Prevalence and Identification of Yeasts Responsible for Mastitis in Dairy Cattle Farms in the Sidi Lahcene Region in the Wilaya of Sidi Bel abbes- Algeria

Mastitis represents one of the main diseases in dairy cows. In Algeria, very few studies have been conducted on the prevalence of fungal mastitis in dairy cattle farms as well as various factors favouring their appearance and development. In most cases, the triggers this infection is bacteria. A growing number of fungi are currently associated with this pathology. This is related to antibiotics too widely used in the treatment of these bacterial agents. So we set as objectives, determining the prevalence of mastitis caused by yeasts and the study of a number of risk factors in some dairy farms in the region of Sidi Lahcène, wilaya of Sidi Bel Abbes. The samples of this study were carried on farms of cows (mastitic cow and clinically healthy cow) belonging to two types of farms (farms with manual milking and mechanical milking farms). The risk factors included, animal secretions, the teat cups, the hands of the milkers, the skin of the mammary gland, the drinker, the manger, the milk storage tank, the milk collection seal. Mycological analysis was conducted at the Laboratory of Parasitology-Mycology from the Higher National Veterinary School-Algiers. The isolated yeasts were identified using microscopic characterization, and auxanogramme realized in biochemical galleries (gallery Pasteur Institut Pasteur Algiers and testing API® AUX BioMerieux, France). Our results showed a prevalence of infection with yeast, a high frequency of isolation was attributed for the genus Candida sp. followed by the genus Rhodotorula sp. followed by the two genera Cryptococcus sp. and Trichosporon sp.


Introduction
Inflammation of the udder is one of the first three pathologies of dairy cows. This pathology can be in apparent (latent infection, subclinical mastitis), it is the most frequent (95 to 98% of the cases) and can be apparent (clinical mastitis), the latter only represents 2 to 5% of the cases [1].
Bovine mastitis is a multifactorial disease involving interrelationship between host, environment and infectious agents. It is considered the most widespread and infectious disease of dairy cows economically important on all continents, with annual losses estimated at $35 trillion in the world dairy industry [2].
Several microorganisms are involved in the etiology of intramammary infections in cattle, represented mainly by bacteria, viruses, mycoplasmas, algae and yeasts [3]. Yeasts are found in moist places that are rich in organic matter, and are easily isolated from teats and milking equipment [4] Mastitis foci caused by yeasts have been reported in intensively managed herds in which there were deficiencies in environmental hygiene or in combination with repeated intramammary treatment [5,6]. Several species of yeasts of the genus Candida, Cryptococcus, Rhodotorula and Trichosporon have been associated with mastitis in dairy cows. Candida is usually the most frequently isolated genus, with large variations in prevalence and species identified [7,8].
Studies on fungal infections of the mammary glands in cows are becoming more frequent due to their increasing incidence. According to the literature, these infections account for 2%-13% of all cases of mastitis in cows [9][10][11]. For this reason we were interested in this pathology by conducting a study in a few dairy cattle farms in the region of Sidi Lahcène, wilaya of Sidi BelAbbes, by distributing a questionnaire to the veterinary practitioners of the region in order to identify this pathology by realizing milk simples and swabs in targeted dairy cattle for mycological analysis to identify responsible yeasts and determine the prevalence of this disease in the intended dairy herds.

Description of the study region
Sidi Lahcene formerly Détrie during the French colonization is one of the 52 communes of the wilaya of Sidi Bel Abbes. Located in the west of Algeria, 150 km from the Moroccan border.

Distribution of a questionnaire
A total of 50 questionnaires were distributed to veterinary practitioners in the region, including the farms covered by our study. The questionnaire was distributed by the veterinary inspector of the wilaya of Sidi Bel Abbas to the veterinarians of the region of Sidi Lahcène which in turn are moved to the farms of the region as well as the farms included in the study and they filled the questionnaire on site, which allowed us to recover all distributed questionnaires (50 questionnaires).
The questionnaire focused on the characteristics of the different livestock farms, the practices of the milkers with regard to the cleaning of the milking equipment, the cleaning of their hands, the preparation of the udders to milking, the environment of dairy cows, litter, the type of housing, their pathological history (mastitis).

The study period
The samples were taken during the second quarter of 2012 (March, April, and May 2012). The mycological analysis was carried out during the first half of 2013 (from January 2013 until July 2013).

Nature and number of samples
A total of 562 samples were collected by the veterinarians' practitioners of Sidi Lahcene's region. That is 280 samples of milk taken from 70 existing cows in 13 farms; all the farms exist in the same region, wilaya of Sidi Bel Abbes. And 275 swabs performed on 13 farms (65 swabs of the breeding equipment and 210 swabs on the cow).
The simples were obtained with different mammary glands health status: 10 cows with healthy mammary glands, 46 cows with subclinical mastitis as determinate by the California Mastitis Test (CMT) (leucocytest, Symbiotic Europe, France) and 14 cows with clinical mastitis was defined by: swelling, reduced milk flow and abnormal milk appearance, additionally, other signs of infection as fever, inappetence, ataxia. CMT was used to identify subclinical mastitis on mammary gland of the cows. For this study, milk simples from gland affected with subclinical mastitis were included when the reaction to CMT was at least grade 2, corresponding with an appearance of viscous milk that does not adhere to the bottom of CMT plate and correlates to 400,000-1,500,000 somatic cells/ml.
On every cow in lactation: 04 takings of milk (a taking of milk of every trayon), an anal swab, a vaginal swab and swabs of the mammary gland, only once during all the period of the taking. In every breeding with manual milking, it made a swabbing of the hands of the milker before the milking (factor of contamination), a swabbing of the feeder, a swabbing of the milk bucket and it made water samples from the water trough. Same taking were made in the breedings with machine milking except swabbing of tumblers milkers of the milking machine (factor of contamination) (

Milk sampling
The correct realization of the sampling procedure was a necessity, regarding the ubiquity of fungi which can contaminate the milk. The characteristics of the atmosphere surrounding were noted: The cow's environment was not loaded of dusts (hays moved nearby, agitated animals). If such was the case, the animals of the dusty premises were taken out. The milk sampling was realized according to the protocol of  [12]. which consists in washing itself hands with a disinfecting soap; identify the flask (in wide opening) with the indelible felt-tip: number of the cow, the mammary gland quarter (FR, FL, RR or RL), date and time; the udder was carefully washed and wiped; the rough drafts of milk to rinse the canal of the udder was eliminate (no more than 2 jets otherwise risk of very poor taking in germs); Disinfect the teat canal of the udder with a compress soaked with alcohol in 70°C; the sterile flask was opened maintaining the opening managed downward. The rubber were kept in the same hand without touching the inside; 08 milliliters of milk were taken and record the flask; every udder were disinfected before taking the milk of the corresponding district; finally. The milk samples were transported to the laboratory of Parasitology and Mycology of the National Veterinary graduate school of Algiers in a cooler (4°C). They were frozen at -20°C until the day of their analysis.

Mycological analyses of the milk
The mycological analysis was realized in the laboratory of Parasitology-Mycology of the National Veterinary graduate school of Algiers. It consists of a direct examination of the samples of milk after colouring with the blue of the lactophenol. Yeasts are recognizable by their budding and their size by several microns, which avoids confusion with bacteria. A few drops of milk are seeded on Sabouraud Dextrose Agar (SDA) (Pasteur institute of Algiers) added of chloramphénicol (cast in petri dishes) and incubated in an oven at 27°C for 48 hours to 72 hours or more a week. Finally, isolated yeasts were identified using macroscopic characterization; this is a first step in the orientation of the mycological diagnosis. We used as criteria the shape, color, consistency and appearance of the colony. The yeast colonies of different appearance are sub cultured on Sabouraud/ chloramphenicol medium at 27°C for subsequent identification. In this phase, various culture media and tests are used for the precise identification of yeasts (Pasteur gallery) (Pasteur institute of Algiers) [13]. This identification was performed taking into consideration morphological characteristics, like formation of chlamydoconidium, pseudohyphae and germinal tube development.
Identification of the species of yeast has been realized by assimilation of carbon sources (auxanogramme): We used the test API ® 20 C AUX (BioMerieux, France) comprising 19 carbon sources [14]. API ® 20 C AUX should not be used directly from clinical sample or other. The microorganisms to be identified must first be isolated on a culture medium adapted according to the usual techniques of bacteriology and mycology. After 48 hours of incubation or 72 hours, observe the growth of the yeast compared to the negative control well 0. A cupule more turbid than the control indicates a positive reaction to note on the results sheet.

Mycological analysis of the water of the drinking trough
A few drops of the water are inoculated on Sabouraud medium with chloramphenicol added. The identification of the pushed colonies is carried out as before for the milk samples.

Mycological analysis of swabs
We followed the same experimental steps as those used in the mycological analysis of milk except direct examination. A seeding on a petri dish by friction of the swab over the entire surface of the medium. The yeast colonies are sub cultured on Sabouraud medium for subsequent identification as mentioned earlier.

Results of the questionnaire
The questionnaire sent to veterinary practitioners allowed us to carry out an initial analysis on the management of dairy cattle farms in the Sidi Lahcène region (Sidi Belabbes wilaya) before going on to laboratory analyses. The veterinarians in charge of follow-up of the bovine breeding note that the measures of hygiene are absent. The breeders change the litter every two days at a percentage of 25% and 20% change the litter once a week (7 days), 39% of the breeders disinfect their milking equipment once a week, but it is noted that there is a certain percentage of the breeders (22%) never disinfects their milking equipment, despite this a few percentage of breeders (11%) disinfecting this equipment twice a day. 78% of milkers do not disinfect their hands before and after each milking. Only 6% of the milkers use an individual cloth to disinfect the teats of the udder and 63% of the milkers use a single cloth for all cows.

Discussion
The survey conducted by questionnaire distributed to veterinarians in the region of Sidi Lahcène revealed the importance of respecting the hygiene of the animal's environment. Indeed, moisture from urine and milk as well as body heat, promote the development of fungi and more specifically the yeasts in the litter, hence the importance of regular renewal of the litter in a stable. Our survey showed that breeders change the litter every other day to 40% and 35% change the litter once a week (from 05 to 07 days).
According to the questionnaire responses, mastitis is common in high-producing cows at 72%, consistent with various studies that have shown positive correlations (0.30 to 0.44) between milk production and susceptibility to mastitis [15,16].
According to Slavolju et al. if no bacteriological research in the milk is carried out, fungal mastitis is suspected when antibiotic treatment fails. This phenomenon is due to the frequent use of antibiotics, which was found by our survey, because in 66.67% of cases, mastitis persists after antibiotic treatment. They are used not only in treatment, but also in the dry period. Ant biotherapy leads to a disturbance in the homeostasis of the udder, inhibition of T lymphocyte and neutrophil activity and consequently to the stimulation of growth of yeast [17][18][19][20]. Half of the cows which the milk is contaminated by yeasts have been The etiological agents of mycotic mastitis are mainly yeasts, especially Candida sp. which is according to several authors found more frequently [21][22][23][24][25][26][27][28][29][30]. Our results are consistent with literature data; indeed, the genus Candida sp. was isolated in 73.86% of the isolated yeasts, which corresponds to about 130 isolated species.
In addition, it is interesting to note that in this study we found that: • The same species was isolated from the four teats of the same animal (example: Candida zeylanoides and Candida tropicalis).
• Different species belonging to the same genus have been isolated from the teats of the same animal (example: Candida zeylanoides, Candida tropicalis, and Candida parapsilosis).
• Different genus of yeasts isolated from the teats of the same animal (example: Candida zeylanoides and Trichosporon cutaneum) • Différents genus of yeasts isolated from the same teat (example: Candida zeylanoides, and Cryptococcus humicola) • Yeasts isolated from two teats and the other two summers free of infection.
This indicates that several fungal agents can infect the same animal as in the bacterial infection and that each quarter of the udder is independent of the other.
According to some authors, animal species and geographical variations may be the reason for this difference in the distribution of yeast species [37].
It is well established that yeasts of the genus Candida, for example, are able to use antibiotics such as penicillin and tetracycline as sources of nitrogen [38]. In addition, high doses of antibiotics can cause a reduction in vitamin A, causing lesions of the udder epithelium, facilitating the invasion of fungi [39]. It has been reported that Candida species can cause clinical mastitis characterized by pain, prolonged fever and inflammatory reaction in the mammary gland and lymph nodes associated with a reduction in milk production in animals [35]. They can also be isolated from animals suffering from subclinical mastitis [40]. Besides the well-known virulence factors in Candida such as hydrolytic enzymes, toxins, dimorphism and hemolysin production.

Conclusion
The literature review and the results we have obtained, allow us to complete the data collected on fungal mastitis in Algeria. Lack of demand for accurate laboratory tests during mastitis and nonspecificity of symptoms in fungal mastitis and bacterial mastitis, results in poor management in this condition since in the field it is systematically treated with antibiotics and anti-inflammatories (diathelic pathway). Training thus, a possible rupture of the mycobacterial equilibrium, already disturbed and thus aggravate the inflammation of the udder of fungal origin. The particularity of fungal mastitis due to the fact that their epidemiological forms depend on the intervention of a number of factors and their combinations together, more than the causal agent. In addition, fungal mastitis most of the time has a tendency to self-limitation and spontaneous healing, these are other factors of intervention which generally ensure the persistence or the extension of the affection.
Analysis of the questionnaire distributed to veterinary practitioners in the region of Sidi Lahcène, wilaya of Sidi Bel Abbas, has highlighted numerous shortcomings in breeding management and in particular the respect of the general rules of the hygiene, the conditions of milking and the general atmosphere. The hygiene in the barns should not be an additional instrument in the conduct of breeding but be constitutive.

Perspectives
Through our investigation, we found that the incidence of fungal mastitis varies widely across regions and investigations. In order to minimize these affections and the economic losses they cause, we propose the following recommendations: A. The practitioner, faced with a problem of mastitis in a dairy livestock, must be alerted to face a next anamnesis: a recent intramammary antibiotic treatment followed by the appearance, persistence or even aggravation of clinical signs. Failure of such treatment should lead to suspicion of a fungal infection of the udder.