Contagious caprine pleuropneumonia: The first isolation and molecular characterization of Mycoplasma capricolum subsp. capripneumoniae in the Kingdom of Saudi Arabia.

Classical contagious caprine pleuropneumonia is one of the most fatal contagious disease of goats listed by World Organization for Animal Health that leads to major economic losses. It is caused by infection with Mycoplasma capricolum subspecies capripneumoniae. In order to isolate the causative agents of CCPP for the first time in the Kingdom of Saudi Arabia, fifteen flocks from Eastern region (Al Ahsa, Dammam and Hafr Albaten) and ten flocks from Riyadh and Al-Kharj regions were selected for this study. A total of 700 samples (400 nasal swabs, 300 pleural fluid samples and lung samples (from necropsied animals)) were collected from goats showing typical signs of CCPP. The clinical signs of diseased cases revealed serous to mucoid nasal discharge, coughing, dyspnea, frothy salivation, and fever (40-42°C). Necropsied animals showed fibrinous pleuropneumonia and increased pleural fluid. Of 400 nasal swabs, 190 pleural fluid, and 110 lung samples, 26 (6.5%), 31 (16.3%) and 19 (17.3%) Mycoplasma isolates were recovered, respectively. Biochemically, all isolates were sensitive to digitonin and fermented glucose. Sixty seven of Mycoplasma isolates were belonged to Mycoplasma mycoides cluster based on detection of 16S rRNA. Polymerase chain reaction screening of Mycoplasma isolates using specific primer for M. capricolum subsp. capripneumoniae confirmed 55 isolates to be M. capricolum subsp. capripneumoniae.

Detection and Isolation of Mycoplasma capricolum Subspecies Capricolum from East Azerbaijan Sheep Flocks.

Mycoplasma capricolum subspecies capricolum (Mcc) is one of the causative agents of contagious agalactia (CA), which is an important disease in sheep and goats in the Mediterranean and Middle East countries. Mycoplasma agalactiae is the classic agent of CA in sheep and goats. Mycoplasma mycoides subspecies Capri (Mmc), Mycoplasma capricolum subspecies capricolum (Mcc), and Mycoplasma putrefaciens (Mp) produce a clinically similar disease, more often in goats. The aim of the present study was to detect Mcc in sheep flocks in East Azerbaijan Province of Iran. Milk, ear canal, and eye swab samples were collected from 49 sheep flocks with clinical signs of CA or a history of a disease. All the samples were examined using both culture and molecular methods. In the molecular method,positive samples for the Mycoplasma genus were tested for M. mycoides cluster and Mcc. From 272 samples, 67, 87, and 62 samples were shown to be positive using the culture method, polymerase chain reaction (PCR) method, and both culture and PCR methods, respectively. Mcc was detected in all the four M. mycoides cluster positive samples, including milk, ear canal, and eye swab samples. This is the first report of Mcc detection from East Azerbaijan. Our results showed that eye, milk, and ear canal samples could be suitable sources for Mcc detection in sheep flocks.

A meta-analysis of contagious caprine pleuropneumonia (CCPP) in Ethiopia.

This systematic literature review was initiated due to lack of comprehensive information on the status and distribution of contagious caprine pleuropneumonia (CCPP) in Ethiopia. The objectives of the review were thus to provide a pooled prevalence estimate of CCPP in the country and asses the level of in between study variance among the available reports. Manual and electronic search was conducted between 8th of January and 25th of June 2015. A total of twelve published articles and one MSc thesis was retrieved from 19 initially identified studies. Twenty five animal level datasets were extracted at regional level considering some hypothesized predictors. The retrieved data were summarized in a meta-analytical approach. Accordingly, the pooled prevalence estimate of CCPP was 25.7% (95% CI:20.9,31.0). The inverse variance square (I(2)) that explains the variation in effect size attributed to reports true heterogeneity was 95.7%.The sub-group analysis was also computed for assumed predictors including, age, sex, type of study population, production systems and regional states. Among these predictors, study population type revealed statistically significant difference (P<0.05). Accordingly, the prevalence estimate for samples collected at abattoir was 39.2%, while that of samples collected at field level was 22.4%. In the final model, type of study population fitted the multivariable meta-regression model accounting for 22.87% of the explainable proportion of heterogeneity among the presumed predictors. Evidence on isolation and confirmation of Mycoplasma capricolum subspp. capripneumonie in the country was obtained from five regional states. In conclusion, it is recommended to further investigate facilities related with transportation and collection premises along with potential role of sheep in the epidemiology of CCPP. Finally, the review emphasizes the need for monitoring the ongoing CCPP control intervention and introduces amendments based on the findings. Besides more surveys are needed in some of the regions where no or few valid data was available.

Field-Applicable Recombinase Polymerase Amplification Assay for Rapid Detection of Mycoplasma capricolum subsp. capripneumoniae.

Contagious caprine pleuropneumonia (CCPP) is a highly contagious disease caused by Mycoplasma capricolum subsp. capripneumoniae that affects goats in Africa and Asia. Current available methods for the diagnosis of Mycoplasma infection, including cultivation, serological assays, and PCR, are time-consuming and require fully equipped stationary laboratories, which make them incompatible with testing in the resource-poor settings that are most relevant to this disease. We report a rapid, specific, and sensitive assay employing isothermal DNA amplification using recombinase polymerase amplification (RPA) for the detection of M. capricolum subsp. capripneumoniae. We developed the assay using a specific target sequence in M. capricolum subsp. capripneumoniae, as found in the genome sequence of the field strain ILRI181 and the type strain F38 and that was further evidenced in 10 field strains from different geographical regions. Detection limits corresponding to 5 × 10(3) and 5 × 10(4) cells/ml were obtained using genomic DNA and bacterial culture from M. capricolum subsp. capripneumoniae strain ILRI181, while no amplification was obtained from 71 related Mycoplasma isolates or from the Acholeplasma or the Pasteurella isolates, demonstrating a high degree of specificity. The assay produces a fluorescent signal within 15 to 20 min and worked well using pleural fluid obtained directly from CCPP-positive animals without prior DNA extraction. We demonstrate that the diagnosis of CCPP can be achieved, with a short sample preparation time and a simple read-out device that can be powered by a car battery, in <45 min in a simulated field setting.

Fatal transmission of contagious caprine pleuropneumonia to an Arabian oryx (Oryx leucoryx).

Contagious caprine pleuropneumonia (CCPP) is an infectious respiratory disease mainly affecting domestic goats. As CCPP has never been documented in grazing antelopes (subfamily hippotraginae), they were not considered susceptible. Mycoplasma capricolum subspecies capripneumoniae (Mccp) was isolated from pleural liquid collected during the necropsy of a severely emaciated Arabian oryx with mild nasal discharge. The Mccp isolate was then genotyped using a multilocus sequence scheme; the sequence type was identical to the Mccp strain previously identified in a sand gazelle from a nearby enclosure. This case shows for the first time that members of the hippotraginae subfamily, here the Arabian oryx, can be affected by CCPP. In addition, genotyping shows that the oryx was most probably infected, at a distance, by sand gazelles.

Fatal outbreak of Mycoplasma capricolum pneumonia in endangered markhors.

A pneumonia outbreak reduced the numbers of a wild population of endangered markhors (Capra falconeri) in Tajikistan in 2010. The infection was diagnosed by histologic examination and bacteriologic testing. Mycoplasma capricolum subsp. capricolum was the sole infectious agent detected. Cross-species transmission from domestic goats may have occurred.

Genome sequence of Mycoplasma capricolum subsp. capripneumoniae strain M1601.

Mycoplasma capricolum subsp. capripneumoniae is the causative agent of contagious caprine pleuropneumonia, a devastating disease of goats listed by the World Organization for Animal Health. Here we report the first complete genome sequence of this organism (strain M1601, a clinically isolated strain from China).

Molecular detection of a mixed infection of Goatpox virus, Orf virus, and Mycoplasma capricolum subsp. capripneumoniae in goats.

The current study investigated an outbreak of mixed infection with Goatpox virus (GTPV), Orf virus (ORFV), and Mycoplasma capricolum subsp. capripneumoniae (MCCP) that occurred on a Chinese goat farm, with a case fatality rate of 60.2%. The observed clinical signs were ecthyma and accelerated respiration with frequent coughing. Specific fragments of the p32 gene of GTPV, B2L gene of ORFV, and 16S ribosomal RNA gene of MCCP were synchronously amplified by polymerase chain reaction (PCR) from the tissues of 12 dead goats. The PCR products were cloned, sequenced, and aligned with related reference sequences in GenBank for further identification of the pathogens. The present study reports a mixed infection with GTPV, ORFV, and MCCP in goats.

First report on the molecular prevalence of Mycoplasma capricolum subspecies capripneumoniae (Mccp) in goats the cause of contagious caprine pleuropneumonia (CCPP) in Balochistan province of Pakistan.

Contagious caprine pleuropneumonia (CCPP) caused by Mycoplasma capricolum subspecies capripneumoniae (Mccp) is a disease of goats which causes high morbidity and mortality and is reported in many countries of the world. There are probably no reports on the molecular prevalence of Mccp, Mycoplasma capricolum subsp. capricolum (Mcc) and Mycoplasma putrefaciens (Mp) in Balochistan and any other part of Pakistan. Thirty goats (n = 30) with marked respiratory symptoms were selected and procured from forty goat flocks in Pishin district of Balochistan in 2008. The genomic deoxyribonucleic acid (DNA) from the lung samples (n = 30) of the slaughtered goats was purified and subjected to polymerase chain reaction (PCR) assays for the presence of Mycoplasma mycoides cluster members and Mp. The PCR-RFLP (restriction fragment length polymorphism) was also used to further confirm the Mccp. Of the thirty lung samples 17 (56.67%) were positive for the molecular prevalence of Mcc, Mccp and Mp. In total the molecular prevalence was observed as 17.65% for Mccp (n = 3), 70.59% for Mcc (n = 12) and 11.76% for Mp (n = 2). The RFLP profile has also validated the PCR results of Mccp by yielding two bands of 190 and 126 bp. The results of PCR-RFLP coupled with the presence of fibrinous pleuropneumonia and pleurisy during postmortem of goats (n = 3) strongly indicated the prevalence of CCPP in this part of world. Moreover the prevalence of Mcc and Mp is also alarming in the study area. We report for the very first time the molecular prevalence of Mcc, Mccp, and Mp in the lung tissues of goats in the Pishin district of Balochistan, Pakistan.

Detection of contagious caprine pleuropneumonia in East Turkey.

A study was implemented to investigate the presence of contagious caprine pleuropneumonia (CCPP) in East Turkey. This study was based on clinical surveillance in the field, surveillance at regional slaughterhouses and regular submission of suspected lesions to regional laboratories. The results showed that the agent of CCPP, Mycoplasma capricolum subspecies capripneumoniae (Mccp), could be detected by culture and specific polymerase chain reaction from 37.5% (12/32) of lung samples taken from goats of ten different herds. This agent was also isolated from two of 13 sheep samples (one from the lung and the other from a nasal swab). Mycoplasma capricolum subsp. capripneumoniae was isolated in pure culture and characterised at a finer molecular level. The East Turkish isolate was found to be closely related to another strain of Turkish origin, as well as to Mccp strains isolated in Tunisia. The isolation of Mccp from sheep lung lesions brings the strict host-specificity of this pathogen into question. It may also indicate that Mccp presents a risk for wildlife in the region. Such results, the authors believe, demonstrate that adequate risk assessments should be undertaken in Turkey and neighbouring countries.

Seroprevalence of contagious caprine pleuropneumonia in Kefta Humera, Alamata (Tigray) and Aba-'ala (Afar), Northern Ethiopia.

A cross sectional study was conducted to determine the sero-prevalence of contagious caprine pleuroneumonia in three districts of Tigray and Afar regions of Ethiopia namely; Kefta Humera, Alamata and Aba-'alla. Proportions and chi-square test statistics were used to analyze the data. From a total of 863 goats and 137 sheep tested, 282 (32.68%) and 25 (18.25%) were positive for antibodies of Mycoplasma capricolum subsp. capripneumoniae respectively using complement fixation test (CFT). The seroprevalence of CCPP in goats among the three districts was statistically significant (x(2) = 76.00, p < 0.001). In this study there was no statistical significant variation in the seroprevalence of CCPP in both sexes (x(2) = 3.619, p = 0.0571) and age (x(2) = 0.990, p = 0.095) groups. The finding of high seroprevalence of CCPP in sheep (18.25%) could indicate that sheep are potential carriers of Mccp.

Participatory investigation of Contagious Caprine Pleuropneumonia (CCPP) in goats in the Hammer and Benna-Tsemay districts of southern Ethiopia.

The study was conducted in two selected districts of Southern Omo zones of Ethiopia, namely Hammer and Benna-Tsemay, during November 2004 and May 2005 to determine the status of contagious caprine pleuropneumonia (CCPP). Participatory disease investigation was conducted in the goat flocks owned by pastoralists of the districts. Participatory methods such as proportionate piling and matrix scoring of diseases were used to characterise major diseases of goats. Clinical and post-mortem examinations and isolation of the causative agent of CCPP were done. Serological tests were conducted using CFT. CCPP (locally termed Sompo) ranked as the first important disease of goats in the study area. Local perception of causes and signs of CCPP were described. Matrix scoring between groups revealed that disease signs and causes showed weak, moderate and good agreement by Kendall's coefficient concordance (W = 0.21-0.99). The overall sero-prevalence of CCPP was 15.5%. The causative agent was isolated from sick animals in the lab. The characteristic clinical signs, gross lesions, bacteriological isolation of the causative agent supported by participatory epidemiological disease investigation revealed that CCPP is a major disease of goats in the study districts. Participatory epidemiology using indigenous knowledge could efficiently be used to generate sufficient information with minimum cost, local materials and within reasonably short period of time, assisting the designing of feasible disease control programme in developing countries.

Specific real-time PCR assays for the detection and quantification of Mycoplasma mycoides subsp. mycoides SC and Mycoplasma capricolum subsp. capripneumoniae.

Contagious bovine pleuropneumonia and contagious caprine pleuropneumonia are two severe respiratory infections of ruminants due to infection by Mycoplasma mycoides subsp. mycoides SC (MmmSC) and Mycoplasma capricolum subsp. capripneumoniae (Mccp), respectively. They are included in the OIE list of notifiable diseases. Here we describe the development of rapid, sensitive, and specific real-time PCR assays for the detection and quantification of MmmSC and Mccp DNA. MmmSC PCR primers were designed after whole genome comparisons between the published sequence of MmmSC strain type PG1(T) and the sequence of an M. mycoides subsp. mycoides large colony strain. For Mccp, previously published conventional PCR primers were applied. SYBR green was used as a detection agent for both assays. The assays specifically detected the targeted species in both cultures and clinical samples, and no cross-amplifications were obtained from either heterologous mycoplasma strain cultures or European field samples. The sensitivity of these new assays was estimated at 3-80 colony forming units per reaction and 4-80fg of DNA, representing a 2-3log increase in sensitivity compared to established conventional PCR tests. These new real-time PCR assays will be invaluable for application in various fields such as direct detection in diagnostic laboratories.

Contagious caprine pleuropneumonia outbreak in captive wild ungulates at Al Wabra Wildlife Preservation, State of Qatar.

Contagious caprine pleuropneumonia (CCPP) caused by Mycoplasma capricolum subsp. capripneumoniae is a highly contagious and serious respiratory disease of domestic goats, characterized by coughing, severe respiratory distress, and high mortality rates. The lesions at necropsy are mainly a fibrinous pleuropneumonia with increased straw-colored pleural fluid. An outbreak of CCPP in wild goat (Capra aegagrus), Nubian ibex (Capra ibex nubiana), Laristan mouflon (Ovis orientalis laristanica), and gerenuk (Litocranius walleri) occurred at Al Wabra Wildlife Preservation in the State of Qatar. The disease was suspected because of the clinical symptoms and the necropsy findings and was confirmed by the isolation and identification of the causative organism. This new finding indicates that CCPP should be considered a potential threat to wildlife and the conservation of endangered ruminant species, especially in the Middle East, where it is enzootic because of its presence in chronic carriers. Susceptible imported animals should be quarantined and vaccinated. The preferred samples for diagnosis are the pleural fluid, which contains high numbers of Mycoplasma, and sections of hepatized lung, preferably at the interface of normal and diseased tissues. Samples must be shipped to diagnostic laboratories rapidly, and appropriate cool conditions must be maintained during shipping.

First isolation of Mycoplasma capricolum subsp. capricolum, one of the causal agents of caprine contagious agalactia, on the island of Lanzarote (Spain).

During an unusually long period of bad weather, several outbreaks of caprine contagious agalactia (CCA) were reported in a number of flocks on the island of Lanzarote (Canary Islands, Spain). Clinical and subclinical mastitis in lactating goats and some cases of arthritis and pneumonia in kids were observed in the affected flocks. Mycoplasma capricolum subsp. capricolum was isolated as the main causal agent of the outbreaks, associated with M. mycoides subsp. mycoides "large colony type" (Mmm LC) in two flocks. This is the first report of an isolation of M. capricolum subsp. capricolum on the island of Lanzarote. The finding is of epidemiological importance and could complicate plans to control the disease. The significance of this mycoplasma species in association with CCA must now be studied in detail.

Isolation of Mycoplasma capricolum-like strains from chickens.

Members of the genus Mycoplasma infect a wide range of hosts, but individual Mycoplasma species tend to exhibit a considerable degree of host specificity. We characterized Mycoplasma strain 700, isolated from a kidney of a layer hen in Spain and Mycoplasma strains ULB-A and ULB-B, isolated from the air sac and from the bile of stunted broiler chickens in Slovenia. The serologic examination showed that these three strains are antigenically unrelated to all of the recognized Mycoplasma species of avian origin, but closely related to the ruminant mycoplasma Mycoplasma capricolum subspecies capricolum (M. capricolum). The comparison of their 16S rRNA gene sequences with the sequence of M. capricolum (California kid) revealed 99.66% sequence identity for the strain 700 and 99.59% identity for strains ULB-A and ULB-B. Moreover, the predicted DnaK sequences of the M. capricolum-like strains, isolated from chickens, were identical to DnaK sequences of M. capricolum. Comparison of their dnaK gene sequences with M. capricolum showed 99.64% sequence identity for strain 700 and 99.27% identity for strains ULB-A and ULB-B. In the flock from which M. capricolum-like strains ULB-A and ULB-B were isolated, the majority of chickens (83% of the chickens examined) raised antibodies reacting with M. capricolum antigens. Notably, the infection of chickens with M. capricolum-like strains represents an unusual exception to the range of Mycoplasma species host specificity.

A specific PCR for the identification of Mycoplasma capricolum subsp. capripneumoniae, the causative agent of contagious caprine pleuropneumonia (CCPP).

Contagious caprine pleuropneumonia is a severe infectious disease of goats in Africa and the Middle East. It is caused by a fastidious mycoplasma, Mycoplasma capricolum subsp. capripneumoniae, a member of the "M. mycoides cluster". Members of this cluster share genomic and antigenic features, which result in common biochemical and serological properties, complicating species identification. Two species of this cluster, M. mycoides subsp. capri and M. mycoides subsp. mycoides large colony biotype, are very often isolated from clinical cases resembling contagious caprine pleuropneumonia. Furthermore, in the laboratory, M. capricolum subsp. capripneumoniae can be easily confused with the closely related capricolum subspecies. Considering these constraints and the scarcity of available methods for identification, a specific polymerase chain reaction was developed. A DNA fragment of 7109 bp containing genes coding for the arginine deiminase pathway (ADI) was chosen as target sequence for the selection of a specific primer pair. The full ADI operon from M. capricolum subsp. capripneumoniae strain GL100 was sequenced. Polymorphism within this locus was analyzed by comparison with the sequence from the closely related IPX strain (M. capricolum subsp. capricolum). It varied from 0.6% to 3.5%. The highest divergence was found in a region coding for arcD. Therefore, this gene was chosen as target for the specific amplification of a 316 bp-long DNA fragment. The specificity of this PCR was validated on 14 M. capricolum subsp. capripneumoniae strains and 27 heterologous strains belonging to the "M. mycoides cluster" and M. putrefaciens. This new PCR will be a valuable tool for the surveillance of contagious caprine pleuropneumonia.