OCCURRENCE OF NOCARDIA SPECIES IN RAW MILK

Dept. Food Hygiene

Faculty of Vet. Med., Assiut University

Occurrence of Nocardia species

in raw milk

(With 5 Tables and 3 Figures)

By

S. Nasr; Nagah Saad; Nahed Wahba^*

 and Walaa Mahmoud^*

* Animal Health Research Institute, Assiut Regional Branch

(Received at 18/3/2009)

تواجد أجناس النوکارديا فى الألبان الخام

سعد نصر , نجاح سعد , ناهد وهبه ،  ولاء محمود

تلعب ميکروبات النوکارديا دوراً خطيراً فى فساد الألبان ومنتجاتها. حيث إنها تسبب نزول اللبن مدمم ومتجلط من الضرع الملتهب بسبب النوکارديا. و أيضا تسبب إنخفاض إنتاج اللبن بنسب واضحه مما يؤدى إلى خساره إقتصاديه کبيره. علاوة على الأثر الکبير على الصحة العامة، لما قد تسببه من إلتهابات بالجهاز الهضمى کله والأمراض الرئوية والإلتهابات الجلديه الشديده وأمراض المخ والجهاز العصبى هذا غير ما قد تسببه من أعراض أخرى مختلفه تؤثر على الإنسان وقد تودى إلى الوفاه وخاصة إن لم يتم معالجتها سريعا. ومن هنا تم عمل هذه الدراسه لمعرفة مدى تواجد هذا الميکروب فى الأنواع المختلفه من الألبان وقد إشتملت هذه الدراسه على التالى : جمع 240 عينه عشوائيه من اللبن الخام والتى جمعت من أماکن مختلفه فى مدينة أسيوط من لبن بقرى وجاموسى ونعاج وماعز بواقع 90عينة لکل من اللبن البقرى والجاموسى مقسمه إلى 30 عينه لکل من لبن المزارع والمحلات والباعه الجائلين و30 عينه لکل من لبن النعاج والماعز. ولقد تبين بالفحص البکتريولوجى أن     43 (8 ,47%) ،39 (3 ,43 %) ,16 (3 ,53) ، 20 (7 ,66%) من العينات المفحوصة کانت ايجابية للميکروب بأستخدام مستنبت النوکارديا بينما کانت النتائج  37 (1 ,41 %) ،  39 (3 ,43) ، 15 (50%) ، 15 (50%) من عينات نفس اللبن السابق ذکرها کانت ملوثه بهذه الميکروبات على التوالى بأستخدام مستنبت البوشنل هاس. وقد بلغ متوسط عدد النوکارديا بأستخدام مستنبت النوکارديا 3 ,1 x ⁴10 ، 6 ,2x  ⁴10 ،  4 ,7 x ³10 ميکروب/مللى من عينات اللبن البقرى المجمعة من المزارع والمحلات والباعه الجائلين على التوالى ومن عينات اللبن الجاموسى 10 x  ³10 ، 2 ,1x ⁴10 ، 9 ,5 x ³10 المجمعه أيضا من مزارع الألبان ومحلات الألبان والباعه الجائلين وأما عينات لبن النعاج والماعز فقد کان متوسط عدد هذا الميکروب 4 ,1x  ⁴10 ،  6 ,7 x ³10 ميکروب/مللى على الترتيب. وبالنسبه لمستنبت البوشنل هاس کان متوسط العد الکلى لهذا الميکروب کالآتى: 2 x ⁴10 ، 3 ,3  x ⁴10 ، 5 ,1 x ⁴10 من عينات اللبن البقرى المجمعه من المزارع والمحلات والباعة الجائلين على التوالى وأيضا 4 ,2 x ⁴10 ، 5 ,2 x ⁴10 ،  5, 9x  ³10 من عينات اللبن الجاموسى المجمعه أيضا من مزارع الألبان ومحلات الألبان والباعة الجائلين 7 x ³10 ، 9, 1 x ³10 ميکروب/مللى من عينات لبن النعاج والماعز على الترتيب. هذا وقد تم تصنيف ميکروبات النوکارديا المعزوله من جميع العينات المفحوصه ووجد ان اعلى نسبة کانت للنوکارديا أستيرويدز تليها النوکارديا برازيلينسـس. هذا وقد تم مناقشة النتائج مع ذکر الشروط الصحية الواجب إتباعها لمنع تلوث الأغذيه وخاصة الألبان بميکروب النوکارديا وذلک لحماية صحة المستهلک وحفاظا على المنتج من الفساد.

Summary

Two hundred and forty random samples of raw milk including cow's (90), buffalo's (90), sheep's (30) and goat's milk (30) were collected from dairy farms, dairy shops and street vendors in Assiut city. These samples were examined for the prevalence of Nocardia spp. using two selective media: Nocardia and Bushnell-Hass media. The recorded data revealed that 43 (47.8%), 39 (43.3%), 16 (53.3%) and 20 (66.7%) of the examined cow's, buffalo's, sheep's and goat's milk samples, respectively were contaminated with Nocardia spp. on Nocardia medium. However, the incidence of Nocardia spp. on Bushnell-Hass medium was 37 (41.1%), 39 (43.3%), 15 (50%), and 15 (50%) in the same samples, respectively. The highest Nocardia positive samples were from goat's and sheep's milk. Different counts of Nocardia spp. from milk samples on both media were recorded. N.asteroides was the predominant species, it could be isolated in percentages of 25.4 and 30.2% on Nocardia and Bushnell-Hass medium respectively, other nocardia species were isolated in different percentages. The public health significance of the organisms and the precautions which should be taken to control this organism in dairy industry as well as the recommended sanitary measures, were also discussed.

Key words: Nocardia spp., cow, buffalo, sheep, goat, raw milk

Introduction

Nocardia species continue to be highly dangerous human pathogens. There is an increase interest of Nocardia species particularly N.asteroides as health risk affecting both human and animal. Nocardiae are found extensively world wide (Brown and McNeil, 2003). They are aerobic, Gram positive, non-motile, non-spore forming rods that usually show branching and aerial hyphae and are usually partially acid-fast and there is considerable confusion in the taxonomy of Nocardia (Carter and Darla, 2004).

About sixteen species of Nocardiae have been implicated in human infection (Saubolle and Sussland, 2003).The most important pathogenic species are "Nocardia asteroides complex" which infect domestic animals and human (Carter and Darla, 2004). The term "N.asteroides complex" is used to include N.asteroides, N.caviae, N.brasiliensis and N.otitidiscaviarum (Saubolle, 2002 and Brown and McNeil, 2003).

Nocardiosis is a bacterial disease of man and animal caused by Nocardia species. Concomitant pulmonary and cerebral nocardiosis have been reported in many cases and there is evidence of hematogenous spread (Emmons et al., 1977 and Patrick et al., 1998). In human, it may be manifested as bacteremia, empyema, brain abscess, pericarditis, synovitis, soft tissue infection, peritonitis and corneal ulcers; Nocardiosis is characterized by an acute inflammatory response terminating in necrosis and abscess formation (Burgert, 1999; Saubolle, 2002 and Brown and McNeil, 2003).

The fatality rate in human from Nocardiosis is high (Carter and Darla, 2004).The medical importance of Nocardiae may cause significant morbidity and mortality, particularly in immunocompromised patients, including transplant recipients, patients infected with human immunodeficiency virus and in those receiving long-term corticosteroid therapies (McNeil and Brown, 1994).

Economically, the most important host is the dairy cow. Mastitis caused by N.asteroides was reported in Hawaii (USA) in a herd from which 100 cows had to be culled and an additional 80 cows died of the infection. The problem arises there not only belongs to the economic coasts to control nocardial mastitis, but mainly due to the potential risk to human health from contamination of milk and the cross reaction to tuberculin test in case of pulmonary nocardiosis (Willett et al., 1982; Costa et al., 1987 and Schoonderwoerd et al., 1990). Moreover Nocardial species were considered as environmental thrive organisms. Pathogenic Nocardiae are saprophytes found in many climates in soils and water, either as indigenous flora or as contaminants. The main routes of infection are inhalation, trauma and ingestion (Dwight et al. 2004).

As, the ability of Nocardia species to grow and multiply in raw milk as well as organisms are potential causative agents of food borne diseases, it is essential to estimate the prevalence of Nocardia species in different types of milk including cow's, buffalo's, sheep's and goat's milk.

Materials and Methods

1-Collection of samples:

A total of 240 random samples of milk including cow's and buffalo's milk were collected from dairy farms, dairy shops and street vendors (30 samples of each). Sheep's milk (30) and goat's milk (30) were collected from different localities in Assiut city. Each milk sample was mixed and tested for heat treatment using Storch test (Lampert, 1975).

2-Preparation of samples:-

             Ten fold serial dilutions were prepared according to A.P.H.A (1992).

3- Isolation and enumeration of Nocardia spp.:-                           

Enrichment of Nocardia spp. was adopted using Bushnell-Haas broth and incubated at 37ºC for 2-7 days (Schaal, 1972). Isolation and enumeration was done using surface spreading technique on Nocardia and Bushnell-Haas agar incubated at 37ºC for 2-7 days but cultures should be examined every 2 days (Waksman, 1967 and Gordon et al., 1974). Suspected colonies should be optimized by seeing filamentous, rosette, white to yellow to orange colonies, may be with aerial and delicate mycelia (Schaal, 1984; Saubolle, 2002 and Brown and McNeil, 2003).

4- Identification of isolates:-

Initial visualization of phenotypic colony coloration and morphology, together with the presence of aerial hyphae, rosette shape often provides initial clues to the genus of the isolates (Carter, 1984). Specific tests include Gram stain, modified Ziehl-Neelsen stain, catalase test, hemolysis on blood agar and other biochemical reactions.

Differentiation between the suspected colonies was done according to Maldonado et al., 2000 and Hamid et al., 2001.Confirmation of the different three Nocardia spp. (N.asteroides, N.brasiliensis and N.caviae) was done according to Larone, 1995 and Brown et al., 1999.

Results

Table 1: Incidence of Nocardia spp. in the examined milk samples.

Table 2: Statistical analytical results of Nocardia spp. in the examined milk samples on Nocardia medium.

Table 3: Statistical analytical results of Nocardia spp. in the examined milk samples on Bushnell-Hass medium.

Table 4: Incidence of the isolated Nocardia spp. recovered from milk samples using Nocardia medium.

Table 5: Incidence of the isolated Nocardia spp. recovered from milk samples using Bushnell-Hass medium

Fig. 1: Differentiation between media used for isolation of          Nocardia spp.

Fig. 2: Incidence of the isolated Nocardia spp. recovered from milk samples using Nocardia medium.

Fig. 3: Incidence of the isolated Nocardia spp. recovered from milk samples using Bushnell-Hass medium.

Discussion

Different types of milk are unique and ideal foods for human. However, they are highly nutritious media in which microorganisms will thrive and lead to deterioration and spoilage. Nocardia spp. is one of the world wide bacterial infections and has been implicated in the pathogenesis of pulmonary nocardiosis, enteritis, arthritis, multiple skin abscesses, central nervous system nocardiosis and endocarditis in all infected humans, including children, immunosuppressed and rare in non-immunocompromised patients (Maria Bernadete et al., 2007).

 Nocardia spp. was isolated from 47.8, 43.3, 53.3 and 66.7% of cow's, buffalo's, sheep's and goat's milk samples on Nocardia medium and 41.1, 43.3, 50 and 50 % of the same samples on Bushnell-Hass medium, respectively (Table 1).

Presence of Nocardia spp. in milk is not surprising in view of fact that it is widely spread in nature and contaminates the milk during milking, handling, storage and transportation. Moreover, dust, soil and plant materials serve as sources of contamination; the problem was complicated by the absence of the cool system that may enhance the multiplication of most pathogenic microorganisms.

 Nocardia medium is preferable than Bushnell-Hass medium for isolation of Nocardia spp. Also N.vaccinii can be isolated on Nocardia medium and not on Bushnell-Hass medium (Fig. 1 and 2). So, for successful detection of different species of Nocardia, using of Nocardia medium is necessary.

Out of the 30 examined raw dairy farms milk samples,              19 (63.3%) and 17 (56.7%) were found to be contaminated with Nocardia spp. on Nocardia and Bushnell-Hass media, respectively. The average count/ml. was 1.3x10⁴ on Nocardia medium while on Bushnell-Hass medium the average value was 2x10⁴ CFU/ml. (Tables 2 and 3). Nearly similar findings were reported by Vaissaire et al. (1984) while, lower values were recorded by Seddek (2001) and Friedman et al. (2004). Nocardia spp. is an uncommon cause of mastitis, but the outbreaks have typically been reported in dairy farms due to poor hygienic and management conditions (Pisoni et al. 2008).

Nocardia spp. were isolated from samples collected from dairy shops and street vendors in lower percentages but nearly similar counts (33.3and 30% for dairy shops and 46.7 and 36.7% for street vendors samples) on Nocardia and Bushnell-Hass media respectively,         (Tables 2 and 3). That may be due to supply using illegal preservatives which inhibit the growth of most organisms including Nocardia spp. present in milk and prolong the shelf life of raw milk in dairy shops.

Nocardia spp. were isolated from teat tips in fore milk stripping (Schinger, 1994). Furthermore, the Nocardia introduced into the udder by insertion of cannula, contaminated preparations, drug mixture and tips of injectors (Battig et al. 1990 and Seddek 2001) this recorded information may support the high incidence of Nocardia spp. in this investigation. The environmental factors facilities for all activities of Nocardia spp.  involving the multiplication of this organism such as hot and wet weathers, bad hygienic measure as contamination of soil from the infected human patient which plays an important role in wide spreading of the pathogens (Pisoni et al., 2008).

Nearly the same result was obtained from buffalo milk samples, The highest incidence (60%) of Nocardia spp. was recorded from the examined dairy farm samples followed by dairy shops (36.7%) and street vendors ( 33.3%) on both media (Tables 2 and 3).

16 (53.3%) and 15(50%) of the examined sheep milk samples proved to harbor Nocardia spp. on both Nocardia and Bushnell-Hass media, respectively. The minimum and maximum counts on Nocardia medium were 7x10² and 7.9x10⁴, respectively with an average value of 1.4x10⁴ CFU/ml. On Bushnell-Hass medium the contamination level ranged from 1x10 to 5.8x10⁴ and the average was 7x10³ CFU/ml. (Tables 2 and 3). Nocardia spp. cause several lesions in ovines as ovine abortion, mastitis and milk infections (Watson, 1977 and John et al., 1980).

Regarding goat's milk, 20 (66.7%) and 15 (50%) of samples were found to be contaminated with Nocardia spp. The average count was 7.6x10³ and 1.9x10³ CFU/ml. on both Nocardia and Bushnell-Hass media, respectively. The high incidence of Nocardia spp. in goat's milk was clarified that the major intramammary pathogens in goats are associated with poor hygienic conditions in housing and in the milking parlous (Contreras et al. 2002). This present study showed that the highest Nocardia positive samples were from goat's and sheep's milk. This emphasized the role of primitive way of producing, bad handling and unhygienic measure of these particular types of milk as an important source of contamination with Nocardia spp. Furthermore, Nocradiae are ubiquitous in the environment and can be found in fresh- and salt water, soil, dust, decaying vegetation and decaying fecal deposits from animals (Diskensoy et al. 2004).

The highest frequency distributed species in all milk samples in both media was N.asteroides followed by N.brasiliensis which considered the most common species prevalent in more temperate climates (Tables 4 and 5, Fig.2 and 3). The same results were demonstrated by Patrick et al. (1998), Petersen et al. (2007) and Yin et al. (2007) who found that the most common species of Nocardia associated with human disease are N.asteroids, N.brasiliensis, N.otitidiscaviarum, and N.farcinica. Also, N.asteroids was the causative agent of an outbreak of mastitis in different animals and in different countries (Al-Bassam et al. 1989 and Seddek 2001).

N.asteroides is the species of Nocardia that is most commonly associated with human disease, which is primarily opportunistic; occurring in immune-compromised patients (Ryan and Ray, 2004). N.brasiliensis is one of the causes of nocardiosis in humans (Carter and Darla, 2004). Although it is associated with tropical environments, it is the second most common isolate after N.asteroides in the United States and has a higher prevalence in the south western and south eastern regions (McNeil and Brown, 1994; Saubolle, 2002 and Brown and McNeil, 2003,). While in Japan, about 303 cases of nocardiosis between 1992 and 2001 were reported, about 72% of the strains belonged to the" N.asteroides complex", including 81 strains of N.farcinica , which was the most frequent isolate, 66 strains were N.brasiliensis (Kageyama       et al., 2004).

Finally, the presence of any Nocardia spp. should be a warning to dairy producers that conditions exist for the growth of pathogenic species of Nocardia and the corrective measures needed to ensure pathogenic-free products. So, great attention must be paid to the problems of these pathogens in our foods. Consequently, more restriction and preventive measures should be taken to improve the quality of raw milk to protect consumers from being infected by this and other organisms.

Reference

A.P.H.A. (1992): Standard Methods for the Examination of Dairy Products 16^th Ed., American Public Health Association ,New York.

Al-Bassam, L.S.; Kastandi, J.D. and Kamalapur, P.N. (1989): Nocardia asteroides as a causative agent of mastitis in dairy cattle in Iraq. Indian Vet. J. 66:10-12.

Battig, U.; Wegmann, P.; Meyer, B. and Penseyres, J.H. (1990): Die Nokardien-Mastitis des Rindes.1. klinische. Arch. Tierheilk. 132: 315-322.

Brown, J.M. and McNeil, M.M. (2003): Nocardia, Rhodococcus, Gordonia, Actinomadura, Streptomyces and other aerobic actinomycetes, Pp. 502-531. In: Murray, P.R.; Baron, E.J.; Pfaller, M.A.; Tenover, F.C. and Yolken, R.H. (ed). Manual of clinical microbiology, 8^th ed. American Society for Microbiology, Washington, D.C.

Brown, J.M.; McNeil, M.M. and Desmond, E.P. (1999): Nocardia, Rhodococcus, Gordonia, Actinomadura, Streptomyces and other actinomycetes of medical importance, p. 370-398. In Murray, P.R.; Baron, E.J.; Pfaller, M.A.; Tenover, F.C. and Yolken, R.H. (ed). Manual of clinical microbiology, 7^th ed. American Society for Microbiology,Washington, D.C.

Burgert, S.J. (1999): Nocardiosis: a clinical review. Infect. Dis. Clin. Pract. 8: 27-32.

Carter, G.R. (1984): Diagnostic procedure in veterinary bacteriology and mycology. Pp. 212-215.

Carter, G.R. and Darla, J.W. (2004): Nocardia and Rhodococcus. Ess. of Vet. Bact. and Mycology: 215-217. Chapter 30.

Contreras, A.; Luengo, C.; Sánchez, A. and Corrales, J.C. (2002): The role of intramammary pathogens in dairy goats. Epidemiología y Enfermedades Infecciosas, Murcia, Spain, October 2002.

Costa, E.; Macedo, M.; Couthinho, S.; Catilho, W.; Texeira, G. and Benesi, J. (1987): Isolation of aerobic Actinomyetes of genus Nocardia from infections of domestic animals. Revista de faculdade de medicina veterinaria e zootecine de unversidade de saopaulo. 24(1): 12-17.

Diskensoy, O.; Filiz, A.; Bayram, N.; Balci, I.; Zer, Y.; Celik, G. and Ekinci, E. (2004): Dwight C. Hirsh; James Maclachlan, N. and Richard L. Walker. (2004): Filamentous bacteria: Actinomyces, Nocardia, Dermatophilus and Streptobacillus. Vet. Microbial. 2^nd ed. Pp. 218-220.

Emmons, CH. W.; Binford, CH. H.; Utz, J. P. and Kwon-Chung, K. J. (1977): Nocardiosis. In medical mycology, 3^rd ed. Lea and Febiger, Philadelphia. Chapter 9.

Friedman, S.; Shoshani, E. and Ezra. (2004): Economical losses from clinical mastitis in 4 dairy herds in Israel. The Nat. service for udder health and milk. 59(1-2).

Gordon, M.A. (1974): Aerobic pathogenic Actinomycetaceae. In Lennette, Spaulding and Truant (eds), Manual of clinical microbiology. American Society for Microbiology, Washington. D.C. Pp.175-188.

Hamid, M. E.; Maldonado, L. G.; Sharaf Eldin, S.; Mohamed, M. F.; Saeed, N. S. and Goodfellow, M. (2001): Nocardia africana sp. nov., a new pathogen isolated from patients with pulmonary infections. J. Clin. Microbiol. 39: 625-630.

John H. Kirk; Huffman, E. M. and Bruce C. Anderson. (1980): Mastitis and Udder Abnormalities as Related to Neonatal Lamb Mortality in Shed-Lambed Range Ewes. J. Anim Sci. 50: 610-616.

Kageyama, A.; Hoshino, Y.; Watanabe, M.; Yazawa, K. and Mikami, Y. (2004): Clinical isolates of Nocardia brasiliensis from Japan exhibit variable susceptibility to the antibiotic imipenem. Mycopathologia 158: 275-278.

Lampert, L. M. (1975): Modern Dairy Products 3^rd Ed., Chemical Publishing Co. Inc. New York.

Larone. (1995): Medically important fungi: a guide for identification, 3^rd ed. American Society for Microbiology, Washington, D.C.

Maldonado, L.; Hookey, J.V.; Ward, A. C. and Goodfellow, M. (2000): The Nocardia salmonicida clade, including descriptions of Nocardia cummidelens sp. nov., Nocardia fluminea sp. nov., and Nocardia soli sp. nov. Antonie Leeuwenhoek 78: 367-377.

Maria Bernadete, F.C.; Marcio, F.C.; Nelson, S.P.; Severo, B.S. and Luizcarlos Severo. (2007): Nocardial infections: report of 22 cases. Rev. Inst. Med. Trop. S. Paulo. 49(4), Jul. / Aug.

McNeil, M.M. and Brown, J. (1994): The medically important aerobic Actinomycetes: epidemiology and microbiology. Clin. Microbiol. Rev. 7: 357-417.

Patrick, R.M.; Rosenthal ken, S.; Kobayashi George, S. and Pfaller Michael, A. (1998): Nocardia, Rhodococcus and related Actinomycetes. In, Medical Microbiology, 3^rd Ed. Mosby, London. Chapter 39.

Petersen, B.E.; Jenkins, S. G.; Yuan, S.; Lamm, C. and Szporn, A.H. (2007): N.farcinica isolated from bronchoalveolar lavage fluid of a child with cystic fibrosis. Pediatr. Infect. Dis. J. 26(9): 858-859.

Pisoni, G.; Locatelli, C.; Alborali, L.; Rosignoli, C.; Allodi, S.; Riccaboni, P.; Grieco, V. and Moroni, P. (2008): Short communication: outbreak of N.neocaledoniensis mastitis in an Italian dairy herds. J. Dairy Sci. 91(1): 136-139.

Ryan K.J. and Ray CG (editors) (2004): Sherris Medical Microbiology, 4th ed., McGraw Hill, pp. 460-2.

Saubolle, M. A. (2002): Aerobic Actinomycetes. In K. D. McClatchey (ed.), Clinical lab medicine, 2^nd ed. Lippincott Williams and Wilkins, Philadelphia, Pa. Pp. 1201-1220.

Saubolle, M. A., and Sussland, D. (2003): Nocardiosis: review of clinical and laboratory experience. J. Clin. Microbiol. 41:4497-4501.

Schaal, K. P. (1972): Zur mikrobiologisher Diagnostik der Nocardiose. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. I Abt. Orig. 220: 242-246.

Schaal, K.P. (1984):  Laboratory diagnosis of Actinomycete diseases. In: Goodfellow, M.; Mordarski, M. and Williams, S. T. (eds). The biology of the Actinomycetes. Academic press, London. Pp. 425-456.

Schinger, M. (1994): Personal communication. Cited by Schuh, G.; Schoder, H.; Weissen Book and Hotter, H. (1994) on the occurance of Nocardial mastitis and resistancy significant mastitis causing pathogens in Austriain in dairy herds. 6^th ed. Sci. Cong. 22 Nov. fac. Vet., Med., Assiut Egypt.

Schoonderwoerd, M.; McFardzen, L.; Manninen and Kand Ollis, G. (1990): Culturing of bulk milk for the presence of Nocardia species. Cana. Vet. J. 6: 453-454.

Seddek, S.R. (2001): Isolation of Nocardia organisms from soil and mastitic milk of dairy cows (Friezian and Holestein) on different media on some governomental farms in Assiut governorate. Assiut Vet. Med. J. 46(92): 72-82.

Vaissaire, J.; Marel, J.; Perreau Belli, P.; Catel, J.; Chenat, M.; Chirol, C.; Legardinier, J.; Montagut, G.; Ramisse, J. and Reverdy, J. (1984): Role of N.asteroides in the aetiology of bovine mastitis and in veterinary medicine. Bulletin de L' Academic Veterinaria de France. 57(1): 119-125.

Waksman, S.A. (1967): The Actinomycetes. Ronald press, New York.

Watson, W. A. (1977): Nocardia species as a possible cause of ovine abortion. Res. In Vet. Science. 23: 171-178.

Willett, G.; Kirpes, D.; Fluharty, D. and Rogers, L. (1982): An economic analysis of mastitis control measure for selected Washington dairy herd. Res. Bull. Agric. Res. Cen. Washington state unit. XBO919.

Yin, X.; Liang, S.; Sun, X.; Luo, S.; Wang, Z. and Li, R. (2007): Ocular nocardiosis: HSP65 gene sequencing for species identification of Nocardia species. Am. J. Ophthalmol. 2007 Aug.13.