Priscilla Anne Melville

Epidemiologic study of environmental sources in a Prototheca zopfii outbreak of bovine mastitis

Bovine mastitis represents the main form of occurrence of protothecosis in animals. The detection of mastitis caused by Prototheca sp. indicates a serious problem which can affect an entire herd. The purpose of this study is to explain some aspects of the epidemiology of mastitis due to Prototheca zopfii with the evaluation of the presence of these microorganisms in samples collected from potential sources in the dairy herd. This study was performed during a Prototheca zopfii outbreak of clinical bovine mastitis in the State of São Paulo, Brazil. The following samples were aseptically collected for microbiological examination: milk (n = 211); rectal swabs (from 15 calves and 2 lactating cows); swabs from teat cup rubbers during milking (n = 2); water (n = 6); soil (n = 6). Prototheca zopfii was isolated from 77 (36.49%) of the 211 milk samples; 11 calves and 2 cows showed Prototheca zopfii in faecal samples; both swabs collected from the teat cup rubbers showed viable forms of Prototheca zopfii; this microorganism was also isolated from 2 water samples, and 1 soil sample collected from the dry cow pasture. Prototheca zopfii seemed to be widespread throughout the dairy herd environment where this outbreak of bovine mastitis occurred.

Evaluation of the susceptibility of Prototheca zopfii to milk pasteurization

Protothecosis has been reported in humans (gastroenteritis, bursitis, etc.) and in many other animal species. Bovine mastitis represents the main form of occurrence of protothecosis in cattle. Milk as well as dairy products, when contaminated with Prototheca spp., represent a potential means of transmission of this zoonosis. The purpose of this study was to evaluate the susceptibility of forty Prototheca zopfii strains isolated from milk from intramammary infections in dairy cows and also from bulk milk tanks of dairy farms, to the different ratios of temperature/time employed in the thermal treatment of milk: 72–75 °C/1 5 seconds, 72–75 °C/20 seconds and 62–65 °C/30 minutes. The samples were subjected to these different temperature/time ratios. The evaluation of the thermal susceptibility of the P. zopfii strains showed that 34 strains were resistant in at least one of the tests. The results point out the need to consider the importance of mastitis caused by Prototheca spp. asrepresenting a public health risk.

Susceptibility and features of the ultrastructure of Prototheca zopfii following exposure to copper sulphate, silver nitrate and chlorexidine

One of the most important forms of the occurrence of protothecosis is bovine mastitis. Studies on the “in vivo” and “in vitro” susceptibility to antimicrobials have shown that the microorganism is resistant to most of them. Looking for alternative treatments this study aimed to study the susceptibility to copper sulphate (which has an important algicide effect) and silver nitrate (used in dairy cattle breeding for the cauterization of mammary glands) and also to chlorexidine (an important post-dipping anti-septic used in dairy practice), and the effect of these antimicrobials in the ultrastructure of Prototheca zopfii before and after the exposure to these drugs. The “in vitro” susceptibility tests to chlorexidine, silver nitrate and copper sulphate of the strains of Prototheca zopfii for the determination of their minimal microbicidal concentrations (MMC), were performed using the tube dilution method in Sabouraud dextrose broth and evaluation of colony growth after plating in Sabouraud dextrose agar. The MMCs of chlorexidine, copper sulphate and silver nitrate of the 50 strains tested were 0.01%, 0.1% and 0.3%, respectively. The tubes containing the material used in the antimicrobial susceptibility tests were prepared for the examination in an electron microscope. The untreated controls of P. zopfii showed a similar ultrastructural appearance with the typical characteristics of the microorganism. Cells exposed to silver nitrate showed changes suggesting thickness of the cell wall. Cells exposed to chlorexidine showed changes suggesting degradation of intra-cellular organelles present in the cytoplasm. P. zopfii treated with copper sulphate showed changes suggesting fibrilation of inner layer of cell wall.

Function of milk polymorphonuclear neutrophil leukocytes in bovine mammary glands infected with Corynebacterium bovis

Corynebacterium bovis is one of the most commonly isolated bacteria from aseptically collected bovine milk samples. The objective of the current study was to characterize the bovine innate immune response by evaluating milk polymorphonuclear neutrophilic leukocytes (PMNL) in mammary glands infected with C. bovis. Twenty quarters infected with C. bovis and 28 culture-negative quarters (with milk somatic cell count <1×10(5) cells/mL) were used. The percentages of milk PMNL and the PMNL expression of L-selectin (CD62L), β2-integrin (CD11b), and one of the endothelial-selectin ligands (CD44), as well as the levels of intracellular reactive oxygen species (ROS) and the phagocytosis of Staphylococcus aureus, were evaluated by flow cytometry. The apoptosis and necrosis rates of the PMNL were quantified using dual-color flow cytometry with fluorescein-labeled annexin and propidium iodide. The present study revealed a higher percentage of PMNL in the milk from C. bovis-infected quarters, although no significant differences were found in levels of CD44, CD62L, or CD11b expression among the PMNL. A lower percentage of apoptotic PMNL was observed in C. bovis-infected quarters, as well as higher percentages of viable PMNL and of PMNL that produced intracellular ROS. However, no alterations were observed in phagocytosis of Staph. aureus by the PMNL or in intensity of intracellular ROS production by PMNL. Thus, results from this investigation of the PMNL function support, at least in part, the fact that intramammary infections by C. bovis may offer protection against intramammary infections by other bacteria.

In vitro algaecide effect of sodium hypochlorite and iodine based antiseptics on Prototheca zopfii strains isolated from bovine milk.

Prototheca zopfii has been considered one of the most important causes of environmental mastitis in Brazil. These algae are refractory to conventional therapy and cause great damage to the mammary gland. The present study evaluated the in vitro algaecide effect of sodium hypochlorite and iodine based antiseptics on 27 P. zopfii strains isolated from the milk of cattle. Low concentrations of sodium hypochlorite (0.0390625-0.15625%) and iodine (0.15625-0.625%) were effective against the isolates. These antiseptics may be recommended for hygiene routines, pre and postdipping and cauterization of bovine mammary glands infected by P. zopfii.

In vitro photoinactivation of bovine mastitis related pathogens

BACKGROUND Bovine mastitis is considered the most important disease of worldwide dairy industry. Treatment of this disease is based on the application intramammary antibiotic, which favors an increase in the number of resistant bacteria in the last decade. Photodynamic inactivation (PDI) has been investigated in different areas of Health Sciences, and has shown great potential for inactivating different pathogens, without any selection of resistant microorganisms. The objective of this study was to investigate the efficacy of PDI in the inactivation of pathogens associated with bovine mastitis. METHODS We tested the effectiveness of PDI against antibiotic resistant strains, isolated from bovine mastitis, from the following species: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Corynebacterium bovis, and the alga Prototheca zopfii. Nine experimental groups were evaluated: control, no treatment; light only, irradiation of a red light-emitting diode (λ=662 (20) nm) for 180 s; exposure to 50 μM methylene blue alone for 5 min; and PDI for 5, 10, 30, 60, 120 and 180 s. RESULTS S. dysgalactiae, S. aureus, and C. bovis were inactivated after 30s of irradiation, whereas S. agalactiae was inactivated after 120 s and P. zopfii at 180 s of irradiation. CONCLUSION These results show that PDI can be an interesting tool for inactivating pathogens for bovine mastitis.

Occurrence of genes coding for MSCRAMM and biofilm-associated protein Bap in Staphylococcus spp. isolated from bovine subclinical mastitis and relationship with somatic cell counts

This study aimed to elucidate aspects of the epidemiology of bovine subclinical mastitis through the assessment of genes encoding MSCRAMM (microbial surface components recognizing adhesive matrix molecules - a group of adhesins) and protein Bap (implicated in biofilm formation), in coagulase-positive (CPS) and coagulase-negative (CNS) Staphylococcus isolated from subclinical mastitis. Milk samples were collected for microbiological exams, somatic cell count (SCC) and a survey of the genes coding for MSCRAMM (cna, eno, ebpS, fnbA, fnbB and fib) and biofilm-associated protein Bap (bap) in 106 Staphylococcus spp. isolates using PCR. The frequencies of occurrence of eno (82.1%), fnbA (72.6%), fib (71.7%) and bap (56.6%) were higher (P < 0.0001) compared with the other assessed genes (cna, ebpS and fnbB). The higher frequency of occurrence (P < 0.005) of the bap gene in CNS compared with CPS suggests that in these species biofilm formation is an important mechanism for the persistence of the infection. The medians of the SCCs in the samples where eno, fnbA, fib and bap genes were detected were higher compared with Staphylococcus without the assessed genes (P < 0.05) and negative samples (P < 0.01), which indicated that the presence of these MSCRAMM may be related to a higher intensity of the inflammatory process.

Comparison of four antibiotics for inactivating leptospires in bull semen diluted in egg yolk extender and experimentally inoculated with Leptospira santarosai serovar guaricura

Inactivation of leptospires in pools of semen from three Holstein Friesian bulls, collected in an artificial vagina, was investigated. Spermatic concentration was adjusted in egg yolk citrate extender, submitted to the following treatments: A (control; without antibiotics); B (penicillin, 1,000 UI/mL - streptomycin, 1,000 µg/mL); C (amoxicillin, 1,000 µg/mL); D (ceptiofur sodium, 1,000 µg/mL); E (amoxicillin 1,000 µg/mL - ceptiofur sodium 1,000 µg/mL). Leptospires (2.0 x 106 leptospires/mL) were added into the diluted semen. Recovery of leptospires was obtained in modified EMJH semi-solid medium with and without antibiotics. The antibiotics in the concentrations used did not affect means of percentage of progressive motility and individual progressive motility of spermatozoids. Penicillin-streptomycin presented the best results in leptospire inactivation (97.1%). Amoxicillin, ceptiofur sodium and their combination at the concentrations studied presented poor results: 59.29%; 32.5% and 60.36% of inactivation, being less effective in leptospire inactivation than penicillin-streptomycin.

Molecular detection of enteropathogenic Escherichia coli in asymptomatic captive psittacines

Psittaciformes are one of the most endangered groups of birds, and several Brazilian species are classified between vulnerable and critically endangered. It is thus necessary to identify agents that cause infections in captive wild animals and to assess the risks posed thereof and to design interventions to minimize the possibility of disease outbreaks, leading to the conservation of endangered species. The purpose of this study was to identify enteropathogenic Escherichia coli (EPEC) cloacal isolates from asymptomatic psittacines in captivity and evaluate the distribution of the EPEC pathotype. Cloacal swabs were obtained from 46 asymptomatic birds, and resulting isolates were tested by polymerase chain reaction (PCR) for the presence of the attaching and effacing gene (eae) and bundle-forming pilus structural gene (bfpA) of EPEC. Samples from several species were tested, and three samples were found to be positive for the eae and bfpA genes and characterized as typical EPEC. This is the first report of this pathotype in asymptomatic psittacines. Although certain E. coli strains are more pathogenic than others, various factors should be considered when determining the potential of E. coli isolates to cause disease in captive psittacines. Birds that are positive for the EPEC (typical) strain could be zoonotic sources of infection, and may have acquired these strains through contact with humans or domestic animals. These findings may also be valuable for the long-term management of endangered species ex situ as one EPEC sample was isolated from a Red-tailed Amazon (Amazona brasiliensis).

Determinação da microbiota presente na cloaca e orofaringe de avestruzes (Struthio camelus) clinicamente sadios

The knowledge of the microbiota present in different parts of the organism is important for the understanding of the infectious diseases of ostriches, although limited literature is available on this matter. The objective of this study was to determine the species of microorganisms (aerobic bacteria and fungi) that form the normal microbiota of ostriches. Samples from cloaca (N=50) and oropharynx (N=50) were collected from healthy ostriches of a breeder. In samples from cloaca, the following microorganisms were isolated: Escherichia coli (76% of the positive samples), Bacillus spp. (60%), Streptococcus spp. (18%), coagulase-negative Staphylococcus (16%), Pseudomonas aeruginosa (8%), Rhodotorula spp. (8%), among others, considering that these microorganisms were isolated in pure culture or associated with other bacteria and/or fungi. In samples collected from oropharynx, the following microorganisms were isolated: E. coli (74% of the positive samples), Candida albicans (44%), Bacillus spp. (38%), coagulase-negative Staphylococcus (32%), Klebsiella pneumoniae (32%), Rhodotorula spp. (8%), Criptococcus spp. (4%), among others, considering that these microorganisms were isolated in pure culture or associated with other bacteria and/or fungi. A predominance of Gram negative bacteria in relation to Gram positive ones was observed in the microbiotas of cloaca and oropharynx. A similar frequence of occurrence between Gram negative bacteria in the microbiotas of cloaca and oropharynx, as well as Gram positive bacteria in these same areas, was verified. A higher occurrence of yeasts was observed in samples of oropharynx when compared to samples from cloaca.