Detection, sequencing, and tissue distribution of piscine orthoreovirus 2-like virus in diseased coho salmon in Alaska.

We performed a diagnostic disease investigation on a cohort of coho salmon (Oncorhynchus kisutch) fingerlings in Alaska exhibiting anorexia, gaping mouths, anemia, and increased mortality. Histologic examination revealed mild-to-severe myocardial degeneration and lymphohistiocytic and neutrophilic myocarditis, moderate splenic histiocytosis, and mild renal histiocytosis. Piscine orthoreoviruses 1 and 3 were not detected by molecular methods, and no other viruses could be cultured on 3 common diagnostic fish cell lines. De novo assembly produced a viral genome of 10 linear segments with >80% homology to piscine orthoreovirus 2 (PRV2) encoding all 11 PRV2 proteins. An in situ hybridization probe using RNAscope was developed against 697 viral nucleotides identified by sequencing, which revealed viral genome in heart, spleen, gill, kidney, liver, blood, and the lamina propria of the intestines. Our findings are supportive of a novel piscine orthoreovirus most closely related to PRV2 associated with morbidity and mortality of coho salmon in the northeastern Pacific.

Genomic characterization of antimicrobial resistance in 61 aquatic bacterial isolates.

Antimicrobial resistance (AMR) in pathogens important to aquatic animal health is of increasing concern but vastly understudied. Antimicrobial therapy is used to both treat and prevent bacterial disease in fish and is critical for a viable aquaculture industry and for maintenance of wild fish populations. Unfortunately, phenotypic antimicrobial susceptibility testing is technically difficult for bacteria recovered from aquatic animal hosts resulting in challenges in resistance monitoring using traditional methods. Whole-genome sequencing provides an appealing methodology for investigation of putative resistance. As part of the ongoing efforts of the FDA CVM Vet-LIRN to monitor AMR, source laboratories cultured and preliminarily identified pathogenic bacteria isolated from various fish species collected in 2019 from across the United States. Sixty-one bacterial isolates were evaluated using whole-genome sequencing. We present here the assembled draft genomes, AMR genes, predicted resistance phenotypes, and virulence factors of the 61 isolates and discuss concurrence of the identifications made by source laboratories using matrix-assisted laser desorption/time-of-flight mass spectrometry.

Investigation of laboratory methods for characterization of aquatic viruses in fish infected experimentally with infectious salmon anemia virus.

Rapid growth in aquaculture has resulted in high-density production systems in ecologically and geographically novel conditions in which the emergence of diseases is inevitable. Well-characterized methods for detection and surveillance of infectious diseases are vital for rapid identification, response, and recovery to protect economic and food security. We implemented a proof-of-concept approach for virus detection using a known high-consequence fish pathogen, infectious salmon anemia virus (ISAV), as the archetypal pathogen. In fish infected with ISAV, we integrated histopathology, virus isolation, whole-genome sequencing (WGS), electron microscopy (EM), in situ hybridization (ISH), and reverse transcription real-time PCR (RT-rtPCR). Fresh-frozen and formalin-fixed tissues were collected from virus-infected, control, and sham-infected Atlantic salmon (Salmo salar). Microscopic differences were not evident between uninfected and infected fish. Viral cytopathic effect was observed in cell cultures inoculated with fresh-frozen tissue homogenates from 3 of 3 ISAV-infected and 0 of 4 uninfected or sham-infected fish. The ISAV genome was detected by shotgun metagenomics in RNA extracted from the medium from 3 of 3 inoculated cell cultures, 3 of 3 infected fish, and 0 of 4 uninfected or sham-infected fish, yielding sufficient coverage for de novo assembly. An ISH probe against ISAV revealed ISAV genome in multiple organs, with abundance in renal hematopoietic tissue. Virus was detected by RT-rtPCR in gill, heart, kidney, liver, and spleen. EM and metagenomic WGS from tissues were challenging and unsuccessful. Our proof-of-concept methodology has promise for detection and characterization of unknown aquatic pathogens and also highlights some associated methodology challenges that require additional investigation.

The fish pathogen Flavobacterium columnare represents four distinct species: Flavobacterium columnare, Flavobacterium covae sp. nov., Flavobacterium davisii sp. nov. and Flavobacterium oreochromis sp. nov., and emended description of Flavobacterium columnare.

Flavobacterium columnare is the causative agent of columnaris disease in freshwater fish and four discrete genetic groups exist within the species, suggesting that the species designation requires revision. The present study determined the taxonomic status of the four genetic groups of F. columnare using polyphasic and phylogenomic approaches and included five representative isolates from each genetic group (including type strain ATCC 23463T; genetic group 1). 16S rRNA gene sequence analysis revealed genetic group 2 isolate AL-02-36T, genetic group 3 isolate 90-106T, and genetic group 4 isolate Costa Rica 04-02-TNT shared less than <98.8 % sequence identity to F. columnare ATCC 23463T. Phylogenetic analyses of 16S rRNA and gyrB genes using different methodologies demonstrated the four genetic groups formed well-supported and distinct clades within the genus Flavobacterium. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (GGDC) values between F. columnare ATCC 23463T, genetic group 2 isolate AL-02-36T, genetic group 3 isolate 90-106T, and genetic group 4 isolate Costa Rica 04-02-TNT were less than 90.84% and 42.7%, respectively. Biochemical and physiological characteristics were similar among the four genetic groups; however, quantitative differences in fatty acid profiles were detected and MALDI-TOF analyses demonstrated numerous distinguishing peaks unique to each genetic group. Chemotaxonomic, MALDI-TOF characterization and ANI/GGDC calculations afforded differentiation between the genetic groups, indicating each group is a discrete species. Herein, the names F. covae sp. nov. (AL-02-36T), F. davisii sp. nov. (90-106T), and F. oreochromis sp. nov. (Costa Rica 04-02-TNT) are proposed to represent genetic groups 2, 3, and 4, respectively.

Clinical and pathologic findings in an outbreak in rabbits of natural infection by rabbit hemorrhagic disease virus 2 in the northwestern United States.

Rabbit hemorrhagic disease virus 2 (RHDV2) causes an often-fatal disease of rabbits that has resulted in outbreaks in rabbitries in Europe, Africa, Australia, and Asia. RHD has historically been characterized as a foreign animal disease in the United States. In July 2019, RHDV2 was detected in rabbits on Orcas Island along the northwestern coast of Washington (WA) State following reports of deaths in multiple feral and domestic rabbits. We document and highlight here the unique clinical presentation and gross and histologic lesions observed in this recent WA outbreak. Affected rabbits died without premonitory signs or displayed hyporexia and/or lethargy for ≤1 d prior to death. The most consistent pathologic finding was random, multifocal hepatocellular necrosis, often with concurrent multifocal-to-diffuse splenic necrosis. The lack of significant clinical signs in conjunction with the random distribution of hepatic necrosis in the WA outbreak contrasts with previous reports of RHDV2 disease progression.

A Novel Neorickettsial Infection in 3 Dogs in the Pacific Northwest.

The genus Neorickettsia includes obligate, intracellular bacteria responsible for diseases including Potomac horse fever caused by Neorickettsia risticii and salmon poisoning disease (SPD) caused by Neorickettsia helminthoeca. The Stellanchasmus falcatus (SF) agent is a member of this genus previously associated only with mild clinical signs in dogs. Between 2013 and 2016, 3 dogs in Washington State (USA) presented with disease suggestive of SPD, but N. helminthoeca was not detected by molecular techniques. Clinical signs included depression, anorexia, and diarrhea. Cytologic examination of aspirates supported a diagnosis of granulomatous lymphadenitis with organisms suggestive of Neorickettsia. Dogs either died or were humanely euthanized due to poor response to therapy. Necropsy findings included lymphadenomegaly and hepatomegaly. Histopathology identified granulomatous and lymphoplasmacytic splenitis, lymphadenitis, enteritis, and hepatitis with extensive necrosis. Neorickettsia DNA was detected using genus-specific primers and direct sequencing showed 100% sequence identity to the SF agent in all 3 dogs. This is the first clinicopathologic description of severe disease in dogs attributed to the SF agent. These findings may suggest the emergence of a novel neorickettsial disease in the Pacific Northwest.

Broad-spectrum antiviral JL122 blocks infection and inhibits transmission of aquatic rhabdoviruses.

The aquaculture industry is growing rapidly to meet the needs for global protein consumption. Viral diseases in aquaculture are quite challenging due to lack of treatment options as well as limited injection-delivery vaccines, which are costly. Thus, water-immersion antiviral treatments are highly desirable. This study focused on broad-spectrum, light-activated antivirals that target the viral membrane (envelope) of viruses to prevent viral-cell membrane fusion, ultimately blocking viral entry into cells. Of the tested small-molecules, JL122, a new broad-spectrum antiviral previously unexplored against aquatic viruses, blocked infection of three aquatic rhabdoviruses (IHNV, VHSV and SVCV) in cell culture and in two live fish challenge models. Importantly, JL122 inhibited transmission of IHNV from infected to uninfected rainbow trout. Further, the effective antiviral concentrations were not toxic to cells or susceptible fish. These results show promise for JL122 to become an immersion treatment option for outbreaks of aquatic enveloped viral infections.

Use of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry for the Identification of Pathogenic Vibrio in Fish.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid, cost-effective method for identification of a broad range of bacterial taxa, but its accuracy for Vibrio spp. from samples of aquatic animal origin is unknown. We used DNA sequence analysis targeting two conserved genes, rpoB and rpoD, as the identification standard for 5 reference strains and 35 Vibrio spp. field isolates obtained from diagnostic aquaculture samples. Overall, MALDI-TOF MS correctly identified 100% of the five reference strains to the genus level and 80% (4 of 5) to the species level. For field isolates, 83% (29 of 35) were correctly identified to the genus level, and 49% (17 of 35) were correctly identified to the species level. Eight (23%) field isolates were incorrectly identified at the species level. The MALDI-TOF MS method produced no identification for 17% (6 of 35) of the field isolates. Using traditional culture identification, 100% of the five reference strains were correctly identified to the species level. All 35 field isolates were correctly identified to the genus level; 51% (18 of 35) of the isolates were identified correctly to the species level, while 29% (10 of 35) were misidentified at the species level. Overall, MALDI-TOF MS was comparable to phenotypic identification, and accuracy will likely improve with enhancement of MALDI-TOF MS database robustness.

Integrative functional analyses using rainbow trout selected for tolerance to plant diets reveal nutrigenomic signatures for soy utilization without the concurrence of enteritis.

Finding suitable alternative protein sources for diets of carnivorous fish species remains a major concern for sustainable aquaculture. Through genetic selection, we created a strain of rainbow trout that outperforms parental lines in utilizing an all-plant protein diet and does not develop enteritis in the distal intestine, as is typical with salmonids on long-term plant protein-based feeds. By incorporating this strain into functional analyses, we set out to determine which genes are critical to plant protein utilization in the absence of gut inflammation. After a 12-week feeding trial with our selected strain and a control trout strain fed either a fishmeal-based diet or an all-plant protein diet, high-throughput RNA sequencing was completed on both liver and muscle tissues. Differential gene expression analyses, weighted correlation network analyses and further functional characterization were performed. A strain-by-diet design revealed differential expression ranging from a few dozen to over one thousand genes among the various comparisons and tissues. Major gene ontology groups identified between comparisons included those encompassing central, intermediary and foreign molecule metabolism, associated biosynthetic pathways as well as immunity. A systems approach indicated that genes involved in purine metabolism were highly perturbed. Systems analysis among the tissues tested further suggests the interplay between selection for growth, dietary utilization and protein tolerance may also have implications for nonspecific immunity. By combining data from differential gene expression and co-expression networks using selected trout, along with ontology and pathway analyses, a set of 63 candidate genes for plant diet tolerance was found. Risk loci in human inflammatory bowel diseases were also found in our datasets, indicating rainbow trout selected for plant-diet tolerance may have added utility as a potential biomedical model.

Inhibition of an Aquatic Rhabdovirus Demonstrates Promise of a Broad-Spectrum Antiviral for Use in Aquaculture.

Many enveloped viruses cause devastating disease in aquaculture, resulting in significant economic impact. LJ001 is a broad-spectrum antiviral compound that inhibits enveloped virus infections by specifically targeting phospholipids in the lipid bilayer via the production of singlet oxygen (1O2). This stabilizes positive curvature and decreases membrane fluidity, which inhibits virus-cell membrane fusion during viral entry. Based on data from previous mammalian studies and the requirement of light for the activation of LJ001, we hypothesized that LJ001 may be useful as a preventative and/or therapeutic agent for infections by enveloped viruses in aquaculture. Here, we report that LJ001 was more stable with a prolonged inhibitory half-life at relevant aquaculture temperatures (15°C), than in mammalian studies at 37°C. When LJ001 was preincubated with our model virus, infectious hematopoietic necrosis virus (IHNV), infectivity was significantly inhibited in vitro (using the epithelioma papulosum cyprini [EPC] fish cell line) and in vivo (using rainbow trout fry) in a dose-dependent and time-dependent manner. While horizontal transmission of IHNV in a static cohabitation challenge model was reduced by LJ001, transmission was not completely blocked at established antiviral doses. Therefore, LJ001 may be best suited as a therapeutic for aquaculture settings that include viral infections with lower virus-shedding rates than IHNV or where higher viral titers are required to initiate infection of naive fish. Importantly, our data also suggest that LJ001-inactivated IHNV elicited an innate immune response in the rainbow trout host, making LJ001 potentially useful for future vaccination approaches. IMPORTANCE: Viral diseases in aquaculture are challenging because there are few preventative measures and/or treatments. Broad-spectrum antivirals are highly sought after and studied because they target common components of viruses. In our studies, we used LJ001, a broad-spectrum antiviral compound that specifically inhibits enveloped viruses. We used the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV) as a model to study aquatic enveloped virus diseases and their inhibition. We demonstrated inhibition of IHNV by LJ001 both in cell culture as well as in live fish. Additionally, we showed that LJ001 inhibited the transmission of IHNV from infected fish to healthy fish, which lays the groundwork for using LJ001 as a possible therapeutic for aquatic viruses. Our results also suggest that virus inactivated by LJ001 induces an immune response, showing potential for future preventative (e.g., vaccine) applications.

Draft Genome Sequences of Three Flavobacterium psychrophilum Strains Isolated from Coldwater Disease Outbreaks at Three Production Hatcheries.

We report here the genome sequences of three Flavobacterium psychrophilum strains causing a bacterial coldwater disease (BCWD) outbreak, isolated from infected rainbow trout from hatcheries in Montana and South Dakota. The availability of these virulent outbreak-causing strain genome sequences will help further understand the pathogenesis of BCWD.

Electrochemical scaffold generates localized, low concentration of hydrogen peroxide that inhibits bacterial pathogens and biofilms.

We hypothesized that low concentrations of H2O2 could be generated through the electrochemical conversion of oxygen by applying an electric potential to a conductive scaffold and produce a low, but constant, concentration of H2O2 that would be sufficient to destroy biofilms. To test our hypothesis we used a multidrug-resistant Acinetobacter baumannii strain, because this species is often implicated in difficult-to-treat biofilm infections. We used conductive carbon fabric as the scaffold material ("e-scaffold"). In vitro experiments demonstrated the production of a maximum constant concentration of ~25 µM H2O2 near the e-scaffold surface. An e-scaffold was overlaid onto an existing A. baumannii biofilm, and within 24 h there was a ~4-log reduction in viable bacteria with an ~80% decrease in biofilm surface coverage. A similar procedure was used to overlay an e-scaffold onto an existing A. baumannii biofilm that was grown on a porcine explant. After 24 h, there was a ~3-log reduction in viable bacteria from the infected porcine explants with no observable damage to the underlying mammalian tissue based on a viability assay and histology. This research establishes a novel foundation for an alternative antibiotic-free wound dressing to eliminate biofilms.

Potential mechanisms of attenuation for rifampicin-passaged strains of Flavobacterium psychrophilum.

BACKGROUND: Flavobacterium psychrophilum is the etiologic agent of bacterial coldwater disease in salmonids. Earlier research showed that a rifampicin-passaged strain of F. psychrophilum (CSF 259-93B.17) caused no disease in rainbow trout (Oncorhynchus mykiss, Walbaum) while inducing a protective immune response against challenge with the virulent CSF 259-93 strain. We hypothesized that rifampicin passage leads to an accumulation of genomic mutations that, by chance, reduce virulence. To assess the pattern of phenotypic and genotypic changes associated with passage, we examined proteomic, LPS and single-nucleotide polymorphism (SNP) differences for two F. psychrophilum strains (CSF 259-93 and THC 02-90) that were passaged with and without rifampicin selection. RESULTS: Rifampicin resistance was conveyed by expected mutations in rpoB, although affecting different DNA bases depending on the strain. One rifampicin-passaged CSF 259-93 strain (CR) was attenuated (4 % mortality) in challenged fish, but only accumulated eight nonsynonymous SNPs compared to the parent strain. A CSF 259-93 strain passaged without rifampicin (CN) accumulated five nonsynonymous SNPs and was partially attenuated (28 % mortality) compared to the parent strain (54.5 % mortality). In contrast, there were no significant change in fish mortalities among THC 02-90 wild-type and passaged strains, despite numerous SNPs accumulated during passage with (n = 174) and without rifampicin (n = 126). While only three missense SNPs were associated with attenuation, a Ser492Phe rpoB mutation in the CR strain may contribute to further attenuation. All strains except CR retained a gliding motility phenotype. Few proteomic differences were observed by 2D SDS-PAGE and there were no apparent changes in LPS between strains. Comparative methylome analysis of two strains (CR and TR) identified no shared methylation motifs for these two strains. CONCLUSION: Multiple genomic changes arose during passage experiments with rifampicin selection pressure. Consistent with our hypothesis, unique strain-specific mutations were detected for the fully attenuated (CR), partially attenuated (CN) and another fully attenuated strain (B17).

Aquacultured rainbow trout (Oncorhynchus mykiss) possess a large core intestinal microbiota that is resistant to variation in diet and rearing density.

As global aquaculture fish production continues to expand, an improved understanding of how environmental factors interact in fish health and production is needed. Significant advances have been made toward economical alternatives to costly fishmeal-based diets, such as grain-based formulations, and toward defining the effect of rearing density on fish health and production. Little research, however, has examined the effects of fishmeal- and grain-based diets in combination with alterations in rearing density. Moreover, it is unknown whether interactions between rearing density and diet impact the composition of the fish intestinal microbiota, which might in turn impact fish health and production. We fed aquacultured adult rainbow trout (Oncorhynchus mykiss) fishmeal- or grain-based diets, reared them under high- or low-density conditions for 10 months in a single aquaculture facility, and evaluated individual fish growth, production, fin indices, and intestinal microbiota composition using 16S rRNA gene sequencing. We found that the intestinal microbiotas were dominated by a shared core microbiota consisting of 52 bacterial lineages observed across all individuals, diets, and rearing densities. Variations in diet and rearing density resulted in only minor changes in intestinal microbiota composition despite significant effects of these variables on fish growth, performance, fillet quality, and welfare. Significant interactions between diet and rearing density were observed only in evaluations of fin indices and the relative abundance of the bacterial genus Staphylococcus. These results demonstrate that aquacultured rainbow trout can achieve remarkable consistency in intestinal microbiota composition and suggest the possibility of developing novel aquaculture strategies without overtly altering intestinal microbiota composition.

Traumatic gastroperitonitis (hardware disease) in an alpaca (Vicugna pacos).

A 5-yr-old, intact male Suri alpaca (Vicugna pacos) presented with acute onset of anorexia, depression, and reluctance to stand. Exploratory laparotomy revealed diffuse peritonitis resulting from penetration of a 5-cm wire through the second gastric compartment (C2). Due to the severity of the alpaca's condition and its deteriorating nature, euthanasia at the time of surgery was elected. This is the first published case of a condition commonly observed in cattle (traumatic gastroperitonitis, "Hardware disease") to be reported in an alpaca.

Assessing the suitability of a partial water reuse system for rearing juvenile Chinook salmon for stocking in Washington State.

To assess the suitability of water reuse technology for raising Pacific salmon Oncorhynchus spp. for stocking purposes, fish health and welfare were compared between two groups of juvenile Chinook salmon O. tshawytscha from the same spawn: one group was reared in a pilot partial water reuse system (circular tanks), and the other group was reared in a flow-through raceway. This observational study was carried out over a 21-week period in Washington State. Reuse and raceway fish were sampled repeatedly for pathogen screening and histopathology; fin erosion and whole-blood characteristics were also evaluated. By the study's end, no listed pathogens were isolated from either cohort, and survival was 99.3% and 99.0% in the reuse and raceway groups, respectively. Condition factor was 1.28 in raceway fish and 1.14 in reuse fish; this difference may have been attributable to occasional differences in feeding rates between the cohorts. Fin indices (i.e., length of the longest dorsal or caudal fin ray, standardized by fork length) were lower in reuse fish than in raceway fish, but fin erosion was not grossly apparent in either cohort. The most consistent histological lesion was gill epithelial hypertrophy in reuse fish; however, blood analyses did not suggest any corresponding physiological imbalances. Overall, results suggest that water reuse technology can be employed in rearing juvenile anadromous salmonids for stocking purposes.

The effects of Myxobolus cerebralis on Apache and Gila trout in laboratory exposures.

Whirling disease has been implicated in salmonid population declines in several western states. To determine the risk of a species or strain of salmonid to whirling disease it is critical to establish its relative susceptibility to Myxobolus cerebralis infection. Gila trout Oncorhynchus gilae and Apache trout Oncorhynchus gilae apache were exposed to various doses of M. cerebralis triactinomyxons (TAMs) in laboratory experiments. In trials conducted in consecutive years, fish were exposed to TAMs in doses ranging from 25 to 2,000/fish at ages ranging from 66 to 201 d posthatch (dph). All fish were held for 900 degree-days, and then the infection intensity of each fish was determined by histological examination. In 2002, 98% of the Gila trout died during exposure or within 48 h postexposure. Seventy-four percent of the Apache trout died before the end of the 90-d study period. Those that survived the entire study period had an average histological score of more than 4.0. In subsequent trials, the TAM dosage was decreased to 25-1,000/fish. Also, the age of the fish was increased from 66-72 dph to 89-201 dph. The survival rate increased from 16.0% to 49.1%. Average histological grades ranged from 1.6 to 4.8. Based on this data, it can be concluded that both Apache and Gila trout are highly susceptible to M. cerebralis in laboratory trials.

An insertion mutation in ABCB4 is associated with gallbladder mucocele formation in dogs.

BACKGROUND: ABCB4 functions as a phosphatidylcholine translocater, flipping phosphatidylcholine across hepatocyte canalicular membranes into biliary canaliculi. In people, ABCB4 gene mutations are associated with several disease syndromes including intrahepatic cholestasis of pregnancy, progressive familial intrahepatic cholestasis (type 3), primary biliary cirrhosis, and cholelithiasis. Hepatobiliary disease, specifically gallbladder mucocele formation, has been recognized with increased frequency in dogs during the past decade. Because Shetland Sheepdogs are considered to be predisposed to gallbladder mucoceles, we initially investigated ABCB4 as a candidate gene for gallbladder mucocele formation in that breed, but included affected dogs of other breeds as well. RESULTS: An insertion (G) mutation in exon 12 of canine ABCB4 (ABCB4 1583_1584G) was found to be significantly associated with hepatobiliary disease in Shetland Sheepdogs specifically (P < 0.0001) as well as other breeds (P < 0.0006). ABCB4 1583_1584G results in a frame shift generating four stop codons that prematurely terminate ABCB4 protein synthesis within exon 12, abolishing over half of the protein including critical ATP and a putative substrate binding site. CONCLUSIONS: The finding of a significant association of ABCB4 1583_1584G with gallbladder mucoceles in dogs suggests that this phospholipid flippase may play a role in the pathophysiology of this disorder. Affected dogs may provide a useful model for identifying novel treatment strategies for ABCB4-associated hepatobiliary disease in people.

Gastric cryptosporidiosis in freshwater angelfish (Pterophyllum scalare).

A freshwater angelfish (Pterophyllum scalare) hatchery experienced variable levels of emaciation, poor growth rates, swollen coelomic cavities, anorexia, listlessness, and increased mortality within their fish. Multiple chemotherapeutic trials had been attempted without success. In affected fish, large numbers of protozoa were identified both histologically and ultrastructurally associated with the gastric mucosa. The youngest cohort of parasitized fish was the most severely affected and demonstrated the greatest morbidity and mortality. The protozoa were morphologically most consistent with Cryptosporidium. All of the protozoan life stages were identified ultrastructurally and protozoal genomic DNA was isolated from parasitized tissue viscera and sequenced. Histological, ultrastructural, genetic, and phylogenetic analyses confirmed this protozoal organism to be a novel species of Cryptosporidium.

Peripheral ovine progressive pneumonia provirus levels correlate with and predict histological tissue lesion severity in naturally infected sheep.

Studies were undertaken to determine whether anti-ovine progressive pneumonia virus (OPPV) antibody responses in serum or OPP provirus levels in peripheral blood associate with the degree of histologically measured tissue lesions in naturally OPPV-infected sheep. Sections of formalin-fixed, paraffin-embedded, and hematoxylin- and eosin-stained lung, mammary gland, carpal synovial membrane, and brain tissues from 11 OPPV-infected ewes (mean age of 8.6 years) and 5 OPPV-uninfected ewes (mean age of 6 years) were evaluated for lesion severity. Ovine progressive pneumonia (OPP) provirus levels and anti-OPPV antibody titers in peripheral blood and serum samples, respectively, were measured upon euthanasia and 3 years prior to euthanasia. Both mean peripheral OPP provirus levels and mean serum anti-surface envelope glycoprotein (anti-SU) antibody titers at the time of euthanasia were significantly higher in ewes with moderate to severe histological lesions than in ewes with no to mild histological lesions. However, although mean peripheral blood OPP provirus levels at euthanasia and 3 years prior to euthanasia significantly correlated with the highest histological lesion score for any affected tissue (two-tailed P values, 0.03 and 0.02), mean serum anti-SU antibody titers, anti-capsid antibody titers, and anti-transmembrane 90 antibody titers at euthanasia did not show a significant correlation with the highest histological lesion score for any tissue (two-tailed P values, 0.32, 0.97, and 0.18, respectively). These data are the first to show that OPP provirus levels predict and correlate with the extent of OPPV-related histological lesions in various OPPV-affected tissues. These findings suggest that peripheral OPP provirus levels quantitatively contribute more to the development of histological lesions than the systemic anti-SU antibody host immune response.