CJVR - April 2023, Vol. 87, No. 2



Effects of concentrated fecal microbiota transplant on the equine fecal microbiota after antibiotic-induced dysbiosis

Rebecca Di Pietro, Luis G. Arroyo, Mathilde Leclere, Marcio Costa (page 85)

Bacterial imbalances are observed in intestinal diseases and fecal microbiota transplantation (FMT) has been used to restore the intestinal microbiota of horses. However, there is evidence that the current methods proposed for FMT in horses have limited efficacy. The objective of this study was to concentrate the bacteria present in the donor stool by centrifugation, and to test the effect in horses with antibiotic-induced dysbiosis. One healthy 11-year-old horse was selected as a fecal donor and 9 horses were given trimethoprim sulfadiazine (TMS) for 5 days to induce dysbiosis. Horses received either a concentrated FMT (cFMT, n = 3), fresh unconcentrated FMT (fFMT, n = 3), or 10% glycerol solution (vehicle, VEH, n = 3) by nasogastric tube for 3 days. Fecal samples were collected on Days 0, 4, 9, 11, and 21 for microbiota analysis (Illumina sequencing). The TMS significantly changed the bacterial composition of horses’ feces (D0 versus D4). The composition of the cFMT and fFMT recipient horses was significantly different after transplantation compared to after antibiotic-induced dysbiosis (D4 versus D11), whereas the microbiota of the vehicle recipients was not, indicating that both protocols induced transient changes. However, preparation of FMT solutions markedly changed the original composition present in the donor’s feces, with significant enrichment of Escherichia genus in the cFMT. Individual susceptibility to restoration of the microbiota was observed in horses, similar to what is known for other species. Our results suggest that concentrating bacteria should not be recommended in preparation of FMT solutions and that further research is required to improve current methods recommended to perform FMT in horses.

Effect of dietary iron supplementation on the equine fecal microbiome

Julia Assis Arantes, Alexandre Secorun Borges, Luiza Stachewski Zakia, Michael Gordon Surette, Jeffrey Scott Weese, Marcio Carvalho Costa, Luis Guillermo Arroyo (page 97)

Iron is an essential element for all living organisms, including bacteria, as several virulence factors and replication components are influenced by iron concentration. The objective of this study was to determine whether the composition and diversity of the fecal microbiota of adult horses are affected by supplemental dietary iron. Ten clinically healthy horses were randomly divided into a control and an iron-supplemented group (n = 5). The treated group was supplemented with oral ferrous sulphate monohydrate (720 ppm of iron), whereas the control group received 320 ppm of iron daily for 15 d. Fecal samples were collected before and 5, 10, 15, and 30 d after supplementation and frozen at −80°C. DNA was sequenced using an Illumina MiSeq platform and data were analyzed using the software Mothur and linear discriminant analysis (LDA) effect size (LEfSe). Iron supplementation caused no change in the overall composition of the fecal microbiota, but some minor changes were observed in the low-abundant bacteria, as well as an increased alpha diversity after 15 d of supplementation. Significant differences in community composition of the fecal microbiota over time were observed in both groups, highlighting the importance of a control group, as there are variables that cannot be controlled in microbiome studies.

Presence of co-infection between bovine leukemia virus and bovine herpesvirus 1 in herds vaccinated against bovine respiratory complex

Ana S. González Méndez, Fernando Cerón-Téllez, Rosa E. Sarmiento Silva, Jorge L. Tórtora Pérez, Edith Rojas-Anaya, Hugo Ramírez Álvarez (page 105)

The aim of this study was molecular identification of bovine leukemia virus and possible co-infection with bovine respiratory disease complex (BRDC) viral agents in Mexican dairy herds. We collected 533 blood samples from cattle vaccinated against the BRDC virus in 9 states across Mexico. Peripheral blood leukocytes were removed and genetic material was extracted to detect bovine leukemia virus (BLV), bovine herpesvirus 1 (BoHV-1), bovine viral diarrhea virus (BVDV), bovine parainfluenza virus 3 (BPIV-3), and bovine respiratory syncytial virus (BRSV) infection using polymerase chain reaction. We identified high BLV infection rates in 270 cattle (50.65%). One hundred and thirty-three cows (24.95%) tested positive for BoHV-1, of which 65 samples were positive for both viruses (BoHV-1 and BLV) and 68 were only positive for BoHV-1. Only 4 samples tested positive for BPIV-3 and no sample was positive for BVDV or BRSV. Relative risk and odds ratio analyses did not identify that the presence of BLV infection favors BoHV-1 co-infection in vaccinated herds.

Response of lymphocytes from pigs naturally infected with porcine respiratory disease complex at 3 different stages of development

Rosa Elvira Nuñez-Anita, Fernando Calderón-Rico, Francisco Pérez-Duran, María Concepción Arenas-Arrocena, Alicia Gabriela Zamora-Avilés, Luis Enrique Franco-Correa, Alejandro Bravo-Patiño, Ilane Hernández-Morales (page 110)

The objective of this study was to analyze the response of lymphocytes from pigs naturally infected with porcine respiratory disease complex (PRDC) at 3 different stages of development. Porcine respiratory disease complexes were isolated from 2 groups: The infected group, consisting of pigs with PRDC and no vaccination against any virus (n = 24), and the control group, consisting of vaccinated and noninfected piglets (n = 24). Both groups were sampled at 3 stages of development: Weaning (WEA) (n = 8), initiation (INI) (n = 8), and growth (GRO) (n = 8). The PRDC status was confirmed by serological testing against porcine circovirus type 2 (PCV-2), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (H1N1), and Mycoplasma hyopneumoniae. PCV-2+ cells were quantified by flow cytometry. Weight gain was registered at each stage. PCV-2+ cells, CD4+ cells, monocytes and lymphocytes populations were measured. Gene expression in CD4+ cells was quantified for interferon-γ (IFN-γ), GATA binding protein 3 (GATA3), T-box transcription factor (T-bet), interleukin-10 (IL-10), and IL-4. Control piglets gained approximately 35% more weight than those infected with PRDC. Specifically, PCV-2+ cells were detected in piglets from the infected group in the following proportions: WEA ≤ INI ≤ GRO. In infected piglets, the CD4+ count increased at WEA and decreased at GRO, CD4+ expression profile showed an overexpression of T-bet at INI and GRO, and the expression of IFN-γ was lower at WEA and GRO. In contrast, IL-4 was overexpressed at all 3 stages. GATA3 was overexpressed at INI and GRO. The infected piglets showed lymphopenia and less CD4+ cells. CD4+ cells showed a different expression profile than the control group, in which IFN-γ was less expressed, whereas IL-4 and T-bet were overexpressed.

Seneca Valley virus induces proinflammatory cytokine and chemokine response in vitro

Jianguo Dong, Mingrui Chen, Linyang Yu, Dan Rao, Ning Zhang, Feng Cong (page 120)

Seneca Valley virus (SVV) is an oncolytic virus, which belongs to the Picornaviridae family, that causes blisters on the nose and hooves, affecting the production performance of pigs. However, the function of proinflammatory cytokines and chemokines in SVV infection is still unclear. In our study, SVV infection could induce a high expression of proinflammatory cytokines interleukin (IL)-1α, IL-1β, and tumor necrosis factor α (TNF-α) and chemokines, including chemokine (C-C motif) ligand 2 (CCL2), chemokine (C-C motif) ligand 5 (CCL5), and chemokine (C-X-C motif) ligand 10 (CXCL10). Interfered genes of IL-1α, IL-1β, and TNF-α inhibited virus replication, but interfered genes of CCL2, CCL5, and CXCL10 promoted virus replication. These results indicate that proinflammatory cytokines and chemokines are involved in SVV infection; this will be beneficial to explore the pathogenesis and cytokine therapy of SVV.

Identification and characterization of potential probiotic lactic acid bacteria isolated from pig feces at various production stages

José D. Uezen, Cecilia Aristimuño Ficoseco, María E. Fátima Nader-Macías, Graciela M. Vignolo (page 127)

Lactic acid bacteria (LAB) were isolated, identified, and characterized from pig feces at various growth stages and feed rations in order to be used as probiotic feed additives. Lactic acid bacteria numbers ranged from 7.10 ± 1.50 to 9.40 log CFUs/g for growing and lactating pigs, respectively. Isolates (n = 230) were identified by (GTG)5-polymerase chain reaction and partial sequence analysis of 16S rRNA. Major LAB populations were Limosilactobacillus reuteri (49.2%), Pediococcus pentosaceus (20%), Lactobacillus amylovorus (11.4%), and L. johnsonii (8.7%). In-vitro assays were performed, including surface characterization and tolerance to acid and bile salts. Several lactobacilli exhibited hydrophobic and aggregative characteristics and were able to withstand gastrointestinal tract conditions. In addition, lactobacilli showed starch- and phytate-degrading ability, as well as antagonistic activity against Gram-negative pathogens and the production of bacteriocin-like inhibitory substances. When resistance or susceptibility to antibiotics was evaluated, high phenotypic resistance to ampicillin, gentamicin, kanamycin, streptomycin, and tetracycline and susceptibility towards clindamycin and chloramphenicol was observed in the assayed LAB. Genotypic characterization showed that 5 out of 15 resistance genes were identified in lactobacilli; their presence did not correlate with phenotypic traits. Genes erm(B), strA, strB, and aadE conferring resistance to erythromycin and streptomycin were reported among all lactobacilli, whereas tet(M) gene was harbored by L. reuteri and L. amylovorus strains. Based on these results, 6 probiotic LAB strains (L. reuteri F207R/G9R/B66R, L. amylovorus G636T/S244T, and L. johnsonii S92R) can be selected to explore their potential as direct feed additives to promote swine health and replace antibiotics.

The impact of freezing and multiple freeze-thaw cycles on Brix refractometry estimates of immunoglobulin concentration in beef cattle colostrum

Jacqueline M. Stalker, Lisa Gamsjäger, Jennifer M. Pearson, Douglas W. Morck, M. Claire Windeyer (page 146)

Evaluation of immunoglobulin G (IgG) concentration in colostrum is important to guide on-farm management. Studies have shown that digital Brix refractometry accurately estimates colostrum IgG concentration in both dairy and beef cattle colostrum. Colostrum is often frozen in both clinical and research settings. The implications of this freezing on the accuracy of Brix refractometry measurements are largely unknown. The first objective of this study was to evaluate the agreement between digital Brix percentage measurements of IgG in beef cattle colostrum taken before and after different durations of freezing. The second objective was to evaluate the effects of multiple freeze-thaw (FT) cycles on Brix percentage measurements of IgG in beef cattle colostrum. There was good agreement between Brix percentages in fresh colostrum and after short (2 to 8 d), medium (4 to 7 mo), and long (3 y) periods of freezing (concordance correlation coefficient: 0.95, 0.96, and 0.96, respectively). Although there was no significant change in mean Brix percentages over 2 FT cycles (P > 0.05), mean Brix percentages decreased with 3 FT cycles (P = 0.017). Samples from the fourth and fifth FT cycles were observably coagulated, and these measurements were therefore deemed inaccurate. Data from this study indicate that freezing had minimal impact on digital Brix refractometer estimates of IgG concentration in beef cattle colostrum, but that samples stored for future testing should not undergo more than 2 FT cycles.

Administration safety of medical-grade honey (MGH) in septic synovial structures in horses: 3 cases

Janine A. Terschuur, Richard P.C. Coomer, Shaun A. McKane (page 153)

The objective of this study was to investigate the postoperative use of intrasynovial honey as an antimicrobial after treatment for synovial sepsis in horses. One colt and 2 mares were presented with acute lameness, with or without an associated wound. All 3 cases were initially managed with surgical endoscopic or tenoscopic debridement and lavage for treatment of different synovial structures. Collection of synovial fluid was consistent with synovial sepsis and this was diagnosed in each case. All horses subsequently underwent arthroscopic lavage under general anesthetic and intraarticular or intrathecal medical-grade honey (MGH) was then instilled. All 3 cases recovered well and were free from lameness at all gaits. Although there is extensive research about the antimicrobial properties of honey and growing interest in the biocompatibility of honey in joints with the use of honey hydrogels in human medicine, the research in veterinary medicine is lacking. There are studies describing the antimicrobial properties of honey in healing wounds in horses, but there are no published studies describing the use of honey within a synovial structure. Further research is necessary to assess the biocompatibility of honey in equine articular cartilage. In the cases described in this article, the use of honey demonstrated a safe adjunctive therapy after conventional surgical treatment for septic arthritis.

Evaluating the outcome after center of rotation of angulation (CORA)-based leveling osteotomy (CBLO) technique to repair unilateral cranial cruciate ligament deficiency using a pressure-sensitive walkway system

Ömer Coskun, Sivert Viskjer (page 157)

The objective of this study was to evaluate the mid-term outcome of center of rotation of angulation (CORA)-based leveling osteotomy (CBLO) in dogs with cranial cruciate ligament (CrCL) deficiency using a pressure-sensitive walkway system. Fifteen dogs with unilateral CrCL deficiency were treated with CBLO for an average of 50 wk (median 46 wk, range: 38 to 91) prior to the evaluation. The contralateral hind limb was confirmed free of any signs of pathology by clinical examination and lateral stifle radiography. A control group of 20 healthy dogs was included to establish reference values for comparison purposes. Spatiotemporal parameters and peak vertical force (PVF) were measured and symmetry index (SI) was calculated between the left and right pelvic limb and between thoracic and pelvic limbs, in both groups. The mean hind limb SI for the 15 CBLO-treated dogs and the 20 dogs in the control group was 1.02 ± 0.1 and 1.03 ± 0.07, respectively, the difference being not significant (P = 0.75). There was no significant difference in the thoracic limb/pelvic limb ratio between the 2 groups (P = 0.42). The dogs’ recovery was objectively measured on a pressure-sensitive walkway and the CrCL-deficient dogs had returned to full functioning within 6 to 12 mo. Center of rotation of angulation-based leveling osteotomy provided normal function of the operated hind limb and should be considered as an option for treating canine CrCL deficiency.