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* Department of Veterinary Pathology, Hygiene and Public Health, University of Milan, via Celoria 10, 20133 Milan, Italy
Istituto Zooprofilattico of Lombardia and Emilia Romagna, Brescia 25100, Italy
Bovine Practitioner, Mantova 46030, Italy
1 Corresponding author: paolo.moroni{at}unimi.it
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ABSTRACT |
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Key Words: Nocardia neocaledoniensis • mastitis • immunohistochemistry
Nocardia genus is recognized worldwide as a cause of disease in human beings and animals, particularly mammals and birds. There has been a recent revolution in the taxonomy of the Nocardia genus. In the last few years, a wide range of biochemical and susceptibility tests, and the application of molecular techniques (for example, 16S rRNA gene sequencing), have expanded the spectrum of the Nocardia genus to more than 30 named species (Chun and Goodfellow, 1995; Conville et al., 2000; Roth et al., 2003).
Nocardia spp. are ubiquitous environmental saprophytes, and they are not a part of the normal flora of mammals, although they may be carried mechanically on the skin. Infection usually arises from direct inoculation of soft tissues after penetrating injuries, by inhalation of aerosols containing organisms, possibly in dust, or through natural routes like the teats of dairy cows (e.g., during milking or mastitis treatment). Fragmentation of branching nocardial filaments late in their growth cycle results in the formation of small, unicellular, spore-like cells that are easily aerosolized from environmental substrates. Infection induces a pyogranulomatous inflammation characterized by lesions mainly reported on the skin and in the lungs, oral cavity, nervous system, soft tissues, and bones.
Nocardia spp. are an uncommon cause of mastitis, and outbreaks have usually been reported only in dairy farms with poor hygienic and management conditions. Clinical features of mastitis induced by Nocardia can be severe, with diffuse fibrosis and firmness of the udder, reduced milk production, altered milk containing clots of exudates and with systemic health problems, anorexia, and fever (Battig et al., 1990; Tarabla et al., 1993).
The outbreak described here involved a dairy farm with 43 lactating cows that, after a long period with low bulk milk SCC (BMSCC, BMSCC < 180,000 cells/mL), experienced an increasing incidence of clinical mastitis with BMSCC greater than 300,000 cells/mL. Quarter milk samples were collected from 43 cows. Teat ends were cleaned with chlorhexidine before sampling. The first streams of foremilk were discharged, and then 10 mL of milk was collected aseptically from each udder into sterile vials. Ten microliters of each milk sample was plated on 5% bovine blood agar and on Sabouraud’s dextrose agar, incubated aerobically at 37°C, and checked daily for bacterial growth.
Pinpoint colonies were generally evident at 24 h, and by 4 d, characteristic aerial hyphae were present on the surface of colonies 2 to 3 mm in diameter. Nocardia colonies were dry, tightly adherent to the surface of the plates and chalky white, somewhat resembling fine cotton, when aerial hyphae were produced. Nocardia spp. were identified by gram staining and biochemical tests (32C Yeast Identification System, BioMerieux, Rome, Italy). Furthermore, we tested for growth at 45°C and for additional phenotypic properties as described by Saintpierre-Bonaccio et al. (2004): growth on sodium citrate and sodium acetate (0.1%, weight/volume), nitrate reduction and urea hydrolysis, and decomposition of casein (1.0%, weight/volume) and tyrosine (5.0%, weight/volume).
Antimicrobial susceptibility tests were performed for all isolates using a disk diffusion method with commercial disks of gentamicin (10 µg), kanamycin (30 µg), neomycin (30 µg), and streptomycin (10 µg), as previously described by Wallace and Steele (1988). A zone size of 
10 mm was defined as the breakpoint indicating resistance to gentamicin and streptomycin after 72 h of incubation at 35°C, whereas the size for kanamycin and neomycin was defined as 20 mm (Wallace and Steele, 1988).
For molecular characterization, DNA was extracted from colonies, milk samples, and paraffin-embedded tissues with QIAamp DNA Mini Kit (Qiagen, Milan, Italy), according to the instructions of the manufacturer, and was subjected to PCR amplification. A 606-bp fragment of the 16S rRNA gene and a 441-bp fragment of the hsp65 gene encoding the 65-kDa heat shock protein were amplified with primers described in Rodriguez-Nava et al. (2006) for comprehensive Nocardia spp. identification.
All epizootic strains were morphologically and phenotypically identical. According to molecular characterization, the representative epizootic strain showed 100% similarity to Nocardia neocaledoniensis for 16S rRNA and hsp65 genes.
Among the 172 cultured milk samples that were evaluated in this study, 55.6% showed the presence of IMI. Coagulase-negative staphylococci were the predominant microorganism, being present in 29.6% of the milk samples. Fifteen mammary quarters (18.5%) from 9 cows were infected by N. neocaledoniensis, and Escherichia coli and Streptococcus spp. together accounted for 7.5% of mammary quarters. For each sample, the quarter milk SCC was determined with an automated fluorescent microscopic somatic cell counter (Bentley Somacount 150, Bentley Instrument, Chaska, MN).
For all statistical procedures, means and their standard errors (SEM) were computed by using statistical software (SPSS 15.0, SPSS Inc., Chicago, IL). Results are expressed as mean values ± standard errors of the mean. Statistical analysis was performed by using 1-way ANOVA, and significant differences were declared at P < 0.01
The mean quarter milk SCC for infected udders was 863 ± 107.2 x 103 cells/mL (range 116 to 3,712 x 103 cells/mL). This was significantly greater (P < 0.01) than the mean value in noninfected quarters (189.7 ± 18.6 ± 103 cell/mL, range 8 to 1,129 x 103 cells/mL).
All epizootic strains showed the same susceptibility pattern. They were resistant to streptomycin and kana-mycin but were susceptible to neomycin and gentamycin.
It is obvious that the in vitro activity of antimicrobial agents is a major criterion for the choice of therapy. The experiences of Gillespie and Timoney (1981), Sears (1986), and Tarabla et al. (1993), however, suggested that the prognosis is unfavorable with antibiotic therapy and that there is a risk of spread of infection to the rest of the herd. Consequently, the affected animals in this herd were culled. At necropsy, the infected quarters were characterized by a diffuse nodular firmness and with thickened linings of teat sinuses and gland cistern. Supramammary and inguinal lymph nodes were edematous but did not contain focal necrotic lesions. Neither gross nor microscopic lesions were observed in other organs or tissues.
Samples from mammary glands and supramammary lymph nodes of slaughtered infected cows were fixed with formalin and embedded in paraffin. Histological examination of 4-µm-thick sections, stained with hematoxylineosin, Gram and Grocott, revealed large, poorly encapsulated, multifocal to coalescing pyogranulomatous lesions. Single pyogranulomas were characterized by a center of necrotic debris and neutrophils, intermixed with epithelioid elements and rare giant cells. Surrounding the necrosis, several eosinophilic colonies of Nocardia-like Gram and Grocott-positive bacteria (Figure 1
) surrounded by epithelioid cells were clearly recognizable. Lymphocytes and plasma cells, intermixed with fibroblasts, constituted the outermost layer of the lesions. In the lymph nodes, only hyperplastic changes were observed, and Gram staining resulted negative.
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). Moreover, numerous positive single bacterial bodies were also recognizable in the necrotic center. Conversely, all the sections of lymph nodes examined gave negative results.
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Effective control of mastitis due to Nocardia spp. is based on early detection, for which definitive and early diagnosis are critical. The need for early detection has encouraged the development of faster and more sensitive diagnostic techniques. Because infected animals could yield negative results on aerobic culture for several weeks before Nocardia can be isolated and because, in a mixed culture, other organisms could plausibly mask the slower growing of Nocardia, diagnostic tests other than bacteriological exams should be considered when Nocardia is suspected.
Moreover, Nocardia spp. identification requires laborious, time-consuming phenotypic and chemotaxonomic methods. Molecular methods offer an alternative, particularly when bacterial cultures are not possible. The advantages of molecular identification of bacteria using 16S rRNA sequencing are rapid turnaround time, reliable identification, and taxonomic meaningfulness. The 16S rRNA gene of Nocardia is highly conserved with constant regions that are identical for all Nocardia spp. and variable regions that are species-specific (Brown-Elliot et al., 2006).
To our knowledge, this is the first report of detection of N. neocaledoniensis, a novel actinomycete isolated from soil, as responsible for bovine mastitis. Progress in the epidemiological analysis of cases of nocardiosis will be achieved when improved diagnostics for these infections (molecular and serological diagnosis) are available, when the genetic diversity of Nocardia spp. isolates is fully understood, and when molecular typing is systematically performed in populations with increased cases of nocardiosis.
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ACKNOWLEDGEMENTS |
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Received for publication June 25, 2007. Accepted for publication October 2, 2007.
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