DNA inhibitor 5-bromo-2'-deoxyuridine, showing that they probably had the same type of nucleic acid--namely RNA. CSIRO 368 was also shown to grow to higher titres in BHK21 cells than in Vero cells. Temperature sensitivity studies at -20, 4 and 37 degrees C showed that the presence of foetal calf serum increased the survival time markedly at -20 degrees C, but only slightly at 4 and 37 degrees C. The virus survived the longest at -20 degrees C in the presence of foetal calf serum.     

Clinical and virological observations on swine experimentally infected with Getah virus

The pathogenicity of Getah virus for swine was examined. All 8 pigs (4 adults and 4 piglets) inoculated with Strains MIP-99 and MI-110 developed pyrexia ranging from 39.4 to 40.7 degrees C and anorexia. Mild depression and diarrhea were observed in 2 of the 4 piglets. These clinical signs were transient. Viremia occurred 1-2 days post-inoculation (p.i.) and the maximum titer was 10(3.0) TCID50 0.1 ml-1. The virus was recovered from a piglet autopsied on Day 3 p.i. from spleen and various lymph nodes. The maximum titer of virus (10(3.75) TCID50 0.1 g-1) was detected in the inguinal lymph node. Seroconversion was demonstrated in all the pigs on Day 6 p.i. These results suggest that Getah virus is mildly pathogenic for swine, which may play a role as an amplifying host in nature.     

Effects of low and high temperatures on infectivity of Cryptosporidium muris oocysts suspended in water

Cryptosporidium muris oocysts suspended in 200 microl of water were pipetted into plastic microcentrifuge tubes which were stored at 4 degrees C or frozen at -5 degrees C for 1, 3, 5, 7, and 10 days and at -20 degrees C for 1, 3, 5, and 8h, respectively. Other samples of C. muris oocysts suspended in water were heated in the metal block of a thermal DNA cycler. Block temperatures were set at 5 degrees C incremental temperatures from 40 to 70 degrees C. At each high temperature setting microcentrifuge tubes containing C. muris oocysts were exposed for 1 min. Both, frozen and heated oocyst suspensions as well as untreated control oocyst suspensions were then inoculated into each of four ICR mice by gastric intubation. Untreated, freeze-thawed or heated oocysts were considered infectious when oocysts of C. muris were found microscopically in the faeces of mice after inoculation. All inoculated mice that received oocysts frozen at -5 degrees C for 3, 5, 7, and 10 days and -20 degrees C for 1, 3, 5, and 8h had no oocysts in faeces. In contrast, C. muris oocysts frozen at -5 degrees C for 1 day remained infective for inoculated mice. Our results also indicated that when water containing C. muris oocysts was exposed at a temperature of 55 degrees C or higher for 1 min, the infectivity of oocysts was lost.     

Properties of an Australian isolate of bovine parvovirus type 1

An Australian bovine parvovirus isolate (BPV 267) was found to haemagglutinate human, guinea-pig, rat and dog erythrocytes, out of a range of 16 species of erythrocyte tested. The haemagglutinating activity was generally found to be both pH and temperature dependent. The virus was found to replicate best in intestinal epithelium, macrophage and lung cells, out of 9 bovine cell types tested. Highest yields of virus were obtained by the use of selected cell strains at low-passage levels which were maintained near neutral pH under conditions of high rates of cell growth. Studies of the rates of thermal inactivation with time showed the virus to be relatively stable at 37 degrees C, 56 degrees C and 70 degrees C, the incorporation of serum proteins, 1 M MgCl2 and 2 M NaCl in the medium having no influence on stability at 56 degrees C. The virus was resistant to the action of CHCl3, ether and 1% trypsin, and its replication was inhibited by BUDR, this effect being reversed by thymidine. Actinomycin D was found to inhibit virus replication, but only when applied during the first 8 h post-infection. Density gradient studies showed infective virus to have a density of 1.41 g cm-3; haemagglutinating non-infective virus with defective morphology having a density of 1.31 g cm-3. In addition, a proportion of morphologically-complete haemagglutinating, but non-infective virus particles was found at a density of 1.36 g cm-3. The virus proved to have a mean diameter of 22 nm.     

Mechanisms contributing to the virus persistence in Aleutian disease

In this review published results and further studies concerning the persistence of Aleutian disease virus (ADV) isolate SL3 are presented. By Southern blot and in situ hybridization with strand-specific RNA probes focal replication of ADV-DNA was demonstrated in spleen, mesenteric lymph nodes, sporadically in mononuclear cells of the peripheral blood and bone marrow cells. These findings further support the concept of the lymphotropism of ADV. All cell culture-adapted ADV strains appear to have a ts-defect. Our in vitro studies indicate that the ADV isolate G(orham) induced the synthesis of comparable amounts of viral replicative DNA and viral proteins VP1 and VP2 at the non-permissive temperature of 37 degrees C. However, the viral progeny DNA synthesis was about threefold less at 37 degrees C compared to the permissive temperature of 32 degrees C. These findings suggest that the reduced level of viral progeny DNA at 37 degrees C accounts for the reduced production of infectious ADV. Finally, we provided experimental evidence that the apparent lack of neutralizing antibodies in AD is due to the masking of critical viral epitopes by cellular phospholipids.     

Survival of Largemouth Bass Iridovirus in Frozen Fish

Abstract

Juvenile largemouth bass Micropterus salmoides were infected with largemouth bass iridovirus by injection and frozen at ?10°C, and the visceral organs and tissues (liver, kidney, spleen, gonads, swim bladder, and muscle) of individual fish were periodically assayed for virus for 155 d postfreezing. The virus was isolated from 35 of 39 (89.7%) of the frozen fish. However, virus loss occurred more rapidly in fish that were dead when frozen than in fish that were alive when frozen. Overall, largemouth bass iridovirus was stable in frozen tissue when the fish were fresh upon freezing.