Osmolality of fortified donor human milk: An experimental study.

BACKGROUND: We quantify the osmolality of human milk fortified with human milk fortifiers (HMFs), powder infant formulas and protein additives. METHODS: Commercial liquid HMFs and powder infant formulas were added to pasteurized pooled donor human milk in triplicate and stirred. The osmolality of unfortified and fortified human milk at 22, 24, 26, 27, 28, and 30 kcal/oz (0.73, 0.8, 0.87, 0.9, 0.93, and 1 kcal/ml, respectively) was determined using freezing-point depression. RESULTS: The osmolality of fortified human milk associated with energy density in a linear relationship regardless of the fortification strategies. Multiple liquid HMFs and every powder infant formula exceeded the osmolality threshold of 450 mOsm/kg H2 O within the energy densities tested. CONCLUSION: The osmolality of fortified human milk is highly variable and should be considered when selecting a fortifying agent for human milk.

Nutrient Composition of Donor Human Milk and Comparisons to Preterm Human Milk.

BACKGROUND: Human milk is the preferred diet for very low birth weight (VLBW, <1500 g) infants. When mother's own milk is unable to meet the needs of VLBW infants, donor human milk (DHM) is the preferred alternative. Unfortunately, the composition of DHM remains elusive and no comparative studies between preterm human milk and DHM have been performed previously. OBJECTIVES: We aimed to analyze the nutrient content of commercial pooled DHM and compare nutrient content in DHM with that of early and mature preterm human milk. METHODS: We analyzed nutrient content in 15 DHM samples provided from 7 commercial milk banks including calories, carbohydrate, fat, protein, sodium, chloride, potassium, zinc, calcium, phosphorus, magnesium, and vitamin D and compared each nutrient to early (7 d of life) and mature (28 d of life) preterm human milk samples (n = 28-36 per nutrient, gestational age = 28 ± 3 wk). Protein-to-energy ratio and carbohydrate-to-nonprotein energy ratio were calculated for each sample and compared. RESULTS: Mean values for all macro- and micronutrients in DHM are reported. In comparison to early or mature preterm human milk, DHM had significantly lower protein, sodium, chloride, potassium, and zinc content. Calorie, carbohydrate, calcium, phosphorus, magnesium, and vitamin D content did not differ statistically between DHM and early or mature preterm human milk. Fat content was modestly lower in early but not mature human milk when compared with DHM. CONCLUSIONS: We provide mean values for several macro- and micronutrients for DHM and identify key differences between DHM and preterm human milk, which may be considered when designing human milk-based feeding plans. This study was registered at clinicaltrials.gov as NCT05742815.

Displacement of human milk during fortification: An experimental study.

BACKGROUND: This study quantified the displacement of human milk by commercial human milk fortifiers (HMFs) and infant formulas. METHODS: Commercial liquid HMFs and powder infant formulas were added to pasteurized pooled donor human milk in triplicate, stirred, and weighed. The difference in weight between unfortified and fortified human milk at 22, 24, 26, 27, 28, and 30 kcal/ounce was calculated. RESULTS: The displacement of human milk by liquid HMFs and powder infant formulas and powder HMF was highly associated with energy density. A human milk-derived HMF displaced significantly more human milk when compared with bovine milk-derived HMFs at equivalent energy densities. Similarly, powder infant formulas displaced less human milk when compared with a powder HMF, and the addition of hydrolyzed powder infant formulas resulted in less human milk displacement when compared with nonhydrolyzed powder infant formulas. CONCLUSIONS: The displacement of human milk by commercial liquid HMFs and infant formulas must be considered when selecting a fortifying strategy.

Acid/base balance in fortified donor human milk: An experimental study.

BACKGROUND: Our goal was to quantify the pH and total acidity of human milk fortified with human milk fortifiers (HMFs), powder infant formulas, and protein additives. METHODS: Commercial liquid HMFs and powder infant formulas were added to pasteurized pooled donor human milk in triplicate and stirred. The pH of unfortified and fortified human milk at 22, 24, 26, 27, 28, and 30 kcal/ounce (624, 680, 737, 765, 794, and 850 kcal/g, respectively) was determined using a pH meter. Phenolphthalein acidity at 24 and 30 kcal/ounce (680 and 850 kcal/g, respectively) was determined using diluted sodium hydroxide. RESULTS: The pH of unfortified human milk increased within the first hour (6.52 ± 0.06 vs 6.62 ± 0.05, P < 0.0001). Changes in pH largely correlated with caloric density; however, directional changes varied considerably between HMFs and powder infant formulas. Two liquid HMFs demonstrated modest reductions in pH with increasing caloric density whereas one liquid HMF alkalinized human milk with increasing caloric density (analysis of variance P < 0.0001). Phenolphthalein acidity was significantly higher for five HMFs and lower for one HMF at 30 kcal/ounce (850 kcal/g) but not 24 kcal/ounce (680 kcal/g). Powder infant formulas generally increased pH with increasing caloric density (analysis of variance P < 0.0001), but no differences in phenolphthalein acidity were noted. CONCLUSION: Changes in acid/base balancefor fortified human milk are variable and may be a consideration when selecting a fortifying agent for human milk.

Perceptions of the impact of a multidose human-milk fortifier on human-milk preparation practices in United States neonatal intensive care units: A survey of nutrition care team members.

BACKGROUND: In 2020, a multidose human-milk fortifier (MDHMF) was designed to improve the process of human-milk (HM) fortification. The bottle of MDHMF (5.5 oz, 163 ml) allows aseptic removal of HMF in a precise measure. This survey aimed to examine the experience of nutrition care team (NCT) members who used the MDHMF in a hospital setting. METHODS: A survey link (Qualtrics XM) was sent to NCT leaders (N = 108) at hospitals who participated in an evaluation of the MDHMF from June 1, 2020, through April 30, 2021. The NCT leaders sent the survey to members at their prospective hospitals (n = 344). The investigators did not know the identities of the recipients of the survey and collected no identifying information on respondents. Respondents were asked to evaluate their experience with the MDHMF compared with their previous practice. RESULTS: The majority of respondents (n = 63, 72%) reported that the MDHMF improved their HM preparation practices and was better than their previous practice for reducing the time to prepare (n = 33, 71.7%), ensuring the accuracy of fortified HM (n = 32, 69.6%), ensuring aseptic preparation (n = 24, 52.2%), reducing HM waste (n = 27, 58.7%), and being easy to use (n = 30, 65.2%). Those responsible for evaluating nutrition status answered that the MDHMF was the same for feeding tolerance (n = 41, 58.6%), weight gain (n = 47, 67.1%), head growth (n = 56, 81.2%), and length growth (n = 53, 76.8%). CONCLUSION: US neonatal intensive care unit NCT members perceived that the MDHMF resulted in improved HM preparation practices while maintaining growth and tolerance.

Novel multinutrient human milk-based human milk fortifier promotes growth and tolerance in premature infants.

BACKGROUND: The objective of this study was to determine whether human milk supplemented with a novel human milk-based human milk fortifier (Novel HMF), compared with a bovine milk-based HMF (Bovine HMF), supports preterm infant growth through 36 weeks' postmenstrual age (PMA). METHODS: This single-center, prospective trial compared growth and nutrition outcomes of preterm infants provided a human milk-based diet (mother's own milk or donor milk) supplemented with a Novel HMF with historic controls provided Bovine HMF. Preterm infants with an estimated gestational age (EGA) between 23 and 33 weeks' PMA and birth weight between 750 and 1800 g were eligible for study inclusion. Weight, length, and head circumference (HC) were monitored weekly. The occurrence of late-onset sepsis, nil per os (NPO) days, necrotizing enterocolitis, metabolic acidosis, and serious adverse events were monitored. RESULTS: Birth weight, length, HC, and EGA were similar between the Novel HMF (n = 37) and Bovine HMF (n = 49) groups. The days to regain birth weight was shorter in the Novel HMF group (9.4 ± 4.0 vs 11.4 ± 4.8, P = .0343), with similar weight gain (g/day) from birth to 36 weeks' PMA. Adjusted weight growth velocity (g/kg/day) was significantly higher in the Novel HMF group at 14 and 21 days but similar at 36 weeks' PMA. The Novel HMF group experienced fewer NPO days with a similar total number of feeding days. CONCLUSIONS: A novel, multinutrient, human milk-based HMF is well tolerated and meets the nutrition needs of preterm infants.

Nutrient composition of preterm mother's milk and factors that influence nutrient content.

BACKGROUND: Breast milk feedings are the optimal feeding choice for premature infants. Clinicians depend on accurate nutrient profiles of the breast milk in order to make informed decisions regarding the need for nutrient supplementation. Existing data for nutrient composition of preterm breast milk are dated and not representative of the current population of women delivering prematurely in the United States. OBJECTIVES: The purpose of this prospective, longitudinal, single-center observational study was to measure the macronutrient and micronutrient composition of breast milk expressed by mothers, including women who self-identify as black, delivering preterm infants at ≤33 completed weeks of gestation. METHODS: We collected breast milk samples from mothers of preterm infants admitted to the neonatal intensive care unit at Augusta University Medical Center from January 2019 through November 2019. Mother's milk samples were collected on postpartum days 7, 14, 21, and 28 and analyzed for macronutrients (energy, fat, protein, and carbohydrates) and micronutrients (sodium, potassium, chloride, calcium, phosphorus, magnesium, vitamin D, and zinc). RESULTS: Thirty-eight mothers, mean age 27 ± 5.1 y and majority black (66%), provided milk for the study. The mean estimated gestational age and birth weight were 28.2 ± 2.8 weeks of gestation and 1098 ± 347 g, respectively, with 42% of mothers in the cohort delivering before week 28 of pregnancy. Differences in protein, sodium, potassium, calcium, phosphorus, and zinc concentrations based on race, day, and milk volume were identified. Dilution effects for protein, sodium, chloride, and vitamin D concentrations over time were identified. CONCLUSIONS: Our study is among the first to characterize breast milk composition from women who delivered extremely preterm infants and adds to the evidence that race, gestational age, and volume influence the composition of preterm mother's milk. These factors should be considered when designing mother's milk-based feeds for premature infants.

Review of Preterm Human-Milk Nutrient Composition.

BACKGROUND: The human milk-fed preterm infant is at risk for growth failure, micronutrient deficiencies, and neurocognitive delay. Although protective and better tolerated than formula, human milk alone cannot meet the high nutrient requirements of this population, and fortification is necessary. Clinicians use assumptions of preterm human-milk composition to determine the type and quantity of fortification. OBJECTIVES: The objectives of this review were to identify evidence of macronutrient and micronutrient concentration in preterm human milk and to identify knowledge gaps regarding composition. METHODS: PubMed and the Cumulative Index to Nursing and Allied Health Literature were used to identify original articles published between January 1950 and December 2019. RESULTS: Twenty-seven articles were found containing original data on macronutrients and micronutrients. Most (67%) of the studies published after 2011 measured the macronutrients and included gestational ages from 28 to 36 weeks. Milk collection methods, experimental design, and analytical methods varied between studies. There are 15 countries represented in this review; all of the American studies (n = 7) were published from 1980 to 1984. CONCLUSIONS: African American women, or women delivering before 28 weeks' gestation are not represented in the literature. Accurate and targeted human-milk fortification depends on comprehensive, complete, and representative human-milk nutrient data. We have aggregated all available preterm human-milk macronutrient and micronutrient data and reported trends associated with lactation stage and gestational age. This report can aid in the design of feeding plans that are appropriate for the gestational age of the preterm infant and the lactation stage of the breastmilk.

Attitudes and Behavior towards Interprofessional Collaboration among Healthcare Professionals in a Large Academic Medical Center.

The increasing rates of comorbidities among patients and the complexity of care have warranted interprofessional collaboration (IPC) as an important component of the healthcare structure. An initial step towards assessing the effectiveness of collaboration requires the exploration of the attitudes and experience of healthcare professionals towards IPC. This online survey aimed to examine the attitudes of healthcare professionals working in a large public academic medical center toward IPC in patient care and the healthcare team, and their behavior and experience regarding IPC. The rankings, according to the perceived importance among the respondents, of the four Interprofessional Education Collaborative (IPEC) core competencies (values/ethics, roles/responsibilities, interprofessional communication, teams/teamwork) were assessed. There were strong but varying levels of consensus among healthcare professionals (N = 551) that IPC facilitates efficient patient care, improves patient problem-solving ability, and increases better clinical outcomes for patients. They acknowledged that IPC promotes mutual respect within the healthcare team and providers' ability to make optimal patient care decisions. However, overall more than 35% of the respondents did not attend multidisciplinary education sessions (grand rounds, seminars, etc.), and about 23% did not participate in bedside patient care rounds. Interprofessional communication was ranked as the most important IPEC core competence. Although the attitude towards IPC among healthcare professionals is strongly positive, many healthcare professionals face challenges in participating in IPC. Institutional policies that facilitate interprofessional learning and interactions for this group of healthcare professionals should be formulated. Online distance learning and interactions, and simulation-enhanced interprofessional education, are options for addressing this barrier. Hospital administrators should facilitate conducive work environments that promote IPC, based on IPEC core competencies, and promote programs that address the challenges of IPC.

IW-3718 Reduces Heartburn Severity in Patients With Refractory Gastroesophageal Reflux Disease in a Randomized Trial.

BACKGROUND & AIMS: Refractory gastroesophageal reflux disease (GERD) reduces quality of life and creates significant financial burden on the health care system. Approximately 30% of patients with GERD who receive label-dose proton pump inhibitors (PPIs) still have symptoms. We performed a trial to evaluate the efficacy and safety of IW-3718, a bile acid sequestrant, as an adjunct to PPI therapy. METHODS: We performed a multicenter, double-blind, placebo-controlled trial, from March 2016 through April 2017, of 280 patients with confirmed GERD. The patients, stratified by esophagitis status, were randomly assigned (1:1:1:1) to groups given placebo or IW-3718 (500, 1000, or 1500 mg) twice daily, with ongoing label-dose PPI. The primary endpoint was percent change from baseline to week 8 in weekly heartburn severity score. We also analyzed percent change from baseline to week 8 in weekly regurgitation frequency score. RESULTS: Mean changes from baseline to week 8 in weekly heartburn severity scores were reductions of 46.0% in the placebo group, 49.0% in the 500 mg group, 55.1% in the 1000 mg group, and 58.0% in the 1500 mg IW-3718 group (dose-response P = .02). The treatment difference was 11.9% between the 1500 mg IW-3718 and placebo groups (P = .04, analysis of covariance). The mean change in weekly regurgitation frequency score from baseline to week 8 in the 1500 mg IW-3718 vs placebo groups was a reduction of 17.5% (95% confidence interval, reductions of 31.4% to 3.6%). The most common adverse event was constipation (in 8.1% of patients receiving IW-3718 and 7.1% of patients receiving placebo). There were no drug-related serious adverse events. CONCLUSIONS: In a randomized trial of patients with refractory GERD, adding 1500 mg IW-3718 to label-dose PPIs significantly reduced heartburn symptoms compared with adding placebo. Regurgitation symptoms also decreased. IW-3718 was well tolerated. (ClinicalTrials.gov, Number: NCT02637557).

Interprofessional Education and Collaboration in Healthcare: An Exploratory Study of the Perspectives of Medical Students in the United States.

Qualified and competent healthcare professionals working in a collaborative team environment is a prerequisite for high quality patient care. In order to be successful in the healthcare working environment, medical students need to be exposed to interprofessional learning early in their education. A single stage online survey was administered to medical students to evaluate their attitudes and perceptions of interprofessional education (IPE) and whether prior exposure to IPE increased their appreciation for interprofessional collaboration. The results suggest that irrespective of prior exposure to IPE, medical students appreciated the importance of interprofessional education and collaboration. Medical students showed a strong interest in attending interprofessional courses in other disciplines. Time constraints, scheduling conflicts, and communication emerged as barriers to IPE. Medical students embraced IPE and welcomed the opportunity to learn with other disciplines. Clinical case studies and simulations were identified as potential methods to integrate with other healthcare disciplines. The positive attitude and perceptions of the medical students toward interprofessional education and collaboration warrants the inclusion of related courses in medical curricula, as this may further increase students' potentials in becoming effective healthcare providers.

Lyplal1 is dispensable for normal fat deposition in mice.

Genome-wide association studies (GWAS) have detected association between variants in or near the Lysophospholipase-like 1 (LYPLAL1) locus and metabolic traits, including central obesity, fatty liver and waist-to-hip ratio. LYPLAL1 is also known to be upregulated in the adipose tissue of obese patients. However, the physiological role of LYPLAL1 is not understood. To investigate the function of Lyplal1 in vivo we investigated the phenotype of the Lyplal1tm1a(KOMP)Wtsi homozygous mouse. Body composition was unaltered in Lyplal1 knockout mice as assessed by dual-energy X-ray absorptiometry (DEXA) scanning, both on normal chow and on a high-fat diet. Adipose tissue distribution between visceral and subcutaneous fat depots was unaltered, with no change in adipocyte cell size. The response to both insulin and glucose dosing was normal in Lyplal1tm1a(KOMP)Wtsi homozygous mice, with normal fasting blood glucose concentrations. RNAseq analysis of liver, muscle and adipose tissue confirmed that Lyplal1 expression was ablated with minimal additional changes in gene expression. These results suggest that Lyplal1 is dispensable for normal mouse metabolic physiology and that despite having been maintained through evolution Lyplal1 is not an essential gene, suggesting possible functional redundancy. Further studies will be required to clarify its physiological role.

Opposing roles of prelimbic and infralimbic dopamine in conditioned cue and place preference.

RATIONALE: Increasing evidence points to the prelimbic (PL) and infralimbic (IL) cortices of the medial prefrontal cortex (mPFC) and their dopaminergic innervations subserving opposing roles in the regulation of instrumental behavior. However, it is at present unclear if they hold similar roles in the regulation of Pavlovian learning. OBJECTIVE: The present study investigated the role of the dopaminergic innervations of the PL and IL in the modulation of Pavlovian appetitive cue and place conditioning, previously shown to be dependent on the basolateral amygdala and hippocampus, respectively. METHODS: Rats received preconditioning microinfusions of D-amphetamine, cis-flupenthixol, or vehicle solution directly into the PL or IL and were trained to simultaneously acquire conditioned cue and place preference in a radial maze. RESULTS: Preconditioning blockade of dopamine neurotransmission in the PL and amphetamine microinfusions in the IL had the same effect of attenuating place conditioning. In contrast, place conditioning remained intact following preconditioning amphetamine microinfusions in the PL and dopamine receptor blockade in the IL. Instead, conditioned cue preference was attenuated following IL dopamine receptor blockade. CONCLUSION: These data indicate that PL dopaminergic mechanisms are critical for the acquisition of appetitive place learning, while IL dopamine may oppose the influence of PL dopamine upon hippocampal-dependent learning. Furthermore, they implicate a functional reciprocity between mPFC and associated subregions of the nucleus accumbens in the regulation of limbic information processing.

Immediate metabolic consequences of intrauterine growth restriction and low birthweight.

Optimal fetal growth resulting in a 'normally grown' term infant is of paramount importance for assuring a healthy start for postnatal growth and development. Fetal, infant and childhood growth restriction is an important clinical problem for obstetricians, neonatologists, pediatricians and globally, for public health. Worldwide, an estimated 20 million infants are born with low birthweight and a substantial proportion are small for gestational age. Many advances have been made in defining growth restriction by prenatal techniques, thus allowing the recognition of intrauterine growth restriction. Distinguishing infants who are small but have appropriate growth potential from those with growth restriction is important in order to apply obstetric surveillance, anticipate neonatal problems and plan for postneonatal guidance. It is clear that the fetus in growth-restricted pregnancies has limited supply of nutrients and oxygen. The resultant changes, if involving the placenta as well, can lead to circulatory and metabolic changes affecting both short- and long-term survival and development. In this paper, the causes and immediate consequence of being born with low birthweight, intrauterine growth restriction or small for gestational age will be discussed.

Bone density in peripubertal boys with autism spectrum disorders.

We determined whether bone mineral density (BMD) is lower in boys with autism spectrum disorders (ASD) than controls, and also assessed variables that may affect BMD in ASD. BMD was measured using dual energy X-ray absorptiometry (DXA) in 18 boys with ASD and 19 controls 8-14 years old. Boys with ASD had lower BMD Z-scores at the spine, hip and femoral neck, and differences at the hip and femoral neck persisted after controlling for maturity and BMI. Vitamin D intake from food and in serum were lower in ASD subjects, as was exercise activity. We conclude that BMD is lower in peripubertal boys with ASD and may be associated with impaired vitamin D status and lower exercise activity.

Effects of sialidase knockout and complementation on virulence of Mycoplasma gallisepticum.

Reannotation of the pathogenic Mycoplasma gallisepticum strain R(low) genome identified the hypothetical gene MGA_0329 as a homolog of the sialidase gene MS53_0199 of Mycoplasma synoviae strain MS53. Potent sialidase activity was subsequently quantitated in several M. gallisepticum strains. Because sialidase activity levels correlate significantly with differing M. synoviae strain virulence, we hypothesized this enzyme may also influence the virulence of M. gallisepticum. MGA_0329 was disrupted in strain R(low) to create mutants 6, 358 and P1C5, which resulted in the loss of sialidase activity in all three mutants. Chickens infected with the knockout mutants had significantly less severe (P<0.05) tracheal lesions and tracheal mucosal thickening than chickens infected with equal doses of strain R(low). Significantly fewer (P<0.05) CCU especially of strains 6 and P1C5 were recovered at necropsy. Mini-Tn4001tet plasmid pTF20 carrying a wild-type copy of MGA_0329 with its native promoter was used to complement the genetic lesion in strain P1C5. Three clones derived from P1C5, each having one copy of MGA_0329 stably transposed into a different site in its genome, expressed sialidase restored to wild-type activity levels (1.58×10(-8)U/CFU). Complementation of P1C5 with MGA_0329 did not restore it to wild-type levels of virulence, indicating that the contribution of sialidase to M. gallisepticum virulence is not straightforward.

Assessment of inactivating stop codon mutations in forty Saccharomyces cerevisiae strains: implications for [PSI] prion- mediated phenotypes.

The yeast prion [PSI(+)] has been implicated in the generation of novel phenotypes by a mechanism involving a reduction in translation fidelity causing readthrough of naturally occurring stop codons. Some [PSI(+)] associated phenotypes may also be generated due to readthrough of inactivating stop codon mutations (ISCMs). Using next generation sequencing we have sequenced the genomes of two Saccharomyces cerevisiae strains that are commonly used for the study of the yeast [PSI(+)] prion. We have identified approximately 26,000 and 6,500 single nucleotide polymorphisms (SNPs) in strains 74-D694 and G600 respectively, compared to reference strain S288C. In addition to SNPs that produce non-synonymous amino acid changes we have also identified a number of SNPs that cause potential ISCMs in these strains, one of which we show is associated with a [PSI(+)]-dependent stress resistance phenotype in strain G600. We identified twenty-two potential ISCMs in strain 74-D694, present in genes involved in a variety of cellular processes including nitrogen metabolism, signal transduction and oxidative stress response. The presence of ISCMs in a subset of these genes provides possible explanations for previously identified [PSI(+)]-associated phenotypes in this strain. A comparison of ISCMs in strains G600 and 74-D694 with S. cerevisiae strains sequenced as part of the Saccharomyces Genome Resequencing Project (SGRP) shows much variation in the generation of strain-specific ISCMs and suggests this process is possible under complex genetic control. Additionally we have identified a major difference in the abilities of strains G600 and 74-D694 to grow at elevated temperatures. However, this difference appears unrelated to novel SNPs identified in strain 74-D694 present in proteins involved in the heat shock response, but may be attributed to other SNP differences in genes previously identified as playing a role in high temperature growth.

Multiplex target enrichment using DNA indexing for ultra-high throughput SNP detection.

Screening large numbers of target regions in multiple DNA samples for sequence variation is an important application of next-generation sequencing but an efficient method to enrich the samples in parallel has yet to be reported. We describe an advanced method that combines DNA samples using indexes or barcodes prior to target enrichment to facilitate this type of experiment. Sequencing libraries for multiple individual DNA samples, each incorporating a unique 6-bp index, are combined in equal quantities, enriched using a single in-solution target enrichment assay and sequenced in a single reaction. Sequence reads are parsed based on the index, allowing sequence analysis of individual samples. We show that the use of indexed samples does not impact on the efficiency of the enrichment reaction. For three- and nine-indexed HapMap DNA samples, the method was found to be highly accurate for SNP identification. Even with sequence coverage as low as 8x, 99% of sequence SNP calls were concordant with known genotypes. Within a single experiment, this method can sequence the exonic regions of hundreds of genes in tens of samples for sequence and structural variation using as little as 1 µg of input DNA per sample.

Systemic adenovirus infection in Sulawesi tortoises (Indotestudo forsteni) caused by a novel siadenovirus.

A novel siadenovirus was identified in the Sulawesi tortoise (Indotestudo forsteni). A group of 105 Sulawesi tortoises was obtained by the Turtle Survival Alliance. Many of the tortoises were in poor health. Clinical signs included anorexia, lethargy, mucosal ulcerations and palatine erosions of the oral cavity, nasal and ocular discharge, and diarrhea. Initial diagnostic tests included fecal testing for parasites, complete blood count and plasma biochemical analysis, mycoplasma serology, and polymerase chain reaction (PCR) testing for intranuclear coccidia and chelonian herpesvirus. Treatment included administration of antibiotics, antiparasitic medications, parenteral fluids, and nutritional support. Tissue samples from animals that died were submitted for histopathologic evaluation. Histopathologic examination revealed systemic inflammation and necrosis associated with intranuclear inclusions consistent with a systemic viral infection in 35 tortoises out of 50 examined. Fecal testing results and histopathologic findings revealed intestinal and hepatic amoebiasis and nematodiasis in 31 animals. Two of 5 tortoises tested by PCR were positive for Chlamydophila sp. Aeromonas hydrophila and Escherichia coli were cultured from multiple organs of 2 animals. The mycoplasma serology and PCR results for intranuclear coccidia and chelonian herpesvirus were negative. Polymerase chain reaction testing of tissues, plasma, and choanal/cloacal samples from 41 out of 42 tortoises tested were positive for an adenovirus, which was characterized by sequence analysis and molecular phylogenetic inference as a novel adenovirus of the genus Siadenovirus. The present report details the clinical and anatomic pathologic findings associated with systemic infection of Sulawesi tortoises by this novel Siadenovirus, which extends the known reptilian adenoviruses to the chelonians and extends the known genera of reptilian Adenoviridae beyond Atadenovirus to include the genus Siadenovirus.

Systemic iridovirus infection in the Banggai cardinalfish (Pterapogon kauderni Koumans 1933).

Iridoviruses infect food and ornamental fish species from a wide range of freshwater to marine habitats across the globe. The objective of the current study was to characterize an iridovirus causing systemic infection of wild-caught Pterapogon kauderni Koumans 1933 (Banggai cardinalfish). Freshly frozen and fixed specimens were processed for histopathologic evaluation, transmission electron microscopic examination, virus culture, molecular virologic testing, microbiology, and in situ hybridization (ISH) using riboprobes. Basophilic granular cytoplasmic inclusions were identified in cytomegalic cells often found beneath endothelium, and hexagonal virus particles typical of iridovirus were identified in the cytoplasm of enlarged cells by transmission electron microscopy. Attempts at virus isolation in cell culture were unsuccessful; however, polymerase chain reaction (PCR)-based molecular testing resulted in amplification and sequencing of regions of the DNA polymerase and major capsid protein genes, along with the full-length ATPase gene of the putative iridovirus. Virus gene sequences were then used to infer phylogenetic relationships of the P. kauderni agent to other known systemic iridoviruses from fishes. Riboprobes, which were transcribed from a cloned PCR amplification product from the viral genome generated hybridization signals from inclusions within cytomegalic cells in histologic sections tested in ISH experiments. To the authors' knowledge, this is the first report of a systemic iridovirus from P. kauderni. The pathologic changes induced and the genomic sequence data confirm placement of the Banggai cardinalfish iridovirus in the genus Megalocytivirus family Iridoviridae. The ISH provides an additional molecular diagnostic technique for confirmation of presumptive infections detected in histologic sections from infected fish.