Seroprevalence and associated risk factors of contagious caprine pleuropneumonia in selected districts of South Wollo Zone Northeast Ethiopia.

Contagious caprine pleuropneumonia (CCPP) is a severe and devastating respiratory disease of goats, which is characterized by severe serofibrinous pleuropneumonia accompanied by high morbidity and mortality. A cross-sectional study was conducted from July 2022 to January 2023 to determine the seroprevalence of CCPP and identify risk factors associated with the occurrence of CCPP in goats in five selected districts of the South Wollo Zone of the Eastern Amhara region. A total of 384 sera samples were collected from goats and examined for antibodies specific to Mycoplasma capricolum subspecies capripneumoniae (Mccp) using Competitive Enzyme-Linked ImmunoSorbent Assay (cELISA) test. Out of the total examined sera, 26 samples were positive for CCPP, giving an overall seroprevalence of 6.7% (95% CI = 6.64-9.77). A seroprevalence of 5.05%, 4.65%, 2.78%, 12.90%, and 10.77% were recorded in Ambasel, Tehuledere, Kalu, Dessie Zuria and Kutaber districts, respectively. However, there was no statistically significant difference among these five districts (p > 0.05). The seroprevalence of CCPP varies significantly between age groups and agroecology (p < 0.05). However, the seroprevalence did not vary with sex, body condition score (BCS), and flock size (p > 0.05). Old-aged goats (OR = 4.10) and goats found in the lowlands (OR = 5.09) were at higher risk of infection with CCPP than young-aged goats and goats found in the highlands, respectively. In conclusion, the present seroprevalence investigation indicated the occurrence of CCPP in those selected study districts of the South Wollo Zone. Therefore, appropriate control measures, including avoiding the mixing of flocks and vaccination should be designed and implemented especially in the lowland areas and older goats to reduce the further spread and magnitude of the disease.

Interactions between Mycoplasma mycoides subsp. mycoides and bovine macrophages under physiological conditions.

We investigated the interactions of unopsonized and opsonized Mycoplasma mycoides subsp. mycoides (Mmm) with bovine macrophages in vitro. Mmm survived and proliferated extracellularly on bovine macrophage cell layers in the absence of Mmm-specific antisera. Bovine complement used at non-bactericidal concentrations did neither have opsonizing effect nor promoted intracellular survival, whereas Mmm-specific antisera substantially increased phagocytosis and Mmm killing. A phagocytosis-independent uptake of Mmm by macrophages occurred at a high multiplicity of infection, also found to induce the production of TNF, and both responses were unaffected by non-bactericidal doses of bovine complement. Bovine complement used at higher doses killed Mmm in cell-free cultures and completely abrogated TNF responses by macrophages. These results provide a framework to identify Mmm antigens involved in interactions with macrophages and targeted by potentially protective antibodies and point towards a pivotal role of complement in the control of inflammatory responses in contagious bovine pleuropneumonia.

A molecular epidemiological investigation of contagious caprine pleuropneumonia in goats and captive Arabian sand gazelle (Gazella marica) in Oman.

BACKGROUND: Contagious caprine pleuropneumonia (CCPP) is a fatal WOAH-listed, respiratory disease in small ruminants with goats as primary hosts that is caused by Mycoplasma capricolum subspecies capripneumoniae (Mccp). Twelve CCPP outbreaks were investigated in 11 goat herds and a herd of captive Arabian sand gazelle (Gazella marica) in four Omani governorates by clinical pathological and molecular analysis to compare disease manifestation and Mccp genetic profiles in goats and wild ungulates. RESULTS: The CCPP forms in diseased and necropsied goats varied from peracute (5.8%), acute (79.2%) and chronic (4.5%) while all of the five necropsied gazelles showed the acute form based on the clinical picture, gross and histopathological evaluation. Colonies of Mccp were recovered from cultured pleural fluid, but not from lung tissue samples of one gazelle and nine goats and all the isolates were confirmed by Mccp-specific real time PCR. Whole genome-single nucleotide polymorphism (SNP) analysis was performed on the ten isolates sequenced in this study and twenty sequences retrieved from the Genbank database. The Mccp strains from Oman clustered all in phylogroup A together with strains from East Africa and one strain from Qatar. A low variability of around 125 SNPs was seen in the investigated Omani isolates from both goats and gazelles indicating mutual transmission of the pathogen between wildlife and goats. CONCLUSION: Recent outbreaks of CCPP in Northern Oman are caused by Mccp strains of the East African Phylogroup A which can infect goats and captive gazelles likewise. Therefore, wild and captive ungulates should be considered as reservoirs and included in CCPP surveillance measures.

Rapid detection of Mycoplasma mycoides subsp. capri and Mycoplasma capricolum subsp. capripneumoniae using high-resolution melting curve analysis.

Mycoplasma capricolum subsp.subsp. capripneumonia (Mccp) and Mycoplasma mycoides subsp.sbusp. capri (Mmc) cause caprine pleuropneumonia (CCPP) and mycoplasmal pneumonia in goats and sheep (MPGS), respectively. These diseases cannot be identified on clinical symptoms alone and it is laborious to distinguish them using biochemical methods. It is therefore important to establish a simple, rapid identification method for Mccp and Mmc. Here, we report a high-resolution melting (HRM) curve analysis using specific primers based on the Mmc 95010 strain MLC_0560 and Mccp F38 strain MCCPF38_00984 gene sequences. The method was highly specific with intra- and inter-batch coefficients of variation < 1%. The lower limit of detection for Mccp and Mmc was 55 copies/µL and 58 copies/µL, respectively. HRM and fluorescence qPCR results were compared using 106 nasal swabs and 47 lung tissue samples from goats (HRM-qPCR coincidence rate 94.8%; 145/153). Mycoplasma isolation and identification was performed on 30 lung tissue samples and 16 nasal swabs (HRM-culturing coincidence rate 87.0%; 40/46). HRM analysis was more sensitive than fluorescence qPCR and Mycoplasma isolation, indicating the practicality of HRM for accurate and rapid identification of Mccp and Mmc, and diagnosis and epidemiology of CCPP and MPGS.

Isolation and identification of Mycoplasma mycoides subsp. mycoides in cattle from south-east Nigeria.

Background: Mycoplasma mycoides subsp. mycoides is the causative organism of Contagious Bovine Pleuropneumonia (CBPP). It is a trans-boundary disease and an endemic in Nigeria having caused serious financial loss for the country's economy. Aim: This study was undertaken to isolate and confirm the presence of M. mycoides subsp. mycoides (Mmm) in cattle, from three selected South-Eastern states of Nigeria. Method: A total of 90 bovine samples (25 pleural fluids and 65 lung tissues) suggestive of CBPP were collected from different abattoirs in the three selected South-eastern states of Nigeria (Anambra, Enugu, and Imo), for the isolation of Mmm by employing cultural method, whereas for confirmation polymerase chain reaction (PCR) approach was used. The collected samples were cultured on Pleuropneumonia like organism (PPLO) agar according to specific protocols. Results: Twenty five of the samples (lungs and pleural fluid) were positive for Mmm on PPLO agar giving an isolation rate of 27.7%. Only 21 of the isolates were further confirmed using PCR. The PCR amplification of the isolates produced a product of 1.1 kbp which is specific for Mmm. No positive isolates were recovered from Imo state. Conclusion: This study confirms the presence of Mmm as the causative organism of CBPP in Southeast Nigeria. It is recommended that active surveillance and vaccination protocol should be undertaken in the region for the control and prevention of this disease.

The first identification of contagious caprine pleuropneumonia (CCPP) in sheep and goats in Egypt: molecular and pathological characterization.

Contagious caprine pleuropneumonia (CCPP) is one of the most fatal and contagious diseases of goats. To date, the occurrence of CCPP in Egypt has not been reported. During the period from 2017 to 2018, 200 goats and 400 sheep from Matrouh Governorate (Al Alamein and El Hammam cities) were suspected to have CCPP; animals were examined to confirm the presence of CCPP infection as well as the epidemiological status, clinical features, and molecular and histopathologic characteristics of lung tissues. Additionally, a treatment trial was performed to assess the efficacy of anti-mycoplasma therapy in the treatment of clinical cases of this disease. The occurrence of CCPP was 32.5% and 5% in goats and sheep, respectively, while case fatality was 30% and 8% in goats and sheep, respectively. The clinical forms of CCPP in both sheep and goats varied from per-acute to acute or chronic cases. Histopathological analysis of lung tissues from dead cases (either sheep or goats) revealed different stages of broncho- and pleuropneumonia ranging from per-acute to acute or chronic stages. Lung tissues showed severe congestion of interalveolar capillaries, flooding of alveoli and bronchi with a fibrinous exudate, a high degree of pleural thickening, and multifocal areas of necrosis that were sometimes sequestered in the fibrous capsule. Isolation of Mycoplasma capricolum subspecies capripneumoniae (Mccp) was confirmed in all dead cases by agar and broth culture methods and polymerase chain reaction. The treatment trial revealed that the marbofloxacin and spiramycin groups had a higher cure rate (70%) than the oxytetracycline group (40%) and a lower fatality rate (30%) than the oxytetracycline group (60%). Conclusively, infection with CCPP in goats and sheep is considered to be novel for Mccp in Egypt, where this species is considered to be the main pathogen in goats, not in sheep. Additionally, it could be concluded that treatment may be effective only if given early. Further comprehensive surveys are required to investigate the risk of CCPP in goats and sheep in all Egyptian governorates.

Evaluation of new generation macrolides for the treatment and metaphylaxis of contagious bovine pleuropneumonia (CBPP) in cattle experimentally infected with Mycoplasma mycoides subspecies mycoides.

BACKGROUND: Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subspecies mycoides (Mmm) is an important disease of cattle that causes serious economic losses. With the known effectiveness of new generation macrolides, tulathromycin and gamithromycin were assessed in comparison with oxytetracycline as a positive control and saline as a negative control for effectiveness in inhibiting lung lesion development, promoting resolution, preventing spread and bacteriological clearance in susceptible local cattle breeds in two separate studies in Kenya and Zambia. Animals were monitored for clinical signs, sero-conversion as well as detailed post-mortem examination for CBPP lesions. RESULTS: Using the Hudson and Turner score for lesion type and size, tulathromycin protected 90%, gamithromycin 80%, and oxytetracycline 88% of treated animals in Kenya. In Zambia, all animals (100%) treated with macrolides were free of lung lesions, while oxytetracycline protected 77.5%. Using the mean adapted Hudson and Turner score, which includes clinical signs, post-mortem findings and serology, tulathromycin protected 82%, gamithromycin 56% and oxytetracycline 80% of the animals in Kenya whereas in Zambia, tulathromycin protected 98%, gamithromycin 94% and oxytetracycline 80%. The saline-treated groups had 93 and 92% lesions in Kenya and Zambia respectively, with Mmm recovered from 5/14 in Kenya and 10/13 animals in Zambia. Whereas the groups treated with macrolides were free from lesions in Zambia, in Kenya 5/15 tulathromycin-treated animals and 6/15 gamithromycin-treated animals showed lesions. Oxytetracycline-treated animals showed similarities with 3/14 and 4/15 showing lesions in Zambia and Kenya respectively and Mmm recovery from one animal in Kenya and six in Zambia. In both studies, lesion scores of saline-treated groups were significantly higher than those of the antibiotic treated groups (p < 0.001). In sentinel animals, CBPP lesions were detected and Mmm recovered from one and two animals mixed with the saline-treated groups in Kenya and Zambia respectively. CONCLUSIONS: This study demonstrated that tulathromycin, a mycoplasmacidal, can achieve metaphylactic protection of up to 80%, while non-recovery of Mmm from sentinels suggests macrolides effectiveness in preventing spread of Mmm. It is recommended that further studies are conducted to evaluate strategies comparing vaccination alone or combining vaccination and antibiotics to control or eradicate CBPP.

An outbreak of Mycoplasma mycoides subspecies capri arthritis in young goats: a case study.

Mycoplasmosis is a well-known cause of morbidity and mortality in small ruminants. Previously recognized outbreaks have involved arthritis, and pneumonia or pleuropneumonia. Modern bacteriology procedures rely less on isolation techniques that require special media for mollicutes given that these species are notoriously difficult to isolate, and rely more on PCR tests. We report an outbreak of arthritis, pleuropneumonia, and mild meningitis affecting dairy goat kids, spanning a period of 3 y, which had unusual epidemiologic characteristics related to husbandry practices. Lesions were characterized by polyarthritis of the appendicular joints, with copious joint fluid and extension of arthritic exudate beyond the joint itself. The cause remained unknown until serendipitous isolation of a mycoplasma on blood agar. Mycoplasmosis was not detected from synovial samples by a general mycoplasma PCR, despite multiple attempts. Isolated colonies were also negative by this general PCR assay. The isolate was identified as Mycoplasma mycoides subspecies capri, using universal 16S primers and amplicon sequencing. Testing of additional isolates from other diseased goats in the herd confirmed that this was the cause of illness. A failure to recognize the distinct nature of organisms of the M. mycoides group of mycoplasmas meant that a PCR test that cannot detect this group of organisms was utilized at first, and the etiology of the illness was overlooked for a period of time. Veterinary pathologists and microbiologists must be aware of the limitations of some PCR assays when confronted with joint disease and pleuropneumonia in small ruminants.

Polymorphism analysis of the apxIA gene of Actinobacillus pleuropneumoniae serovar 5 isolated in swine herds from Brazil.

The bacterium Actinobacillus pleuropneumoniae is the etiological agent of Contagious Porcine Pleuropneumonia, a disease responsible for economic losses in the swine industry worldwide. A. pleuropneumoniae is capable of producing proteinaceous exotoxins responsible for inducing hemorrhagic lesions, one of which is ApxI. Few studies have conducted an in-depth evaluation of polymorphisms of the nucleotides that make up the ApxI toxin gene. Here we analyze the polymorphisms of the apxIA gene region of A. pleuropneumoniae serovar 5 isolated from swine in different regions in Brazil and report the results of molecular sequencing and phylogenetic analysis. Analysis of the apxIA gene in 60 isolates revealed the presence of genetic diversity and variability. The polymorphisms in the nucleotide sequences determined the grouping of the Brazilian sequences and five more sequences from the GenBank database into 14 different haplotypes, which formed three main groups and revealed the presence of mutations in the nucleotide sequences. The estimation of selection pressures suggests the occurrence of genetic variations by positive selective pressure on A. pleuropneumoniae in large groups of animals in relatively small spaces. These conditions presumably favor the horizontal dissemination of apxIA gene mutations within bacterial populations with host reservoirs. As a result, the same serovar can demonstrate different antigenic capacities due to mutations in the apxIA gene. These alterations in sequences of the apxIA gene could occur in other areas of countries with intense swine production, which could lead to differences in the pathogenicity and immunogenicity of each serovar and have implications for the clinical status or diagnosis of A. pleuropneumoniae.

Improving Quality Control of Contagious Caprine Pleuropneumonia Vaccine with Tandem Mass Spectrometry.

Vaccines to protect livestock against contagious caprine pleuropneumonia (CCPP) consist of inactivated, adjuvanted antigens. Quality control of these vaccines is challenging as total protein quantification provides no indication of protein identity or purity, and culture is not an option. Here, a tandem mass spectrometry approach is used to identify the mycoplasma antigen contained in reference samples and in commercial CCPP vaccines. By the same approach, the relative amounts of mycoplasma antigen and residual proteins originating from the production medium are determined. Mass spectrometry allows easy and rapid identification of the peptides present in the vaccine samples. Alongside the most probable mycoplasma species effectively present in the vaccines, a very high proportion of peptides from medium constituents are detected in the commercial vaccines tested.

Polymorphonuclear cells and reactive oxygen species in contagious bovine pleuropneumonia: New insight from in vitro investigations.

Reactive oxygen species (ROS) are suggested to play a role in the pathogenesis of contagious bovine pleuropneumonia, a severe respiratory disorder caused by Mycoplasma mycoides subsp. mycoides (Mmm). The present study investigated the generation of ROS by different strains of Mmm, as well as their effect on the oxidative response of bovine neutrophils. The production of ROS was indirectly measured using a luminol-based chemiluminescence assay. Our results confirm that Mmm can produce ROS via the metabolism of glycerol, significant differences existing between African and European strains. Mmm was capable of adhering to the external surface of neutrophils. Interestingly, Mmm enhanced the respiratory burst of bovine neutrophils. This activity was particularly pronounced with the African field strain and in presence of glycerol. Taken together, our data argue in favour of a major role for neutrophils as the main source of ROS in contagious bovine pleuropneumonia.

Respiratory explants as a model to investigate early events of contagious bovine pleuropneumonia infection.

Contagious bovine pleuropneumonia (CBPP) is a severe disease caused by Mycoplasma mycoides subsp. mycoides (Mmm). Knowledge on CBPP pathogenesis is fragmented and hampered by the limited availability of laboratory animal and in vitro models of investigation. The purpose of the present study is to assess respiratory explants as useful tools to study the early stages of CBPP. Explants were obtained from trachea, bronchi and lungs of slaughtered cattle, tested negative for Mycoplasma spp. and for the major bacterial and viral respiratory pathogens. The interaction of Mmm with explant cells was studied by immunohistochemistry (IHC), double-labelling indirect immunofluorescence (DLIIF) and laser scanning confocal microscopy (LSCM). Mmm capability to survive and proliferate within the explants was evaluated by standard microbiological procedures. Finally, the putative cellular internalization of Mmm was further investigated by the gentamicin invasion assay. IHC and DLIIF indicated that Mmm can colonize explants, showing a marked tropism for lower airways. Specifically, Mmm was detected on/inside the bronchiolar and alveolar epithelial cells, the alveolar macrophages and the endothelial cells. The interaction between Mmm and explant cells was abolished by the pre-incubation of the pathogen with bovine anti-Mmm immune sera. Mmm was able to survive and proliferate in all tracheal, bronchial and lung explants, during the entire time course of the experiments. LSCM and gentamicin invasion assay both confirmed that Mmm can enter non-phagocytic host cells. Taken together, our data supports bovine respiratory explants as a promising tool to investigate CBPP, alternative to cattle experimental infection.

Identification of targets of monoclonal antibodies that inhibit adhesion and growth in Mycoplasma mycoides subspecies mycoides.

Mycoplasma mycoides subspecies mycoides (Mmm) adhesion is tissue and host specific. Inhibition of adhesion will prevent Mmm from binding to lung cells and hence prevent colonization and disease. The aim of this study was to develop a panel of Mmm monoclonal antibodies against Mmm and use these antibodies to investigate their inhibitory effect on the adherence of Mmm to bovine lung epithelial cells (BoLEC), and to further identify an antigen to any of the inhibitory antibodies. Thirteen anti-Mycoplasma mycoides subsp. mycoides (AMMY) monoclonal antibodies (mAbs) inhibited adhesion by at least 30% and ten of the mAbs bound to multiple bands on Western blots suggesting that the antibodies bound to proteins of variable sizes. AMMY 10, a previously characterized Mmm- capsular polysaccharide (CPS) specific antibody, inhibited growth of Mmm in vitro and also caused agglutination of Mmm total cell lysate. AMMY 5, a 2-oxo acid dehydrogenase acyltransferase (Catalytic domain) (MSC_0267) specific antibody, was identified and polyclonal rabbit serum against recombinant MSC_0267 blocked adhesion of Mmm to BoLEC by 41%. Antigens recognized by these antibodies could be vaccine candidate(s) and should be subsequently tested for their ability to induce a protective immune response in vivo.

Resistance mechanisms against quinolones in Mycoplasma capricolum subsp. capricolum.

Quinolones interact with bacterial DNA gyrase and topoisomerase IV, the subunits of which are encoded by gyrA/gyrB and parC/parE, respectively. The aim of this study was to evaluate the relationship between changes in these genes and quinolone susceptibility of Mycoplasma capricolum subsp. capricolum (Mcc). Using in vitro selected resistant mutants and field isolates from goats, predicted amino acid changes in gyrA, gyrB and parC were associated with higher minimum inhibitory concentration values for quinolones. Alterations in parC predicted amino acid sequences were most frequently associated with quinolone resistance in Mcc.

Mmm-derived lipid-associated membrane proteins activate IL-1ß production through the NF-κB pathway via TLR2, MyD88, and IRAK4.

Mycoplasma mycoides subsp.mycoides (Mmm) is a pathogen that causes pneumonia, otitis media, and arthritis in young calves. Its pathogenesis is attributed in part to excessive immune responses. Mmm-derived lipid-associated membrane proteins (LAMPs) are potent inducers of the host innate immune system; however, interactions between Mmm-derived LAMPs as pathogenic agents, toll-like receptors (TLRs), and the signaling pathways responsible for activating inflammation and nuclear factor (NF)-κB have not been fully elucidated. Here, we analyzed the expression kinetics of interleukin (IL)-1ß in Mmm-derived LAMP-stimulated embryonic bovine lung (EBL) cells and found that Mmm-derived LAMPs induced IL-1ß expression. Subcellular localization analysis revealed the nuclear translocation of the NF-κB p65 subunit after EBL cells were stimulated with Mmm-derived LAMPs. Furthermore, a specific inhibitor assay demonstrated that NF-κB is required for Mmm-derived LAMP-induced IL-1ß expression. Additionally, overexpression of TLR2, myeloid differentiation primary response gene 88 (MyD88), and IL-1 receptor-associated kinase 4 (IRAK4) increased IL-1ß expression during LAMP stimulation, and TLR2-neutralizing antibodies reduced IL-1ß expression in EBL cells during LAMP stimulation. Furthermore, LAMPs inhibited IL-1ß expression following transfection with dominant-negative MyD88 and IRAK4 variants. These results suggested that Mmm-derived LAMPs activate IL-1ß production through the NF-κB pathway via TLR2, MyD88, and IRAK4.

Search for OIE-listed ruminant mycoplasma diseases in Afghanistan.

BACKGROUND: Little is known about the occurrence of important diseases of ruminants in Afghanistan because of the conflict affecting the country over the last 40 years. To address this discrepancy, ruminant herds in Afghanistan were screened for OIE-listed mycoplasma diseases, contagious bovine (CBPP) and caprine pleuropneumonias (CCPP). RESULTS: Of the 825 samples from 24 provinces tested for serological evidence of CBPP caused by Mycoplasma mycoides subsp.mycoides, 20 (3.4%) had ELISA values greater than the positive threshold of 50% though all were less than 55%. Repeat testing of these suspect sera gave values below 50. A smaller number of sera (330) from cattle in nine provinces were also tested by the rapid latex agglutination test (LAT) for CBPP, 10 of which were considered suspect. However, no positive bands were seen when immunoblotting was carried out on all sera that gave suspect results. Serological evidence of Mycoplasma bovis was detected in half of 28 herds in eight provinces. The cause of CCPP, M. capricolum subsp. capripneumoniae was not detected in any of the 107 nasal swabs and lung tissue collected from goats in seven provinces though sample handling and storage were not optimal. However, strong serological evidence was detected in goat herds in several villages near Kabul some of which were over 50% seropositive by LAT and ELISAs for CCPP; immunoblotting confirmed positive results on a selection of these sera. CONCLUSIONS: The data presented here provide a first assessment of the occurrence of the two OIE listed mycoplasma diseases in Afghanistan. From the results of the testing bovine sera from the majority of provinces there is no evidence of the presence of CBPP in Afghanistan. However the samples tested represented only 0.03% of the cattle population so a larger survey is required to confirm these findings. Serological, but not bacterial, evidence was produced during this investigation to show that CCPP is highly likely to be present in parts of Afghanistan.

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.

Selected ethno-medicinal plants from Kenya with in vitro activity against major African livestock pathogens belonging to the "Mycoplasma mycoides cluster".

ETHNOPHARMOCOLOGICAL RELEVANCE: Members of 'Mycoplasma mycoides cluster' are important ruminant pathogens in Africa. Diseases caused by these Mycoplasma negatively affect the agricultural sector especially in developing countries through losses in livestock productivity, mortality and international trade restrictions. There is therefore urgent need to develop antimicrobials from alternative sources such as medicinal plants to curb these diseases. In Kenya, smallholder farmers belonging to the Maasai, Kuria and Luo rely on traditional Kenyan herbals to treat respiratory symptoms in ruminants. In the current study extracts from some of these plants were tested against the growth of members of Mycoplasma mycoides cluster. AIM: This study aimed at identifying plants that exhibit antimycoplasmal activities using an ethnobotanical approach. MATERIALS AND METHODS: Kenyan farmers of Maasai, Luo and Kuria ethnic groups were interviewed for plant remedies given to livestock with respiratory syndromes. The plant materials were thereafter collected and crude extracts prepared using a mixture of 50% of methanol (MeOH) in dichloromethane (CH2Cl2), neat methanol (MeOH), ethanol (EtOH) and water to yield four crude extracts per plant part. The extracts were tested in vitro against five strains of Mycoplasma mycoides subsp. capri, five strains of Mycoplasma mycoides subsp. mycoides and one strain of Mycoplasma capricolum subsp capricolum using broth micro-dilution assays with an initial concentration of 1mg/ml. Minimum inhibitory concentration (MIC) of the most active extracts were determined by serial dilution. RESULTS: Extracts from five plants namely: Solanum aculeastrum, Albizia coriaria, Ekebergia capensis, Piliostigma thonningii and Euclea divinorum exhibited the highest activities against the Mycoplasma strains tested. Mycoplasma mycoides subsp. mycoides were more susceptible to these extracts than Mycoplasma mycoides subsp. capri and Mycoplasma capricolum susp. capricolum. The activities of the crude extracts varied with the solvent used for extraction. The MICs mean values of the active extracts varied from 0.02 to 0.6mg/ml. CONCLUSIONS: The results suggested that these plants could potentially contain antimicrobial compounds that might be useful for the treatment of respiratory diseases in ruminants. Future work should focus on the isolation and identification of the active compounds from the plant extracts that showed interesting activities and evaluation of their antimicrobial and cytotoxic potential.

In vitro assessment of the antimicrobial susceptibility of caprine isolates of Mycoplasma mycoides subsp. capri.

The minimum inhibitory concentration (MIC) and minimum mycoplasmacidal concentration (MMC) of 17 antimicrobials against 41 Spanish caprine isolates of Mycoplasma mycoides subsp. capri (Mmc) obtained from different specimens (milk, external auricular canal and semen) were determined using a liquid microdilution method. For half of the isolates, the MIC was also estimated for seven of the antimicrobials using an epsilometric test (ET), in order to compare both methods and assess the validity of ET. Mutations in genes gyrA, gyrB, parC and parE conferring fluoroquinolone resistance, which have been recently described in Mmc, were investigated using PCR. The anatomical origin of the isolate had no effect on its antimicrobial susceptibility. Moxifloxacin and doxycycline had the lowest MIC values. The rest of the fluoroquinolones studied (except norfloxacin), together with tylosin and clindamycin, also had low MIC values, although the MMC obtained for clindamycin was higher than for the other antimicrobials. For all the aminoglycosides, spiramycin and erythromycin, a notable level of resistance was observed. The ET was in close agreement with broth microdilution at low MICs, but not at intermediate or high MICs. The analysis of the genomic sequences revealed the presence of an amino acid substitution in codon 83 of the gene gyrA, which has not been described previously in Mmc.