Mycobacterium avium subsp. avium distribution studied in a naturally infected hen flock and in the environment by culture, serotyping and IS901 RFLP methods

Mycobacterium avium subsp. avium (MAA) of serotype 2 and genotype IS901+ and IS1245+ was cultured from 21 naturally infected hens (Gallus domesticus) from one smallholder aviary. From a total of 330 samples taken from hens, 124 mycobacteria were detected. Out of which MAA was detected in 103 (35.7%) of 288 tissues, in 4 (19.0%) of 21 swabs of cloacae and in 9 (42.9%) of 21 faeces samples, 8 other conditionally pathogenic mycobacterial species were also isolated. Tuberculous (TB) lesions were found in the liver, spleen and intestinal organs of seven hens. The isolates of MAA (n=58) from 16 infected hens (7 with TB lesions and 9 without TB lesions) were found to be of 3 IS901 RFLP types AE (n=48), AD (n=4) and E (n=6), where these MAA isolates are highly virulent to hens. Mixed infections with IS901 RFLP types (AE and AD) and (AE and E) were also evident in seven hens. From a total of 35 examined environmental samples, 23 mycobacterial isolates were detected. Out of which four (17.4%) MAA isolates of IS901 RFLP type AE and 19 (82.6%) other isolates of conditionally pathogenic mycobacteria were detected. The finding of identical IS901 RFLP types from both tissues and faecal isolates confirms that infected domestic hens are the principal source of infection for other susceptible hosts and lead to the contamination of the surrounding environment. The presence of different IS901 RFLP types in tissue isolates may indicate the repeated incidence of MAA infection and the occurrence of polyclonal infection.     

Paratuberculosis and avian tuberculosis infections in one red deer farm studied by IS900 and IS901 RFLP analysis

As the attempt to eradicate paratuberculosis in one red deer (Cervus elaphus) farm failed, all 167 red deer of different age groups were slaughtered and examined by culture for mycobacteria, and the farm was closed down. Spleen and hepatic lymph nodes, mediastinal lymph node, ileocecal lymph node, and ileum were collected from each animal and examined (a total of 835 organs). Neither tuberculosis lesions nor pathognomic signs of paratuberculosis were detected. Among all microscopically negative for mycobacteria organs, Mycobacterium avium subsp. paratuberculosis alone was isolated from 165 organs, M. a. avium alone from 41 organs, and both pathogens from four organs. M. a. paratuberculosis alone was detected in 71 red deer, M. a. avium alone in 13 red deer and both pathogens in 18 red deer. Using standardised RFLP methods, three IS900 RFLP types B-C1, B-C16, and B-C32 were identified among 40 M. a. paratuberculosis isolates and four IS901 RFLP types N-B1, N-B3, N-B4, and P-B3 among 17 M. a. avium isolates.     

Serotypes of Erysipelothrix rhusiopathiae in Australian pigs, small ruminants, poultry, and captive wild birds and animals

Serotypes of 93 Australian isolates of Erysipelothrix rhusiopathiae from diseased domestic animals and poultry and a variety of captive wild birds and animals were determined by double diffusion gel precipitation. Two isolates, from the faeces of a swallow were also examined. Serotypes 1a, 1b and 2 were isolated from pigs and serotypes 1a, 1b, 2, 5, 15 and 21 from sheep or goats. Erysipelas in poultry was attributed to serotypes 1b, 5, 15 and 16. In captive wild birds serotypes 1b, 5, 6, 8, 14, 21 and an isolate reactive with antiserum to strain Seehecht were associated with septicaemic deaths. Single isolates from tissues of a bilby (Macrotis lagotis), black rat (Rattus rattus), brown snake (Pseudechis australis) and a bandicoot (Isoodon macrouris) were classified as serotypes 4, 4, 7, and 10 respectively. Six isolates were not able to be typed. Serotype 1b was the most widely distributed and most common (28%), being associated with disease in pigs, sheep, poultry and wild birds. Serotypes 1a or 2 were found in a more restricted range of animals, being commonly associated with erysipelas in pigs, less commonly in sheep and infrequently in other species. From diseased pigs, 26 of 33 isolates (79%) were serotypes 1a and 1b.     

Development and validation of a real-time Taqman polymerase chain reaction assay for the detection of Mycoplasma gallisepticum in naturally infected birds

In this study, we report the development and validation of a real-time polymerase chain reaction (PCR) assay using a Taqman-labeled probe for the detection of Mycoplasma gallisepticum (MGLP assay). The MGLP assay was highly specific with a detection limit of 25 template copies per reaction and a quantification limit of 100 template copies per reaction. Validation of the assay was completed with 1247 samples (palatine cleft and tracheal swabs) from M. gallisepticum-positive and -negative chicken flocks. The MGLP assay was compared to an enzyme-linked immunosorbent assay (ELISA), a conventional polymerase chain reaction assay (mgc2 PCR), and isolation of M. gallisepticum from naturally infected flocks. A total of 805 samples collected from negative flocks, as verified by ELISA and/or mgc2 PCR, were negative by the MGLP assay. A total of 442 samples were collected from positive flocks, of which a total of 228 samples were positive by the MGLP assay. These results agreed for 98.87% of the samples when tested by mgc2 PCR. When comparing the MGLP assay with M gallisepticum isolation, the MGLP assay was more sensitive than isolation for detecting positive birds from a positive flock, 172/265 and 50/265, respectively. Overall, the MGLP assay and M. gallisepticum isolation agreed for 52.8% of the samples tested. In conclusion, the MGLP assay was highly specific, sensitive, and reproducible, and allowed the quantification of template copies directly from clinical samples.     

Histopathological,histochemical and immunohistochemical findings of the small intestine in goats naturally infected by Trichostrongylus colubriformis

Gastrointestinal (GI) strongyle infection remains one of the main constraints to goat production worldwide. Samples of small intestine from 15 Syrian goats naturally infected with Trichostrongylus colubriformis were examined by routine histology, histochemistry and immunohistochemistry to describe the histological changes and the phenotypes of inflammatory cellular components of the mucosa. Results indicated that the immune response to infection by T. colubriformis was characterized by an increased rate of the severity of the histologic lesions, an increase rate of T cell lymphocytes recruitment to the intestinal mucosa and quantitative and qualitative changes in the histochemical composition of mucin in goblet cells.     

Evolution of clinical, haematological and biochemical findings in young dogs naturally infected by vector-borne pathogens

Longitudinal studies evaluating the evolution of clinical, haematological, biochemical findings in young dogs exposed for the first time to multiple vector-borne pathogens have not been reported. With the objective of assessing the evolution of clinical, haematological and biochemical findings, these parameters were serially monitored in naturally infected dogs throughout a 1-year follow-up period. Young dogs, infected by vector-borne pathogens based on cytology or polymerase chain reaction, were examined clinically and blood samples were obtained at seven different follow-up time points. Dogs were randomized to group A (17 dogs treated with a spot-on formulation of imidacloprid 10% and permethrin 50%) or to group B (17 dogs untreated). In addition, 10 4-month-old beagles were enrolled in each group and used as sentinel dogs. At baseline, Anaplasma platys was the most frequently detected pathogen, followed by Babesia vogeli, Bartonella spp., Ehrlichia canis and Hepatozoon canis. Co-infections with A. platys and B. vogeli, followed by E. canis and B. vogeli, A. platys and H. canis and A. platys and Bartonella spp. were also diagnosed. In dogs from group B, abnormal clinical signs were recorded at different time points throughout the study. No abnormal clinical signs were recorded in group A dogs. Thrombocytopenia was the most frequent haematological alteration recorded in A. platys-infected dogs, B. vogeli-infected dogs and in dogs co-infected with A. platys and B. vogeli or A. platys and Bartonella spp. Lymphocytosis was frequently detected among dogs infected with B. vogeli or co-infected with A. platys and B. vogeli. Beagles were often infected with a single pathogen rather than with multiple canine vector-borne pathogens. There was a significant association (p<0.01) between tick infestation and A. platys or B. vogeli, as single infections, and A. platys and B. vogeli or A. platys and Bartonella spp. co-infections. This study emphasizes the clinical difficulties associated with assigning a specific clinical sign or haematological abnormality to a particular canine vector-borne disease.     

Evaluation of the sensitivity and specificity of bovine tuberculosis diagnostic tests in naturally infected cattle herds using a Bayesian approach

Test-and-slaughter strategies have been the basis of bovine tuberculosis (BT) eradication programs worldwide; however, eradication efforts have not succeeded in certain regions, and imperfect sensitivity and specificity of applied diagnostic techniques have been deemed as one of the possible causes for such failure. Evaluation of tuberculosis diagnostic tools has been impaired by the lack of an adequate gold standard to define positive and negative individuals. Here, a Bayesian approach was formulated to estimate for the first time sensitivity (Se) and specificity (Sp) of the tests [single intradermal tuberculin (SIT) test, and interferon-gamma (IFN-γ) assay] currently used in Spain. Field data from the first implementation of IFN-γ assay (used in parallel with SIT test 2-6months after a first disclosure SIT test) in infected beef, dairy and bullfighting cattle herds from the region of Castilla and Leon were used for the analysis. Model results suggested that in the described situation: (i) Se of SIT test was highly variable (40.1-92.2% for severe interpretation, median=66-69%), and its Sp was high (>99%) regardless interpretation criteria; (ii) IFN-γ assay showed a high Se (median=89-90% and 83.5% for 0.05 and 0.1 cut-off points respectively) and an acceptable Sp (85.7% and 90.3% for 0.05 and 0.1 thresholds) and (iii) parallel application of both tests maximized the combined Se (95.6% using severe SIT and 0.05 cut-off point in the IFN-γ assay). These results support the potential use of the IFN-γ assay as an ancillary technique for routine BT diagnosis.     

Analyses of Dutch Haemophilus parasuis isolates by serotyping, genotyping by ERIC-PCR and Hsp60 sequences and the presence of the virulence associated trimeric autotransporters marker

In this study, 117 isolates of Haemophilus parasuis from organs and tissues from pigs showing clinical signs, were characterised and compared with 10 H. parasuis reference strains. The isolates were subjected to the 16S rRNA gene PCR and subsequently serotyped, genotyped by 60-kDa heat shock protein (Hsp60) gene sequences, the enterobacterial repetitive intergenic consensus (ERIC) PCR and a multiplex PCR for the detection of the vtaA virulence associated trimeric autotransporter genes. Serotyping revealed the presence of 13 H. parasuis serovars. Serovars 3 and 10 were not detected, and 16 of the 117 H. parasuis isolates could not be typed by specific antisera. All isolates were positive in the 16S rRNA gene specific H. parasuis PCR. ERIC-PCR revealed a very heterogeneous pattern with 61 clusters; based on a 90% agreement. In total, 46 different Hsp60 sequence types were detected. Using 98% sequence similarity, as threshold for separation, 22 separate Hsp60 sequence clusters were distinguished. There was no correlation between H. parasuis serovars and ERIC-PCR clusters or Hsp60 sequence types, but both the ERIC-PCR and the Hsp60 sequence typing are suited as markers for H. parasuis molecular-epidemiology studies. In total, 102 H. parasuis swine isolates corresponded to the virulence associated group 1 vtaA type. The group 1 vtaA was detected in 12 different serovars. Only four of the 46 Hsp60 sequence types were not associated with the group 1 vtaA. This study shows that Dutch H. parasuis isolates from pigs with clinical signs have both a high serovar and genotypic lineage diversity. A majority of the known serovars contain the group 1 vtaA.     

Prevalence and molecular typing of Giardia spp. in captive mammals at the zoo of Zagreb, Croatia

A total of 131 faecal samples from 57 mammalian species housed at the zoo of Zagreb, Croatia, were tested for the presence of Giardia spp. cysts using epifluorescence microscopy. The overall prevalence (29%) was high, yet all animals were asymptomatic at the time of sampling. Positive samples were characterized by PCR and sequence analysis of both conserved and variable loci, for the identification of Giardia species and G. duodenalis assemblages and genotypes. Assemblages A and C were identified in Artiodactyla, assemblage B in Primates, Rodentia and Hyracoidea, and assemblages A, B, C and D, as well as Giardia microti, in Carnivora. Genotyping at the ITS1-5.8S-ITS2 region, at the triose phosphate isomerase, glutamate dehydrogenase and beta-giardin genes revealed extensive polymorphisms, particularly among assemblage B isolates. A phylogenetic analysis of concatenated sequences showed that isolates from captive mammals housed at the zoo are genetically different from isolates of human and domestic animal origin. This is the first survey in a zoological garden to include a molecular characterization of the parasite, and provides novel sequence data of G. duodenalis from many previously uncharacterized hosts.     

IgG, IgM and IgA in the serum of cattle naturally infected with Mycobacterium paratuberculosis

Serum IgG, IgM and IgA antibody response in 20 cattle naturally infected with Mycobacterium paratuberculosis and in 15 non-infected cattle were measured by enzyme-linked immunosorbent assay. A strong IgG response was detected in 16 (80%) of the infected animals. Diagnostic levels of IgM were detectable in all of the infected animals as well as in 8 (53%) of the non-infected animals. Animals with paratuberculosis had a very weak specific serum IgA response and this appears to be of little value in detection of infection in these animals.     

Species-specific polymerase chain reactions for the detection of Mycoplasma buteonis, Mycoplasma falconis, Mycoplasma gypis, and Mycoplasma corogypsi in captive birds of prey

Mycoplasmas are pathogens of different avian species, but the role of Mycoplasma in raptors is not yet completely determined. As Mycoplasma isolation and identification present several difficulties, species-specific polymerase chain reactions (PCRs) for the detection of mycoplasmas found in birds of prey (Mycoplasma buteonis, Mycoplasma corogypsi, Mycoplasma falconis, and Mycoplasma gypis) were established. The specificity of the PCR methods were investigated using known avian Mycoplasma reference strains and isolates as well as related bacteria and was found to be specific. Amplificons obtained with these PCRs from field samples showed no false-positive results in restriction enzyme analysis and sequencing. The sensitivities of the different PCR assays varied between 50 fg and 1 pg DNA. Twenty-five tracheal swabs from healthy captive birds of prey were investigated by culture and immunobinding assay as comparison to the PCRs. Mycoplasmal DNA was detected in 88% of the samples, with negative results only from vultures. Mycoplasma falconis and M. buteonis were regularly found in falcons, and M. gypis was found in a common buzzard. Mycoplasma corogypsi was not demonstrated. Several isolates could not be differentiated using an immunobinding assay as well as the described PCR methods.     

Immunodetection of coproantigens for the diagnosis of amphistomosis in naturally infected Indian Water Buffalo,Bubalus bubalis

The infection of gastrointestinal helminths in livestock is routinely diagnosed by microscopical examination of faecal samples for the presence of ova/eggs but this approach becomes ineffective for the seasonally egg producing trematodes. Therefore, an alternative approach to detect the coproantigens of liver and rumen amphistomes, Gigantocotyle explanatum and Gastrothylax crumenifer respectively, infecting Indian water buffalo Bubalus bubalis, was undertaken using ELISA, immunodot and countercurrent immunoelectrophoresis (CCIEP). The hyperimmune polyclonal antisera were separately raised in rabbits against excretory/secretory (ES) antigens of both the flukes under study. An overall 70% buffalo faecal samples were tested positive for G. crumenifer and 75% for G. explanatum in Aligarh region. The ELISA results reflected higher infection intensity among individual buffaloes that was also observed at necropsy. Using the respective homologous hyperimmune antiserum, 55% buffaloes tested positive for G. crumenifer and 65% positive for G. explanatum in immunodot assay. Further, the faecal samples with high absorbance values in ELISA and strong immunodot reaction tested positive in CCIEP. The analysis of CCIEP result revealed two and one precipitin bands in G. crumenifer and G. explanatum respectively, indicating prominent antigenic differences in the coproantigens of these two parasites. Taken together, it is suggested that polyclonal antibodies could be conveniently used for the detection of coproantigens by ELISA and immunodot methods, particularly during the non-egg producing phase of the seasonally regulated reproductive cycle of the rumen amphistome G. crumenifer. It is concluded that the coproantigen detection is a good alternative over conventional method for the diagnosis of amphistomosis in livestock; however, further studies are required on a larger sample size of field buffaloes to augment the reproducibility of the present results.     

Evaluation of cercarial antigen for the serodiagnosis of fasciolosis in experimentally and naturally infected sheep

The value of cercarial antigen for diagnosis of experimental and natural sheep fasciolosis was studied by enzyme linked immunosorbent assay (ELISA) and enzyme linked immunotransfer blot (EITB). In ELISA, the antibody levels of experimentally infected sheep with Fasciola gigantica appeared at 2 weeks post infection (PI), gradually increased till 7 weeks PI and nearly remained at the same level from 7 to 13 weeks PI (the end of experiment). Also, the sensitivity and specificity of cercarial antigen for diagnosis of naturally sheep fasciolosis were 100 and 90%, respectively.In EITB, in the sheep experimentally infected with F. gigantica, the band of 32.5kDa molecular weight polypeptide appeared at 2 weeks PI and continued till the end of experiment. Also, the cercarial antigen recognized 32.5kDa molecular weight band with all sera from naturally infected sheep with fasciolosis (n = 25). This band did not cross-react when tested with sera from infected sheep with Cysticercus tenuicollus (n = 20). This study suggests that, the 32.5kDa molecular weight polypeptide could be used as sensitive and specific epitope for the serodiagnosis of sheep fasciolosis.     

不同毒力结核分支杆菌感染RAW264.7巨噬细胞不同时间细胞因子转录水平标准曲线的建立及应用

为检测不同毒力结核分枝杆菌感染巨噬细胞后细胞因子转录水平的变化,构建IL-6、IL-10、TNF-α重组标准质粒,建立了实时荧光定量PCR标准曲线。应用该方法对不同毒力结核分枝杆菌感染RAW264.7巨噬细胞6个时间点细胞因子的转录水平进行分析,结果显示H37Rv组、BCG组相比于对照组刺激后均使三种细胞因子的转录水平发生变化,其中IL-6和TNF-α发生明显变化。本试验通过对不同时间点细胞因子转录水平研究的分析为结核分枝杆菌对巨噬细胞凋亡机制的研究提供新思路。     

Comparison of IS900 tissue PCR, bacterial culture, johnin and serological tests for diagnosis of naturally occurring paratuberculosis in goats

Comparative efficacy of an IS900 tissue PCR, bacterial culture, johnin, agar-gel immunodiffusion (AGID) and absorbed-ELISA tests was investigated in 43 goats naturally infected with paratuberculosis. On histological examination, tissue sections from all animals showed typical granulomatous inflammatory changes. The lesions were classified as multibacillary (MB) (n=30), which had diffuse granulomatous lesions with abundant acid-fast bacilli (AFB), and paucibacillary (PB) (n=13), which had focal or multifocal granulomatous lesions with few AFB. The sensitivities of johnin test, tissue culture, faecal culture, tissue PCR, AGID and ELISA were 68% (17/25), 100% (30/30), 84.6% (22/26), 100% (30/30), 96.2% (25/26) and 100% (26/26) in MB goats, and 88.8 (8/9), 46.1% (8/13), 40% (4/10), 61.5% (8/13), 50% (5/10), and 70% (7/10) in PB goats, respectively. Except for the johnin test, which showed higher sensitivity in PB goats, all other tests displayed significantly higher sensitivities in MB goats. The results indicate the usefulness of tissue PCR, culture and serological tests in the diagnosis of clinically affected paratuberculous goats, especially with multibacillary pathology.     

IgG, IgM and IgA in the serum of cattle naturally infected with Mycobacterium paratuberculosis

Serum IgG, IgM and IgA antibody response in 20 cattle naturally infected with Mycobacterium paratuberculosis and in 15 non-infected cattle were measured by enzyme-linked immunosorbent assay. A strong IgG response was detected in 16 (80%) of the infected animals. Diagnostic levels of IgM were detectable in all of the infected animals as well as in 8 (53%) of the non-infected animals. Animals with paratuberculosis had a very weak specific serum IgA response and this appears to be of little value in detection of infection in these animals.     

Evaluation of the ELISA test for the antibody detection in cattle naturally and experimentally infected with Cysticerccus bovis

The ELISA test was evaluated for the diagnosis of bovine cysticercosis using heterologous antigens from the larvae of T. solium and T. crassiceps, by using different types of positive and negative control sera, to allow a broader analysis of the results. The ELISA test showed low sensitivity under natural conditions of bovine cysticercosis manifestation, but high rates (up to 90%) under experimental conditions. The high specificity of the test (81-100%) made evident its capacity to differentiate cysticercosis from other bovine diseases. No difference in performance was found among the antigens studied. It was concluded that the ELISA test has deficiencies in detecting anti-cysticercosis antibodies of animals at slaughterhouse. However, it can be useful in detecting experimentally infected animals and differentiating cysticercosis from other bovine diseases.     

Evaluation of the pharmacokinetic profile of artesunate, artemether and their metabolites in sheep naturally infected with Fasciola hepatica

The pharmacokinetic (PK) parameters of artesunate, artemether and their metabolites dihydroartemisinin (DHA) and dihydroartemisinin-glucuronide (DHA-glucuronide) were determined in sheep naturally infected with Fasciola hepatica. Sheep were treated either with artesunate (intramuscular (i.m.): 40 and 60 mg/kg) or artemether (i.m.: 40 and 160 mg/kg; oral: 80 mg/kg). Blood samples were withdrawn at selected time points post treatment and the artemisinins were quantified in plasma by liquid chromatography and tandem mass spectrometry (LC-MS/MS). The in vitro effect of the metabolites against F. hepatica was investigated using a phenotype-based assay and scanning electron microscopy (SEM). Following artesunate applications (40 and 60 mg/kg), comparable C(max) (maximal plasma concentration) and AUCs (area under the plasma concentration-time curve) were observed for artesunate (C(max): 8.4×10(3) and 9.4×10(3)ng/ml; AUC: 6.9×10(5) and 9.7×10(5) ng min/ml), DHA (C(max): both 2.4×10(3)ng/ml; AUC: 3.7×10(5) and 5.0×10(5) ng min/ml), and DHA-glucuronide (C(max): 1.7×10(4) and 1.6×10(4)ng/ml; AUC: 2.6×10(6) and 3.3×10(6) ng min/ml). Mean elimination half-lifes (t(1/2)) of artesunate, DHA and DHA-glucuronide ranged between 58 and 63 min, 94 and 113min, and 89 and 98 min, respectively. The i.m. oil-based drug formulation liberated artemether slowly and constant levels of artemether and its metabolites were observed during the entire sampling period (24 h). The AUCs of all analytes were significantly higher for the i.m. 160 mg/kg dose compared to i.m. 40 and oral 80 mg/kg doses (P=0.018). Mean C(max) of artemether (2126 and 426 ng/ml) and DHA-glucuronide (3477 and 1587 ng/ml) were higher following oral compared to i.m. (160 mg/kg) treatments (P>0.068), whereas C(max) of DHA was significantly higher following i.m. applications (P=0.0062). DHA rapidly reduced the viability of F. hepatica in vitro, whereas DHA-glucuronide showed no activity. SEM observations revealed only minor and focal tegumental alterations in few of the DHA treated worms. The calculated PK parameters reflect the anthelmintic activity of artesunate and artemether following different routes of application and will aid in the design of future studies with these drugs.     

Sensitivity and specificity of various serological tests for the detection of Toxoplasma gondii infection in naturally infected sheep

Comparative serological examination of 300 serum samples from sheep slaughtered in the main abattoir in Cairo, Egypt revealed a higher prevalence of toxoplasmosis (43.7%) with the modified agglutination test (MAT), followed by the enzyme linked immune-sorbant assay (ELISA) (41.7%) and the indirect fluorescent antibody test (IFAT) (37%), while the lowest prevalence was detected with the dye test (DT) (34%). When the data from the first three serological tests were compared with that of the DT test, which was used as a reference test for toxoplasmosis, MAT had the highest sensitivity (96%), followed by ELISA (90.1%) and IFAT, which demonstrated the lowest sensitivity (80.4%). Conversely, IFAT had the highest specificity (91.4%), followed by MAT (88.9%) and ELISA (85.9%).