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Summary: The alpaca classic grey phenotype is of particular interest to the industry. Until now, there were only indirect data suggesting that the KIT gene was involved in the classic grey phenotype. All exons of KIT in three black and three classic silvergrey alpacas were sequenced. Five non‐synonymous SNPs were observed. There was only one SNP found that was present only in the silvergrey alpacas, and this was also the only SNP predicted to be damaging. This variant results in a change of a glycine (Gly) to an arginine (Arg) at amino acid position 126 (c.376G>A), occurring in the second Ig‐like domain of the extracellular domain of KIT. Basic protein modelling predicted that this variant is likely destabilising. Therefore, an additional 488 alpacas were genotyped for this SNP using the tetra‐primer amplification refractory mutation system PCR (Tetra‐primer ARMS‐PCR). All classic grey alpacas were observed to be heterozygous, and 99.3% of non‐grey dark base colour alpacas were found to be homozygous for the wildtype allele in this position. These results confirm that the classic grey phenotype in alpacas is the result of a c.376G>A (p.Gly126Arg) SNP in exon 3 of KIT. These data also support the hypothesis that the grey phenotype is autosomal dominant and that the mutation is most likely homozygous lethal. [ABSTRACT FROM AUTHOR]

The coding region of the alpaca Agouti signalling protein (ASIP) gene was sequenced. It was determined to be 402 nucleotides long and code for a protein that is 133 amino acids long. Eight mutations were identified in a sample of 15 alpaca, five in the coding region and three in the introns flanking the exons. In silico analysis showed that three of the five mutations in the coding sequence, c.325_381del57, c.292C>T and c.353G>A are probable loss-of-function mutations. The three mutations were strongly associated with black fibre colour, with 0·90 of black alpacas in the current study having two copies of one or another of the mutations. However, not all black animals displayed the putative ‘aa’ genotype, and almost half of the non-black animals did display that genotype. Contributing factors such as regulatory region mutations, interactions of ASIP with melanocortin-1 receptor (MC1R) and α-melanocyte stimulating hormone (α-MSH), the effect of dilution genes and subjective phenotype assignment are discussed. These mutations will allow alpaca breeders to select for or against black, but they do not explain all black phenotypes in this species. [ABSTRACT FROM PUBLISHER]

The Warehouse beetle, Trogoderma variabile (Coleoptera: Dermestidae), is an internationally significant invasive pest of packed goods and stored grain. When it was first documented in Australia at Griffith, New South Wales, in 1977, an eradication campaign was initiated. After several years and considerable effort, the eradication campaign was abandoned. To monitor the presence and spread of T. variabile, surveys were carried out by government agencies in 1992 and 2002. When survey data was compared, it was concluded that the distribution of morphologically identified T. variabile had doubled in most Australian states. Here, we used samples from the 2002 survey to conduct a phylogenetic study using partial sequences of mitochondrial genes Cytochrome oxidase I and Cytochrome B, and the nuclear gene 18S, to examine the distribution and dispersal of T. variabile and detect the presence of misidentified species. Based on our molecular results, we show that only 47% of the samples analysed were T. variabile, and the remaining were a mixture of six putative species. In addition, T. variabile was found in only 78% of the trapping sites. We discuss the importance of correct diagnosis in relation to the eradication campaign. [ABSTRACT FROM PUBLISHER]

Human cytomegalovirus (HCMV) is carried lifelong by ∼80 % of adults worldwide, generating distinct disease syndromes in transplant recipients, people with HIV (PWH) and neonates. Amino acids 15-23 encoded by the HCMV gene UL40 match positions 3-11 of HLA-A and HLA-C, and constitute a "signal peptide" able to stabilise cell surface HLA-E as a restriction element and a ligand of NKG2A and NKG2C. We present next generation sequencing of UL40 amplified from 15 Australian renal transplant recipients (RTR), six healthy adults and four neonates, and 21 Indonesian PWH. We found no groupwise associations between the presence of multiple sequences and HCMV burden (highest in PWH) or HCMV-associated symptoms in neonates. Homology between UL40 and corresponding HLA-C and HLA-A peptides in 11 RTR revealed perfect matches with HLA-C in three individuals, all carrying HCMV encoding only VMAPRTLIL - a peptide previously associated with viremia. However indices of the burden of HCMV did not segregate in our cohort., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 American Society for Histocompatibility and Immunogenetics. All rights reserved.)

Around 80% of adults worldwide carry human cytomegaloviris (HCMV). The HCMV gene UL18 is a homolog of HLA class I genes and encodes a protein with high affinity for the NK and T-cell cytotoxicity inhibitor LIR-1. UL18 was deep sequenced from blood, saliva or urine from Indonesian people with HIV (PWH) ( n = 28), Australian renal transplant recipients (RTR) ( n = 21), healthy adults ( n = 7) and neonates ( n = 4). 95% of samples contained more than one variant of HCMV UL18 , as defined by carriage of nonsynonymous variations. When aligned with immunological markers of the host's burden of HCMV, the S318N variation associated with high levels of antibody reactive with HCMV lysate in PWH over 12 months on antiretroviral therapy. The A107T variation associated with HCMV antibody levels and inflammatory biomarkers in PWH at early timepoints. Variants D32G, D248N, V250A and E252D aligned with elevated HCMV antibody levels in RTR, while M191K, E196Q and F165L were associated with HCMV-reactive T-cells and proportions of Vδ2 - γδ T-cells-populations linked with high burdens of HCMV. We conclude that UL18 is a highly variable gene, where variation may alter the persistent burden of HCMV and/or the host response to that burden.

Human cytomegalovirus (HCMV) is a beta-herpesvirus carried by ~80% of adults worldwide. Acute infections are often asymptomatic in healthy individuals but generate diverse syndromes in neonates, renal transplant recipients (RTR), and people with HIV (PWH). The HCMV gene UL111a encodes a homolog of human interleukin-10 (IL-10) that interacts with the human IL-10 receptor. Deep sequencing technologies were used to sequence UL111a directly from 59 clinical samples from Indonesian PWH and Australian RTR, healthy adults, and neonates. Overall, 93% of samples contained more than one variant of HCMV, as defined by at least one nonsynonymous variation. Carriage of these variants differed between neonates and adults, Australians and Indonesians, and between saliva and blood leukocytes. The variant alleles of N41D and S71Y occurred together in Australian RTR and were associated with higher T-cell responses to HCMV pp65. The variant P122S was associated with lower levels of antibodies reactive with a lysate of HCMV-infected fibroblasts. L174F was associated with increased levels of antibodies reactive with HCMV lysate, immediate-early 1 (IE-1), and glycoprotein B (gB) in Australian RTR and Indonesians PWH, suggesting a higher viral burden. We conclude that variants of UL111a are common in all populations and may influence systemic responses to HCMV.

In order to characterize the phenotype and to examine the effects of sun exposure on the color and structure of eumelanin (EM) and pheomelanin (PM) in alpaca fibers, we applied Soluene-350 solubilization, alkaline hydrogen peroxide oxidation (AHPO) and hydroiodic acid (HI) hydrolysis to the base and tip fibers of 20 true-black (TB) and 20 warm-black (WB) alpacas. We analyzed absorbances at 500 nm (A500) and 650 nm (A650), Free and Total pyrrole-2,3,5-tricarboxylic acid (PTCA), 2,3,4,5-tetracarboxylic acid (PTeCA) as degradative products from EM, and 4-amino-3-hydroxyphenylalanine (4-AHP), 3-amino-4-hydroxyphenylalanine (3-AHP) and thiazole-2,4,5-tricarboxylic acid (TTCA) as degradative products from PM. We found that the ratio of PTeCA/Total PTCA increased significantly from the base to the tip in both colors of alpaca fibers, while the ratios of A650/A500 and 4-AHP/3-AHP decreased significantly. These results show that structures made of both EM and PM in alpaca fibers are modified significantly by sun exposure inducing color change. This study indicates that the ratios of A650/A500, PTeCA/Total PTCA and 4-AHP/3-AHP are highly sensitive markers of color change and photodegradation of EM and PM, respectively., (© 2021 American Society for Photobiology.)

Human cytomegalovirus (HCMV) is a beta-herpesvirus carried by ∼80% of the world's population. Acute infections are asymptomatic in healthy individuals but generate diverse syndromes in neonates, solid organ transplant recipients, and HIV-infected individuals. The HCMV gene US28 encodes a homolog of a human chemokine receptor that is able to bind several chemokines and HIV gp120. Deep sequencing technologies were used to sequence US28 directly from 60 clinical samples from Indonesian HIV patients and Australian renal transplant recipients, healthy adults, and neonates. Molecular modeling approaches were used to predict whether nine nonsynonymous mutations in US28 may alter protein binding to a panel of six chemokines and two variants of HIV gp120. Ninety-two percent of samples contained more than one variant of HCMV, as defined by at least one nonsynonymous mutation. Carriage of these variants differed between neonates and adults, Australian and Indonesian samples, and saliva samples and blood leukocytes. Two nonsynonymous mutations (N170D and R267K) were associated with increased levels of immediate early protein 1 (IE-1) and glycoprotein B (gB) HCMV-reactive antibodies, suggesting a higher viral burden. Seven of the nine mutations were predicted to alter binding of at least one ligand. Overall, HCMV variants are common in all populations and have the potential to affect US28 interactions with human chemokines and/or gp120 and alter responses to the virus. The findings relied on deep sequencing technologies applied directly to clinical samples, so the variants exist in vivo . IMPORTANCE Human cytomegalovirus (HCMV) is a common viral pathogen of solid organ transplant recipients, neonates, and HIV-infected individuals. HCMV encodes homologs of several host genes with the potential to influence viral persistence and/or pathogenesis. Here, we present deep sequencing of an HCMV chemokine receptor homolog, US28, acquired directly from clinical specimens. Carriage of these variants differed between patient groups and was associated with different levels of circulating HCMV-reactive antibodies. These features are consistent with a role for US28 in HCMV persistence and pathogenesis. This was supported by in silico analyses of the variant sequences demonstrating altered ligand-binding profiles. The data delineate a novel approach to understanding the pathogenesis of HCMV and may impact the development of an effective vaccine.

Chronic psychosocial stress is increasingly being recognised as a risk factor for sporadic Alzheimer's disease (AD). The hypothalamic-pituitary-adrenal axis (HPA axis) is the major stress response pathway in the body and tightly regulates the production of cortisol, a glucocorticoid hormone. Dysregulation of the HPA axis and increased levels of cortisol are commonly found in AD patients and make a major contribution to the disease process. The underlying mechanisms remain poorly understood. In addition, within the general population there are interindividual differences in sensitivities to glucocorticoid and stress responses, which are thought to be due to a combination of genetic and environmental factors. These differences could ultimately impact an individuals' risk of AD. The purpose of this review is first to summarise the literature describing environmental and genetic factors that can impact an individual's HPA axis reactivity and function and ultimately AD risk. Secondly, we propose a mechanism by which genetic factors that influence HPA axis reactivity may also impact inflammation, a key driver of neurodegeneration. We hypothesize that these factors can mediate glucocorticoid priming of the immune cells of the brain, microglia, to become pro-inflammatory and promote a neurotoxic environment resulting in neurodegeneration. Understanding the underlying molecular mechanisms and identifying these genetic factors has implications for evaluating stress-related risk/progression to neurodegeneration, informing the success of interventions based on stress management and potential risks associated with the common use of glucocorticoids., (© 2021 Cambridge Philosophical Society.)

Chestnut coat color in horses is determined by a missense mutation within the MC1R gene. However, the intensity of the chestnut color can vary widely within individuals possessing this genotype. Here, we investigated this variation using standardized photographs of 96 horses. Each horse was ranked lightest to darkest within the cohort for phenotype by 3 blinded observers. A genome-wide association study utilizing the relative shade ranking as the phenotype and using 268 487 single-nucleotide polymorphisms (SNPs) genotyped using the Affymetrix Equine 670k array identified a single significantly associated region on chromosome 3 (P = 2.934 × 10-8). Analysis of whole-genome sequences for horses spanning the diverse range of chestnut color identified candidate SNPs within the coding sequence of the only gene in the region: SALL1. The function of SALL1 is largely unknown, though it is predicted to interact with the Hermansky-Pudlak Syndrome type 1 (HPS1) protein, which causes partial albinism in humans. However, with only one study suggesting a circumstantial influence of the SALL1 protein on pigmentation, additional work is needed to confirm this new coat color locus in larger populations and investigate the function of this protein for impacts on equine health., (© The American Genetic Association. 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Sponges represent one of the most species-rich hosts for commensal barnacles yet host utilisation and diversity have not been thoroughly examined. This study investigated the diversity and phylogenetic relationships of sponge-inhabiting barnacles within a single, targeted host group, primarily from Western Australian waters. Specimens of the sponge order Dictyoceratida were surveyed and a total of 64 host morphospecies, representing four families, were identified as barnacle hosts during the study. Utilising molecular (COI, 12S) and morphological methods 42 molecular operational taxonomic units (MOTUs) of barnacles, representing Acasta, Archiacasta, Euacasta and Neoacasta were identified. Comparing inter- and intra-MOTU genetic distances showed a barcode gap between 2.5% and 5% for COI, but between 1% and 1.5% in the 12S dataset, thus demonstrating COI as a more reliable barcoding region. These sponge-inhabiting barnacles were demonstrated to show high levels of host specificity with the majority being found in a single sponge species (74%), a single genus (83%) or a single host family (93%). Phylogenetic relationships among the barnacles were reconstructed using mitochondrial (12S, COI) and nuclear (H3, 28S) markers. None of the barnacle genera were recovered as monophyletic. Euacasta was paraphyletic in relation to the remaining Acastinae genera, which were polyphyletic. Six well-supported clades of molecular operational taxonomic units, herein considered to represent species complexes, were recovered, but relationships between them were not well supported. These complexes showed differing patterns of host usage, though most were phylogenetically conserved with sister lineages typically occupying related hosts within the same genus or family of sponge. The results show that host specialists are predominant, and the dynamics of host usage have played a significant role in the evolutionary history of the Acastinae., (Copyright © 2021. Published by Elsevier Inc.)

Human cytomegalovirus (HCMV) infections are common following renal transplantation and may have long-lasting effects. HCMV can be measured directly by viral DNA or indirectly via host immune responses. HCMV-encoded microRNA (miRNA) may alter the pathobiology of HCMV infections and contribute to the progression of HCMV disease. HCMV-encoded miRNAs can be detected in blood but have not been sought in saliva. We investigated saliva samples from 32 renal transplant recipients (RTR) and 12 seropositive healthy controls for whom immunological data was available. Five HCMV-encoded miRNAs (miR-UL112-5p, miR-US5-2-3p, miR-UL36, miR-US25-2-3p and miR-UL22A) were sought using primer probe assays. HCMV miRNA species were detected in saliva from 15 RTR and 3 healthy controls, with miR-US5-2-3p most commonly detected. The presence of HCMV miRNAs associated with increased T-cell responses to HCMV IE-1 in RTR, suggesting a link with frequent reactivations of HCMV.

Background: Comparative genomics studies are central in identifying the coding and non-coding elements associated with complex traits, and the functional annotation of genomes is a critical step to decipher the genotype-to-phenotype relationships in livestock animals. As part of the Functional Annotation of Animal Genomes (FAANG) action, the FR-AgENCODE project aimed to create reference functional maps of domesticated animals by profiling the landscape of transcription (RNA-seq), chromatin accessibility (ATAC-seq) and conformation (Hi-C) in species representing ruminants (cattle, goat), monogastrics (pig) and birds (chicken), using three target samples related to metabolism (liver) and immunity (CD4+ and CD8+ T cells)., Results: RNA-seq assays considerably extended the available catalog of annotated transcripts and identified differentially expressed genes with unknown function, including new syntenic lncRNAs. ATAC-seq highlighted an enrichment for transcription factor binding sites in differentially accessible regions of the chromatin. Comparative analyses revealed a core set of conserved regulatory regions across species. Topologically associating domains (TADs) and epigenetic A/B compartments annotated from Hi-C data were consistent with RNA-seq and ATAC-seq data. Multi-species comparisons showed that conserved TAD boundaries had stronger insulation properties than species-specific ones and that the genomic distribution of orthologous genes in A/B compartments was significantly conserved across species., Conclusions: We report the first multi-species and multi-assay genome annotation results obtained by a FAANG project. Beyond the generation of reference annotations and the confirmation of previous findings on model animals, the integrative analysis of data from multiple assays and species sheds a new light on the multi-scale selective pressure shaping genome organization from birds to mammals. Overall, these results emphasize the value of FAANG for research on domesticated animals and reinforces the importance of future meta-analyses of the reference datasets being generated by this community on different species.

A new species of sponge-inhabiting barnacle, Membranobalanus porphyrophilus sp. nov. , is described herein. This species can be distinguished from all other congeners by a combination of characters, in particular by the shapes of the tergum and scutum and the armament of the cirri. COI sequence data from the type specimens have been lodged with GenBank and a morphological key to the species of Membranobalanus is provided to aid future research. The host of the new species is the southern Australian endemic demosponge Spheciospongia purpurea . The new species of barnacle is thought to be host species specific.

The development of high-quality chromosomally assigned reference genomes constitutes a key feature for understanding genome architecture of a species and is critical for the discovery of the genetic blueprints of traits of biological significance. South American camelids serve people in extreme environments and are important fiber and companion animals worldwide. Despite this, the alpaca reference genome lags far behind those available for other domestic species. Here we produced a chromosome-level improved reference assembly for the alpaca genome using the DNA of the same female Huacaya alpaca as in previous assemblies. We generated 190X Illumina short-read, 8X Pacific Biosciences long-read and 60X Dovetail Chicago ® chromatin interaction scaffolding data for the assembly, used testis and skin RNAseq data for annotation, and cytogenetic map data for chromosomal assignments. The new assembly VicPac3.1 contains 90% of the alpaca genome in just 103 scaffolds and 76% of all scaffolds are mapped to the 36 pairs of the alpaca autosomes and the X chromosome. Preliminary annotation of the assembly predicted 22,462 coding genes and 29,337 isoforms. Comparative analysis of selected regions of the alpaca genome, such as the major histocompatibility complex (MHC), the region involved in the Minute Chromosome Syndrome (MCS) and candidate genes for high-altitude adaptations, reveal unique features of the alpaca genome. The alpaca reference genome VicPac3.1 presents a significant improvement in completeness, contiguity and accuracy over VicPac2 and is an important tool for the advancement of genomics research in all New World camelids.

There is emerging evidence of the utility of virus-like particles (VLPs) as a novel model for the study of receptor-ligand interactions in a native plasma membrane environment. VLPs consist of a viral core protein encapsulated by portions of the cell membrane with membrane proteins and receptors expressed in their native conformation. VLPs can be generated in mammalian cells by transfection with the retroviral core protein (gag). In this study, we used Chinese hamster ovary (CHO T10) cells stably overexpressing the insulin receptor (IR) to generate IR bearing VLPs. The diameter and size uniformity of VLPs were estimated by dynamic light scattering and morphological features examined by scanning electron microscopy. The presence of high affinity IR on VLPs was demonstrated by competitive binding assays (K D : 2.3 ± 0.4 nM, n = 3), which was similar to that on the parental CHO T10 cells (K D : 2.1 ± 0.4 nM, n = 3). We also report that increases or decreases in membrane cholesterol content by treatment with methyl-β-cyclodextrin (MBCD) or cholesterol pre-loaded methyl-β-cyclodextrin (cMBCD), respectively, substantially decreased insulin binding (> 30%) to both VLPs and cells, and we speculate this is due to a change in receptor disposition. We suggest that this novel finding of decreases in insulin binding in response to changes in membrane cholesterol content may largely account for the unexplained decreases in insulin signalling events previously reported elsewhere. Finally, we propose VLPs as a viable membrane model for the study of insulin-IR interactions in a native membrane environment., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Historically, the immune environment was not considered an important target for breast cancer treatment. However, the association of lymphocytic infiltrates in triple negative and HER-2 over-amplified breast cancer subtypes with better outcomes, has provoked interest in evaluating the role of the immune system in the luminal B subtype that accounts for 39% of breast cancers and has a poor patient prognosis. It is unknown which immunosuppressive cell types or molecules (e.g., checkpoint molecules) are relevant, or where measurement is most informative. We hypothesize that a profound immunosuppressive tumor and/or lymph node milieu is prognostic and impacts on responses to therapies.

African Animal Trypanosomiasis (AAT) is endemic in at least 37 of the 54 countries in Africa. It is estimated to cause direct and indirect losses to the livestock production industry in excess of US$ 4.5 billion per annum. A century of intervention has yielded limited success, owing largely to the extraordinary complexity of the host-parasite interaction. Trypanotolerance, which refers to the inherent ability of some African livestock breeds, notably Djallonke sheep, N'Dama cattle and West African Dwarf goats, to withstand a trypanosomiasis challenge and still remain productive without any form of therapy, is an economically sustainable option for combatting this disease. Yet trypanotolerance has not been adequately exploited in the fight against AAT. In this review, we describe new insights into the genetic basis of trypanotolerance and discuss the potential of exploring this phenomenon as an integral part of the solution for AAT, particularly, in the context of African animal production systems., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: Conditioned pain modulation (CPM) encompasses the effects of inhibitory and facilitatory pain modulatory systems and is inefficient in some chronic pain states. A proportion of healthy subjects also exhibit little or no CPM, perhaps suggesting that inherent factors such as gender or genetics may be influential. However, there is no consensus on how best to determine a meaningful CPM effect. This study aimed to determine the proportion of pain-free subjects exhibiting a meaningful CPM effect. Analyses of associations between 5HTTLPR (serotonin transporter-linked polymorphic region) polymorphisms on the serotonin transporter gene (SLC6A4), gender, and CPM effect were also carried out. A total of 125 healthy subjects (47 male; 78 female) underwent pressure pain threshold testing before, during, and after a cold pressor conditioning stimulus. A buccal cell sample was collected for analysis of 5HTTLPR genotype. Meaningful CPM effect was determined as an increase in pressure pain threshold values from baseline greater than the inherent error of measurement, calculated as 5.3%. During the conditioning stimulus, 116 subjects (92.8%) exhibited a CPM effect whereas 9 did not. CPM effect did not differ significantly between genders or between 5HTTLPR genotypes. This provides a clear basis on which to determine the proportion of patients with a chronic pain disorder that exhibit a meaningful CPM effect., Perspective: This study proposes a method for calculating meaningful CPM effect and reports the proportion and magnitude of effect elicited in a large sample. Associations between CPM, gender, and genotype were also analyzed. Clarification of normal CPM response may help to elucidate the mechanisms driving CPM inefficiency in chronic pain., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

To date, a molecular phylogenetic approach has not been used to investigate the evolutionary structure of Trogoderma and closely related genera. Using two mitochondrial genes, Cytochrome Oxidase I and Cytochrome B, and the nuclear gene, 18S, the reported polyphyletic positioning of Trogoderma was examined. Paraphyly in Trogoderma was observed, with one Australian Trogoderma species reconciled as sister to all Dermestidae and the Anthrenocerus genus deeply nested within the Australian Trogoderma clade. In addition, time to most recent common ancestor for a number of Dermestidae was calculated. Based on these estimations, the Dermestidae origin exceeded 175 million years, placing the origins of this family in Pangaea.

The Complement Factor B gene (CFB) of the alternative complement pathway has been identified in the sheep Major Histocompatibility Complex (MHC) and its genomic sequence determined. CFB is located approximately 600 bp upstream of the complement C2 gene, contains 18 exons, and manifests the domain signature characteristic of CFB protein. Thirteen single nucleotide polymorphisms were identified in merino sheep and interbreed variation was identified by comparison with International Sheep Genomics Consortium data. Two predicted non synonymous substitutions were observed and in-silico analysis indicates that these are likely to have a destabilizing effect on the protein structure. Sheep and cattle CFB were compared and shown to contain a common nine nucleotide deletion in exon 18 relative to human CFB. Predicted CFB amino acid sequences for these two species contain 761 aa relative to 764 aa in the human orthologue. Sequencing of the cosmid and BAC clones used in this study permitted the relative positions of three adjacent loci to be determined and showed that the previously described microsatellite locus (BfMs) is located within SKIV2L., (Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.)

The microorganisms in rumen contents were physically separated into five fractions on the basis of size using counter-flow centrifugal elutriation (CCE). The use of CCE allowed the microbial population to be separated in a highly repeatable manner into two protozoal and three bacterial fractions with minimal loss of material (dry weight), and with no visible damage to the microorganisms. A Coulter counter was used to determine the sizes of the organisms in each fraction. The modified CCE method is suitable for studies of the rumen microbial ecosystem that require separation of defined fractions of the population.