Name : Randa Mohamed Ibrahim Ismail Alarousy Department ... · Randa. M. Alarousi et al/International Journal of ChemTech Research, 2016,9(5),pp 200-206. 201 Routine yeast distinguishing

Name : Randa Mohamed Ibrahim Ismail Alarousy Department: Medical Laboratories Publications:

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Molecular Characterization of Rhodotorula Spp. Isolated From Poultry Meat

Randa. M. Alarousi1*, Sohad M. Dorgham1,El-Kewaiey I. A.2, Amal A. Al-Said3

1Department of Microbiology, National Research Center, Dokki, Giza, Egypt.*Department of Medical Laboratories, College of Applied Medical Sciences,

Majmaah University, KSA2Food Hygiene Unit,Damanhour Provincial Lab., Animal Health

Research Institute, Egypt .3Microbiology Unit, Damanhour Provincial Lab., Animal Health Research Institute, Egypt .

Abstract: A total of 50 Rhodotorula spp strains were isolated from 200 fresh Chicken and quail products, represented by chicken breast and thigh (Chicken: n=50 breast, n=50 thigh and quail: n=50 breast, n=50 thigh) with an incidence 25%. PCR was applied on 5 positive samples out of 15 samples derived from chicken breasts; 5 positive samples out of 12 samples from chicken thigh meat; 5 positive samples out of 13 from quail breasts and 5 samples out of 10 positive of quail thigh meat samples, CrtR gene was detected at 560bp. High incidence of Rhodotorula spp, it may be due to un hygienic measures during rearing and slaughtering of poultry, whichrepresent potential public health risk.Key words: Rhodotorula, PCR, CrtR gene, quail.

Introduction

Microbial nourishment security and sustenance borne diseases are critical general wellbeing concern worldwide.There have been various sustenance borne illuness coming about because of ingestion of contaminated nourishments, for example, chicken meats. Poultry meat is one of the exceedingly expended creature started nourishment thing. With high nutritive quality, having both vital large scale and micronutrients. The vast majority of the pathogens that assume a part in sustenance borne maladies have a zoonotic starting point1. Most wellbeing powers have considered the importance of yeasts in nourishments, including meat items, in perspective of the general wellbeing. Rhodotorula is a basidiomycetous yeast in the fungal family Sporidiobolaceae (Phylum Basidiomycota)2. The family Rhodotorula consolidates 8 species, of which R. mucilaginosa, R. glutinis, and R. minuta are known not infection in individuals, it was beforehand considered non-pathogenic; nonetheless, it has developed as a deft etiologic operator amid the most recent two decades 3.

Among the few references to the pathogenicity of Rhodotorula spp. in animals, there are several reports of an outbreak of skin infections in (chickens and sea animals), lung infections and otitis in (sheep and cattle), dermatitis in a cat that had crusted lesions and mastitis 4. Most of the cases of infection due to Rhodotorula in humans were fungemia associated with central venous catheter (CVC) use. Not at all like fungemia, a portion of the other limited infections caused by Rhodotorula, including meningeal, skin, visual, peritoneal, and prosthetic joint diseases, are not as a matter of course connected to the utilization of CVCs or immunosuppression5.

International Journal of ChemTech Research CODEN (USA): IJCRGG, ISSN: 0974-4290, ISSN(Online):2455-9555

Vol.9, No.05 pp 200-206, 2016

Randa. M. Alarousi et al /International Journal of ChemTech Research, 2016,9(5),pp 200-206. 201

Routine yeast distinguishing proof taking into account phenotypic qualities is frequently deceptive and uncertain, and for the most part should be underlined by sub-atomic strategies.

Differentiation of all types of the family Rhodotorula in view of morphological characters and physiological tests is conceivably tedious, costly and requires extensive aptitude. Subsequently, a more adequate strategy rose through the presentation of a coordinated perspective of the variety taking into genus based on molecular data and this, however not a simple errand.

Use of improved methods for identification such as the application of molecular techniques to species identification, has also contributed significantly. Since the study of 6 through which they described seven more new species of the genus Rhodotorula, according to differences in the D1and D2 region of the large-subunit rDNA, many studies were conducted. 7 demonstrated the effectiveness of using cytochrome b gene sequence for both species identification and the investigation of phylogenetic relationships among basidiomycetous yeasts.

Therefore, the present work aimed to: (a) Isolation and identification of Rhodotorula spp. field isolates from chicken and quail meat (b) Detection of cytochrome oxidase (crtR) gene (c) Evaluate their public health significance through the incidence of isolation.

Material and Methods

(i) Samples collection and preparation:

Two hundred fresh poultry products (Chicken and quails), represented by chicken breast and thigh (Chicken: n=50 breast, n=50 thigh and quail: n=50 breast, n=50 thigh). All samples were collected from different markets at Bahaira governorate from January to March 2015. The samples were transferred directly to the laboratory in ice boxes without any delay. Twenty-five grams of each sample were mixed with 225 ml of sterile peptone water 0.1% and thoroughly homogenized under aseptic conditions 8.

(ii) Isolation and identification of yeast:

A loop full of each sample were inoculated on Sabouraud`s dextrose agar then incubated at 25°C for 3 days 9.

Yeast colonies of different morphological appearance were re-cultivated on Sabouraud´s dextrose agar slopes and incubated at 25°C for 5 days and kept for identification.

Identification of isolated yeasts morphologically and biochemically were carried out according to methods recommended by 10.

(iii) DNA extraction and PCR Amplification:

Genomic DNA of Rhodotorula spp. was extracted by using an extraction kit (QIAamp mini kit, Qiagen). Specific oligonucleotide primer for Cytochrome oxidase (crtR) gene was used as described in table (2). The amplification conditions for crtR gene included initial denaturation at 95°C for 3 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, synthesis at 72°C for 3 min and a final extension step at 72°C for 10 min. Samples were kept at 4°C until checked. The amplicons were separated by 0.8 % agarose gel electrophoresis in TAE buffer containing 0.5 μg /ml Ethidium bromides 11.

Table.1: Primer sequences and product size of crtR gene:

Target Primer sequence Product size Reference

crtR F.CARACTGGKACDGCHGARGATTR. WGGDCCRATCATGAYRACTGG

560 bp Alcaíno et al. [12]


(iv) CrtR gene detection:

A total of 20 Rhodotorula spp. strains have been selected randomly for detection of CrtR gene.

(v) Statistical analysis:

Data were analyzed statistically by Chi-square test using statistical package of social science SPSS (version 18.0).

Result

1. Isolates:

All suspected orange colored colonies were examined under light microscope. The results of traditional biochemical tests indicated that all suspected isolates are Rhodotorula spp. The incidence of isolation was mentioned in tables (2&3).

Table (2): Number and percentage of Rhodotorula spp. in chicken and quail:

Groups +ve -veChicken (n=100) 27

(27%)73

(73%)Quail (n=100) 23

(23%)77

(77%)Total (n=200) 50

(25%)150

(75%)Rhodoturola was isolated from 25% of chicken and quail samples. There were no statistical differences (P> 0.05) between number of isolated samples from chicken (27/200) and quail (23/200).

Table: 3. Number and percentage of Rhodotorula spp.in chicken

Chicken samples Rhodotorula +ve samples Rhodotorula -ve samples

Breast (n=50)15

(30%)35

(70%)

Thigh (n=50)12

(24%)38

(76%)

Total (n=100)27

(27%)73

(73%)

Table: 4. Number and percentage of Rhodotorula spp. in quail:

Quail samples Rhodotorula +ve samples Rhodotorula -ve samples

Breast (n=50)13

(26%)37

(74%)

Thigh (n=50)10

(20%)40

(80%)

Total (n=100)23

(23%)77

(77%)


Table.5. classification of Rhodotorula spp. In chicken:

Rhodotorula species Breast (n=15) Thigh (n=12)Total(n=27)

R. glutinis8

(53%)6

(50%)14*

(51.9%)

R. pallida7

(47%)Not isolated

7*(25.9%)

R. minuta Not isolated6

(50%)6*

(22.2%)* Significant difference at P> 0.05

It is clear from table (5) that the percentage of R. glutinis (51.9%) isolated from chicken samples were significantly (P> 0.05) higher than both R. pallida and R. minuta (25.9% and 22.2%, respectively).

Table.6. classification of Rhodotorula spp. in quail

Rhodotorula species Breast (n=13) Thigh (n=10)Total(n=23)

R. glutinis6

(46%)6

(60%)12*

(52.2%)

R. pallida Not isolated4

(40%)4*

(17.4%)

R. flava7

(54%)Not isolated

7*(30.4%)

* Significant difference at P> 0.05

1. It is clear from table (6) that the percentage of R. glutinis (52.2%) isolated from chicken samples were significantly (P> 0.05) higher than both R. pallida and R. flava (17.4% and 30.4%, respectively).

2. As show in table 5 and table 6, R. glutinis and R. pallida was isolated from both chicken and quail Rhodotorula +ve samples. While R. minuta was isolated from chicken samples, R. flava was isolated from quail samples.

CrtR gene detection:

Twenty selected strains of Rhodotorula spp. carried CrtR gene as showed in figure (1).

Figure (1): Ethidium bromide-stained agarose gel of PCR assay of Rhodotorula spp. strains. The position corresponding to CrtR PCR amplicons was at 560bp, an appropriate size marker in a 100 bp ladder.


Discussion

Most wellbeing powers have considered the criticalness of yeasts in nourishments, including meat items, in perspective of the general wellbeing. The determination of the defilement of nourishment fixings and of prepared sustenance with yeasts is a fundamental part of any quality affirmation or quality control program in the sustenance business 13.

According to our outcomes, morphological, cultural and biochemical recognizable proof uncovered that our suspected detaches were Rhodotorula spp., this result was affirmed by Fell et al.[6] who mentioned that Rhodotorula is a yeast which produces mucoid colonies with a characteristic carotenoid pigment.

Previously, Rhodotorula is considered a low virulence organism in comparison to Candida or Trichosporon. Recently, Rhodotorula must be considered as a potential pathogen14.

Rhodotorula mucilaginosa has been getting expanding consideration since it can be secluded from normally matured milk 15 and other nourishment lattices16,17, in addition to, different and extreme ecosystems, including the complex core gut microbiota of carnivore wild fish18, marine shores, glacial core cold environments 19 and hydrocarbon-contaminated soil20.

As indicated by our outcomes, R. glutinis, and R. palida were detached from chicken's breast with an occurrence 53%, 47%, and thigh with a frequency 50% for R. glutinis separately. The frequency rate of R. glutinis separation in quail's breast and thigh was 46% and 60% respectively. R. palida was confined from quail's thigh with an occurrence 40%. The past result can't help contradicting 21 who segregated R. glutinis, and R. palida from chicken filet test with low rate 5%. On the other side, our outcomes were in amicability with 22

who disengaged Rhodotorula spp. from chicken carcass and thigh with an incidence6 (60 %) and 4 (40 %) separately. These results show that our examples were more debased with yeast strains; this might be because of terrible hygienic measures amid the preparing steps and taking care of. At the season of butchering, the quills, bolster and assemblages of the flying creatures have been observed to be tainted with yeasts 23.

In expansion to that, yeasts have been withdrawn from the air and soil beginning from poultry repeating and raising houses, old litter and litter-containing water, wet support and winged creature droppings 24. The high danger of Yeasts sullying is making a noteworthy commitment to the general microbial biology of poultry andmight likewise add to the progressions prompting decay 25. Molecular methodologies are currently being produced to give a faster and target identification of yeasts contrasted with conventional phenotypic strategies.

Carotenoids are natural pigments of yellow, orange or red color. More than 600 different chemical structures have been described to date26. They are terpenoids with the isopentenyl- pyrophosphate (IPP) molecule as the basic unit. Astaxanthin is a carotenoid with a high commercial interest due to its use as a food additive for trout and salmon flesh pigmentation in aquaculture27. Its biosynthesis is limited to a few microorganisms such as the microalgae Haematococcus pluvialis; the basidiomycetous yeast Xanthophyllomyces dendrorhous 28, 29 and Rhodotorula spp7.

The biosynthesis of astaxanthin requires a lot of sophisticated steps, cytochrome P reductase enzyme encoded by the cpr gene are one of the most important requirements for this pathway 30. Albeit a few genes for various cytochrome P450 enzymes can exist in a living being, in many species one and only cpr gene exists. A few special cases have been seen in plants and zygomycetes that contain a few cpr genes 31.

Our study, a specific primer pair based on the sequence of cpr gene12 was used as conserved primers for all Rhodotorula spp. In this work we tried to reach a rapid diagnosis of Rhodotorula by purifying the DNA sample directly from grown colonies. The PCR amplification revealed 20 (100 %) positive samples out of 20 samples were chosen to apply PCR to confirm the primary conventional identification assays used on all the samples. PCR was applied on 5 positive samples out of 15 samples derived from chicken breasts; 5 positive samples out of 12 samples from chicken thigh meat; 5 positive samples out of 13 from quail breasts and 5 samples out of 10 positive of quail thigh meat samples.


Conclusion

In conclusion, this result was obtained within hours with a highly sensitive and specific result and required less efforts and time in comparison to the conventional methods used for identification of the organism; hence time factor for diagnosis of any infectious disease is very important to start as early as possible in treatment of the infection. Future prospective studies will be carried out for further studying of molecular characterization of CrtR gene in Rhodotorula species.

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4. Kadota K, Uchida K, Nagatomoet T. Granulomatous epididymitis related to Rhodotorula glutinisinfection in a dog Veterinary Pathology 1995; 32 (6):716–718.

5. Wilth F and Goldani LZ Epidemiology of Rhodotorula: An Emerging Pathogen. Interdisciplinary Perspectives on Infectious Diseases 2012, Article ID 465717: 7. http://dx.doi.org/10.1155/2012/465717

6. Fell J W, Boekhout T, Fonseca A, Scorzetti G and Statzell-Tallman A. Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/ D2 domain sequence analysis. Int J Syst Evol Microbiol 2000; 50:1351-1371.

7. Biswas S K, Yokoyama K, Nishimura K and Miyaji M. Molecular phylogenetics of the genus Rhodotorula and related basidiomycetous yeasts inferred from the mitochondrial cytochrome b gene, Int J Syst and Evol Microbiol. (2001); 51: 1191–1199.

8. Russell SM, Cox NA and Bailey JS Microbiological methods for sampling poultry messing plant equipment. J Appl Poultry Res 1997; 6: 229-233.

9. Beuchat LR (ed.) Food and Beverage Mycology, 2nd ed. Van Nostrand Reinhold, New York 1987, Chapter 17: 609.

10. Kreger-Van Rij N J W The Yeasts, a Taxonomic Study. 3rd ed. Amsterdam, Elsevier, 1984.11. Sambroock J and Russell DW Molecular cloning. A laboratory manual 3rd edition. Cold Spring Harbor

NY: Cold Spring Harbor Laboratory. Press; 2001.12. Alcaíno J, Barahona S, Carmona M, Lozano C, Marcoleta A, Niklitschek M, Sepúlveda D, Baeza M

and Cifuentes V. Cloning of the cytochrome p450 reductase (crtR) gene and its involvement in the astaxanthin biosynthesis of Xanthophyllomyces dendrorhous. BMC Microbiology 2008; 8:169.

13. Deak T and Beuchat LR Handbook of Food Spoilage Yeasts. CRC Press, Inc., Baton, FL. 210 pp. 1996.14. Gomez-Lopez A, Mellado E, Rodriguez-Tudela JL, Cuenca-Estrella M. Susceptibility profile of 29

clinical isolates of Rhodotorula spp. and literature review. J Antimicrob Chemo Ther 2005; 55:312-316.15. Bai M, Qing M, Guo Z, Zhang Y, Chen X, Bao Q, Zhang H, Sun TS Occurrence and dominance of

yeast species in naturally fermented milk from the Tibetan Plateau of China. Can J Microbiol 2010; 56:707–714. http://dx.doi.org/10.1139/w10-056.

16. Sabate J, Cano J, Esteve-Zarzoso B, Guillamón JM Isolation and identification of yeasts associated with vineyard and winery by RFLP analysis of ribosomal genes and mitochondrial DNA. Microbiol Res 2002; 157: 267–274. http://dx.doi.org/10.1078/0944-5013-00163.

17. Lucena-Padrós H, Caballero-Guerrero B, Maldonado-Barragán A and Ruiz-Barba JL Microbial diversity and dynamics of Spanish-style green table-olive fermentations in large manufacturing companies through culture-dependent techniques. Food Microbiol 2014; 42:154 –165. http://dx.doi.org/10.1016/j.fm.2014.03.020.

18. Raggi P, Lopez P, Diaz A, Carrasco D, Silva A, Velez A, Opazo R, Magne F and Navarrete PA Debaryomyces hansenii and Rhodotorula mucilaginosa comprised the yeast core gut microbiota of wild and reared carnivorous salmonids, croaker and yellowtail. Environ Microbiol 2014; 16: 2791–2803. http://dx.doi.org/10.1111/1462-2920.12397.


19. Singh P, Tsuji M, Singh SM, Roy U, Hoshino T Taxonomic characterization, adaptation strategies and biotechnological potential of cryophilic yeasts from ice cores of Midre Loven breen glacier, Svalbard, Arctic. Cryobiology 2013; 66:167–175. http://dx.doi.org/10.1016/ j.cryobiol. 2013.01.002.

20. Chandran P, Das N. Role of plasmid in diesel oil degradation by yeast species isolated from petroleum hydrocarbon-contaminated soil. Environ Technol 2012; 33:645– 652. http://dx.doi.org/10.1080/09593330.2011.587024.

21. Hemly A and Mahmoud YA Anti-Yeast Effects of Some Plant Extracts on Yeasts Contaminating Processed Poultry Products in Egypt. Czech. J Food Sci 2005; 23 (1): 12–19.

22. Abd-Elrahman HA, Soliman SA and Rahal EG Prevalence of yeast in chicken meat and their products. 2013: SCVMJ, XVIII (2).

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24. Bryan FL Poultry and poultry meat products. In: SILLIKER JH (ed.): Microbial Ecology of Foods. Food Commodities. Academic Press, London. 1980 (2): 410–458.

25. Viljoen BC, Geornaras I, Lamprecht A, Von Holy A Yeast populations associated with processed poultry. Food Microbiology 1998; 15:113–117.

26. Takaichi S, Sandmann G, Schnurr G, Satomi Y, Suzuki A, Misawa N The carotenoid 7,8-dihydro-ψ end group can be cyclized by the licopenecyclases from the bacterium Erwini auredovora and the higher plant Capsicum annuum Eur J Biochem 1996; 241:291-296.

27. Johnson E, Conklin D, Lewis M The yeast Phaffiarhodozyma as a dietary pigment source for salmonids and crustaceans. J Fish Res Board Can 1997; 34: 2417-2421.

28. Andrewes AG, Phaff HJ, Starr MP Carotenoids of Phaffiarhodozyma. A red pigmented fermenting yeast. Phytochem 1976; 15: 1003-1007.

29. Fraser P, Miura Y, Misawa N In vitro characterization of astaxanthin biosynthetic enzymes. J Biol Chem 1997; 272: 6128-6135.

30. Van den Brink HM, van Gorcom RF, van den Hondel CA and Punt PJ Cytochrome P450 enzyme systems in fungi. Fungal Genet Biol 1998; 23:1-17.

31. Malonek S, Rojas MC, Hedden P, Gaskin P, Hopkins P, Tudzynski B The NADPH-cytochrome P450 reductase gene from Gibberellafujikuroiis essential for gibberellin biosynthesis. J Biol Chem 2004; 279:25075-25084.

*****

In Vivo Study on the Hindrance Activity of Cinnamon Extract Against Aspergillus niger in Mice

Randa Mohamed Alarousy1,4, Sherein IsmailAbd El-Moez1,

Kawkab Ahmed Ahmed2 and Hany Mohamed Hassan3

1Department of Microbiology and Immunology, National Research Centre, Giza, Egypt 2Department of Pathology, Faculty of Veterinary Medicine, Cairo University,

Giza, Egypt 3Department of Immunology, Animal Reproduction Research Institute

4Department of Medical Laboratories, College of Applied Medical Sciences, Majmaah University, KSA

Abstract : Aim: The antimycotic effect and immune stimulating capacity of natural extract of Cinnamomum zeylanicum plant was evaluated against Aspergillus niger strain.

Methods and results: the herbal extract was prepared from cinnamon park to be examined

against Aspergillus niger (ATCC16404)species fungal cell suspension. Mice were injected with both fungal cell suspensions and Cinnamomum zeylanicum extract in a certain regime.

Histopathological examination was applied on lung, liver and brain tissues extracted from the

experimental animals and histopathological findings revealed the strong fungicidal effect of the herbal extract in the mice's tissues. Phagocytic activity, interleukins 2 & 6 (IL 2& IL6) and

tumor necrosis factor(TNF) were measured in mice's blood samples as immunity stimulating

efficacy parameters and also results revealed the potent immune stimulating efficacy of the

extract and its safetyin vivo. Conclusion: According to the results of the study, Cinnamomum zeylanicum extract has a

strong fungicidal activity against A. niger(ATCC16404)in addition to potent immune

stimulating action confirmed experimentally. Results are supporting the efficacy of C. zeylanicum extract as prophylactic agent as well as fungicide.

Significance and impact of the study: our study spotted a light on the advantages of the

examined herbal extract as antimycotic substance, the promising results revealed encourages the use of C. zeylanicum extract as pharmaceutical preparation for treating and prevention of

mycosis in immunocompromised patients instead of using some chemical antimycotic

preparations available for commercial use to avoid the disadvantages of these chemically

prepared medications. Keywords: Cinnamomum zeylanicum– Interleukin 2 – Interleukin 6 – Antimycotic extract –

Immunostimulant.

Introduction:

Fungal infection is considered among the main causes of morbidity in immunocompromised patients. The epidemiology of invasive mycotic infections has changed within the last decade (Lehrnbecher et al.,

1

2010)

International Journal of ChemTech Research CODEN (USA): IJCRGG, ISSN: 0974-4290, ISSN(Online):2455-9555

Vol.9, No.12 pp 962-972, 2016

Randa Mohamed Alarousy et al /International Journal of ChemTech Research, 2016,9(12): 962-972. 963

About 20 Aspergillus species out of 185 have been reported as opportunistic infectious agent in both

man and animals (Stevenset al., 2 2000)

Although opportunistic fungal agent leads to life – threatening infection in immunosuppressed patients,

fungal infection affects both immunosuppressed patients as well immune-competent persons in developing

countries (Del Poeta and Chaturvedi, 3 2012) The rising incidence in fungal infections observed in the last

decade correlates with increases in invasive medical interventions, long-term hospitalization and with large

numbers of immunosuppressed patients due to acquired infection due to human immunodeficiency viral

infection or treatment-induced immunodeficiency as prolonged using of cortisone before organ trans-plantation

or during anticancer therapy (Pfaller andDiekema,4,5

2007 and2010)

It is very hard to distinguish between systemic mycosis from other microbial infection depending on the

clinical symptoms. This often leads to delayed appropriate intervention using antifungal therapy(Pfaller,6 2012)

Antimycotic agents are associated with inverse results of systemic aspergillosis; the ascending

problems due to azoles – resistance and in addition, currently used antifungals have substantial economic burden (Karthaus,

7 2011).So, presenting safe and economic antimycotic would be an alternative approach.

Certain plant extracts have been examined experimentally to study the immuno-stimulating activities;

they revealed great stimulating efficacy of immune system through activating macrophages, T and B lymphocyte, natural killer (NK) cells, as well as interleukins, tumor necrosis factor and interferon(Puriet al.,

8

1993 and Suresh and Vasudevan 9 1994).The aim of the present work is to evaluate invivo fungicidal and

immunity stimulating capacity of Cinnamomum zeylanicum extract against Aspergillus nigerreference strain (ATCC16404)that used to induce an experimental infection of lab. animals.

Materials and methods:

Microorganism:

Aspergillus niger ATCC 16404 referencestrain was used in this study.Strain was spread onto Sabouraud's dextrose agar with Chloramphenicol (0.5 g L

-1) to inhibit bacterial growth and incubated at 30

0C

for 1 to 3 days.

Black colonies with fluffy down can be transferred to Czapek's solution agar (Difco Lab, Detroit, MI)

for a definitive confirmation. A piece of colony was teased apart with a needle, stained, marked and mounted

with a cover slip. Species confirmationwas achieved on the basis of morphological criteria upon microscopic examination (Richard and Beneke,

10 1989)

Cell-counting:

Spore concentration of different spore suspensions was adjusted using haemocytometer, according to

the classical procedure.

Spores were harvested by flooding the agar surface with PBS (Sigma) and then filtered and suspended

in PBS with 0.01 % Tween 80 (Difco), in serial concentrations. Spore suspensions were stored at 4 0C for up to

5 days till use.

Hundred micro liters of fungal stock solutions from Aspergillus nigerATCC 16404 spores were

inoculated into 10 ml of (YPD) broth and cultured for 48 h. at 30oC with agitation. Fungal pellets were re-

suspended in sterile saline solution and were adjusted to 105 - 10

7 spore/ml using haemocytometer to prepare

working solution from microorganism spores.

Experimental infection:

C. zeylanicum extraction from Cinnamon park:

C. zeylanicum, the active principle of cinnamon, was extracted from cinnamon park using steam distillation

according to Nandam and Vangalapati11

(2012) the extracted material in liquid form was then diluted 10:100

in ddH2O to be injected intravenously in experimental mice.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Pfaller%20MA%5BAuthor%5D&cauthor=true&cauthor_uid=17223626



Lab. Animals:

(experimental infection complied with relevant professional and institutional animal welfare policies)

Eight to ten week-old male mice were purchased from laboratory animal house of Ramad hospital,

Giza, Egypt. The mice were provided with clean water and solid feed. All animal studies were performed in accordance with the guidelines and permission of the animal experiment care, feeding, changing bedding and

general good health was observed regularly.

Fifty mice were divided randomly into 5 groups in 5 cages each contains 10 mice and treated as follow:

A1: mice were injected I/Vwith 100µl of 105 - 10

7 spore/ml of A. niger ATCC 16404 reference strain as control

positive.

A2: mice were injected with 100µl of 105 - 10

7 spore/ml of A. nige rATCC 16404 followed by 100µl of C.

zeylanicum extract that was prepared previously three days post infection.


7 spore/ml of A. niger ATCC 16404 followed by 100µl of plant

extract seven days post infection.


7 spore/ml of A. niger ATCC 16404 followed by 100µl of plant

extract ten days post infection.

A5: mice were injected with 100µl of normal saline as negative control group.

Mortalities were determined and recorded daily.

Sampling:

After 14 days, serum and heparinized blood samples were collected via heart puncture from all mice for

immunological assays. All mice then were euthanized; lungs, livers and brains of sacrificed mice were removed

and fixed in 10% formalin, the organs then were cut and embedded in paraffin then these blocks were cut into 4 µm thick sections and were stained with Hematoxylin and Eosin (H&E) stain for histopathological examination

microscopically(Bancroft et al., 12

1996)

Blood samples were used to measure certain immunological parameters as phagocytic activity (phage);

killing activity of macrophages; lymphocyte transformation (Lyt1); interleukin 2 (IL2); interleukin 6 (IL6) and

tumor necrosis factor (TNF).

Lymphocyte transformation:

Lymphocyte transformation assay was carried out as described by Denizot and Lang 13

(1986) with some modifications(Maslak and Reynolds,

14 1995)

Determination of phagocytic killing and chemotaxis activities of PMN:

Polymorph nuclear cells were isolated from blood by the method described by Rouse et al15

(1998).The

mixture of PMN and bacteria (S. aureus) was incubated at 370C for 2 hours with regular stirring and then the

mixture was centrifuged at 200 g for 5 minutes at 40C. The supernatant was used to estimate the percentage of

bacteria phagocytosed (Woldehiwet and Rowan, 16

1990). The mixture of bacteria and PMN was treated with

one cycle of freezing and thawing and the percentage of bacteria killed was estimated according to the formula

described by Woldehiwet and Rowan16

(1990).The chemotactic index of PMN was calculated using the chemotaxis under agarose technique based upon migration patterns to chemotactic factor (E. coli

filtrate)Nelson, 17

1974)

Measurement of IL-2; IL-6 and TNF by enzyme linked immunosorbent assay:

Serum IL-2, IL-6 and TNF-α level was measured by using a polyclonal ELISA kits (Uscn, Life Science Inc., USA) following the manufacturer’s instructions. Briefly, the anti-IL-6 capture polyclonal antibody was

absorbed on a polystyrene 96-well plate and the IL-6 present in the sample was bound to the antibody coated


wells. The biotinylated anti-IL-6 detecting pAb was added to bind the IL-6 captured by the first antibody.

Avidin-peroxidase (Sigma, USA) was added to the wells to detect the biotinylated detecting antibody and

finally 2,2´- azinobis (ABTS; Sigma, USA) substrate was added and a colored product was formed and measured at optical density 405 nm (OD 405) with an ELISA MICROPLATE READER (MODEL 450, Bio-

Rad, Chicago, Illinois, USA). A standard curve was generated and calculated. The measurement of TNF- α and

IL-2 are similar to that of IL-6. All determinations were performed by full-time technical personnel. Statistical

analysis was applied using One Way ANOVA SPSS, POSTHOC C ALPHA (0.05).

Results:

The gross findings of liver, lung and brain those were extracted from mice were depicted in Table (1).

Table (1): Showed the gross findings of extracted organs from experimental animals:

Group Liver Lung Brain

A1 (+ve) Pale/severe pale Fleshy/semi normal Fragile-granulated-

calcified/highly fragile to semi

normal.

A2 (3rd

) Slight pale Normal Normal

A3 (7th

) Slight normal Lobulated lungs Slight normal

A4 (10th

) Slight pale Normal Normal

A5 (-ve) Normal Normal Normal

Remark: Total mortalities were 30%

Blood samples were normal in all cases.

Histopathological profile:

The histopathological findings of lung tissues from the experimentally infected mice were shown in

figures 1, 2, 3, 4, 5 and 6. The profile of liver histopathology is revealed in figures number 7, 8, 9, and 10, while the histopathological findings of mice brain were clear in figures 11, 12, 13 and 14. Comparing the

histopathological profiles among the mice groups were applied in order to correlate the fungicidal efficacy of C.

zeylanicum extract and the intervention time of its use.

Histopathological examination of lung tissues:

Fig. (1): Lung of mice from group A1 showing

marked dilatation and congestion of pulmonary

blood vessels (H & E X 100)


focal interstitial inflammatory cells infiltration (H

& E X 400).



dilatation and congestion of pulmonary blood

vessels (H & E X 400).


focal pulmonary emphysema (H & E X 400).


congestion of perialveolar blood capillaries and

focal mononuclear inflammatory cells aggregation

(H & E X 400).


congestion of pulmonary blood vessels and

interstitial pneumonia (H & E X 400).

Histopathological findings of liver tissues:

Fig. (7): Liver of mice from group A1

showing congestion of central vein and

perivascular inflammatory cells infiltration

(H & E X 400).


showing hydropic degeneration of

hepatocytes

(H & E X 400).


showing focal hepatic necrosis associated

with inflammatory cells infiltration (H & E


showing focal hepatic necrosis associated

with inflammatory cells infiltration as well


X 400).

as Kupffer cells activation (H & E X 400).

Histopathological profile of brain tissues:

Fig. (11): Brain of mice from group A1 showing

necrosis of neurons and neuronophagia

(H & E X 400).


focal gliosis (H & E X 400).


focal necrosis infiltrated with glia cells (H & E X

400).


focal necrosis of neurons infiltrated with glia

cells (H & E X 400).

Immunological profile:

Phagocytic activity percentages (Phage); lymphocytes transformation capacity (Lyt1); values of

interleukin 2 (IL2); interleukin 6 (IL6) and tumor necrosis factor (TNF) were measured to assess the

immunological stimulation efficacy of C. zeylanicum extract and to evaluate the significance of intervention time for treating the mycosis using it, results were depicted in tables 2 and 3.Statistical analysis was applied

using one way ANOVA/SPSS.

Table (2): Immunological parameters values among all tested groups:

A1 (n= 3) A2 (n= 3) A3 (n= 3) A4 (n= 3) A5 (n= 3)

Phage

Mean 76.4667 76.5067 80.2667 80.8433 89.3333

SD 3.86307 3.47327 4.42982 4.76593 4.30968

Kill

Mean 70.9333 74.1000 75.5000 76.2667 84.9333

SD 3.95517 2.75136 5.15655 4.37531 5.21568

Lyt 1

Mean 1.1833 1.2100 1.4033 1.4600 1.6333

SD .12662 .08185 .17559 .10149 .61460

IL6

Mean 496.0833 465.4633 374.6167 405.5433 238.8800

SD 64.98828 .48014 13.83812 14.78130 11.62476

IL2

Mean 193.3133 186.5567 187.4100 179.9467 167.4667

SD 9.46088 7.75016 11.24827 9.66580 5.09912

TNF

Mean 182.1400 168.7167 164.4667 169.6900 138.7267

SD 11.26012 10.40734 7.79219 3.09264 10.29692


Table (3): Correlation between immunological parameters and intervention time:

Dependent

Variable

(I) group (J) group Mean Difference (I-J) Std. Error Sig.

Phagocytic

Activity

A1

(untreated)

A2 -.00190- .14282 1.000

A3 -.18095- .16159 .908

A4 -.20841- .16867 .864

A5 (Control) -.61270- .15912 .104

A2

A3 -.17905- .15476 .894

A4 -.20651- .16213 .847

A5 (Control) -.61079- .15217 .096

A3

A4 -.02746- .17889 1.000

A5 (Control) -.43175- .16992 .316

A4 A5 (Control) -.40429- .17666 .396

Killing Activity A1

(untreated)

A2 -.13533- .11888 .899

A3 -.19516- .16034 .871

A4 -.22792- .14552 .711

A5 (Control) -.59829- .16150 .126

A2

A3 -.05983- .14421 1.000

A4 -.09259- .12752 .989

A5 (Control) -.46296- .14549 .223

A3

A4 -.03276- .16686 1.000

A5 (Control) -.40313- .18096 .417

A4 A5 (Control) -.37037- .16797 .428

Lymphocyte Transformation

(Lyt1)

A1

(untreated)

A2 -.02186- .07135 1.000

A3 -.18033- .10245 .620

A4 -.22678- .07679 .226

A5 (Control) -.36885- .29696 .848

A2

A3 -.15847- .09168 .645

A4 -.20492- .06170 .166

A5 (Control) -.34699- .29342 .869

A3

A4 -.04645- .09598 .999

A5 (Control) -.18852- .30249 .994

A4 A5 (Control) -.14208- .29479 .999

Interleukin 6

(IL6)

A1 (untreated)

A2 .09892 .12121 .969

A3 .39240 .12393 .285

A4 .29249 .12431 .451

A5 (Control) .83089 .12314 .070

A2

A3 .29348* .02583 .029

A4 .19357 .02758 .073

A5 (Control) .73198* .02170 .003

A3 A4 -.09991- .03777 .288

A5 (Control) .43850* .03371 .001

A4 A5 (Control) .53841* .03507 .001

Interleukin 2 (IL2)

A1

(untreated)

A2 .16917 .17679 .955

A3 .14781 .21247 .993

A4 .33467 .19552 .640

A5 (Control) .64714 .15536 .116

A2

A3 -.02137- .19746 1.000

A4 .16550 .17909 .962

A5 (Control) .47797 .13411 .151

A3

A4 .18686 .21439 .973

A5 (Control) .49933 .17853 .308

A4 A5 (Control) .31247 .15797 .538

Tumor Necrosis Factor (TNF)

A1

(untreated)

A2 .20977 .13834 .736

A3 .27619 .12355 .427

A4 .19456 .10536 .609

A5 (Control) .67844* .13767 .047

A2

A3 .06642 .11730 .998

A4 -.01521- .09796 1.000

A5 (Control) .46867 .13209 .132

A3

A4 -.08163- .07564 .910

A5 (Control) .40225 .11651 .153

A4 A5 (Control) .48388 .09700 .110


Discussion:

Cinnamon is famous in traditional Chinese medicine as a carminative, it is used for most

gastrointestinal complaints (Nadkarni, 18

1954). In addition, it is often present in traditional Chinese formulas

to facilitate the action of the other herbs present, cinnamon is frequently used as a cardio-tonic, which helps to

stimulate blood flow Bensky and Gamble19

(1993).Antioxidants properties of cinnamon extract were reported in many previous studies confirming its expressed ferric reducing antioxidant power (FRAP)(Tenoreet al.,

20

2011).

The current study describes the effectiveness of C. zeylanicum whole plant extract against Aspergillus

nigerATCC 16404 reference strain invivo in experimental laboratory animals.

Preliminary experiments were carried out in vitro using C. zeylanicum essential oil on solid medium

through solid medium drop diffusion technique in order to evaluate the effect of some essential oils, including

it, on the fungal growth(Alarousy,21

2004). Results showed that C. zeylanicum had the strongest fungicidal

effect on fungal cells.

Aspergillus niger, that is known as environmental fungal species, was used as a model in this

preliminary study and other species will be used in next studies aspathogenic models.

The antimycoticactivity of phytochemicals found in C. zeylanicum extractmay involve inhibition

ofextracellular enzymes synthesis of the cellwall structure of fungal cells, cellular damage that leads to cellular death (Brul and Coote,

22 1999; Burt,

23 2004 and Atandaet al.,

24 2006).Atanda, et al.

24(2006)and Rasooli,

et al.25

(2006) also reported that C. zeylanicum extract is able to interfere with the enzymatic reactions, suchas

respiratory electron transport, protein transport and coupledphosphorylation that take place in the mitochondrial membrane.

It was necessary to explore whether the fungicidal effect is restricted on the essential oils only or not, as

it would be difficult somehow to assess the fungicidaleffect of C. zeylanicum essential oil in the laboratory animals (in vivo) due to the difficulties of injecting oils in living bodies. In our study, we tried to test this

concept and to approve that the fungicidal effect is due to the entire compounds of the plant as well as its

essential oil. So, we used the whole plant extract to inject the pre-infected mice with it in order to track the efficacy of the plant extract inside the animal body for its fungicidal effect as well as its capacity for immune

system stimulation. Beside, we designed the experiment to use the extract after the infection of mice with

Aspergillus nigerATCC 16404 reference strain with different time intervals (three, seven and ten days post infection).

Histopathological profile:

Histopathological examination of lung tissues:

Histopathological lesions are the phenotypic profile representing the direct correlation between defense mechanisms of the host and virulence of infectious agent (Tochigiet al.,

26 2013)

Results depicted in table (1) revealed that the lungs from group A1 mice who were injected with the fungal spores only, were the most damaged lungs in comparison to the other four groups used in this study,

followed by lungs from group A3 and A4 who received the C. zeylanicum extract seven days and ten days' post

infection respectively. Histopathological results confirmed the harmful effect of the mold on lung tissues, examined lung tissues of mice from group A1 revealed marked dilatation and congestion of pulmonary blood

vessels (Fig. 1), focal interstitial inflammatory cells infiltration (Fig. 2) and focal pulmonary emphysema.

However, examined sections from group A2, who received the C. zeylanicum extract three days post

infectionshowed improvement in histopathological picture as the lung showed dilatation and slight congestion of pulmonary blood vessels (Fig. 3) and focal pulmonary emphysema (Fig. 4). Meanwhile, lung of mice from

group A3 revealed congestion of perialveolar blood capillaries and focal mononuclear inflammatory cells

aggregation (Fig. 5). Examined sections of lung of mice from group A4 showed severecongestion of all pulmonary blood vessels, focal emphysema and interstitial pneumonia (Fig. 6). These findings agreed to those

explained by Tochigi et al26

(2013), they identified two distinct patterns of histopathological findings in lung

tissues, one of those pattern involves a distinct nodule consisting of demarcated round-shaped coagulation


necrosis, while the other pattern involves fused lobular integration, which corresponds to the usual

bronchopneumonia which is characterized histologically by the filling of acute inflammatory exudates with a

fungal proliferation in alveoli.

Histopathological findings of liver tissues:

findings of group A1 revealed congestion of central vein, perivascular inflammatory cells infiltration

(Fig. 7), Kupffer cells activation and focal hepatic necrosis associated with inflammatory cells infiltration.

Meanwhile, liver of mice from group A2 revealed no histopathological changes except hydropic degeneration

of hepatocytes (Fig. 8). In spite of the slightly normal gross appearance of livers extracted from groups A3& A4, livers showed more or less similar histopathological changes confined as Kupffer cells activation and focal

hepatic necrosis associated with inflammatory cells infiltration (Figs. 9 & 10).

No liver abscesses were detected in the current study, in contrary to the expected results. According to

Kumar et al., 27

(2010), the organisms reach the liver either through ascending infection in biliary tract, or

vascular seeding, either portal or arterial, or direct invasion of the liver from a nearby source or through penetrating injury.

Histopathological profile of brain tissues:

Histological findings of brain tissues were completely different, the brain tissues from the groups: A1,

A3 and A4 showed variable degrees of necrosis as shown in figures (11, 12 and 14). However, 20% only of

group A2 showed gliosis as shown in figure 13. These results elucidated the limited fungicidal effect of the examined herbal extract in brain. This might be due to rapid growth of lesions caused by Aspergillus species if

reached the brain tissue.

The rapiddevelopment of lesions could be explained by three possible mechanisms. First, the origins of

the multiple perforator arteries are in proximity along the parent artery and are presumably vulnerable to an

extending vasculo-pathy of the parent artery wall. There is an evidence suggesting that the necessary conditions for the vasculo-pathy are present. First, endothelial cells in culture have been shown to engulf the

organism(Paris et al., 28

1997)and Aspergillus has been seen to infiltrate and destroy the internal elastic lamina

of major cerebral arteries (Chouet al., 29

1993). Second, any infarction, thromboembolic or septic, may have

associated secondary edema, which increases during the first few days after the arterial occlusion. Third, Aspergillus hyphae were frequently observed within the parenchymal lesions at autopsy; infection of the

infarcted tissue may be aggressive, and direct extension into the surrounding brain may progress quickly

(DeLoneet al., 30

1999).

Aspergillosis as an invasive fungal infection has an inflammatory properties as revealed through the

histopathological profile of the current studyand C. zeylanicum extract showed good fungicidal capacity less or more the expected result, hence cinnamon park extract was reported previously to have strong antimicrobial

activity (Shan et al., 31

2007).Comparing the histopathological profiles between groups A1 and A5 revealed the

fungicidal activity of C. zeylanicumin vivo and comparing the histopathological profiles of the three treated

groups (A2, A3 and A4) using C. zeylanicum extract also showed the significance of early intervention with this herbal extract, as all the results showed the marked improvement of histopathological profile in group A2 which

received the treatment three days post infection. Absence of toxicity signs on experimental animals indicated

the safety of using C. zeylanicum extract in vivo.

Immunological profile:

To assess the primary capacity of C. zeylanicum extract for stimulating the immune system, values of

phagocytic activity (phage); killing activity of macrophage; lymphocyte transformation activity (Lyt1); IL2; IL6

and TNF were measured and compared between the untreated group (A1) and all treated groups (A2; A3; A4

and A5), regardless the intervention time, from one side and withinthe three treated groups (A2; A3 and A4) from the other side.

Results of multiple comparison between all the treated groups (A2; A3 and A4), regardless intervention time, and A1 group were significant except for TNF (p value = 0.047) between A1 and A5 (the accepted mean

difference is significant at the 0.05 value). Significance was the least between untreated group (A1) and control


negative group (A5),as for phage (p value = 0.10); for killing capacity (p value = 0.126); for IL6 (p value =

0.07) and for IL2 (p value = 0.116), while for TNF, it didn’t show accepted significance (p value = 0.047) as

mentioned before. These results were matching with the reported ones byCarballo et al. 32

(1998).

In the other hand, another factor was correlated to the efficacy of medicinal application of the extract

which is the initiation time of using C. zeylanicum extract in vivo. The relation between the immune stimulating capacity of C. zeylanicum and the intervention time of its use was clarified in table (3), as for phage (F= 4.703);

killing activity (F= 4.244); Lyt1 (F= 1.178); IL2 (F= 3.689) and TNF (F= 9.296) all values showed significant

differences within the three treated groups (A2, A3 and A4). Meanwhile IL6 values were significant only

between (A4 & A2) and between (A4 & A3) and there wasn’t significance between the values in groups A2 and A3 (P value = 0.029). Hence, these results indicated the capacity of immune stimulation of the extract in

correlation to the intervention beginning time and this is a novel character of C. zeylanicum extract application

medicinally for treating latent and/ or chronic fungal infection as well as its usage as prophylaxis by immunocompromised patients. Further studies will be conducted to investigate deeper the time factor effect on

the efficacy of the therapeutic application of the extract.

The conclusion from the current study revealed that Cinnamomum zeylanicum extract has strong

fungicidal activity and great immune system stimulating efficacy with correlation to intervention time as well.

Results proved that the tested natural herbal extract is more advantageous than the synthetic agents due to its

biodegradability, potency, absence of toxicity, efficacy as a prophylactic agent for immunocompromised patients and future studies will be done to analyze the herbal components of Cinnamomum zeylanicum and

compare them with the currently available antimycotic pharmaceutical preparations.A. nigerATCC 16404

reference strainwas chosen in this study as preliminarytested strain and further investigation will be carried out on other fungal species.

Conflict of interest:

The authors declare that there is no conflict of interest.

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European Journal of Academic Essays 4(8): 202-223, 2017 ISSN (online): 2183-1904 ISSN (print): 2183-3818 www.euroessays.org

Cryptococcosis in Animals and Birds: A Review

Mohamed K. Refai 1, Mahmoud El-Hariri

1and Randa Alarousy

2,3

1 Department of Microbiology, Faculty of Veterinary Medicine Cairo University 2 Department of Microbiology and Immunology, National Research Center, Dokki, Giza, Egypt.

3 Department of Medical Laboratories, College of Applied Medical Sciences, Majmaah University, KSA

Abstract: Cryptococcus infections in bovines was mostly reported in association with mastitis, mainly in cattle. Cryptococcus

neoformans was recognized as the cause of severe outbreaks of mastitis in cattle and also in sporadic cases in buffaloes. Bovine

systemic cryptococcosis was rarely diagnosed. In camels, cryptococcosis is very rare. It has been reported in South American

camelids (llamas, alpacas and vicunas). In equines, cryptococcosis is uncommon. The nasal cavity is the most common site of

infection. Sporadic cases have been associated with granulomatous pneumonia, nasal granuloma, endometritis and placentitis with

neonatal cryptococcal pneumonia, abortion and mesenteric lymph node abscesses. Cryptococcal meningitis in equines is almost

associated with pneumonia and disseminated infection. Cryptococcus species were reported in sheep and goats as causes of

mastitis. Experimental mastitis in goats was induced by unilateral intramammary inoculation of Cryptococcus neoformans. Nasal

cryptococcosis is frequently seen as clinical signs in cats and dogs. With time, infections involving the nasal cavity can spread to

adjacent structures and disseminate to the brain and other organ and even the skin. Cryptococcal pneumonia and meningitis due to

Cryptococcus gatti was reported also in goats. In wild animals, cryptococcosis occurs in different animals and Cryptococcus

species. can affect the gastrointestinal and respiratory systems, nasal cavity and eyes.

Keywords: Cryptococcosis, Cryptococcus neoformans, Cryptococcus gatti, bovines, equines, camels, sheep and

goats, cats and dogs, wild animals, birds.

Corresponding author: Randa [email protected] -

[email protected]

1. Introduction

Cryptococcosis in animals is a systemic fungal infection of

worldwide significance that usually initially infects the nasal

cavity, paranasal tissues, or lungs. It can then disseminate,

most commonly to the skin, eyes, or central nervous system.

Cryptococcosis is a fungal disease caused by C. neoformans

and C. gattii, which are ubiquitous, saprophytic, round,

basidiomycetous yeasts (5 to 10 μm) with a large

heteropolysaccharide capsule (1 to 30 μm) that does not take

up common cytologic stains.

According to the current classification, the species complex

comprises two species, namely C. neoformans and C.

gattii [1& 2] with serotypes A, D and AD for the former,

and B and C for the latter species. Cryptococcus

neoformans currently consists of two varieties: C.

neoformans variety grubii (serotype A) [3] and C.

neoformans variety neoformans (serotype D) [4]

Recently, Hagen et al. [5] proposed the

genus Cryptococcus to include 3 separate species, C.

mailto:[email protected]


European Journal of Academic Essays 4(8): 202-223, 2017

204

neoformans (Cryptococcus neoformans var. grubii)

represented by genotypes VNI and VNII, C. deneoformans

(Cryptococcus neoformans var. neoformans) represented by

genotypes VNIII and VNIV, and Cryptococcus

gattii (represented by genotypes VGI, VGII, VGIII, and

VGIV).

The environmental reservoir of C. neoformans is usually

related to bird faeces, particularly pigeon droppings.

However, this yeast has also been found in decaying trees,

wood and plant debris, waterways and soil, all usually

contaminated with bird excrement [6, 7, 8, 9, 10, 11, 12].

The epidemiology of clinical disease depends largely on the

species of infecting organism. Cryptococcus neoformans

var. grubii (serotype A) and C. neoformans var. neoformans

(serotype D) are globally distributed and infect

predominantly immunocompromised hosts. Cryptococcus

gattii (serotypes B and C) has recently been recognized as a

species distinct from C. neoformans based on molecular and

mating type characteristics.

2. Cryptococcosis in domestic animals

2.1. Cryptococcosis in bovines:

Cryptococcus infections in bovines was mostly reported in

association with mastitis, mainly in bovines. Klein [13] was

the first to isolate a yeast from a case of mastitis, which he

reported to be identical with strains of Cryptococcus

neoformans of human and plant origin. Almost 50 years

later, Cryptococcus neoformans was recognized as the cause

of severe outbreaks of mastitis in cattle [14, 15, 16, 17].

Pounden et al. [18] reported the clinical aspects of an

outbreak in which 106 cows were affected in a 235 cow-

herd. They stated that during the outbreak, Cryptococcus has

been isolated from samples where no visible changes were

noted in either the gland or milk, and the cases with visible

signs varied from mild and transient swelling of one or more

quarters of the udder to severe swelling and distention of the

affected glands

Cryptococcal mastitis was detected also in sporadic cases by

Abdel Ghani et al. [19], Rahman et al., [20], Moawad,

[21], Hassan et al. [22], mostly, following treatment with

antibiotics [23, 24, 25, 26]. On the other hand, Rippon [27]

emphasized that cryptococcal mastitis in dairy cows is

worldwide in distribution.

Cryptococcus neoformans was the most commonly recorded

species in cows [28, 29, 8]. Cr .neoformans was also

recorded as a cause of mastitis in buffaloes [30, 31]. Other

species like C. albidus, C. laurentii, C. flavus, C.

lactativorus, C. luteolos, C. terreus, C. uniguttulatus and

Cryptococcus species were also reported in few reports [28,

32, 33, 34, 35, 36, 37, 38, Table 1].

Cryptococcal pneumonia was infrequently reported in

bovines [39, 22]. On the other hand, bovine systemic

cryptococcosis was rarely diagnosed. Only two reports could

be found in the available literature [40, 41]. Similar findings

were obtained in case of abortion caused by Cryptococcus

species [40, 41].


205

Table 1: Cryptococcosis in bovines

Cryptococcus References

Cryptococcal mastitis

C. neoformans

8,9,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27.28,

29,30,31,34,116,117,118,119,120,121,122,123,124,125

C. albidus 28, 32

C. laurentii 32,33, 34 , 35,37

C. flavus 32

C. luteolos 32

C.uniguttulatus 36

C. lactativorus 12 129,130,1316

C. terreus 38

Cryptococcus species 30, 32, 127.128

Cryptococcal pneumonia

C. neoformans 22,39,

C. laurentii 39

Cryptococcal abortion

C. neoformans 22,39

C. laurentii 39

Systemic cryptococcosis

C. neoformans 40

C. species 41

2.2. Cryptococcosis in equines

In equines, cryptococcosis is uncommon and sporadic cases

have been reported, however it is relatively common in

Western Australia, and there is an epidemiologic

relationship between C. gattii and the Australian river red

gum tree (Eucalyptus camaldulensis) and between C.

neoformans var neoformans and bird (particularly pigeon)

excreta. From the literature it appears that the nasal cavity is

the most common site of equine cryptococcosis. Sporadic

cases have been associated with granulomatous pneumonia,

nasal granuloma, endometritis and placentitis with neonatal

cryptococcal pneumonia, abortion, mesenteric lymph node

abscesses , intestinal polypoid granulomas, osteomyelitis

and meningitis [Table 2] .

Cryptococcal nasal infections in equines involved also the

frontal and maxillary sinuses and the corresponding lymph

nodes and in most cases a polyp or granuloma that almost

completely occupied the nasal cavity was reported. Zoppa et

al. [42] reported a case of nasal obstruction caused by fungal

granuloma in a 9-year-old horse with serosanguineous nasal

discharge, absence of breath out through the right nostril,

and respiratory noise. Endoscopic and radiographic

examinations revealed a six centimeter diameter mass,

covered by yellowish mucosa, which was obstructing the

entire right nasal cavity and part of the left one. The mass

was excised through a right frontal sinusotomy. The

microscopic examination and the culture revealed a

cryptococcal granuloma.

Equine cryptococcal pneumonia was reported mostly in

single cases characterized by diffuse, severe interstitial


206

pneumonia [43], often as granulomatous pneumonia [44,

45].

Ryan and Wyand [43] reported neonatal cryptococcal

pneumonia in a foal, followed by placentitis and abortion in

the mare at her next pregnancy. Histologic examination of

lung sections showed diffuse, severe interstitial pneumonia,

while Blanchard and Filkins [46] reported Cryptococcal

pneumonia in a 9-month-old equine foetus aborted by a

healthy American Paint mare. Endometritis was diagnosed

on biopsy, and vaginal specimens obtained for culture were

Cryptococcus-positive.

The clinical, radiographic and post-mortem findings in 6

horses with cryptococcal pneumonia were described by

Riley et al. [47] and Begg et al. [48]. The infected animals

were reported to suffer from chronic cough, fever, weight

loss that developed later into dyspnoea, tachypnoea and

exercise intolerance. The diagnosis in most cases was

established using lateral thoracic radiography and

transthoracic ultrasonography and confirmed by the

detection of encapsulated, budding yeasts in smears made

from transtracheal washings and needle aspirates of the

pulmonary lesions, demonstrated in Wright's-stained

preparations.

Cryptococcal meningitis in equines is almost associated with

pneumonia and disseminated infection [49, 50, 51, 52, 53,

54, 55, 45].

Cho et al. [55] presented extensive gross and microscopic

cavitary lesions of cerebral cryptococcosis in a 5-year-old,

quarter horse mare. The mare was dehydrated, became

markedly depressed and ataxic, and violently resisted any

attempt of restraint. Hart et al. [54] reported a 4-year-old

Tennessee Walking Horse with episodic fever and acute

bilateral blindness of approximately 7 days’ duration and

Del Fava et al. [45] described a case of cryptococcal

granulomatous pneumonia and meningitis in a mature

Thoroughbred horse stabled at an equine racing center

located in an urban area of Brazil.

The diagnosis in cryptococcal meningitis was achieved by

cytologic evaluation of the CSF for detection of

encapsulated Cryptococcus neoformans or serology by

serum latex agglutination test and by histological sections of

the brain that showed granulomatous encephalitis with the

classic "soap-bubble" appearance associated with the

characteristic encapsulated yeast cells [54, 45].

Disseminated cryptococcosis was diagnosed by Lenard et

al. [56] in a 4-year-old Arab mare, including osteomyelitis

of the proximal phalanx of the left hind limb, osteomyelitis

with associated soft tissue granuloma of a rib and

disseminated, large cryptococcal nodules in the lungs. The

mare presented had a large swelling over the right

caudodorsal thorax and a marked swelling of the left hind

fetlock. The mass was hypoechoic and round, with the 17th

rib visible as an echogenic structure that casts an acoustic

shadow in the centre of the mass.The lesion in the

dorsoproximal aspect of the proximal phalanx had a large

area of cortical lysis with speculated periosteal new bone

and extensive soft tissue swelling. The affected rib had a

pathological fracture. Multiple, well circumscribed, soft


207

tissue opacity nodules (1 to 8 cm diameter) are present in the

lung.

Table 2 Cryptococcosis in equines

Cryptococcus References

Cryptococcal nasal infections

C. neoformans 15,42,83,132,133,134


C. neoformans 43, 44,45,46,47,48,135,136

Cryptococcal meningitis

C. neoformans 45,49,50,51,52,53,54,55

Cutaneous cryptococcosis

C. species 127,138

Cryptococcal endometritis and placentitis

C. neoformans 46,139

Abdominal cryptococcosis

C. neoformans 47

Disseminated cryptococcosis

C. species 56

2.3. Cryptococcosis in camels

The only case of cryptococcosis in camels found in the

available literature was reported by Ramadan et al. [57] in a

camel (Camelus dromedarius) in Saudi Arabia. The tissue

reaction in cryptococcosis varied depending on the organ

affected. Two basic histological patterns were described:

gelatinous and granulomatous. The granulomatous lesion

consisted of histiocytes, giant cells and lymphocytes. The

pathology in this camel was modified by the secondary

bacterial infection. All other cases were reported in vicuna

[58], alpaca [59, 60] and llama [61]. Most of these cases

suffered from meningitis caused by Cryptococcus gattii.

The case described by Bildfell et al. [61] was in a pastured

17-year-old male llama, which was found in lateral

recumbency, was undergoing continuous tonic clonic

convulsions of all limbs with periodic spasms of the head

and neck. The animal was hypothermic and unresponsive to

stimuli. A complete neurologic examination could not be

performed due to the clinical status of the animal. The llama

was in good body condition with no history of serious health

problems and no known exposure to toxins. The owners had

noted this llama to be mildly anorexic during the preceding 3

days but had not attempted any therapy. A health program

for the 16 animals on the premises included periodic

deworming, vaccination, and health checks. Differential

diagnoses at the time of presentation included various

neurologic diseases such as trauma, organophosphate

toxicity, hypomagnesemia, visceral larval migrans, equine

herpes virus type 1 (EHV1) encephalomyelitis, rabies, and

bacterial infections of the CNS such as listeriosis or brain

abscessation. After considering the prognosis, the owner

requested that the llama be euthanatized without further

clinical work-up. Tissues affected included the brain, spinal

cord, lung, and kidney. The character of the leukocytic

response varied from minimal to pyogranulomatous


208

meningitis with intralesional yeast that were bordered by a non-staining halo.

2.4. Cryptococcosis in sheep and goats

Cryptococcus species were reported in sheep and goats as

causes of mastitis, pneumonia, meningitis and abortion

[Table 3].

Experimental mastitis in goats was reported by unilateral

intramammary inoculation of 10 goats with 2 x 106 cells of

Cryptococcus neoformans [62]. The infection resulted in the

development of mastitis, with gross and microscopic lesions

being restricted to the infected udder halves only and there

was no dissemination of infection to the opposite uninfected

udder halves as well as to other organs of the body. The

experiment was continued for 40 days, with 2 animals, each

from the infected and control groups being killed on 5th,

10th, 20th, 30th and 40th day post-inoculation (DPI). Initial

enlargement of the infected udder halves was followed by

marked decrease in size leading to very small, firm and

nodular udder halves. After infection, there was also sharp

fall in the milk yield. Cryptococcal organisms were

demonstrated in the mastitic milk and udder impression

smears with special stains. C. neoformans was re isolated

from the milk of the only infected udder halves up to 25th

DPI. Microscopically, there was initially acute diffuse

purulent mastitis which later on became chronic,

characterized by marked infiltration of lymphocytes,

macrophages, extensive fibrosis and development of

multiple granulomas. The cryptococcal organisms could be

demonstrated in the udder sections only up to 30th DPI.

Mycotic pneumonia in goats due to Cryptococcus gatti was

reported in Spain by Baro et al. [63]. The strains were

isolated from lung (10 samples), liver (1 sample), and brain

(2 samples) tissue specimens from six goats suffering from

predominantly severe pulmonary disease that were

autopsied. Biotyping was performed by culturing the isolates

on L-canavanine-glycine-bromothymol blue medium and

testing them for the assimilation of D-proline and D-

tryptophan. Serotyping by agglutination tests confirmed the

characterization of all strains as C. gattii.

C. gattii was reported to cause 5 epidemic outbreaks of

cryptococcosis in goats grazing freely in west Spain

grasslands [64] . The goats belonged to various milking breeds and

were grazing with variable status of health and husbandry. Goats affected

by cryptococcosis showed similar respiratory symptoms,

consisting of mucopurulent nasal discharge, cough, dyspnea

and progressive cachexia, causing death in a period of 2 to 4

weeks. In three outbreaks many animals also showed ataxia,

midriasis, blindness and progressive paralysis. Clinical

prevalence varied from 2 to 12% in the different outbreaks.

On the other hand, Gutiérrez and García Marin [65]

presented an adult Blanca-Celtibérica doe originating from a

goat herd with a high prevalence of tuberculosis with

respiratory signs. At necropsy, this goat had a diffuse and

severe mycotic pneumonia associated with the presence of

Cryptococcus neoformans concomitant with pulmonary

focal caseous nodules from which Mycobacterium bovis was


209

isolated. Microscopically, the mycotic lesion was a

granulomatous pneumonia with many large foamy

macrophages containing intracellular yeast bodies. The

extensive mycotic changes, their granulomatous nature, and

the lack of positive response to different immunologic tests

for mycobacterial infection suggested an impaired immune

status in this animal.

Cryptococcal meningitis in goats was described by

Luvizotto et al. [66], who euthanized four-year-old male

goat with a history of neurological disorder. It presented

uncommon nodules in the brain and lungs associated with

multiple abscesses, predominantly in the spleen and liver.

Histological examination of brain and lung sections revealed

yeast forms confirmed to be Cryptococcus gattii. On the

other hand, Stilwell and Pissarra [67] described a case of a

five year old buck showing severe neurological signs,

including paraplegia and strong pain reaction to touch of the

hindquarters region. Postmortem examination revealed

lumbar meningitis, lung nodules and caseous lymphadenitis

lesions. Encapsulated Cryptococcus neoformans were

identified from the lungs and meninges, showing that

cryptococcal meningitis should be included in the

differential diagnosis of goats showing paresis and

hyperesthesia. Both Cryptococcus gattii genotype

AFLP4/VGI and Cryptococcus neoformans var. neoformans

genotype AFLP2/VNIV were incriminated as causes of

meningoencephalitis in goats by Maestrale et al. [68].

Cryptococcal meningitis in goats was described by da Silva

et al. [69], who performed a study aimed to report a 5-year-

old goat showing intermittent dry cough, ruminal tympany,

anorexia, fever, tachycardia and tachypnea in State of São

Paulo, Brazil. Postmortem examination revealed numerous

2.0-6.0 cm diameter yellow gelatinous pulmonary masses.

Tissues were evaluated by a combination of pathological,

mycological, and molecular diagnostic techniques.

Microscopically, pneumonia granulomatous, multifocal to

coalescing, moderate, with many intralesional carminophilic

yeasts was observed. The immunohistochemistry and

mycological culture confirmed Cryptococcus spp. Internal

transcribed spacers and orotidine monophosphate

pyrophosphorylase nucleotide differentiation demonstrated

that the isolate corresponds to the C. gattii VGII molecular

subtype.

Table 3: Cryptococcosis in sheep and goats

Cryptococcus Animal References

Cryptococcal mastitis

C. neoformans goats 62,140


C. neoformans

goats 22,63,65

sheep 22,141

C. gattii goats 69,142,143,144


C. neoformans goats 67,68

sheep 68

C. gattii goats 66,68

sheep 68

Cryptococcal abortion

C. gattii goats 144


210

2.5. Cryptococcosis in cats and dogs

In cats and dogs, cryptococcosis can be either focal or

disseminated, affecting a single organ system or many

[Table 4]. It can begin insidiously, and may gradually

become more severe over weeks or months. Fever may be

absent, and if present, is often mild. Other nonspecific signs

can include lethargy, anorexia and weight loss. The most

common site of localized infections is the nasal cavity. Nasal

cryptococcosis is frequently seen clinical signs including

sneezing, snoring or snorting, dyspnea, nasal deformities

and/ or a mucopurulent, serous or sero-sanguineous nasal

discharge. Polyp-like masses sometimes protrude from one

or both nostrils. Cutaneous or subcutaneous swellings and

nodules may be seen on the face, particularly the bridge of

the nose, side of the face, upper lip or nostril. Some of these

lesions may ulcerate. In addition, the submandibular lymph

nodes are often enlarged. Rhinitis in cats was reported to be

caused by C. neoformans var. neoformans [70, 71, 72, 73].

With time, infections involving the nasal cavity can spread

to adjacent structures and disseminate to other organs [74,

75, 76, 77, 78], including the brain [79, 80] and eyes [79,

81] and even the skin [82]. Cutaneous involvement usually

appears as fluctuant or firm papules and nodules. Some skin

lesions may ulcerate, but there is little or no pruritus. Direct

inoculation of organisms into the skin can occasionally

cause solitary lesions.

Cryptococcus gattii has emerged since 1999 as an important

pathogen of humans and animals in southwestern British

Columbia. Historically thought to be restricted to the tropics

and subtropics, C. gattii has posed new diagnostic and

treatment challenges to veterinary practitioners working

within the recently identified endemic region. Clinical

reports of canine and feline cryptococcosis caused by C.

gattii diagnosed between January 1999 and December 2003

were reported. The most common manifestations of disease

were respiratory and central nervous system signs.

Multivariate survival analysis revealed that the only

significant predictor of mortality was the presence of central

nervous system signs upon presentation or during therapy.

Case fatality rates in both species were high. [70, 73, 83,

84].

Trivedi et al. [85] mentioned that Cryptococcosis,

principally caused by Cryptococcus neoformans and

Cryptococcus gattii, is the most common systemic mycosis

of cats worldwide. Cats may be infected following inhalation

of spores from the environment, with the nasal cavity

suspected as being the initial site of colonization and

subsequent infection. Other sites of infection in cats are the

skin, lungs, lymph nodes, central nervous system (CNS),

eyes and, occasionally, periarticular connective tissue.

Cryptococcosis can be diagnosed using serology (antigen

testing), cytologic examination of smears, histopathology or

culture. Treatment of localized disease is generally

successful using azole antifungal drugs; however, cats with

CNS involvement or disseminated disease require additional

treatment with amphotericin B, with or without flucytosine.

The prognosis is variable, depending on host and pathogen

factors. Some cats require long-term (>1 year) treatment or

indefinite therapy. Cats of any breed, gender and age may be


211

affected. Retroviral status does not appear to be a risk factor

for developing cryptococcosis and indoor cats are not

protected from disease.

Feline cryptococcosis occurs worldwide, but is most

frequently reported in Australia, western Canada and the

western United States. Species and molecular type vary in

different geographical regions and may affect clinical

presentation and antifungal susceptibility patterns. Serologic

tests that detect cryptococcal antigen in serum are sensitive

and specific, but false negatives can occur in cats with

localized disease. Long-term drug therapy can be expensive

and has the potential for toxicity.

Pennisi et al. [86] mentioned that Cryptococcosis is

worldwide the most common systemic fungal disease in

cats; it is caused by the Cryptococcus neoformans–

Cryptococcus gattii species complex, which includes eight

genotypes and some subtypes (strains) with varying

geographical distribution, pathogenicity and antimicrobial

susceptibility. Cats acquire the infection from a

contaminated environment. The prognosis is favourable in

most cases, provided a diagnosis is obtained sufficiently

early and prolonged treatment is maintained. Basidiospores

are the infectious propagules of Cryptococcus species as

they penetrate the respiratory system and induce primary

infection. Asymptomatic colonisation of the respiratory tract

is more common than clinical disease.

Cryptococcosis caused by C neoformans or C gattii is

indistinguishable clinically. The disease can present in nasal,

central nervous system (which can derive from the nasal

form or occur independently), cutaneous and systemic

forms. An easy and reliable test for cryptococcosis diagnosis

is antigen detection in body fluids. Only isolation and

polymerase chain reaction allow identification of the species

genotype. Amphotericin B, ketoconazole, fluconazole and

itraconazole have all been used to treat cats. Surgical

excision of any nodules in the skin, nasal or oral mucosa

assists recovery. Continued treatment is recommended until

the antigen test is negative. Efficient preventive measures

have not been demonstrated. Vaccines are not available.

Cryptococcus species other than C. neoformans/C. gattii

complex were reported in cats by Danesi et al. [87], who

sampled cats from 162 urban and rural feral cat colonies

over 3 years. Of 766 cats from which nasal swabs were

obtained, Cryptococcus spp. were recovered from 95

(12.6%), including 37 C. magnus (4.8%), 16 C. albidus

(2.0%), 15 C. carnescens (1.9%), 12 C. neoformans (1.6%),

as well as C. oeirensis (n = 3), C. victoriae (n = 3), C.

albidosimilis (n = 2),Filobasidium globisporum (n = 2), C.

adeliensis (n = 1), C. flavescens(n = 1), C. dimnae (n = 1),

C. saitoi (n = 1), and C. wieringae (n = 1) with prevalence

<1%. Thirteen Cryptococcus species were identified by

polymerase chain reaction and sequencing of internal

transcribed spacer amplicons. Statistical analysis did not

identify any predisposing factors that contributed to nasal

colonization (eg, sex, age, season, or habitat). Results

suggested that asymptomatic feral cats may carry C.

neoformans and other Cryptococcus species in their

sinonasal cavity. Genotyping of the specific cryptococcal

isolates provides a better understanding of the epidemiology

of these yeasts.


212

Table 4: Cryptococcosis in cats and dogs

Cryptococcus Animal References

Cryptococcal rhinitis

C. neoformans var. neoformans cats 70,71,72,73

dogs 71,145

C. gattii cats 70,73,83, 146,147,148

dogs 83,145

C. species Cats 149

Disseminated cryptococcosis

C. neoformans var. neoformans dogs 74

C. gattii cats 78

dogs 76

C. albidus cats 75

C. magnus 77


C. neoformans var. grubii cats 80

dogs 80

C. gattii cats 80

dogs 80

C. species cats 79, 150

Cryptococcal skin infection

C. species cats 82

Cryptococcal ophthalmitis

C. neoformans var. neoformans cats 81

C. species 79

Cryptococcal urinary tract infection

C. species cats 150.

3. Cryptococcosis in wild animals

In wild animals, cryptococcosis occurs in different animals

and Cryptococcus spp. can affect the gastrointestinal and

respiratory systems and eyes [Table 5]. The koala often

have primary infection of the upper or lower respiratory tract

as the first disease feature. Among captive wild animals,

cryptococcosis has been documented in Marmoset monkey

[88], Rhesus monkey [89], Squirrel monkey [90], Short-

eared elephant shrews, Large tree shrews Lesser tree shrews

[91], Common marmoset [92] Eastern gray squirrels [93],

wild rabbit [94], Bandicota indica [95], Common Toad [96],

Cheetah [97], Spinner dolphin [98], Harbor Porpoise [99],

Koala [100, 101, 102], Ferret [103], California sea lion

[104], Gorilla gorilla [105].

Krockenberger et al. [106] mentioned that Cryptococcus

gattii has been shown to have a strong association with

eucalypts frequently used by koalas and, not surprisingly, it

has been shown to colonize the nasal cavities of koalas. The

progression from nasal colonization to tissue invasion is

critical for understanding the pathogenesis

of cryptococcosis in this species and provides a model for

pathogenesis of cryptococcosis in other species.

Krockenberger et al. [107] studied the relationship among

Cryptococcus gattii, koala and the environment. The koala

https://www.google.com.eg/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwj6vcbsodTSAhUDvxQKHQuSBMMQjhwIBQ&url=http%3A%2F%2Fwww.alamy.com%2Fstock-photo-common-toad-bufo-bufo-in-profile-a-common-british-and-european-toad-93057149.html&psig=AFQjCNEGwNSDvzq_FUqEtClC7Dt5p0YrXQ&ust=1489521412086132


213

was used as a natural biological sampler in an attempt to

understand the dynamics of Cryptococcus gattii in

Australian environments. Evidence of asymptomatic nasal

and skin colonization for extended periods by large numbers

of Cryptococcus gattii was obtained and geographical

factors assessed. The key finding was the ability of koalas to

amplify numbers of Cryptococcus gattii in certain

environments. Koalas were not found to be obligatory for

the survival of the organism in all environments.

Geographical factors alone could not explain differing rates

of nasal and skin colonization in koalas in different

environments. A strong association between healthy koalas

and Cryptococcus gattii was confirmed and Cryptococcus

gattii was isolated from novel sources, including the

turpentine gum tree (Syncarpia glomulifera), tallowwood

(Eucalyptus microcorys) and flooded gum (E. grandis).

Table 5. Cryptococcosis in wild animals

Cryptococcus species Animals References

C. neoformans

var. neoformans

Koalas 102,151

monkeys 89

Shrews 91

common toad 96

C. neoformans

var grubii

Ferrets 80,106

bandicoot 95

Gorilla 105

C. gattii Cheetah 97,152,155

Koalas 101,106,107,154

Ferrets 80,103,106

Dolphins 156,157

Porpoises 99,158

C. albidus Sea lion 104

C. yokohamensis Koalas 100

C. species Ferrets 103

Monkeys 88,90

Koalas 153

Marmosets 92

4. Cryptococcosis in birds

Cryptococcus neoformans is known to inhabit natural

environments such as soil and grows in bird excreta,

especially that of pigeons [108, 109, 110]. Infections in birds

are rare. C. neoformans has been isolated from the faeces of

canaries (26%), carrier pigeons (18%), budgerigars (2%) and

psittacine birds (1%), apart from domestic poultry [111,

112]. Cryptococcus species have been reported to be

associated with the trachea of fowls, isolated from broilers

of a poultry-processing plant [113].

Cryptococcus laurentii was reported to be associated with

feather loss in a glossy starling (Lamprotornis chalybaeus).

The bird exhibited patchy feather loss, especially around the

back and beak area, and greyish crusts sticking quite firmly


214

to the underlying skin. The feathers had a greasy appearance

and disseminated a musty odour.

Malik et al. [114] analyzed clinical and laboratory findings

in 15 unreported cases of avian cryptococcosis from

Australia contrasted with 11 cases recorded in the literature.

Cryptococcus species produced localized invasive disease of

the upper respiratory tract of captive parrots living in

Australia. This resulted in signs referable to mycotic rhinitis

or to involvement of structures contiguous with the nasal

cavity, such as the beak, sinuses, choana, retrobulbar space

and palate. Cryptococcus appeared to behave as a primary

pathogen of immunocompetent hosts. One tissue specimen

was available from an Australian racing pigeon with

minimally invasive subcutaneous disease; immunohistology

demonstrated a Cryptococcus infection, presumably

subsequent to traumatic inoculation of yeast cells into the

subcutis.

Cryptococcus neoformans var. grubii was isolated from a

tissue specimen from an Australian racing pigeon with

minimally invasive subcutaneous disease; and was

demonstrated immunohistologically in the subcutis tissues

[114].

Cryptococcus gattii produced localized invasive disease of

the upper respiratory tract of captive parrots living in

Australia. This resulted in signs referable to mycotic rhinitis

or to involvement of structures contiguous with the nasal

cavity, such as the beak, sinuses, choana, retrobulbar space

and palate. Cryptococcus appeared to behave as a primary

pathogen of immunocompetent hosts [114]. Cryptococcus

gattii was reported in a 14-yr-old female Pesquet's parrot

(Psittrichas fulgidus) with lethargy and decreased ability to

fly. Radiographs revealed an irregular osteolytic lesion

isolated to the distal right humerus. Bone biopsy of the

lesion, cytology, and histopathology were diagnostic for

osteomyelitis with intralesional yeasts confirmed to be on

fungal culture [115]. Most of the studies on Cryptococcus in

avian species are concerned with bird droppings as seen in

[Table 6].

Table 6: Cryptococcus species isolated from avian droppings

Cryptococcus species References

C. neoformans

var neoformans

6,7,8,11,110,159,160,161,162,163,164,165,166,167,168,169,170,171,17

2,173,174,175,176

C. neoformans var. grubii 177,178,179,180

Cryptococcus gattii 159,170,174

Cryptococcus laurentii 6,162,165,166,174,181

Cryptococcus luteolus 174

Cryptococcus ater 174

Cryptococcus species 182

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Dr. Mohamed K. Refai Professor Emeritus, Deparetment of Microbiology, Faculty

of Vet. Med. Cairo Univ.e-mail [email protected]

Dr.mahmoud D. El-Haeiri, Ass. Prof

Deparetment of Microbiology, Faculty of Vet. Med. Cairo

Univ.e-mail [email protected]

.

Dr. Randa M. Alarousy

Researcher of Microbiology and Immunology, National

Research Center. .e-mail [email protected]


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