CONTAGIOUS CAPRINE PLEUROPNEUMONIA

To My Quyen, Tran Duy Thanh, Pham Trang Thanh Nguyen, 

Nguyen Khanh Thuan, Nguyen Phuc Khanh, Nguyen Thanh Lam*

  1. Introduction

  Contagious caprine pleuropneumonia (CCPP) is an important respiratory disease of small ruminants and causes major economic losses in many geographical regions of the world, particularly in Africa and Asia. The disease is caused by Mycoplasma capricolum subsp. capripneumoniae (Mccp), formerly known as Mycoplasma strain F38—a member of the genus Mycoplasma which belongs to the Mycoplasma mycoides cluster (Leach et al., 1993). CCPP is highly contagious and is spread rapidly by close contact and through inhalation of contaminated droplets. The disease is often associated with high morbidity (100%) and mortality (80%) (Rurangirwa et al., 1981). The disease is included in the list of notifiable diseases by the Office International des Epizooties (OIE, 2020). Given the passive surveillance approaches, limited resources, and limited diagnostic capacity in many parts of Africa and Asia, the current prevalence figures are probably an underestimate. Nevertheless, prevalence data retrieved from the OIE specific WAHID interface (Figure 1) over the last 10 years suggests that there has not been any progress towards control of CCPP, especially on the African continent (Jores et al., 2020). 

2. Aetiology

CCPP is caused by Mycoplasma capricolum subspecies capripneumoniae abbreviated as Mccp. Previously, it was known as Mycoplasma biotype F38 (Leach et al., 1993). These pathogenic bacteria belong to the class Mollicutes, which lack cell wall but have galactan and small genomes (0.58–1.35 Mb). They have limited biosynthetic capability and cause a number of infections in animals. The four lineages of mycoplasma correspond to different geographic regions. Mccp is placed in Mycoplasma mycoides cluster (Suliman, 2015) and has different species and subspecies, namely Mycoplasma mycoides subsp. mycoides large colony strains (MmmLC), Mycoplasma mycoides subsp. mycoides small colony strains (MmmSC), Mycoplasma sp. bovine group 7 of Leach (Mbg7), Mycoplasma capricolum subsp. capricolum (Mcc), and Mycoplasma mycoides subsp. capri (Mmc). Of these members, some cause similar diseases in sheep and goats but have extrapulmonary involvements also. Disease caused by M. mycoides subsp. capri are different than CCPP as was previously erroneously believed (Cottew et al., 1987; Leach et al., 1989). 

Table 1: Mycoplasma mycoides cluster (Suliman, 2015) 

3. Epidemiology

3.1 Susceptible hosts

Goats (Capra hircus) are considered naturally susceptible domestic species, and sheep can also be affected (Bölske et al., 1995), which is yet to be well established. Bovidae family are also considered susceptible. In addition, many wild animal species have been found naturally susceptible to CCPP. Various wild animal families or orders among ungulates are quoted as susceptible too (Velarde, 2017). CCPP has been reported from various wild animals like wild sheep, wild goat, gazelle, Tibetan antelope, Arabian oryx and sand gazelles. Though CCPP may affect other wild species, the susceptibility of these species to Mccp has not been worked out (Velarde, 2017).

3.2 Transmissions

Inhalation of infected aerosols is the main route of transmission. The main source of contamination is direct contact with affected animals (Thiaucourt and Bölske, 1996). Airborne transmission can result in distant spread (Lignereux et al. 2018) with a 50 m distant transmission reported. Infected objects, vectors, fomites and animal products are yet to be known in transmission role (Velarde, 2017). Under cold, moist and overcrowded environment pathogen can persist longer and may lead to severe outbreaks. Shorter survival time (3–14 days) in external environment limits transmission of Mccp. Higher temperature inactivates Mccp rapidly (within 60 min at 56°C and within 2 min at 60°C). However, low temperature prolongs survival. Mccp can survive for 10 years in frozen infected pleural fluid. Moisture and humidity (Wright et al., 1968) also affect survival and hence transmission of mycoplasma. The details of the transmission of  Mccp are given in Figure 2 (Iqbal Yatoo et al., 2019). 

4. Pathogenesis

Mycoplasma are extracellular pathogens of mucous membranes, and it is believed that they may attach to epithelial cells (Nicolet, 1996). Adhesion of pathogen to host cells favors colonization for setting up of infection. Metabolic activity of mycoplasma releases free radicals like hydrogen superoxide and super oxide radicals, which can damage cilia or the membranes of cells. These events may be related as in other mycoplasma species (Almagor et al., 1984; Tanaka et al., 2014). Capsules noted in some mycoplasmas may play an important role in this aspect, especially the galactan of Mccp. The role of other mycoplasmal structures, for example, biological membranes is yet to be elucidated in the pathogenesis of the disease. 

Though the activation of the host immune system by Mccp is yet to be elucidated, it has the strong support of playing an important role in the pathogenesis of CCPP. Immune cell stimulation by antigens of Mccp can aggravate or suppress immunity. The mitogenic stimulation of host immune cells especially lymphocytes, their antiphagocytic activity, suppression of immunity and auto-immune phenomena by antigens are well studied. Following stimulation, production of inflammatory mediators like proinflammatory cytokines, such as the tumour necrosis factor alpha (TNF-α), interleukins and interferon γ by immune cells (macrophages and monocytes) due to interaction with mycoplasma (Sacchini et al., 2012) has been reported for some but is yet to be worked out for Mccp. Mycoplasmal structures are mainly involved in this interaction and cytokine production. Biological membranes of mycoplasmas including plasma membrane, and their components like lipoproteins and lipids are believed to induce cytokine secretion. However, this has not been elaborated for Mccp yet though for other mycoplasmas a mechanism considered different than the bacterial lipopolysaccharides are thought to be involved (Nicolet, 1996). In this preliminary studies, an increase was noticed in levels of proinflammatory cytokine TNF-α, increase in total oxidant status and associated decrease in total antioxidant status in CCPP affected Pashmina goats suggesting inflammatory reaction and oxidative stress. This entire pathological mechanism might be initiated by antigens of Mccp which need to be explored. The pathogenesis of Mccp has been detailed in Figure 2 (Iqbal Yatoo et al., 2019).

5. Clinical signs and pathology

5.1 Clinical signs

The clinical signs were almost similar in most cases with slight variations. The encountered clinical signs were fever (40.5–41.5°C), anorexia, depression, severe respiratory distress in forms of dyspnoea, painful breathing, mouth breathing, frequent painful coughing and grunting. Death of many cases was observed within five days and few cases recovered.

5.2 Pathology

5.2.1 Macroscopic pathology

In most cases, the post-mortem lesions were observed confined to the thoracic cavity, which were considered indicative of CCPP. The majority of them were with one lung affected, which is covered by thick layer of fibrin. The prevailing lesions encountered were: fibrinous pleuropneumonia, pleurisy, consolidation and hepatization associated with pleural adhesions and effusions in some cases, and hydrothorax and straw colored pleural exudates (Saeed and Osman, 2018). 

Experimental study: 

In the peracute form, 9 peracute cases suddenly died (5 goat and 4 sheep), and the lungs of these animals showed marked lung marbling (Figure 4a). However, 30 animals in the acute stage of the disease suffered many clinical manifestations, including fever (42°C), coughing, dyspnoea, labored respiration, copious nasal discharge, and corneal opacity (Figure 4b); 20 goats and 2 sheep died within 9 days. The postmortem lesions in these animals showed lung marbling, straw-colored exudate within the thoracic cavity, and thickening of interlobular septa. Moreover, 44 animals showed a chronic stage of disease, exhibiting a non-feverish condition, coughing, emaciation, and stunted growth; 5 goats and 2 sheep died after 40 days (not responding to treatment). Sequestra (Figure 4c) and adhesions between the lungs and pleurae were detected upon postmortem examination of animals chronically infected with CCPP (Abd-Elrahman et al., 2020).

5.2.2 Microscopic pathology

Pathologic examination of lung tissues from CCPP infected animals revealed different stages of inflammation ranging from per-acute to acute, sub-chronic, or chronic broncho- and pleuropneumonia (Figure 5). Severe congestion of interalveolar capillaries with flooding of alveolar spaces and bronchioles with serofibrinous or fibrinous exudate and acute inflammatory cells were evident in peracute and acute infected tissues (Figure 5a, b). Examination of pleurae in different cases revealed that the visceral layer of the pleura was distended by a serofibrinous exudate, while the parietal layer showed fibrous tissue organization admixed with inflammatory infiltration (Figure 5c).

The interlobular septum was distended by a serofibrinous exudate, congested blood vessels, and inflammatory infiltrates (Figure 5d). Additionally, necrosis and fusion of the bronchial epithelium with extensive peribronchial and sub-epithelial infiltration of lymphocytes was also observed (Figure 5e). Additionally, interstitial lymphoplasmacytic infiltrations and multifocal infiltrations of lymphocytes in the form of folliclelike aggregates in the peribronchial and perivascular areas were observed (Figure 5f), with mild vasculitis and formation of intravascular thrombi of adjacent blood vessels observed in subchronically and chronically infected animals (Figure 5g). In addition, activation of intra-alveolar and intra-bronchial macrophages was described in lung tissues from chronically infected animals (Figure 5h). Formation of multifocal areas of caseous necrosis as a result of vasculitis and thrombus formation was noticed; these necrotic areas contained eosinophilic necrotic tissue, necrotic neutrophils, and cellular debris and were surrounded by a zone of inflammatory cells and encapsulated in thick fibrous tissue capsules (Figure 5i, j) (Abd-Elrahman et al., 2020). 

6. Diagnosis

6.1 Isolation and identification

Isolation of Mccp is considered as a confirmatory diagnosis but is a difficult task and requires technical expertise for proper isolation and identification. This being a tedious job not only because of the practical difficulty of obtaining appropriate samples but also of the fragile and fastidious nature of the pathogen (Thiaucourt and Bölske, 1996). Besides difficult isolation and highly technical expertise, Mccp requires a very special growth medium, well-equipped and sophisticated laboratory facility as the pathogen is very fastidious and requires a prolonged initial incubation for at least 5–7 days (average 4 days) at 37 °C with up to 5% carbon dioxide under sterile laboratory environment (Freundt, 1983). Sampling, culture, and isolation are done as per standard procedures (OIE, 2014) or depending on regional modifications. Nasal discharges, swabs, pleural fluid, lung tissue or pleural tissue are appropriate samples for isolation. Media, liquid broth or solid agar either readymade or prepared in laboratory generally contains nutrients or supplements that can support amino acids, nucleotides and lipid synthesis as Mccp lacks the capability of synthesizing these essential nutrients. Media usually contains beef heart, infusion (6.0 g), peptone (10.0 g), sodium chloride (5.0 g), agar (14.0 g) and supplemented by yeast extract (0.09 g) and horse serum (22.8 ml).

6.2 Haemato-biochemical parameters

Hematological changes were studied in CCPP-affected goats (Abdelsalam et al., 1988). Usually, in field cases, hematological findings are not so much relevant to the diagnosis of the disease, but anemia and leukocytosis followed by leucopenia have been noted in animals affected with mycoplasmosis (Mondal et al., 2004). Lower blood total protein, albumin and elevated ASAT, ALAT, calcium, glucose, and globulin have been reported in mycoplasma affected goats in comparison to control ones (Mondal et al., 2004).

6.3 Sero-molecular tests

Molecular tests have become the novel diagnostic interventions for CCPP not only because of high specificity and sensitivity, but also because of difficulties in culturing of Mccp. Globally, various molecular tests are being employed for diagnosis of CCPP. Broadly they are serological and amplification-based tests, and their diagnostic capability depends on antigens, antibody, and genes/DNA either of Mccp or against it (antibody). Agglutination tests (non-specific and specific), immunoassays (enzyme-linked immunosorbent assay [ELISA]), fluorescent antibody assay (FAT), complement fixation assay (CFT), passive or indirect haemagglutination test (IHT), amplification tests (PCR, hybridization, sequencing) are the currently employed diagnostic tests with latex agglutination, cELISA, and PCR being of prime importance and routinely used in global studies. However, the field level availability or even absence in research facilities in most of the countries is a major limitation. Moreover, cost involvement is another binding on use in limited spheres (Liljander et al., 2015). Nevertheless, novel sensitive and specific, field applicable and cost-effective versions of these diagnostic tests are utmost important and focused on for development.

7. Treatment

Several antibiotics have been used against Mccp for the treatment of CCPP (Onoviran, 1974; Yatoo et al., 2018). However, macrolides especially tylosin is considered to be the drug of choice against Mccp. Tylosin is usually rarely available in the areas where CCPP is prevalent. The use of oxytetracycline against CCPP has proven effective and has been used for quite long time but usage over long period in most of the areas can predispose to side effects (teratogenicity in goat kids) besides the risk of antibiotic resistance with additional risk being imposed by prophylactic usage that too in minimal doses enabling mycoplasma to better cope up the antibiotics. Though other antibiotics like fluoroquinolone (enrofloxacin, danofloxacin), aminoglycosides (streptomycin), and pleuromutilin (tiamulin) are being used and newer ones among these classes being explored against Mccp but cost, availability, acting period and convenience in use and creation of carrier state are major determinants in the application of antibiotics in CCPP prevalent areas. Earlier combination of dihydrostreptomycin sulphate with penicillin G procaine was used for treatment of CCPP at a dose rate of 30 mg/kg BW for each intramuscularly (Rurangirwa et al., 1981). Dihydrostreptomycin sulphate at a single dose of 20, 30, 40 or 50 mg/kg BW intramuscularly was able to cure goats without creating carriers. Streptomycin was able to cure CCPP affected goats on day 3 both in natural and experimental infections besides that treated goats completely developed immunity to Mccp (Rurangirwa et al., 1981). Then CCPP inoculated goats were treated with oxytetracycline or tylosin which reduced severity, but 20% remained infectious. They used short acting oxytetracycline and tylosin at a dose rate of 10 and 20 mg/kg BW intramuscularly, respectively daily for 6 days, and single dose of long acting oxytetracycline at 20 mg/kg BW intramuscularly. Afterwards several antibiotics were used in clinical cases and outbreaks. Danofloxacin has shown good effectiveness in clinical CCPP cases when administered at 6 mg/kg BW subcutaneously repeated at 48-h interval (Ozdemir et al., 2006). Long acting oxytetracycline has prevented disease and deaths and also controlled CCPP outbreak (Giadinis et al., 2008).

8. Vaccination

Development of vaccines from local isolates, specific species, subspecies or strains and their potent antigens need to be explored for better and highly specific immune response. Many trials have also been conducted in this direction globally and few have been successful. Initially, vaccine trials has been started for the disease using a high passage mycoplasma strain F38 and studied the effect on disease course. This was followed by a series of trials which developed inactivated and saponin adjuvanated vaccines whose effectiveness have been proved under field conditions (Litamoi et al., 1989). However, the main drawback of these vaccines was their cost of production which was due to the fastidious nature of Mccp requiring rich and costly medium, slow growth to ensure a large quantity of medium for sustenance and requirement of heavy doses of antigen (0.15 mg per goat) for appropriate immune responses. Formalinized culture was used as vaccine and reported that optimum age for vaccination of kids should be beyond 10 weeks of age. A lyophilised killed F38 vaccine for CCPP was explored and found that this vaccine provided full protection against mortality and against clinical disease (95% protection). As CCPP usually occurs in severe form and requires an early management of acute cases, some researchers have recommended the raising of antiserum against the pathogen (Wise et al., 1995).

Considering the limitations of whole culture vaccines, subunit fractions of Mccp as vaccines were studied for any inhibitory effects (March et al., 2000). Purification of IgG was also studied. Humoral immune responses were studied in experimentally Mccp infected goats. Aluminum hydroxide [Al(OH)2] terpene vaccine has also been recommended (Radostitis et al., 2000). In current times, novel vaccines based on strains or antigenic proteins are being evaluated. An inactivated vaccine (M1601 strain) was developed for the disease. An inactivated whole culture of the CCPP was evaluated to be used as a trial vaccine. For evaluating vaccine quality control, tandem mass spectrometry was used for identifying the mycoplasma antigen in reference to commercial CCPP vaccines.

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