Outbreaks of abortions by Coxiella burnetii in small ruminant flocks and a longitudinal serological approach on archived bulk tank milk suggest Q fever emergence in Central Portugal

Q fever is a worldwide zoonotic infectious disease caused by Coxiella burnetii and sheep and goats are known to be the main reservoir for human infection. This study describes the epidemiological and laboratory findings of C. burnetii outbreaks affecting sheep and goat flocks and also provides the results of a prospective serosurvey in bulk tank milk samples to assess C. burnetii circulation in a population of sheep living in close contact to the human population in Central Portugal. In the epizooties, C. burnetii was identified in tissues of the resulting abortions by qPCR . As for the serological survey, 10.2% (95%CI : 4.5‐19.2) of the 78 bulk tank milk samples collected in 2015 presented IgG antibodies against C. burnetii . The same farms were visited and sampled in 2016 and 25.6% (95%CI : 16.4‐36.8) were positive. This steep increase in the number of anti‐C. burnetii farms between the 2015 and 2016 collections showed to be statistically significant (p  = 0.020) and is strongly suggestive of Q fever emergence in Central Portugal. Measures on animal health and on disease spread control to the human population should be considered.     

Tuberculosis outbreak caused by Mycobacterium caprae in a rabbit farm in Spain

Animal tuberculosis remains a great source of socioeconomic and health concern worldwide. Its main causative agents, Mycobacterium bovis and Mycobacterium caprae, have been isolated from many different domestic and wild animals. Naturally, occurring tuberculosis is extremely rare in rabbits, and implication of M. caprae has never been reported earlier. This study describes a severe tuberculosis outbreak caused by M. caprae in a Spanish farm of rabbits raised for meat for human consumption. The disease was first identified in a cachectic dam, and then it was confirmed in ten does with similar clinical signs. Subsequently, a depopulation operation was ordered for public health, animal welfare and environmental reasons. To broaden knowledge of spontaneous tuberculosis in rabbits, a study focused on pathological, epidemiological and diagnostic aspects was carried out on 51 does and 16 kittens after receiving the necessary authorizations. These animals were subjected to a modified intradermal test. After being euthanized, rabbits were examined for the presence of visible tuberculosis‐compatible lesions. Lung, kidney, caecal appendix and sacculus rotundus samples underwent microbiological and anatomopathological analysis. Infection was revealed by at least one of the methods used in 71% of dams and in 44% of kittens. The intradermal test was shown to be a good indicator of infection. Lung was the tissue for which more animals were positive but renal and intestinal tissues were also affected in many cases. Apparently, M. caprae spread mainly through the aerogenous route. Infection was pathologically characterized by the absence of evident fibrous capsules surrounding granulomas. A spoligotype (SB0415) frequently found in this area was considered responsible for the outbreak but the source could not be established. Regardless of the exceptional nature of animal tuberculosis in this host, rabbit industry might not escape from its effects and therefore, current biosafety and surveillance strategies should also consider this disease.     

Molecular prevalence of Chlamydia and Chlamydia‐like bacteria in Tunisian domestic ruminant farms and their influencing risk factors

Chlamydia and Chlamydia ‐like bacteria are well known to infect several organisms and may cause a wide range of diseases, particularly in ruminants. To gain insight into the prevalence and diversity of these intracellular bacteria, we applied a pan‐Chlamydiales real‐time PCR to 1,134 veterinary samples taken from 130 Tunisian ruminant herds. The true adjusted animal population‐level prevalence was 12.9% in cattle, against 8.7% in sheep. In addition, the true adjusted herd‐level prevalence of Chlamydiae was 80% in cattle and 25.5% in sheep. Chlamydiales from three family‐level lineages were detected indicating a high biodiversity of Chlamydiales in ruminant herds. Our results showed that Parachlamydia acanthamoebae could be responsible for bovine and ovine chlamydiosis in central‐eastern Tunisia. Multivariable logistic regression analysis at the animal population level indicated that strata and digestive disorders variables were the important risk factors of bovine and ovine chlamydiosis. However, origin and age variables were found to be associated with bovine and ovine chlamydiosis, respectively. At the herd level, risk factors for Chlamydia positivity were as follows: abortion and herd size for cattle against breeding system, cleaning frequency, quarantine, use of disinfectant and floor type for sheep. Paying attention to these risk factors will help improvement of control programs against this harmful zoonotic disease.     

Development of a non‐lethal hydrogen peroxide treatment for surveillance of Gyrodactylus salaris on trout farms and its application to testing wild salmon populations

This study documents the development of a non‐lethal sampling method to recover gyrodactylid parasites from large numbers of fish that will underpin an improved surveillance strategy for Gyrodactylus salaris. A review of published literature identified over 80 compounds that have previously been tested against gyrodactylids or closely related parasite species. Five safe and relatively fast‐acting compounds were selected for testing to determine their efficiency in removing gyrodactylids from host fish in small‐scale aquaria trials using three‐spined stickleback infected with Gyrodactylus gasterostei as a model host–parasite system. The most effective compound was hydrogen peroxide; short‐duration exposure (3 min) achieved a parasite detection sensitivity of 80%–89%. The practicality of exposing farmed salmonids to hydrogen peroxide for G. salaris surveillance was tested in the field by conducting a parasite recovery trial using a brown trout stock endemically infected with G. truttae and G. derjavinoides and comparing this to the whole‐body examination procedure currently conducted by UK authorities. Significantly more parasites were recovered after exposing fish to hydrogen peroxide and filtering the treatment solution than by direct whole‐body examination of killed fish (mean: 225 vs. 138 parasites per fish). The gyrodactylid recovery rate of the two methods was 84.6% and 51.9%, respectively. A comparison of timings for the two methods indicated scope for significant time savings in adopting the chemical screening method. The study demonstrated that hydrogen peroxide bath treatment may be successfully applied to the surveillance of gyrodactylid parasites and established as a non‐lethal method for sampling farmed and wild fish. This approach has the potential to reduce resources required to collect and isolate parasites for diagnostic testing and improve the sensitivity and confidence of surveillance programmes designed to demonstrate freedom from disease, thus underpinning a robust and defensible surveillance strategy for G. salaris for the UK aquatic animal disease contingency plan.     

Monitoring human cytomegalovirus infection in pediatric hematopoietic stem cell transplant recipients: using an affordable in‐house qPCR assay for management of HCMV infection under limited resources

Quantitative real‐time PCR (qPCR) assay is accepted as the method of choice for monitoring human cytomegalovirus (HCMV) infection in hematopoietic stem cell transplant recipients, but the high cost of commercial kits has hampered its use in many developing countries. In this study, an affordable in‐house qPCR was used to manage HCMV infection in pediatric patients and the diagnostic value of this method was compared with the conventional pp65 antigenemia assay. A total number of 1179 samples from 82 recipients were used in this study, and the effect of some potential risk factors on HCMV reactivation was evaluated. The qPCR was able to detect HCMV reactivation earlier and with higher sensitivity than antigenemia assay. Forty‐six episodes of reactivation were detected in 39 patients, of which all were detected by the qPCR assay, while only 21 episodes were diagnosed by antigenemia. The DNAemia level of 1284 IU/ml plasma was defined as the optimal cutoff value for starting pre‐emptive therapy. It was shown that the acute GVHD severity and the relationship of donor and recipient are the most significant risk factors for HCMV reactivation. The data suggest that the antigenemia method for monitoring HCMV reactivation could be substituted by the qPCR assay.     

Free‐living Waterfowl as a Source of Zoonotic Bacteria in a Dense Wild Bird Population Area in Northeastern Spain

Salmonella spp. and Campylobacter spp. are zoonotic bacteria that represent an economic and public health concern worldwide. Due to the difficulty to collect samples from free‐living waterfowl, little is known on their importance as a reservoir of zoonotic agents. Thus, a study was conducted to determine the prevalence, genotypic diversity and antimicrobial susceptibility of Salmonella and Campylobacter from waterfowl in Ebro Delta (northeastern Spain), a geographical area with a dense wild bird population. Samples were collected from 318 adult waterfowl belonging to nine fowl species. All the samples were taken during the hunting season from 2008 to 2010. None of the birds were positive for Salmonella, while the overall Campylobacter prevalence was 12.58% (40/318). A much higher Campylobacter coli prevalence than Campylobacter jejuni was found (11.64% versus 0.94%). The species Fulica atra showed the highest Campylobacter prevalence (78.05%). ERIC‐PCR of the isolates showed a high diversity of strains. Antimicrobial susceptibility testing of Campylobacter isolates showed that all the isolates were susceptible to the seven antibiotics tested.     

The uretero-vesical jet as a functional diagnostic tool in childhood hydronephrosis

 Childhood hydronephrosis (HN) is accurately detected by ultrasound (US), but its functional work-up remains a domain of scintigraphy, the Whitaker-test, and clinical follow-up. We utilized color doppler US (CD-Jet) of uretero-vesical jets (UVJ) to visualize the postobstructive ureteral flow in childhood HN. A total of 177 standardized CD-Jet were performed in 132 children aged 1 day to 14.9 years. Sixty-nine investigations in 43 patients with unilateral HN were compared with a standardized technetium 99m-mercaptotriacetylglycine nephrogram; in 10 infants both procedures were performed consecutively on the same day. UVJ were visible in 96% of all investigations. Ureteral obstruction resulted in absence of UVJ in 85% of examinations; in the remaining 15% the jet frequency was less than 10% observed in the contralateral control. Results are highly significant in both uretero-pelvic (P<0.00007) and uretero-vesical lesions (P<0.00005, Wilcoxon test) and are reproducible in sequential investigations. In nonobstructive distal HN the jet frequency averaged 70% of the unaffected side (P<0.05). In proximal lesions it is equal. Jet evaluation in reflux nephropathy did not differ from controls. Compared with scintigraphy, CD-US identifies obstruction with a specificity of 94.2% and sensitivity of 94.8%. CD-US of UVJ therefore may serve as a reliable screening parameter in unilateral childhood HN. Received June 10, 1996; received in revised form October 8, 1996; accepted October 18, 1996     

Cultural competency of a mobile,customized patient education tool for improving potential kidney transplant recipients’ knowledge and decision‐making

Patients considering renal transplantation face an increasingly complex array of choices as a result of the revised kidney transplant allocation system. Decision aids have been shown to improve patient decision‐making through the provision of detailed, relevant, individualized clinical data. A mobile iOS‐based application (app) including animated patient education and individualized risk‐adjusted outcomes following kidney transplants with varying donor characteristics and DSA waiting times was piloted in two large US transplant programs with a diverse group of renal transplant candidates (N = 81). The majority (86%) of patients felt that the app improved their knowledge and was culturally appropriate for their race/ethnicity (67%‐85%). Patients scored significantly higher on transplant knowledge testing (9.1/20 to 13.8/20, P < .001) after viewing the app, including patients with low health literacy (8.0 to 13.0, P < .001). Overall knowledge of and interest in living and deceased donor kidney transplantation increased. This pilot project confirmed the benefit and cultural acceptability of this educational tool, and further refinement will explore how to better communicate the risks and benefits of nonstandard donors.     

The red fox (Vulpes vulpes) as a potential natural reservoir of human cryptosporidiosis by Cryptosporidium hominis in Northwest Spain

Giardia duodenalis and Cryptosporidium spp. are ubiquitous intestinal protozoa that parasitize domestic and wild animals, as well as human beings. Due to their zoonotic potential, the objective of the present study was to determine the presence of these pathogens in the fox population (Vulpes vulpes) located in Northwest Spain. A total of 197 faecal samples from legally hunted foxes were collected in the autonomous region of Galicia. The presence of G. duodenalis and Cryptosporidium spp. was investigated by PCR‐based methods amplifying the small subunit ribosomal RNA (ssu rRNA) gene of the parasites. Attempts to genotype obtained positive samples were subsequently conducted at the glutamate dehydrogenase (gdh) and β‐giardin (bg) genes of G. duodenalis, and the 60 kDa glycoprotein (gp60) gene of Cryptosporidium. Giardia duodenalis and Cryptosporidium spp. were identified in 19 (9.6%) and 12 (6.1%) of the investigated samples, respectively. However, five Cryptosporidium species were detected at the ssu rRNA locus: C. hominis (33.4%, 4/12), C. canis (25.0%, 3/12), C. parvum (16.7%, 2/12), C. ubiquitum (8.3%, 1/12) and C. suis (8.3%, 1/12). An additional Cryptosporidium‐positive sample was identified at the genus level only. Typing and subtyping of Giardia‐ and Cryptosporidium‐positive samples were unsuccessful. The detection of C. hominis in wild foxes indicates the probable overlapping of sylvatic and domestic cycles of this parasite in rural settings. Besides, this finding raises the question of whether red foxes may act as natural reservoirs of C. hominis. The detection of C. parvum and C. suis is suggestive of active transmission events between farm and wild animals, opening up the possibility of transmission to human beings.     

Evaluation on a Streptococcus suis Vaccine Using Recombinant Sao‐L Protein Manufactured by Bioreactors as the Antigen in Pigs

Streptococcus suis (S. suis) can be classified into 33 serotypes based on the structure of capsular polysaccharides. Recent research indicated that a new surface protein designated as Sao (surface antigen one) reacts with 30 serotypes of convalescent‐phase sera during S. suis infections, which makes Sao a good potential antigen for developing S. suis vaccines. The objectives of this study were to produce recombinant Sao‐L protein (rSao‐L) from a strain of S. suis serotype 2 by a prokaryotic expression system in bioreactors and to use rSao‐L as the antigen for a S. suis vaccine in mouse and swine models. The antibody titres in mice and pigs immunized with rSao‐L were significantly (P < 0.05) increased. After challenge with live S. suis serotype 1 bacteria, the anatomical lesions in pigs immunized with rSao‐L were reduced by 60%. These data indicated that immunization with rSao‐L can confer cross‐serotype protection against S. suis. Moreover, percentages of CD8⁺ and CD4⁺/CD8⁺ double‐positive T cells in immunized pigs were significantly higher than those of the control group (P < 0.01). Using bioreactors to produce rSao‐L as the antigen for S. suis vaccines may broaden protective efficacy and reduce production costs.     

Risks from disease caused by Mycobacterium orygis as a consequence of Greater one‐horned Rhinoceros (Rhinoceros unicornis) translocation in Nepal

The greater one‐horned rhinoceros (Rhinoceros unicornis) is listed as vulnerable by the IUCN Red List. Mycobacterium orygis–associated disease was identified in a single greater one‐horned rhino in Chitwan National Park in February 2015 prior to a planned translocation of five greater one‐horned rhinoceros from Chitwan National Park to Bardia National Park for conservation purposes. This paper describes a qualitative disease risk analysis conducted retrospectively post‐translocation for Mycobacterium orygis and this translocation, with the aim to improve the understanding of disease threats to the conservation of greater one‐horned rhino. The disease risk analysis method used was devised by Sainsbury & Vaughan‐Higgins (Conservation Biology, 26, 2017, 442) with modifications by Bobadilla Suarez et al (EcoHealth, 14, 2017, 1) and Rideout et al (EcoHealth, 14, 2017, 42) and included the use of a scenario tree and an analysis of uncertainty as recommended by Murray et al. (Handbook on import risk analysis for animals and animal products. Volume 1. Introduction and qualitative risk analysis, 2004), and the first time this combination of methods has been used to assess the risk from disease in a conservation translocation. The scenario tree and analysis of uncertainty increased the clarity and transparency of the analysis. Rideout et al.’s (EcoHealth, 14, 2017, 42) criteria were used to assess the source hazard and may be useful in comparative assessment of source hazards for future conservation translocations. The likelihood of release into the destination site of Mycobacterium orygis as a source hazard was estimated as of low risk, the risk of exposure of populations at the destination was of high risk and the likelihood of biological and environmental consequences was low. Overall, the risk from disease associated with Mycobacterium orygis as a result of this translocation was found to be low. Recommendations on disease risk management strategies could be improved with a better understanding of the epidemiology including the presence/absence of Mycobacterium orygis in greater one‐horned rhino to develop effective disease risk management strategies.     

Lack of Evidence of Spill‐Over of Salmonella enterica Between Cattle and Sympatric Iberian ibex (Capra pyrenaica) from a Protected Area in Catalonia,NE Spain

Salmonella enterica is a zoonotic agent of worldwide importance found in a wide range of wild hosts. However, its prevalence in many popular game species has never been assessed. Iberian ibex (Capra pyrenaica) is the main game caprinae of the Iberian Peninsula and around two thousand individuals are hunted every year for trophy or for home consumption. In this work, 313 Iberian ibexes from the Ports de Tortosa i Beseit National Game Reserve (NE Spain) were tested for Salmonella enterica in faeces, and anti microbial susceptibility was determined. The exact location of shooting or capture was recorded with a GPS device to study the links of Salmonella infection with cattle presence and human proximity. Additionally, samples were taken from cattle grazing inside this reserve (n = 73). Only three Iberian ibexes (0.96%, 95% CI 0.2–2.8) were positive to Salmonella (serotype Enteritidis, Bardo and 35:r:z35), while prevalence was moderate in cattle: 21.92% (95% CI 13.10–33.14, serotype Meleagridis, Anatum, Kedougou and Othmarschen). All isolates were susceptible to the anti microbial agents tested. Moreover, a case of fatal septicaemic salmonellosis in an 11‐year‐old male Iberian ibex is described where Salmonella enterica serotype Enteritidis was isolated from the lung, liver and spleen samples. The low prevalence of Salmonella in Iberian ibex and the lack of shared serotypes suggest no association to cattle. Despite this, game meat aimed for human consumption should be examined, and it is strongly recommended that hunters and game keepers manipulate animals and carcasses under maximal hygienic conditions to avoid environmental contamination and human contagion.     

Cytomegalovirus infection and graft rejection as risk factors for pneumocystis pneumonia in solid organ transplant recipients: A systematic review and meta‐analysis

A growing number of publications have reported the outbreaks of post‐transplant pneumocystis pneumonia (PJP). In most studies, the onset of PJP was beyond 6‐12 months of prophylaxis. Cytomegalovirus (CMV) infection and allograft rejection have been repeatedly reported as probable risk factors for post‐transplant PJP. In this systematic review and meta‐analysis, we determined the pooled effect estimates of these 2 variables as risk factors. Data sources included PUBMED, MEDLINE‐OVID, EMBASE‐OVID, Cochrane Library, Networked Digital Library of Theses and Dissertations, World Health Organization, and Web of Science. We excluded publications related to hematopoietic stem cell transplantation (HSCT) or Human Immunodeficiency Virus (HIV) patients. Eventually, 15 studies remained for the final stage of screening. Cytomegalovirus infection (OR: 3.30, CI 95%: 2.07‐5.26, I²: 57%, P = 0.006) and allograft rejection (OR:2.36, CI95%: 1.54‐3.62, I2: 45.5%, P = 0.05) significantly increased the risk of post‐transplant PJP. Extended prophylaxis targeting recipients with allograft rejection or CMV infection may reduce the risk of PJP.     

Development of a Gene Expression Assay for the Diagnosis of Mycobacterium bovis Infection in African Lions (Panthera leo)

Mycobacterium bovis infection, the cause of bovine tuberculosis (BTB), is endemic in wildlife in the Kruger National Park (KNP), South Africa. In lions, a high infection prevalence and BTB mortalities have been documented in the KNP; however, the ecological consequences of this disease are currently unknown. Sensitive assays for the detection of this infection in this species are therefore required. Blood from M. bovis‐exposed, M. bovis‐unexposed, M. tuberculosis‐exposed and M. bovis‐infected lions was incubated in QuantiFERON^®‐TB Gold (QFT) tubes containing either saline or ESAT‐6/CFP‐10 peptides. Using qPCR, selected reference genes were evaluated for expression stability in these samples and selected target genes were evaluated as markers of antigen‐dependent immune activation. The abundance of monokine induced by gamma interferon (MIG/CXCL9) mRNA, measured in relation to that of YWHAZ, was used as a marker of ESAT‐6/CFP‐10 sensitization. The gene expression assay results were compared between lion groups, and lenient and stringent diagnostic cut‐off values were calculated. This CXCL9 gene expression assay combines a highly specific stimulation platform with a sensitive diagnostic marker that allows for discrimination between M. bovis‐infected and M. bovis‐uninfected lions.     

Supplementary feeding as a source of multiresistant Salmonella in endangered Egyptian vultures

Wild birds have repeatedly been highlighted as vectors in the dissemination of livestock and human pathogens. Here, the occurrence, serotypes and antimicrobial resistance of Salmonella were assessed in adult Egyptian vultures (Neophron percnopterus ), to test the hypothesis that infection is associated with the consumption of swine carcasses provided at supplementary feeding stations (SFS s). Faeces of year‐round resident griffon vultures (Gyps fulvus ) were also tested to assess whether infection was acquired in the breeding grounds of both species or in the African wintering quarters of Egyptian vultures. Depending on the shedding rate criteria considered, the occurrence of infection in Egyptian vultures varied between the three consecutive sampling days in a range with a minimum of 23%–41% and a maximum of 64%–92% of individuals (n  =  11–14 individuals, 27–39 faeces). The occurrence in the single sampling of griffon vultures was 61% of faeces (n  =  18). Vultures mostly fed on pig carcasses, which together with their predominant infection with multiresistant serotypes (mostly the monophasic 4,12:i:‐ variant resistant to aminopenicillins, aminoglycosides and tetracyclines) typically found in pigs from Spain, strongly supports a carcass‐to‐vulture transmission and cross‐infection routes at SFS s. Efforts are encouraged to avoid discarding carcasses of pigs with Salmonella at SFS s established for the conservation of threatened scavengers. This could contribute to reducing the long‐distance transmission of resistant pathogens with an impact on livestock and human health while avoiding infection risk and its effects on wildlife.     

Genetic characterization of African swine fever virus isolates from soft ticks at the wildlife/domestic interface in Mozambique and identification of a novel genotype

African swine fever virus (ASFV ) is one of the most threatening infectious diseases of pigs. There are not sufficient data to indicate the importance of the sylvatic cycle in the spread and maintenance of the disease locally and potentially, globally. To assess the capacity to maintain ASF in the environment, we investigated the presence of soft tickreservoirs of ASFV in Gorongosa National Park (GNP ) and its surrounding villages. A total of 1,658 soft ticks were recovered from warthog burrows and pig pens at the wildlife/livestock interface of the GNP and viral DNA was confirmed by nested PCR in 19% of Ornithodoros porcinus porcinus and 15% of O. p. domesticus . However, isolation of ASFV was only achieved in approximately 50% of the PCR ‐positive samples with nineteen haemadsorbing virus isolates recovered. These were genotyped using a combination of partial sequencing of the B646L gene (p72 ) and analysis of the central variable region (CVR ) of the B602L gene. Eleven isolates were classified as belonging to genotype II and homologous to contemporary isolates from southern Africa, the Indian Ocean and eastern Europe. Three isolates grouped within genotype V and were similar to previous isolates from Mozambique and Malawi. The remaining five isolates constituted a new, previously unidentified genotype, designated genotype XXIV . This work confirms for the first time that the virus currently circulating in eastern Europe is likely to have a wildlife origin, and that the large diversity of ASFV maintained in wildlife areas can act as a permanent sources of different strains for the domestic pig value chain in Mozambique and beyond its boundaries. Their genetic similarity to ASFV strains currently spreading across Europe justifies the need to continue studying the sylvatic cycle in this African country and other parts of southern Africa in order to identify potential hot spots of ASF emergence and target surveillance and control efforts.