Bovine mastitis, an evolving disease: Application of infrared thermography for the diagnosis of subclinical forms and presence of methicillin resistant Stahpylococcus aureus in dairy farms

Subclinical mastitis in dairy cows is a big economic loss for farmers. The monitoring of subclinical mastitis is usually performed through Somatic Cell Count (SCC) in farm but there is the need of new diagnostic systems able to quickly identify cows affected by subclinical infections of the udder. In the last years the fight against mastitis has seen the emergence of mastitis cases caused by multiresistant bacteria which poorly respond to antibiotic treatments. One of the most important pathogens because of this aspects of antimicrobial resistance and for its potential as zoonotic pathogen is the Methicillin Resistant Staphylococcus aureus (MRSA). The aim of this thesis is to investigate two important aspects of bovine mastitis, in a first study we evaluated the potential of infrared thermography in the diagnosis of subclinical mastitis and in a second study it was investigated the presence and the prevalence of MRSA in the territory of the Veneto Region. In the first study we evaluated the udder health status of 98 Holstein Friesian dairy cows with high SCC in 4 farms. From each cow a sample of milk was collected from all the functional quarters and submitted to bacteriological culture, SCC and Mycoplasma spp. culture. Thermographic images were taken from each functional udder quarter and nipple. Pearson’s correlations and Analysis of Variance were performed in order to evaluate the different diagnostic techniques. The most frequent pathogen isolated was Staphylococcus aureus followed by Coagulase Negative Staphylococci (CNS), Streptococcus uberis, Streptococcus agalactiae and others. The Somatic Cell Score (SCS) was able to discriminate (p<0.05) cows positive for a pathogen from cows negative at the bacteriological culture except for cows with infection caused by CNS. Infrared thermography was correlated to SCS (p<0.05) but was not able to discriminate between positive and negative cows. Thermographic imaging seems to be promising in evaluating the inflammation status of cows affected by subclinical mastitis but seems to have a poor diagnostic value. In the second study we investigated the presence and prevalence of MRSA in the Veneto Region. In order to do that 70 farms holding at least 100 animals were selected. In each farm were collected composite milk samples and nasal swabs from lactating cows. The samples collected were submitted for specific bacteriological culture for MRSA. The isolated strains were characterized to discover the presence of genes codifying for the resistance to several antimicrobial classes and to evaluate the epidemiological characteristics. 15 MRSA strains were isolated from 6 farms. 13 from milk samples and 2 from nasal swabs. The prevalence found was similar to what obtained in previous studies conducted in other European countries. The prevalence inside the farms was low and no farm was simultaneously positive for MRSA in milk and in nasal swabs. The low prevalence of colonization of the upper respiratory tract in somewhat surprising, considered the high prevalence of colonization in veal calves farms, in which are raised male calves coming from dairy farms. The genetic characterization has determined the belonging of the isolated strains to well known clones (CC1, CC97, CC398) adapted to livestock (Livestock Associated MRSA) and different from the more dangerous strains associated to healthcare environment (Hospital Associated MRSA). The clones found has anyway already proven their zoonotic potential and represent not only a new challenge in the fight against bovine mastitis but also a risk for the exposed personnel.La mastite subclinica è causa di importanti perdite economiche per gli allevatori di vacche da latte. Il monitoraggio dello stato di salute della mammilla negli allevamenti è generalmente effettuato tramite la Conta delle Cellule Somatiche (SCC) ma a causa della scarsa sensibilità di questa metodica c’è la necessità di trovare nuove tecniche diagnostiche in grado di identificare rapidamente infezioni subcliniche della mammella. Ad aggiungersi alle difficolta nella diagnosi di mastite negli ultimi anni sono comparse forme di mastite causate da batteri multiresistenti che causano un fallimento delle terapie antibiotiche. Uno dei batteri di primaria importanza sia per gli aspetti di antibioticoresistenza che per l’importanza quale agente zoonosico è lo Staphylococcus aureus meticillino-resistente (MRSA). Lo scopo di questa tesi è stato quello di investigare due tematiche relative alla mastite bovina, in un primo studio è stata valutata l’applicazione di un nuovo strumento diagnostico per la diagnosi delle infezioni subcliniche, mentre nel secondo studio è stata valutata la presenza nel territorio Veneto di un patogeno emergente, L'MRSA. Nel primo studio è stato valutato lo stato sanitario della mammella di 98 vacche di razza Frisona con elevata SCC, suddivise in 4 allevamenti della Regione Veneto. Da ogni vacca sono stati raccolti campioni di latte raccolti in maniera asettica su cui è stata eseguita la conta cellulare, l’esame batteriologico e la cultura per Mycoplasma spp.. Sono state inoltre raccolte immagini termografiche per la rilevazione delle temperature superficiali di tutti i quarti e i capezzoli funzionali. Al fine di valutare le differenti techinche diagnostiche sono state effettuate Correlazioni di Pearson, Analisi della Varianza (ANOVA) e curve ROC. Il patogeno isolato con maggiore frequenza è lo Staphylococcus aureus seguito dagli Stafilococchi coagulasi negativi (CNS), Streptococcus uberis, Streptococcus agalactiae e altri isolati con minore frequenza. Il Somatic Cell Score (SCS), calcolato sulla base della conta delle cellule somatiche è stato in grado di discriminare (p<0.05) i quarti positivi per un patogeno rispetto ai quarti negativi, con l’eccezione dei quarti infetti a causa di CNS. Lae temperature rilevate con l’analisi delle immagini termografiche sono positivamente correlate al SCS (p