Survey of selected pathogens in free-ranging pinnipeds in Uruguay.

Marine mammals, regarded as sentinels of aquatic ecosystem health, are exposed to different pathogens and parasites under natural conditions. We surveyed live South American fur seals Arctocephalus australis and South American sea lions Otaria flavescens in Uruguay for Leptospira spp., canine distemper virus (CDV), Mycobacterium spp., Toxoplasma gondii, and Neospora caninum. Samples were collected from 2007 to 2013. The seroprevalence of Leptospira spp. was 37.6% positive, 50.9% negative, and 11.5% suspect for A. australis (n = 61) while for O. flavescens (n = 12) it was 67% positive, 25% negative, and 8% suspect. CDV RNA was not detected in any of the analyzed samples. Most animals tested seropositive to tuberculosis antigens by WiZo ELISA (A. australis: 29/30; O. flavescens: 20/20); reactivity varied with a novel ELISA test (antigens MPB70, MPB83, ESAT6 and MPB59). Seroprevalence against N. caninum and T. gondii was 6.7 and 13.3% positive for O. flavescens and 0 and 2.2% positive for A. australis respectively. To evaluate possible sources of infection for pinnipeds, wild rats Rattus rattus and semi-feral cats Felis catus were also tested for Leptospira spp. and T. gondii respectively. Water samples tested for Leptospira revealed saprofitic L. bioflexa. Pathogenic Leptospira were detected in the kidneys of 2 rats, and cats tested positive for T. gondii (100%). These results represent a substantial contribution to the study of the health status of wild pinnipeds in Uruguay.

Piscine mycobacteriosis in the ornamental fish trade in Trinidad and Tobago.

The freshwater ornamental fish trade represents a major contributor to the livelihoods of many producers in Trinidad and Tobago, with stocks destined for local, regional and international markets. A review of clinical cases presented to the Aquatic Animal Health Unit at the University of the West Indies, School of Veterinary Medicine for the period September 2010 to December 2012 suggested that piscine mycobacteriosis may be widespread throughout the local ornamental fish industry. Thus, to determine the prevalence of mycobacteriosis in ornamental fish sold in pet stores, a total of 122 specimens were sourced from 24 retail suppliers across Trinidad. Fish were killed and internal organs were examined for lesions suggestive of granulomas. All wet-mount slides were acid-fast stained, regardless of the presence or absence of observed granuloma-like lesions. Histological analysis was performed on one randomly selected whole specimen from each facility. Mycobacterium sp. was identified using real-time PCR detecting the 16S rRNA gene in tissue samples. Associations between parasitism, facility biosecurity and presence of positive animals were determined. The prevalence of Mycobacterium sp. infection was 61 ± 7% (74/122), with positive specimens being acquired from 54.2% (13/24) of facilities examined. Further, 100% of facilities did not employ optimum biosecurity measures.

[Genetic Engineering in Mycobacteria].

Genetic tools for targeted modification of the mycobacterial genome contribute to the understanding of the physiology and virulence mechanisms of mycobacteria. Human and animal pathogens, such as the Mycobacterium tuberculosis complex, which causes tuberculosis, and M. leprae, which causes leprosy, are of particular importance. Genetic research opens up novel opportunities to identify and validate new targets for antibacterial drugs and to develop improved vaccines. Although mycobacteria are difficult to work with due to their slow growth rate and a limited possibility to transfer genetic information, significant progress has been made in developing genetic engineering methods for mycobacteria. The review considers the main approaches to changing the mycobacterial genome in a targeted manner, including homologous and site-specific recombination and use of the CRISPR/Cas system.

Isolation and identification of nontuberculous mycobacteria from specimens of lower respiratory tract of transplanted patients based on the evaluation of 16SrRNA gene.

BACKGROUND: Nontuberculous mycobacteria are pervasive microorganisms and are often present as saprophytes in humans, animals, and the environment. Today, these bacteria are known as the most important environmental opportunists and, in the last decades, infections by nontuberculous mycobacteria have multiplied, due to increased immunodeficiency (cancer, transplant recipients, HIV). STUDY DESIGN: This study aimed to investigate the infections by nontuberculous mycobacteria in transplanted patients. METHODS: The study was performed on 57 samples from respiratory secretions of transplant recipients taken by standard methods. Nontuberculous mycobacteria were identified by culture method and molecular identities of clinical isolates were investigated by PCR amplification using 16SrRNA gene and sequence analysis and Blast of the sequences. Demographic data were evaluated by Spss software. RESULTS: The prevalence of nontuberculous mycobacteria in transplant patients was 22.8%, the age of patients was between 23 and 52 years. The most common involvement of nontuberculous mycobacteria in our transplanted individuals were 6 strains of M avium-intracellulare Complex (42.87%), followed by 2 strains of M marinum (14.29%) and 1 strain each (7.14%) of M xenopi, M chelonae, M intracellulare, M kansasii, M simiae. At the conclusion of the tests, one final strain was identified as M tuberculosis (7.14%). CONCLUSION: The prevalence of nontuberculous mycobacteria indicates their importance in the fate of these patients. The identification of nontuberculous mycobacteria is a neglected part of microbiology laboratories, due to the lack of sufficient facilities and the risk associated with their culture. Therefore developing routine methods for the identification of these infections appears to be critical, especially in hospitals with the transplantation ward.

Evidence of fish and human pathogens associated with doctor fish (Garra rufa, Heckel, 1843) used for cosmetic treatment.

Doctor fish (Garra rufa, Heckel, 1843) are increasingly used for cosmetic treatment raising particular concerns regarding the potential transmission of infections to clients. Investigations of microbial causes undertaken in two outbreaks of mortality among G. rufa used for cosmetic treatment revealed the presence of multiple bacteria, including both fish and human pathogens such as Aeromonas veronii, A. hydrophila, Vibrio cholerae, Shewanella putrefaciens, Mycobacterium marinum and M. goodii. This range of bacteria indicates an intense microbial proliferation involving multiple pathogens, most likely induced by the poor health condition of the fish. Most of the detected pathogens are well-known agents of zoonosis. Indeed, M. goodii is an emerging nosocomial human pathogen that has never been detected in fish to date, nor in other animals. This first detection of M. goodii associated with fish infection points out a new zoonotic potential for this pathogen. These findings point out that handling, poor environmental conditions and the presence of fish pathogens, that can compromise the immune system of fish, can result in a mixed microbial proliferation and increase the spread of waterborne bacteria, including zoonosis agents. Accordingly, the microbiological surveillance of fish used for cosmetic treatment is extremely important, particularly in association with mortality outbreaks.

Dibasic Derivatives of Phenylcarbamic Acid against Mycobacterial Strains: Old Drugs and New Tricks?

In order to provide a more detailed view on the structure⁻antimycobacterial activity relationship (SAR) of phenylcarbamic acid derivatives containing two centers of protonation, 1-[2-[({[2-/3-(alkoxy)phenyl]amino}carbonyl)oxy]-3-(dipropylammonio)propyl]pyrrolidinium oxalates (1a⁻d)/dichlorides (1e⁻h) as well as 1-[2-[({[2-/3-(alkoxy)phenyl]amino}carbonyl)oxy]-3-(di-propylammonio)propyl]azepanium oxalates (1i⁻l)/dichlorides (1m⁻p; alkoxy = butoxy to heptyloxy) were physicochemically characterized by estimation of their surface tension (γ; Traube's stalagmometric method), electronic features (log ε; UV/Vis spectrophotometry) and lipophilic properties (log kw; isocratic RP-HPLC) as well. The experimental log kw dataset was studied together with computational logarithms of partition coefficients (log P) generated by various methods based mainly on atomic or combined atomic and fragmental principles. Similarities and differences between the experimental and in silico lipophilicity descriptors were analyzed by unscaled principal component analysis (PCA). The in vitro activity of compounds 1a⁻p was inspected against Mycobacterium tuberculosis CNCTC My 331/88 (identical with H37Rv and ATCC 2794, respectively), M. tuberculosis H37Ra ATCC 25177, M. kansasii CNCTC My 235/80 (identical with ATCC 12478), the M. kansasii 6509/96 clinical isolate, M. kansasii DSM 44162, M. avium CNCTC My 330/80 (identical with ATCC 25291), M. smegmatis ATCC 700084 and M. marinum CAMP 5644, respectively. In vitro susceptibility of the mycobacteria to reference drugs isoniazid, ethambutol, ofloxacin or ciprofloxacin was tested as well. A very unique aspect of the research was that many compounds from the set 1a⁻p were highly efficient almost against all tested mycobacteria. The most promising derivatives showed MIC values varied from 1.9 µM to 8 µM, which were lower compared to those of used standards, especially if concerning ability to fight M. tuberculosis H37Ra ATCC 25177, M. kansasii DSM 44162 or M. avium CNCTC My 330/80. Current in vitro biological assays and systematic SAR studies based on PCA approach as well as fitting procedures, which were supported by relevant statistical descriptors, proved that the compounds 1a⁻p represented a very promising molecular framework for development of 'non-traditional' but effective antimycobacterial agents.

Comparison of Saramis 4.12 and IVD 3.0 Vitek MS Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Mycobacteria from Solid and Liquid Culture Media.

During the last decade, many investigators have studied matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for identification of mycobacteria. Diverse and contradictory results indicated that optimal level for routine testing has not been reached yet. This work aimed to assess Vitek MS through two distinct versions, Saramis v4.12 RUO and the IVD v3.0, under conditions close to routine laboratory practice. Overall, 111 mycobacterial isolates were subjected to protein extraction and same spectra were matched against both databases. The IVD v3.0 database proved to be superior to Saramis v4.12 and its identification rates remarkably increased, from 67% to 94% for isolates grown on Middlebrook 7H10 solid medium and from 62% to 91% for isolates grown on mycobacterial growth indicator tube (MGIT) liquid medium. With this new version, IVD v3.0, MALDI-TOF MS might be integrated into routine clinical diagnostics, although molecular techniques remain mandatory in some cases.

Pathologic features of mycobacteriosis in naturally infected Syngnathidae and novel transcriptome assembly in association with disease.

Syngnathidae (seahorses, seadragons and pipefish) suffer significant losses from non-tuberculous mycobacteria. However, they produce markedly different lesions in response to the disease compared to other teleost species, notably infrequent granuloma formation. This study evaluated 270 syngnathid fish, from which 92 were diagnosed with mycobacteriosis by histopathology, culture or both. Microscopic lesions variably consisted of random foci of coagulative necrosis in multiple organs, containing high numbers of free bacteria and large aggregates or sheets of macrophages with cytoplasm laden with acid-fast bacilli. Mycobacterial associated granulomas were identified in only six seahorses. Five fish had positive cultures with no observed microscopic changes. RNA-seq of the head kidney was performed to investigate the transcriptome of two infected and six non-infected lined seahorses Hippocampus erectus. Assembled and annotated putative transcripts serve to enrich the database for this species, as well as provide baseline data for understanding the pathogenesis of mycobacteriosis in seahorses. Putative components of the innate immune system (IL-1ß, IL-6, TNF, NOS, Toll-like receptor 1, MHC Class I, NF-κß, transforming growth factor beta, MyD88) were identified in the RNA-seq data set. However, a homolog for a key component in the TH1 adaptive immune response, interferon-gamma, was not identified and may underlie the unique pathologic presentation.

Parasite prevalence in Mycobacterium spp. infected dairy goats in the region of Murcia (south-east Spain).

Eighty-four Murciano-Granadina dairy goats slaughtered in the region of Murcia (South-East Spain) were necropsied to evaluate parasitic infections. The majority of the animals (94.0%) were parasitised and multiple infections were present. Twenty-one parasite species were found, including 18 nematode species (Muellerius capillaris, Dictyocaulus filaria, Neostrongylus linearis, Cystocaulus ocreatus, Teladorsagia circumcincta, T. occidentalis, T. trifurcata, Marshallagia marshalli, Camelostrongylus mentulatus, Trichostrongylus capricola, Nematodirus abnormalis, N. filicollis, N. spathiger, T. vitrinus, T. colubriformis, Trichuris spp., Chabertia ovina and Skrjabinema ovis); one trematode species (Dicrocoelium dendriticum); one arthropod species (Oestrus ovis); and one protozoa genus (Eimeria spp.). Additionally, 17.85% of the animals were Mycobacterium spp. positive. Therefore, comparison between parasite prevalence, intensity and abundance in tuberculosis-positive and -negative animals was performed. Statistically significant differences between the prevalence of lungworms and gastrointestinal nematodes in Mycobacterium spp. infected and free goats were found. The paper discusses this co-infection between Mycobacteriaceae and endoparasites.

Une étude a été conduite sur 84 chèvres laitières de race Murciano­Granadina abattues dans la région de Murcia (sud-est de l'Espagne) afin d'évaluer la présence d'infections parasitaires. Dans leur grande majorité (94,0 %), les chèvres étaient infestées de parasites et le nombre de pluri-infections était important. Au total, 21 espèces de parasites ont été observées, dont 18 espèces de nématodes (Muellerius capillaris, Dictyocaulus filaria, Neostrongylus linearis, Cystocaulus ocreatus, Teladorsagia circumcincta, T. occidentalis, T. trifurcata, Marshallagia marshalli, Camelostrongylus mentulatus, Trichostrongylus capricola, Nematodirus abnormalis, N. filicollis, N. spathiger, T. vitrinus, T. colubriformis, Trichuris spp., Chabertia ovina et Skrjabinema ovis), une espèce de trématode (Dicrocoelium dendriticum), une espèce d'arthropode (Oestrus ovis) et des membres d'un genre de protozoaire (Eimeria spp.). En outre, la présence de Mycobacterium spp. a été détectée chez 17,85 % des chèvres examinées. Par conséquent, une étude comparative a été réalisée pour déterminer la prévalence, l'intensité et l'abondance des parasites chez les animaux ayant donné des résultats respectivement positifs et négatifs à la détection de la tuberculose. Des différences significatives au plan statistique ont été constatées entre les chèvres infectées et indemnes de Mycobacterium spp., respectivement, pour ce qui concerne la prévalence des vers pulmonaires et des nématodes gastro-intestinaux. Les auteurs font le point sur cette co-infection par des mycobactéries et des endoparasites.

Tras ser sacrificadas, ochenta y cuatro cabras lecheras murciano­granadinas de la región de Murcia (sudeste de España) fueron sometidas a necropsia para determinar la presencia de eventuales parasitosis. La mayoría de los animales (un 94,0%) presentaban parásitos y múltiples infecciones. Se descubrieron veintiuna especies de parásito: 18 especies de nemátodo (Muellerius capillaris, Dictyocaulus filaria, Neostrongylus linearis, Cystocaulus ocreatus, Teladorsagia circumcincta, T. occidentalis, T. trifurcata, Marshallagia marshalli, Camelostrongylus mentulatus, Trichostrongylus capricola, Nematodirus abnormalis, N. filicollis, N. spathiger, T. vitrinus, T. colubriformis, Trichuris spp., Chabertia ovina y Skrjabinema ovis); una especie de tremátodo (Dicrocoelium dendriticum); una especie de artrópodo (Oestrus ovis) y un género de protozoo (Eimeria spp.). Además, el 17,85% de los ejemplares eran positivos para Mycobacterium spp. A partir de ahí se procedió a comparar la prevalencia, intensidad y abundancia de parásitos en los animales positivos para la tuberculosis con respecto a los negativos. Se observaron diferencias estadísticamente significativas entre las cabras que estaban infectadas por Mycobacterium spp. y las que no lo estaban en cuanto a la prevalencia de nemátodos pulmonares y gastrointestinales. Los autores examinan esta infección concomitante entre micobacteriáceas y endoparásitos.

Granulomatous lesions in a wild mullet population from the eastern Ligurian Sea (Italy): mycobacteriosis vs. pseudotuberculosis.

Mycobacterium spp. and Photobacterium damselae subsp. piscicida are recognized as the most frequent causative agents of granulomatous lesions in fish. Although frequent episodes of mycobacterial infections have been reported in wild fish worldwide, only sporadic cases have been documented to date in Italy. To investigate for the presence of lesions referable to mycobacteriosis and to identify the mycobacterial species involved, a total of 159 wild mullets were fished from the eastern coast of the Ligurian Sea, killed and necropsied. Liver and spleen samples were collected from all fish for histopathological and microbiological analyses. Molecular investigations for identification of Photobacterium damselae subsp. piscicida were performed. Gross examination revealed granulomatous lesions in one animal; microscopically, 42.14% of fish displayed granulomas with various histological features, 19.50% resulted positive at Ziehl-Neelsen staining, and were confirmed as mycobacterial lesions by culture. The identified colonies were characterized as M. fortuitum, M. abscessus, M. flavescens, M. chelonae, M. septicum and M. nonchromogenicum. In all, 35% of animals resulted positive for Photobacterium damselae subsp. piscicida. These data suggest widespread mycobacterial infection also by Photobacterium damselae subsp. piscicida infections in wild fish. Moreover, the pathogenicity of some mycobacterial species, previously considered as saprophytic, was demonstrated.

Serum proteomics analysis for differentiation among Mycobacterium tuberculosis infection categories.

Inhalation of Mycobacterium tuberculosis (Mtb) bacilli can lead to a range of TB categories including early clearance (EC), latent TB infection (LTBI) and active TB (ATB). There are few biomarkers available to differentiate among these TB categories: effective new biomarkers are badly needed. Here, we analyzed the serum proteins from 26 ATB cases, 20 LTBI cases, 34 EC cases and 38 healthy controls (HC) using label-free LC-MS/MS. The results were analyzed using MaxQuant software and matched to three different bacterial proteomics databases, including Mtb, Mycobacterium spp. and normal lung flora. PCA of protein candidates using the three proteomics databases revealed 44.5% differentiation power to differentiate among four TB categories. There were 289 proteins that showed potential for distinguishing between each pair of groups among TB categories. There were 50 candidate protein markers specifically found in ATB and LTBI but not in HC and EC groups. Decision trees using the top five candidate biomarkers (A0A1A2RWZ9, A0A1A3FMY8, A0A1A3KIY2, A0A5C7MJH5 and A0A1X0XYR3) had 92.31% accuracy to differentiate among TB categories and the accuracy was increased to 100% when using 10 candidate biomarkers. Our study shows that proteins expressed from Mycobacterium spp. have the potential to be used to differentiate among TB categories.

"Trained Immunity" from Mycobacterium spp. exposure (BCG vaccination and environmental) may have an impact on the incidence of early childhood leukemia.

Preventive variables for childhood leukemia incidence (LI) remain unknown. Past assertions that childhood vaccinations, especially BCG, may be potentially protective have remained disputed for over five decades because of the lack of a unifying framework to explain variable outcomes in different studies. An examination of the early childhood LI for 2020 in European Region countries with supposedly similar underlying confounders but differential childhood vaccination coverage displays negative covariation with prevailing Mycobacterium spp. exposure in BCG-vaccinated children. The childhood LI in 0-4-year-old populations with >90% childhood BCG vaccination coverage is found to be strongly but negatively correlated with prevailing tuberculin immunoreactivity [r(24): -0.7868, p-value: < 0.0001]. No such correlation existed for the LI in 0-4-year-old populations without BCG vaccinations, though weak associations are hinted at by the available data for MCV2, PCV3, and DTP3 vaccinations. We hypothesize that early childhood BCG vaccination "priming" and subsequent "trained immunity" augmentation by "natural" boosting from Mycobacterium spp. exposure play a preventive and protective role in childhood LI. The non-consideration of prevailing "trained immunity" could have been a cause behind the conflicting outcomes in past studies. Exploratory studies, preferably performed in high-burden countries and controlling for the trained-immunity correlate and other potential confounders, would be warranted in order to establish a role for BCG vaccination and early-life immune training (or lack thereof) in childhood LI and help put the current controversy to rest.

BCG vaccination policy, natural boosting and pediatric brain and CNS tumor incidences.

Bacille Calmette-Guérin (BCG) vaccination supposedly imparts and augments "trained immunity" that cross-protects against multiple unrelated pathogens and enhances general immune surveillance. Gradual reductions in tuberculosis burden over the last 3-5 decades have resulted in the withdrawal of BCG vaccination mandates from developed industrialized countries while reducing to a single neonatal shot in the rest. Concurrently, a steady increase in early childhood Brain and CNS (BCNS) tumors has occurred. Though immunological causes of pediatric BCNS cancer are suspected, the identification of a causal protective variable with intervention potential has remained elusive. An examination of the countries with contrasting vaccination policies indicates significantly lower BCNS cancer incidence in 0-4-year-olds (per hundredthousand) of countries following neonatal BCG inoculations (n=146) vs. non-BCG countries (n=33) [Mean: 1.26 vs. 2.64; Median: 0.985 vs. 2.8; IQR: 0.31-2.0 vs. 2.4-3.2; P=<0.0001 (two-tailed)]. Remarkably, natural Mycobacterium spp. reexposure likelihood is negatively correlated with BCNS cancer incidence in 0-4-year-olds of all affected countries [r(154): -0.6085, P=<0.0001]. Seemingly, neonatal BCG vaccination and natural "boosting" are associated with a 15-20-fold lower BCNS cancer incidence. In this opinion article, we attempt to synthesize existing evidence implying the immunological basis of early childhood BCNS cancer incidence and briefly indicate possible causes that could have precluded objective analysis of the existing data in the past. We draw the attention of the stakeholders to consider the comprehensive evaluation of immune training as a potential protective variable through well-designed controlled clinical trials or registry-based studies as feasible for its potential applications in reducing childhood BCNS cancer incidence.

Antibiotic Susceptibility of Mycobacteria Isolated from Ornamental Fish.

Introduction: Nontuberculous mycobacteria (NTM) are increasingly recognised as causative agents of opportunistic infections in humans for which effective treatment is challenging. There is very little information on the prevalence of NTM drug resistance in Poland. This study was aimed to evaluate the susceptibility to antibiotics of NTM, originally isolated from diseased ornamental fish. Material and Methods: A total of 99 isolates were studied, 50 of them rapidly growing mycobacteria (RGM) (among which three-quarters were Mycobacterium chelonae, M. peregrinum, and M. fortuitum and the rest M. neoaurum, M. septicum, M. abscessus, M. mucogenicum, M. salmoniphilum, M saopaulense, and M. senegalense). The other 49 were slowly growing mycobacteria (SGM) isolates (among which only one was M. szulgai and the bulk M. marinum and M. gordonae). Minimum inhibitory concentrations for amikacin (AMK), kanamycin (KAN), tobramycin (TOB), doxycycline (DOX), ciprofloxacin (CIP), clarithromycin (CLR), sulfamethoxazole (SMX), isoniazid (INH) and rifampicin (RMP) were determined. Results: The majority of the isolates were susceptible to KAN (95.95%: RGM 46.46% and SGM 49.49%), AMK (94.94%: RGM 45.45% and SGM 49.49%), CLR (83.83%: RGM 36.36% and SGM 47.47%), SMX (79.79%: RGM 30.30% and SMG 49.49%), CIP (65.65%: RGM 24.24% and SGM 41.41%), and DOX (55.55%: RGM 9.06% and SGM 46.46%). The majority were resistant to INH (98.98%: RGM 50.50% and SGM 48.48%) and RMP (96.96%: RGM 50.50% and SGM 46.46%). Conclusion: The drug sensitivity of NTM varies from species to species. KAN, AMK, CLR and SMX were the most active against RGM isolates, and these same four plus DOX and CIP were the best drugs against SGM isolates.

Isothermal inactivation of Mycobacterium avium subsp. paratuberculosis in curd simulating the stretching phase in pasta-filata cheese process.

Raw milk and dairy products are usually considered the major sources of Mycobacterium avium subsp. paratuberculosis (MAP) exposure for humans. During the production process of mozzarella cheese, as well as of other pasta-filata cheeses made with pasteurized or raw milk, curd is heated and stretched by addition of hot or boiling water. This step is the critical point for the inactivation of MAP during the production process, but, to our knowledge, no studies have been published about the thermal death time values of MAP in curd. The aim of this study was to determine the inactivation kinetics of MAP in curd used to produce pasta-filata cheese in six independent experiments. The milk was inoculated with a mix of MAP strains (field and registered strains) and, with the aim to simulate the thermal treatment of the curd during the stretching step, samples of 10 g of contaminated curd were vacuum packed and treated separately at six different temperatures from 60°C to 75°C in a water bath. MAP survival was then evaluated by plate count method and inactivation parameters were estimated for determining the thermal resistance of the pathogen directly in the curd. D-values increased from 0.15 min (D75-value) to 4.22 min (D60-value) and the calculated z-value was 10.2°C. These data aid: (i) to design food thermal process treatments defining acceptance limits of critical control points to ensure safety against MAP; (ii) to predict the time/temperature combinations needed to obtain a certain MAP log reduction during the curd stretching step; (iii) to optimize or validate pasta-filata cheese process.

Cryptobia iubilans Infections in Discus Fish in Trinidad and Tobago.

Discus (Symphysodon spp.) are costly and prized specimens in the international ornamental fish trade. The majority of discus submitted to the Aquatic Animal Health Unit at the University of the West Indies School of Veterinary Medicine for necropsy between September 2010 and September 2015 had lesions consistent with Cryptobia iubilans infection, thus prompting this study. To determine the prevalence of the flagellated gastrointestinal protozoan C. iubilans in discus fish, 32 discus were sourced from 10 suppliers, including breeders, importers, and hobbyists across Trinidad. Fish were euthanized, and the internal organs, particularly the stomach and intestine, were observed under a light microscope for characteristic granulomatous lesions and/or live C. iubilans parasites. All wet-mount slides on which granulomas were observed were also Ziehl-Neelsen acid-fast stained to presumptively exclude the presence of Mycobacterium spp., the main differential when diagnosing C. iubilans-associated granulomatous gastritis or to determine the presence of dual infections. Further histological analyses were performed on stomach and intestinal sections, and transmission electron microscopy was used to confirm the parasite in stomach sections. The prevalence of C. iubilans infection was found to be 81.3%, and the prevalence of presumptive dual infections with Mycobacterium spp. was found to be 21.9%. To the best of our knowledge, this is the first documented study of C. iubilans infections in the wider Caribbean region.

Occurrence of Mycobacterium spp. in ornamental fish.

INTRODUCTION AND OBJECTIVE: Fish mycobacteriosis is a chronic granulomatous disease caused by several species of bacteria from the genus Mycobacterium, described as nontuberculous mycobacteria (NTM). The most important species causing fish mycobacterioses are M. chelonae, M. fortuitum, and M. marinum. Mycobacteria infecting fish also include zoonotic pathogens. M. marinum is the cause of most cases of fish-related mycobacterial infection in humans. The disease occurs more frequently in workers in the fishing industry, people whose hobbies involve water activities, and aquarists. The aim of the present study was to examine the occurrence of different species of mycobacteria in freshwater ornamental fish. MATERIAL AND METHODS: The occurrence of Mycobacterium spp. in freshwater ornamental fish was studied from January 2015 - December 2016. Material isolated from skin scrapings, contents of the digestive tracts, and internal organs of ornamental fish was stained with Ziehl-Neelsen (ZN) and inoculated on Lowenstein-Jensen medium. All isolates found positive by ZN were identified by amplification of the gene encoding the Hsp65 protein. A total of 408 samples obtained from 136 ornamental fish from 36 species were tested. RESULTS: Using the culture method Mycobacterium was isolated from 69 fish (50.1%) and 99 samples (24.3%). Sequence analysis of gene fragments coding for the Hsp65 protein of 99 isolates revealed occurrence of 13 species of mycobacteria: M. abscessus, M. chelonae, M. fortuitum, M. gordonae, M. marinum, M. mucogenicum, M. neoaurum, M. peregrinum, M. salmoniphilum, M. saopaulense, M. senegalense, M. septicum, and M. szulgai. CONCLUSIONS: The obtained results indicate a significant role of ornamental fish as a source of mycobacteria which are potentially dangerous,especially to humans.

Mycobacterium caprae in a dairy farm in Northeast Italy.

Veneto region, Northeast Italy, has been declared officially free from bovine tuberculosis since 2008, although the disease is sporadically detected in association with cattle trade. In September 2015, bovine tuberculosis was detected in a dairy cattle farm of the region, in a holding with 69 animals. The herd underwent single intradermal tuberculin testing as part of the regional surveillance plan, and 24 animals resulted positive. Mycobacterium caprae was evidenced in 22 samples, further genotyped by PCR-based assays, as Allgäu type. Epidemiological investigation reported that sixteen animals were introduced from an officially tuberculosis free Member State in previous years. Nevertheless, spoligotyping and multilocus variable tandem repeat analysis (MLVA) indicated that M. caprae was strictly related to the strain circulating in 2007-2009 in Trento province, although no at-risk contacts were described. M. caprae is a zoonotic pathogen and further analyses are warranted in order to control its spread and impact on public health and animal trade.

Naturally Avian Influenza Virus-Infected Wild Birds Are More Likely to Test Positive for Mycobacterium spp. and Salmonella spp.

Wild birds often harbor infectious microorganisms. Some of these infectious microorganisms may present a risk to domestic animals and humans through spillover events. Detections of certain microorganisms have been shown to increase host susceptibility to infections by other microorganisms, leading to coinfections and altered host-to-host transmission patterns. However, little is known about the frequency of coinfections and its impact on wild bird populations. In order to verify whether avian influenza virus (AIV) natural infection in wild waterbirds was related to the excretion of other microorganisms, 73 AIV-positive samples (feces and cloacal swabs) were coupled with 73 AIV-negative samples of the same sampling characteristics and tested by real-time PCR specific for the following microorganisms: West Nile virus, avian avulavirus 1, Salmonella spp., Yersinia enterocolitica, Yersinia pseudotuberculosis, Mycobacterium avium subspecies, Mycobacterium tuberculosis complex, and Mycobacterium spp. Concurrent detections were found in 47.9% (35/73) of the AIV-positive samples and in 23.3% (17/73) of the AIV-negative samples (P = 0.003). Mycobacterium spp. and Salmonella spp. were found to be significantly more prevalent among the AIV-positive samples than among the AIV-negative samples (42.9% vs. 22.8%; P = 0.024 and 15.2% vs. 0.0%; P = 0.0015, respectively). Prevalence of concurrent detections differed significantly among sampling years (P = 0.001), host families (P = 0.002), host species (P = 0.003), AIV subtypes (P = 0.003), and type of sample (P = 0.009). Multiple concurrent detections (more than one of the tested microorganisms excluding AIV) were found in 9.6% (7/73) of all the AIV-positive samples, accounting for 20% (7/35) of the concurrent detection cases. In contrast, in AIV-negative samples we never detected more than one of the selected microorganisms. These results show that AIV detection was associated with the detection of the monitored microorganisms. Further studies of a larger field sample set or under experimental conditions are necessary to infer causality in these trends.

Las aves silvestres frecuentemente albergan microorganismos infecciosos. Algunos suponen un riesgo por su posible transmisión a animales domésticos o representar un problema de salud pública si son zoonóticos. Se ha relacionado la detección de algunos microorganismos microbianos con una mayor susceptibilidad del hospedador a la infección por otros, llevando a una coinfección y a una alteración de los patrones de transmisión entre hospedadores. Sin embargo, poco se sabe sobre la frecuencia y el impacto de estas coinfecciones en la epidemiologia de las enfermedades en las aves silvestres. Con el ánimo de determinar si una infección natural con el virus de la influenza aviar (VIA) en aves acuáticas se relaciona con la excreción de otros microorganismos, se seleccionaron 73 muestras positivas a VIA y un número igual de negativas de similares características y se sometieron a análisis por PRC a tiempo real para la detección de los siguientes agentes: virus del Nilo occidental, avulavirus aviar de tipo 1, Salmonella spp., Yersinia enterocolitica, Yersinia pseudotuberculosis, subspecies de Mycobacterium avium, complejo Mycobacterium tuberculosis y Mycobacterium spp. Se detectaron otros agentes concurrentes en el 48% (35/73) de las muestras positivas a VIA frente al 23.3% (17/73) en las negativas (p=0.003). La prevalencia de Mycobacterium spp. y Salmonella spp. fue significativamente mayor entre las muestras positivas a VIA que entre las negativas (42.9% vs. 22.8%; p=0.024 y 15.2% vs. 0.0%; p=0.0015 respectivamente). La prevalencia de otros agentes difirió significativamente entre el año de recogida, la familia (p=0.002), la especie (p=0.003), los subtipos de VIA (p=0.003) y el tipo de muestra (p=0.009). Se detectaron múltiples microorganismos en el 9.6% (7/73) de las muestras positivas a VIA, lo que se correspondió con un 20% (7/35) de las detecciones concurrentes. Sin embargo en las muestras negativas a VIA no detectamos más de uno de los microorganismos estudiados. Estos resultados confirman que la detección de los agentes microbianos monitorizados se vio incrementada en presencia del VIA. Consideramos necesario la realización de estudios con un mayor número de muestras o en condiciones experimentales para inferir causalidad sobre estas tendencias.

Seroprevalence and risk factors for selected respiratory and reproductive tract pathogen exposure in European bison (Bison bonasus) in Poland.

After the complete extinction from the wild of European bison (Bison bonasus) at the beginning of the twentieth century, the worldwide species population was restored to approximately 5500 individuals, with the species however remaining endangered. Despite numerous studies on the ecology and genetics of European bison, the threats of infectious diseases have been largely unexamined. The aim of this study was to screen the exposure of the world's largest population of European bison to the pathogens, which may influence the condition and development of the endangered species. A total of 240 free-ranging and captive European bison from eight main Polish populations sampled were tested for the presence of specific antibodies against ten different viruses, bacteria or protozoan. The samples were collected from chemically immobilized, selectively culled or found dead animals. Based on serology, the exposure to bovine viral diarrhea virus (BVDV), bovine herpesvirus type 1 (BoHV-1), Mycoplasma and Brucella spp. was determined as rather accidental. Using gamma-interferon assay followed by Mycobacterium tuberculosis subs. caprae detection in tissues, diagnosis of bovine tuberculosis was made for 6 out of 78 (7.7%) bison from one captive herd. The highest seroprevalence was found for bovine adenovirus type 3 (BAdV-3) -60.2% and bovine parainfluenza type 3 (PIV-3) -34.0%, while the antibodies against bovine respiratory syncytial virus (BRSV), Toxoplasma gondii and Leptospira spp. were found in 10.4%, 10.4% and 8.7% of samples, respectively. In the multivariable statistical analysis using generalized linear mixed models (GLMMS), the risk factors for PIV-3 seropositivity included population type (free-living/captive), age and health status (apparently healthy/eliminated due to the poor condition). Higher risk of BAdV-3 seropositive result was observed in free-living female European bison. The high BAdV-3 and PIV-3 seroprevalences may suggest involvement of these pathogens in the most frequently observed respiratory disorders in European bison. Moreover, this is the first study demonstrating BAdV-3 exposure in the species.