Bovine respiratory disease (BRD) is a multifactorial disease complex that causes significant economic loss in the cattle industry. Bovine respiratory syncytial virus (BRSV), one of the primary viruses included in BRD, primarily targets the lower respiratory tract and causes a wide variety of subclinical and clinical signs. Current diagnosis techniques require off-site laboratory testing, creating delays that allow BRSV to spread through herds and highlighting the need for rapid diagnostic methods. The objective of the study was to evaluate near-infrared spectroscopy as a diagnostic tool for BRSV detection across varying infection severities. Twenty-two Holstein bull calves were reared in research pens at Mississippi State University from 1 week old until 12 weeks of age when the study began. At 12 weeks old, the calves were randomly divided into 4 groups and challenged via aerosol with 103, 104, and 105 TCID50 units of BRSV, creating low, medium, and high dose groups respectively. Negative control was nebulized with cell culture media. Biofluid samples, including serum, plasma, nasal secretions, saliva, and breath condensate, were collected daily for 14 days following infection. The presence and severity of infection was confirmed by quantitative real-time PCR (qPCR) threshold (Ct) values. Near-infrared spectra of all sample types were acquired using an ASD FieldSpec 4 NIR spectrometer (350–2500 nm) and analyzed within the 1300–1600 nm region (first overtone of water). Data was pre-processed using different techniques and classified using Soft Modeling of Class Analogies (SIMCA) to distinguish BRSV-positive (Ct < 35) from negative (Ct > 35) samples. Preliminary results showed variation in accuracy, sensitivity, and specificity across biofluids, with overall classification accuracy ranging from 42.8% to 76.4%. Nasal secretions produced the highest accuracy, while plasma demonstrated a more balanced sensitivity–specificity relationship among blood-derived samples. This approach demonstrates potential for a rapid alternative to conventional diagnostic methods for BRSV, suggesting that NIRS can detect biochemical changes in biofluids in cattle.

Abstract The tropical house cricket, Gryllodes sigillatus , is a mass-produced insect that is used as a protein source for pets and livestock. However, intensive mass-rearing conditions, coupled with high genetic relatedness, create an ideal environment for the spread of pathogenic microbes that severely impact production. Cricket iridovirus (CrIV) is a pathogen that impedes cricket growth and causes significant losses for cricket farmers. Interestingly, recent studies have shown that CrIV is often present asymptomatically, yet the molecular basis of the emergence of disease symptoms remains unknown. To address this, we sampled healthy and diseased crickets and examined differences in cricket and CrIV gene expression via RNAseq. Using differential gene expression analysis and functional enrichment analysis, we found significant differences in host and viral gene expression between healthy and diseased crickets, including genes involved in immunity. Interestingly, while we observed high CrIV gene expression across the entire CrIV genome in sick populations, healthy asymptomatic populations showed elevated expression at a single viral locus. Our results shed light not only on the cricket immune response to CrIV infection but also identify a viral gene that is highly expressed during covert infections, suggesting its potential role in suppressing the host’s immune response. These findings enhance our understanding of how CrIV interacts with our cricket host, providing essential insights for developing targeted strategies to manage CrIV outbreaks in cricket mass-rearing facilities.

Understanding the tripartite interaction between diet, the microbiome, and host physiology is essential for optimizing the bioconversion efficiency of black soldier fly larvae (Hermetia illucens). These interactions were investigated by analyzing life-history traits and metatranscriptomes of larvae fed carbohydrate-rich (1P:5C), protein-rich (5P:1C), and balanced ratio macronutrient (1P:1C) diets. The results showed that dietary macronutrients correlated with shifts in the microbial community and gene expression. In particular, optimal larval performance, characterized by the highest weight and survivorship, was achieved on the balanced ratio diet. While the carbohydrate-rich diet increased microbial alpha diversity and enriched microbial transcripts for carbohydrate metabolism and transport, it significantly delayed pupation, reduced larval weight, and induced host immune genes related to pathogen recognition. Larval guts were predominantly composed of the fungal genus Magnusiomyces, no matter the diet, whereas the frass microbiome was primarily bacterial. In the frass, Klebsiella dominated the carbohydrate-rich diet, while Streptococcus, Lactobacillus, and Klebsiella were the most prevalent taxa in the balanced and protein-rich treatments. Significant correlations were identified between host gene expression and microbial alpha diversity and transcript expression, suggesting host-microbe crosstalk in response to nutritional stress. Ultimately, these findings demonstrate that balanced macronutrient ratios are required to synchronize host-microbiota metabolic synergy and mitigate physiological stress, providing a definitive mechanistic framework for optimizing industrial black soldier fly rearing outcomes.

Black soldier fly (BSF) larvae are increasingly valued as a sustainable source of proteins and essential minerals in animal feed and potentially food, yet their physiological response to substrate iron fortification is poorly defined. Here, integrated transcriptomic, proteomic, and tissue iron detection approaches were used to characterize responses of BSF larvae reared on diets containing 323 (control), 1255, and 6970 mg Fe/kg dry matter (DM). Larval growth at day 12 was unaffected, while prepupal emergence after 15 days showed a statistically non-significant increase at the highest iron level, suggesting only subtle developmental effects. Prussian blue staining showed a dose-dependent iron accumulation in the midgut epithelium, consistent with known insect iron responsive regions of entoferritin-based sequestration. An elevated iron signal in the peritrophic matrix indicated a complementary defensive barrier. Multi-omics profiling revealed oxidative stress responses, suppression of mitochondrial and translational pathways, and activation of exoskeleton biosynthesis. Entoferritin levels rose by ~70% for both protein subunits despite insignificant transcript changes, pointing to a post-transcriptional regulation mechanism. These results suggest a gut-centered "accumulate-and-store" physiological strategy enabling BSF larvae to tolerate high dietary iron. This entoferritin-based high iron accumulation capacity highlights the potential of this insect as a sustainable source of a protein bound iron in feeds.

Understanding the complex interplay between a host, its diet, and its microbiome is crucial for comprehending an organism's health and adaptability. Diet impacts both the host and microbiome, which then influence each other. We used black soldier fly larvae (Hermetia illucens) as a model to investigate this tripartite interaction due to its resilience and bioconversion capabilities. We analyzed life-history traits and metatranscriptomics in larvae fed three diets: carbohydrate-rich, protein-rich, and balanced. Our results showed that dietary macronutrients correlated with shifts in the microbial community and gene expression. The carbohydrate-rich diet, in particular, led to increased microbial diversity and carbohydrate metabolism transcripts. However, this diet also negatively affected larval weight and development, suggesting potential host control over the microbiome. Overall, black soldier fly performance was highest on the balanced diet. This study highlights the black soldier fly's resilience and its value as a model for exploring host-diet-microbe interactions. ### Competing Interest Statement The authors have declared no competing interest. U.S. National Science Foundation, https://ror.org/021nxhr62, 2052454, 2052565, 2057288

Mycoplasma bovis (M. bovis) causes serious disease in cattle world­wide, characterized by pneumonia, conjunctivitis, arthritis and mastitis. Antemortem diagnostics may help guide management of calves with M. bovis pneumonia, but detailed results have not been described. This report described the clinical and diagnostic progression of a naturally occurring M. bovis outbreak.

BED BUGS: (Hemiptera: Cimicidae) are a globally distributed hematophagous pest that routinely feed on humans. Unlike many blood-sucking arthropods, they have never been linked to pathogen transmission in a natural setting, and despite increasing interest in their role as disease vectors, little is known about the viruses that bed bugs naturally harbor. Here, we present a global-scale survey of the bed bug RNA virosphere. We sequenced the metatranscriptomes of 22 individual bed bugs (Cimex lectularius and Cimex hemipterus) from 8 locations around the world. We detected sequences from two known bed bug viruses (Shuangao bedbug virus 1 and Shuangao bedbug virus 2) which extends their geographical range. We identified three novel bed bug virus sequences from a tenui-like virus (Bunyavirales), a toti-like virus (Ghabrivirales), and a luteo-like virus (Tolivirales). Interestingly, some of the bed bug viruses branch near to insect-transmitted plant-infecting viruses, opening questions regarding the evolution of plant virus infection. When we analyzed the viral sequences by their host's collection location, we found unexpected patterns of geographical diversity that may reflect humans' role in bed bug dispersal. Additionally, we investigated the effect that Wolbachia, the primary bed bug endosymbiont, may have on viral abundance and found that Wolbachia infection neither promotes nor inhibits viral infection. Finally, our results provide no evidence that bed bugs transmit any known human pathogenic viruses.

The mass rearing of animals in close quarters can be highly conducive to microbe transmission, including pathogens. This has been shown multiple times in the case of important industrial insects such as crickets, silkworms, and honeybees. One industrial insect of increasing importance is the black soldier fly (Diptera: Hermetia illucens), as it can convert organic waste into high-quality protein and fatty acids. Along with this, they take up far less space than traditional protein sources, as millions of black soldier flies can be reared in a relatively small facility. Because of this, there is a growing interest in the pathogens that could impact black soldier fly-rearing efforts. So far, only three black soldier fly-associated viruses have been identified. We used metatranscriptomic sequencing to survey black soldier fly guts, frass, and diet for viruses. We detected sequences from two novel viruses. One, which we name Hermetia illucens sigma-like virus 1, is phylogenetically related to viruses of the genus Sigmavirus, which have been highly studied in Drosophila. The other novel virus, which we name Hermetia illucens inse-like virus 1, is the second double-stranded RNA virus of the order Ghabrivirales described in the black soldier fly, and groups within a new family of insect viruses called the Inseviridae. We also detected two black soldier fly-associated viruses previously identified by our group: BSF nairo-like virus 1 and BSF uncharacterized bunyavirus-like 1. Consistent with our previous study, these two viruses are found primarily in frass samples and occur together more often than expected at random. When analyzing host transcription, we found significant differences in gene expression for eight candidate antiviral genes in the black soldier fly when comparing samples with and without viral sequences. Our results suggest that black soldier fly–virus interactions are ongoing, and they could be of interest to black soldier fly producers.

Bovine herpesvirus type 1 (BoHV-1) is an important agricultural pathogen that infects cattle and other ruminants worldwide. Though it was first sequenced and annotated over twenty years ago, the Cooper strain, used in this study, was sequenced as recently as 2012 and is currently said to encode 72 unique proteins. However, tandem mass spectrometry has identified several peptides produced during active infection that align with the BoHV-1 genome in unannotated regions. One of these abundant peptides, “ORF M”, aligned antisense to the DNA helicase/primase protein UL5. This study characterizes the novel transcript and its protein product and provides evidence to support the existence of homolog protein-coding genes in other Herpesviruses.

The black soldier fly (Hermetia illucens, BSF) has emerged as an industrial insect of high promise because of its ability to convert organic waste into nutritious feedstock, making it an environmentally sustainable alternative protein source. As global interest rises, rearing efforts have also been upscaled, which is highly conducive to pathogen transmission. Viral epidemics have stifled mass-rearing efforts of other insects of economic importance, such as crickets, silkworms, and honeybees, but little is known about the viruses that associate with BSF. Although BSFs are thought to be unusually resistant to pathogens because of their expansive antimicrobial gene repertoire, surveillance techniques could be useful in identifying emerging pathogens and common BSF microbes. In this study, we used high-throughput sequencing data to survey BSF larvae and frass samples, and we identified two novel bunyavirus-like sequences. Our phylogenetic analysis grouped one in the family Nairoviridae and the other with two unclassified bunyaviruses. We describe these putative novel viruses as BSF Nairovirus-like 1 and BSF uncharacterized bunyavirus-like 1. We identified candidate segments for the full BSF Nairovirus-like 1 genome using a technique based on transcript co-occurrence and only a partial genome for BSF uncharacterized bunyavirus-like 1. These results emphasize the value of routine BSF colony surveillance and add to the number of viruses associated with BSF.

Each year, bovine respiratory disease (BRD) results in significant economic loss in the cattle sector, and novel metabolic profiling for early diagnosis represents a promising tool for developing effective measures for disease management. Here, 1 H-nuclear magnetic resonance ( 1 H-NMR) spectra were used to characterize metabolites from blood plasma collected from male dairy calves (n = 10) intentionally infected with two of the main BRD causal agents, bovine respiratory syncytial virus (BRSV) and Mannheimia haemolytica (MH), to generate a well-defined metabolomic profile under controlled conditions. In response to infection, 46 metabolites (BRSV = 32, MH = 33) changed in concentration compared to the uninfected state. Fuel substrates and products exhibited a particularly strong effect, reflecting imbalances that occur during the immune response. Furthermore, 1 H-NMR spectra from samples from the uninfected and infected stages were discriminated with an accuracy, sensitivity, and specificity ≥ 95% using chemometrics to model the changes associated with disease, suggesting that metabolic profiles can be used for further development, understanding, and validation of novel diagnostic tools.

Bovine respiratory syncytial virus (BRSV) is a major contributor to respiratory disease in cattle worldwide. Traditionally, BRSV infection is detected based on non-specific clinical signs, followed by reverse transcriptase-polymerase chain reaction (RT-PCR), the results of which can take days to obtain. Near-infrared aquaphotomics evaluation based on biochemical information from biofluids has the potential to support the rapid identification of BRSV infection in the field. This study evaluated NIR spectra (n = 240) of exhaled breath condensate (EBC) from dairy calves (n = 5) undergoing a controlled infection with BRSV. Changes in the organization of the aqueous phase of EBC during the baseline (pre-infection) and infected (post-infection and clinically abnormal) stages were found in the WAMACS (water matrix coordinates) C1, C5, C9, and C11, likely associated with volatile and non-volatile compounds in EBC. The discrimination of these chemical profiles by PCA-LDA models differentiated samples collected during the baseline and infected stages with an accuracy, sensitivity, and specificity >93% in both the calibration and validation. Thus, biochemical changes occurring during BRSV infection can be detected and evaluated with NIR-aquaphotomics in EBC. These findings form the foundation for developing an innovative, non-invasive, and in-field diagnostic tool to identify BRSV infection in cattle.

This study evaluated near infrared (NIR) spectra (n = 970) of nasal secretions (NS) from dairy calves (n = 5) challenged with bovine respiratory syncytial virus (BRSV). This pathogen is a common cause of respiratory disease in young calves worldwide and is typically diagnosed by evaluation of the clinical signs, followed by time-consuming serological and molecular methods. More rapid diagnostic methods could improve outcomes for infected calves. The near infrared aquaphotomics evaluation of this biofluid unveiled changes between the spectra (1300–1600 nm) of samples collected during the uninfected (n = 200) and infected (n = 200) stages, specifically identified in the WAMACS (water matrix coordinates) C1, C9, C10, and C11, where water molecules are highly associated with chaotropic solutes in water asymmetrical stretching vibrations (ν3) and with kosmotropic solutes in water clusters with 2, 3, and 4 hydrogen bonds (S2, S3, S4). These chemical differences were discriminated by PCA-LDA using a leave-one-animal-out approach with averaged percentages of accuracy, sensitivity, and specificity of 90.1 ± 4.3, 88.1 ± 3.8, 92.0 ± 5.5 in the calibration process, respectively. By collecting spectra from nasal secretions, we revealed the potential of NIR spectroscopy in combination with aquaphotomics and chemometrics for the detection of this viral infection in-vivo; as a first step toward developing a rapid in-field diagnostic tool for BRSV infection.

Bovine Respiratory Disease (BRD) is a multifactorial condition affecting cattle worldwide resulting in high rates of morbidity and mortality. The disease can be triggered by Bovine Herpesvirus-1 (BoHV-1) infection, stress, and the subsequent proliferation and lung colonization by commensal bacteria such as Mannheimia haemolytica, ultimately inducing severe pneumonic inflammation. Due to its polymicrobial nature, the study of BRD microbes requires co-infection models. While several past studies have mostly focused on the effects of co-infection on host gene expression, we focused on the relationship between BRD pathogens during co-infection, specifically on M. haemolytica's effect on BoHV-1 replication. This study shows that M. haemolytica negatively impacts BoHV-1 replication in a dose-dependent manner in different in vitro models. The negative effect was observed at very low bacterial doses while increasing the viral dose counteracted this effect. Viral suppression was also dependent on the time at which each microbe was introduced to the cell culture. While acidification of the culture medium did not grossly affect cell viability, it significantly inhibited viral replication. We conclude that M. haemolytica and BoHV-1 interaction is dose and time-sensitive, wherein M. haemolytica proliferation induces significant viral suppression when the viral replication program is not fully established.

In this work, a long-read sequencing (LRS) technique based on the Oxford Nanopore Technology MinION platform was used for quantifying and kinetic characterization of the poly(A) fraction of bovine alphaherpesvirus type 1 (BoHV-1) lytic transcriptome across a 12-h infection period. Amplification-based LRS techniques frequently generate artefactual transcription reads and are biased towards the production of shorter amplicons. To avoid these undesired effects, we applied direct cDNA sequencing, an amplification-free technique. Here, we show that a single promoter can produce multiple transcription start sites whose distribution patterns differ among the viral genes but are similar in the same gene at different timepoints. Our investigations revealed that the circ gene is expressed with immediate–early (IE) kinetics by utilizing a special mechanism based on the use of the promoter of another IE gene (bicp4) for the transcriptional control. Furthermore, we detected an overlap between the initiation of DNA replication and the transcription from the bicp22 gene, which suggests an interaction between the two molecular machineries. This study developed a generally applicable LRS-based method for the time-course characterization of transcriptomes of any organism.

Each year, Bovine Respiratory Disease (BRD) results in significant economic loss in the cattle sector, and novel metabolic profiling and early diagnosis techniques represent a promising tool for developing effective measures for disease management. Here, proton - Nuclear Magnetic Resonance ( 1 H - NMR) spectra were used to characterize metabolites from blood plasma collected from dairy calves intentionally infected with the main BRD causal agents, bovine respiratory syncytial virus (BRSV) and Mannheimia haemolytica (MH), to generate a well-defined metabolomic profile under controlled conditions. In response to infection, 42 metabolites (BRSV = 27, MH = 24) changed in concentration compared to the Baseline (non-infected) state. Fuel substrates and products exhibited a particularly strong effect, reflecting imbalances that occur during the immune response. Glucose levels decreased only during bacterial infection, suggesting that the clinical signs of bacterial BRD are more energetically taxing than those of viral BRD. Furthermore, 1 H - NMR spectra from Baseline and Infected samples were discriminated with an accuracy, sensitivity, and specificity ≥ 95% using chemometrics to model the changes associated with disease, suggesting that metabolic profiles can be used for further development and validation of diagnostic tools.

Fire ants (Solenopsis spp.) have increasingly been reported from carrion in the southeastern United States and are now a part of the normal succession community. There have been previous observations of these ants altering carrion and preying on other carrion-attendant fauna; however, the overall effects of these activities on carrion decomposition rates, community composition, and blow fly larval development are poorly understood. Alteration of these ecological processes by fire ants could affect the forensic interpretation of entomological data. We conducted a study in Mississippi and Florida whereby portions of the succession fauna were excluded from access to pig carrion to study the relative effects of fire ants and blow flies on carrion decomposition and succession: a control with all fauna having access, a second treatment where fire ants and other geophilic taxa were excluded, and a third treatment in which blow flies and other large organisms were excluded. Fire ants inflicted lesions in the carrion, buried portions that touched the ground, and preyed on some members of the succession fauna. Their exclusion did not affect carrion decomposition rates that were measured but slightly affected the overall carrion community, and strongly affected the oviposition and development of blow flies. Despite the presence of fire ants early in the control, blow flies were eventually able to overcome predation of eggs and larvae, continue colonization, and complete development; however, the delay in the colonization of blow flies on carrion could affect the determination of postmortem intervals when development rates of blow flies are considered in the calculation.

During a recent pig carrion succession study in northwest Florida, United States, we noticed an abundance of spiders but found that literature on spider ecology at carrion is sparse and scattered. We compiled a literature review of 569 carrion succession studies, of which 37 studies specifically mentioned the presence of spiders, with less than a third providing species-level identifications and only half providing family-level identifications. Nineteen spider families have been reported at carrion in the literature. Spiders are recognized as generalist, opportunistic predators in carrion succession studies, but only 38% of the studies that mentioned spiders during carrion succession included any ecological information. Data on spiders in the present experiment were compared to background samples, finding that the difference in abundance was statistically significant for all spider species combined. Seven species of spiders from five families were identified from both background and carrion succession samples; statistical differences in abundance were found for three species: the linyphiid Florinda coccinea and the lycosids Tigrosa annexa and Pirata seminolus. The family Corinnidae is reported from carrion for the first time. This research reviews the existing literature on spider ecology during carrion succession, provides additional data on species identity and abundance, and demonstrates the potential significance of spiders in carrion ecology and forensic investigations.

Bovine respiratory disease (BRD) linked with Mannheimia haemolytica is the principal cause of pneumonia in cattle. Diagnosis of BRD traditionally relies on visual assessment, which can be untimely, insensitive, and nonspecific leading to inadequate treatment and further spread of disease. Near Infrared Spectroscopy (NIRS) is a rapid acquisition vibrational spectroscopy that can profile changes in biofluids, and when used in combination with multivariate analysis, has potential for disease diagnosis. This study characterizes the NIR spectral profile of blood plasma from dairy calves infected with M. haemolytica and validates the spectral biochemistry using standardized clinical and hematological reference parameters. Blood samples were collected for four days prior to (baseline), and 23 days after, a controlled intrabronchial challenge. NIR spectral profiles of blood plasma discriminated and predicted Baseline and Infected states of animal disease progression with accuracy, sensitivity, and specificity ≥ 90% using PCA–LDA models. These results show that physiological and biochemical changes occurring in the bloodstream of dairy calves during M. haemolytica infection are reflected in the NIR spectral profiles, demonstrating the potential of NIRS as a diagnostic and monitoring tool of BRD over time.

We present an annotated list of 22 species of blow flies (Diptera: Calliphoridae) and one species of cluster flies (Polleniidae) known from the state of Mississippi, with one additional blow fly species likely to occur there based on a record in neighboring Alabama. New state records include five species from the subfamily Calliphorinae: Angioneura flavescens (Reinhard), Angioneura obscura (Townsend), Opsodexia bicolor (Coquillett), and Opsodexia nox (Downes), and Lucilia eximia (Wiedemann). The number and density of taxa per 100,000 km(2) is consistent with that of blow fly taxa recorded for other states across the contiguous United States.