Isolation, Molecular Identi cation of Mycoplasma Putrefaciens Local Strains and Its Associated Pathological Lesions in Goats

Muhammad Saeed The University of Agriculture Peshawar Farhan Anwar (  farhan82@aup.edu.pk ) The University of Agriculture Peshawar https://orcid.org/0000-0001-6024-1269 Farhan Anwar Khan The University of Agriculture Peshawar Faiz Ur Rehman The University of Agriculture Peshawar Hayatullah Khan The University of Agriculture Peshawar Faisal Ahmed The University of Agriculture Peshawar Mehboob Ali The University of Agriculture Peshawar Mushtaq Ahmed The University of Agriculture Peshawar Qudrat Ullah The University of Agriculture Peshawar Aamir Khan The University of Agriculture Peshawar


Introduction
In Pakistan, livestock is a major subsector of agriculture that contributes 60.54% to the agriculture gross domestic product (GDP) (Economic survey, 2018(Economic survey, -2019. Pakistan have the largest population of goats among livestock. Goat is considered as poor man's cow in Indo-Pak sub-continent (Rahman et al., 2003). The population of goat is facing diversi ed problems in the country because of poor management practices, low feeding and infectious and non-infectious diseases. Among the infectious diseases, Mycoplasmosis causes great loss to animals because of high morbidity and mortality (Siddique et al., 2012).The Mycoplasma belongs to class Mollicutes having no cell wall, prokaryotes, small size and have unusually small genomes approximately 0.58 to 2.20 Mb. (Khan et al., 2016;2018). . In small ruminants it cause respiratory disease, arthritis, eye lesions, genital lesions and mastitis (Nicholas, 2002;Sharif and Muhammad, 2009).
Mycoplasma putrefaciens is responsible for great economic loses in developing countries in term of decrease milk production, meat, hide and hair in most countries (OIE, 2000). Mycoplasma putrefaciens adversely affect the goat population throughout the world causing morbidity and an acute illness with mortality. Mycoplasma putrefaciens is considered as an etiological agent of the contagious agalactiae syndrome in goats by the world organization (OIE) for animal health (Manual, Paris, France 2000) characterized by Mastitis, Arthritis, Keratoconjunctivitis, Pneumonia, and Septicemia (MAKePS) (Thiaucourt et al., 1996). Mycoplasma putrefaciens also found in ear canal as a commensal organisms (Cottew GS et al., 1981). Mycoplasma putrifaciens is closely related to mycoplasma mycoides cluster that consist of 5 major members, highly pathogenic to ruminants including M. mycoides subsp. capri, M. mycoides subsp . mycoides, M. capricolum subsp. capricolum, M. capricolum subsp. capripneumoniae, and M. leachii (Manso-Silván L et al., 2007) but different from these mycoplasmas because the rst sign which appear in Mp infection is mastitis (Adler et al., 1980).The most impressive biochemical characteristic that differentiate Mycoplasma putrifaciens from all other mycoplasmas is the production of bed smell that occur in cultured broth (Tully et al 1974).The gross pathology macroscopic study revealed arthritis (increase in the size of the carpel joints) and respiratory signs such as serous to mucoid nasal discharge and temperature range from 38.1 to 40C° (Rodriguez et al., 1995).
Looking at the economic losses, high morbidity and mortality of small ruminants due to Mycoplasma putrifaciens a rapid, speci c and sensitive detection test was developed called Polymerase chain reaction in order to isolate, identify and characterize the Mycoplasma putrifaciens that has never been reported in northern regions of Pakistan. This study will be helpful for the researcher to eradicate this fatal disease.
The Histopathological study Mp showed intestinal lesions characterized by a multi focal necrotichemorrhagic enteritis and areas of catarrhal enteritis with fusion of villi and in ltration of cells which are composed of mainly neutrophils and eosinophil's. The joints showed acute brinopurulent arthritis with hyperplasia of the synovial epithelium (DaMassa and others 1984(DaMassa and others , 1987(DaMassa and others , 1992.

Study Area
The study was approved by the ethical committee of College of Veterinary Sciences, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar. A total of 600 samples were collected from Northern region (Chitral, Gilgit Baltistan, Swat) of kpk, Pakistan during November 2017 to December 2018. All the samples were collected from naturally infected small ruminants having respiratory signs like coughing, muco-purulent nasal discharge, along with decrease milk production in lactating animals.

Sample size and Sampling
The samples for the bacterial isolation were collected that comprised of nasal cavity through nasal swab, synovial uid from live animals by aspiration, pleural uid and lungs tissue were taken from dead animals suspected for Mycoplasma putrifaciens. Total 600 samples were collected, 200 from each region. Sterile cotton swab were used for collection of nasal discharge which are inserted gently and touch nasal passage of the goat to get the secretion. Pleural uid was collected with the help of sterile 5ml syringe and pour into screw caped sterile 15ml falcon tube and placed in icebox immediately. Lung sample were collected aseptically between both normal and morbid part (consolidation, red hepatization) of lung. Lung tissues were also preserved in 10% neutral buffered formalin for Histopathological examination. All the samples were immediately preserved in icebox at -4C° and transported to Pathology laboratory, College of Veterinary Sciences, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar for further processing.

Cultivation of Samples
Nasal swab, Synovial uid and Pleural uid were cultured in PPLO broth media with the composition of PPLO broth (Sigma, USA) 2.1% supplemented with 0.5% Sodium pyruvate (Sigma, USA), 0.09% Yeast extract (Sigma, USA), 0.2% glucose (Sigma, USA), 20% Horse Serum (New Zealand). Lung tissues were rst minced and then cultured in PPLO broth media. All the tubes were placed at 37C° in 5% CO 2 incubator (New Brunswick, Galaxy48S, UK) for 3-4 days. The samples were checked every 12 hours for color change, whirling movement and turbidity. One tube containing Sterile PPLO broth having no sample were also kept as a negative control.

Isolation and Puri cation of Mycoplasma putrifaciens
Mycoplasma putrifaciens positive cultured samples were processed further for isolation and puri cation. Primary positive culture was passed through PVDF syringe lter having pore size 0.45µm for removing large debris and other eld bacteria. After ltration positive samples were cultured and sub-cultured in PPLO broth and Agar, incubated for 3-4 days. This process was repeated 4-5 times to get a single pure colony of Mycoplasma putrifaciens. The plates were then examined under CCD microscope (Olympus, Japan) at 4X, and 10X on daily basis for 3-4 days.

DNA Extraction
The culture showing turbidity, whirling movement were transferred to 1.5ml eppendorf tube and centrifuged at 10000rpm for 15 minutes. Supernatant was discarded and genomic DNA was extracted through DNA extraction kit (Thermoscienti c, USA). Concentration and purity was checked through Nanodrop (Multiskan, Thermoscienti c, USA) which was in the range of 30-50ng/µl.

Polymerase Chain Reaction
Polymerase chain reaction (PCR) tests for the detection of Mycoplasma Putrifaciens was performed on puri ed DNA samples by using specie speci c primers Mp primers: Forward: (5 / -GCGGCATGCCTAATACATGC-3 / ) Reverse: (5 / AGCTGCGGCGCTGAGTTCA-3 / ) with the amplicon size is 540bp (Shankster et al, 2002). The primer used in this study were synthesized by Invitrogen (USA). About 1.5µl sample of each puri ed genomic DNA was ampli ed in 25µl of PCR with a composition of 10µl master mix "1.5 mM Mgcl2, 200µM of each dNTP. 1 unit Taq polymerase and 10µM of each forward and reverse primers". All the PCR tubes were subjected to Gradient Thermal cycler machine (Bio-Rad,USA) with a initial denaturation at 95°C for 3 minutes followed by 34X (cycles) consisting of 60 seconds of denaturation at 94°C, 60 seconds of annealing at 59.5°C. Extension for 60 seconds at 72°C and Final extension for 5 minutes at 72°C. Gel red (Thermo, USA) was added 1% agrose gel for visualization the PCR products by using ultraviolet gel documentation system (Fasgene, Germany).

Results
Clinical signs and lesions observed in Mycoplasma putrifaciens in suspected goats Clinically suspected goats for Mycoplasma putrifaciens shows Hyperthermia, coughing, mucopurulent nasal discharge, conjunctivitis, arthritis and mastitis were the prominent signs that recorded during this study. The postmortem examination shows unilateral hepatization, consolidation and straw color uid present in lungs (Fig. 1) Cultivation of eld samples Out of total 600 samples 317 samples were positive upon cultivation showing whirling movement by shaking, turbidity and color change (Fig. 2). Separately, of nasal samples (52.66%), synovial uid (42.66%), pleural uid (59.33%) and lung tissues (56.66%) showed growth movement for mycoplasma putrifaciens when incubated for 2-3 days in 5% CO2 incubator. Each of the positive culture broth from nasal swab, synovial uid, pleural uid and lung tissues gives a circle like colonies having a very small dot in the center of the colony post incubation when cultured on PPLO agar media (Fig. 4).

Discussion
This works describes the isolation and identi cation of Mycoplasma putrifaciens for the rst time in southern regions of Pakistan from goats having respiratory signs along with mastitis, arthritis, keratocunjunctivitis, septicemia, and pneumonia ((MAkePS). Mp is also the causative agent of contagious agalactia syndrome (OIE, 2008). M. agalactiae, M. putrefaciens which are not members of Mycoplasma mycoids cluster mostly cause complications when occur along with cluster. (Sadique et al., 2012,) .The isolation and identi cation of Mp is very di cult work because of slow growing rate and require a special media for culturing.
In present study the molecular prevalence of Mp was undertaken by using the PCR assay for the rst time in southern regions of kpk, Pakistan. Undoubtedly, the previous study on Mycoplasma con rm that it is widely spread in Pakistan. For identi cation of Mycoplasma there are many techniques such as conventional and non-conventional but most of the time gives false negative result. For proper con rmation of Mycoplasma it is important to culture the suspected samples but in most cases it is failed because of lack of diagnosis of infected animals and second one is the media which are used for culturing the Mycoplasma is expensive and takes time to grow.
Mp was isolated successfully when cultured on modi ed PPLO media from different samples comprised of nasal swab, synovial uid, Pleural uids and lungs tissue. The most appropriate sample for the isolation of Mycoplasma is pleural uid. Same ndings were also observed by (Sadique et al., 2012, Thiacourt et al., 1994. Such investigations are con rmed by the fact that Mycoplasma cell membrane have lipoglycan that triggers the acute in ammation leads to severe exudation. The Mycoplasma livability increases as it is deeply present in host tissue. The presence of disease in different selected areas con rms that Gilgit Baltistan have high prevalence of Mp by comparing to swat and chitral. This above statement is proved by the physical features and location of Gilgit Baltistan that are mostly hilly areas and harsh climatic change due to rain on daily basis. The local people of Gilgit Baltistan migrated from their areas because of small ruminants looking for pasture. Due to migration of the local peoples the small ruminants comes in stress that leads to predisposed of the animals to Mp. Due to lack of awareness, education about proper vaccination, diagnosis and treatment Mp is spreading from neighbour's countries due to freely movement of nomads from one place to another. The same observations also reported by (Mondal et al., 2004).
For isolation of Mp a special media was used called PPLO (Pleuropneumonia like organism). Mycoplasma is completely dependent on host because to get fatty, amino and nucleic acids, precures of lipid and vitamins. The medium must contain cholesterol which may replace by other sterols like cholesterol or orgosteroll (Rodwell., 1979). Antifungal (Fluconazole) was also added to the medium because of chances of fungal growth in medium is a common problem. Typical fried egg colonies were appeared when streaked on PPLO agar. Thermo scienti c gene jet genomic DNA kit was used for the extraction of DNA from cultured broth showing color change, turbidity and whirling and then perform PCR to con rm the presence of Mp. By DNA extraction or culturing the Mp positive broth on PPLO agar there was a putrid smell of culture. The ndings were also noticed by (Tully et al 1974). PCR is the most sensitive method as compared to biochemical tests as most of the time biochemical tests gives false negative results.
Typical fried egg like colonies were appeared from PCR positive culture broth when streaked on PPLO agar. Same ndings were also observed by (shah et al., 2017). The colonies were appeared after 2 to 3 days post culturing. Every single colony was picked up with sterile needle of syringe and pour in PPLO broth in order to obtain pure culture broth and then place in Incubator having 5% CO2 at 37C°. Out of 200 samples, the total prevalence of MP in Gilgit Baltistan through PCR was 38.85%; in which the percentage of nasal swab, synovial uid, pleural uid and lung tissues were 9, 2, 13 and 16 respectively. In Chitral, 25% samples were positive having nasal swab 5%, synovial uid 2%, pleural uid 9% and lung tissues 13%. The total positive samples in Swat were 19.38%, comprised of 7% positive samples of nasal swab, 1% synovial uid, 5% pleural uid and 6% lung tissues. Same primers were used as used by (Shankster et al., 2002) having amplicon size of 540bp.
Gross pathology during postmortem examination there was polyarthritis, various degree of pneumonia, red hepatization of one lung, pleural uid was seen that was also reported by (Rodriguez et al., 1994, Peyraud et al., 2003, Awan et al., 2009. The Histopathological study showed arthritis having acute brinopurulent hyperplasia of the synovial epithelium. There is congestion in lungs of septal vessels and also alveolar edema along with cell debris and alveolar macrophages. The Mp is highly prevalent throughout Pakistan and cause high economic loss to goat population in northern and southern regions of the country (Fauzia et al., 2016;Shahzad et al., 2012;Saddique et al., 2012). Declarations 1. Acknowledgment: Shansster, S., Ayling, R. D., Lenfart, D. D., Mercier, P., & Nicholas, R. A. J. 2002. 74 Figure 1 (A) Shows conjunctivitis and mucopurulent nasal discharge, (B) shows swelling of joints in kid, (C) postmortem examination shows unilateral hepatization (D) shows pleural uid aspirated through sterile syringe.

Figure 2
Cultivation of eld samples collected from naturally infected goats in PPLO broth: from left to right rst 6 samples shows Mycoplasma putrifaciens growth on 2nd day post incubation while last tube placed as a negative control.
Lane 4 indicates as a positive control.

Figure 4
Puri ed colonies of Mycoplasma putrifaciens having a very small center on PPLO agar post 2-3 days incubation.