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,1
* Instituto de Ciências Biomédicas de Abel Salazar (ICBAS),
Unidade Multidisciplinar de Investigação Biomédica (UMIB), and
Centro de Investigação em Biodiversidade e Recursos Genéticos (CIBIO), Universidade do Porto, Porto, Portugal
1 Corresponding author: saramarques{at}mail.icav.up.pt
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ABSTRACT |
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Key Words: bovine mastitis • antimicrobial susceptibility • Prototheca
Bovine mastitis in both its clinical and subclinical forms continues to result in substantial loss of milk production and reduced milk quality. The use of antibiotics and the implementation of effective control programs for contagious pathogens are contributing to the predominance of environmental mastitis pathogens, especially in well-managed herds (Cattell et al., 2001). In many instances, the indiscriminate use of antimicrobial agents in the treatment of mastitis in dairy cattle is increasing the selection pressure toward antimicrobial resistance. A single strain may predominate because of antimicrobial resistance, host adaptation, or other factors (Lehtolainen et al., 2003). Bovine mastitis caused by algae of the genus Prototheca is gaining economic and public health importance, and its increasing incidence may be explained by these mechanisms (Benites et al., 1999; Melville et al., 2002; Roesler and Hensel, 2003).
Members of the genus Prototheca are unicellular algae related to the green algae of the genus Chlorella, but without chlorophyll (Pore, 1998; Hollingsworth, 2000; DiPersio, 2001; Rodriguez, 2003). They are resistant and ubiquitous and can be isolated from a great variety of environmental sources (Melville et al., 1999; Bexiga et al., 2003; Zhao et al., 2004). Only 2 species are pathogenic, Prototheca wickerhamii and Prototheca zopfii (Pore, 1998; Malinowski et al., 2002; Melville et al., 2002). Protothecosis in cattle is generally associated with P. zopfii, but recently, and for the first time, we identified one case of bovine mastitis caused by P. wickerhamii. In humans, protothecosis is almost exclusively associated with P. wickerhamii (DiPersio, 2001; Roesler et al., 2001; Roesler and Hensel, 2003; Linares et al., 2005). Prototheca as a cause of mastitis in dairy cows depends on predisposing factors such as poor environmental conditions and insufficient milking hygiene (Roesler et al., 2001; Roesler and Hensel, 2003). Infection by these algae causes acute to chronic granulomatous mastitis, leading to reduced milk production and atresia of the udder. Because of its capacity to infect and survive in macrophages and to invade the mammary tissue, they are responsible for a persistent infection with intermittent shedding (Costa et al., 1997; Benites et al., 1999; Roesler et al., 2001; Roesler and Hensel, 2003), and transmission may be by direct contact (Costa et al., 1997; Roesler et al., 2001; Bexiga et al., 2003). These algae do not respond to routine mastitis therapy, and the only control method to date has been elimination of the infected animals (Costa et al., 1997; Benites et al., 1999; Melville et al., 1999; Roesler et al., 2001).
Reports of in vivo and in vitro susceptibility studies of these algae to the usual mastitis antimicrobial agents indicate that they are resistant in the majority of cases (Melville et al., 2002; Bexiga et al., 2003; Zhao et al., 2004). However, Segal et al. (1976) found in vitro susceptibility of P. zopfii isolates to 2 polyenic antifungal agents commonly used in human protothecosis therapy. These findings led us to test the in vitro susceptibility of both P. zopfii and P. wickerhamii obtained from bovine mastitis to these antifungal drugs, with the objective of studying their antimicrobial resistance patterns.
For this study, one P. wickerhamii and 8 P. zopfii isolates previously obtained from clinical and subclinical bovine mastitis were revived by subculture on Sabouraud 4% dextrose agar (Merck, Darmstadt, Germany) and incubated for 48 to 72 h at 37 ° C. Speciation was performed using routine culture, macro- and microscopic morphological characterization, and API 20C AUX (bioMérieux, Marcy l’Etoile, France) methods (Pore, 1998), and confirmed by PCR (G. Thompson, unpublished data). Although there are no guidelines or interpretative criteria specific for Prototheca spp., the National Committee for Clinical Laboratory Standards (NCCLS, Clinical and Laboratory Standards Institute) M27-A2 guidelines for preparation of media and inoculum were followed (NCCLS, 2002). For MIC determination, macrodilution in brain heart infusion broth (Merck) and agar diffusion in Mueller-Hinton agar acc. NCCLS (Merck) were used. The selected antimicrobials were amphotericin B (AMB) 250 µg/mL (Sigma-Aldrich, Sintra, Portugal) and nystatin (NYT) 5000 IU/mL (manufactured by Lemos Pharmacy, Oporto, Portugal). Both antifungal drugs were diluted (1:2) in brain heart infusion broth with a concentration range from 0.00763 to 125 µg/mL for AMB and from 0.0305 to 500 µg/mL for NYT. The colony forming units of each isolate were between 0.74 x 106 and 9.1 x 106 cfu/mL for P. zopfii and were 2.8 x 105 cfu/mL for P. wickerhamii. A volume of 20 µL from each concentration of each Prototheca isolate was then inoculated into each dilution of the antifungal drug. Following an incubation period of 48 to 72 h at 37 ° C, 100 µL of each dilution was spread on Sabouraud 4% dextrose agar and incubated under the same conditions to confirm growth inhibition. In the macrodilution method, the MIC corresponds to the last tube without turbidity. To determine the growth inhibition diameter, the agar diffusion method was used. Each Prototheca spp. inoculum was streaked on Mueller-Hinton agar plates and blank susceptibility disks were placed on these plates after being submerged in the drug dilution tubes. The plates were incubated for 48 to 72 h at 37 ° C and the inhibition diameters were measured. The MIC obtained for P. zopfii and P. wickerhamii are summarized in Table 1
. For P. zopfii, the AMB and NYT minimum growth inhibition was measured at 10 and 8 mm in diameter, respectively, and for P. wickerhamii, this value corresponded to a growth inhibition diameter of 22 and 10 mm for AMB and NYT, respectively.
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). In a study by Malinowski et al. (2002), all P. zopfii strains obtained from the udders of cows were also sensitive to NYT at 100 IU, which is in agreement with our results. In another study (Buzzini et al., 2004), NYT was found to inhibit the growth of 58% of the Prototheca strains, compared with 33% growth inhibition for AMB, both at 50 µg/mL. Bovine mastitis by P. wickerhamii is a rare event and, although based in one isolate, our results suggest that growth of this Prototheca spp. is less affected than P. zopfii by both antifungals studied, as indicated by a systematic smaller diameter of the inhibition zone. These results demonstrate the need for a greater number of samples to achieve more conclusive results. There are still no drugs with proven clinical efficacy against protothecal bovine mastitis (Bexiga et al., 2003). Although our results indicate some sensitivity to these antifungal drugs in vitro, this does not guarantee that the drugs can be used for therapy against infections with these pathogens in cows. In other in vitro susceptibility studies, these algae have shown variable but low sensitivity to some antibiotics (Bexiga et al., 2003; Zhao et al., 2004). Amphotericin B is widely used in human medicine to treat protothecal infections (Di-Persio, 2001; Rodriguez, 2003; Linares et al., 2005) and demonstrates good activity, with MIC ranging from 0.09 to 3.12 µg/mL (Segal et al., 1976). Recently, Voriconazole demonstrated greater activity (MIC90 = 0.5 µg/mL) than AMB (MIC90 = 1 µg/mL) against all P. wickerhamii strains isolated from humans (Linares et al., 2005), and testing on bovine Prototheca sp. isolates is warranted. The different patterns of susceptibility to AMB and NYT shown by P. wickerhamii and P. zopfii in this study imply the need for further investigation into drugs that can be used with similar efficacy and, at the same time, be approved for use for intramammary or intramuscular administration in cattle. Mastitis caused by P. wickerhamii is very rare, and the pattern of susceptibility seen here needs to be confirmed with further studies.
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ACKNOWLEDGEMENTS |
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Received for publication March 30, 2006. Accepted for publication May 26, 2006.
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