Introduction: Vascular cell adhesion molecule 1 (VCAM-1) is a member of Ig superfamily. The aim of this study was to prepare highly specific polyclonal antibodies against bovine VCAM-1 and to evaluate the expression of VCAM-1 in the mammary lymph nodes of cows with subclinical mastitis.Material and Methods: The VCAM-1 gene was cloned from bovine Peyer’s patches and inserted into the pGEX-4T-1 and pET-28a vectors. The recombinant plasmids pGEX-4T-1/VCAM-1 and pET-28a/VCAM-1 were transferred into Escherichia coli BL21 and the recombinant strains were induced by isopropyl-D-thiogalactoside to produce fusion proteins tagged with polyhistidine (His) and glutathione S-transferase (GST), respectively. The expressed fusion proteins His-VCAM-1 and GST-VCAM-1 were identified by SDS-PAGE and Western blot. His-VCAM-1 protein was used as an antigen to immunise Wistar rats and polyclonal antibody serum against VCAM-1 was obtained.Results: The serum titre tested by indirect ELISA was 128,000 using GST-VCAM-1 as the well coating antigen. Western blots indicated that the antibody recognised recombinant VCAM-1 protein as well as endogenous VCAM-1. In addition, using qPCR and Western blot, VCAM-1 mRNA and protein expression levels were measured in dairy cows with subclinical mastitis. It was demonstrated that VCAM-1 levels in the mammary lymph nodes of the cows were significantly higher than those from healthy controls (P < 0.05).Conclusion: These results are to our knowledge the first report that VCAM-1 expression in the mammary lymph nodes is elevated in dairy cows with subclinical mastitis.

Introduction: The aim of this study was to use TaqMan real-time PCR technique to investigate Jaagsiekte sheep retrovirus (JSRV) proviral DNA in whole blood samples of sheep, and compare the results to those of histopathological examinations. Material and Methods: Eighty blood samples from clinically healthy sheep were randomly collected before the animals were slaughtered. Ten tissue samples from each lung and associated caudal mediastinal lymph node were taken. Results: Fifteen (18.75%) blood samples were found to contain proviral DNA, and 11 (13.75%) corresponding lung samples showed microscopic changes consistent with ovine pulmonary adenocarcinoma. None of the samples displayed metastases to the caudal mediastinal lymph nodes. The prominent pattern of neoplastic nodules consisted of acinar (alveolar) form. Conclusion: The results indicated the higher sensitivity of real-time PCR compared to histopathological examinations in detection of ovine pulmonary adenocarcinoma.

Bovine tuberculosis, caused by M. bovis, is endemic in Mexico and has had a big impact on public health. Jalisco is considered to be an important dairy region in the country, accounting for approximately 19% of the total milk production. Within Jalisco, the region of Altos Sur holds the largest proportion of the cattle inventory of the state. To determine the frequency of bovine tuberculosis in Altos Sur, Jalisco, as well as M. bovis genetic diversity, sampling of tissue (lymph nodes, lungs, and liver) from Holstein cattle was performed in four abattoirs belonging to three municipalities of this region (Tepatitlán de Morelos, San Miguel el Alto, and Arandas). Spoligotyping and whole-genome sequencing were carried out to assess the genetic relationships of M. bovis strains circulating in this area, as well as a comparison to isolates from other places in Mexico. Prevalence was 15.06%, and distribution similar among the three municipalities. The most frequent spoligotypes were SB0673, SB121, and SB0145. Whole-genome sequencing revealed three main clades (I, II, III), but isolates did not show clustering by region. Phylogenetic analysis suggested ongoing transmission between herds of the different regions, and no unique source of infection was determined. This hinders efforts under the national program for the control and eradication of the disease, so serious attention must be paid to rural regions such as Altos Sur in order to improve its success.

OBJECTIVE To characterize abdominal lymphatic drainage in cats after thoracic duct ligation (TDL) and cisterna chyli ablation (CCA). ANIMALS 7 purpose-bred research cats. PROCEDURES Baseline CT lymphangiography was performed. A popliteal lymph node was injected with iohexol, and images were acquired at 5-minute intervals for 15 minutes. Cats underwent TDL and CCA; methylene blue was used to aid in identifying lymphatic vessels. The CT lymphangiography was repeated immediately after and 30 days after surgery. All cats were euthanized and necropsied. RESULTS Results of baseline CT lymphangiography were unremarkable for all 7 cats. Only 5 cats completed the study. Leakage of contrast medium at the level of the cisterna chyli was seen on CT lymphangiography images obtained from all cats immediately after surgery. Evaluation of 30-day postoperative CT lymphangiography images revealed small branches entering the caudal vena cava in 2 cats, leakage of contrast medium into the caudal vena cava with no visible branches in 1 cat, and no contrast medium in the caudal vena cava in 2 cats. Contrast medium did not flow beyond the level of the cisterna chyli in any cat. Gross examination during necropsy revealed that all cats had small lymphatic vessels that appeared to connect to local vasculature identified in the region of the cisterna chyli. CONCLUSIONS AND CLINICAL RELEVANCE Abdominal lymphaticovenous anastomoses formed after TDL and CCA in cats. This would support use of these procedures for treatment of cats with idiopathic chylothorax, although additional studies with clinically affected cats are warranted.

OBJECTIVE: To investigate the distribution of T-cell markers (CD4 and CD8α) in lymphoid organs of newborn, juvenile, and adult yaks. ANIMALS: 15 healthy male yaks of various ages from highland plateaus. PROCEDURES: Yaks were allocated to groups on the basis of age (newborn [1 to 7 days old; n = 5], juvenile [5 to 7 months old; 5], and adult [3 to 4 years old; 5]). The thymus, spleen, 5 mesenteric lymph nodes, and 5 hemal nodes were harvested from each yak within 10 minutes after euthanasia. Morphological characteristics of those lymphoid organs were assessed by histologic examination; expression of CD4 and CD8α mRNAs and proteins were measured by quantitative real-time PCR assay and immunohistochemical staining. RESULTS: Among the lymphoid organs evaluated, expressions of CD4 and CD8α mRNAs were highest in the thymus in all age groups. In newborn lymphoid organs, CD4 mRNA expression and CD4+ cell distribution were more predominant, whereas in juvenile and adult lymphoid organs, CD8α mRNA expression and CD8α+ cell distribution were more predominant. The CD4+ and CD8α+ cells were mainly located in the cortex and medulla of the thymus, the medulla of the hemal nodes and mesenteric lymph nodes, the periarteriolar lymphoid sheaths, and the red pulp of the spleen. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that the CD4 mRNA expression and CD4+ T-cell distribution in yak lymphoid organs decreased and CD8α mRNA expression and CD8α+ T-cell distribution increased with age. Moreover, CD8α+ cells were present in the follicles of yaks’ secondary lymphoid organs, which differs from findings for other mammals.

In order to study the ability of Lactobacillus casei to ameliorate murine enteritis, 18 mice were randomly divided into 3 groups: the enteritis group, intervention group, and control group. The interleukin (IL)-6 and transforming growth factor-β (TGF)-β content in mouse peripheral blood and duodenum was detected using an enzyme-linked immunosorbent assay (ELISA). The number of CD4+CD25+Foxp3+ T-regulatory cells (Tregs) and CD4+IL-17A+ Th17 cells in the mesenteric lymph nodes (MLN) and spleen were detected using flow cytometry, and quantitative reverse transcription polymerase chain reaction (PCR) and western blot analysis were used to measure Foxp3 and retinoid-related orphan receptor-γ (RORγt) mRNA and protein expression in the MLN. Histological changes in the duodenum were observed. Results indicate that in the intervention group, IL-6 content in mouse peripheral blood and duodenum was significantly lower than in the enteritis group (P < 0.05), while TGF-β content was significantly increased compared to the enteritis group (P < 0.05). For the intervention group, the percentages of CD4+CD25+Foxp3+ Tregs in spleen and MLN were increased (P < 0.05), while the percentages of CD4+IL-17A+ Th17 cells were decreased compared to the enteritis group (P < 0.05). The expression of Foxp3 mRNA and protein in the intervention group was higher than in the enteritis group, while RORγt mRNA and protein were significantly lower (P < 0.05). After mice in the enteritis group were treated with L. casei, duodenal inflammation was relieved. This study demonstrated that L. casei could have possible implications for the enterotoxigenic Escherichia coli (ETEC) induced intestinal inflammation by regulating the ratio imbalance of Th17/Treg cells.

Tumor necrosis factor alpha (TNF-α) plays an important role in the immune system. In this study, TNF-α expression was analyzed in 11 tissues of 8 piglets resistant to enterotoxigenic Escherichia coli (ETEC) F18 and 8 ETEC F18-susceptible piglets from the Large White breed. The expression levels of TNF-α were high in immune organs (spleen, lung, thymus, and lymph nodes). The levels were higher in ETEC F18-resistant piglets than in ETEC F18-susceptible piglets, with significant differences in spleen, kidney, thymus, lymph node, and duodenum (P < 0.05). The mutation TNF-α −791(C→T) and 3 genotypes (CC, CT, and TT) were identified. The TNF-α expression levels in the spleen, kidney, lymph nodes, and duodenum were significantly higher in the TT pigs than in the CC pigs (P < 0.05). Thus, TNF-α −791(C→T) has significant effects on mRNA expression and may regulate ETEC F18 resistance of weaning piglets. Therefore, the −791(C→T) mutation of the TNF-α gene could be considered an important potential genetic marker of ETEC F18 resistance.

Objective-To develop a method for experimental induction of equine rhinitis A virus (ERAV) infection in equids and to determine the clinical characteristics of such infection. Animals-8 ponies (age, 8 to 12 months) seronegative for antibodies against ERAV. Procedures-Nebulization was used to administer ERAV (strain ERAV/ON/05; n = 4 ponies) or cell culture medium (control ponies; 4) into airways of ponies; 4 previously ERAV-inoculated ponies were reinoculated 1 year later. Physical examinations and pulmonary function testing were performed at various times for 21 days after ERAV or mock inoculation. Various types of samples were obtained for virus isolation, blood samples were obtained for serologic testing, and clinical scores were determined for various variables. Results-ERAV-inoculated ponies developed respiratory tract disease characterized by pyrexia, nasal discharge, adventitious lung sounds, and enlarged mandibular lymph nodes. Additionally, these animals had purulent mucus in lower airways up to the last evaluation time 21 days after inoculation (detected endoscopically). The virus was isolated from various samples obtained from lower and upper airways of ERAV-inoculated ponies up to 7 days after exposure; this time corresponded with an increase in serum titers of neutralizing antibodies against ERAV. None of the ponies developed clinical signs of disease after reinoculation 1 year later. Conclusions and Clinical Relevance-Results of this study indicated ERAV induced respiratory tract disease in seronegative ponies. However, ponies with neutralizing antibodies against ERAV did not develop clinical signs of disease when reinoculated with the virus. Therefore, immunization of ponies against ERAV could prevent respiratory tract disease attributable to that virus in such animals.

Objective: To determine various measurements of medial retropharyngeal lymph nodes (MRPLNs) in healthy cats via ultrasonography and CT. Animals: 45 cats (age range, 2 to 8 years). Procedures: Cats underwent CT of the head and ultrasonography of the cervical region. Various measurements of MRPLNs were obtained, and parenchymal heterogeneity, presence of a hilus, appearance of margins, and attenuation of MRPLNs were determined. Results: Data for 7 cats were excluded because they did not meet inclusion criteria; data for 38 cats were evaluated. Measurements of left and right MRPLNs were not significantly different. Mean length × rostral height × rostral width dimensions of MRPLNs were 20.7 × 12.4 × 3.7 mm and 20.7 × 13.1 × 4.7 mm in ultrasonographic and CT images, respectively. Maximum MRPLN dimensions were approximately 32 × 20 × 7 mm. Mean attenuation of MRPLNs was 40.2 Hounsfield units. Parenchyma of MRPLNs was mildly (via CT) to moderately (via ultrasonography) heterogeneous. A hilus was identified in 95% (via ultrasonography) and 24% or 92% (via CT [depending on criteria used to define a hilus]) of MPRLNs. Lymph node margins were smooth in CT images and mildly irregular in ultrasonographic images. A negative linear correlation was detected between age of cat and MRPLN volume. Conclusions and Clinical Relevance: MRPLNs in cats were easily imaged via ultrasonography and CT. Left and right MRPLNs were symmetric, and MRPLNs were larger in young adult cats versus old cats. Data were intended to serve as references for evaluation of MRPLNs in healthy cats.

Objective: To assess the prevalence of Mycobacterium bovis infection in cattle and wild ruminants (WRs) in a wildlife-livestock interface area (WLIA) of the Mexican highland plateau. Animals: 24,400 cattle from 793 herds (including 17,351 commercially slaughtered cattle) and 142 WRs (110 white-tailed deer [Odocoileus virginianus], 20 red deer [Cervus elaphus], and 12 North American elk [Cervus canadensis]) harvested via controlled hunting. Procedures: Cattle were serially tested for M bovis infection via caudal fold tuberculin and comparative cervical tuberculin tests during field surveillance. Carcasses of cattle and WRs were inspected for gross lesions; samples suggestive of tuberculosis were analyzed via histologic evaluation and mycobacterial culture (HMC). A PCR assay to detect Mycobacterium tuberculosis complex organisms was performed to confirm positive results of HMC. Results: WRs had inflammatory lesions in lungs and lymph nodes, although HMC results did not indicate M bovis infection. Eight cattle had positive results for both tuberculin tests, and 31 had positive results for HMC of grossly detected lesions; all were from 7 herds, and ≥ 1 cow in each herd had positive PCR assay results. These 7 herds were depopulated; adjacent herds and herds related via commerce were quarantined. Calculated true prevalence of M bovis infection was 0.86% (95% confidence interval, 0.24% to 1.49%) in cattle; M bovis was not detected in any WRs. Conclusions and Clinical Relevance: M bovis infection was present in cattle. Although transmission to WRs in this WLIA was not detected, diagnosis and prevention activities should be implemented and consolidated to prevent potential M bovis transmission between cattle and WRs.