Research in Veterinary Science 88 (2010) 211–213
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Research in Veterinary Science
journal homepage: www.elsevier.com/locate/rvsc
In vitro algaecide effect of sodium hypochlorite and iodine based antiseptics on
Prototheca zopfii strains isolated from bovine milk
Tatiana Salerno a,*, Márcio Garcia Ribeiro a, Hélio Langoni a, Amanda Keller Siqueira a,
Elizabeth Oliveira da Costa b, Priscilla Anne Melville b, Válter Ferreira Félix Bueno c,
Aline Artioli Machado Yamamura d, Uwe Roesler e, Aristeu Vieira da Silva f
a
Department of Veterinary Hygiene and Public Health, School of Veterinary and Animal Science, São Paulo State University, Botucatu, São Paulo, Brazil
Research on Mammary Gland and Milk Production (NAPGAMA), Department of Preventive Veterinary, University of São Paulo, São Paulo, Brazil
Center of Research for Foods, Veterinary School, Goiás Federal University, Brazil
d
Department of Preventive Veterinary, Londrina State University, Paraná, Brazil
e
Institute of Animal and Environmental Hygiene, Freie Universität of Berlin, Germany
f
Executive Management of Administration the Research, Paranaense University, Umuarama, Paraná, Brazil
b
c
a r t i c l e
i n f o
Article history:
Accepted 4 August 2009
Keywords:
Prototheca zopfii, antiseptics
Bovine mastitis
Milk
Brazil
a b s t r a c t
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.
Ó 2009 Elsevier Ltd. All rights reserved.
Prototheca spp. are unicellular, achlorophyllous and ubiquitous
algae (Quinn et al., 1994). The taxonomic status of Prototheca includes six species: P. zopfii, P. wickerhamii, P. blaschkeae, P. stagnora,
P. ulmea and P. moriformis (Pore, 1985; Roesler et al., 2006). Among
these species, P. zopfii, P. wickerhamii and P. blaschkeae have been
associated with human and bovine infections (Thiele and
Bergmann, 2002; Roesler et al., 2006; Marques et al., 2008). In
Brazil, P. zopfii was described as an emergent cause of bovine environmental mastitis (Costa et al., 2004; Yamamura et al., 2007;
Marques et al., 2008).
P. zopfii mammary infections are characterized by clinical manifestation, presenting watery secretion and reduced milk production (Radostitis et al., 2007). Currently, protothecal mastitis cause
pyogranulomatous processes, tissue alterations and destruction
of glandular parenchyma (Melville et al., 2002).
Recently, P. zopfii was classified in three biotypes, namely I, II
and III. Based on genotypic evidence, biotypes I and II were re-classified as genotypes 1 and 2, respectively, and biotype III as a new
species, called P. blaschkeae (Roesler et al., 2006). P. zopfii genotype
* Corresponding author. Address: Faculdade de Medicina Veterinária e Zootecnia,
Departamento de Higiene Veterinária e Saúde Pública (DHVSP), Universidade
Estadual Paulista (UNESP), Distrito de Rubião Júnior s/n, CEP 18.618-000, Botucatu,
São Paulo, SP, Brazil. Tel.: +55 14 3811 6270; fax: +55 14 3811 6075.
E-mail address: [email protected] (T. Salerno).
0034-5288/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.rvsc.2009.08.001
1 strains are found in liquid cattle manure; genotype 2, in bovine
intramammary infections; and P. blaschkeae, in piggeries.
P. blaschkeae causes human onychomycosis (Möller et al., 2007)
and has recently been reported to be a causative agent of bovine
mastitis (Marques et al., 2008).
Studies on the in vitro and in vivo susceptibility profile of the Prototheca genus have revealed great resistance to conventional antimicrobial, antifungal and antiseptic drugs (Costa et al., 1996a,b;
Bueno et al., 2006; Marques et al., 2006; Buzzini et al., 2008).
Sodium hypochlorite is efficient against a wide range of microorganisms. This antiseptic of halogens group affects microbial
enzymatic systems, producing changes in the cell wall (Hoffmann
et al., 1995). In Brazil, Ribeiro (1999) used sodium hypochlorite
for pre (0.8–1.2%) and postdipping (4.0%) in the control of mammary protothecosis in dairy farms.
Likewise, iodine is recognized as an efficient antiseptic from the
family of halogen elements. Iodine has high cell penetrating power,
causes protein precipitation and oxidation of essential enzymes,
and interferes in vital metabolic reactions of microorganisms
(Taniyama et al., 1994). In dairy farms, iodine is generally indicated
in pre (0.1%) and postdipping (0.5–1.0%) for the control of bovine
mastitis (Ribeiro, 1999).
The aim of present study was to evaluate the in vitro algaecide
effect of sodium hypochlorite and iodine based antiseptics on
P. zopfii strains isolated from bovine milk in Brazil.
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T. Salerno et al. / Research in Veterinary Science 88 (2010) 211–213
Twenty seven P. zopfii strains isolated from clinical or subclinical bovine mastitis cases in different states of Brazil, and one bulk
milk strain were used in this trial. Strains were cultured on defibrinated bovine blood agar (5%) and incubated aerobically at
37 °C for 48–72 h. All strains were identified as P. zopfii based on
morphological, physiological, staining aspects, and on the following cultural characteristics: size, presence of capsule and assimilation of trehalose, dextrose, sucrose and n-propanol (Camargo and
Fischman, 1979).
Molecular analysis of 27 strains was performed based on a
highly conserved small-subunit 18S rDNA gene sequence. The following cycle conditions were used: 30 s denaturation (94 °C),
annealing (58 °C for genotypes 1 and 2, and 63 °C for P. blaschkeae)
and 40 s extension (72 °C). P. zopfii genotype 1 (RZI-1, RZI-2 and
SAG 2063), genotype 2 (SAG 263-4T, SAG 2021, RZII-2 and RZII-3)
and P. blaschkeae (RZIII-1, RZIII-2 and SAG 2064T) were used as
control strains (Roesler et al., 2003). Gene sequence analysis
showed that all isolates belonged to genotype 2.
Antiseptics were tested based on the minimal microbicidal concentration – MMC (M27-A2) (Melville et al., 2002), using the tube
dilution test (Pfaller, 2002), with some modifications. Minimal
algaecide concentration (MAC) of isolates was defined as the lowest disinfectant concentration able to prevent post exposure algae
growth on bovine blood agar. For the preparation of the inoculums,
all isolates were cultured on defibrinated bovine blood agar
(OxoidTM), incubated aerobically at 37 °C for 48–72 h. Next, two
or three colonies of each strain were transferred to tubes containing 10 mL of brain–heart infusion broth – BHI (OxoidTM), and
incubated in the same conditions. Turbidity was adjusted using
tube 1 of McFarland scale.
Sodium hypochlorite and iodine in an initial concentration of
10.0% were used in the evaluation of MAC. One mL of 10.0% sodium
hypochlorite solution was added to the first tube of all strains and
0.5 mL of sterilized Milli-Q water was placed in nine subsequent
tubes. After that, 0.5 mL of sodium hypochlorite was transferred
from the first to the second tube, which was homogenized, and
the same process was repeated successively in the other tubes;
in tube 10, 0.5 mL were discarded. Next, 0.5 mL of P. zopfii inoculum was added to each tube to obtain the following concentrations: 5.0%, 2.5%, 1.25%, 0.625%, 0.3125%, 0.15625%, 0.078125%,
0.0390625%, 0.01953125% and 0.009765625%. Tubes were incubated at 37 °C for approximately 12 h. After homogenization,
10 ll of each tube were streaked on defibrinated bovine blood agar
(5.0%) and incubated aerobically at 37 °C for 7 days. Results were
read every 24 h in order to assess the minimal algaecide concentration. The same modified dilution process was carried out using
10.0% iodine solution. Positive control suspensions were prepared
in sterilized tubes containing 0.5 mL of sterilized Milli-Q water
and 0.5 mL of the inoculums, adjusted McFarland scale with optical
density equivalent to tube one following plated and finally cultured as described above. All in vitro tests were performed in
duplicates and showed high repeatability of the results.
Minimum and maximum values to MAC for sodium hypochlorite and iodine were determined by Mann–Whitney test. Statistical
analysis was carried out in EpiInfo 6 software (Centers for Disease
Control, Atlanta) with a = 0.05 (Triola, 2005).
Bovine mastitis caused by Prototheca genus has increased significantly in several countries, including Brazil (Costa et al., 2004;
Yamamura et al., 2007). P. zopfii genotype 2 have been identified
in bovine mastitis described in Europe (Roesler et al., 2006; Möller
et al., 2007). Molecular analysis of our 27 P. zopfii strains enabled
the classification of the isolates as genotype 2, reinforcing the predominance of this genotype in bovine mammary protothecosis
(Radostits et al., 2007).
Due to resistance of algae to conventional therapy, different
studies have investigated in vitro susceptibility profiles against
antimicrobial, antiseptic or algaecide products in Prototheca spp.
strains isolated from bovine mastitis (Melville et al., 2002; Marques et al., 2006). Several antiseptics have been used as alternatives in the treatment of mammary protothecosis (Costa et al.,
1996b; Melville et al., 2002; Marques et al., 2006).
In Brazil, hypochlorite is widely used in dairy farms for the disinfection of floors, walls, utensils and equipment, as well as in the
control of microorganisms in water, and in pre and postdipping
antisepsis of mammary glands, because of the low cost, wide availability and efficient microbicidal action of the product (Domingues
and Langoni, 2001; Pedrini and Margatho, 2003). In our study, MAC
was 0.078125% for hypochlorite against 19 (70.4%) from 27 isolates; 0.0390625% against 7 (25.9%) strains, and 0.15625% against
1 (3.7%) isolate. MAC was 0.15625% against P. zopfii genotype 2
(SAG 2021) positive control. Pedrini and Margatho (2003) observed
statistical significant effect of 2.0% sodium hypochlorite against
several microorganisms responsible for environmental and contagious mastitis. However, these same authors did not observe
in vitro microbicidal effect when concentrations used were equal
or lower than 0.5%. Ribeiro (1996) was successful using 4% sodium
hypochlorite from 30 s to 1 min against 11 P. zopfii strains isolated
from mastitis in Brazil. In our study, satisfactory algaecide effect
was obtained using low concentrations of sodium hypochlorite
against 27 strains. Variations in the concentration threshold of sodium hypochlorite effectiveness reported by others study may be
justified by different in vitro methods and exposure times used.
In present study MAC for iodine was 0.3125% against 22 (81.5%)
strains; 0.625% against 3 (11.1%) strains, and 0.15625% against 2
(7.4%) isolates. MAC for iodine against P. zopfii genotype 2 positive
control was 0.3125%. Great microbicidal in vitro effect against several mastitis pathogens was reported using 1% and 2% iodine solution (Pedrini and Margatho, 2003). Algaecide effect of iodine was
observed against all our P. zopfii isolates, in concentrations ranging
from 0.15625% to 0.625%. Ribeiro (1996) reported in vitro algaecide
effect using 0.5% and 1.0% iodine against P. zopfii isolated from
cows with mastitis.
Average values observed by disinfectants revealed statistical
differences (p < 0.0001) between in vitro efficacy of sodium hypochlorite and iodine. Our results suggest that these antiseptics may
be used as alternatives in predipping solutions for cattle affected
by mammary protothecosis. In vitro susceptibility of P. zopfii
strains to sodium hypochlorite also suggests that this antiseptic
may be indicated to be used in milking routine and cleaning of
equipment in farms where protothecosis occur. Furthermore, higher concentrations of sodium hypochlorite may be recommended in
the chemical cauterization of mammary glands infected by P. zopfii.
Similarly, iodine revealed good in vitro algaecide effect against the
isolates, and may also be used as an antiseptic, mainly in postdipping solutions, as a control measure against contagious mastitis in
bovines. However, presence of organic material (feces), milk protein content, temperature, humidity and other environmental factors may reduce the effectiveness of disinfectant products.
The present study highlighted the effectiveness of iodine as an
antiseptic agent for postdipping or in chemical cauterization of udder quarters affected by P. zopfii, and revealed that sodium hypochlorite may be an alternative for the control of mammary
protothecosis in dairy herds.
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