Isolation of Cryptococcus Laurentii Associated with Bovine Mastitis Cases in India

Mycotic mastitis mainly caused by yeasts like Cryptococcus spp. and Candida spp. have been emerging since last decade due to several factors like indiscriminate use of antibiotics, immune suppression, corticosteroid therapy, teat injuries and faulty milking machineries. The fungus, Cryptococcus laurentii is a non-neoformans, encapsulated, basidiomycete, which was earlier considered to be saprophytic and non-pathogenic. Now it is increasingly being reported in humans, especially in the immune compromised patients. In this study, Cryptococcus laurentii was isolated and identied from bovine mastitic milk samples. This probably might be the rst report of isolation and identication of Cryptococcus laurentii from mastitic milk sample of buffalo from India to best of our knowledge. On milk culture examination, typical creamy white color colonies were appeared on Sabouraud's Dextrose agar, which on Gram’s staining gave budding yeast cells appearance. India ink staining revealed bright halo of capsules surrounding the yeast cells. All the isolates were positive for urease production and biolm formation. Further conrmation was done using VITEK 2 compact system (BioMerieux) which was based on their biochemical tests proles. Molecular conrmation was done by the PCR assay. Isolation and identication of this rare fungus from milk samples in present study raises a potential threat of zoonosis.


Introduction
Bovine mastitis is a multifactorial disease causing huge economic losses to the dairy sector. Mastitis can be caused by over 250 microorganisms (Shome et al. 2011) which include bacteria, fungi, viruses and protozoa. Relative importance of different infections is likely to vary in different geographical regions. In the past, very little importance was given to fungal/mycotic mastitis due to very less percent prevalence but since last decade increasing trend is seen in reports of bovine mycotic mastitis. This could be attributed to several factors like indiscriminate use of antibiotics, immune suppression, corticosteroid therapy, teat injuries and faulty milking machines (Gupta et al. 2018).
The most common fungal species isolated from cases of bovine mastitis are the Candida species. However, there have been reports of isolation of different other yeast like Cryptococcus species as well. The genus Cryptococcus is a heterogenous group of encapsulated fungi including important pathogenic species for human and animals, most commonly the Cryptococcus neoformans and non-neoformans Cryptococcus species is considered to be non pathogenic. Eight percent of non-neoformans Cryptococcus species include Cryptococcus laurentii (C. laurentii) and Cryptococcus albidus (Khawcharoenporn et al. 2006). Earlier C. laurentii was considered to be saprophytic and non-pathogenic and has also been diagnosed as the etiological pathogen able to cause human infections mainly in  Gupta et al. 2018). Their ndings explored its pathogenicity in multiple hosts and considered it as a pathogen of major concern. C. laurentii has been reported to be a rare and opportunistic yeast pathogen isolated from bovine mastitic milk samples. It has also been associated with bio lm formation and refractory to most of the antibiotic treatments (Ajesh and Sreejith, 2012). The present study is probably the rst report of C. laurentii isolated from bovine mastitic milk samples from India.

Isolation and biochemical identi cation
The College Central Laboratory (CCL), LUVAS, Hisar, Haryana, India receives milk samples from all over the state of Haryana for routine examination of mastitis. A total of 600 milk samples from buffaloes and cows with a history of chronic mastitis and prolonged antibiotic use were processed for isolation of isolation and identi cation of rare C. laurentii. Mastitic milk samples were plated onto blood agar, McConkey agar, and Sabouraud dextrose agar and incubated at 37 0 C for 48-72 h. Fungi were identi ed phenotypically, on the basis of colony morphology and further stained by Gram's stain and Indian ink capsular stain. The presumptive isolates were also tested for urease activity and bio lm production on Congo red agar plate.
The isolates were identi ed and con rmed using VITEK 2 Compact system (bioMerieux) and different biochemical tests results of each isolates were analyzed.
C. laurentii var. laurentii MTCC 2898 (Microbial Type Culture Collection) and Cryptococcus neoformans ATCC 14116 strain (American Type Culture Collection) were used as positive and negative control strains for identi cation, respectively.

Molecular con rmation of Cryptococcus laurentii
Phenotypically and biochemically con rmed isolates were subjected to DNA samples from all the isolates were extracted using Quick -DNA Miniprep PLUS kit (Zymo Research) and PCR ampli cation was performed targeting D1-D2 28S rDNA and 18S rRNA gene Internal transcribed sequence (ITS) region sequences (Barton, 2010). PCR using standard procedure was carried out using primer sets (Table 1)

Results And Discussion
Isolation and biochemical identi cation A total of four isolates (Isolate number 421, 657, 1848, 2085) of C. laurentii were con rmed using phenotypic and biochemical test pro le generated by automated VITEK 2 compact system. Automated VITEK 2 compact system revealed that isolate number 421, 657, 1848 and 2085 had 89%, 87%, 90%, and 88% con dence level (probability), respectively of being C. laurentii species. The isolates have typical creamy white colonies on Sabouraud's Dextrose agar (Fig. 1a) and blood agar plate. Gram's staining showed budding yeast cells (Fig. 1b) and India ink staining revealed bright halo of capsules surrounding the yeast cells (Fig. 1c). All the isolates were detected as bio lm producers (Fig. 1d) on Congo red agar plate by producing black coloured colonies and also tested positive for urease production (Fig. 1e). Bio lm formation is the important virulence factor of many microorganism including Candida and other fungi which are usually associated with chronic and recurrent infections. There are various methods to detect bio lm production, of them Congo red agar method is most convenient one (Saxena et al. 2014). Fourteen distinguished biotype patterns were observed on the basis of biochemical test result of each isolates by VITEK 2 compact system ( Table 2). Biotype 1 comprised of 26 types of tests which were tested positive by all the isolates. Biotypes 2 to 14 were tested positive by variable numbers of isolates ( Table 2).

Molecular con rmation of Cryptococcus laurentii
All the four phenotypically and biochemically con rmed isolates ampli ed species speci c 600 bp amplicon size of D1-D2 28S rDNA and 18S rRNA gene Internal transcribed sequence (ITS) region sequences producing variable size amplicon of C. laurentii by PCR using primer set reported earlier (Sugita et al. 2000). Table 2 Biotyping of Cryptococcus laurentii isolates on the basis of biochemical test results pro le generated by The PCR ampli ed products of 18S rRNA ITS of each isolates were sequenced by Sanger's Sequencer (Applied Biosystems). All of them showed 100% similarity with ITS sequences of C. laurentii species on nucleotide BLAST at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Similar sequences with different geographical regions were retrieved from public domain and aligned using Bio Edit (Hall et al. 2011).
Phylogenetic tree was constructed using MEGA6 (Tamura et al. 2013) by maximum likelihood method based on whole ITS region of C. laurentii and related species with 1000 bootstrap replications. While tracing the sequences for fungi on NCBI, none of them was found to be of Indian origin.
Of the four isolates reported in the present study, two isolates (isolate number 421 and 1848) were in close homology with each other while two isolates (isolate number 647 and 2085) were clustered distinctly ( Figure 3). None of them were clustered or grouped with C. laurentii isolates originating from the rest of the world. Phylogenetic analysis revealed their unique identities. All other isolates from USA and European origin were mixed and grouped in separate cluster. To best of our knowledge, none of the isolates were reported earlier from India. Probably this is the rst report of C. laurentii isolated from mastitic milk of bovine in India.
Fungal infections, by both yeast and lamentous, are now considered as opportunistic agents causing severe illness in immune compromised hosts (both humans and animals) demonstrating its public health importance. They were previously considered to be non-pathogenic in most of the cases or their pathogenesis was not clearly understood. Various researchers have been working to characterize both most common as well as rare fungal agents in both animals and humans (Spanamberg et al. 2009;Dalanezi et al. 2018). C. laurentii has been associated with fungemia and pulmonary infection in humans ( (Banerjee et al. 2013). Low birth weight neonate in humans from India has also been reported earlier (Gupta et al. 2018).
Bovine mastitis caused by fungi is considerably less frequent than that caused by bacteria and sometimes the infection can become chronic and more di cult to treat. With the wide use of antibiotics across the world, more and more yeast species are being reported from clinical samples. Researcher detected 41.33% of all the tested samples as positive for mycotic mastitis (Yassein et al. 2016). Of them, 55.64% were yeast isolates and 7.24% were Cryptococcus neoformans. Seven isolates of C. laurentii were reported in blood and urine samples of clinically suspected cases by VITEK 2 Compact system (Nath et al. 2018). Rare opportunistic fungal species has also been found in milk samples nowadays causing risk to public health (Krukowski and Saba 2003). Accurate diagnosis and differentiating bacterial infections from infections in cases of bovine mastitis has become very crucial to minimize potential risks and epidemiological importance. Automated microbial identi cation such as VITEK 2 compact system has been frequently in use for diagnosis of uncommon fungal pathogens. However their sensitivity and speci city towards variable pathogens may be altered due to numerous reasons. Some organisms are misidenti ed by commercial automated systems (Nath et al. 2018 To the best of our knowledge, this might be the rst report of rare C. laurentii species from bovine mastitic milk sample in India. The pathogenic potential of the organism is unknown. However, more studies are needed to understand its pathogenicity as primary pathogen causing mastitis in bovines. Additionally, understanding the common patterns of resistance against non-neoformans cryptococcal infections especially C. laurentii will prevent further treatment failure and economic losses associated with bovine mycotic mastitis.    Phylogenetic tree (UPGMA) constructed by sequence analysis of 18S rRNA internal transcribed sequence (ITS)