Clinicopathological and Sero-Molecular Detection of Mycoplasma capricolum subsp. capripneumoniae in Goats in Southern Areas of Pakistan

Contagious caprine pleuropneumonia (CCPP) is a highly fatal infectious disease of goats, caused by Mycoplasma capricolum subsp. capripneumoniae (Mccp). This disease is causing huge economic losses to the goat industry in Pakistan. However, little is known about the epidemiology of CCPP, especially in the hard areas of Khyber Pakhtunkhwa (KP), Pakistan, despite having a huge population of goats. Therefore, this study aimed to elucidate sero-molecular epidemiology and pathology associated with Mccp infection in goats in southern areas of KP including Dera Ismail Khan (DI Khan), Bannu, Karak, and Kohat. A total of 200 (50 from each area) serum samples were collected from clinically infected goats, whereas 600 various samples (nasal swab n = 50, pleural fluid n = 50, lungs n = 50 at each selected area of study) were collected from live goats showing respiratory clinical signs and dead/slaughter goats having lesions in the lungs/pleura. A commercial competitive ELISA kit confirmed anti-Mccp antibodies in altogether 17% of serum samples, while area-wise seroprevalence was recorded as follows: Kohat, 28%, Bannu, 18%, DI Khan, 14%, and Karak, 8%. Moreover, a total of 5.5% of samples collected from clinically positive live and dead goats for Mccp were found by species-specific PCR, whereas area-wise molecular prevalence of Mccp was found in 3% samples from Kohat, 7.33%, Bannu, 6%, Khan, 5.33%, and Karak, 3.33%. Of 400 clinically examined goats, 242 (60%) had nasal discharge, 207 (51%) had pyrexia, 50.75% (203) had coughing, 48.25% (193) had pneumonia, 23% (92) had lacrimation, 7.75% (31) had pneumonia with lacrimation, and 10 (2.5%) showed all signs. Of the total 200 dead/slaughtered goats, pleural fluid was found in 36 goats and consolidation and red hepatization were observed in 40 and 42 goats, respectively. The present study found the presence of prevailing Mccp strain in the goat population of the study area. The highest prevalence of Mccp was found in collected samples from Kohat by PCR. The highest seroprevalence of Mccp was found in serum samples collected from Kohat by ELISA.


Introduction
In Pakistan, the livestock sector is dominated by the largest population of goats (Pakistan Economic Survey 2020-2021). erefore, the goat is known as "poor's man cow" in the subcontinent [1]. e most important threats to livestock population, especially goats, are respiratory diseases worldwide. Among respiratory infections, Mycoplasma-linked infections are responsible for massive economic losses in small ruminants in developing countries [2]. Mycoplasmas are the simplest self-replicating microorganisms, lacking a cell wall; however, they are highly species-specific and successful pathogens [3][4][5]. Mycoplasma-related infections are widely distributed all over the world, almost in all developing countries of Middle East Asia, South East Asia, and Africa [6,7]. e most dreadful respiratory mycoplasmosis in goats is contagious caprine pleuropneumonia (CCPP). CCPP was first clinically reported in 1873 in Algeria [8]. en, in 1881, CCPP was proved as a contagious infection in goats [9]. After a century, in 1976, the actual causative agent of CCPP, Mccp, was first isolated and characterized [10][11][12]. Mccp has been isolated from 13 countries but reported in 40 countries so far [13]. CCPP caused by Mccp is responsible for 100% morbidity and 60-80% mortality in goat flocks [14]. Mccp belongs to the Mycoplasma mycoides cluster. ere are six species and subspecies in the Mycoplasma mycoides cluster [15], which causes disease in small ruminants as well as large ruminants. It shares multiple genomic properties or multiple phenotypic properties [16]. Mycoplasma mycoides cluster is further divided into two subgroups: Mycoides and Capricolum. Mycoides are further divided into three subspecies; Mycoplasma mycoides subsp. mycoides small colony (MmmSC), Mycoplasma mycoides subsp. mycoides large colony (MmmLC), and Mycoplasma mycoides capri (Mmc). Capricolum includes three subspecies: Mycoplasma capricolum subsp. capricolum (Mcc), Mycoplasma capricolum subsp. capripneumoniae (Mccp), and Mycoplasma subsp. bovine 7 th group (BG7) [12,17]. e non-Mycoplasma cluster subspecies are Mycoplasma ovipneumoniae, Mycoplasma putrefaciens, and Mycoplasma agalactiae [7,18].
Mycoplasma capricolum subsp. capripneumoniae is a very fastidious slow-growing Mycoplasma with incubation time ranging from 7 to 10 days. e incubation may vary between 5 and 28 days. e first clinical sign of Mccp infection in goats is high body temperature (41°C) and reluctance to walk but the animal continues to feed intake. en, the respiratory signs appear prominently with painful and deep respiration and frequent coughing. In the advanced stages, the animals are reluctant to move, continue salivation, and exhibit mucopurulent nasal discharge. In some cases, the animals have marked lameness, diarrhea, and nervous signs [19]. e gross pathological lesions associated with Mccp infection are restricted to the pleura and lung. e lungs are usually infected unilaterally, but bilateral infection has also been reported with CCPP [20]. ere is massive red hepatization and pleurisy with fibrinous pleuropneumonia and straw-color pleural fluid. e necrotic areas on the lungs are sequestered and of black discoloration [19]. Histopathological lesions associated with Mccp infections are pulmonary emphysema, condensing of interlobular septa almost in all cases, and atelectasis [19,21]. e identification of Mccp infection is difficult on the basis of clinical signs and symptoms because there is variation in clinical signs and symptoms [22]. Serological tests are most commonly used for the diagnosis of mycoplasmas. e common serological tests are growth inhibition test (GIT), indirect or passive haemagglutination assay (IHA/ PHA), enzyme-linked immunosorbent assay (ELISA), complement fixation test (CFT), latex agglutination tests (LAT), and fluorescent antibodies test (FAT) [23][24][25][26].
Although the PCR is the most sensitive and accurate technique for the diagnosis of Mccp, it is a time-consuming, expensive technique and hardly detects Mccp in treated animals with antimicrobials. On the other hand, ELISA could diagnose the Mccp infection in treated animals as well as recovered animals. erefore, we used a combination of serological, molecular, and pathological techniques for the detection of actual prevalence of Mccp local strain in goats in study areas.

Study Area.
e current study was conducted in southern areas of Khyber Pakhtunkhwa including Khan, Bannu, Karak, and Kohat. ese areas were visited for collection of samples from goats clinically suspected of CCPP.

Collection of Samples.
Samples were collected from the suspected goat population in districts of Khan, Bannu, Karak, and Kohat of Khyber Pakhtunkhwa. A total of 800 samples were collected from clinically suspected and dead or slaughtered goats for CCPP. A total of 200 (50 from each area) serum samples were collected from clinically infected goats, whereas 600 various samples (nasal swab n � 50, pleural fluid n � 50, lungs n � 50 at each selected area of the study) were collected from live goats showing respiratory clinical signs and dead/slaughter goats having lesions in the lungs/pleura. Blood samples were collected from the jugular vein for serodiagnosis of Mccp infection, whereas nasal swabs, lung tissue, and pleural fluid were collected from CCPP suspected goats for molecular detection of Mccp by PCR. Pleural fluid was collected in a sterile tube. Sterile cotton nasal swabs were inserted deep into the nasal passage to get the secretions of the goat. e lung tissue was collected in sterile sealable plastic bags and transported in an ice box to Pathology Laboratory, College of Veterinary Sciences, the University of Agriculture, Peshawar, and stored at −20°C or −86°C freezer until used. For histopathology, lung tissues were collected and transported in 10% buffered formalin to the laboratory.

Sero-Epidemiological Analysis of Mccp Infection by ELISA.
e serum samples were tested with a commercially available competitive ELISA kit (IDEXX-USA). e manufacturer's protocol was followed for cELISA.

Genomic DNA Extraction and Quantification.
Genomic DNA (gDNA) was extracted using GeneJET Genomic DNA Purification Kit made by ermo Scientific, USA.
e DNA extraction procedure was followed according to manufacturer instructions. e extracted gDNA was quantified with Nanodrop ( ermofisher-Finland). e DNA was diluted according to the desired level for PCR as reported elsewhere [27,28].

Selection of Primers for PCR.
e following set of primers was used for the detection of Mccp as reported previously [28][29][30]. Specific primers of Mycoplasma capricolum subsp. capripneumoniae were used for targeting a specific Mccp gene and generating an amplicon of 316 bp: Mccp.spe-F: 5′-ATC ATT TTT AAT CCC TTC AAG-3′  Mccp.spe-R: 5′-TAC TAT GAG TAA TTA TAA TAT  ATG CAA-3′ 2.4.3. Preparation of PCR and Conditions. PCR was performed using a PCR ermal Cycler (Bio-Rad T100 USA). A total of 25 µl PCR reaction was prepared consisting of 1.75 µl forward primer and 1.75 µl reverse primer, 8 µl nuclease-free water, 10 µl PCR Master Mix, and 3.5 µl DNA template [28,31]. Initial denaturation of template DNA was done at 94°C for 5 min followed by cycles of denaturation at 94°C for 30 sec, annealing at 53°C for 60 sec, and extension at 72°C for 90 sec and final extension was performed at 72°C for 5 min.

Gel Electrophoresis.
PCR product was run on 1.5% agarose gel. After loading 6 µl of PCR products, including samples, positive control, negative control, and DNA ladder of 1 kb or 100 bp, agarose gel was run on the gel for 35 min at 120 V. e PCR products were visualized by the gel documentation system (FastGene, Germany).

Histopathological Study.
Tissue samples (Trachea and lungs) were collected and preserved in 10% buffered formalin. Tissues were processed according to the standard protocol as adapted in [32,33].

Statistical Analysis.
All the data was collected and arranged in a Microsoft Excel worksheet. e collected data were then subjected to Statistical Package SPSS v20. A Chi-Square test was performed for analyzing the data. e overall seroprevalence of Mccp was found at 17% in southern areas of KP Pakistan. Statistical analysis by χ2 showed a significant association (P > 0.05) among four districts (Tables 1-2, Figure 1).

. Clinical Manifestation of Mccp Infection in Goats.
A total of 400 goats (100 in each district) were examined for clinical signs of CCPP including body temperature, coughing, nasal discharge, lacrimation, conjunctivitis, and arthritis. In a total of 400 goats, 242 (60.5%) animals showed nasal discharge, 51% exhibited pyrexia (103-104°F), 51% showed cough, and 48% showed signs of pneumonia. Conjunctivitis was recorded in 24% of animals, while lacrimation and arthritis were recorded in 23% and 3% animals, respectively. Only 8% of animals showed both pneumonia and lacrimation concomitantly, whereas 3% of animals showed all clinical signs mentioned above (Table 5, Figure 5).

Gross Pathology.
Pathological investigations (gross and histopathological) were carried out on tissue samples from a total of 200 necropsied/slaughtered animals. e tissue samples were collected from different slaughterhouses. A total of 50 tissue samples were collected from each area (Dera Ismail Khan, Bannu, Karak, and Kohat). Lung tissue samples from animals (n � 26) at DI Khan were grossly normal, whereas samples from animals (n � 24) showed various gross lesions including consolidation of the affected lungs, red hepatization (unilateral), and adhesion of the lungs with the thoracic cavity. Tissue samples from animals (n � 34) at Bannu were grossly normal, whereas samples from 16 animals were infected showing pleural effusion (n � 9), red hepatization (n � 7), and adhesion of the lungs with the thoracic cavity (n � 9) from infected animals. From Karak, tissue samples from animals (n � 41) were grossly normal, while 9 animals who were grossly infected exhibited different lesions that includes pleural fluid (n � 4), red hepatization (n � 5), and adhesion of the lungs (n � 5). From Kohat, samples from 23 animals were normal, while 27 had gross lesions (Table 6, Figure 6).

Discussion
Mycoplasmosis causes serious threat and massive economic losses in small ruminants (sheep and goats) in developing countries [2,34,35]. Mycoplasmosis causes high morbidity and mortality [2]. Mycoplasmosis is a pathogenic bacteria for multisystems and is collectively caused by Mycoplasma mycoides cluster. Mycoplasma is highly prevalent all over the world almost in all developing countries of Middle East Asia, South East Asia, and Africa [6,7]. Mycoplasma is the smallest and slow-growing bacteria. It can cause disease in different species of animals and also cause disease in humans. Mycoplasma causes respiratory disorders, genital disorders, eye lesions, arthritis, and mastitis [36,37]. Mycoplasmosis in goats is known as CCPP. e targeted area for Mccp in the host is the respiratory system and is restricted to the thoracic cavity [15].  Veterinary Medicine International e species-specific primers have enabled an advanced technique to be applied directly to the clinical samples, i.e., nasal swabs, fluid samples, and tissue samples [38,39]. A total of 600 different samples were collected from naturally dead and slaughtered goats from different study areas. ose samples were subjected to PCR for the identification of Mycoplasma capricolum subsp. capripneumoniae. Of 600, only 33 samples were from nasal discharges, pleural fluids, and lung tissues. 10 (5%), 15 (7.5%), and 8 (4%) were detected with Mycoplasma capricolum subsp. capripneumoniae. Of 150 samples, 8 (5.33%) from Khan, 9 (6%) from Bannu, 5 (3.33%) from Karak, and 11 (7.33%) from Kohat were positive from nasal discharges, pleural fluid, and lung tissues on PCR analysis. e total positive percentage of Mycoplasma capricolum subsp. capripneumoniae on PCR was 5.5%. Similar studies were also conducted in [40][41][42][43]. e remaining samples were found negative on PCR. at might be due to other Mycoplasma cluster species that cause diseases in goats and them presenting similar clinical signs.
e cELISA results showed 17% overall seroprevalence at the southern areas of KP. Of 50 serum samples, 7 (14%) from DI Khan,9 (18%) from Bannu, 4 (8%) from Karak, and 14 (28%) from Kohat were positive. e remaining 166 serum samples were negative on cELISA. e same study was reported in [34,44,45] in the northern areas (Swat and Buner) of KPK, Pakistan. But the results were in contrast because samples were collected randomly from goats, while in the current study, the serum samples were collected from suspected goats. It means that the disease is prevalent throughout the country. Area-wise distribution of the Mccp was more prevalent in Kohat as compared to Khan, Bannu, and Karak. e statement is justified by the inhabitant of the small ruminants of former nomads, constantly moving from place to place in search of pastures. e reason contributed to stress, which is predisposed to Mccp infection in goats. And these nomads cross the border easily and enter the area which leads to transboundary transmission of the disease; the same observation was also reported by the finding in [42]. e third objective of the work was the clinicopathological study of Mccp in southern areas of Khyber Pakhtunkhwa Pakistan. In the present study, a total of 400 goats were examined for clinical signs and symptoms. e respiratory signs were common features for infected goats followed by pyrexia found in 207 (51%) goats, coughing in 203 (50.75%), pneumonia in 193 (48.25%), nasal discharge in 242 (60.5%), lacrimation in 92 (23%), arthritis in 97 (24.25%), and pyrexia, coughing, nasal discharge, and lacrimation combinedly found in 31 (7.75%), while all of the above signs were found in 10 (2.5%) goats. Similar signs were reported by many researchers [15,34,40,46]. ese findings are further supported by that of mycoplasma-infected goats showing high body temperature, painful respiration, and persistent cough [42]. It is justified that most of the Mycoplasma species present similar signs and symptoms.
Pathological lesions play an important role in the proper diagnosis of the disease. Pathological lesions provide evidence for pathologists to evaluate the severity of infection. e present study was carried out for the necropsy of a total   Veterinary Medicine International of 200 goats across the southern areas (Khan, Bannu, Karak, and Kohat) of Khyber Pakhtunkhwa. e lung lesion was recorded in 38% of dead/slaughtered goats comprising accumulation of straw-color fluid in the pleural cavity called pleural fluid (36 (18%)), consolidation, red hepatization (40 (20%)), cranio-ventral pneumonia, and unilateral infected lungs (42 (21%)) recorded in the thoracic cavity. e similar lesions were recorded in [19,20,40,47,48]. e Mccp infection is restricted to the thoracic cavity, and this is why the lesions are mainly limited to lung tissues. e histopathological lesions were found different in different lungs tissue samples from various number of goats including pulmonary emphysema was recorded in lung tissues rom 33 dead goats, leucocytic infiltration in 72, atelectasis in 30, and thickening of interlobular septa were observed in lung tissues from 34 goats . ese observations were closely related to the findings of many researchers [19,40,42,[48][49][50][51]. Data Availability e data presented in this study are deposited and made publicly available in an acceptable repository, prior to publication.

Conflicts of Interest
e authors declares that they have no conflicts of interest.

Authors' Contributions
FUR, and FAK designed and conceived the study. FUR, HK, FA, MS, QU, and MA carried out the research. FUR, FAK, and MA analyzed the data. FUR, FAK, and HK wrote the manuscript. FAK, and FUR critically reviewed and revised the manuscript.