Introduction: Several Mycoplasma species can cause severe diseases in ruminant hosts, some of which are the diseases listed by the World Organisation for Animal Health (OIE). The role of the Cervidae family in carrying and transmitting ruminant mycoplasma infections in Poland is unknown. Material and Methods: Antibody and antigen detection tests for the main mycoplasma species that can affect wild ruminants were performed on 237 samples (serum, nasal swab, bronchoalveolar lavage, and lung) collected from 161 animals during 2011-2014. The samples were obtained from a cull of healthy population of deer which included: 96 red deer (Cervus elaphus elaphus), 19 fallow deer (Dama dama), and 46 roe deer (Capreolus capreolus). Results: Serological screening tests revealed positive reactions to Mycoplasma bovis in one sample and to Mycoplasma capricolum subsp. capripneumoniae in three samples; however, these three samples were negative by immunoblotting. Other antibody and antigen detection tests demonstrated negative results. Conclusion: Currently wild cervids in Poland do not play a significant role in transmitting mycoplasma infections to domestic animals, but they remain a potential risk.

OBJECTIVE To characterize the expression of monocarboxylate transporters (MCTs) 1 and 4 and the ancillary protein CD147 in the intestinal tract of healthy equids and determine the cellular location of CD147 in the intestinal epithelium. ANIMALS 12 healthy horses and ponies slaughtered for meat production or euthanized for reasons unrelated to gastrointestinal tract disease. PROCEDURES The entire gastrointestinal tract was removed from each equid within 45 minutes after slaughter or euthanasia. Tissue samples were obtained from the antimesenteric side of the duodenum, jejunum, ileum, middle part of the cecum, sternal flexure of the ventral colon, pelvic flexure, sternal flexure of the dorsal colon, and descending colon (small colon). Expressions of MCT1, MCT4, and the ancillary protein CD147 were examined in tissue samples from each of the 8 intestinal locations by means of quantitative PCR assay, immunoblotting, and immunohistochemical analyses. RESULTS Expression of MCT1 was most abundant in the cecum and colonic sites, whereas expression of MCT4 was predominantly in the proximal section of the intestine (small intestinal sites and cecum). Immunohistochemical analysis revealed that MCT1 and CD147 were present in the membranes of enterocytes (in crypts and villi). CONCLUSIONS AND CLINICAL RELEVANCE The anatomic distribution of MCT1 and MCT4 in the equine intestinal tract determined in this study together with the previous knowledge of the sites of substrate absorption indicated that MCT1 might predominantly contribute to the uptake of short-chain fatty acids in the large intestine and MCT4 might predominantly contribute to the uptake of lactate in the small intestine.

Objective-To establish a sensitive test for the detection of autoantibodies against thyroid peroxidase (TPO) in canine serum samples. Sample Population-365 serum samples from dogs with hypothyroidism as determined on the basis of serum concentrations of total and free triiodothyronine (T3), total and free thyroxine (T4), and thyroid-stimulating hormone, of which 195 (53%) had positive results for at least 1 of 3 thyroid autoantibodies (against thyroglobulin Tg, T4, or T3) and serum samples from 28 healthy dogs (control samples). Procedure-TPO was purified from canine thyroid glands by extraction with detergents, ultracentrifugation, and precipitation with ammonium sulfate. Screening for anti-TPO autoantibodies in canine sera was performed by use of an immunoblot assay. Thyroid extract containing TPO was separated electrophoretically, blotted, and probed with canine sera. Alkaline phosphatase-conjugated rabbit anti-dog IgG was used for detection of bound antibodies. Results-TPO bands were observed at 110, 100, and 40 kd. Anti-TPO autoantibodies against the 40-kd fragment were detected in 33 (17%) sera of dogs with positive results for anti-Tg, anti-T4, or anti-T3 autoantibodies but not in sera of hypothyroid dogs without these autoantibodies or in sera of healthy dogs. Conclusions and Clinical Relevance-The immunoblot assay was a sensitive and specific method for the detection of autoantibodies because it also provided information about the antigen. Anti-TPO autoantibodies were clearly detected in a fraction of hypothyroid dogs. The value of anti-TPO autoantibodies for use in early diagnosis of animals with thyroid gland diseases should be evaluated in additional studies.

Poly(methacrylic acid) hydrogels were tested for oral delivery of a vaccine against Pasteurella haemolytica infection in cattle. Culture supernatants of P haemolytica, the most common bacterium associated with pneumonia in cattle, were used as the antigens in the vaccine. Hydrogels containing culture supernatants were administered orally to calves. Calves were then challenge-exposed with virulent P haemolytica. Calves were euthanatized 3 days after challenge exposure. The lungs of each calf were scored for severity and size of pneumonic lesions. Results indicated that vaccinated calves had smaller, less severe pneumonic lesions and lived longer than nonvaccinated calves. These results indicated that hydrogels can be used to deliver vaccines orally to calves to enhance resistance to pneumonia caused by P haemolytica.

A highly sensitive enzyme-linked immunoelectrotransfer blot (EITB) assay, capable of detecting aphthovirus-specific antibodies to replicating virus in sera from cattle with persistent infection, was developed. The assay uses a set of purified recombinant DNA-derived nonstructural viral antigens as serologic probes in lieu of the traditionally used virus infection-associated antigen(s) partially purified from baby hamster kidney-infected cells. Sera from cattle with experimentally induced aphthovirus infection were analyzed sequentially by EITB at various postinoculation days, and the results were compared with those obtained by currently used techniques. It was established that, in aU cases, EITB results remained positive at late stages of infection. At these times, results of virus infection-associated antigen-antibody determinations were negative by use of the conventional immunodiffusion in agarose gel test, and virus was recovered only occasionally from esophageal-pharyngeal fluid. Specificity of the EITB test was indicated by negative results for sera from cattle in aphthovirus-free areas, including samples from cattle infected with a variety of bovine viruses. Moreover, the test eliminated a substantial number of false-positive results (on the basis of the immunodiffusion in agarose gel assay) caused by reactivity of sera from vaccinated cattle. Use of additional nonstructural viral antigens, other than RNA polymerase, is proposed to differentiate between seropositivity resulting from vaccination or infection. This procedure may be considered to have potential applications as a sensitive, safe, rapid, and economic field test for specific diagnosis of persistent aphthovirus infection in affected animals.

We purified porcine whey lactoferrin by affinity chromatography on a heparin-sepharose column, followed by high-performance liquid chromatography. Molecular mass of purified lactoferrin (PLF) is 78,000 daltons. The iron-binding activity of PLF had a UV/ visible-light absorption spectrum indistinguishable from that of human and bovine lactoferrins (absorbance ratio [465 nm/280 nm] approx 0.046). The growth ratio of WIL-2 cells in PLF-supplemented medium is 70% of that in serum-containing medium. The aforementioned characteristics are similar to those of human and bovine lactoferrins. Immunoblot analysis, using polyclonal antibody raised in rabbits against porcine whey lactoferrin, revealed high specificity for PLF, and low cross-reactivity with commercial human and bovine lactoferrins.

In this study, we described water-insoluble proteins extracted from the germinative regions (stratum internum and coronary band epithelium) and the cornified outer surface (stratum medium) of the equine hoof wall. Two major types of polypeptides were identified: the intermediate filaments (IF) and the IF-associated proteins. The IF, including keratins, composed a major portion of this fraction, had electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the range of 40 to 80 kDa, and reacted with acidic or basic keratinspecific monoclonal antibodies. Differences in the composition of keratins between germinative layers and the stratum medium were seen. Another less well-characterized group of polypeptides associated with the IF also were extracted with the water-insoluble polypeptide fraction. These associated proteins had an apparent molecular weight between 10 and 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and contained a higher percentage of sulfur-containing amino acids than did the IF. Water-insoluble protein fractions compared favorably with those found in other less-specialized keratinizing tissue with respect to size, immunoreactivity with monoclonal antibody, and amino acid composition.

A double-antibody sandwich ELISA for detection of antibodies directed against the exotoxin of Corynebacterium pseudotuberculosis, the cause of caseous lymphadenitis (CL) in small ruminants, was developed. A concentrated exotoxin was used. For interpretation of ELISA results, these sera were tested: sequentially obtained sera of C pseudotuberculosis-inoculated goats and sheep that were monitored for 68 weeks; sequentially obtained sera from 80 goats of 3 flocks with CL; sera from 652 goats of 7 flocks without CL; sera from 160 sheep of 4 flocks without CL; and 2,265 caprine and 208 ovine sera submitted for diagnostic testing. Data regarding the infection status and history of 10,454 of the 23,302 animals were collected after testing; most of these were goats that had been part of a CL control program. Specificity and sensitivity of the ELISA were nearly 100%. Subsequently, 31,978 animals from which no data on infection status of flocks had been collected were then tested. It was concluded that the ELISA is a useful diagnostic test for CL eradication programs. Sera with doubtful or inconclusive ELISA results were examined by use of immunoblot analysis. Proteins from C pseudotuberculosis culture supernatant were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose. Six proteins with molecular mass of 68, 65, 39, 38, 31, and 29 kDa reacted with sera from goats and sheep with experimentally induced or naturally acquired infection. Immunoblot analysis was valuable in classifying sera with doubtful or inconclusive results by ELISA.

Two African swine fever virus (ASFV) antigens were tested for use in an ELISA to detect antibody to ASFV. Antigens used were the cytoplasmic soluble fraction (CS-P) of infected cells grown in the presence of porcine serum and the semipurified viral structural protein VP73 (SVP73). Both antigens were tested by ELISA against 72 sera obtained during several ASF field episodes and from ASFV-inapparent carriers. Of the 72 sera, only 2.8% had positive results by ELISA against CS-P antigen; 60% of positive-reacting sera (to both antigens) had higher ELISA values when the CS-P antigen was used. Samples (with positive results) that reacted only to CS-P antigen had results confirmed by immunoblot analysis. Such sera reacted against ASFV-infection proteins IP25, IP25.5, and IP30, but not against IP73. In time-course experiments to detect appearance of ASFV-antibodies in infected miniature pigs, antibodies were detected by immunoblot analysis on postinoculation day (PID) 8. At that time, only the polypeptides IP25, IP25.5, IP30, and IP31 were recognized; IP73 and IP12 were first detected 3 and 4 days later, respectively. In the same experiments, ASFV antibodies were detected by ELISA, using CS-P or SVP73 antigens, on PID 7 and 9, respectively. These results could explain the percentage of sera not having positive results by ELISA using SVP73 antigen, if the sera were obtained from ASFV-infected pigs during the first days of infection before induction of antibody response against the IP73 protein. This feature makes the use of CS-P antigen advantageous in early serologic detection of AFSV-infected pigs.

Objective—To examine the expression and distribution of tight junction (TJ) and adherens junction (AJ) proteins in canine duodenal and colonic mucosa. Sample—Mucosa obtained from 4 healthy Beagles. Procedures—Biopsy specimens of the duodenum and colon were obtained via endoscopy from 4 healthy dogs. The expression patterns and subcelluar localization of claudin-1, -2, -3, -4, -5, -7, and -8; E-cadherin; and β-catenin in the duodenum and colon were analyzed by use of immunoblotting and immunofluorescence microscopy. Results—In the duodenum, there was clear expression of claudin-3 and -5, E-cadherin, and β-catenin proteins and weak expression of claudin-7 protein. In contrast, there was clear expression of claudin-2 and -3, E-cadherin, and β-catenin proteins and weak expression of claudin-5 and -7 proteins in the colon, as determined by use of immunoblotting. As determined by the use of immunofluorescence microscopy, the duodenum and colon had staining for claudin-3 and -5, E-cadherin, and β-catenin in the most apical region and staining for claudin-7 in the basolateral region. Staining for claudin-2 was also observed in the colon. Conclusions and Clinical Relevance—Information was provided about the expression patterns of TJ and AJ proteins in the duodenum and colon of clinically normal dogs. These results may provide valuable information for use in evaluating the importance of these TJ and AJ proteins in the pathogenesis of inflammatory bowel disease in dogs.