Survey of Bovine Mycotic Mastitis in

Different Mammary Gland Statuses in Two

North-Eastern Regions of Algeria

Samir Ksouri, Somia Djebir, Youcef

Hadef & Ahmed Benakhla

Mycopathologia

ISSN 0301-486X
Volume 179
Combined 3-4

Mycopathologia (2015) 179:327-331

DOI 10.1007/s11046-014-9845-2

1 23
Your article is protected by copyright and all
rights are held exclusively by Springer Science
+Business Media Dordrecht. This e-offprint
is for personal use only and shall not be self-
archived in electronic repositories. If you wish
to self-archive your article, please use the
accepted manuscript version for posting on
your own website. You may further deposit
the accepted manuscript version in any
repository, provided it is only made publicly
available 12 months after official publication
or later and provided acknowledgement is
given to the original source of publication
and a link is inserted to the published article
on Springer's website. The link must be
accompanied by the following text: "The final
publication is available at link.springer.com”.

1 23
 Author's personal copy
Mycopathologia (2015) 179:327–331
DOI 10.1007/s11046-014-9845-2

Survey of Bovine Mycotic Mastitis in Different Mammary

Gland Statuses in Two North-Eastern Regions of Algeria
Samir Ksouri • Somia Djebir • Youcef Hadef •

Ahmed Benakhla

Received: 7 June 2014 / Accepted: 30 November 2014 / Published online: 7 December 2014
Ó Springer Science+Business Media Dordrecht 2014

Abstract The aim of this study was to evaluate the and Saccharomyces fragilis. Mould species have also
prevalence of mycotic mastitis in different mammary been isolated from samples of both healthy milk and
gland statuses. The study was conducted on 304 dairy clinical mastitis milk. Aspergillus amstelodami (from
cows from ten farms in two north-eastern regions in glaucus group), A. fumigatus and Geotrichum candi-
Algeria; Guelma and Souk Ahras with 922 and 199 dum were identified, while the other species including
samples, respectively, forming thus a total number of Penicillium sp. and Cladosporium sp. were not
1,121 milk samples. A total of 321 milk samples were identified.
collected from clinical mastitis, 544 milk samples
from subclinical mastitis and 256 milk samples from Keywords Candida
Mammary gland
Mastitis

healthy mammary glands. Mycological analyses Milk
Yeast
revealed that 10.17 % of the treated samples were
positive recording 114 species of fungi including 88
yeasts and 26 moulds. The most frequent species was
Candida kefyr followed by C. albicans, C. guillier- Introduction
mondii, C. famata, C. tropicalis, C. colliculosa, C.
krusei, C. rugosa, C. glabrata, C. parapsilosis, C. Mastitis is a mammary gland inflammation that results
inconspicua, Trichosporon sp., Rhodotorula glutinis from a biological conflict between the existing
microorganisms and the animal’s udder. The eco-
nomic consequences of this disease are very impor-
S. Ksouri (&)
A. Benakhla tant. Besides the decrease in production, mastitis
Institut des Sciences Vétérinaires, Université Chadli
causes biochemical changes in milk [1]. In the
Bendjedid d’El-Tarf, BP 73, 36000 El-Tarf, Algeria
e-mail: ksourisamir@gmail.com majority of mastitis cases, bacteria are the etiological
agents while mycotic mastitis represents only a small
S. Djebir portion [2]. Since Rolle’s princeps observation of
Faculté des Sciences de la Nature et de la Vie et des
mycotic mastitis [3], several species of fungi were
Sciences de la Terre et de l’Univers, Université de 8 Mai
1945 de Guelma, BP 401, 24000 Guelma, Algeria isolated. A predominance of the genus Candida was
recorded in most studies and surveys [4–10]. In
Y. Hadef Algeria, no data are available about the existence of
Laboratoire de Chimie Analytique, Département de
mycotic mastitis. Indeed, the objectives of this study
Pharmacie, Faculté de Médecine, Université Bedji
Moukhtar d’Annaba, Route de Zaafrania BP 205, are to identify the species of fungi, to determine their
23000 Annaba, Algeria isolation frequencies and the prevalence of mycotic

123
 Author's personal copy
328 Mycopathologia (2015) 179:327–331

mastitis in dairy cows in two north-eastern regions in Cycloheximide 0.1 % (Pasteur Institute of Algiers),
Algeria and finally to assess their impact on economy urea hydrolysis and the auxanographic characters (the
and on public health. system API 20 C Aux). The identification of yeast
species was performed by apiweb software (bio-
MerieuxTM) and the key described by Kurtzman and
Materials and Methods Fell [13], while mould identification was based on the
key of Botton et al. [14].
The study was conducted on 304 dairy cows of
different breeds, ameliorated ones (Holstein and
Montbeliard) and local ones (Guelmoise and Cheurfa). Results
A total of 1,121 samples were collected during
23 months from July 2010 to May 2012. The selected The mycological analyses showed that 10.17 % of the
dairy cows are from ten farms in two north-eastern examined samples were positive (Table 1), 89/922 in
regions in Algeria; Guelma (six municipalities) and Guelma and 25/199 in Souk Ahras. Fungi were
Souk Ahras (two municipalities) with 922 and 199 detected in 12.50 % of the healthy milk samples, in
samples, respectively. Each sample corresponds to a 5.88 % of subclinical mastitis milk samples and in
quarter of a different animal. In this study, samples 15.57 % of clinical mastitis milk samples. A variety of
were collected in different mammary glands condi- fungal species are recorded in the three categories of
tions. Actually, there were 256 milk samples from milk. We also noted a predominance of Candida yeast
healthy mammary glands and 544 milk samples from with 71.93 % that represents eleven species, espe-
cows with subclinical mastitis detected by California cially Candida kefyr which is the most frequent one
Mastitis Test, Schalm and Noorlander [11] and with 16.67 % isolates, followed by Candida albicans
Schneider et al. [12]. The CMT score included in the with 13.16 % isolates. In moulds species, Aspergillus
study is higher than or equal to 1. Finally, 321 milk was the most frequent one with a rate of 14.92 %.
samples from cows suffering from clinical mastitis In 256 milk samples from healthy mammary
diagnosed after a general check-up of cows and a glands, only 32 were positive. We recorded the
special examination of the mammary gland. prevalence of yeasts with 17/32 isolates, especially
The samples were taken after udder-washing with the genus Candida with 16/32 isolates and C. kefyr
bleach and disinfection with alcohol at 70°. Samples with 4/32 isolates. Moulds isolated from this milk
of 14 ml of milk were put in sterile tubes, immediately samples represent 15/32 of the isolates.
closed and placed in isothermal cooler at 4 °C. Milk Only 32 isolates from 544 subclinical mastitis milk
samples were centrifuged at 4,000 rpm for 5 min [8], samples were recorded. We have noticed the predom-
and then, a direct examination of a pellet drop was inance of Candida yeast with 30/32 isolates repre-
essential for the detection of fungal elements, yeast, sented by nine different species. The identification
pseudohyphae and hyphae. 0.5 ml of the milk pellet revealed a co-dominance of C. famata and C. kefyr
was homogenized in 4.5 ml of Sabouraud broth with 6/32 isolates.
(FleukaTM) at a pH of 3.7 and incubated at 37 °C In 321 milk samples of clinical mastitis, 50 isolates
[13]. After 10 days of incubation, 50 lL of the culture were recorded. The three quarters of the fungi species
was inoculated in Sabouraud chloramphenicol agar were yeasts of the genus Candida with eight different
(bioMérieuxTM), and then, the dishes were incubated species. The most frequently isolated species were C.
at 37 °C for 5 days. albicans with 12/50 isolates. In mould species,
Fungi isolated in this study were identified based on Aspergillus fumigatus was relatively frequent with
the observed morphological characteristics. Chlamy- 6/50 isolates.
doconidium, pseudohyphae and hyphae are screened
on Rice cream medium (Pasteur Institute of Algiers).
Blood serum was used to detect germinative tubes. Discussion
Cryptococcus capsules were observed after being
stained with India ink. Biochemical characteristics Many studies conducted in different countries on
taken into account include: the sensitivity of fungi to mycotic mastitis revealed that the frequency of fungi

123
 Author's personal copy
Mycopathologia (2015) 179:327–331 329

Table 1 The genera and species of yeasts isolated from cases of bovine mastitis in different mammary gland statuses
Genera Species Mastitis status of mammary glands Frequency isolation
Healthy Subclinical Clinical Nombre %

Candida C. kefyr 4 6 9 19 16.67

C. albicans 0 3 12 15 13.16
C. guilliermondii 3 4 7 14 12.28
C. famata 3 6 3 12 10.53
C. tropicalis 2 3 2 7 6.14
C. colliculosa 0 4 0 4 3.51
C. krusei 1 1 1 3 2.63
C. rugosa 0 1 1 2 1.75
C. glabrata 1 0 1 2 1.75
C. parapsilosis 2 0 0 2 1.75
C. inconspicua 0 2 0 2 1.75
(16)a (30)a (36)a (82)a (71.93)b
Trichosporon Not identified 1 1 1 3 2.63
Rhodotorula R. glutinis 0 1 1 2 1.75
Saccharomyces S. fragilis 0 0 1 1 0.88
Aspergillus A. amstelodami 8 0 1 9 7.90
A. fumigatus 2 0 6 8 7.02
(10)a (0)a (7)a (17)a (14.92)b
Penicillium sp. Not identified 3 0 4 7 6.14
Geotrichum G. candidum 1 0 0 1 0.88
Cladosporium sp. Not identified 1 0 0 1 0.88
Total 32/256 (12.50 %) 32/544 (5.88 %) 50/321 (15.57 %) 114/1,121 (10.17 %) 100
a
The sum of the species in a genus
b
Percentage rate of the genus

isolation is continuously changing. Zaragoza et al. yeast species use antibiotics as a nitrogen source for
[10] reported a rate of 25.75 % in Mexico, a rate of growth, so this can explain their abundance.
6.1 % was reported in Egypt [15], 1.3 % in Denmark In our study, the fungi species isolated from milk
[16] and South Korea [17] and 9.6 % in Poland [9]. collected from cows with clinical and subclinical
Moreover, this variability is also observed in the same mastitis are considered pathogens. The pathogeneity
country; in Brazil, isolation rates of 12.07, 25.40 and of these fungi results in an inflammation of the
27.42 % were recorded, respectively, by Costa et al. mammary gland. In addition, a high fungal density
[18] Casia Moacir Dos Santos and Marin [19] and found after the inoculation on appropriate media
Spanamberg et al. [20]. In our study, fungi isolation proves the infection. On the other hand, the absence of
frequency in all milk samples collected in three clinical signs and the low CMT score (equal to 0)
mammary gland statuses was at the rate of 10.17 %. observed in the milk of healthy mammary glands lead
We recorded 88 yeast isolates against 26 mould us to think that the fungi species found in this category
isolates. Yeast abundance may be explained by their of milk are non-pathogenic fungi or saprophytes of the
pre-existence in the mammary glands. The pre-exist- mammary gland.
ing yeasts can be disrupted by several factors such as According to the results of mycological analysis,
antibiotherapy and corticotherapy that are highly we noticed that Candida kefyr was relatively frequent.
recommended in mastitis for curative or prophylactic Lagneau et al. [8] have found the same results in a
purpose [21]. According to Mehnert et al. [22], some similar study performed in Belgium. In Poland,

123
 Author's personal copy
330 Mycopathologia (2015) 179:327–331

Krukowski et al. [9] reported a predominance of this the milk samples of mastitis. This species, which is
species in their studies on the prevalence of mycotic rarely isolated in diseases, was reported for the first
mastitis. Candida kefyr was also isolated in the time in a hemoculture from a patient with a gastro-
mastitis milk samples by several authors, Aalbaek intestinal cancer. It is also found worldwide in many
et al. [16], Guilhon et al. [3], Pengov [23], McDonald foods, mainly fermented ones. Candida colliculosa
et al. [24] and Richard et al. [7]. was also isolated from the liver and the spleen of wild
In our study, C. kefyr isolation frequency was higher rodents in Venezuela. In 1954, Candida colliculosa
in the milk samples of subclinical and clinical mastitis was isolated from a skin lesion in a child of 3 months
than its isolation frequency in healthy milk samples. in Brazil [28]. Recently, this yeast was isolated from a
Indeed, since its first observation in France by Guilhon fungal endocarditis in a man of 71 years [29]. This
et al. [3], the pathogeny of this yeast is well known and patient was interested in gardening and in the produc-
demonstrated experimentally by Aalbaek et al. [16]. It tion of his own dairy products. According to these
can grow at temperatures around 42 °C [8]. This authors, the consumption of dairy products may be a
represents a necessary condition to infect the mammary risk factor for the infection by Candida colliculosa.
gland. C. kefyr is among the Candida species that The extremely rare pathogenicity of Candida colli-
assimilate lactose [25], which is a crucial character for culosa can be explained by the results of Dworecka-
the growth in milk medium. According to Guilhon et al. Kaszak et al. [30]. In their study about yeasts isolated
[3], the source of Candida kefyr is the cattle faeces that on mastitis and identified by PCR, they indicate that
are perpetually present in the mammary glands. strains of fungi phenotypically classified as the same
Candida albicans is the second most frequent Candida species can have different genotypes.
species recorded in our study. This yeast has been According to our results, C. parapsilosis and C.
isolated only in milk samples of clinical and subclin- inconspicua can be considered as opportunistic patho-
ical mastitis. Similar results are mentioned in a recent gens, because they are detected only in healthy milk
study by Zaragoza et al. [10]. The absence of Candida samples and subclinical mastitis milk samples. On the
albicans in the healthy milk samples can be explained other hand, C. parapsilosis is reported as a pathogen
by its pathogeny in mammary glands. It causes an responsible for clinical mastitis in several studies [9,
inflammatory reaction resulting from the increase of 10, 18, 19, 30].
the CMT score. Indeed, this yeast was isolated by Numerous authors reported the presence of Tricho-
several authors [4–7, 16, 19, 23]. sporon sp. [18, 31], Rhodotorula glutinis [9, 18, 20]
In our study, C. guilliermondii, C. famata, C. and Saccharomyces fragilis [3] in mycotic mastitis. In
tropicalis and C. Krusei are isolated from the three the present study, these species were also recorded,
categories of milk samples. Several studies reported especially in clinical and subclinical mastitis cases.
that these Candida species were often found in the Concerning the isolated mould species, we have
milk samples of clinical and subclinical mastitis, and noted their exclusive presence in the milk samples of
even in healthy milk samples [8, 10, 18, 26]. clinical mastitis and especially in the healthy milk
Yet, Candida rugosa was isolated from the milk samples. They may represent just contaminations, but
samples of clinical and subclinical mastitis. A similar this is not confirmed or proved because many studies
result was recently declared by Şeker in Turkey [26]. about mycotic mastitis reported their presence [4, 6, 18,
However, C. glabrata was only isolated from the 31–33], except for Cladosporium sp. This species has
samples of healthy milk and clinical mastitis milk not been considered as a mastitis agent. However, it was
samples. Its absence in the milk samples of subclinical frequently isolated as a laboratory contaminant [34, 35].
mastitis can be explained by the presence of many The detection of healthy carriers in the studied
bacteria producing metabolites that act as antagonists dairy cows was significant with a rate of 12.5 %. In the
and inhibitors of yeast growth. Moreover, these most studied farms, milk is intended for human
bacteria compete for substrates in milk medium [27]. consumption. This can be considered as a danger for
Candida colliculosa is an ascomycete yeast with a public health. The direct fungi transmission from milk
great ability to ferment sugars. It is widely used in the to man has never really been proven, although it
production of white beer and fermented drinks [28]. In cannot be completely excluded. Besides, yeasts resis-
our study, Candida colliculosa was not isolated from tance is important; they were found viable in butter

123
 Author's personal copy
Mycopathologia (2015) 179:327–331 331

and cheese made from the milk of sick cows, even 17. Yeo G, Choi WP. Studies on yeast-like fungi associated with
after more than 7 years [36]. Therefore, to prevent any bovine mastitis, 1: epidemiological study; 2: sensitivity of yeast-
like fungi to antifungal agents. Korean J Vet Res. 1982;22:121–4.
risk of human or animal infection, it is essential to 18. Costa EO, Gandra CR, Pires MF, Coutinho SD, Teixeira
diagnose subclinical mastitis milk, especially in the CM. Survey of bovine mycotic mastitis in dairy herds in the
healthy carriers, to avoid their direct consumption or state of São Paulo, Brazil. Mycopathologia. 1993;124:13–7.
their use in dairy products. This illustrates the 19. Santos RC, Marin JM. Isolation of Candida spp. from mastitic
bovine milk in Brazil. Mycopathologia. 2005;159:251–3.
economic consequences of this disease. 20. Spanambercg A, Wünder EA Jr, Brayer Pereira DI, Argenta
J, Cavallini Sanches EM, Valente P, Ferreiro L. Diversity of
Acknowledgments The authors are particularly grateful for yeasts from bovine mastitis in Southern Brazil. Rev Iberoam
assistance from Dr. Bentourki Aymen. Micol. 2008;25:154–6.
21. Guilhon J. Antibiotiques et perturbations de la flore mamm-
aire des femelles laitières. Bull Acad Vet. 1965;149:241–7.
22. Mehnert B, Ernst K, Gedek W. Hefen als Mastitiserryer bein
References Rind. Zentralblatt Veterinarmed. 1964; lla:97–121, apud
Costa EO, Gandra CR, Pires MF, Coutinho SD, Teixeira
1. Schalm OW. Pathologic changes in the milk and udder of CM. Survey of bovine mycotic mastitis in dairy herds in the
cows with mastitis. JAVMA. 1977;170(10 Pt 2):1137–40. state of São Paulo, Brazil. Mycopathologia. 1993;124:13–7.
2. Kirk JH, Bartlett PC. Bovine mycotic mastitis. Compend 23. Pengov A. Prevalence of mycotic mastitis in cows. Acta
Food Animal. 1986;8:106–10. Veterinaria (Beograd). 2002;52(2–3):133–6.
3. Guilhon J, Charton A, Drouhet E, Kahn J, Lecoanet J. 24. Mc Donald JS, Richard JL, Anderson AJ. In vitro antimy-
Mammite de la vache due à Candida pseudotropicalis. Vigot cosic sensitivity of yeasts isolated from infected bovine
Frères Editeurs. Bul Acad Vét. 1961; Tome XXXIV:367–70. mammary glands. Am J Vet Res. 1980;41(12):1980–90.
4. Monga DP, Kalra DS. Prevalence of mycotic mastitis among 25. Garcı́a-Garibay M, Gómez-Ruiz L. Uses of microbial beta-
animals in Haryana. Indian J Animal Sci. 1971;41(9):813–6. galactosidases to reduce lactose content in milk and dairy
5. Fransworth RJ, Sorensen DK. Prevalence and species dis- products. Rev Invest Clin. 1996;48(Suppl):51–61.
tribution of yeasts in mammary glands of dairy cows in 26. Şeker E. Identification of candida species isolated from
Minnesota. Can J Comp Med. 1972;36:329–32. bovine mastitic milk and their in vitro hemolytic activity in
6. Kumar-Jand S, Dhillon SS. Mastitis caused by fungi. Indian Western Turkey. Mycopathologia. 2010;169:303–8.
Vet J. 1975;52(1):125–8. 27. Moretti A, Pasquali P, Mencaroni G, Boncio L, Piergili F.
7. Richard JL, McDonald JS, Fichtner RE, Anderson AJ. Relationship between cell counts in bovine milk and the
Identification of yeasts from infected bovine mammary presence of mastitis pathogens (yeasts and bacteria). Zen-
glands and their experimental infectivity in cattle. Am J Vet tralbl Vet Med B. 1998;45:129–32.
Res. 1980;41(12):1991–4. 28. Dromer F, Lortholary O, Desnos-Olliver M, Garcia-Hermoso D,
8. Lagneau PE, Lebtahi K, Swinne D. Isolation of yeasts from Hoinard D, Raoux-Barbot D, Bretagne S, Dannaoui E, Gantier
bovine milk in Belgium. Mycopathologia. 1996;135:99–102. JC. Rapport d’activité 2009 du CNR Mycologie et Antifongi-
9. Krukowski H, Tietze M, Majewski TR, Ró_zański P. Survey ques [technical reports]. CNRMA: Institut Pasteur; 2010.
of yeast mastitis in dairy herds of small-type farms in the 29. Kaygusuz I, Mulazimoglu L, Cerikcioglu N, Toprak A,
Lublin region, Poland. Mycopathologia. 2000;150:5–7. Oktay A, Korten V. An unusual native tricuspid valve
10. Zaragoza CS, Cervantes Olivares RA, Ducoing Watty AE, De endocarditis caused by Candida colliculosa. Clin Microbiol
La Peña Moctezuma A, Tanaca LV. Yeasts isolation from Infect. 2003;9:319–22.
bovine mammary glands under different mastitis status in the 30. Dworecka-Kaszak B, Krutkiewicz A, Szopa D, Kleczkow-
Mexican High Plateu. Rev Iberoam Micol. 2011;28(2):79–82. ski M, Biegańska M. high prevalence of candida yeast in
11. Schalm OW, Noorlander DO. Experiments and observa- milk samples from cows suffering from mastitis in Poland.
tions leading to the development of the California Mastitis Sci World J. 2012;. doi:10.1100/2012/196347.
test. J Am Vet Med Assoc. 1957;130:199–204. 31. Ramisse J, Brement AM, Lamarre C, Viaud MA, Breard A.
12. Schneider E, Jasper DE, Elde RN. The relationship between Résultats d’une enquête sur les mammites en Vendée. Point
bulk tank microscopic cell counts and the individual California Vét. 1982;13(63):63–73.
Mastitis test reactions. Am J Vet Res. 1966;27:1169–75. 32. Schällibaum M, Nicolet J, König H. Aspergillus nidulans
13. Kurtzman CP, Fell JW. The Yeasts: a taxonomic study. 4th and Aspergillus fumigatus as causal agents of bovine mas-
ed. Amsterdam: Elsevier Science B V; 1998. titis. Sabouraudia. 1980;18:33–8.
14. Botton B, Breton A, Fevre M, Guy PH, Larpent JP, Veau P. 33. Simaria MB, Dholakia PM. Incidence and diagnosis of
Moisissures utiles et nuisibles: importance industrielle. mycotic mastitis in cattle. Indian J Animal Sci.
Paris: Masson Centre National des Lettres; 1985. 1986;56(10):995–1000.
15. Awad FI, El Molla A, Fayed A, Abd El-Halim M, Refai M. 34. Chabasse D, Guigen CL, Contet-Audonneau N. Mycologie
Studies of mycotic mastitis in Egypt. J Egypt Vet Med Ass. médicale. Paris: Masson; 1999.
1980;40(3):35–41. 35. Koenig H. Guide de mycologie médicale. Paris: Ellipses;
16. Aalbaek B, Stenderup J, Jensen HE, Valbak J, Nylin B, 1995.
Huda A. Mycotic and algal bovine mastitis in Denmark. 36. Chermette R. Mycoses des ruminants et santé publique. Le
APMIS. 1994;102:451–6. point Vét. Numéro Spéc. 1994;26:43–8.

123