Prevalence of latent, neuropathogenic equine herpesvirus-1 in the Thoroughbred broodmare population of central Kentucky.

REASON FOR PERFORMING STUDY: An emerging problem of equine herpesvirus-1 (EHV-1) infection in horses in the USA is a high-mortality myeloencephalopathy that commonly occurs where large numbers of horses are stabled. EHV-1 isolates recovered from recent neurological outbreaks represent a mutant virus strain that possesses enhanced neuropathogenicity. A central question of EHV-1 myeloencephalopathy is the latency carriage rate for these mutants of EHV-1 in USA horse populations. OBJECTIVE: To estimate the prevalence of neuropathogenic strains of EHV-1 as latent infections in the Thoroughbred broodmare population of central Kentucky. METHODS: Submandibular lymph nodes (SMLN) were collected during post mortem examination of 132 Thoroughbred broodmares. Total DNA purified from SMLN tissue was tested for the presence of latent EHV-1 DNA by an ultrasensitive magnetic bead-based, sequence-capture, nested PCR method. Differentiation of active from latent infections by EHV-1 was achieved by detection of transcripts of EHV-1 glycoprotein B by reverse transcription PCR. RESULTS: Latent EHV-1 DNA was detected in the SMLN tissues of 71 (54%) of the 132 mares submitted for necropsy. Thirteen (18%) of the 71 latently infected horses harboured the neuropathogenic biovar of EHV-1. Of the 13 horses latently infected with an ORF30 mutant strain of EHV-1, 11 also carried a latent, wild-type strain of the virus in their SMLN tissues. CONCLUSIONS: Neuropathogenic strains of EHV-1 have established a significant presence in the Thoroughbred broodmare population of central Kentucky as latently infected carrier horses. The data also indicate that a highly sensitive DNA detection method is required to identify many instances of EHV-1 latency. POTENTIAL RELEVANCE: The presence of a relatively large biological reservoir of latent, neuropathogenic EHV-1 has the potential for posing emerging equine health and economic threats to the future prosperity of the USA horse industry.

Regulation of SP60 mRNA during development of Dictyostelium discoideum.

The accumulation of mRNA recognized by oligonucleotides coding for a portion of the spore coat protein, SP60, was determined throughout development of Dictyostelium discoideum. The 1.8 kb mRNA first appears at the tipped aggregate stage and accumulates until culmination. This mRNA is present in pre-spore cells but absent from pre-stalk cells. A cDNA clone was selected by the oligonucleotides and found to be homologous to this mRNA. Although the oligonucleotides were designed to match the sequence coding for a hexapeptide repeat at the amino-terminus of SP60, they were able to recognize a similar repeated region at the carboxy-terminus of the protein coded by the cDNA clone. The SP60 gene appears to be subject to temporal and cell-type-specific transcriptional controls that are coordinate with those of SP96, another spore coat gene.

Microbiologic and pathologic findings in an epidemic of equine pericarditis.

During the spring and summer of 2001 and in association with the mare reproductive loss syndrome, 22 terminal and 12 clinical cases of equine pericarditis were diagnosed in central Kentucky. Actinobacillus species were the principal isolates from 8 of 10 nontreated, terminally affected and 3 of 10 clinically affected horses. Enterococcus faecalis and Streptococcus zooepidemicus were cultured from the remaining 2 nontreated terminal cases. No viruses were isolated in tissue culture. Nucleic acid of equine herpesvirus-2 was detected in pericardial and tracheal wash fluids of 3 and 1 individuals, respectively. Microscopic alterations in sections of heart and parietal pericardium were consistent with chronic fibrinous bacterial pericarditis. This report confirms a significant role of Actinobacillus species in equine pericarditis and describes an epidemic of this infrequently observed syndrome in the horse.

Antiapoptotic compound to enhance hypothermic liver preservation.

BACKGROUND: Apoptosis (programmed cell death) occurs as a consequence of global organ ischemia during isolation and storage prior to transplantation. If apoptosis is inhibited during ischemia, organ preservation should be improved, and the length of time for permissible storage may be increased. The objective of this study was to test the effect of a newly developed antiapoptotic compound, LXR-015, during extended hypothermic liver preservation. METHODS: Three groups of 12 rats each were studied. In the normal group, liver function was studied immediately after harvesting. In the study group, harvested livers were flushed with Euro-Collins solution (30 ml/kg body weight) containing LXR-015 at a concentration equivalent to 9 mg/kg animal body weight (300 microg/ml). The livers were then stored at 4 degrees C for 24 hr before liver function was studied. In the control group, harvested livers were flushed with Euro-Collins solution without LXR-015 and then stored at 4 degrees C for 24 hr before liver function was studied. RESULTS: Portal venous flow was higher (P<0.05) in the normal and study groups compared with the control group. Portal venous resistance was lower (P<0.05) in the normal and study groups compared with the control group. Liver tissue oxygen consumption in the study group was significantly higher than in both the normal and control groups (P<0.05). Liver enzyme production (aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, creatine kinase) was higher in the control group than in either the study or normal group (P<0.05). Bile production in both the normal and study groups was higher than in the control group (P<0.05). The liver tissue wet to dry weight ratio in both the normal and study groups was lower than in the control group (P<0.05). Histopathology studies revealed fewer apoptotic bodies (P<0.05) in both the normal (1.70+/-0.15 per high-power field) and study groups (2.08+/-0.10 per high-power field) than in the control group (7.92+/-.33 per high-power field). CONCLUSIONS: Adding an antiapoptotic compound, LXR-015, to Euro-Collins solution significantly improves hypothermic preservation of the rat liver compared with Euro-Collins solution alone.

Comparison of the structure and expression pattern for a low molecular weight heat-shock protein cDNA clone from Nicotiana tabacum.

We isolated a cDNA clone, named TLHS-1, for a low molecular weight heat-shock protein (LMW HSP) from tobacco. The nucleotide sequence determination of the clone identified an open reading frame for 159 amino acids. To the upstream of the open reading frame, a sequence of 124 nucleotides was determined. To the 3' downstream of the open reading frame, 212 nucleotides were identified which carried a poly(A) tail. A comparison of the open reading frame of TLHS-1 with the previously reported class I LMW HSPs showed a high identity which classified the TLHS-1 as a class I LMW HSP cDNA clone. Reexamination of the amino acid sequences for LMW HSPs including the TLHS-1 argued for the necessity to redraw the consensus region. Six consensus regions were proposed instead of the two consensus regions suggested previously. A RNA blot hybridization for TLHS-1 showed the typical expression pattern of a heat-shock inducible gene from 3 common tobacco cultivars. The genomic DNA blot hybridization for the TLHS-1 gene from the 3 tobacco cultivars showed identical patterns. These results implied that the expression control and structure of the TLHS-1 gene have been well conserved in the tobacco cultivars which are not bred for high temperature stress resistance.

Structure of a polyubiquitin gene in Nicotiana tabacum.

Using a tobacco ubiquitin cDNA clone as a probe, a genomic clone in EMBL3 coding for a tobacco polyubiquitin protein was isolated. Southern blot hybridization of the genomic clone with the cDNA clone identified a BamHI/EcoRI fragment of 2.5 kb to contain the coding region of polyubiquitin, and thus the fragment was subcloned into a plasmid vector. Nucleotide sequence determination of the clone identified an open reading frame for the four head-to-tail repeats of ubiquitin monomer of 76 amino acids interrupted by an intron sequence of 55 nucleotides. The four ubiquitin units were completely conserved except for the extra glutamine at the carboxy terminus of the last ubiquitin monomer. At the 5'-region upstream of the open reading frame, a sequence of 630 nucleotides was determined. In this region, well-known regulatory sequences such as the CCAAT box, TATA box and heat-shock elements could not be located; instead, a region very rich in C and T and repeats of CA was noticed. In the 3'-downstream region of the open reading frame, a sequence of 474 nucleotides was determined which contained putative polyadenylation signals and a GU-rich region.

High temperature stress resistance of Escherichia coli induced by a tobacco class I low molecular weight heat-shock protein.

TLHS1 is a class I low molecular weight heat-shock protein (LMW HSP) of tobacco (Nicotiana tabacum). For a functional study of TLHS1, a recombinant DNA coding for TLHS1 with a hexahistidine tag at the amino-terminus was constructed and expressed in Escherichia coli. An expressed fusion protein, H6TLHS1, was purified using a Ni2+ affinity column and a Sephacryl S400 HR column. A polyclonal antibody against H6TLHS1 was produced to follow the fate of H6TLHS1 in E. coli. The fusion protein in E. coli maintained its solubility at a temperature of up to 90 degrees C and most of the proteins in the E. coli cell lysate with H6TLHS1 were prevented from thermally induced aggregation at up to 90 degrees C. We compared the viability of E. coli cells expressing H6TLHS1 to the E. coli cells without H6TLHS1 at a temperature of 50 degrees C. After 8 h of high temperature treatment, E. coli cells with H6TLHS1 survived about three thousand times more than the bacterial cells without H6TLHS1. These results showed that a plant class I LMW HSP, TLHS1, can protect proteins of E. coli from heat denaturation, which could lead to a higher survival rate of the bacterial cells at high temperature.

Nucleotide sequence of a ubiquitin-extension protein gene in Nicotiana tabacum and its expression pattern upon heat shock.

Using a tobacco cDNA clone as a probe, a genomic clone coding for a tobacco ubiquitin extension protein was isolated. Nucleotide sequence determination of the clone identified an open reading frame for an ubiquitin unit of 76 amino acids and an extension tail of 80 amino acids. To the 5' upstream of the open reading frame, a sequence of 1,110 nucleotides was determined and a putative CCAAT box and TATA box were located. An intron sequence was not apparent as the open reading frame for the ubiquitin-extension protein was continuous for 468 nucleotides. In the 3' downstream region of the gene, a putative polyadenylation signal and GU rich region were observed. Northern blot analysis of tobacco transcripts with the tail part of the gene as a probe specifically identified the 1.3 kb transcript. The Northern signal at 1.3 kb indicated that the expression of the ubiquitin-extension protein gene in tobacco is downregulated upon heat-shock.

Dietary vitamin E and selenium and toxicity of nitrite and nitrate.

Nitrites and nitrates are important antimicrobial and flavoring/coloring agents in meat and fish products. However, nitrites and nitrates may cause methemoglobinemia and other illness, and may react with certain amines to form carcinogenic nitrosamines. The nutritional status of vitamin E and selenium has long been associated with nitrite and nitrate toxicity, although the mechanism involved is not yet clear. Information available recently shows that nitrites and nitrates are both oxidation products and ready sources of nitric oxide (NO*), that NO* reacts rapidly with superoxide to form highly reactive peroxynitrite (ONOO-), and that vitamin E may mediate the generation and availability of superoxide and NO*. Increased formation of ONOO- resulting from nitrite treatment and low intake of vitamin E and selenium may thus be the critical event leading to tissue damage and animal mortality observed previously. The protection against the adverse effects of nitrites/nitrates by vitamin E is attributed to its ability to reduce ONOO- formation, while selenium exerts its protective effects via seleno-enzymes/compounds, which reduce ONOO- formed.

Effect of dietary vitamin E on nitrite-treated rats.

The effect of dietary vitamin E on cellular responses to nitrite was studied in rats. One-month-old male Sprague-Dawley rats were fed a basal vitamin E-deficient diet with or without 100 ppm vitamin E and 1000 ppm sodium nitrite (NaNO2) for 9 weeks. In addition to a high mortality rate, nitrite-fed rats maintained on a vitamin E-deficient diet exhibited a marked increase in liver necrosis, tubular nephrosis and myodegeneration, as well as greater biochemical and hematological alterations when compared to the control animals. No animal mortality or histopathologic lesions in any tissues were observed in rats receiving a vitamin E-supplemented diet with or without nitrite. The results suggest that depletion of vitamin E renders rats more susceptible to the adverse effect of nitrite, and that nitrite administration potentiates deficiency of vitamin E in rats.

Equine abortion and stillbirth in central Kentucky during 1988 and 1989 foaling seasons.

Pathologic and microbiologic examinations were performed on 1,211 aborted equine fetuses, stillborn foals, and placentas from premature foals in central Kentucky during the 1988 and 1989 foaling seasons to determine the causes of reproductive loss in the mare. Placentitis (19.4%) and dystocia-perinatal asphyxia (19.5%) were the 2 most important causes of equine reproductive loss. The other causes (in decreasing order) were contracted foal syndrome and other congenital anomalies (8.5%), twinning (6.1%), improper separation of placenta (4.7%), torsion of umbilical cord (4.5%), placental edema (4.3%), equine herpesvirus abortion (3.3%), bacteremia (3.2%), fetal diarrhea (2.7%), other placental disorders (total of 6.0%), and miscellaneous causes (1.6%). A definitive diagnosis was not established in 16.9% of the cases submitted. Streptococcus zooepidemicus, Escherichia coli, Leptospira spp., and a nocardioform actinomycete were organisms most frequently associated with bacterial placentitis, and Aspergillus spp. was the fungus most often noted in mycotic placentitis. No viral placentitis was noticed in this series. Dystocia-perinatal asphyxia was mostly associated with large foals, maiden mares, unattended deliveries, and malpresentations. The results of this study indicate that in central Kentucky, the noninfectious causes of equine reproductive loss outnumber the infectious causes by an approximate ratio of 2:1, placental disorders are slightly more prevalent than nonplacental disorders, Leptospira spp. and a nocardioform actinomycete are 2 new important abortifacient bacteria in the mare, the occurrence of contracted foal syndrome is unusually frequent, the incidence of twin abortion has sharply declined, and torsion of the umbilical cord is an important cause of abortion in the mare.

Etiology and pathology of equine placentitis.

Placentas from aborted, stillborn, and premature foals were examined during the 1988 and 1989 foaling seasons, and 236 of 954 (24.7%) had placentitis. Microorganisms associated with placentitis were isolated or demonstrated from 162 of 236 (68.6%) placentitis cases. Leptospira spp. and a nocardioform actinomycete were 2 important, newly emerging bacteria associated with equine placentitis. Major pathogens identified in decreasing order were Streptococcus zooepidemicus, Leptospira spp., Escherichia coli, a nocardioform actinomycete, fungi, Pseudomonas aeruginosa, Streptococcus equisimilis, Enterobacter agglomerans, Klebsiella pneumoniae, and alpha-hemolytic Streptococcus. Pathogens were not recovered in 64 cases (27.1%) and overgrowth by saprophytic bacteria was recorded in 10 cases (4.2%). Twenty-seven cases (16.6%) had mixed bacterial growth and 93 cases (57.4%) had bacteria cultured from both placenta and fetal organs. The majority of the placentitis cases caused by bacteria, with the exception of Leptospira spp. and the nocardioform actinomycete, occurred in 2 forms. One was acute, focal or diffuse; had an infiltration of neutrophils in the intervillous spaces or necrosis of chorionic villi; was associated with bacteremia; and frequently occurred in the placenta from fetuses expelled before or at midgestation. The other was observed from foals expelled at late gestation, was mostly chronic and focal or focally extensive, and occurred mostly at the cervical star area. Chronic placentitis was characterized by the presence of 1 or a combination of the following lesions: necrosis of chorionic villi, presence of eosinophilic amorphous material on the chorion, and infiltration of mononuclear inflammatory cells in the intervillous spaces, villous stroma, chorionic stroma, vascular layer, and allantois.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationships between acute sediment toxicity in laboratory tests and abundance and diversity of benthic infauna in marine sediments: a review.

Acute sediment toxicity tests have become important in regulatory, monitoring, and scientific programs, partly because it has been assumed that they are indicative of ecological damage to benthic infaunal resources. Data from tests of sediment toxicity and measures of benthic community structure were examined from > 1,400 saltwater samples to determine the relationships between acute toxicity and changes in the abundance and diversity of infauna resources. Data were compiled from studies conducted along portions of the Atlantic, Gulf of Mexico, and Pacific coasts of the United States. There was considerable variability among the data sets in the relationships between laboratory results and benthic measures. However, in 92% of the samples classified as toxic, at least one measure of benthic diversity or abundance was < 50% of the average reference value. In 67% of these samples, at least one measure of benthic infauna abundance or diversity was < 10% of average reference conditions. No amphipods were found in 39% of samples that were classified as toxic, whereas amphipods were absent from 28% of the nontoxic samples. In many survey areas, the abundance of crustaceans (notably the amphipods) decreased in the infauna as amphipod survival decreased in the laboratory tests. There appeared to be a break point in the data indicating that, generally, amphipod abundance in the field was lowest when survival in the laboratory tests dropped below 50% of controls. Based on the weight of evidence from all the data analyses, we conclude that ecologically relevant losses in the abundance and diversity of the benthic infauna frequently corresponded with reduced amphipod survival in the laboratory tests.

Congenital polyalveolar lobe in three foals.

Three cases of congenital polyalveolar lobe (pulmonary hamartoma) were diagnosed in female Thoroughbred foals. Foal 1 was born at full term but died shortly afterwards. Foal 2 was aborted at the seventh month of gestation. Parturition was induced at the tenth month of gestation in foal 3 because it developed hydrops of the amnion and ascites. In all three foals, the polyalveolar lobe occurred on the right side and affected the entire right lung. In each case, the right lung formed a tumour-like mass, and expanded into the left chest cavity. The lung masses were pink to dark red and spongy to rubbery, with marked lobular patterns on the pleural and cut surfaces. The left lung was compressed and small. The right and left lungs weighed 0.9-6.3 kg and 80 g-0.3 kg, respectively. Microscopically, the polyalveolar lobe consisted of normal alveoli, bronchioli and blood vessels, but the alveolus:artery ratio was greater than normal. In the polyalveolar lobe of two foals the numbers of alveoli per artery were 65.2 and 52.5; in contrast, the corresponding values for three control lungs were 26.9, 26.5 and 27.6. Chronic passive congestion with generalized oedema was observed in foals 2 and 3.

Rhodococcus equi-associated necrotizing lymphadenitis in a llama.

A case of Rhodococcus equi-associated necrotizing lymphadenitis in a 2-year-old male llama is described. Caseous necrosis, resembling macroscopically that seen in ovine caseous lymphadenitis, was observed diffusely in the tracheobronchial and mediastinal lymph nodes, and in an extensive lesion in the lungs. Necrosis was present to a lesser extent in the spleen and hepatic and gastric lymph nodes. Numerous bacteria-laden macrophages were present around the necrotic areas. The findings suggest that, as in cattle and pigs, the primary targets of R. equi infection in the llama are the lymphoid organs.

Malignant rhabdoid tumour in the orbit of a horse.

A malignant rhabdoid tumour was diagnosed in the orbit of a 2-year-old Thoroughbred filly. The neoplasm, which was very aggressive, was present in nearly every part of the ocular and periocular structures and had spread to the lymph nodes of the head and neck, the salivary glands and the subcutaneous tissues around the eye. The neoplasm was composed of polygonal cells with abundant eosinophilic cytoplasm. Many cells had a large, vesiculate, indented nucleus and contained a paranuclear globular inclusion. Ultrastructurally, the inclusions were seen to consist of whorls of intermediate filaments. The neoplastic cells were immunoreactive to vimentin and cytokeratin antisera, but were negative for desmin and actin.

Glomerular lesions in male rabbits treated with aluminium lactate: with special reference to microaneurysm formation.

Novel glomerular lesions were seen in male rabbits after intravenous administration of aluminum lactate. Eight rabbits in the treated group were given 0.1 mmol/kg of aluminum lactate 5 days a week for 4 weeks. The control group of 8 rabbits was given 0.3 mmol/kg of sodium lactate by the same injection protocol. In the treated group, the mesangial cells in the glomerular tufts in 6 of 8 rabbits were distended with grayish blue granular material, which was identified by laser microprobe mass spectrometry and acid solochrome azurine stain as an aluminum compound. Other consistent findings in the glomeruli included microaneurysm in 6 of 8 rabbits and segmental sclerosis in 6 of 8 rabbits. Less frequently observed glomerular changes included crescent formation, necrosis with calcification, fibrosis of the Bowman's capsule, cystic dilation of the Bowman's space, and exudation of erythrocytes into the Bowman's space. The mechanism by which aluminum lactate induces the glomerular changes is not certain. However, the pathogenesis may involve the deposition of aluminum in the mesangial cells, resulting in mesangiolysis which in turn causes microaneurysm. The sclerotic change is interpreted as a sequela of microaneurysm. The findings suggest that aluminum induces glomerular lesions in rabbits. This may serve as a good animal model to study mesangiolysis and microaneurysm formation.

Campylobacteriosis in an aborted equine fetus.

Abortion caused by Campylobacter fetus subsp fetus was diagnosed in a 7-month-old equine fetus. The fetus was small for its gestational age. Macroscopically, the proximal portion of the small intestine was hemorrhagic and its wall was thick. Histologically, the Brunner glands were distended with neutrophils, and the submucosa was thick, owing to fluid accumulation and/or cellular infiltrates. Curved bacteria were observed in the Brunner glands and intestinal glands. Campylobacter fetus subsp fetus was isolated from stomach contents, liver, and lungs, and was detected by dark-field microscopic examination of ocular fluid and stomach contents. Placenta was not available for examination.

Sarcocystosis in an aborted bovine fetus.

Sarcocystosis was diagnosed in an aborted bovine fetus. Immature and mature schizonts of Sarcocystis were disseminated in the vascular endothelium of all organs, but especially the brain. Microscopic granulomas, focal gliosis, and petechial hemorrhages in the neuropil were scattered in the brain. Multifocal collections of mononuclear cells were observed in the kidney, liver and heart. Organisms in sections of frozen tissues were demonstrated by immunofluorescent techniques to be Sarcocystis.

Myodegeneration in Kentucky white-tailed deer.

Skeletal or cardiac myodegeneration, or both, was observed in 4 free-ranging female white-tailed deer that were found dead or recumbent and unable to rise. Gross pathologic findings included white or chalky streaks in heart or skeletal musculature. Degeneration, necrosis, and mineralization were observed microscopically in affected striated muscles. Although the precise cause of myodegeneration was not determined, changes were considered compatible with nutritional or exertional myopathy.