Determination of Anti-Mycoplasma Capricolum Subsp. Capripneumoniae Antibodies For The Sero-Epidemiology of Contagious Caprine Pleuropneumonia

Contagious caprine pleuro-pneumonia (CCPP) is fatal disease of goats and causes huge economic losses due to high morbidity and mortality. CCPP is enlisted as notiable animal disease by OIE. The causative agent of CCPP is Mycoplasma capricolum subsp. capripneumoniae (Mccp). The present study was aimed to investigate the seroprevalence of CCPP in northern Pakistan. The study areas were divided into three zones; northern zone, central zone and tribal zone. A total of 1300 serum samples were collected during November 2017 – April 2019 from goats of different age and sex and were analysed by monoclonal antibody based cELISA. The analyses revealed 227 (17.5%) samples positive for anti-Mccp antibodies. The zone wise distribution of CCPP in goats was signicantly different (P(cid:0)0.05), indicated by positive sera for Mccp of 23% animals from northern zone followed by 15% and 13% animals from tribal and central zones, respectively. The analysis of data showed non-signicant values in the seroprevalence among bucks and doe(s), indicated by anti-Mccp sera from 16.6% bucks and 18.3% doe (s). Moreover, among different age groups the prevalence of disease in adult goats (20%) was signicantly (P(cid:0)0.05) higher than kids (10.8%). It is evident from the present study that CCPP caused by Mccp is prevalent in Pakistan and both sex of animals are equally susceptible to Mccp infection. Furthermore the disease is more prevalent in northern zone.


Introduction
Goats farming in developing countries like Pakistan face several problems including poor husbandry practices, extreme environmental stresses, and de ciency of good quality feed stuff and various types of infectious, parasitic and various other non-corrosive diseases. Among the infectious bacterial diseases, the mycoplasmal diseases are well known all over the world for its pathogenicity and high economic loss directly affecting farmers (Regassa et al., 2010). The prevalence of caprine mycoplasmosis has been documented from different region of the globe, however the occurrence of these diseases is more frequent in Asia and Africa and are considered the major constraint to goat industry in terms of high morbidity and mortality (Tigga et  putrefaciens are involved to induce manifold pathological condition in small ruminants like mastitis, arthritis, keratoconjunctivitis, pneumonia and septicaemia, which is usually term as 'MAKePS' (Thiaucourt and Bolske, 1996).
In Pakistan, Mmc was rst detected among Mm cluster by applying various tests on clinical samples from infected goats suspected for CCPP (Khan et al., 1989). In district Pishin of Balochistan, goats ocks were investigated for CCPP based on clinical examinations, by biochemical, growth inhibition and PCR on the collected samples (nasal swabs, lungs, liver, and intestinal tissues), two mycoplasma species i.e. Mcc and Mycoplasma putrefaciens (MP) were found prevalent in the affected goats (Awan et al., 2008). The prevalence of respiratory mycoplasmal infection in different region of Pakistan was reported by late agglutination (LAT) test for Mccp, growth inhibition test (GIT) for Mmc and PCR for Mm cluster and found forty eight samples positive for Mmc on GIT, none of the serum sample was detected positive for Mccp, whereas thirty-ve samples were con rmed as Mm cluster by molecular technique (Shahzad et al., 2012). Similarly, Mmc was also found in samples collected from goats having similar signs of CCPP This project was designed to investigate the serological prevalence of CCPP the Khyber Pakhtunkhwa Province and northern areas of Pakistan for effective therapeutic interventions and control strategy to prevent CCPP outbreak in goat's population.

Study Area
This study was performed across the Khyber Pakhtunkhwa, Gilgit-Baltistan, Pakistan. The selected districts along with tribal districts were divided into three different zones namely, northern zone, central zone, tribal zone. Northern zone includes Gilgit-Baltistan, Chitral, Swat, Buner, and Hazara. Central zone includes Charsadda, Mardan, Swabi, Peshawar, and Nowshera districts. The tribal zone includes the tribal districts, Khyber, Bajour, and Mohmand. The climatic condition of the northern zone is extremely cold with heavy rainfall and snowfall in winter season. The weather is pleasant in summer days and extremely cold in winter season. The tribal region containing federally administrated areas (Now part of Khyber Pakhtunkhwa, Province) has borders with Afghanistan and has extreme climate in summer and winter.
The climatic condition of the Central zone is hot humid (Fig. 1).

Sample size
A total of 1300 serum samples were collected from goats during November 2017 to April 2019. All the samples were collected based on clinical signs i.e.mucopurulent nasal discharges, productive cough, deep abdominal respiration (Fig. 2), pyrexia (40-41 o C) and history of respiratory infection and no vaccination record against CCPP. The samples were collected from different age and sex groups of animals. The number of samples collected from each zone is presented in ( Table 1). The Lab work and analysis of the results were performed in pathology laboratory, College of Veterinary Sciences, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture Peshawar.

Harvesting of serum
The blood samples were collected from the jugular vein of animal's adopting aseptic condition. The blood was poured to gel containing tube and centrifuged for 5 minutes at 5000 rpm. The serum was transferred to sterile eppendorf tube in aseptic environment and was stored at -20 o C until further processing.

IDEXX CCPP Antibody Test
The serum was subjected to cELISA test for the detection of Mccp speci c antibodies. The procedure was performed following the manufacturer instructions (IDEXX). The optical density (OD) values of samples and controls were measured and record at 450 nm wavelength using the ELISA plate reader.

2.4.1: Serum samples absorbance (OD value) analysis:
The absorbance values of Controls, were calculated by the following formulas:

Statistical Analysis
All the data obtained was compiled into the Microsoft Excel spread sheet and analysed through Chi square statistical test. Con dence level was taken at 95% and p value less than 0.05 for signi cance in all analysis.

Results
A total of 1300 samples were collected in different zones including northern, central, and tribal zones from naturally respiratory infected goats which were considered suspected for CCPP. The screening of the samples through monoclonal antibody based cELISA test revealed 227 (17.5%) samples positive for CCPP.

Prevalence of CCPP in different zones
The region wise prevalence of the disease in goats was recorded as 23% in northern zone followed by 15% tribal zone, and 13% central zone. The analysis of the data through Chi square statistical test revealed signi cantly difference (P 0.05), in the prevalence of disease, among different zones of the studied areas (Table 2).

Gender based prevalence of CCPP in different zones
Out of total 1300 samples, 650 serum samples were collected from bucks and 650 from Doe(s). The processing of the samples by cELISA revealed 108 (16.6%) male and 119 (18.3%) female goats positive for anti-Mccp antibodies. The analysis of the data by Chi square test revealed non-signi cant difference (P>0.05), in the prevalence of disease, between different sexes of goats (Table 3).

Age wise prevalence of CCPP in goats
Out of 900 samples collected from adult goats having age above 180 days, the anti-Mccp antibodies by cELISA were observed in 184 (20%) samples. While in young animals (age: day1 up to 6 months), 43 (10.8%) samples revealed reactive antibodies against Mccp pathogen. The prevalence was signi cantly (P 0.05) different recorded between different age group of animals (Table 4).

District wise prevalence of CCPP in studied area
In the northern zone the highest prevalence of the disease was found in animals from Gilgit Baltistan (27.4%), followed by animals from Chitral, Hazara, Buner and Swat 24.4%, 23%, 19.7%, and 16.8% respectively ( Table 5).
The central zone is divided into 05 districts (Charsadda, Mardan, Swabi, Peshawar, Nowshera). The prevalence of Mccp recorded in the present study was higher in Charsadda 17.5% followed by Mardan, Swabi, Nowshera and Peshawar 15%, 13.7%, 11.2% and 7.5% respectively. The lowest occurrence of the disease was recorded in District Peshawar (Table 6). In tribal zone the maximum frequency of occurrence of the CCPP was recorded in tribal district Khyber (18.6% and tribal district Bajour (14%), while the lowest occurrence of the disease was noted in tribal district Mohmand (11%) ( Table 7). respectively. The highest frequency of the disease in the present study was recorded in northern region of Pakistan. This could be due to pastoral practices, extreme cold environmental condition, improper management system for large ock (up to 2000 animals in single ock), and movement of animals in winter season (last of October to start of April) for grazing. These factors may lead immunosuppression and make the goats susceptible for the proliferation of pathogenic Mycoplasma to cause infection. It has been reported that the ock size, management and production system of the small ruminants, carrier animals in the vicinity play a key role in extent of the disease in small ruminants (Sherif et al., 2012).