A new formula for estimation of standard liver volume using computed tomography-measured body thickness.

The objective of this article is to derive a more accurate and easy-to-use formula for finding estimated standard liver volume (ESLV) using novel computed tomography (CT) measurement parameters. New formulas for ESLV have been emerging that aim to improve the accuracy of estimation. However, many of these formulas contain body surface area measurements and logarithms in the equations that lead to a more complicated calculation. In addition, substantial errors in ESLV using these old formulas have been shown. An improved version of the formula for ESLV is needed. This is a retrospective cohort of consecutive living donor liver transplantations from 2005 to 2016. Donors were randomly assigned to either the formula derivation or validation groups. Total liver volume (TLV) measured by CT was used as the reference for a linear regression analysis against various patient factors. The derived formula was compared with the existing formulas. There were 722 patients (197 from the derivation group, 164 from the validation group, and 361 from the recipient group) involved in the study. The donor's body weight (odds ratio [OR], 10.42; 95% confidence interval [CI], 7.25-13.60; P < 0.01) and body thickness (OR, 2.00; 95% CI, 0.36-3.65; P = 0.02) were found to be independent factors for the TLV calculation. A formula for TLV (cm3 ) was derived: 2 × thickness (mm) + 10 × weight (kg) + 190 with R2 0.48, which was the highest when compared with the 4 other most often cited formulas. This formula remained superior to other published formulas in the validation set analysis (R2 , 5.37; interclass correlation coefficient, 0.74). Graft weight/ESLV values calculated by the new formula were shown to have the highest correlation with delayed graft function (C-statistic, 0.79; 95% CI, 0.69-0.90; P < 0.01). The new formula (2 × thickness + 10 × weight + 190) represents the first study proposing the use of CT-measured body thickness which is novel, easy to use, and the most accurate for ESLV. Liver Transplantation 23 1113-1122 2017 AASLD.

Determinants of the source of cytomegalovirus in murine renal allograft recipients.

Cytomegalovirus is present in a latent state in renal allografts and may be reactivated in recipients. While human and murine strains are alike in that a primary infection occurs in seronegative recipients of a kidney from a seropositive donor, they may differ when the recipient is seropositive. In a murine transplant model, superinfection of a seropositive recipient with a second strain is unusual. Reports in human transplantation indicate that superinfection of a seropositive recipient does occur, however the frequency is unknown. Our studies examine the potential importance of specific viral strains in host and recipient, the contribution of prior humoral immunity in the recipient, and the technical ability to identify distinctive strains of virus in the presence of each other. Our results indicate that reversal of the viral strains in donor or recipient animals does not alter our previous observation that reactivation of the endogenous recipient viral strain predominates as the infecting strain in the posttransplant period. Further, the presence of antibody in nearly all donors and recipients confirms that all animals were originally infected prior to transplantation. Finally, we demonstrated the technical ability to detect one virus in the presence of the other, thus excluding this variable as possibly confounding. We conclude that the endogenous, latent recipient strain of cytomegalovirus in the murine model is preferentially reactivated in the posttransplant interval.

Enhanced green fluorescent protein as a marker for localizing murine cytomegalovirus in acute and latent infection.

A recombinant murine cytomegalovirus (mCMV) that expresses enhanced green fluorescent protein (EGFP) under control of the native immediate-early 1/3 promoter was constructed to detect directly sites of viral activity in latent and reactivated infections. The recombinant virus had acute and latent infection characteristics similar to those of wild-type mCMV. Rare green-fluorescing foci were observed in paraffin sections from lungs and spleens infected latently. Positive immunoperoxidase staining for EGFP in sections of the same lung tissues suggests that these cells may be sites of restricted viral gene expression. EGFP was detected easily in tissue explants reactivating from latent infection in vitro. Morphology and adhesion characteristics of fluorescing cells suggest that viral reactivation occurs in tissue macrophages in explant cultures. The observations presented in this study demonstrate the usefulness of EGFP-expressing recombinants as tools for direct tracking of mCMV activity in vivo and in vitro.

Pleuropneumonia caused by Actinobacillus pleuropneumoniae biotype 2 in growing and finishing pigs.

Actinobacillus pleuropneumoniae biotype 2 was isolated in pure culture or as the predominant isolate from the lungs of 9 growing and finishing pigs with pleuropneumonia. Gross and microscopic lesions resembled those caused by A. pleuropneumoniae biotype 1 serotypes (Nos. 1, 5, and 7) traditionally seen in the United States. The overall mortality rate for growing and finishing pigs on this 1,200-sow farrow-to-finish farm ranged from 0.37% to 0.84% per month from July 1990 to February 1991, and mortality due to respiratory disease ranged from 0.17% to 0.52% per month for the same period. This Actinobacillus species did not require V factor (no satellitism on blood agar with a Staphylococcus streak), was strongly beta-hemolytic, and demonstrated restriction fragment length polymorphisms in hybridization studies with A. suis, A. lignieresii, and A. equuli. Biochemically, the isolate most closely resembled A. pleuropneumoniae, and a DNA fragment considered specific for A. pleuropneumoniae biotypes 1 and 2 was demonstrated using polymerase chain reaction. Necrohemorrhagic pleuropneumonia similar to that caused by A. pleuropneumoniae biotype 1 was reproduced experimentally in 2 4-week-old pigs inoculated intratracheally with broth cultures of the A. pleuropneumoniae biotype 2. This study demonstrated the presence of A. pleuropneumoniae biotype 2 in the United States.

Mortality in swine herds endemically infected with Haemophilus pleuropneumoniae: effect of immunization with cross-reacting lipopolysaccharide core antigens of Escherichia coli.

The benefit of increased immunity to cross-reacting lipopolysaccharide core antigens of gram-negative bacteria induced by vaccination with an Rc mutant of Escherichia coli 0111:B4 (strain J5) was evaluated in commercial swine herds endemically infected with Haemophilus pleuropneumoniae. Weanling pigs were vaccinated IM with E coli J5 (group 1) before the expected time of H pleuropneumoniae infection. Clinical signs, antibiotic treatment frequency, mortality, growth performance (days to market weight), and serologic responses of the pigs were monitored for approximately 5 months after vaccination. The results were compared with those of pigs vaccinated IM with a commercial H pleuropneumoniae bacterin (group 2) and with those of nonvaccinated control pigs of the same age (group 3). The treatment frequency and growth performance were similar in the 3 groups. However, vaccination with E coli J5 or with the H pleuropneumoniae bacterin lowered mortality, compared with mortality in the controls. Serum titers against E coli J5 increased after vaccination with the E coli J5 bacterin, but were not increased by vaccination with the H pleuropneumoniae. In contrast, serum titer to E coli J5 increased in all treatment groups as a result of H pleuropneumoniae infection or exposure. The protection against lethal H pleuropneumoniae infections in swine that was provided by vaccination with the E coli J5 and the H pleuropneumoniae bacterin appeared to be immunologically distinct on the basis of serologic analysis, indicating the possibility of different mechanisms of protection.

Clinical observations on eperythrozoonosis.

Eperythrozoonosis was diagnosed in 23 herds of swine. Icterus and anemia were the major diagnostic criteria, with low packed cell volumes confirming the clinical impression of anemia. Indirect hemagglutination titers provided an indirect measure of infection rates and appeared to correlate positively with severity of signs. Treatment with oxytetracycline and arsanilic acid controlled the disease, but only when combined with efforts to limit transmission of the causative organism through control of lice.

Interactions of management and animal performance in a swine feedlot.

The factors of feed efficiency, growth rate, carcass trim at slaughter, amount of antibiotic treatment, and death rate were compared among groups of pigs fed ("finished") in an open-front feedlot in Kansas. The factor having the greatest influence on performance was the month of entry of the pigs into the feedlot. Carcass trim at slaughter was greater in groups wherein each pig was given more than 4 ml of antibiotic than in groups wherein less than 4 ml of antibiotic was given to each pig. Death rates of pigs that remained in the feedlot longer than 150 days were nearly twice those of pigs that had been sent to slaughter before 150 days.

Effects of supplemental vitamin D3 on serum 25-hydroxycholecalciferol and growth of preweaning and nursery pigs.

Four experiments were conducted to investigate the effects of varying concentrations of supplemental vitamin D3 on pig growth, feed preference, serum 25-hydroxycholecalciferol [25(OH)D3] , and bone mineralization of nursing and weanling pigs. In Exp. 1, 270 pigs (1.71 ± 0.01 kg BW) were administered 1 of 3 oral vitamin D3 dosages (none, 40,000, or 80,000 IU vitamin D3) on d 1 or 2 of age. Increasing oral vitamin D3 increased serum 25(OH)D3 on d 10 and 20 (quadratic, P < 0.01) and d 30 (linear, P < 0.01). No differences were observed in ADG before weaning or for nursery ADG, ADFI, or G:F. Vitamin D3 concentration had no effect on bone ash concentration or bone histological traits evaluated on d 19 or 35. In Exp. 2, 398 barrows (initially 7 d of age) were used in a 2 × 2 split plot design to determine the influence of vitamin D3 before (none or 40,000 IU vitamin D3 in an oral dose) or after weaning (1,378 or 13,780 IU vitamin D3/kg in nursery diets from d 21 to 31 of age) in a 45-d trial. Before weaning (7 to 21 d of age), oral vitamin D3 dose did not influence growth but increased (P < 0.01) serum 25(OH)D3 at weaning (d 21) and tended (P = 0.08) to increase 25(OH)D3 on d 31. Increasing dietary vitamin D3 concentration from d 21 to 31 increased (P < 0.01) serum 25(OH)D3 on d 31. Neither the oral vitamin D3 dose nor nursery vitamin D3 supplements influenced nursery ADG, ADFI, or G:F. In Exp. 3, 864 pigs (initially 21 d of age) were allotted to 1 of 2 water solubilized vitamin D3 treatments (none or 16,516 IU/L vitamin D3 provided in the drinking water from d 0 to 10) in a 30-d study. Providing vitamin D3 increased serum 25(OH)D3 concentrations on d 10, 20, and 30; however, vitamin D3 supplementation did not affect overall (d 0 to 30) ADG, ADFI, or G:F. In Exp. 4, 72 pigs were used in a feed preference study consisting of 2 feed preference comparisons. Pigs did not differentiate diets containing either 1,378 or 13,780 IU vitamin D3/kg but consumed less (P < 0.01) of a diet containing 44,100 IU vitamin D3/kg compared with the diet containing 1,378 IU vitamin D3/kg. Overall, these studies demonstrate that supplementing vitamin D3 above basal concentrations used in these studies is effective at increasing circulating 25(OH)D3, but the supplement did not influence growth or bone mineralization. Also, concentrations of vitamin D3 of 44,100 IU/kg of the diet may negatively affect feed preference of nursery pigs.

Detection of two porcine circovirus type 2 genotypic groups in United States swine herds.

In late 2005, sporadic cases of an acute onset disease of high mortality were observed in 10- to 16-week-old growing pigs among several swine herds of the United States. Tissues from the affected pigs in Kansas, Iowa, and North Carolina were examined, and porcine circovirus type 2 (PCV2) was detected consistently among these tissues. Phylogenetically, PCV2 can be divided into two major genotypic groups, PCV2-group 1 and PCV2-group 2. Whereas PCV2-group 1 isolates were detected in all the diseased animals, only two of the diseased animals harbored PCV2-group 2 isolates. This observation is important because PCV2-group 1 isolates had never been reported in the United States before (GenBank as of May 16, 2006), and they are closely related to the PCV2-group 1 isolates that have been described in Europe and Asia, previously. Our analysis revealed that each genotypic group contains a distinct stretch of nucleotide or amino acid sequence that may serve as a signature motif for PCV2-group 1 or PCV2-group 2 isolates.

Detection of murine cytomegalovirus immediate early 1 transcripts in the spleens of latently infected mice.

The spleens of mice latently infected with murine cytomegalovirus were examined by reverse transcription followed by the polymerase chain reaction to assay for transcriptional activity of the viral immediate early 1 gene. Transcripts were detected in 6 of 8 animals at a level between 50 and 500 copies in 200 ng of total cellular RNA. This level appears to be roughly 10-fold higher than that of viral genome even though viral DNA is more readily detectable. These data suggest that a significant fraction of latent murine cytomegalovirus is transcriptionally active in the spleen, which may be indicative of an abortive infection or possibly low-level persistence or a transient reactivation.

Stones in orthotopic, non-obstructing ureteroceles.

A case is presented in which stones were found bilaterally in orthotopic, non-obstructing ureteroceles in a child. This is apparently the first reported instance of this condition occurring in the presence of sterile urine. A review of related literature gives clues to the possible etiology of such an occurrence.

Three-dimensional configuration of the mitochondria in cultured heart cells.

The mitochondria of chick heart cells grown in monolayer culture occupy a relatively large percentage (30%) of the cell cytoplasmic volume, as determined by thin section morphometry, and vary in their ultrastructural configuration in response to different functional states of the cell. The present study was undertaken to determine the three-dimensional structure of the mitochondria in cardiac cells, since reconstruction experiments have demonstrated the existence of a mitochondrial network in cardiac and other tissues, e.g. diaphragm, soleus and vastus muscle, kidney and urinary bladder, in vivo. Confluent monolayers of synchronously beating embryonic chick heart cells were fixed, stained with a mitochondria-specific heavy metal complex (Pb-Cu citrate), and processed for either transmission electron microscopy (TEM) or scanning electron microscopy (SEM) and backscatter electron imaging (BSI). Both secondary and backscatter images revealed the presence of many thin, extremely elongate structures which in stereo views appeared as branching, anastomosing networks. TEM demonstrated localization of the electron dense stain to either nuclear or mitochondrial membranes, structures which were easily distinguished with SEM or BSI alone. These morphological results indicate that many mitochondria of the cultured heart muscle cell are interconnected and suggest that this morphological network may represent a parallel functional unit which maintains the energy state of the cell.

Expression of a murine cytomegalovirus early-late protein in "latently" infected mice.

Monoclonal antibodies were isolated from the spleen of a latently infected mouse to identify possible antigenic markers for latent murine cytomegalovirus infection. One antibody, AM3, was selected for further study. Characterization of the antigen recognized by AM3 confirmed that it is the early-late phosphoprotein pp50. The antigen was readily detected by immunoautoradiography in the spleens of acutely and latently infected mice. Low levels of the AM3/pp50 transcript were also detected in latently infected tissues by in situ hybridization. Presence of AM3/pp50 mRNA, as well as IE-1 transcripts, was confirmed by reverse transcription-polymerase chain reaction in 3 of 5 latently infected spleens. The expression of both immediate-early and early-late classes of viral genes suggests that persistent infection may be a component of the MCMV life cycle operationally defined as latency and that this gene is not specific for latency.

The murine cytomegalovirus M25 open reading frame encodes a component of the tegument.

The murine cytomegalovirus (MCMV) monoclonal antibody 5C7:6 was used in Western analysis to probe MCMV infected murine embryo cells (MEC). This antibody recognizes three virus specific polypeptides of 130, 105, and 95 kDa and pulse-chase experiments demonstrated that these three proteins, although antigenically related, are distinct. The 105- and 95-kDa species were expressed with early kinetics, whereas the 130-kDa protein was synthesized as a true late. By screening a lambdagt11 MCMV cDNA library, the gene encoding these proteins was identified as the M25 open reading frame previously reported by Dallas et al. (Dallas, P. B., Lyons, P. A., Hudson, J. B., Scalzo, A. A., and Shellam, G. R., 1994, Virology 200, 643-650). Immunofluorescent studies monitored the location of pM25, present in the nucleus at 15 h after infection, condensing around the periphery of the nucleus at 18 h, before finally accumulating in the cytoplasm. Immunoelectron microscopy detected gold particles associated with the viral tegument of enveloped virions located in the cytoplasm and extracellular space but not with naked nucleocapsids. Western analysis of MCMV purified virions depicted the presence of the 130-kDa protein, the predominant M25 species, in mature virus particles. Together these findings provide compelling evidence that the 130-kDa M25 polypeptide is a component of the viral tegument.

Protection against Mycobacterium avium by DNA vaccines expressing mycobacterial antigens as fusion proteins with green fluorescent protein.

Mycobacterium avium causes disseminated disease in humans with AIDS, paratuberculosis in ruminants, lymphadenopathy in swine, and tuberculosis in birds. We constructed DNA vaccines expressing mycobacterial antigens as fusion proteins with enhanced green fluorescent protein (EGFP). Plasmids p65K-EGFP, p85A-EGFP, and p85B-EGFP expressed the M. avium 65-kDa antigen, the Mycobacterium bovis BCG 85A antigen, and the M. avium 85B antigen, respectively, as EGFP fusion proteins. We visualized protein expression directly in cultured murine fibroblasts and intact muscle. p65K-EGFP expressed fusion protein in a diffuse cytoplasmic pattern, and p85A-EGFP and p85B-EGFP produced a speckled pattern. We vaccinated C57BL/6 mice with three doses of plasmid DNA and then challenged them intraperitoneally with M. avium. Negative controls received saline, and positive controls received one dose of BCG vaccine. Mice in all groups developed disseminated infection with a high burden of organisms. Compared to negative controls, mice vaccinated with p85A-EGFP had an eightfold reduction in spleen M. avium CFU at 4 weeks after infection and a fourfold reduction at 8 weeks, reductions similar to those generated by BCG vaccine. Mice vaccinated with p65K-EGFP had a fourfold CFU reduction at 4 weeks and no effect at 8 weeks. This is the first report of DNA vaccines expressing foreign antigens as fusion proteins with EGFP and the first report of successful DNA vaccination against M. avium.

Detection of mouse cytomegalovirus nucleic acid in latently infected mice by in vitro enzymatic amplification.

Transmission of human and murine cytomegalovirus (CMV) with transfusions and organ transplantation suggests that the latent virus is located in multiple organs and perhaps multiple cell types. The direct identification and localization of the latent virus in the normal host has been difficult using standard culture and hybridization techniques. In vitro amplification using the polymerase chain reaction followed by oligonucleotide hybridization can be used to detect murine CMV DNA. When this method was applied to DNA extracted from latently infected mice, it allowed detection of viral nucleic acid not detected by standard Southern hybridization. The results of these studies support the presence of latent murine CMV in multiple organs including the salivary gland, spleen, and kidney. Amplification and detection of viral DNA in purified renal tubule preparations suggest that this may be a site of viral latency and potential source of the virus during renal transplantation.