Isolamento di agenti mastitogeni e riflessi ambientali sull'allevamento bufalino

The milk is universally recognized a complete diet due to its chemical-nutritional characteristics and can be considered one of the most important human food. However, its quality can worse after mastitis occurrence in both dairy cattle and buffalo. Mastitis are largely widespread in dairy herds and cause severe milk yield and quality losses, paid directly by milk producers and indirectly by consumers. Both cattle and buffalo are susceptible to mastitis, in particular in developing countries, resulting in health hazards: in fact, pathogens bacteria that cause mastitis can be conveyed through milk and be responsible of diseases in humans (zoonosis). The clinical mastitis is an individual problem and it is characterized by changes in udder and milk. On the contrary, subclinical mastitis can be considered a herd problem, since the animals become healthy carriers of pathogens that can spread in the herd. The purpose of the research carried out during the PhD was to detect: a) The seasonality of the mammary gland health status in buffaloes undergone milk recording; b) The frequency of isolation of the main germs isolated from the milk of clinically healthy buffaloes or buffaloes with clinical/subclinical mastitis, according to the seasonality. Furthermore, the antimicrobial resistance of these bacteria was tested over a period of six years (2010-2015); c) The timing and the histo-pathological modifications of the mammary gland following mastitis by specific agents. Experiment 1. In the first phase of the trial a statistical analysis was carried out on about 700,000 specimens, collected from about 100,000 buffaloes recorded by the National Association of Buffalo Breeders (ANASB) during the last 10 years. Milk yield and somatic cell count measured by Milkoscan were recorded. Experiment 2. A total of 3,585 milk samples (1745 positive to mastitis agents) were collected and cultured on different media for isolation and identification of bacterial species. The cultural and colony characteristics of the growing bacteria were examined and isolated bacteria were tested for sensitivity to antibiotics using the Kirby-Bauer’s method. Experiment 3. In order to assess the hystopathological damages of the mammary gland and the pathogens involved, a trial was carried out in a farm located in Foggia province. Buffaloes with more than 300,000 somatic cells and without a history of clinical mastitis, were selected and slaughtered in three periods: February, May and July. Mammary glands were collected and swabs were carried out for each quarter for microbiological examination. The swabs were processed for infectious bacteria, zoonotic agents and environmental or other opportunists germs. The isolated colonies underwent biochemical, enzymatic and serological tests. A statistical analysis was performed by Chi-square test. Experiment 1. Data recorded over a period of 10 years from 100,000 buffaloes undergone milk recording clearly showed the results regarding the application of the “out of season breeding mating” (OBSM) technique. In fact, a drastic reduction of milk production was recorded in primiparous buffaloes from August/September, while a less severe reduction was observed in pluriparous counterparts. It is also worth pointing out that milk production recorded in primiparous animals was about 10% lower than that recorded in pluriparous. The application of the OBSM technique is also highlighted by somatic cell count, that was affected by milk yield and days in milk. In fact somatic cell count was constant and lower than 300,000 (limit of MD 882/2004) throughout the year, except in December, when a higher incidence of both subjects at the end of lactation and subjects at the beginning of lactation is recorded, compared to the other months. In any case, the level lower of 300,000 somatic cells suggests that health of the udder. Experiment 2. The most common bacteria isolated in milk samples positive to mastitis agents were Staphylococcus aureus, Staphylococcus spp., Streptococcus aglactiae, Streptococcus dysgalactiae, Streptococcus uberis, Escherichia coli. Experiment 3. An analysis of the udders recovered at the slaughterhouse from buffaloes with more than 300,000 somatic cells revealed no signs due to acute mastitis after a macroscopic examination. However, in some cases, a teat and milk ducts thickening was observed, together a significant fibrosis. The microbiological examination revealed the presence of different Gram positive and negative bacteria examined. Staphylococcus spp. was isolated in 100% of the samples, it was possible to identify S. aureus in over 23% of cases (7/30): this bacterium is widely recognized as one of the main mastitis agents in both in cattle and buffalo. Clear signs of mastitis were observed by the histological analysis in 18/19 samples of udders collected in February and May (94.5%): in 12 of these (63.2%) significant lesions in the mammary gland ducts, interstitium and teats were assessed. In 10/11 mammary glands collected in July (90.1%) marked interstitial lesions characterized by lymphoplasmocytic infiltration and fibrosis, together with teats lesions were highlighted. Immunohistochemical and immunofluorescence investigations, showed the presence of CD8 positive lymphocytes in the interstices and in the epithelium. Mastitis is one of the main diseases found buffalo species. The data reported in this study can be a wake-up call if the somatic cell count is considered. In fact higher values than those considered pathological for bovine were observed in more than 20% of the samples analyzed from the population subjected to milk recording. Further studies are needed to debug a preventive approach for mastitis control and their diffusion in the herd. Furthermore, the analysis of mammary gland samples revealed no signs of acute mastitis. Only in some cases a thickening was visible, localized especially in the teats and milk ducts, where a significant fibrosis was appreciated and the microbiological examination revealed the presence of both Gram positive and negative bacteria. Finally, major attention would be paid to the agronomic and climatic conditions of the region, variations in socio-cultural practices, milk market, feeding and management, which can be considered critical points in increasing the incidence of subclinical mastitis