Comparative Study of Mycobacterium bovis and Mycobacterium avium subsp. paratuberculosis In Vitro Infection in Bovine Bone Marrow Derived Macrophages: Preliminary Results.

Bovine tuberculosis and paratuberculosis are endemic in many areas worldwide. This work aims to study cytokines production and gene expression profiles of bovine macrophages infected with Mycobacterium bovis and Mycobacterium paratuberculosis subsp. avium (MAP) strains to identify potential diagnostic biomarkers. Bovine bone marrow stem cells were differentiated into macrophages and subsequently infected in vitro with different spoligotypes of M. bovis and MAP field strains (as single infections and coinfections), using different multiplicity of infection. Supernatant and cell pellets were collected 24 h, 48 h, and one week post-infection. Preliminarily, gene expression on cell pellets of IL-1ß, IL-2, INFγ, IL-6, IL-10, IL-12, and TNFα was assessed by qRT-PCR one week p.i. Subsequently, IL-1ß and IL-6 were measured by ELISA and qRT-PCR to investigated their production retrospectively 24 h and 48 h p.i. A variability in macrophages response related to the concentration of mycobacteria, the coinfection with MAP, and M. bovis spoligotypes was identified. An early and constant IL-6 increase was observed in the M. bovis infection. A lower increase in IL-1ß was also detected at the highest concentration of the two M. bovis spoligotypes one week post-infection. IL-6 and IL-1 ß production was reduced and differently expressed in the MAP infection. IL-6 appeared to be the earliest cytokines produced by bovine macrophages infected with M. bovis.

Large-scale serological survey on Mycobacterium avium subsp. paratuberculosis infection in sheep and goat herds in Sicily, Southern Italy.

Introduction: Paratuberculosis (PTB) is a worldwide chronic, contagious enteric disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) mainly affecting ruminant species. PTB is a WOAH-listed disease with direct and indirect economic losses in the livestock sector, negative impact on animal welfare and significant public health concerns. In spite of this, MAP prevalence in small ruminants is still unknown and the prevalence appears to be underestimated in many countries. The aim of this study is providing a first large-scale serological survey on MAP infection in small ruminants in Sicily, a region of Southern Italy with the 11.3 and 8.9% Italian national heritage of sheep and goats, respectively. Methods: For this purpose, we analyzed a total of 48,643 animals reared in 439 flocks throughout Sicily. MAP seroprevalence was estimated both at herd-level and animal-level within breeds reared in all the nine sampled provinces. Results: Our results revealed a high overall apparent prevalence at herd-level of 71.8% in sheep and 60.8% in goat farms with an animal-level prevalence of 4.5 and 5.1% in sheep and goats, respectively. Significant statistical differences were found between the provinces and within the breeds both in sheep and goats. Discussion: Our study provides the first large-scale serological survey on PTB infection in small ruminants in Sicily and showed a high prevalence of disease depending to the species, breed and province. This study represents the first step to better understand the MAP epidemiology in a typical Mediterranean breeding context, suggesting the need of in-depth study on the herds risk factors, including the eventual presence of candidate genes for resistance/susceptibility to PTB in native breeds.

Development and evaluation of a multi-antigen serological assay for the intra-vitam diagnosis of Tuberculosis caused by Mycobacterium bovis in pigs.

Bovine Tuberculosis (bTB) is a chronic disease caused by Mycobacterium bovis, affecting cattle and other mammalian species, such as pigs. In the present work, we developed a novel multi-antigen assay (The TB-Luminex multiplex test) to diagnose bTB in pig sera. Moreover, we investigated the seroreactivity to the different antigens employed (MPB83, MPB70, CFP10 and ESAT6) and the possible correlation with bTB lesions distribution in the positive pigs. The serum samples were collected from 59 bTB positive pigs and 186 pigs reared in an officially Tuberculosis free area. Sera were processed according to an optimized protocol for the detection of antibodies by a multiantigen assay using Luminex technology. The positive group showed visible lesions with localized (54.2%) or generalized (45.8%) distribution. Culture confirmed the infection in 62.7% of the cases, and histopathology and intra-vitam assays were used as additional confirmatory tests. Within the set of antigens tested, the immunodominant was MPB83 (positive in 94.9% of the affected pigs), followed by CFP10, MPB70 and ESAT6 (positivity shown in 81.3%, 67.8% and 25.4% of the positive pigs tested, respectively). The best antigens combination was MPB83/CFP10, with a 96.6% sensitivity and 96.8% specificity. Overall, the test showed high sensitivity (98.3% and 86.4%) and specificity (96.2% and 97.8%), if sera were considered positive according to the positivity to a single antigen or at least two antigens, respectively. The TB-Luminex multiplex test results did not give significantly different outcomes according to lesions distribution. Given the present study results, the TB-Luminex multiplex test is a reliable test capable of detecting bTB in most infected pigs with good Se and Sp, regardless of the stage of the disease. In conclusion, multi-antigen tests can be used as individual tests and screening tools for domestic and wild suids within bTB eradication programs.

Aujeszky's disease in hunting dogs after the ingestion of wild boar raw meat in Sicily (Italy): clinical, diagnostic and phylogenetic features.

BACKGROUND: Aujeszky's disease is caused by Suid Herpes Virus-1 and species belonging to the genus Sus scrofa are the main reservoir hosts. This virus, however, is capable of infecting and causing severe disease, with an almost constant fatal outcome in other species, both domestic and wild (carnivores, monogastric herbivores and ruminants). Moreover, the possibility of transmission to humans has been demonstrated. This study reports and describes the clinical, diagnostic, pathological and phylogenetic aspects of two cases of Aujeszky's disease in two hunting dogs following the ingestion of infected wild boar raw meat. These cases are contextualized in the province of Messina (Sicily), where a high prevalence of Aujeszky's disease has been recorded (average of 12,20% in the period 2010-2019) in farmed pig, and with evidence of spread to other species. A severe outbreak in cattle has recently been reported in these areas. Nevertheless, cases of Aujeszky's disease in dogs are rarely reported and this study represents the first well-documented report in this species in Sicily. CASE PRESENTATION: After a wild boar hunt, two dogs showed neurological symptoms and intense itching unresponsive to therapy. Diagnosis of Aujeszky's disease was made based on clinical suspicion, anamnestic information and confirmed by the isolation of the virus from the brain of both dogs. In addition, molecular typing, sequencing and phylogenetic analysis of the Real-Time PCR products were performed. The sequences studied were placed in the Italian Clade 1 along with the sequences obtained from wild boars and hunting dogs from Italy and France. CONCLUSIONS: The finding of this disease in non-natural hosts in Sicilian multi-host epidemiological contexts suggests that the risk of inter-species transmission is concrete and that attention should be paid to developing disease control programs in these territories. The data obtained from genome sequencing of the two SuHV-1 isolates contribute to the enrichment of the GenBank with unknown sequences and the phylogenetic analysis implementation.

Retrieving Historical Cases of Aujeszky's Disease in Sicily (Italy): Report of a Natural Outbreak Affecting Sheep, Goats, Dogs, Cats and Foxes and Considerations on Critical Issues and Perspectives in Light of the Recent EU Regulation 429/2016.

Aujeszky's disease is caused by Suid alphaherpesvirus 1, and its main reservoir host is the pig. However, other species are also susceptible. Infection with this virus causes a severe neurological clinical picture named Aujeszky's disease, usually accompanied by itching and death a few days after the onset of symptoms. This study reports a multi-species outbreak of Aujeszky's disease that occurred in Sicily, which led to the death of 2 goats, 15 sheep, 2 dogs, 2 cats and 2 foxes. The diagnosis was made by culture, indirect immunofluorescence on brain samples and confirmed by biological test on rabbits. This study reports the first cases of Aujeszky's disease in Italy in cats, goat and sheep. The finding of Aujeszky's disease in several species in Sicily suggests a potential epizootic risk. In such areas where a multi-host system is recognised, an analysis of the risk factors should be carried out in order to develop targeted strategies for the control and eradication of the disease. The critical issues that hinder the control of Aujeszky's disease in the studied territory and perspectives for eradication in the light of EU regulation 429/2016 are also discussed.

Small-scale pig producers and disease surveillance: key threats.

A series of meetings for small-scale pig producers raised awareness of surveillance for pig diseases in Great Britain and highlighted different types of disease threat. This focus article summarises some key messages from those meetings and two of the threats discussed.

Intra-vitam Diagnosis of Tuberculosis in Pigs: Concordance Between Interferon-Gamma Release Assay and Comparative Tuberculin Skin Test.

The role of pigs in the maintenance of bovine tuberculosis caused by Mycobacterium bovis has been demonstrated in many settings; however, the current control programs usually do not state any intra-vitam diagnostic procedure in this species, as for the cattle. Carcass inspection has shown to be insufficient to detect infection in swine; thus, the assessment of intradermal tuberculin test and interferon-gamma release assay (IGRA) in this species is mandatory. The current study compares the performances of the single intradermal comparative cervical tuberculin (SICCT) test and IGRA. A total of 628 Nebrodi Black pigs raised in free-roaming farms were subjected to the two tests simultaneously. Besides, 124 animals underwent postmortem examination for the detection of tuberculous lesions and isolation of mycobacteria from target organs. The two tests showed a concordance of 94.42% with a Cohen's k coefficient of 0.786 and McNemar chi-square of 4.83 (P = 0.03). Slightly lower levels of concordance (90.32%) between SICCT and IGRA were obtained in the group of 124 animals, with a Cohen's k = 0.797 and McNemar chi-squared value of 0.69 with a non-significant P = 0.41. Moreover, the results showed how IGRA tends to result positive in higher rates, mostly when non-tuberculous mycobacteria (NTM) were isolated, suggesting a possible impairment of specificity in the event of coinfections in the swine. In conclusion, the results obtained support the possibility of the strategic use of IGRA or SICCT in combination or alternatively one to the other, particularly IGRA which showed lower specificity but has evident advantages over SICCT.

Cistus salviifolius Toxicity in Cattle.

Toxicity related to consumption of Cistus sp. pl. has been described in ruminants in some countries. This report describes the clinical and pathological findings of Cistus salviifolius toxicosis in 3 beef cattle herds located in 2 different areas of Sicily, Italy. Outbreaks were observed after grazing in poor winter pasture where C. salviifolius was abundant. Mean morbidity and mortality were 29% and 21%, respectively. Most of the affected animals (6 to 36 months old) showed anorexia, weight loss, and pollakiuria culminating in recumbency and death. Occasionally, abortion and neurological signs were observed. In animals with acute signs, there was a moderate decrease of sodium and chloride concentrations in serum. Animals with chronic signs showed an increase of serum urea, creatine phosphokinase (CPK), aspartate transaminase (AST), lactate dehydrogenase (LDH), and phosphorus and a decrease in total serum protein, calcium, chloride, and magnesium concentrations. Moderate anemia and slight neutropenia, lymphocytosis, and eosinophilia were detected in all groups. At necropsy, the main lesion was severe distention of the urinary bladder with turbid hemorrhagic urine and crystalluria. Histologically, chronic cystitis, interstitial nephritis, eosinophilic enteritis, and nonsuppurative necrotizing hepatitis were observed. To our knowledge, this is the first report of C. salviifolius toxicosis in cattle in Italy.

Genotype diversity and distribution of Mycobacterium bovis from livestock in a small, high-risk area in northeastern Sicily, Italy.

Bovine tuberculosis (bTB) caused by Mycobacterium bovis is an important re-emerging disease affecting livestock, wildlife and humans. Epidemiological studies are crucial to identifying the source of bTB infection, and its transmission dynamics and host preference, and thus to the implementation of effective strategies to contain it. In this study, we typed M. bovis isolates from livestock, and investigated their genetic diversity and distribution. A total of 204 M. bovis isolates were collected from cattle (n = 164) and Sicilian black pigs (n = 40) reared in a limited area of the province of Messina, northeastern Sicily, an area that had previously been identified as having the highest incidence of bTB in livestock on the island. All M. bovis isolates were typed by both spoligotyping and 12-loci MIRU-VNTR analysis. Results from both methods were then combined in order to improve the discriminatory power of M. bovis typing. We identified 73 combined genetic profiles. Thirty-five point six percent of the profiles were common to at least two animals, whereas 64.4% of profiles occurred in only one animal. A number of genetic profiles were predominant in either cattle or black pigs. We identified common genetic patterns in M. bovis isolates originating not only from neighboring districts, but also from non-neighboring districts. Our findings suggest that bTB is widespread in our setting, and is caused by a large number of genetically diverse M. bovis strains. The ecology and farming practices characteristic of the area may explain the substantial M. bovis heterogeneity observed, and could represent obstacles to bTB eradication.

A case of bovine trypanosomiasis caused by Trypanosoma theileri in Sicily, Italy.

Despite some researchers reporting clinical signs in cattle associated with Trypanosoma theileri, its role as a pathogen is still unclear. We describe here the isolation of Trypanosoma theileri during a routine laboratory investigation. Mature and immature vital parasitic forms were observed within hematopoietic cell cultures from the bone marrow of one cow for monocyte isolation. The animal was submitted to clinical examination and blood sample counting (CBC). Postmortem analysis included gross and histological examination and PCR in the liver, spleen, brain, lymph nodes, and lungs. PCR and Giemsa staining were used for parasite identification. A second cow belonging to the same farm was positive for Trypanosoma theileri by PCR performed on blood sample. In this case, the postmortem analysis included also testis. Clinical examination showed only a reduction in body weight in both cases. The CBC revealed an increase of lymphocytes and neutrophils while red blood cells were within the normal range. Spleen was slightly increased in volume and the histology revealed a proliferative activity of the white and red pulp. The biomolecular analysis identified the parasite as Trypanosoma theileri and its DNA was detected in the bone marrow, testis, and brain. The unusual finding of parasite in the brain, testis, and bone marrow raises new clinical implication on disease course and also possible sexual transmission.

Lesions Consistent with Tuberculous Spondylitis in Domestic and Wild Swine and Their Potential Use as a Model for Pott Disease in Humans.

During a routine abattoir inspection of pig carcasses and control activities of hunted wildlife in 2013, 118 large white pigs Sus scrofa domesticus, 474 Nebrodi black pigs and 135 wild boars Sus scrofa scrofa were submitted to anatomopathological examination to evaluate the presence of tuberculosis-like lesions. Localized and generalized granulomatous lesions were detected with a prevalence of about 25% in large white pigs, 13% in Nebrodi black pigs and 8.15% in wild boars. Localized lesions involved mainly the submandibular lymph nodes, but when the disease was spread throughout the body, the inner organs and, also, in some cases, udders and/or bones were injured. The highest prevalence of generalized lesions (15/30) was observed in large white pigs, the only ones in which tuberculous granuloma affected also the spine. The bovine tuberculous spondylitis cases observed showed some similarities with Pott disease in humans regarding aspect and localization of lesions and age of the affected animals.Tissue samples of the positive animals were collected and submitted to bacteriological analysis, and the bone samples were also subjected to histological and immunohistochemistry analysis. M. bovis was isolated in all the analysed samples, and the granuloma encapsulation was found often incomplete indicating that the disease was in an active phase. The presence of lesions associated to tuberculous spondylitis in pigs suggests the possibility to use this animal species as model for the study of Pott disease in humans.

M. bovis infection in pigs: Improvement of the γ-IFN assay efficiency in this species using a maintenance medium.

The interferon-gamma (IFN-γ) test measures cell mediated immune response (CMI) during the early stages of tuberculosis infection. Although Bovine Tuberculosis (BT) spread in feral pigs is widely documented in literature, the effectiveness of IFN-γ in this species has been only recently reported. One of the major obstacle of this assay is that whole blood samples should be stimulated with purified protein derivative (PPD) cocktail within 8 h from the blood sampling. This study set up a defined broth culture in which lymphocytes, the cell population predominantly responsible for IFN-γ production, are maintained in a steady-state and their vitality is preserved. The IFN-γ production measured from the samples added with the maintenance medium and stored at 4 °C was similar to the enzyme-linked immunosorbent assay (ELISA) optical density values obtained from the same assay performed within 8 h from sampling.

Human leptospirosis cases in Palermo Italy. The role of rodents and climate.

Many regions of the world are increasingly exposed to leptospirosis due to poverty, global warming and high urban density. Here, we report a molecular survey for pathogenic Leptospira spp. in rodents and two symptomatic human cases of leptospirosis in the city of Palermo, Italy. Four rodent species were captured in six areas of the city, and a molecular analysis for pathogenic Leptospira spp. on DNA from the kidney samples showed a different prevalence of leptospirosis in all the species of rodents. In addition, two human cases that occurred in May and October of 2009 in the city were also reported. A 67-year-old woman recovered after antibiotic treatment, whereas a 71-year-old woman did not survive. The weather during both of those times was notable for a violent cloudburst that caused street flooding. For the past several years, the incidence of leptospirosis has remained steady at 9 human cases every 10 years across the entire island of Sicily, with a population of almost 5 million inhabitants. The high prevalence of leptospirosis in rodents and the simultaneous presence of known risk factors, such as a mild/wet climate, street flooding and garbage accumulation, could explain the two cases of leptospirosis within the same city in the same year. This occurrence should raise awareness of this under-estimated zoonosis among public health authorities, especially given the potential fatality among elderly and immune-compromised individuals in urban settings in developed countries.

Intramammary infusion of a live culture of Lactococcus lactis in ewes to treat staphylococcal mastitis.

PURPOSE: Alternatives to antibiotic therapy for mastitis in ruminants are needed. We present an evaluation, in two trials, of the efficacy of an intramammary infusion of a live culture of Lactococcus lactis for the treatment of subclinical and clinical mastitis in ewes. METHODOLOGY: In total, 67 animals were enrolled: 19 lactating ewes (study 1), including healthy (N=6) and coagulase-negative staphylococci (CNS)-infected ewes (N=13); and 48 lactating ewes (study 2) with either CNS mastitis (N=32), or Staphylococcus aureus mastitis (N=16), for a total of 123 mammary glands. Intramammary infusions were performed with either L. lactis or PBS for 3 (study 1) or 7 (study 2) consecutive days. Antibiotic-treated and untreated control glands were included. Milk samples for microbiology, somatic cell analysis and milk production were collected before and after treatment.Results/Key findings.L. lactis rapidly activated the mammary glands' innate immune response and initiated an inflammatory response as evidenced by the recruitment of polymorphonuclear neutrophils and increased somatic cell counts. But while leading to a transient clearance of CNS in the gland, this response caused mild to moderate clinical cases of mastitis characterized by abnormal milk secretions and udder inflammation. Moreover, S. aureus infections did not improve, and CNS infections tended to relapse. CONCLUSION: Under our experimental conditions, the L. lactis treatment led to a transient clearance of the pathogen in the gland, but also caused mild to moderate clinical cases of mastitis. We believe it is still early to implement bacterial formulations as alternatives in treating mastitis in ruminants and further experimentation is needed.

Pathology and genetic findings in a rare case of Mycobacterium caprae infection in a sow.

Bovine tuberculosis, a reemerging zoonosis in diverse ecological scenarios, has been reported in the autochthonous Nebrodi black pig breed population used for meat production in Italy. During a routine abattoir inspection in 2013, 24 of 299 carcasses (8%) of Nebrodi black pigs presented tuberculosis-like lesions at pathologic examination. Mycobacterium bovis was isolated from 23 animals and M. caprae from a 3-year-old sow. The sow showed severe diffuse lesions involving the visceral organs, right coxofemoral joint, and mammary glands. Isolation of M. caprae from mammary glands is uncommon, with only one other case involving a sow reported so far; however, Mycobacteria infection of the mammary glands may be transmitted from lactating sows to piglets, contributing to the spread and maintenance of bovine tuberculosis in swine. Genotyping analysis showed M. caprae spoligotype SB0866 and profile 4,1,5,4,4,11,4,2,4,3,8,7 MIRU-VNTR (mycobacterial interspersed repetitive units-variable number of tandem repeats). The worldwide prevalence of this spoligotype is very low. The finding of severe, diffuse tuberculous lesions strongly suggests that Nebrodi black pigs are susceptible for Mycobacterium spp. and that they might act as a distributor for these microorganisms. Since natural ecosystems with multiple contacts among different livestock species and wild animals are very common in Mediterranean regions, current surveillance and eradication plans for bovine tuberculosis will need to be extended to other potential reservoir species in regions where extensive and traditional breeding systems are operated.

An outbreak of bovine tuberculosis in a fallow deer herd (Dama dama) in Sicily.

Wild ruminants have an important role in the epidemiology of bovine tuberculosis (bTB). This study describes an outbreak of bovine tuberculosis occurring in a fallow deer herd in Sicily. In 2012 a Sicilian herd of 47 animals was referred for cachexia. Pathological examination of 2 dead animals revealed disseminated granulomas predominantly involving the skin and subcutaneous tissues. Tissue samples were submitted for histological analysis, bacteriological culture, and biomolecular assay. PCR analysis identified Mycobacterium strains. Genotyping by spoligotyping and MIRU-VNTR profiles identified Mycobacterium bovis spoligotype SB0120 in both animals. In 2014, bTB skin testing of 28 fallow deer from the same group was positive in 4 and inconclusive in another 4. All 8 positive/inconclusive reactors were euthanized. Disseminated granulomatous lesions were noted in 6 of these animals, 3 of which (2 positive and 1 negative to skin tests) also presented cutaneous lesions. M. bovis spoligotype SB0120 was identified from all animals in which tuberculous-like lesions were observed, including 2 negative reactors. Many of the animals involved in this outbreak presented diffuse skin lesions, a potential route of transmission of M. bovis infection. Given the epidemiological role wildlife play in the maintenance of bTB infection and its potential risk for humans, a comprehensive monitoring plan for this zoonosis in wildlife species in Sicily is needed.