Potential application of serological tests on fluids from carcasses: detection of antibodies against Toxoplasma gondii and Sarcoptes scabiei in red foxes (Vulpes vulpes)

Background

Serological surveys for disease investigation of wild animal populations require obtaining blood samples for analysis, which has logistic, ethic and economic difficulties. Applying serological test to fluids collected from dead animals is an alternative. The aim of this study was to assess if antibodies could be detected in two types of fluids collected from 56 carcasses of red foxes (Vulpes vulpes): pleural fluid and lung extract.

Findings

In 22 (39%) foxes antibodies against Sarcoptes scabiei were detected in both fluid types by ELISA and Western blot. In 46 (82%) foxes, antibodies against Toxoplasma gondii were detected in pleural fluid and in 41 (73%) in lung extract applying a Toxo-screen test (DAT). Antibodies were still detectable in the same fluids kept at room temperature for 28 days, although in fewer foxes (16 and 14 foxes tested for T. gondii in lung extract and pleural fluid respectively; and 1 and 4 tested for S. scabiei in lung extract and pleural fluid respectively.

Conclusions

These results indicate the potential utility of using fluids from carcasses for antibody screening of wild animals at the population level.     

Atypical colony-like structures developing in control media and in clinical L-form cultures containing serum

Solid and liquid media designed to support growth of cell wall deficient variants were evaluated for the presence of endogenous variants. The media compared in this study consisted of brain heart infusion base containing 10% sucrose, 3% NaCl + 5% sucrose, 3% NaCl, or 5% NaCl + 20% sucrose, and supplemented with filtrates of 10–20% horse serum - Sources A, B, C, suppliers of slaughterhouse (Type I) serum; and S, a supplier of standing herd (Type II) serum - and yeast extract. Three types of yeast supplements used in this study were aqueous extracts of: (1) fresh, or (2) active dry yeast; and (3) 0.5% commercial dehydrated yeast extract. Liquid control media prepared from one supplier gave rise to a pleomorphic Gram positive rod on extended incubation ( > 10 days) in spite of rigorous controls for asepsis during preparation and use. The second and more serious problem concerned repeated encounters with ‘pseudocolony’-like structures in work with clinical material. Correlation with severity of disease, different rates of appearance, and promotion of development by high osmolarity, led to a comparison of this phenomenon in control Type I- vs. Type II-BYE medium. Type I gave rise to numerous ‘pseudocolony‘- like bodies within 3 weeks, while Type II media containing standing herd serum developed < 20 per plate. Media variations known to promote growth of L-phase variants and mycoplasmas were noted to affect time of appearance and numbers of atypical colonies in control, uninoculated Type II-BYE. These included serum level, quality of yeast extract, pH, osmolar supplements, and the O₂ tension. The development of these structures in only certain Type II-BYE cultures of clinical fluids does not support the view that disturbance of an agar surface, or the presence of cells (or nuclei) promotes ‘growth’. No atypical colony-like structures or amber bodies developed in plates incubated for 7 weeks in the presence of vapors of phenol + formaldehyde, nor during an additional 10-day interval in a normal gaseous environment. It is suggested that all horse sera tested in this study contained unclassified atypical L-forms with unusual resistance to heat and chemicals.     

Molecular Monitoring of the Main Changes in Bacterial Floral Diversity in the Gastrointestinal Tract of a Thoroughbred Foal with Catarrhal Enteritis by Using PCR-DGGE

In this study, the main changes in bacterial floral diversity in the gastrointestinal tract of a Thoroughbred foal were monitored by using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). The foal died of catarrhal enteritis of the cecum and large colon. Diarrheal feces and gastrointestinal contents were compared with normal feces. The closest relatives of the bacterium in the samples were Lactobacillus johnsonii (100% similarity), uncultured Bacteroides sp. (92.5% similarity), Bacteroides fragilis (96.3% similarity), and Enterococcus faecium/Enterococcus durans (100% similarity); these were detected by PCR-DGGE using a universal primer set. Monitoring revealed that the numbers of Escherichia coli/Shigella sonnei (97.9% similarity) were significantly higher in the diarrheal feces. Thus, PCR-DGGE is a useful tool for monitoring the main changes in bacterial floral diversity occurring in the gastrointestinal tracts of Thoroughbreds.     

Recovery and identification of anaerobes in veterinary medicine: A 2-year experience

The examination of 300 specimens over a 2-year period in a veterinary diagnostic laboratory (1975–1977) revealed that of the specimens yielding bacteria, 56.3% contained anaerobes while 71.8% yielded facultative bacteria. Of the total specimens 26.6% proved to be sterile and 4.6% were unsuitable. The anaerobes which were isolated with the highest frequency from diseased animals belonged to the following genera: Clostridium, 46.0%; Bacteroides 15.1%; Fusobacterium, 14.3%; Actinomyces, 11.1%; and Propionibacterium, 5.6%. The culture method used in this study was based upon reducible solid media for primary isolation. Its efficiency compared favorably with what is commonly accepted as an effective recovery rate for anaerobes. However, the relative proportion of anaerobic genera obtained from veterinary clinical specimens was in direct contrast with specimens obtained in medical diagnostic laboratories. Because of this difference and the kinds of anaerobes most frequently encountered in veterinary medicine, it is believed that the culture method used in this study is more suitable to veterinary medicine than other methods more commonly employed in medical diagnostic laboratories.     

The Effect of Pneumostrongylus tenuis (Nematoda: Metastrongyloidea) on Kids

Kids, 6-28 weeks of age, infected with 200-1000 infective larvae of meningeal worm (Pneumostrongylus tenuis) of white-tailed deer (Odocoileus virginianus), developed colitis and peritonitis. Bacteroides sp., Bacillus sp., Escherichia coli, Proteus sp. and Enterococcus sp. were found in the peritoneal cavity. Most kids were moribund or dead 4-11 days after infection and frequently the colon had ruptured. Only two kids (of 11) in which the disease was allowed to run its course survived colitis and peritonitis but these two animals developed severe neurologic signs 18 and 38 days after infection. Numerous developing worms and various traumatic and other lesions were found in the central nervous system of kids which developed neurologic signs.     

Detection of Bartonella sp. and a novel spotted fever group Rickettsia sp. in Neotropical fleas of wild rodents (Cricetidae) from Southern Brazil

The Neotropical region shows a great diversity of fleas, comprising more than 50 genera. The importance of the study of fleas is linked to their potential role as disease vectors. The aim of this study is to investigate the presence of Rickettsia spp. and Bartonella spp. in Neotropical fleas collected from wild rodents in Southern Brazil. From 350 rodents captured, 30 were parasitized by fleas. A total of 61 fleas belonging to two genera and six different species were collected (Craneopsylla minerva minerva, Polygenis occidentalis occidentalis, Polygenis platensis, Polygenis pradoi, Polygenis rimatus, and Polygenis roberti roberti). In 13 % of fleas of three different species (C. minerva, P. platensis, and P. pradoi) Rickettsia sp. DNA was found. Phylogenetic analysis of concatenated sequences of gltA, htrA, and ompA genes showed that Rickettsia sp. found in rodent fleas (referred as strain Taim) grouped together with Spotted Fever Group Rickettsia. In reference to Bartonella spp., five genotypes were identified in seven fleas of two species (C. minerva and P. platensis) and in five rodent spleens. Also, 207 frozen samples of wild rodents were screened for these pathogens: while none was positive for Rickettsia spp.; five rodent spleens were PCR-positive for Bartonella spp.. Herein, we show the detection of potential novel variants of Bartonella sp. and Rickettsia sp. in fleas collected of wild rodents from Southern Brazil. Further studies are needed to fully characterize these microorganisms, as well as to improve the knowledge on the potential role of Neotropical flea species as diseases vectors.     

Influence of dietary mushroom Agaricus bisporus on intestinal morphology and microflora composition in broiler chickens

In this study, we evaluated the intestinal morphology and bacteria populations in broiler chickens fed for six weeks diets that contained different amount of the mushroom Agaricus bisporus. Ninety day-old female chicks were randomly divided into three dietary treatments, each with three replicates kept in floor pens and fed a basal diet supplemented with the dried mushroom at levels of 0, 10 or 20 g/kg fresh feed. Feed and water were offered to birds ad libitum. The morphological examinations of the intestine were carried out on 1-cm long excised segments from duodenum, jejunum and ileum. The populations of total aerobes, total anaerobes, Lactobacilli spp., Bifidobacteria spp., Escherichiacoli, Bacteroides spp. and Enterococci were enumerated in ileum and caecum by conventional microbiological techniques using selective agar media. The results of the study showed that dietary mushroom supplementation did not significantly affect intestinal morphology at either level of inclusion. Morphometrical parameters of depth of duodenum, jejunum and ileum crypt and height of villi revealed no differences amongst dietary treatments. In the ileum, Lactobacilli spp. were higher in birds supplemented at the level of 20 g/kg compared to controls; however, other measurements of bacteria loads were similar amongst the three dietary treatments. In the caecum, Lactobacilli spp. and Bifidobacteria spp. loads were higher in birds supplemented at either level of inclusion compared to control birds, although these did not differ between the two levels of supplementation. In conclusion, dietary mushroom supplementation may beneficially affect intestinal health of broiler chickens.     

Identification of bacteria in the tracheal swabs of farmed ostriches and their effect on the viability of influenza A virus

Avian influenza surveillance is a requirement for commercial trade in ostrich products, but influenza A viruses (IAVs) have proven difficult to isolate from ostrich tracheal swabs that test positive using molecular methods. We hypothesized that microbes unique to the ostrich trachea propagate in the transport medium after sampling and affect viral viability. We cultured tracheal swabs from 50 ostriches on 4 farms in South Africa, and recovered and identified 13 bacterial, 1 yeast, and 2 fungal species. Dietzia sp. had not been identified previously in the oropharyngeal tract of a bird, to our knowledge. The bacteria were tested for antimicrobial susceptibility, and most aerobic species, except for Streptococcus sp. and Pseudomonas sp., were sensitive to enrofloxacin; all were susceptible to sulfonamide. Virus inhibition experiments determined that ostrich-source Streptococcus sp., Pantoea sp., and Citrobacter freundii produced extracellular metabolites that caused a substantial reduction in the IAV titers of 99.9%. Streptomyces, Corynebacterium, Staphylococcus, Arthrobacter gandavensis, Pseudomonas putida, and Acinetobacter spp. similarly reduced the viability of IAV from 77.6% to 24.1%. Dietzia appeared to have no effect, but Rothia dentocariosa, Rhodotorula spp., and Clostridium spp. slightly increased the viability of IAV by 25.9, 34.9, and 58.5%, respectively.     

Age-associated changes in caecal microbiome and their apparent correlations with growth performances of layer pullets

The microbiome in gastrointestinal tracts play an important role in regulating nutrient utilization and absorption, gut immune function, and host growth or development. This study was conducted to investigate the composition and dynamic distribution of caecal microbiota in pullets during the first 16 weeks. Growth performance, immune organs index, and intestinal morphology of pullets were analyzed at 3, 6, 12 and 16 weeks of age. The caecal contents were collected for microbiota analysis by 16S rRNA gene sequencing method. With advancing ages in pullets, the gradually increased average daily feed intake (ADFI), feed conversion ratio (FCR) and intestinal villus height, but the gradually decreased organs index of thymus and bursa were determined. Meanwhile, more abundant caecal bacterial communities were determined from pullets at 12 and 16 weeks of age than those at 3 and 6 weeks of age. Furthermore, the dominant microflora of pullets from different weeks of age were analyzed by using LEfSe: The higher abundance of Blautia, Prevotella, Alistipes, and Eggerthella were found at 6 weeks; Anaerostipes, Oscillospira, Enterococcus and Methanobrevibacter were determined at 12 weeks; and the higher abundance of Parabacteroides, Anaerofustis, Lactobacillus and Butyricimonas were determined at 16 weeks. Further functional predicted analysis by PICRUSt revealed that the endocrine system and carbohydrate metabolism were significantly developed at 3 weeks. The development of the immune system was predicted to be mainly during 6 to 12 weeks, while cardiovascular diseases and circulatory system were during 12 to 16 weeks. In addition, the significantly negative correlation between Bacteroides and villus height, the significantly negative correlation between growth parameters (ADFI and FCR) and Bacteroides, Oscillospira and Alistipes; and the significantly positive relations between growth parameters (ADFI and FCR) and Bilophila, Lactobacillus, Rikenella and Anaerofustis were determined by using Pearson analyses. In conclusion, our data demonstrated that growth performance and intestinal morphology correlate well with caecal microbiota, which could provide new insights to establish or develop nutritional strategies to manage the intestinal health or development of laying pullets.     

Isolation and characterisation of bacteria from pyothorax (empyaemia) in cats

Samples from 19 cases of feline empyaemia were examined. All cases had no prior treatment and specimens were collected by thoracocentesis after preparation of the skin as for aseptic surgery. Of the 87 bacterial strains isolated, 70 (80.5%) were anaerobes and 17 (19.5%) were facultative anaerobes. Fourteen cases contained mixtures of anaerobes and facultative anaerobes, and five contained anaerobes only. Bacteroides was the most commonly isolated genus (42.5% of all isolates). Clostridium villosum (16.1%) was the most frequently isolated bacterial species followed by Peptostreptococcus anaerobius and Pasteurella multocida (each 12.6%). The most commonly isolated anaerobic species was Clostridium villosum sp.nov. (20% of anaerobic isolates and 16.1% of all isolates) and Pasteurella multocida was the most commonly isolated facultatively anaerobic species (64.7% of facultative isolates and 12.6% of all isolates).     

Microscopic and molecular identification of hemotropic mycoplasmas in South American coatis (Nasua nasua)

Hemoplasmas were detected in two apparently healthy captive South American coatis (Nasua nasua) from southern Brazil during an investigation for vector-borne pathogens. Blood was subjected to packed cell volume (PCV) determination, a commercial real-time PCR panel for the detection of Anaplasma spp., Babesia spp., Bartonella spp., Hepatozoon spp., Leishmania spp., Mycoplasma haemofelis, ‘Candidatus Mycoplasma turicensis’, ‘Candidatus Mycoplasma haemominutum’, Neorickettsia risticii, Rickettsia rickettsii and Leptospira spp., and a pan-hemoplasma conventional PCR assay. PCV was normal, but both coatis tested positive for hemoplasmas and negative for all the remaining pathogens tested. Using different techniques for microscopy (light, confocal or SEM), structures compatible with hemoplasmas were identified. Sequencing of the 16S rRNA gene identified an organism resembling Mycoplasma haemofelis and another hemotropic Mycoplasma sp., with a sequence identity of 96.8% to a Mycoplasma sp. previously detected in capybaras.     

Comparison of seven nucleic acid amplification tests for detection of Taylorella equigenitalis

Taylorella equigenitalis causes contagious equine metritis. Here we compared seven nucleic acid amplification tests for T. equigenitalis to select a rapid and reliable diagnostic method. The 95% detection limits of each assay varied greatly: real-time PCR had the lowest detection limit (0.77 fg/reaction); those of some of the conventional PCRs (cPCRs) were >100 fg/reaction. In experimentally infected samples, real-time PCR and semi-nested PCR showed the highest positive numbers (33 out of 42 samples), but two of the cPCRs detected only 2 and 7 positive results. Our results indicate that the use of sensitive molecular assays is important for the efficient detection of T. equigenitalis in clinical samples.     

Levels of nicotinamide adenine dinucleotide in extracellular body fluids of pigs may be growth-limiting for Actinobacillus pleuropneumoniae and Haemophilus parasuis

During infection, nutrient deprivation can alter bacterial phenotype. This, in turn, may have implications for pathogenesis and prophylaxis. Actinobacillus pleuropneumoniae (biotype 1) and Haemophilus parasuis, respiratory tract pathogens of swine, are both V-factor-dependent. The concentrations of V factor in the extracellular fluids of pigs are unknown and may limit the growth of these bacteria in vivo. The aim of this study was to determine the concentrations of nicotinamide adenine dinucleotide (NAD) in select porcine body fluids and to compare the availability of NAD in vivo with the affinities of the organisms for this compound. Levels in plasma, tissue fluids (peritoneal, pleural, synovial, and cerebrospinal), and laryngeal, tracheal, and lung washings were determined with an enzymatic cycling assay. We concluded that, although the NAD supply in the respiratory tract is probably not growth-limiting, it may become limiting if the organisms are disseminated.     

Targeted sequencing of candidate gene regions for myelofibrosis in dogs

BackgroundMyelofibrosis often lacks an identifiable cause in dogs. In humans, most primary myelofibrosis cases develop secondary to driver mutations in JAK2, CALR, or MPL.ObjectivesTo determine the prevalence of variants in JAK2, CALR, or MPL candidate regions in dogs with myelofibrosis and in healthy dogs.AnimalsTwenty‐six dogs with myelofibrosis that underwent bone marrow biopsy between 2010 and 2018 and 25 control dogs matched for age, sex, and breed.MethodsCross‐sectional study. Amplicon sequencing of JAK2 exons 12 and 14, CALR exon 9, and MPL exon 10 was performed on formalin‐fixed, decalcified, paraffin‐embedded bone marrow (myelofibrosis) or peripheral blood (control) DNA. Somatic variants were categorized as likely‐benign or possibly‐pathogenic based on predicted impact on protein function. Within the myelofibrosis group, hematologic variables and survival were compared by variant status (none, likely‐benign only, and ≥1 possibly‐pathogenic). The effect of age on variant count was analyzed using linear regression.ResultsEighteen of 26 (69%) myelofibrosis cases had somatic variants, including 9 classified as possibly‐pathogenic. No somatic variants were detected in controls. Within the myelofibrosis group, hematologic variables and survival did not differ by variant status. The number of somatic variants per myelofibrosis case increased with age (estimate, 0.69; SE, 0.29; P = .03).Conclusions and Clinical ImportanceSomatic variants might initiate or perpetuate myelofibrosis in dogs. Our findings suggest the occurrence of clonal hematopoiesis in dogs, with increasing incidence with age, as observed in humans.     

Determination of the Anthelmintic Efficacy of Albendazole in the Treatment of Chickens Naturally Infected with Gastrointestinal Helminths

In the spring of 2006, 60 naturally infected hens obtained from a broiler-breeder farm in northwest Arkansas were used in a controlled titration study to determine the anthelmintic efficacy of albendazole in the treatment of both nematode and cestode infections. Albendazole was used at the dose rates of 0.0, 5.0, 10.0, and 20.0 mg/kg of BW, with all treatments given individually as an oral suspension on d 0 (split doses) and with necropsies for parasite collection conducted on d 7. There were 15 birds per treatment group. Statistically significant (P < 0.05) reductions in worm burdens from control levels were seen at the 5.0 mg/kg dose level for adult and larval stages of Ascaridia galli, Heterakis gallinarum, and Capillaria obsignata. A significant (P < 0.05) reduction in the numbers of Raillietina cesticillus (scolexes) from control group levels was seen only at the 20.0 mg/kg rate of treatment. For albendazole given at the rates of 5.0, 10.0, and 20.0 mg/kg, respective anthelmintic efficacies based on geometric means were 87.7, 91.2, and 98.2% (A. galli larvae); 100.0, 100.0, and 100.0% (A. galli adults); 96.9, 95.7, and 98.9% (H. gallinarum larvae); 92.7, 95.4, and 94.9% (H. gallinarum adults); 90.3, 91.3, and 95.1% (C. obsignata larvae and adults combined); and 73.1, 73.1, and 96.2% (R. cesticillus). No adverse reactions to albendazole were observed in this study.     

Polymerase chain reaction typing of genes of the locus of enterocyte effacement of ruminant attaching and effacing Escherichia coli

The variability of the tir, espA, and espD genes of the locus of enterocyte effacement (LEE) in 185 attaching and effacing Escherichia coli (AEEC) strains isolated from healthy and diarrheic cattle, sheep, and goats was investigated by polymerase chain reaction. Nineteen of the strains were enterohemorrhagic E. coli (EHEC); the other 166 were enteropathogenic E. coli (EPEC). The combinations of the tir and esp genes were associated with the variants of the eae gene but not with a strain’s belonging to the EPEC or EHEC group, animal species, or health status (healthy or diarrheic) of the animal. In addition, most of the strains showed the same combinations of LEE genes and serogroups as have been found in AEEC strains isolated from humans, which indicates that ruminants seem to be an EPEC reservoir for humans.     

Isolation and identification of bacteriophages infecting ayu Plecoglossus altivelis altivelis specific Flavobacterium psychrophilum

In order to investigate methods for controlling systemic bacterial coldwater disease (CWD), bacteriophages that infect Flavobacterium psychrophilum were isolated by the enrichment method from pond water collected from Japanese ayu farms. The five phages isolated were classified as members of Myoviridae (PFpW-3, PFpC-Y), Podoviridae (PFpW-6, PFpW-7), and Siphoviridae (PFpW-8) and had highly variable patterns of infectivity for different F. psychrophilum isolates (n = 128). The stability tests of the phages in different waters, pHs and temperatures were assessed, and the results indicated that none of the phages were affected by ayu farm conditions. Among the phages, PFpW-3 had high infectivity for F. psychrophilum isolated from ayu and other fish and demonstrated sufficient survivability in the stability tests. Thus, PFpW-3 and its indicator strain N2-3 were inoculated into cytophaga broth at different doses of multiplicity of infection (MOI) and proved to be efficient for the reduction of bacterial growth. This study may be the basis for a further evaluation of phage therapy in the treatment of CWD in Japanese ayu farms.     

Occurrence of Giardia and Cryptosporidium in pigs on Prince Edward Island, Canada

In a cross-sectional study of 633 pigs from 21 herds on Prince Edward Island, Canada (PEI), the prevalence of infection with Cryptosporidium and Giardia, and the genotypes and species of isolates were determined in order to establish the zoonotic potential of pigs in this region. As determined by direct immunofluorescence microscopy (DFA), 18 herds (86%) and 163 animals (26%; 95% CI: 22-29%) tested positive for Cryptosporidium, while just 3 herds (14%) and 6 animals (1%; 95% CI: 0.4-2%) tested positive for Giardia. Cryptosporidium spp. isolates were detected in 39% (95% CI: 34-44%) of weanlings (1-3 months of age) and 9% (95% CI: 6-13) of sows (>8 months of age). Molecular characterization using the 18S rDNA and HSP70 gene fragments revealed the presence of Cryptosporidium sp. pig genotype II, C. suis, C. parvum, and Cryptosporidium sp. mouse genotype. Among the 113 isolates of Cryptosporidium spp. successfully genotyped, pig genotype II (61%) predominated, with C. suis (36%) being the next most prominant isolate. C. parvum (2%; two isolates) and Cryptosporidium sp. mouse genotype (0.9%; one isolate) were only occasionally isolated. The only two Cryptosporidium-positive genotyped isolates from sows included one each of C. suis and Cryptosporidium sp. pig genotype II.All but one of the six Giardia positive isolates were detected in weanling pigs. None of the Giardia-positive isolates was amenable to PCR. This study demonstrates that Cryptosporidium spp. are highly prevalent in pigs on PEI, Canada, are found mostly in weanlings (1-3 months of age). Furthermore, the pigs are primarily infected by the host-specific genotypes and species, Cryptosporidium sp. pig genotype II and C. suis, whereas the zoonotic C. parvum is rare. Giardia duodenalis is only occasionally found in pigs. These findings suggest that domestic pigs on PEI, Canada, likely do not pose a significant health risk to humans from these parasites.     

Molecular investigation of zoonotic intracellular bacteria in Chilean bats

Intracellular pathogens were investigated for the first time in 55 Chilean bats belonging to six species. Using a conventional PCR protocol targeting a fragment of the ITS region, 21 bats (38 %) were positive for DNA of Bartonella sp. Molecular characterization of fragments of the gltA, rpoB and fstZ genes and subsequent phylogenetic analysis indicated the presence of diverse genotypes related to Bartonella from bats worldwide. DNA from C. burnetii was investigated using a real-time PCR (qPCR) protocol targeting the IS1111 gene and yielded positive results for 5 individuals (9%), being the first report of C. burnetii in wildlife in Chile. All bats were negative for Rickettsia sp., evaluated by qPCR for the gltA gene, confirming that bats do not act as important reservoirs for Rickettsia. This preliminary survey calls for more comprehensive studies on the epidemiology of these agents, including larger sample sizes, the evaluation of potential transmission routes and spillover potential.