PJZ_57_5_2393-2405

Introduction

Contagious caprine pleuropneumonia (CCPP) is an infectious disease of caprine caused by a tiny gram negative bacterium belonging to the genus Mycoplasma (Cottew et al., 1987; Fischer et al., 2012). The infection is characterized by acute respiratory distress accompanied by coughing with purulent discharges, fever and anorexia (Radostitis et al., 2009; OIE, 2014). The disease results in the huge economic losses to the livestock industry due to its high morbidity and mortality rate in the endemic areas especially in Asia and Africa (Nicolas et al., 2008; OIE, 2014; Sandip et al., 2014).

Several Mycoplasma species are known to be associated with the diseases including CCPP and contagious agalactia (CA) in small ruminants and contagious bovine pleuropneumonia (CBPP) in large ruminants (Quinn et al., 2002; OIE, 2008). While, the small ruminants are known to be affected by the Mycoplasma species belonging to the Mycoplasma mycoides cluster including Mycoplasma mycoides subsp. capri (MMC), M. capricolum subsp. capripneumoniae (MCCP) (Cottew et al., 1987; Manso-Silván et al., 2007; Fischer et al., 2012). However, a variety of less clinically important species are also being involved in the infections such as M. arginine, M. ovipneumoniae, M. agalactiae and M. putrifaciens (Ayling and Nicholas, 2007; Manso-Silván et al., 2007).

These Mycoplasma species are highly contagious in nature which makes them easy spread from animal to animal through direct contact or aerosols (Thiaucourt et al., 1996; Lignereux et al., 2018) thereby achieving almost 100% morbidity with mortality can reach up to 80% in affected herds (DaMassa et al., 1997; OIE, 2014; Yatoo et al., 2018). Several factors are considered for the continuous persistence of the infection including environmental conditions, breed type, immunity of individual host, infection burden and interaction of herds (Thiaucourt et al., 1996; Wesonga et al., 2004). However, the cold and moist weather further adds up in the persistence and reoccurrence of the infection in endemic areas (OIE, 2009).

Clinically, CCPP is manifested by respiratory sign with acute to chronic forms. The symptoms may become evident with pyrexia followed by coughing along with nasal discharges (Radostitis et al., 2009; OIE, 2014). In addition to the respiratory infections, Mycoplasma can spread to the other organs causing arthritis, mastitis, ocular lesions and reproductive disorders (Bergonier et al., 1997; Kumar et al., 2013; Semmate et al., 2022). Further, the respiratory infections caused by the Mycoplasma species can pave the way for other microbes including bacterial and viral pathogens which further can complicate the disease and leads an animal to the death (Chu et al., 2011; Kgotlele et al., 2019).

Recent development in the molecular diagnostic tools are of great importance due to several advantages over traditional tools. The formerly used culture-based techniques require a long procedural time due to complexity and slow growth of Mycoplasma species which results in the delay in diagnosis and treatment of the disease (Nicholas et al., 2008). While, the serological tests widely used for the diagnosis of mycoplasmas are rapid but mostly avoided because of cross reactivity due to similarity among Mycoplasma species that again restrict the use of such techniques when species specific identification is required (Thiaucourt et al., 1994; Nicholas and Baker, 1998; Al-Harthi et al., 2018). The PCR based molecular tools can provide a more precise and sensitive detection and identification of specific Mycoplasma species infecting animals. Up to date, these molecular tools remained rapid and robust method for species specific detection of Mycoplasma which further can provide the basis for treatment and prevention of infections (Dominique et al., 2004; Halium et al., 2019; Semmate et al., 2022).

Livestock population in Pakistan is always dominated by goat population with currently almost 82.5 million heads are being domesticated in the country (GOP, 2022). Despite the huge investment and employment opportunities created, the goat production system is still facing several constraints particularly lack of vaccination and treatment against commonly prevalent diseases. Keeping in mind, the present study aimed to determine the prevalence and molecular characterization of Mycoplasma species infecting goats from Sindh Pakistan which can help in generating baseline data for prevention of the disease.

Materials and Methods

Study area

The study area targeted during to sample collection comprised four different districts of Sindh Province including Thatta, Tharparkar, Jamshoro and Larkana. Geographically these area are located between 27.8514 to 24.4498° N latitude and 68.2944 to 70.5315° E longitude (Fig. 1). The areas were selected as representative zones of the province having humid to dry, warm to moderate cool, and with rainfall fall during summer months. These areas comprises various agro-climatic zones and are rich in goat population with nomadic behavior also observed during dry seasons.

Animals and samples collection

A total of eight hundred (800) mix breed goats (6 months to 4 years) from different goat farms located in different zones of Sindh province of Pakistan (Thatta, Tharparkar, Jamshoro and Larkana) were examined and random nasal samples were collected during the period from September 2018-April 2019. During the sampling, clinical signs of the disease were also observed for further characterization of the clinical disease. The nasal swabs were collected using sterile swabs in duplicate. The swabs used for culture and isolation of organism were transported to the laboratory in PPLO broth (Oxoid Limited, UK). While, swabs used for DNA extraction were transported in ice box in order to avoid deterioration.

Phenotypic characterization of Mycoplasma

For isolation and phenotypic characterization of Mycoplasma, the nasal swabs inoculated PPLO broth (supplemented with 0.5% glucose, 0.5g/L thallium acetate, 0.9g/L yeast extract, 0.03% penicillin G and 20% inactivated horse serum) was filtered through 0.22 µM pore size. Afterwards, the filtered broth was inoculated initially in PPLO broth and cultured at 37 °C for 3–7 days followed by streaking on PPLO agar medium and incubated at 37 °C for 14 days in a humidified incubator supplemented with 5% CO2. The positive colonies were identified through the typical “fried egg” colonies in the specified time. Further, the growth inhibition test was employed using the protocol described by OIE (2008) and Hernandez et al. (2006) to differentiate the Mycoplasma with other related organisms. Briefly, the tenfold dilutions of the bacterial culture in the broth were prepared. Each dilution of culture was spread on the Mycoplasma agar plates. After even spreading, the excessive material was removed. The plates were incubated at 30° C for 50 min for drying. Disks impregnated with polyclonal rabbit anti-sera against Mycoplasma mycoides subsp. capri (VPU, Central Veterinary Diagnosis Laboratory, Tandojam) were carefully placed on the agar plates. The plates were incubated at 37° C for 2-6 days. The zones of inhibition were recorded.

Genomic DNA extraction

The genomic DNA from the nasal swabs/bacterial pellet was collected through phenol-chloroform method (Sambrook and Russell, 2006). Briefly, the swabs/pellet was taken into a fresh 1.5 ml Eppendorf tube and mixed with 200 µl of lysis buffer (10 mM NaCl, 20 mM Tris-HCL (pH:8.0), 1mM EDTA, 1% SDS) through vertexing followed by the addition of 50 µg of proteinase K. The samples were then incubated at 56°C for overnight. Afterwards, 500 µl of a solution of phenol-chloroform-isoamyl alcohol (25:24:1) was added, vortexed and centrifuged at 12000 rpm for 10 min. After centrifugation, clear supernatants were carefully collected and transferred to a new tube. The DNA precipitation was performed using 40 µl of 3 M sodium acetate (pH 5.0) and 0.8 ml ethanol through incubating samples at -80°C for 1 h. Then samples were centrifuged and supernatant was discarded and washed once with 1 ml each of 100% ethanol and then by 75% ethanol. The supernatants were discarded and pellets were air dried. Finally, 100 ul of TE buffer was added to dissolve pellet and then the extracted DNA was stored at -20°C until further use.

Polymerase chain reaction (PCR)

The PCR amplification was carried out using the extracted gDNA to accurately identify the Mycoplasma species. The reaction was set up with a total volume of 50 µL containing 25 µL 2× Absolute Master Mix (MOLEQULE-ON®, New Zealand), 1 µL (10 pmol/µL) of each primer, 2 µL (50 ng/µL) DNA with the remaining volume adjusted with double distilled water (ddH2O). The PCR reaction was carried out using the primers Mm-F: 5′-CGAAAGCGGCTTACTGGCTTGTT-3′ along with Mm-R: 5′-TTGAGATTAGCTCCCCTTCACAG-3′ (Hotzel et al., 1996), MM450: 5′-GTATTTTCCTTTCTAATTTG-3′ along with MM451: 5′-AAATCAAATTAATAAGTTTG-3′ (Bashiruddin et al., 1994) in a thermal cycler (2720 Thermal Cycler, Applied Biosystems, USA). The reaction was performed with an initial denaturation at 94°C for 3 min, followed by 45 cycles of denaturation (94°C) for 30 sec, annealing (54°C and 58°C, respectively) for 30 sec, extension (72°C) for 45 sec and a final extension step at 72°C for 10 min. The PCR products were separated on 1.5% agarose gel and visualized under UV light in a gel documentation system.

Sequencing and phylogenetic analysis

The PCR products were further sequenced by Bionics Pvt. lmt. through Sanger dideoxy sequencing technology. The obtained DNA sequences were searched on National Center for Biotechnology Information (NCBI) database by using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) tool for comparison and identification of each species. Further, the sequences were analyzed by using Chromas Lite version 2.0 (http://www.technelysium.com.au). The genomic sequences of target genes from different Mycoplasma species were retrieved from NCBI database for comparison and alignments using Clustal W (http://www.clustalw.genome.jp). The sequence homology was searched used sequence identity and similarity (SIAS) tool (http://imed.med.ucm.es/Tools/sias.html). Phylogenetic tree was constructed for the gene sequences of Mycoplasma species through Neighbor-joining (NJ) analyses with 10000 bootstraps using MEGA 6 (Tamura et al., 2013).

Data submission

The DNA sequences obtained from the field isolates were submitted to the GenBank sequence database of NCBI for assigning the accession numbers.

Results

Prevalence of Mycoplasma species in goats

A total of 800 randomly collected nasal samples during the study period were subjected to the bacteriological examination for confirmation of the Mycoplasma infection. Initially, the nasal samples were inoculated into Mycoplasma specific PPLO broth and subsequently after observing the bacterial growth, the broth were filtered through 0.22 µM pore size filter in order to achieve purity in the samples through filtering large sized bacteria. Further, the filtered media were streaked on PPLO agar plates. The growth of the Mycoplasma was observed and differentiated through typical “fried egg” with an average colony diameter of 0.1–0.5 mm on day 14 (Fig. 2). For further confirmation of the presence of Mycoplasma infection, the growth inhibition test was employed on all samples which shows any growth on PPLO media. Considering the typical fried egg colonies and growth inhibition test, a total of 48 nasal samples out of 800 were found positive for the presence of either species of Mycoplasma which indicates 6.0% prevalence in the study area. Furthermore, the data regarding the prevalence of Mycoplasma among positive samples indicates that the higher prevalence was recorded in Tharparker district (35%) followed by Thatta (27%), Jamshoro (21%) and Larkana (17%) (Fig. 3, Table I).

Table I. The prevalence of Mycoplasma infection in goats from different districts of Sindh Pakistan.

Risk factors	Districts
Risk factors	Thatta	Tharparkar	Jamshoro	Larkana
Sex
Male	4.16%	8.33%	4.16%	2.08%
Female	22.91%	27.08%	16.66%	14.58%
Age
<1 year	4.16%	6.25%	4.16%	2.08%
1-2 year	10.41%	12.50%	6.25%	8.33%
>2 year	12.50%	16.66%	10.41%	6.25%
Breeds
Teddy	8.33%	8.33%	4.16%	2.08%
Tapri	12.50%	16.66%	6.25%	6.25%
Pateri	4.16%	6.25%	4.16%	4.16%
Kamori	2.08%	4.16%	6.25%	4.16%

Molecular detection of isolated Mycoplasma

Following the traditional techniques based identification, all the positive samples were subjected to the PCR amplification for molecular identification using different sets of primers targeting Mycoplasma species. The results shows that PCR successfully amplified the target fragments in all isolated samples with two sets of primers (Fig. 4). During the PCR, initially the universal sets of primers targeting all Mycoplasma species were used for confirmation of the presence of either species of Mycoplasma. The data indicates that the primer pair targeting 30S ribosomal protein S7 gene successfully amplified a 574 bp fragment from 31 out of 48 samples while the primer pair targeting 16S rRNA gene found amplifying 548 bp fragment in 17 out of 48 samples. While, unfortunately no any species specific primer amplified any samples which makes more complicated the identification of specific Mycoplasma species involved in the infection (data not shown).

Sequence analysis

To further confirm the species involved in the infection in study area, the amplified fragments were further sequenced for DNA based identification. The obtained sequences shows that all the samples amplified using primer set targeting 30S ribosomal protein S7 gene resulted in the yield of identical sequences belonging to the M. mycoides subsp. capri (Fig. 4), while the primer set targeting 16S rRNA gene resulted in the two highly similar sequences belonging to M. capricolum subsp. capripneumoniae (Fig. 5). Due to identical sequences, one representative sequence from M. mycoides and two from M. capricolum were deposited in the GenBank of National Center for Biotechnology Information (NCBI) with the accession numbers; OP352532 (M. mycoides), OP243453 and OP243454 (M. capricolum).

The sequences obtained from the current study were aligned with the sequences of other isolates of Mycoplasma for comparisons. The multiple alignment of sequences shows that all the obtained sequences possess high sequence similarity with the strains isolated from different parts of the world (Figs. 4, 5). Further, the sequence similarity and identity analysis indicate that both the species identified in the current study found to have high nucleotide sequence similarity with M. mycoides and M. capricolum isolated from different sources and regions of the world. M. mycoides shows a high sequence similarity with the M. mycoides subsp. capri strain GM12 originally isolated from nasal samples of goat from Turkey and Switzerland (Table II). However, M. capricolum showed a high sequence similarity with M. capricolum subsp. capripneumoniae strain 033C1 isolated from lung samples of goat also from Turkey (Table III).

Phylogenetic tree analysis

The phylogenetic analysis indicate that the isolates belonging to the M. mycoides were sub-clustered in the same clade as with the isolates from M. mycoides subsp. capri isolated from nasal discharges of the goat (Fig. 6A). While, the isolates from the M. capricolum were clustered along with the isolates from M. capricolum subsp. capripneumoniae (Fig. 6B).

Table II. Sequence identity percentage of the 30S ribosomal protein S7 gene of isolated Mycoplasma mycoides as compared to the reference and field strains of other related species.

		1	2	3	4	5	6	7	8	9	10	11	12	13
OP352532-M. mycoides subsp. capri strain GM12 (This study)	1	100.0
LS483519-M. mycoides subsp. capri strain GM12	2	100.0	100.0
CP065581-M. mycoides subsp. capri strain PG3	3	100.0	100.0	100.0
FQ377874-M. mycoides subsp. capri LC str. 95010	4	99.8	99.8	99.8	100.0
CP011264-M. mycoides subsp. mycoides strain Ben468	5	99.3	99.3	99.3	99.1	100.0
CP002107-M. mycoides subsp. mycoides SC str. Gladysdale MU	6	99.3	99.3	99.3	99.1	100.0	100.0
CP041712-M. capricolum subsp. capripneumoniae strain 033C1	7	96.3	96.3	96.3	96.5	96.3	96.3	100.0
CP041711-M. capricolum subsp. capripneumoniae strain AMRC-C758	8	96.3	96.3	96.3	96.5	96.3	96.3	100.0	100.0
CP000123-M. capricolum subsp. capricolum ATCC 27343	9	96.3	96.3	96.3	96.5	96.3	96.3	99.7	99.7	100.0
FR668087-M. leachii 99/014/6	10	96.0	96.0	96.0	96.2	96.0	96.0	97.9	97.9	97.6	100.0
LR739237-M. feriruminatoris 14/OD_0492	11	97.6	97.6	97.6	97.7	96.9	96.9	95.1	95.1	95.1	95.1	100.0
CP003513-M. gallisepticum NC08_2008.031-4-3P	12	67.1	67.1	67.1	67.1	66.7	66.7	66.6	66.6	66.4	66.6	66.7	100.0
CP010546-M. pneumoniae FH	13	57.3	57.3	57.3	57.5	57.8	57.8	57.8	57.8	57.8	57.3	58.0	60.5	100.0

Table III. Sequence Identity percentage of the 16S rRNA gene of isolated Mycoplasma capricolum as compared to the reference and field strains of other related species.

Organism		1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
OP243453-M. capricolum subsp. capripneumoniae (This study)	1	100
OP243454-M. capricolum subsp. capripneumoniae (This study)	2	99.6	100
CP041712-M. capricolum subsp. capripneumoniae strain 033C1	3	99.5	99.1	100
EF373061-M. capricolum subsp. capripneumoniae strain TR01	4	99.5	99.1	100	100
CP041711-M. capricolum subsp. capripneumoniae strain AMRC-C758	5	99.5	99.1	100	100	100
AF202927-M. capricolum subsp. capripneumoniae	6	99.3	98.9	99.8	99.8	99.8	100
MK692954-M. mycoides subsp. mycoides isolate PAK/C499	7	99.1	98.7	99.6	99.6	99.6	99.5	100
MK692953-M. mycoides subsp. mycoides isolate PAK/C354	8	99.1	98.7	99.6	99.6	99.6	99.5	99.6	100
CP065584.M. mycoides subsp. capri strain 7730	9	99.1	98.7	99.6	99.6	99.6	99.5	99.6	99.6	100
HQ661812-M. mycoides subsp. capri LC strain 37	10	93.2	92.9	93.8	93.5	93.8	93.6	93.8	93.8	94.2	100
BX293980-M. mycoides subsp. mycoides SC strain PG1	11	98.7	98.4	99.3	99.3	99.3	99.1	99.3	99.3	99.6	93.8	100
LR739236-M. feriruminatoris 14/OD 0535	12	98.7	98.4	99.3	99.3	99.3	99.1	99.3	99.6	99.6	93.8	99.3	100
FR668087-M. leachii 99/014/6	13	97.6	97.3	98.2	98.2	98.2	98	98.2	98.2	98.5	93.1	98.2	98.2	100
CP001872-M. gallisepticum	14	24	24.3	24	24	24	24	24	23.6	24	24	24.3	23.6	24.3	100
CP039761-M.pneumoniae strain 16-734	15	22.8	23.2	22.5	22.5	22.5	22.5	22.5	22.1	22.5	22.5	22.8	22.1	22.8	89.1	100

Discussion

CCPP is a multifactorial disease of the goats that can cause acute disease and mortality especially in kids which results in economic losses in goat industry (Nicolas et al., 2008; OIE, 2014). The highly contagious nature, chronic and persistent nature of infection and high resistance to antibiotics make this pathogen more successful in causing outbreaks in the host animals (Thiaucourt et al., 1996; Wesonga et al., 2004; Lignereux et al., 2018). Among the goat diseases, CCPP has a long history of affecting goats throughout the Pakistan with outbreaks in each season thus leading to the severe consequences for the farmers (Rahman et al., 2006; Awan et al., 2009; Sadique et al., 2012; Banaras et al., 2016). Due to lack of available data regarding the genetic diversity of Mycoplasma species infecting goats, the current study was designed to explore the species diversity infecting goats in different zones of southeastern province of Pakistan.

During the study, out of 800 randomly collected samples from different districts, only 48 (6%) samples showed growth on PPLO culture media which were further confirmed as either species of Mycoplasma through growth inhibition test and typical fried egg colonies observed on day 7th post incubation. These typical characteristics of the colonies on the similar time duration was also observed by several researchers with the identical colony shape observed for M. mycoides subsp. capri, M. capricolum subsp. capripneumoniae, M. putrefaciens (Houshaymi et al., 2002; Wang et al., 2014; Kabir and Bari, 2015). The lower prevalence rate may be attributed due to random sampling where most of the animals were apparently healthy with no clinical signs. Similarly, another study in cattle from Nigeria also showed 6.27% infection rate of Mycoplasma mycoides small colony (Nwankpa et al., 2008).

During the data analysis, it was found that the highest infection rate was observed in Tharparker and Thatta districts which are supported by the statements from the previous researchers that harsh weather and nomadic nature in these areas may contribute to the persistence of the infection (Mekuria et al., 2008; Shahzad et al., 2012).

Despite the limitations, still culture-based techniques including colony morphology analysis, several biochemical tests are widely used for the identification of Mycoplasma species (OIE, 2004; Adehan et al., 2006). However, arriving at the conclusion that which specific species is responsible for the infection in an area, it is imperative to determine the accurate species level identification in order to properly treat the infection or vaccination against prevalent species.

Due to sophisticated nature and low sensitivity of the serological tests used for the detection of Mycoplasma species in the nasal secretions with cross reactivity makes more difficult to diagnose the infection caused by different species (Samiullah, 2013). The development of DNA based detection of Mycoplasma infection by PCR is used as an accurate and feasible tool for monitoring the infection in goats (Abraham et al., 2015).

In the current study, the culture positive samples were further subjected to the PCR based molecular identification of individual species prevalent in the study area. Herein, all the cultured samples were confirmed by PCR using universal primer sets targeting different genes. The data revealed that only two universal primers targeting 30S ribosomal protein S7 gene and 16S rRNA gene successfully amplified target fragments. Using the universal sets of primers specified for Mycoplasma genus, a total of 31 samples found positive for one primer pair yielding 574 bp fragment targeting 30S ribosomal protein S7 gene, while the 17 samples were successfully amplified through another primer pair yielding a band of 548 bp through targeted amplification of 16S rRNA gene. The sequence analysis revealed that all the samples amplified through the targeted amplification of 30S ribosomal protein S7 gene are of M. mycoides subsp. capri, while the primer set targeting 16S rRNA gene belong to M. capricolum subsp. capripneumoniae. Interestingly, both of the primer pairs are genus specific but the results indicated that these primers amplified only targeted species. The data showed a higher abundance of M. mycoides in goats from the study area. However, the findings of the some researchers confirmed through PCR based identification in the country indicates a higher prevalence rate (20.59%) of Mycoplasma in different zones of Khyber Pakhtunkhwa (Shah et al., 2016). While, the molecular detection of infection in goats from Balochistan indicates almost similar pattern of prevalence (maximum of 9%) in different districts of the province (Awan et al., 2012; Ejaz et al., 2015). Moreover, another study focusing on different districts of Punjab and Khyber Pakhtunkhwa provinces reported only 2% infection rate through PCR based confirmation with the goats being infected by M. mycoides subsp. capri (Shahzad et al., 2012). Furthermore, the similar study focusing on molecular diagnosis of mycoplasmosis in goats from Gilgit-Baltistan and Khyber Pakhtunkhwa provinces indicates 8% prevalence rate (Ahmed et al., 2021). In contrast to the present study, previous reports indicates the prevalence of M. putrefaciens in Khyber Pakhtunkhwa and Balochistan provinces which was although very low (⁓1-8%) but at least shown a presence of infection in the country (Awan et al., 2012; Ejaz et al., 2015; Shah et al., 2017). The variation in the prevalence rate may be attributed to the different geographic locations, climatic conditions, sampling seasons, breeds and husbandry practices.

Further, the molecular analysis based on sequence similarity showed 99% similarity of identified Mycoplasma mycoides with different strains of M. mycoides isolated from different regions of the world, while M. capricolum shares almost 96% similarity with the identified M. mycoides isolates. However, 16S rRNA gene sequences amplified from the M. capricolum from the present study showed a highest similarity (99%) among both Mycoplasma species infecting goats. This may be due higher sequence identity of the target gene amplified.

Although, lack of available data regarding DNA sequences obtained from different regions of the country, this is rather difficult to infer an evolutionary relationship among isolates from different vicinities. However, the obtained sequences from Mycoplasma mycoides showed a close relationship with the isolates from Turkey and Switzerland (accession no. LS483519, CP065581 and CP041712). In addition, M. capricolum isolates obtained during the current study showed a close relationship with the M. mycoides (accession no. MK692953 and MK692953) isolated of from the Punjab province as these are clustered in the same clade (Anjum et al., 2020). These data indicates that both of these species might be originated from the same species and geographical location. However, this may be confirmed through the availability of more sequences from different geographical origins. The PCR based assay followed by sequencing made more precise identification of different strains of a variety of Mycoplasma species which will further add in the development of vaccine against commonly prevalent species.

In addition, the goats in the study area are usually kept in open entities with grazing in different pastures which further predispose them to the infection due to interaction with different herds during grazing. Further, the nomadic nature of the farmers and animal trade play a key role in the maintenance and spread of the infection in the endemic area. Such type of findings are also observed by different researchers that the husbandry practices play a key role in the transmission of the infection among herds (Mekuria et al., 2018; Sadique et al., 2012).

Up to date, there is only one vaccine against M. mycoides subsp. capri is available in Pakistan which maybe the reason of failure to protect infection as the other members of the group may cause infection (Sadique et al., 2012). This was also confirmed by sequencing of the organism from the killed vaccine available in the province.

Conclusion

Taken together, data revealed that CCPP is an endemic disease of goats in the Sindh Province. The disease in the studied population is caused by two species namely M. mycoides subsp. capri and M. capricolum subsp. capripneumoniae. The results indicate that there are multiple genotypes and species associated with this disease in the study areas as well as in the country which may vary depending on region as well as other risk factors such as husbandry practices or environmental conditions which can influence its spread or severity within an area or population group.

Declarations

Acknowledgement

We are highly thankful to the Dr. M. Bachal Bhutto Chairman Department of Veterinary Parasitology for facilitating the research work in the Molecular Biology Laboratory. We are also grateful of the staff at Vaccine Production Unit Tandojam for providing assistant in Mycoplasma culture.

Funding

The study received no external funding.

Ethical statement and IRB approval

All the experiments conducted were pre-approved through Ethical Committee of Directorate of Advanced Studies, Sindh Agriculture University Tandojam, Pakistan through letter no. DAS/1884/of 2021 dated: 18-06-2021.

Data availability

The datasets generated and submitted during the current study are included in this published article. While, the raw data can be made available by the authors upon request.

Statement of conflict of interest

The authors have declared no conflict of interest.

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