The influence of a phytogenic additive in the control of proliferative enteropathy and the assessment of production results in weaned pigs naturally infected with Lawsonia intracellularis

Uzgoj svinja predstavlja jednu od najvažnijih grana stočarstva. Neadekvatna primena biosigurnosnih mera i loši ambijentalni uslovi, uz prisustvo patogenih prouzrokovača bolesti, mogu onemogućiti uspešnu proizvodnju svinja. U preko 50% slučajeva enterične bolesti su odgovorne za ograničavanje rentabilne proizvodnje. Jedan od važnih uzročnika koji dovodi do enteričnih poremećaja i velikih ekonomskih gubitaka je i Lawsonia intracellularis, obliganta intracelularna Gram negativna bakterija, koja prouzrokuje proliferativnu enteropatiju (PE). „Zlatni standardi“ za identifikaciju L. intracellularis su imunohistohemijska i PCR metoda. Razvojem kvantitativne real-time (qPCR) metode omogućeno je rutinsko otkrivanje subkliničkih formi bolesti, a kvantifikovanje uzročnika je omogućilo da se odredi težina kliničke slike inficiranih svinja. Zbog često pogrešne dijagnostike ove bolesti na farmama, posledično prekomerne upotrebe antibiotika i razvoja antimikrobne rezistencije uzročnika, postoji potreba za pronalaženjem alternativnih rešenja u kontroli PE. Takođe, dobre biosigurnosne mere na farmama, za koje je pokazano da pozitivno utiču na zdravlje i proizvodne rezultate svinja, mogu predstavljati bitan faktor u prevenciji nastanka PE. U skladu sa tim cilj istraživanja ove doktorske disertacije bio je ispitivanje uticaja komercijalnog fitogenog aditiva (Patente Herba® Plus) u kontroli PE kod odlučene prasadi prirodno inficirane bakterijom L. intracellularis, uspostavljanje i optimizacija protokola za molekularnogenetičku identifikaciju bakterije L. intracellularis, kao i kvantifikaciju stepena infekcije kod prasadi putem real-time qPCR i IHC metoda. Dodatno je ispitivan i uticaj različitih biosigurnosnih nivoa i Patente Herba® Plus fitogenog aditiva na proizvodne rezultate i broj izlučenih bakterija L. intracellularis u fecesu prasadi. Eksperiment je sproveden na četiri različite farme svinja na kojima je određen nivo eksternih, internih i ukupnih biosigurnosnih mera. Na svakoj od farmi (farma BS1, farma BS2, farma BS3 i farma BS4), prasad stara sedam nedelja, ujednačenih telesnih masa, bila je raspoređena u kontrolne grupe koje u hrani nisu dobijale preparat Patente Herba® Plus (K-BS1, K-BS2, K-BS3 i K-BS4) i tretman grupe koje su hrani dobijale preparat Patente Herba® Plus u koncentraciji od 2 kg/t hrane (T-BS1, T-BS2, T-BS3 i T-BS4). Kontrolne i tretman grupe formirane su sa po 72 jedinke, raspoređene u devet bokseva sa po osam prasadi, odnosno 144 prasadi po farmi, ukupno 576 prasadi. Za potrebe histoloških analiza izveden je eksperiment sa individualno gajenom prasadi, po šest u kontrolnoj i tretman grupi. Tokom 28 dana eksperimenta (0., 14. i 28. dan) uzimani su uzorci fecesa za molekularnogenetička ispitivanja, praćeni su proizvodni rezultati prasadi (telesna masa, dnevni i ukupni prorast, dnevna i ukupna konzumacija i konverzija), dok na kraju eksperimenta su uzimani uzorci ileuma za histološke (mikroskopske promene i histomorfometrijski parametari) i imunohistohemijske analize (ekspresija antigena L. intracelullaris). Na sve četiri farme, u okviru eksternih biosigurnosnih mera, najbolje su bile ocenjene potkategorije koje se odnose na kupovinu životinja i semena i zaposlene i posetioce, dok najniže ocene su dobile biosigurnosne mere koje se odnose na hranu, vodu i opremu. U okviru internih biosigurnosnih mera potkategorije kontrola bolesti i prasilište i period dojenja bile su najbolje ocenjene na farmama, a najlošije su bile ocenjene biosigurnosne mere koje se odnose na čišćenje i dezinfekciju, odgajivalište i mere između odeljaka i korišćenje opreme. Ukupne vrednosti biosigurnosnih mera na sve četiri farme (BS 1 60%, BS 2 64%, BS 3 77% i BS 4 86%) koje su analizirane u ovoj doktorskoj disertaciji bile su više od prosečnih vrednosti biosigurnosnih mera na farmama u Srbiji (58%) i svetu (64%), izuzev farme BS 1 čija ocena ukupnih biosigurnosnih mera je bila niža u poređenju sa prosečnim vrednostima u svetu (60%). Na farmi BS1 utvrđen je značajno manji broj bakterija L. intracellularis u fecesu prasadi tretman grupe u odnosu na kontrolnu grupu 14. i 28. dana eksperimenta (P=0,0080 i P=0,0015, pojedinačno). Takođe, prasad tretman grupe imala je značajno manji broj bakterija L. intracellularis u fecesu 14. i 28. dana u odnosu na 0. dan eksperimenta (P0,05), niti je uočen značajan pad broja bakterija L. intracellularis u fecesu prasadi tretman grupa tokom trajanja ogleda (P>0,05). Uočena je značajna negativna korelaciona zavisnost (r=-0,664, P0,05). Histološke promene na preparatima ileuma kontrolne i tretman grupe prasadi su deskriptivno analizirane, dok je histomorfometrijskim ispitivanjima pokazano da su u tretman grupi prasadi kripte bile značajno pliće (P=0,0284), a odnos visina resica/dubina kripti značajno veći (P=0,0040) nego u kontrolnoj grupi. Nije uočen efekat dodavanja fitogenog aditiva u hrani prasadi na visinu resica (P=0,0607), širinu resica (P=0,0728), površinu resica (P=0,7676) i broj peharastih ćelija/100 enterocita (P=0,0575). Semikvantitativnom analizom, na osnovu imunohistohemijskih nalaza, 33,33% uzoraka ilealnog tkiva tretman grupe prasadi bilo je ocenjeno ocenom 0, a preostalih 66,67% ocenom 1, dok je u kontrolnoj grupi 50% uzoraka bilo ocenjeno ocenom 1, 33,33% ocenom 2, a 16,67% uzoraka ocenom 3. Primena fitogenog aditiva u hrani nije značajno uticala na telesnu masu prasadi nakon 28 dana eksperimenta na svim ispitivanim farmama (P>0,05). Ukupni i dnevni prirast prasadi tretman grupe bio je značajno veći na farmi BS 2 u poređenju sa kontrolnom grupom (P=0,0349 i P=0,0345, pojedinačno), dok na farmama BS 1, BS 3 i BS 4 razlike u ukupnom i dnevnom prirastu tretman i kontrolne grupe nisu uočene (P>0,05). Najbolji prirast je postigla grupa koja je u hrani dobijala preparat Patente Herba® Plus na farmi BS 4, koji je bio značajno viši u poređenju sa K-BS1, K-BS2 i T-BS2 grupom prasadi (P<0,0001). Na kraju eksperimenta (28. dan) nije uočena razlika u ukupnoj i dnevnoj konzumaciji hrane između kontrolne i tretman grupe prasadi u okviru svake farme (P>0,05). Značajno bolja konverzija tretman grupe prasadi u odnosu na kontrolnu grupu utvrđena je na farmi BS 1 (P=0,0121), dok na drugim farmama nije uočena razlika u konverziji hrane prasadi kontrolne i tretman grupe (P>0,05). Dvofaktorijalnom analizom varijanse utvđen je značajan efekat primene preparata Patente Herba® Plus i biosigurnosnih nivoa farmi na ukupan i dnevni prirast prasadi (P0,05).Pig breeding is one of the most important branches of animal husbandry. Inadequate application of biosecurity measures and poor environmental conditions, in the presence of pathogens, could disable successful pig production. In over 50% of cases, enteric diseases could effect profitable production. One of the important enteric pathogens associated with large economic losses is Lawsonia intracellularis, an obligate intracellular Gram-negative bacterium, which causes proliferative enteropathy (PE). The “gold standards” for identifying L. intracellularis are immunohistochemical and PCR methods. The development of the quantitative real-time (qPCR) method enabled the routine detection of subclinical forms of the disease, and the quantification of the bacteria gives the possibility to determine the severity of the clinical symptoms in infected pigs. Since this infection is often misdiagnosed on pig farms, this results in excessive use of antibiotics and the ability of bacteria to develop resistance to antimicrobial agents. Therefore, it is necessary to find alternative solutions in PE control. Also, good biosecurity measures on farms, for which it is shown to have positive effect on health and production results of pigs, can be an important factor in the prevention of PE. Accordingly, the aim of this doctoral dissertation was to investigate the effect of commercial phytogenic additive (Patente Herba® Plus) in PE control in weaned piglets naturally infected with L. intracellularis, to establish and optimize protocols for molecular genetic identification of L. intracellularis, as well as to quantify the degree of infection in piglets via real-time qPCR and IHC methods. Additionally, the effect of different biosecurity levels and Patente Herba® Plus phytogenic additive on the production results and the number of excreted L. intracellularis in the faeces of piglets were investigated. The experiment was conducted on four different pig farms on which the levels of external, internal and total biosecurity measures were determined. On each of the farms (farm BS1, farm BS2, farm BS3 and farm BS4), seven-week-old piglets of uniform body weight were assigned to control groups that did not receive Patente Herba® Plus (K-BS1, K-BS2, K-BS3 and K-BS4) and the treatment groups that received Patente Herba® Plus in their feed at a concentration of 2 kg/t feed (T-BS1, T-BS2, T-BS3 and T-BS4). Control and treatment groups consisted of 72 piglets each, distributed in nine pens with eight piglets each, i.e. 144 piglets per farm, a total of 576 piglets. For histological analyzes, an additional experiment with individually reared piglets was performed, which consistet of six piglets in each, control and treatment group. During the 28 days of the experiment (days 0, 14, and 28) faeces samples were taken for molecular genetic analysis, the production results of piglets (body weight, daily and total weight gain, daily and total consumption and feed conversion ratio) were monitored, while at the end of the experiment ileum samples were taken for histological (microscopic changes and histomorphometric parameters) and immunohistochemical analyzes (L. intracelullaris antigen expression). On all four farms, within the external biosecurity, the subcategories related to the purchase of animals and semen and personnel and visitors were rated the best, while the biosecurity measures related to the feed, water and equipment supply were rated the lowest. Within the internal biosecurity, the subcategories of disease management and farrowing and suckling period had the highest score on farms, while the lowest score had biosecurity measures related to cleaning and disinfection, nursery unit and measures between compartments and use of equipment. The total biosecurity values of all four examined farms (BS 1 60%, BS 2 64%, BS 3 77% and BS 4 86%) in this doctoral dissertation were higher than the average biosecurity values on farms in Serbia (58%) and in the world (64%), with exception of farm BS 1 where total biosecurity value was lower compared to the average values in the world (60%). On the farm BS1, a significantly lower number of L. intracellularis was found in the faeces of piglets in the treatment group compared to the control group on days 14 and 28 of the experiment (P=0.0080 and P=0.0015, respectively). Also, the piglets of the treatment group had a significantly lower number of L. intracellularis in the faeces on days 14 and 28 compared to day 0 of the experiment (P0.05), also a significant decrease in the number of L. intracellularis in the faeces of piglets in the treatment groups during the experiment was not observed (P>0.05). A significant negative correlation was determined (r=-0.664, P0.05). Histological changes on ileum preparations of the control and the treatment groups of piglets were descriptively analyzed, while histomorphometric examinations showed that in the treatment group the crypts were significantly shallower (P=0.0284) and the villus height/crypt depth ratio significantly higher (P=0.0040) than in the control group. No effect of the addition of phytogenic additive was observed in piglet feed on villus height (P=0.0607), villus width (P=0.0728), villus surface area (P=0.7676) and the number of goblet cells/100 enterocytes (P=0.0575). Using semiquantitative analysis, based on immunohistochemical findings, 33.33% of ileal tissue samples of the treatment group piglets were rated with a grade 0, and the remaining 66.67% with a grade 1, while in the control group 50% of samples were rated with a grade 1, 33.33% with grade 2, and 16.67% of samples with grade 3. The use of phytogenic additive in feed did not significantly affect the body weight of piglets after 28 days of the experiment on all tested farms (P>0.05). The total and daily weight gain of piglets in the treatment group was significantly higher compared to the control group on the farm BS 2 (P=0.0349 and P=0.0345, respectively), while on farms BS 1, BS 3 and BS 4 the differences in total and daily weight gain in treatment and control groups of piglets were not observed (P>0.05). The best weight gain was achieved in the group that received the preparation Patente Herba® Plus in feed on farm BS 4, which was significantly higher compared to K-BS1, K-BS2 and T-BS2 group of piglets (P<0.0001). At the end of the experiment (day 28), no differences in total and daily feed consumption were observed between the control and treatment groups of piglets within each farm (P>0.05). Significantly better feed conversion ratio of piglet in the treatment group compared to the control was found on farm BS 1 (P=0.0121), while on other farms no differences were observed in the feed conversion ratio of piglets in control and treatment groups (P>0.05). Two-way analysis of variance revealed a significant effect of the application of Patente Herba® Plus and biosecurity levels of farms on the total and daily weight gain of piglets (P0.05)