Introduction

Contagious caprine pleuropneumonia (CCPP) is a fatal caprine disease caused by Mycoplasma capricolum subsp. capripneumoniae (MCCP), formerly known as Mycoplasma sp. strain F38. The disease was firstly reported in 1873 in Algeria (McMartin et al., 1980). It is a devastating disease of goats (Bascunana et al., 1994) included in the list of notifiable diseases of the World Organisation for Animal Health (OIE) (Manso-Silvan et al., 2011). CCPP is a major threat to goat farming industry in developing countries (Lorenzon et al., 2002) and is pandemic in Africa, Middle East and Asia (Manso-Silvan et al., 2011; Nicholas et al., 2012). Previously, only 20 countries have reported the isolation of the MCCP organism due to inefficient laboratory expertise (Nicholas et al., 2002b), but it has now been isolated in China, Mauritius, Tajikistan (Chu et al., 2011 & Srivastava et al., 2010), etc. CCPP causes economic losses in goat industry globally as these organisms can infect domestic as well as wild breeds of goat (Nicholas et al., 2002b, Arif et al., 2007; Ostrowski et al., 2011), with 100% morbidity and 60–80% mortality rates. This disease is characterised by fibrinous pleuropneumonia with increased straw coloured pleural fluid in the infected lung (Rurangirwa et al., 2012).

MCCP belongs to the Mycoplasma mycoides cluster (Heldtander et al., 2001), which comprises of five taxa and three subclusters, which corresponds to the M. mycoides subspecies, the M. capricolum subspecies and the novel species M. leachii sp. (formerly known as Mycoplasma sp. bovine group 7). M. mycoides subsp. mycoides Large Colony (MmmLC) and M. mycoides subsp. Capri (Mmc) are grouped into the single subspecies M. mycoides subsp. capri, as both agents cause similar diseases in small ruminants and have the same morphological, cultural, biochemical and genotypic properties (Manso-Silvan et al., 2009). All of these are pathogenic and can cause disease in ruminants. The members of the M. mycoides cluster are closely related and are therefore difficult to identify by the conventional serological methods used for Mycoplasma typing. These classical methods are time consuming and complicated by serological cross reactions between the closely related organisms. Molecular diagnosis has improved their detection and identification, specifically the polymerase chain reaction (PCR) for Mycoplasma “mycoides cluster” coupled with restriction enzyme analysis (REA) (Bolske et al., 1996).

CCPP is endemic in the state of Jammu and Kashmir particularly in Kashmir province due to its temperate climate. The cold winter and poor husbandry practices are important predisposing factors (Awan et al., 2009). The extreme weather conditions prevalent in the region contribute to high mortality observed in the outbreaks. In recent times, the severity of the disease has increased and attained significant importance in the goat industry. The Changthangi or Pashmina goats are inhabiting Changthang and adjoining regions of Leh district of Kashmir province.

These goats are reared for their world famous ultra-fine cashmere wool known as Pashmina. Shawls made from the Pashmina wool are exported worldwide. The Pashmina goats have revitalised the poor economy of Changthang, Leh and Ladakh region. CCPP causes huge mortality in Pashmina goats particularly during winter season and incurs colossal losses to farmers’ and the goat industry of the region, thereby affecting their socio-economic status. The present study attempted to detect and isolate MCCP in Pashmina and local goat breeds in Kashmir, India.

Materials and Methods

Collection of Clinical Samples

The samples were collected from Feb. 2013 to Dec. 2015, from five districts of Kashmir province viz. Leh, Kargil, Bandipora, Ganderbal and Kupwara, respectively (Table 1). A total of 55 lung samples and 70 nasal swabs were collected from naturally infected Pashmina as well as local breeds of goats showing primary clinical signs of CCPP prior to death. The post mortem examination was carried out immediately in dead goats and whole lung was sampled (Fig. 2) for study and subsequently frozen. The nasal swabs were also taken from sick goats showing typical respiratory signs of classical CCPP including coughing, anorexia, labored breathing and rise in temperature up to 41°C. All the samples (lungs and nasal swabs) were kept on ice packs in a cool box and transported to the laboratory within 48 hrs of collection. The sampling was done by trained field veterinarians to minimise contamination of specimens.

Table 1: Distribution of samples in five districts of Kashmir province

*L= lungs, N.S= Nasal swabs

Fig. 2: Goat lungs showing congestion and grey areas of hepatisation characteristic of CCPP

Growth Media

The growth medium used for MCCP contained the following ingredients: 17.5 g of Bacto PPLO Broth (Difco), 2.5 g of yeast extract (Difco), 2.5 g of Lactalbumin Hydrolysate (LAH), 200 ml of horse serum (Sigma Aldrich) inactivated at 56°C for 30 min, 2.0 g of dextrose, 2.0 g of sodium pyruvate (Sigma Aldrich), 2.5 ml of 10%thallium acetate (Sigma Aldrich), 250 mg of ampicillin (Sigma Aldrich), 5 ml of 0.5% phenol red (HiMedia, Mumbai)and glass-distilled water upto 1000 ml. The pH was adjusted to 7.8 with NaOH or HCl. 1.75% of agar (Difco) was separately autoclaved, cooled to 45 °Cand added to above filtered broth to prepare PPLO agar.

Isolation and Identification of MCCP from Clinical Samples

The suspected lungs, positive for MCCP were processed for its isolation and identification. Briefly, 5-10 g of lung tissue was taken from the interface between consolidated and unconsolidated areas of whole lung and cut into small pieces with the help of sterile scissor. The tissue was homogenized using the homogenizer (Polytron-PT- MR 2100, Kinematica AG, Switzerland) and PPLO broth was added to the homogenized tissue and mixed thoroughly. The suspension recovered was syringe filtered and poured onto sterile PPLO agar plates and into culture tubes containing sterile PPLO broth. Similarly, the nasal swabs were vortexed briefly to suspend swabbed material into PPLO broth and swabs were then removed and suspension was syringe filtered and poured onto sterile PPLO agar plates and broth. Incubation was

done at 37°C in a humid anaerobic jar with 5% carbon dioxide supplied by candle. Humidity was maintained by placing cotton wool soaked in water. The PPLO agar plates were examined daily for evidence of growth, which was manifested by a colour change from red to yellow due to acid production from fermentation and the appearance of floccular materials at the bottom of the culture tube or a swirl from the bottom when it was agitated. The solid media was observed under stereomicroscope (40 X, Labomed-CZM-4, U.S.A) from day 3 upto 21 days for the evidence of micro-colonies with a “fried-egg” appearance.

Extraction of DNA

The suspension of nasal swabs left after pouring onto PPLO agar plates and broth were subjected to centrifugation at 10,000 × g for 10 min. The supernatant was discarded and suspensions of the pelleted material were prepared in 1.5 ml microcentrifuge tubes in 150 µl of sterile phosphate buffer saline (PBS) by gentle vortexing. The samples were boiled for 5 min, cooled on ice for 10 min and centrifuged at 10,000 × g for 5min. Three microlitres of the supernatant was used as the template for each polymerase chain reaction (PCR). DNA was also extracted for PCR from lung tissues using DNA extraction kit (PROMEGA Wizard genomic DNA Purification Kit, U.S.A) as per the manufacturer’s instruction. The concentration and purity of the extracted DNA was checked at λ260 using BioPhotometer Plus (Eppendorf, Germany) and stored at -20°C until use.

Detection of MCCP by PCR

All the samples (lungs and nasal swabs) were subjected to PCR. A fragment of 16S rRNA gene from the members of M. mycoides cluster was amplified to obtain a 548 bp PCR product with the forward primer MmF and the reverse primer MmR as detailed by (Bascunana et al., 1994). The PCR reaction consisted of 3.0 µl template DNA, 2.5 µl of 10X buffer, 0.2 mM concentrations of each 2′-deoxynucleoside 5′-triphosphate (dNTP) mix, 0.5 μM concentration of each primer, 1.5 U of Taq DNA Polymerase (Fermentas Life Sciences), MgCl2 was used at 2.0 mM concentration and sterile distilled water. The amplification cycles in thermal cycler (Mastercycler Gradient, Eppendorf, Germany) consisted of initial denaturation of 95 °C for 4 min, followed by 35 cycles of 95°C for 45 s, 55 °C for 1 min, 72°C for 1 min, and final extension of 72 °C for 5 min. The PCR products were analysed in 1.5%-2% agarose gel, stained with ethidium bromide, visualized under ultraviolet illumination and photographed using Gel Documentation System (Biospectrum 500 Imaging system, UVP, U.K).

Restriction Digestion

The PCR products were then cleaved without further purification by restriction enzyme PstI (Fermentas Life Sciences) to differentiate MCCP from the other members of the M. mycoides cluster.

Cloning of Amplicon

Representative amplicon with unique cleavage pattern in restriction digestion were purified using the MinElute PCR Purification Kit (Qiagen, Hilden, Germany) as per the manufacturer’s instructions. The PCR products were ligated into the p-Drive Cloning Vector (Qiagen) according to the manufacturer’s recommended protocol. The ligation mixture was used to transform DH5α Escherichia coli cells by electroporation as per the standard protocol. Five convergent positive colonies for each transformation were picked up and transferred into Luria Bertani (LB) broth (HiMedia, India) then incubated with shaking (200rpm) overnight at 37 °C in a rotary shaking incubator (JEIO TECH, Korea).

DNA Sequencing of 16SrRNA Fragment

The plasmid DNA from selected colonies were extracted using a QIA prep Miniprep Kit (Qiagen, Hilden, Germany) and the concentration of the DNA adjusted to 200 ng/µl. The DNA was sequenced commercially by Macrogen Inc., Korea, using M13universal primers.

Sequence Analysis

Sequence alignments and translation were performed using Basic Local Alignment Search Tool (BLAST), FastPCR (PrimerDigitalLtd, Helsinki Finland) and ClustalX (GenomeNet Japan).

Results

Polymerase Chain Reaction

Out of 55 lung samples received, 33 (60%) were positive as revealed by the specific amplification of 548 base pair (bp) amplicon characteristic of the 16S rRNA gene fragments of M. mycoides cluster. Restriction enzyme analysis of PCR products: REA with PstI differentiated MCCP from other members of the cluster. The presence of three bands, the uncleaved 548-bp fragment and the two cleavage products of 420 and 128 bp, showed that MCCP was present in the sample (Fig. 3). All the 33 positive samples revealed three typical bands characteristic of MCCP. The district wise distribution of MCCP during the period of study is depicted in Table 1.

Isolation of MCCP from Clinical Samples

Out of 55 lung samples, only five (9.0%) yielded the isolates in pure culture. These five lung samples were collected from Leh district. The microcolonies of MCCP showing characteristic fried egg appearance on PPLO agar is depicted in Fig. 4. No isolates was obtained from nasal swabs.

Fig. 3:  Restriction digestion of 16S rRNA gene fragment of M. mycoides with PstI

Lane 1:100 bp DNA ladder (Ferment as Life Sciences)
Lane 2-4: Cleavage products after restriction digestion

Fig. 4: Unstained microcolonies of Mccpon PPLO agar with characteristic friedegg appearance understereomicroscope(40X)

Cloning and DNA Sequencing of Amplicon

The representative amplicon showing characteristic cleavage pattern (three bands) in gel when subjected to REA was cloned (Fig. 5) and subsequently out-sourced for DNA sequencing. The DNA sequencing of 16SrRNA gene fragment confirmed the organism as MCCP. The sequence has been deposited into the NCBI GenBank with the accession number JX844648.1.

Fig. 5: Cloningof 16S rRNA gene fragment of M. mycoides cluster in p-Drive cloning vector

Discussion

Ladakh is the home tract of Pashmina goats that is famous for its ultra-fine cashmere wool known as Pashmina. The outbreaks of CCPP have been reported in Pashmina goats particularly during winter season resulting in their high mortality and morbidity, thereby putting economic burden on Pashmina goat rearers of the region. This paper describes the detection and isolation of MCCP in Pashmina and local goats of Ladakh region and northern parts of Kashmir valley. In this study, molecular test like PCR- REA was employed for detection and identification of MCCP from lung samples and nasal swabs. This molecular technique is specific for MCCP as all members of the M. mycoides cluster have two rRNA operons, and there are sequence differences in the 16S rRNA genes of the two operons (Pettersson et al., 1998). This PCR system amplifies segments of the two 16S rRNA genes from all members of the M. mycoides cluster. The PCR product from the two operons of this gene when subjected to restriction enzyme digestion with PstI reveals unique cleavage pattern for MCCP distinguishing it from the other members in the M. mycoides cluster based on a single nucleotide substitution. In the present investigation 33 (60%) lung samples and 24 (34.2%) nasal swabs were detected positive for MCCP by PCR-REA test. However, it is notable that a large portion of nasal swabs (65.71%) were negative for MCCP. This would suggest either that MCCP load in nasal discharge is very low particularly during later stages of the disease as the nasal swabs in most of the cases were taken from sick goats prior to death and PCR is not sensitive

enough to amplify the DNA if bacterial cell number is less than 10 3 cells per swab sample (Fontaine

et al., 1993) or due to crude method of DNA extraction such as boiling method or the presence of PCR inhibitory substance like humic acid which is present in swabs soiled with dirt is another possibility (Tsai et al., 1992). This is in agreement with the findings of (Bolske et al., 1996) who also reported failure to detect MCCP in any of the dried nasal swabs originating from a herd with CCPP due to low prevalence of the organism in the nasal cavity particularly during latter stages of the disease and presence of PCR inhibitory substances.

Overall, the high incidence of MCCP in lung samples (60%) and in nasal swabs (34.2%) of Pashmina

and local goats is alarming in the present investigation therefore, stressing further screening of goat herds for CCPP to determine its prevalence and hence propensity of disease in the region. In the present study, the highest percentage of MCCP was observed in lung samples (70%) and nasal swabs (39.4%) of Pashmina goats in Leh district which suggests that the Pashmina goats in this region are highly vulnerable to MCCP and the lowest percentages were observed in both Kupwara and Ganderbal districts, respectively. There seems no other reports available from India or abroad with which to compare these results. The high incidence rate of MCCP in Leh and Kargil districts could be due to severe cold (sub-zero temperatures) as goats are kept in close proximity during cold season and poor husbandry practices. During confinement period Pashmina goats have to share housing (without proper ventilation), water and feed which facilitates the spreads of CCPP from carriers to susceptible goats. The outbreaks of disease often occur after cold spells and the recovered carrier animals start shedding the mycoplasmas after the stress of sudden climatic change. It is believed that a long-term carrier state may exist.

The isolation of MCCP from the PCR positive samples were attempted which yielded five isolates. These isolates were obtained from lung tissues of Pashmina goats of Leh district and no isolate was recovered from the nasal swabs. This could be because of the fastidious growth requirements of MCCP, fragility of the organism, the ease of being overgrown by other bacteria and fungi making isolation difficult (Thiaucourt et al., 1996), less expertise in isolation techniques and possibly also because of a high proportion of dead organisms in the tissue due to freeze-thawing of lung samples. However, it is also possible that the frequent use of antibiotics by goat rearers as observed in the study areas might have contributed to the low level of isolation since antimicrobial (especially the macrolides, tetracyclines and fluoroquinolones) treatment significantly lowers mycoplasma recovery rates. Therefore, there is a need to improve isolation techniques and characterization methods for these microorganisms. Further, it is the need of hour to screen larger goat population of the region for CCPP and determine its prevalence which will help to understand its epidemiology and devise proper control measures including autogenous vaccine formulation against such economically important disease of goats.

Conflict of Interest Statement

The authors declare there is no conflict of interest.

Conclusion

In this study, highest prevalence of CCPP in goats was observed in cold arid regions of Leh and Kargil districts of Kashmir province particularly during winters, causing huge mortality in Pashmina goats and incurs colossal losses to farmers’. The extreme weather conditions and poor husbandry practices were important predisposing factors contributing to high mortality observed in the outbreaks. There is an immediate need to characterize the organism and formulate area specific autogenous vaccine against the disease.

Acknowledgements

The authors wish to thank the National Agricultural Innovation Project (NAIP) of the Indian Council of Agricultural Research (ICAR), New Delhi, Grant No. 4132 for financial assistance. The authors are also highly grateful to Disease Investigation Laboratory (DIL), Department of sheep husbandry, Government of Jammu and Kashmir, India for providing necessary assistance.

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