Introduction: The aim of the study was to optimise and compare two multiplex PCR assays for the detection of Listeria spp. and Listeria monocytogenes in biological samples including the liver, brain, and blood. Material and Methods: Three strains of L. monocytogenes and single strains of each of the species: L. ivanovii, L. innocua, L. grayi, L. welshimeri, and L. seeligeri were used. Additionally, five other species of bacterium were used to evaluate the specificity of the tests. Results: Specific amplification products were obtained for both multiplex PCR assays, which confirmed the tested strains as Listeria spp. and L. monocytogenes, respectively. Isolates of other species did not yield PCR products. Conclusion: Both multiplex PCR assays proved to be significantly sensitive and highly-specific methods for the detection of Listeria strains.

Introduction: The giant liver fluke, Fascioloides magna, has spread across Europe over the years posing a serious threat to the Polish cervid population. Material and Methods: Macroscopic and histopathological studies of the liver of 22 roe deer (Capreolus capreolus), 10 red deer (Cervus elaphus), and 6 fallow deer (Dama dama) were performed. Species determination of the recovered liver flukes and eggs was performed by PCR protocol amplifying fragments of ribosomal DNA (ITS2), according to a standard method. Results: The presence of F. magna was confirmed in three (13.6%) roe deer, seven (70.0%) red deer, and two (33.3%) fallow deer. The fluke eggs were found only in the stools of five red deer and one fallow deer. Conclusion: This study presents detailed pathological and histopathological changes in the liver of wild Polish cervids, including roe deer, which were subjected to such study for the first time. The hepatic lesions typical for different stages of liver cirrhosis varied depending on the host species and stage of the disease.

Introduction: The paper describes identification of Trichinella species isolated from wild boars (Sus scrofa) in the most popular hunting region of the West Pomeranian Province of Poland.Material and Methods: The Trichinella larvae were identified by digestion method. For species identification of the larvae, multiplex PCR was used according to the European Reference Laboratory for Parasites Multiplex PCR protocol. The results were confirmed by molecular amplification of 5S rDNA gene and sequence analysis.Results: Prevalence of 0.54% Trichinella–positive wild boars in the West Pomeranian Province was recorded. Examination of the larvae showed the occurrence of T. spiralis in 79 %, T. britovi in 16.5 %, mixed infection with T. spiralis/T. britovi in 3.5%, and T. pseudospiralis in 1.0% of the boars.Conclusion: This is the first record of wild boar infected with non-encapsulated larvae of T. pseudospiralis in Poland. The species is very difficult to determine, especially using trichinoscopic method. The discovery of the larvae in the animals which may be intended for human consumption confirms that digestion technique should be the only method used for the inspection of meat, especially that from wild boars..

Introduction: The current study was designed to detect Mycoplasma agalactiae (Ma), Mycoplasma mycoides subsp. capri (Mmc), Mycoplasma capricolum subsp. capricolum (Mcc) and Mycoplasma putrefaciens (Mp) in sheep and goats with clinical signs consistent with contagious agalactia.Material and Methods: A total of 299 samples were collected from 55 mixed herds in Azarbaijan-e-Sharghi province, Iran. Samples were examined using PCR and culture methods.Results: The findings showed that in 40 herds at least one sample was positive by PCR or culture method. Moreover, out of 274 sheep samples, 101 were proved to be positive using the PCR technique and 76 were found positive using the culture method. Out of 25 goat samples, 10 were found positive using PCR and 9 were positive through the culture method. Less than 20% of isolated mycoplasmas were Ma. Ma was detected from almost all studied regions in the province while Mmc, Mcc, and Mp were detected only in a very limited area that was deemed by the research group the mixed infection zone.Conclusion: In vaccination or eradication projects, it would be more economical to focus on mixed infection zones. Further investigation on mixed infection zones could facilitate better understanding of contagious agalactia epidemiology.

Introduction: The aim of this study was to carry out a genetic analysis of Babesia canis isolates detected in dogs in eastern Poland and to study the correlation of the protozoa variant with a specific geographical region. Material and Methods: PCR was used to identify strains of B. canis from naturally infected animals (240 dogs from four provinces: Mazowieckie, Lublin, Podlasie, and Podkarpacie) by amplifying and sequencing a fragment of the 18S rRNA gene. Results: Sequencing the PCR products led to the identification of four variants of B. canis. Two previously described protozoa variants (18S rRNA-A and 18S rRNA-B) were observed in all provinces. Additionally, in the Mazowieckie and Lublin provinces a B. canis variant which contributed to the development of acute or atypical babesiosis was observed. The fourth variant of B. canis was detected only in dogs from the Lublin province, and the course of the disease was subclinical in all dogs infected with this variant. Conclusion: These results indicate the appearance of a new fourth B. canis genotype in Poland and confirm that it is still necessary to study the relationships between the genetic structure of protozoa, geographical distribution of the parasites, and clinical course of the disease.

Introduction: The aim of the study was to investigate the occurrence of goose haemorrhagic polyomavirus (GHPV) in wild birds inhabiting Poland.Material and Methods: Samples from 508 birds of different species were obtained between 2010 and 2015. The internal organ sections were homogenised and then total cellular DNA was isolated. The study was performed by means of PCR assay using primers complementary to the VP1 gene of the GHPV.Results: The presence of genetic material of GHPV was detected in 22 (4.33%) samples.Conclusion: It was the first such study in Poland to emphasise the role of wild birds as a potential source of GHPV infection for farmed geese.

Introduction: In the present study, some of the commercial fish farms located in the Black Sea region of Turkey, were screened for bacteria between 2012 and 2014.Material and Methods: The bacterial agents isolated from fish were identified by classical biochemical tests and the rapid diagnostic tests (API 20 E and API 20 Strep). All strains were further identified by sequencing of the 16S rRNA genes. The strains were also investigated for resistance to different antimicrobials by the disc diffusion method. Antibiotic resistance genes, including tetracycline (B), β-lactam (ampC, blaTEM, blaPSE), florfenicol (floR), erythromycine (ereA, ereB), sulphonamide (sulI, sulII), and trimethoprim (dhfr1) genes, were determined by the PCR method.Results:Vibrio anguillarum, Vibrio fluvialis, Photobacterium damselae subsp. piscicida, Pseudomonas luteola, Lactococcus garvieae, Streptococcus iniae, Aeromonas hydrophila, and Yersinia ruckeri were isolated from marine and freshwater cultured fish. According to the results of disc diffusion, all isolates were sensitive to florfenicol, trimethoprim+sulfamethoxazole, oxitetracycline, and enrofloxacin, and resistant to lincomycin, penicillin G, and amoxicillin. Also, sulI, sulII, and floR resistance genes were detected in the bacteria.Conclusion: The results of the study open up the opportunity to perform further investigations which could determine the possible role of ARGs in fish pathogens.

OBJECTIVE To determine the effects of topical ocular application of 1% trifluridine ophthalmic solution in dogs with experimentally induced recurrent ocular canine herpesvirus-1 (CHV-1) infection. ANIMALS 10 specific pathogen–free Beagles. PROCEDURES 12 months prior to the beginning of the randomized, masked, placebo-controlled 30-day trial, latent ocular CHV-1 infection was experimentally induced in each dog by topical ocular inoculation of both eyes with a field strain of CHV-1. Recurrent ocular CHV-1 infection was induced by oral administration of prednisolone for 7 days (starting day 1). Starting on the fourth day of prednisolone administration, each dog received 1% trifluridine solution or artificial tears (placebo) topically in both eyes 6 times daily for 2 days and then 4 times daily for 12 days. Ophthalmic examinations were performed every 2 days, and ocular disease scores were calculated. Ocular samples for CHV-1 PCR assays and blood samples for clinicopathologic analyses and assessment of CHV-1 serum neutralization antibody titers were collected at predetermined intervals. RESULTS Conjunctivitis was clinically detected in all dogs by day 4. Compared with dogs receiving placebo, mean and total clinical ocular disease scores were significantly lower and median CHV-1 shedding duration was significantly shorter for the trifluridine-treated dogs. Both groups had increasing CHV-1 serum neutralization antibody titers over time, but no significant differences between groups were detected. Clinicopathologic findings were unremarkable throughout the study. CONCLUSIONS AND CLINICAL RELEVANCE Topical ocular application of 1% trifluridine ophthalmic solution was well tolerated and effective at reducing disease scores and viral shedding duration in dogs with experimentally induced ocular CHV-1 infection, but may require frequent administration.

OBJECTIVE To determine effects of in ovo administration of a probiotic on development of the intestinal microbiota of 2 genetic lineages (modern and heritage) of chickens. SAMPLE 10 newly hatched chicks and 40 fertile eggs to determine intestinal microbiota at hatch, 900 fertile eggs to determine effects of probiotic on hatchability, and 1,560 chicks from treated or control eggs. PROCEDURES A probiotic competitive-exclusion product derived from adult microbiota was administered in ovo to fertile eggs of both genetic lineages. Cecal contents and tissues were collected from embryos, newly hatched chicks, and chicks. A PCR assay was used to detect bacteria present within the cecum of newly hatched chicks. Fluorescence in situ hybridization and vitality staining were used to detect viable bacteria within intestines of embryos. The intestinal microbiota was assessed by use of 16S pyrosequencing. RESULTS Microscopic evaluation of embryonic cecal contents and tissues subjected to differential staining techniques revealed viable bacteria in low numbers. Development of the intestinal microbiota of broiler chicks of both genetic lineages was enhanced by in ovo administration of adult microbiota. Although the treatment increased diversity and affected composition of the microbiota of chicks, most bacterial species present in the probiotic were transient colonizers. However, the treatment decreased the abundance of undesirable bacterial species within heritage lineage chicks. CONCLUSIONS AND CLINICAL RELEVANCE In ovo inoculation of a probiotic competitive-exclusion product derived from adult microbiota may be a viable method of managing development of the microbiota and reducing the prevalence of pathogenic bacteria in chickens.

OBJECTIVE To determine the minimum infectious dose of porcine epidemic diarrhea virus (PEDV) in virus-inoculated feed. ANIMALS 30 crossbred 10-day-old pigs. PROCEDURES Tissue culture PEDV was diluted to form 8 serial 10-fold dilutions. An aliquot of stock virus (5.6 × 105 TCID50/mL) and each serial PEDV dilution were mixed into 4.5-kg batches of feed to create 9 PEDV-inoculated feed doses; 1 virus-negative dose of culture medium in feed was also created. Pigs were challenge exposed via oral administration of PEDV-inoculated feed, and fecal swab specimens were collected. All pigs were euthanized 7 days after challenge exposure; fresh tissues were collected and used for PCR assay, histologic examination, and immunohistochemical analysis. RESULTS The PCR cycle threshold (Ct) decreased by approximately 10 when PEDV was added to feed, compared with results for equivalent PEDV diluted in tissue culture medium. Pigs became infected with PEDV when challenge exposed with the 4 highest concentrations (lowest concentration to cause infection, 5.6 × 10(1) TCID50/g; Ct = 27 in tissue culture medium and 37 in feed). CONCLUSIONS AND CLINICAL RELEVANCE In this study, PEDV in feed with detectable Ct values of 27 to 37 was infective. The Ct was 37 for the lowest infective PEDV dose in feed, which may be above the limit of detection established for PEDV PCR assays used by some diagnostic laboratories. Overall, results indicated 5.6 × 10(1) TCID50/g was the minimum PEDV dose in feed that can lead to infection in 10-day-old pigs under the conditions of this study.