Immunophenotyping of mononuclear cell infiltrate in swine liver naturally infected with hepatitis E virus

U ovoj disertaciji ispitano je 233 rektalna brisa od klinički zdravih svinja, različitih uzrasnih kategorija, poreklom sa četiri farme, na prisustvo nukleinske kiseline virusa hepatitisa E metodom RT-PCR. Pored toga, RT-PCR, bakteriološkom i PCR analizom su ispitani uzorci jetre od 12 svinja kod kojih je RT-PCR nalaz iz uzorka rektalnog brisa pokazao prisustvo HEV RNK. Kod 12 svinja kod kojih je metodom RT-PCR u uzorku rektalnog brisa utvrđena nukleinska kiselina virusa hepatitisa E, izvršena je punkcija vena cava cranialis i uzeta je krv za biohemijska ispitivanja. Nakon koagulisanja krvi i izdvajanja seruma uz pomoć poluautomatskog biohemijskog spektrofotometra određeni su sledeći parametri: ukupni proteini, albumini, aspartat-aminotransferaza (AST), alanin-aminotransferaza (ALT), γ- glutamil-transpeptidaza (GGT) i ukupni bilirubin. Histopatološka i imunohistohemijska analiza urađena je na uzorcima jetre koji su fiksirani u 10% neutralnom formalinu, obrađeni u automatskom tkivnom procesoru i uklopljeni u parafin. Parafinski isečci debljine oko 3-5μm bojeni su hematoksilineozin i Masson trihrom metodom. Imunofenotipizacija ćelija mononuklearnog infiltrata izvršena je streptavidin-biotin (LSAB2) metodom i tehnikom dvostrukog imunohistohemijskog bojenja, uz upotrebu antitela za CD3 i CD79. Ekspresija TGFβ1 molekula postignuta je streptavidin-biotin (LSAB2) metodom. Za procenu gustine i distribucije limfocitno – plazmaćelijskog infiltrata u jetri korišćena je semikvantitativna analiza. Broj CD3 i CD79 pozitivnih ćelija u jetri određen je morfometrijskom analizom. Pored deskriptivnih statističkih parametara upotrebljeni su ANOVA i Tukey test. Ukupno je ispitano 233 svinje, od kojih je kod dvanaest u uzorku rektalnog brisa dokazana HEV RNK (5,15%). Istovremeno prisustvo HEV RNK u uzorku rektalnog brisa i jetre je ustanovljeno kod pet svinja (41,67%). U dvanaest uzoraka jetre nije dokazano prisustvo aerobnih i anaerobnih bakterija i genoma svinjskog cirkovirusa tip 2...In this thesis from clinically healthy pigs of different ages, originating from 4 farms, a total of 233 rectal swabs were tested for the presence of HEV RNA by RTPCR. In addition, liver samples of 12 pigs which were RT-PCR positive to HEV RNA were tested by RT-PCR, bacteriological and PCR analysis. A total of 12 pigs which rectal swabs tested positive by RT-PCR analysis were further on sampled by puncture of vena cava cranialis, as well. After coagulation blood sera were analyzed on a biochemical spectrophotometer and the following parameters were determined: total protein, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ – glutamil transpeptidase (GGT) and total bilirubin. Histopathological and immunohistochemical analyses were performed on liver samples fixed in 10% neutral formalin, processed in an automated tissue processor and embedded in paraffin. Paraffin sections about 3-5μm thick were stained with hematoxilin-eosin and by Masson trichrome method. Immunophenotypisation of mononuclear infiltrate cells was carried out by the streptavidin – biotin method (LSAB2) and by the double immunohistochemical staining technique with antibodies for CD3 and CD79. Expression of TGF-β1 molecule was obtained by the streptavidin – biotin method (LSAB2). Estimation of the density and distribution of lymphocyticplasmacytic infiltrate in the liver was done by semiquantitative analysis. The number of CD3 and CD79 positive cells in the liver was determined by morphometric analysis. In addition to descriptive statistical parameters ANOVA and Tukey test were used, as well. A total of 233 pigs were tested. Out of this number in 12 rectal swabs HEV RNA (5.15%) was confirmed. Concurrent presence of HEV RNA in both rectal and liver samples was determined in 5 pigs (41.67%). In 12 liver samples the presence of aerobic and anaerobic bacteria, as well as circovirus type 2 genome were not confirmed. Results of blood serum biochemical testing did not show statistical significance between values for total proteins, albumins, and AST between the infected pigs and the control group. A statistically very significant difference (p<0.01) between the infected and the control group was determined for ALT, and a significant difference (p<0.05) was recorded for GGT and total bilirubin..

Viral hepatitis E: A disease of humans and animals

The hepatitis E virus is ubiquitous in all parts of the world where pig production exists. The infection occurs in several animal species and its course is mostly asymptomatic. Viral strains isolated from pigs and humans are genetically similar, which indicates a potential zoonotic nature of the disease, and the possibility that pigs, and perhaps also other species of animals diseased with viral hepatitis E are a source of infection to humans. The pig hepatitis E virus, which is similar to the hepatitis E virus in humans, was isolated and described for the first time in the USA in 1997. The infection of pigs with hepatitis E virus occurs through faeco-oral transmission, by ingestion of feed and water contaminated with the virus, or through direct contact between infected and healthy animals. The pathogenesis of this infection in pigs differs from its pathogenesis in humans and it has not been sufficiently examined in all its aspects. Even though viral hepatitis E in pigs has been described as a subclinical disease, some authors describe changes in the concentration of certain biochemical parameters in blood serum of the infected pigs. Histologically, a mild to moderate lymphotic-plasma cellular infiltration is observed in livers of infected pigs, as well as focal areas of hepatocyte necrosis. Viral hepatitis E is an endemic disease of humans in Asia, Africa, and Latin America. In developed countries, hepatitis E sporadically occurs in humans, but it is becoming of increasing importance in particular in Japan, North America, and Europe, because the populations of these areas travel extensively to the endemic regions or as a result of the consumption of thermally untreated meat of wild boar and products made from thermally untreated meat. Pork products can be contaminated with hepatitis E virus. Further proof that indicates the zoonotic potential of this virus and places this diseases among the group of professional diseases of farmers and veterinarians is the finding of antibodies to hepatitis E virus in farmers and veterinarians who work on pig farms without showing any clinical signs of the disease. Having in mind the fact that viral hepatitis E has been proven in pig farms in Serbia and neighboruign countries, there should be strict respect of biosecutiry measures from the episootiological and epidemiological aspects, and the principle of good production and hygiene practice should be adhered to on pig farms. This disease should in future also be included in the legal regulations of our country in order to ensure the production of products of animal origin that are safe from the aspect of hygiene. [Projekat Ministarstva nauke Republike Srbije, br. III 46009 i br. TR 31062

Cutaneous fibroma in the roe deer (Capreolus capreolus)

Fibromas present very frequent skin neoplasms in different species of wild game of the family Cervidae. Viral etiology of skin neoplasms was proven in certain species of wild game from this family, with the most frequent diagnoses being: fibromas, ossifying fibromas, fibrosarcomas, multiple neurofibromatosis, fibropapillomas, and papillomas. The diagnozed tumor in the roe deer had the histological characteristics of a polimorphous fibroblast, which is not the case with domestic animals. This finding can be considered as a characteristic of fibromas in animals of the family Cervidae. Solitary fibroma or multiple fibroma (fibromatosis) does not present a significant cause of deer deaths, but they cause concern among hunters who are in direct contact with them. Although fibromas do not lead to spoilage of game meat, they are esthetically repellent and people are reluctant to consume meat of such game

Isolation and detection of Listeria monocytogenes in poultry meat by standard culture methods and PCR

Listeria is the genus of a bacteria found in soil and water and some animals, including poultry and cattle. It can be present in raw milk and food made from raw milk. It can also live in food processing plants and contaminate a variety of processed meats. Microscopically, Listeria species appear as small, Gram-positive rods, which are sometimes arranged in short chains. In direct smears, they can be coccoid, so they can be mistaken for streptococci. Longer cells can resemble corynebacteria. Flagella are produced at room temperature but not at 37 degrees C. Haemolytic activity on blood agar has been used as a marker to distinguish Listeria monocytogenes among other Listeria species, but it is not an absolutely definitive criterion. Further biochemical characterization is necessary to distinguish between the different Listeria species. The objective of this study was to detect, isolate and identify Listeria monocytogenes from poultry meat. Within a period of six months from January to June 2017, a total of 15 samples were collected. Three samples were positive for the presence of Listeria monocytogenes. Biochemical and microbiological tests as well as PCR technique using specific primers were used to confirm L. Monocytogenes in the samples

Prevalence of Salmonella enterica in slaughtered pigs in Serbia: Serotyping, PFGE-genotyping and antimicrobial resistance

Introduction: The aim of this study was to determine the prevalence of Salmonella along the slaughter line and to identify possible critical control points in one slaughterhouse facility located in the city of Belgrade. Methodology: In total, 700 samples were tested: two swabs from both sides of carcass were taken from each of 100 pigs. In this way, 200 pig skin swab samples were taken after stunning, 200 after processing and 200 after chilling. Additional 100 samples of ileal contents were also taken from the same pigs to obtain a collection of 270 isolates. All samples were analyzed using standard culture methods and serotyping. PFGE was performed for 27 isolates. Determination of antimicrobial resistance was performed by E-test. Results: In total, 47 (23.5%) swab samples were positive for the presence of Salmonella after stunning. After processing, Salmonella was isolated in two swab samples (1%), whereas all samples which were collected after chilling were negative for Salmonella. The sampling of ileal contents was positive for five Salmonella isolates (5%). The most frequently isolated serotypes were S. Derby (90.74%), S. Infantis (5.56%) and S. Typhimurium (3.7%). All tested isolates were resistant to tetracycline. Resistance was recorded to nalidixic acid (23.3%), ciprofloxacin (20%), ampicillin (10%) and chloramphenicol (14.4%), as well. The PFGE results indicated that isolates had a high genetic similarity. Conclusions: The investigation has confirmed that bacteriological examinations of carcass swabs, as well as ileal content, could be used to assess the carriage of salmonellae in pigs at the time of slaughter

Listeria monocytogenes contamination in ready to eat foods

Listeria monocytogenes is a food-borne pathogen responsible for listeriosis, a sickness with a high mortality rate. Listeriosis is largely associated with ready-to-eat (RTE) foods. It is well established that foods that pose the greatest risk of foodborne listeriosis are those RTE foods that have intrinsic characteristics such as pH and water activity that support the growth of L. monocytogenes. RTE foods can also become re-contaminated during further processing and handling. Increased handling leads to a higher probability of contamination. Sources of contamination can be food contact surfaces, processing machinery and workers. In our research, L. monocytogenes was detected in a RTE salad. Food safety criteria for Listeria monocytogenes in RTE foods have been applied from 2006 (Commission Regulation (EC) 2073/2005). Still, human invasive listeriosis was reported to increase during 2009-2013 in the European Union and European Economic Area. Time series analysis for the 2008-2015 period in this area showed an increasing trend of the monthly notified incidence rate of confirmed human invasive listeriosis of the over 75 age groups and female age group between 25 and 44 years old (probably related to pregnancies)

Salmonellosis in poultry breeding in view of introducing vaccination

With the increased demand of consumers for safe poultry products, efficient control of pathogenic microorganisms that cause diseases in humans is becoming the chief challenge in contemporary poultry production. Poultry meat, eggs and related products present the main source of salmonellosis in humans that occurs as a result of the consumption of contaminated food. Any serotype of Salmonella spp. that is not strictly linked to the host is capable of causing gastrointestinal disorders of various degrees in humans. However, according to the World Health Organization (WHO), Salmonella enterica serotype enteritidis has been the leading cause of salmonellosis in humans over the past 10 years. One of the available prophylactic measures is resorting to the vaccination of poultry against salmonellosis. Several vaccines against poultry salmonellosis, produced by different manufacturers, have been registered in Serbia. In order for these vaccines to become widely used in our country, it is necessary that the factors in primary poultry production are aquainted with the mechanisms of action of these vaccines, the effects achieved by their implementation, the economic feasibility, and the possibilities for monitoring in keeping with the valid legal regulations. Certainly, vaccination should not be taken as the only method in the prophylaxis of salmonellosis, but as one of the very important methods in a series of biosafety and other measures that should be applied on poultry farms for the successful curbing of salmonellosis infections

Salmoneloza u živinarstvu u svetlu uvođenja vakcinacije

With the increased demand of consumers for safe poultry products, efficient control of pathogenic microorganisms that cause diseases in humans is becoming the chief challenge in contemporary poultry production. Poultry meat, eggs and related products present the main source of salmonellosis in humans that occurs as a result of the consumption of contaminated food. Any serotype of Salmonella spp. that is not strictly linked to the host is capable of causing gastrointestinal disorders of various degrees in humans. However, according to the World Health Organization (WHO), Salmonella enterica serotype enteritidis has been the leading cause of salmonellosis in humans over the past 10 years. One of the available prophylactic measures is resorting to the vaccination of poultry against salmonellosis. Several vaccines against poultry salmonellosis, produced by different manufacturers, have been registered in Serbia. In order for these vaccines to become widely used in our country, it is necessary that the factors in primary poultry production are aquainted with the mechanisms of action of these vaccines, the effects achieved by their implementation, the economic feasibility, and the possibilities for monitoring in keeping with the valid legal regulations. Certainly, vaccination should not be taken as the only method in the prophylaxis of salmonellosis, but as one of the very important methods in a series of biosafety and other measures that should be applied on poultry farms for the successful curbing of salmonellosis infections.Sa povećanim zahtevima potrošača za bezbednim živinskim proizvodima, efikasna kontrola patogenih mikroorganizama koji izazivaju oboljenja kod ljudi, postaje glavni izazov u savremenoj živinarskoj proizvodnji. Živinsko meso, jaja i proizvodi od njih predstavjaju glavni izvor salmoneloze kod ljudi koja nastaje usled konzumiranja kontaminirane hrane. Bilo koji serotip Salmonella spp. koji nije striktno vezan za domaćina sposoban je da kod ljudi izazove gastrointestinalne poremeć aje različitog stepena. Međutim, po navodima Svetske zdravstvene organizacije (WHO) Salmonella enterica serotip enteritidis je vodeći uzročnik salmoneloze kod ljudi tokom prethodnih 10 godina. Jedna od profilaktičkih mera je i pristupanje vakcinaciji živine protiv salmoneloze. U Srbiji je registrovano nekoliko vakcina protiv salmoneloze kod živine, različitih proizvođača. Da bi ove vakcine ušle u širu primenu u našoj zemlji, neophodno je da proizvođači u primarnoj živinarskoj proizvodnji budu upoznati sa mehanizmima delovanja tih vakcina, efektima koji se postižu njihovom primenom, ekonomskom isplativošću i moguć nošću monitoringa u skladu sa postojećom zakonskom regulativom. Svakako, vakcinaciju ne treba shvatiti kao jedini metod u profilaksi salmoneloze, već kao jedan od veoma važnih metoda u nizu biosigurnosnh i drugih mera koje bi trebalo primenjivati na farmama živine za uspešno suzbijanje salmoneloznih infekcija

Detection of Mycoplasma bovigenitalium and Mycoplasma tauri in Holstein Friesian dairy cows with subclinical endometritis

Mycoplasma spp. is the cause of serious cattle health disorders that lead to poor reproductive efficiency. Chronic seminal vesiculitis, infertility, vulvovaginitis and dystocia were observed in Mycoplasma spp. infection. The objectives of the present study were to investigate the frequency of Mycoplasmas in the uterus of postpartum dairy cows and its potential role in the occurrence of subclinical endometritis. Our study included 102 Holstein Friesian dairy cows 22-32 days postpartum. Uterine samples for cytological, bacteriological, and molecular analysis were provided using Cytoprint AI®. Metricheck® was used for the assessment of cervicovaginal mucus. After detection of Mycoplasma spp. genome in uterine samples, the swabs of the vulva, vagina, nasal swab, and samples of milk were sampled in order to exclude possible contamination with other microorganisms as potential trigger of subclinical endometritis. The genome of Mycoplasma spp. in the uterus was confirmed in 4 cows (2.40%). Sequencing of the 16S RNA revealed that detected mycoplasmas belonged to Mycoplasma bovigenitalium and Mycoplasma tauri species, with a prevalence of 1.80% and 0.6%, respectively. The cytological evaluation showed a 69.90%, 54.87% and 48.33% of polymorphonuclear cells for M. bovigenitalium positive cows, and 61.64% for M. tauri positive cow. Bacteriological examination revealed Trueperella pyogenes in uterine samples in all 4 observed cows. This is the first study where M. tauri was detected in the uterus of a live cow with subclinical enodmetritis. In this paper we hypothesized that M. bovigenitalium and M. tauri may have a certain role in the etiology of subclinical enodmetritis

Cardiopulmonary parasitic nematodes of the red fox (Vulpes vulpes) in Serbia

Among the wild canids, the red fox (Vulpes vulpes) is recognised as an important reservoir species for a range of parasites, including cardiopulmonary nematodes of public health and veterinary importance. As cross-host transmission between the red fox and domestic carnivores can play an important role in the epizootiology of cardiopulmonary parasitic diseases, the aim of the present investigations was to obtain data on the geographical distribution of cardiopulmonary nematodes of the red fox. The material for examination consisted of 83 foxes which were legally hunted at different locations during a three-month period from December 2017 to February 2018. The presence of four emerging species in Europe (Angiostrongylus vasorum, Crenosoma vulpis, Dirofilaria immitis and Eucoleus aerophilus) was revealed in red foxes of Serbia. Crenosoma vulpis and E. aerophilus were detected in foxes in both the plain and the mountainous areas across the country. Dirofilaria immitis is distributed in red foxes near alluvial rivers in Vojvodina province (northern Serbia). Angiostrongylosis caused by A. vasorum was demonstrated to exist in two enzootic foci with a high percentage of infected foxes in a plain area of northern Serbia. To the best of our knowledge, C. vulpis and A. vasorum were discovered for the first time in red foxes in central Serbia. The results provide strong evidence for veterinarians to take into consideration the parasitic nematodes discovered in red foxes in the differential diagnosis of diseases of companion animals. In the context of the ‘One Health’ approach the results related to the distribution of the zoonotic species E. aerophilus and D. immitis can be useful for medical epidemiology