Fecal microbiome profiles of neonatal dairy calves with gastrointestinal disease

Primary author: Giovana Slanzon
Co-author(s): Lindsay Parrish; Sophie Trombetta; William Sischo; Craig McConnel
Faculty sponsor: Dr. Craig Stephen McConnel

Primary college/unit: College of Veterinary Medicine
Campus: Pullman

Abstract:

Gastrointestinal disease is the most common illness in preweaned dairy calves. Studies have associated fecal microbiome composition with health status, but changes in the microbiome across varying levels of GI disease remain unclear. Our objective was to show a correlation between clinical symptoms and shifts in the microbiome composition. Calves (n=360) were enrolled in a sampling scheme of repeat fecal collections throughout 5-15 d of age. Daily evaluations assessed health status based on clinical signs and treatments. Calves with fecal scores of 3 (loose) or 4 (watery) were diagnosed with diarrhea and classified as bright-sick (BS) or depressed-sick (DS) according to their behavior and appetite. Calves with fecal scores <3 and no clinical illness were classified as healthy. Sixty-one fecal samples from healthy calves and 30 samples from diarrheic calves were selected to represent different breeds and ages. The V3-V4 region of the 16S rRNA gene was sequenced and analyzed using DADA2 pipeline. The ASV table was used to compare the taxonomic profiles, differences were identified by LEfSe (p2). Healthy calves had greater enrichment of the families Bifidobacteriaceae, Bacteroidaceae, Prevotellaceae, Eubacteriaceae, Ruminococcaceae, Erysipelotrichaceae. In BS calves Listeriaceae, Clostridiaceae and Lachnospiraceae were more enriched. DS calves had enriched Lactobacillaceae, Streptococcaceae and Enterobacteriaceae in their microbiome. Breed differences included the association of Bacteroidaceae, Eubacteriaceae, Erysipelotrichaceae and Fusobacteriaceae with healthy Jersey calves. Bifidobacteriaceae was more enriched in healthy Holstein calves. On the other hand, Enterobacteriaceae was associated with DS Holstein calves. For DS Jersey calves, the order Lactobacillales was enriched.

The Sea as a Source of Novel Antiparasitic Compounds

Primary author: Rachel Relat
Faculty sponsor: Roberta O’Connor

Primary college/unit: College of Veterinary Medicine
Campus: Pullman

Abstract:

Cryptosporidium, an intracellular parasite that causes severe diarrhea, is now recognized as a leading cause of waterborne disease worldwide, infecting both humans and valuable livestock. Cryptosporidium infection is particularly devastating in children as it causes life-threatening diarrhea, along with developmental delays including growth stunting and cognitive impairment. Cryptosporidium is an especially important disease of immunocompromised individuals of all ages, leading to chronic, potentially fatal diarrhea. No effective treatment exists for many human or veterinary patients diagnosed with Cryptosporidium.

To address this medical imperative, we designed a high throughput screen to test thousands of unique compounds, with the goal of discovering new, potent, anti-Cryptosporidium drugs and drug scaffolds. Our collaborators at the Harbor Branch Oceanographic Institute (HBOI) have amassed a library of over 125 natural compounds, and 6500 highly enriched fractions from extracts of marine organisms. We began screening this library as it contains compounds and fractions produced by sessile oceanic animals which frequently use chemical defenses. Additionally, numerous sessile oceanic organisms are known to have endosymbionts that are likely to produce additional bioactive metabolites.

We screened greater than 3,600 partially purified extracts from the HBOI library and identified 10 extracts that effectively inhibit Cryptosporidium growth without damaging the host cells in which these parasites live. Ongoing studies indicate that some of the identified fractions may also inhibit the growth of Toxoplasma gondii, a closely related parasite. This work will significantly contribute to the goal of discovering effective treatments against Cryptosporidium specifically, and further the search for novel antiparasitic drugs.

Identification of two noncanonical members to the CCp gene family of Babesia bovis and Babesia bigemina

Primary author: Sezayi OZUBEK
Co-author(s): Heba F. Alzan; Reginaldo G. Bastos; Carlos E. Suarez
Faculty sponsor: Carlos Suarrez; Scientific and Technological Research Council of Turkey(TUBITAK)

Primary college/unit: College of Veterinary Medicine
Campus: Pullman

Abstract:

Babesia bovis and B. bigemina are responsible for bovine babesiosis, a tick borne disease that causes important economic losses in many tropical and semi-tropical regions worldwide. The CCp protein family, containing LCCL motifs, is differentially expressed on gametocytes of apicomplexans, including Plasmodium and Babesia. Knocking out Plasmodium CCp genes blocks development of the parasite in the mosquito vector, making the CCp proteins potential targets for transmission-blocking vaccines. Although six LCCL protein family members (CCp1-5 and FNPA) have been identified in Plasmodium falciparum, only genes encoding for homologous of the CCp1-3 proteins have so far been reported in B. bovis and B. bigemina. In this study, we describe the presence of two additional CCp-related genes (CCp5 and FNPA) in the genomes of B. bovis and B. bigemina. Similar to other apicomplexan CCp proteins, the newly identified CCp proteins also have a signal peptide (B. bigemina CCp5 and FNPA) and a transmembrane domain (B. bovis FNPA), consistent with surface expression. Consistent with their putative role during sexual reproduction of the parasite in the tick gut, RNAseq analysis sugests that none of the CCp genes are highly expressed in the blood stage, and at negligible levels in kinete stages. The pattern of expression of the new CCp members during distinct developmental stages of B. bovis and B. bigemina will be explored in further studies.

Natural Genetic Variation in the Drosophila Genetic Reference Panel Reveals Genes Associated with Susceptibility or Tolerance to Coxiella burnetii Infection

Primary author: Rosa Guzman
Faculty sponsor: Alan Goodman

Primary college/unit: College of Veterinary Medicine
Campus: Pullman

Abstract:

The Gram-negative bacterium Coxiella burnetii (C. burnetii) is the causative agent of Query (Q) fever in humans and coxiellosis in livestock. Association between host genetic background and C. burnetii pathogenesis has been demonstrated both in humans and animals; however, specific genes associated with severity of infection remain unknown. Our lab has shown that the Drosophila melanogaster model is suitable for studying host-pathogen interactions during C. burnetii infection. In this study, we employed the Drosophila Genetics Reference Panel (DGRP), a library of 209 inbred wild-type fly lines, to identify host genetic variants that affect susceptibility or tolerance to C. burnetii infection. We infected each DGRP line, monitored survival, and calculated a hazard ratio for each line to perform a genome-wide association study (GWAS). We tested 25 GWAS candidate genes by infecting flies carrying a null mutation or RNAi knockdown of each gene and monitoring survival. Of these 25 genes, 6 validated in null mutants only, 5 in RNAi knockdown only, and 4 in both null mutants and RNAi. One gene that validated in both null mutants and RNAi knockdown flies was taranis, which is essential for regeneration and JNK signaling. The human ortholog of taranis is SERTAD1, which has been recently linked to immunity. Our results demonstrate that from genes identified and validated in our genetic screen, mechanism of action studies should be performed to determine the genes’ role in the host immune response to infection.

Type IV Secretion System Effectors of Anaplasma phagocytophilum

Primary author: Deirdre Fahy
Co-author(s): Jason Park; Michael Dodd; Kelly Brayton

Primary college/unit: College of Veterinary Medicine
Campus: Pullman

Abstract:

Anaplasma phagocytophilum (Ap) is a tick-transmitted obligate intracellular zoonotic pathogen. Ap achieves intracellular infection by delivering effector proteins into the host cytosol utilizing a Type IV secretion system (T4SS). In contrast to other intracellular pathogens e.g. Legionella pneumophilia and Coxiella burnetii, where 100s of T4SS effectors have been found, only 4 have been identified in Ap. Our goal was to extend the repertoire of known Ap effectors. To predict effectors, we used the recently published T4SS effector prediction software OPT4e to identify Ap proteins containing features characteristic of T4SS substrates. To screen for T4SS translocation, effector candidates were fused to a reporter enzyme, which if translocated to the host cell cytoplasm generates a product detectable by ELISA. To overcome the genetic intractability of Ap, fusion constructs were assayed for translocation by the heterologous T4SS of Legionella pneumophila. Although only 7 of 70 screened proteins were translocated in a T4SS dependent manner, there are likely more effectors since a negative result may be due to the heterologous nature of the reporter system. Three of the translocated substrates localize to the host cell nucleus when ectopically expressed. Bioinformatic analysis identified putative C-terminal nuclear localization signals (NLS) in all three of these T4SS substrates. Deletion of these NLS sequences abrogated both nuclear-specific localization and also T4SS translocation, which is consistent with the requirement for specific C-terminal features in translocation of known effectors. Functional characterization of these and other effectors will inform their role in Ap replication, pathogenesis, and transmission.

Construction of a Recombinant Bovine Herpesvirus 4 as a Delivery System for Ovine Herpesvirus 2 Glycoprotein B

Primary author: Emily Cole
Faculty sponsor: Dr. Cristina Cunha

Primary college/unit: College of Veterinary Medicine
Campus: Pullman

Abstract:

Ovine herpesvirus 2 (OvHV-2) causes a frequently fatal disease called malignant catarrhal fever (MCF) in several ungulates, such as bison, cattle, pigs and deer. Vaccine development is a major goal for MCF research because there are no treatment options. However, the inability to propagate OvHV-2 in in-vitro systems has hindered the development of a vaccine. Since the virus cannot be modified or attenuated in-vitro, alternative approaches for delivering OvHV-2 antigens for immunization are of utmost importance. OvHV-2 glycoproteins, gB, gH, and gL, are necessary for viral entry and can stimulate neutralizing antibody responses capable of protecting animals from disease. This makes them ideal vaccine candidates. Additionally, bovine herpesvirus 4 (BoHV-4) has been evaluated as a vaccine vector for several viral diseases with promising results in delivering heterologous antigen that confer immunity.The purpose of this study was to construct and evaluate a recombinant BoHV-4 for the expression of OvHV-2 gB. To do this, the bacterial artificial chromosome recombineering galK selection system was used and confirmed with PCR, sequencing, and restriction enzyme digestion. Then viral growth curves were used to assess reconstitution of the infectious virus in various cell types. Also, western-blot analysis and immune-fluorescence assays were used to confirm OvHV-2 gB expression. The construction of this recombinant BoHV-4 virus will allow further MCF research regarding vaccine efficacy, as this vector virus could provide a means of delivering OvHV-2 gB in vivo. Vaccine development is vital considering this is an untreatable, global disease that economically effects agriculture, particularly bison production.