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Journal of Clinical Microbiology, June 2008, p. 1941-1945, Vol. 46, No. 6
0095-1137/08/$08.00+0     doi:10.1128/JCM.00323-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Bovine Mastitis Associated with Prototheca blaschkeae

Sara Marques,^1,2* Eliane Silva,^1,2 Christine Kraft,³ Júlio Carvalheira,^1,4 Arnaldo Videira,^1,5 Volker A. R. Huss,³ and Gertrude Thompson^1,2

Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Largo Prof. Abel Salazar, 2, 4099-003 Porto, Portugal,¹ Unidade Multidisciplinar de Investigação Biomédica, Universidade do Porto, Largo Professor Abel Salazar, 2, 4099-003 Porto, Portugal,² Department für Biologie—Molekulare Pflanzenphysiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, D-91058 Erlangen, Germany,³ Centro de Investigação em Biodiversidade e Recursos Genéticos, Universidade do Porto, Rua Padre Armando Quintas, 4485-661 Vairão, Portugal,⁴ Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal⁵

Received 15 February 2008/ Returned for modification 16 March 2008/ Accepted 13 April 2008

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Bovine mastitis is an important and complex disease responsible for economic losses in the dairy industry. Biotype II strains of the green alga Prototheca zopfii can be involved, most often resulting in chronic mastitis of difficult treatment associated with reduced milk production. This type of infection is rare, but the number of reported cases is increasing worldwide. In order to determine the kind of species involved in mastitis by Prototheca in northwest Portugal, 41 Prototheca isolates were genetically characterized. The algae are part of Prototheca isolates that were collected during a 6-year period, isolated from the milk of 41 dairy cows in a total of 22 herds with a history of increasing somatic cell counts, mild clinical signs of udder infection, and unsuccessful response to the usual therapy. PCR amplification of the 18S ribosomal DNA (rDNA), amplified rDNA restriction analysis, and phylogenetic analyses of the 18S rDNA sequences were performed. Thirty-seven isolates were identified as P. zopfii var. hydrocarbonea and four as Prototheca blaschkeae. These data suggest a high incidence of P. zopfii var. hydrocarbonea mastitis in the region and demonstrate for the first time the involvement of P. blaschkeae with bovine mammary gland infections.

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Green algae of the genus Prototheca, and more rarely Chlorella, are the only known plant-like organisms that cause infectious diseases in humans and animals (6, 11-13). The genus Prototheca consists of microscopical, unicellular, achlorophyllic algae with asexual reproduction by autosporulation with variable numbers of autospores (5, 10). These algae are ubiquitous and saprophytic, but some species may turn into unusual opportunists causing pathology when the host immunological defenses are impaired (16, 18, 21) or when predisposing factors occur, such as, in the case of dairy cows, poor animal care and poor milking hygiene (8, 11, 18). The incidence of bovine mastitis associated with Prototheca infections is steadily increasing and gaining more and more economic and public health importance (18). Prototheca is included in the class Trebouxiophyceae (14) and is closely related to green algae of the genus Chlorella (13, 23), which are among the best-studied unicellular green algae (7). Within the last decade, Prototheca taxonomy has developed and five species are currently assigned to the genus, namely Prototheca zopfii, P. wickerhamii, P. stagnora, P. ulmea, and most recently, P. blaschkeae (19). The validation of another species, Prototheca moriformis (1, 17), has been questioned by molecular studies (19, 25). Until recently, the only species known to cause infectious diseases in humans and animals were P. wickerhamii and P. zopfii, respectively (4, 20, 28). The new species P. blaschkeae was identified in 2006 from a previous case of human onychomycosis (19). This species was previously defined as biotype III of P. zopfii, which, together with biotype I, was reported to be nonpathogenic, while biotype II has been associated with bovine mastitis (20). Biotype classification was based on phenotypic characteristics, as well as auxonographical and biochemical analyses (3, 20). Serological analyses revealed differences in the pattern of immunogenic structures between the biotypes (20). Further phylogenetic studies of 18S ribosomal DNA (rDNA) sequences were able to determine discriminatory molecular characteristics to define the new species P. blaschkeae and reclassify the P. zopfii biotypes into two genotypes (19).

The present study aimed to elucidate the epidemiology of bovine mastitis by molecular characterization of Prototheca isolates obtained from 41 dairy cows of 22 herds in Portugal. We report the predominant occurrence of Prototheca zopfii var. hydrocarbonea in milk of cows with mastitis associated with Prototheca and for the first time the association of P. blaschkeae with bovine mastitis.

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Isolation of algae. Prototheca isolates in this study belong to a major collection compiling several milk pathogens that belong to the Laboratory of Infectious Diseases of the Veterinary Medicine School of Porto University. These represent a small number of pathogenic organisms that were recovered from about 3,500 mastitic milk analyses during a 6-year period (from January 2002 to December 2007). Prototheca cells were retrieved from milk of 41 cows with mastitis originating from 22 different dairy herds from northwest Portugal, which was collected under sterile conditions. The affected cows had a history of persistently high somatic cell counts or clinical mastitis that was not responsive to intramammary antibiotic treatment. To avoid confounding effects due to the therapy, only milk samples collected after the treatment safety period were analyzed. For diagnostic purposes, 40-µl aliquots of milk samples collected from individual quarters of the udder were streaked onto Columbia agar plates supplemented with 5% sheep blood (bioMérieux, Marcy l'Etoile, France). After 42 to 72 h at 37°C, plates were examined for Prototheca growth and any colonies resembling this alga were subcultured once on Sabouraud dextrose agar medium (Merck Laboratories, Darmstadt, Germany). After macroscopical and microscopical identification, Prototheca isolates were spread and grown on Sabouraud dextrose agar medium.

Preparation of genomic DNA. Algal cultures were grown on Sabouraud dextrose agar for 48 h at 37°C. Cells were harvested by centrifugation (1,600 x g, 10 min) and mechanically ground with glass beads and extraction buffer (200 mM Tris-HCl, pH 7.5, 250 mM NaCl, 25 mM EDTA, 0.5% sodium dodecyl sulfate) in 1.5-ml Eppendorf tubes in a cell mill (Qiagen tissue lyser) for 10 min at 30 Hz. The efficiency of cell disruption was controlled by microscopical observation. Subsequently, 200 µl of chloroform was added and the suspension was shaken for 1 min. After centrifugation (21,000 x g, 5 min), the supernatant was transferred to a new tube and mixed with 200 µl of isopropanol for DNA precipitation. The DNA was pelleted by centrifugation (21,000 x g, 5 min) and washed with 70% ethanol. The pellet was air dried and resuspended in a maximum of 50 µl of Milli-Q sterilized water for further use.

rDNA amplification and amplified rDNA restriction analysis. For amplification of the 18S rDNA, the conserved eukaryote-specific primers previously designed by Huss et al. (7) (forward primer, 5' WACCTGGTTGATCCTGCCAGT 3'; reverse primer, 5' GATCCTTCYGCAGGTTCACCTAC 3') were used. Thirty-five cycles were run in a Biometra T3000 thermal cycler with 60 s of denaturation at 95°C, 60 s of annealing at 54°C, and 150 s of extension at 72°C. The amplification products were analyzed on a 0.8% (wt/vol) agarose gel after staining with ethidium bromide. The amplified rDNA was submitted to restriction analysis with HaeIII (NEB, Inc., Frankfurt, Germany) and analyzed on a 1.8% (wt/vol) agarose gel after staining with ethidium bromide. Several of the amplified rDNAs were purified with a PCR purification kit (Qiagen, Inc., Hilden, Germany) and directly sequenced using the following primers: 300 F (5' AGGGTTCGATTCCGGAG 3'), 1055 F (5' GGTGGTGCATGGCCG 3'), 536 R (5' GWATTACCGCGGCKGCTG 3'), and 1200 R (5' GGGCATCACAGACCTG 3') (7). Nucleotide sequence determination was carried out by Macrogen, Ltd. (Korea).

Phylogenetic analyses. The 18S rDNA sequences determined in this study were manually aligned on a MicroVAX computer with the sequence editor program distributed by G. Olsen (15). For the phylogenetic analyses, the sequences were aligned with sequences of representative trebouxiophycean green algae extracted from a larger alignment and with two chlorophycean algae that were used as an outgroup. To improve the alignment of the data set, secondary structure models were constructed according to the model provided by Wuyts et al. (27). Highly variable regions that could not be aligned unambiguously for all sequences were excluded from the analyses together with PCR primer binding regions, resulting in a total of 1,782 positions. Phylogenetic trees were inferred from the aligned sequence data by the neighbor-joining (NJ), maximum parsimony (MP), and maximum likelihood (ML) method using PAUP* 4.0 (22). One thousand bootstrap replicates each were carried out for NJ and MP, as well as 100 replicates for ML.

Nucleotide sequence accession numbers. The 18S rRNA gene sequences determined in this study have been deposited in GenBank under accession no. EU439262 and EU439263.

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The recovery of the 41 Prototheca isolates from mastitic milk from 41 different dairy cows was achieved by usual microbiological methods. After milk samples were streaked on Columbia agar supplemented with 5% sheep blood or on Sabouraud dextrose agar and incubated for 48 to 72 h, grayish and white to cream-colored small colonies of 1 mm or 1 to 2 mm, respectively, with yeast-like appearance and odor were detected. No hemolysis was observed on the Columbia sheep blood agar plates. Four of the Prototheca isolates, obtained from cows of different herds, showed slower growth and paler and smoother colonies. The microscopical observation of Prototheca cells stained with lactophenol cotton blue showed a typical appearance with ovoid to globose sporangia with sporangiospores in several developmental stages (Fig. 1A). However, some differences in the internal organizations of sporangiospores of the four isolates mentioned above were observed. Their sporangiospores were more consistently and regularly organized than those of the other isolates (Fig. 1B), suggesting that they could belong to a different Prototheca species. The morphological appearance observed for the Prototheca spp. was in concordance with the previous description of Pore (16) and DiPersio (5) for P. zopfii and of Roesler et al. (19) for P. blaschkeae.

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FIG. 1. Photomicrographs obtained by light microscopy of wet mount preparations of Prototheca spp. stained with lactophenol cotton blue. (A) Characteristic morphology of P. zopfii sporangiospores within sporangia (arrow). (B) Regularly organized sporangiospores of P. blaschkeae within the sporangia (arrow). Magnification, x1,000. Bar, 20 µm.

To determine the species of Prototheca associated with bovine mastitis in this study, the 18S rRNA genes of all isolates were amplified (Fig. 2). For each isolate analyzed, a PCR fragment of about 1,800 bp was detected as expected, since the sequence length of P. zopfii 18S rDNA is about this size (24). Each amplified rDNA was submitted to restriction analysis with HaeIII, resulting in two different restriction patterns (Fig. 3). For 37 isolates, a restriction pattern defined as restriction pattern 1 (RP 1) was identified. The four morphologically distinct isolates showed a different restriction pattern referred to as RP 2, which could be clearly distinguished from RP 1 by the lack of a 400-bp fragment (Fig. 3). The 18S rRNA genes from eight isolates representing restriction pattern RP 1 and four with RP 2 were completely sequenced. All sequences with RP 1 had a length of 1,807 bp and were identical to each other and also to the published sequence of Prototheca zopfii var. hydrocarbonea strain RND16 (GenBank accession no. AB066502). The four isolates displaying RP 2 were also identical among themselves, had a length of 1,815 bp, and showed 99.8% identity (with three differences) within the 1,449 bp available for the 18S rDNA sequence of P. blaschkeae SAG 2064 (AY973041). The three differences were found in variable regions of the 18S rRNA. Similarity between the two types of sequences that displayed different restriction patterns was only 98.0%, corresponding to 40 differences including indels (insertions/deletions). Phylogenetic analyses with the NJ, MP, and ML method confirmed that most of our isolates belong to the P. zopfii clade, specifically to P. zopfii var. hydrocarbonea and P. zopfii SAG 2021, while there is high bootstrap support that four isolates are more closely related to P. blaschkeae (Fig. 4).

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FIG. 2. 18S rDNA amplification of some Prototheca isolates (P1 to P7) with eukaryote-specific PCR amplification primers. All PCR fragments have a size of approximately 1,800 bp. Lane ClaI, molecular weight standard; lane –C, negative control without DNA.

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FIG. 3. Restriction patterns of amplified 18S rDNA fragments of four Prototheca isolates. PCR fragments were digested with HaeIII, yielding two different patterns, RP 1 and RP 2. RP 2 lacks the 400-bp fragment observed in RP 1. Lanes 50 bp, molecular weight standard (50-bp ladder).

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FIG. 4. Phylogenetic tree inferred from complete or almost complete 18S rRNA gene sequences of trebouxiophycean green algae showing species affiliation of Prototheca isolates representing RP 1 and RP 2 (see Fig. 3). The tree topology is derived from an ML analysis. Bootstrap values are shown at the internal nodes for each 1,000 resamplings of NJ and MP and 100 resamplings for ML, if values are higher than 50%. Thick lines indicate 100% bootstrap support for all three methods. Asterisks indicate sequences determined in this study. Branch lengths correspond to evolutionary distances. Bar, 0.01 nucleotide substitution per site.

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Prototheca species are known to be widely dispersed within dairy farmlands. The occurrence of mastitis due to these algae usually takes place during periods of warm weather with high rainfall (8, 12, 16), reflecting poor management and hygiene combined with insufficient premilking cleaning and disinfection of the teats. Generally, infections are maintained in a herd by clinically healthy shedders (8, 18). The objective of this work was to determine the species of Prototheca that are associated with bovine mastitis in the northwest of Portugal. The results of morphological characterization and of the molecular analyses of the 18S rRNA genes demonstrated that most of our isolates, with RP 1, belong to biotype II of P. zopfii represented by strain SAG 2021 in Fig. 4, as suggested by Roesler et al. (20). This was not unexpected, as all algae associated with bovine mastitis have been identified as P. zopfii biotype II (2, 13, 18) and are generally considered to be its causative agent (13). Surprisingly, these isolates share complete 18S rDNA sequence identity with P. zopfii var. hydrocarbonea RND16, a variety of P. zopfii which was first described in 2002 by Ueno et al. (26). This thermotolerant strain was isolated from a hot spring and was never associated with bovine mastitis. It would be interesting to know if this strain is potentially infectious or not. Differences in infectivity combined with the knowledge of characteristic physiological differences could be used to specify prerequisites for the pathogenicity of biotype II strains. In this context, determination of fermentation patterns of our isolates might reveal essential differences, as heterogeneities for strains of a single species of P. zopfii are well known (9).

In a recent study (19), biotype I and II strains were reclassified as two genotypes of P. zopfii by 18S rRNA gene sequence analysis and determination of cellular fatty acids, and biotype III strains were defined as a new species, P. blaschkeae. Interestingly, four of our isolates, those with RP 2, share much more sequence similarity with P. blaschkeae than with P. zopfii (99.8% compared to 98%). As P. blaschkeae so far had been isolated only from a case of human onychomycosis (19), the results of our study demonstrate for the first time the involvement of P. blaschkeae in the etiology of bovine mastitis. To quickly discern between the two species, we also want to point out the important additional information provided by amplified rDNA restriction analysis, which allows fast screening of large numbers of isolates for the detection of different infectious species.

 
This work was supported by Fundação para a Ciência e Tecnologia, Portugal, grant SFRH/BD/28892/2006.

We thank Isabel Santos and Luís Pinho for providing some of the samples used in this study.

 
* Corresponding author. Mailing address: Laboratório de Doenças Infecciosas do ICBAS, ICAV, Rua Padre Armando Quintas, 4485-661 Vairão, Portugal. Phone: (351) 252 660 400. Fax: (351) 252 661 780. E-mail: saramarques{at}mail.icav.up.pt

Published ahead of print on 23 April 2008.

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Journal of Clinical Microbiology, June 2008, p. 1941-1945, Vol. 46, No. 6
0095-1137/08/$08.00+0     doi:10.1128/JCM.00323-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Marques, S. Articles by Thompson, G. Search for Related Content PubMed PubMed Citation Articles by Marques, S. Articles by Thompson, G.