Molecular Survey of Anaplasma and Ehrlichia of Red Deer and Sika Deer in Gansu,China in 2013

Anaplasma and Ehrlichia are important emerging tick‐borne pathogens in both humans and animals. Here, we conducted a molecular surveillance study in Gansu, China to assess the prevalence of Anaplasma and Ehrlichia spp. in red deer and sika deer based on polymerase chain reaction (PCR) analysis and sequencing of 16S rRNA or msp genes. PCR revealed that the prevalence of Anaplasma ovis, Anaplasma bovis and Anaplasma platys of the Qilian Mountain samples was 32%, 9% and 9%, respectively; the prevalence of Anaplasma ovis, Anaplasma bovis, Anaplasma platys was 20%, 15% and 15% among the Long Mountain samples, respectively. Of the Long Mountain samples, two (5%) of the 40 samples were positive for Ehrlichia canis, but all 44 of the Qilian Mountain samples were negative for E. canis, and no other Anaplasma or Ehrlichia spp. were found in the samples. The phylogenetic tree showed that the newly isolated Anaplasma and Ehrlichia spp. could be classified as belonging to four clades, including an A. bovis cluster, A. ovis cluster, A. platys cluster and E. canis cluster. In addition, Bartonella schoenbuchensis was firstly identified in blood samples from red deer in Gansu, China. Our results provide important data to increase the understanding of the epidemiology of anaplasmosis and ehrlichiosis of red deer and sika deer and will assist with the implementation of measures to control anaplasmosis and ehrlichiosis transmission to red deer, sika deer and other animals in Gansu, China.     

Persistence of Mycobacterium bovis bacillus Calmette–Guérin (BCG) Danish In White‐tailed Deer (Odocoileus virginianus) Vaccinated with a Lipid‐Formulated Oral Vaccine

Mycobacterium bovis, the causative agent of tuberculosis in animals, has a broad host range, including humans. Historically, public health concerns prompted programs to eradicate tuberculosis from cattle in many nations. Eradication efforts decreased the prevalence of bovine tuberculosis; nevertheless, some countries encountered significant obstacles, not least of which was a wildlife reservoir of M. bovis. Efforts to decrease the size of the affected wildlife populations have neither eliminated disease nor eliminated transmission to cattle. Consequently, the use of a vaccine for wildlife is being explored. The vaccine most studied is M. bovis BCG, an attenuated live vaccine, first developed 100 years ago. The most efficient and effective means of vaccinating wildlife will be an oral vaccine. White‐tailed deer in Michigan, USA, constitute a reservoir of M. bovis. White‐tailed deer are a popular game species, and as such, represent a food animal to many hunters. BCG persistence in deer tissues could result in human exposure to BCG. Although non‐pathogenic, BCG exposure could induce false‐positive skin test results, confounding the central component of public health surveillance for TB. The objective of the present study in white‐tailed deer was to evaluate persistence of lipid‐encapsulated BCG and a liquid suspension of BCG after oral administration at two different dosages. Vaccine was not recovered at any time after oral consumption of a bait containing a single dose (1 × 10⁸ CFU) of lipid‐encapsulated BCG. However, persistence was consistent in deer consuming 10 lipid‐encapsulated baits (1 × 10⁹ CFU), with BCG recovered from at least one deer at 1, 3, 6, 9 and 12 months after consumption. Persistence of up to 9 months was seen in deer vaccinated with orally with a liquid suspension. Persistence of BCG was limited to lymphoid tissue and never found in samples of muscle collected at each time point. Although the risk of exposure to hunters is low, BCG persistence should be considered prior to field use in white‐tailed deer.     

Clinical and Serological Dynamics of Besnoitia besnoiti Infection in Three Endemically Infected Beef Cattle Herds

The dynamics of bovine besnoitiosis were studied in an area where the disease is endemic. A four‐year longitudinal study was conducted for the first time in three infected beef cattle herds located in the Urbasa‐Andía Mountains (Navarra, Spain). Each herd was visited four to seven times, and clinical and serological prevalence rates and incidence rates were estimated. Clinical inspections to identify compatible clinical signs with the disease stages were conducted at the beginning and end of the study. Serological assessment was initially performed by ELISA. Seronegative animals with clinical signs and seropositive animals with relative index per cent (RIPC) values lower than 30 that did not increase during the study period were analysed by Western blot to optimize the sensitivity and specificity of the ELISA test. Clinical prevalence rates were slightly higher (62% on average) than the seroprevalence rates (50% on average), and tissue cysts located in the vestibulum vaginae and sclera were the most frequently detected clinical signs. The proportion of seropositive animals with clinical signs varied from 16.7% to 73.6% among the herds, and 17% of cattle with clinical signs proved to be seronegative by both serological tests. An average 22% serological incidence rate was also reported in addition to clinical incidence rates that varied from 12.5% to 16.7%. Additionally, parasitemia was investigated in the herd that showed the highest clinical and seroprevalence rates. Only one PCR positive blood sample was detected. Thus, the role that blood may play in parasite transmission needs to be further investigated. Infected herds maintained both high prevalence and incidence rates in the absence of control measures and a high number of parasite carriers. Finally, economic impact studies on reproductive and productive losses associated with besnoitiosis need to be performed to implement a cost–benefit control programme.     

Naturally acquired bovine besnoitiosis: Disease frequency,risk and outcome in an endemically infected beef herd

The recent spread of bovine besnoitiosis warrants further epidemiological investigations to improve the knowledge on disease development. Thus, a 4‐year longitudinal open cohort study was conducted in the first German cattle herd naturally infected with Besnoitia besnoiti . At seven herd‐visits between 2008 and 2012, fourteen breeding bulls (>1.5 years) and 131 females (>1 year) were examined clinically and serologically. In females, clinical and serological prevalences, incidence and remission rates were determined. In addition, the association of age, antibody levels and number of visible parasitic cysts with clinical and serological outcome was investigated. The seroprevalence (89.4%–100%) and serological incidence rate (140.5 per 100 animal‐years) were considerably higher than the clinical prevalence (23.5%–36.6%) and clinical incidence rate (16.7 per 100 animal‐years). Of 33 new clinical and 12 new serological cases, only 6.7% (3/45) attracted attention with clinical signs of acute bovine besnoitiosis. The apparent serological remission rate (1.9 per 100 animal‐years) was considerably lower than the clinical remission rate (37.3 per 100 animal‐years). A median cyst score of <1 and mean immunofluorescent antibody test (IFAT ) titre of ≤1,600 over the entire observation period was significantly associated with a negative clinical outcome at the end. Overall cyst score was not significantly associated with serological outcome and age had no significant influence on clinical and serological outcome. Within 4 years, there was a significant reduction in cyst scores and IFAT titres in the same animals, leading to eight clinically and serologically negative animals in the end. Two initially negative animals achieved clinical and apparent serological remission in about 2.5 years. In bulls, the time between herd entry and seroconversion was 7–30 months and the serological incidence rate was nearly identical to the rate in females (142.0 per 100 animal‐years). This shows that a high B. besnoiti prevalence leads to infection of bulls within a short time period.     

An Inter‐Laboratory Comparative Study of Serological Tools Employed in the Diagnosis of Besnoitia besnoiti Infection in Bovines

Bovine besnoitiosis is considered an emerging chronic and debilitating disease in Europe. Many infections remain subclinical, and the only sign of disease is the presence of parasitic cysts in the sclera and conjunctiva. Serological tests are useful for detecting asymptomatic cattle/sub‐clinical infections for control purposes, as there are no effective drugs or vaccines. For this purpose, diagnostic tools need to be further standardized. Thus, the aim of this study was to compare the serological tests available in Europe in a multi‐centred study. A coded panel of 241 well‐characterized sera from infected and non‐infected bovines was provided by all participants (SALUVET‐Madrid, FLI‐Wusterhausen, ENV‐Toulouse, IPB‐Berne). The tests evaluated were as follows: an in‐house ELISA, three commercial ELISAs (INGEZIM BES 12.BES.K1 INGENASA, PrioCHECK Besnoitia Ab V2.0, ID Screen Besnoitia indirect IDVET), two IFATs and seven Western blot tests (tachyzoite and bradyzoite extracts under reducing and non‐reducing conditions). Two different definitions of a gold standard were used: (i) the result of the majority of tests (‘Majority of tests’) and (ii) the majority of test results plus pre‐test information based on clinical signs (‘Majority of tests plus pre‐test info’). Relative to the gold standard ‘Majority of tests’, almost 100% sensitivity (Se) and specificity (Sp) were obtained with SALUVET‐Madrid and FLI‐Wusterhausen tachyzoite‐ and bradyzoite‐based Western blot tests under non‐reducing conditions. On the ELISAs, PrioCHECK Besnoitia Ab V2.0 showed 100% Se and 98.8% Sp, whereas ID Screen Besnoitia indirect IDVET showed 97.2% Se and 100% Sp. The in‐house ELISA and INGEZIM BES 12.BES.K1 INGENASA showed 97.3% and 97.2% Se; and 94.6% and 93.0% Sp, respectively. IFAT FLI‐Wusterhausen performed better than IFAT SALUVET‐Madrid, with 100% Se and 95.4% Sp. Relative to the gold standard ‘Majority of test plus pre‐test info’, Sp significantly decreased; this result was expected because of the existence of seronegative animals with clinical signs. All ELISAs performed very well and could be used in epidemiological studies; however, Western blot tests performed better and could be employed as a posteriori tests for control purposes in the case of uncertain results from valuable samples.     

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.     

Genetic Characterization of Circulating African Swine Fever Viruses in Nigeria (2007–2015)

Sequencing and analysis of three discrete genome regions of African swine fever viruses (ASFV) from archival samples collected in 2007–2011 and active and passive surveillance between 2012 and 2015 in Nigeria were carried out. Analysis was conducted by genotyping of three single‐copy African swine fever (ASF) genes. The E183L and B646L genes that encode structural proteins p54 and p72, respectively, were utilized to delineate genotypes before intragenotypic resolution by characterization of the tetrameric amino acid repeat region within the hypervariable central variable region of the B602L gene. The results showed no variation in the p72 and p54 gene regions sequenced. Phylogeny of p72 sequences revealed that all the Nigerian isolates belonged to genotype I, while that of the p54 recovered the Ia genotype. Analysis of B602L gene revealed the differences in the number of tetrameric repeats. Four new variants (Tet‐15, Tet‐17a, Tet‐17b and Tet‐48) were recovered, while a fifth variant (Tet‐20) was the most widely distributed in the country displacing Tet‐36 reported previously in 2003–2006. The viruses responsible for ASF outbreaks in Nigeria are from very closely related but mutated variants of the virus that have been circulating since 1997. A practical implication of the genetic variability of the Nigerian viral isolates in this study is the need for continuous sampling and analysis of circulating viruses, which will provide epidemiological information on the evolution of ASFV in the field versus new incursion for informed strategic control of the disease in the country.     

Factors that Influence Mycobacterium bovis Infection in Red Deer and Wild Boar in an Epidemiological Risk Area for Tuberculosis of Game Species in Portugal

Bovine tuberculosis (bTB) is a worldwide zoonotic disease of domestic and wild animals. Eradication has proved elusive in those countries with intensive national programmes but with ongoing transmission between wildlife and cattle. In Portugal, a high‐risk area for bTB was defined and specific measures implemented to assess and minimize the risk from wildlife. Data from the 2011 to 2014 hunting seasons for red deer (Cervus elaphus) and wild boar (Sus scrofa) were analysed with bovine demographic and bTB information to assess factors that determined the occurrence and distribution of bTB in both species. The likelihood of bTB‐like lesions in wild boar was positively associated with density of red deer, wild boar and cattle, while for red deer, only their density and age were significant factors. The likelihood of Mycobacterium bovis isolation in wild boar was associated with density of cattle and red deer and also with the anatomical location of lesions, while for red deer, none of the variables tested were statistically significant. Our results suggest that, in the study area, the role of red deer and wild boar may be different from the one previously suggested by other authors for the Iberian Peninsula, as red deer may be the driving force behind M. bovis transmission to wild boar. These findings may assist the official services and game managing bodies for the management of hunting zones, what could also impact the success of the bTB eradication programme.     

Pathogenic Rickettsia in ticks of spur‐thighed tortoise (Testudo graeca) sold in a Qatar live animal market

The dissemination of vector arthropods harbouring zoonotic pathogens through the uncontrolled transboundary trade of exotic and pet animals poses an important threat to Public Health. In the present report, we describe the introduction of pathogenic Rickettsia africae and R. aeschlimanni in ticks removed from imported tortoises in Qatar. A total of 21 ticks were collected from pet spur‐thighed tortoises (Testudo graeca) from Doha, May 2018, and studied for species identification and characterization of Rickettsia spp. Morphological and molecular analysis of ticks allowed their identification as Hyalomma aegyptium. Molecular analysis of partial ompA and gltA genes showed that Rickettsia sequences found on these ticks clustered with sequences classified as R. aeschilimanii and R. africae. Since pre‐adult stages of H. aegyptium also feed on humans, this tick species may play a role in the transmission of R. aeschilimanii and R. africae. We alert for the introduction of non‐native pets as vehicles for tick importation, known vectors for animal and human pathogenic agents. Importation of exotic species into non‐autochthonous countries deserves strict control to enforce robust surveillance and mitigate potential exotic diseases epidemics.     

Spatiotemporal monitoring of selected pathogens in Iberian ibex (Capra pyrenaica)

An epidemiological surveillance programme was carried out to assess exposure and spatiotemporal patterns of selected pathogens (Brucella spp., Mycobacterium avium subsp. paratuberculosis (MAP), Mycoplasma agalactiae, Pestivirus and bluetongue virus (BTV)) in Iberian ibex (Capra pyrenaica) from Andalusia (southern Spain), the region with the largest population of this species. A total of 602 animals in five distribution areas were sampled during 2010–2012 (P1) and 2013–2015 (P2). The Rose Bengal test (RBT) and complement fixation test (CFT) were used in parallel to detect anti‐Brucella spp. antibodies. Commercial ELISAs were used to test for antibodies against the other selected pathogens. Sera positive for BTV and Pestivirus by ELISA were tested by serum neutralization test (SNT) to identify circulating serotypes/genotypes. The overall seroprevalences were as follows: 0.4% for Brucella spp. (2/549; CI 95%: 0.1–1.3) (14/555 positive by RBT; 2/564 by CFT), 0.5% for MAP (3/564; CI 95%: 0.1–1.5), 5.7% for M. agalactiae (30/529; CI 95%: 3.9–8.0), 11.1% for Pestivirus (58/525; CI 95%: 8.5–14.1) and 3.3% for BTV (18/538; CI 95%: 2.0–5.2). Significantly higher seropositivity to both M. agalactiae and BTV was observed in P1 compared with P2. Spatiotemporal clusters of high seroprevalence were also found for M. agalactiae in four of the five sampling areas in 2010, and for BTV in one of five areas in 2012. Specific antibodies against BTV‐4, BDV‐4 and BVDV‐1 were confirmed by SNT. Our results indicate that the Iberian ibex may be considered spillover hosts of Brucella spp. and MAP rather than true reservoirs. The prevalence of antibodies against M. agalactiae and BTV suggests spatiotemporal variation in the circulation of these pathogens, while Pestivirus has a moderately endemic circulation in Iberian ibex populations. Our study highlights the importance of long‐term surveillance for a better understanding of the spatiotemporal distribution of shared infectious diseases and providing valuable information to improve control measures at the wildlife–livestock interface.     

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.     

Liver fluke (Fasciola hepatica) co‐infection with bovine tuberculosis (bTB) in cattle: A retrospective animal‐level assessment of bTB risk in dairy and beef cattle

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, remains a persistent problem for cattle industries in endemic countries. The frequency, quality, and performance of tests, and the presence of wildlife reservoirs, have been identified as impediments to eradication. Recently, exposure to helminth infection (Fasciola hepatica) has been associated negatively with the disclosure of bTB. Here, for the first time, we assess impact of concurrent infections of Fasciola hepatica and the disclosure of bTB at the animal‐level using large surveillance datasets. We utilized a dataset of 138,566 animal records from an abattoir from Northern Ireland (2011–2013). The presence of F. hepatica infection was assessed from macroscopic tissue inspection at abattoir. Multivariable models were developed to assess co‐infection associations with bTB status based on: Single Intradermal Comparative Tuberculin Test (SICTT), lesion, bacteriological confirmation, including either all animals, or only skin‐test negative animals (lesions at routine slaughter; LRS; confirmed nonreactors at routine slaughter; cNRs) or positive (reactors) animals alone, respectively. The relationship between skin tuberculin reaction sizes and fluke status was also explored for a subset of animals with field recordings (n = 24,680). Controlling for known risk factors (e.g., climatic, herd, and individual level characteristics), we did not find significant associations between the SICTT (standard or severe interpretation), lesion, nor confirmation status of animals and their liver fluke status. The only exception was a negative association between liver fluke positivity, and LRS or cNRs, respectively; though effect‐sizes were small (e.g., LRS Odds‐Ratio: 0.87; 95% CI: 0.76–1.00). There was limited evidence of a relationship between tuberculin reaction sizes during SICTT testing and liver fluke infection status. These data do not support the contention that the detection of bTB using skin‐tests or reactor postmortem follow‐up may be compromised by co‐infection at a population level, but the relationship with lesion formation (pathogenesis) may indicate an impact for postmortem surveillance.     

Molecular detection and characterization of carnivore parvoviruses in free‐ranging Eurasian otters (Lutra lutra) in southern Italy

The most important Italian population of the Eurasian otter (Lutra lutra) occurs in the southern part of the peninsula with two isolated sub‐populations of about 250 adult individuals. The Eurasian otter is considered to be near threatened and it is a fully protected species. The aims of this study were to investigate for the first time the occurrence and characterize the parvoviruses included in the species Carnivore protoparvovirus 1 in seven carcasses of road‐killed Eurasian otters from the southern Italy. Carnivore protoparvovirus 1 are responsible for acute gastroenteritis and leukopenia in pets and free‐ranging carnivores. Initial screening of tissue samples by real‐time PCR revealed CPV/FPV DNA in tissue samples of five Eurasian otters; three of them, showed co‐infections by both CPV and FPV. Among the five positive Eurasian otters, we successfully obtained six DNA sequences from four individuals including two CPV‐2a, one CPV‐2b, one CPV‐2c, and two FPV sequences. Comparison of these sequences with 250 VP2 gene sequences deposited in the GenBank database, showed 10 nt differences resulting in two synonymous and eight non‐synonymous substitutions. On the basis of these results, two sequences here found were characterized as new CPV‐2a, one was characterized as new CPV‐2b variant, and one was characterized as FPV‐like mutant. The last two sequences belong to a FPV and CPV‐2c strain respectively. Carnivore protoparvovirus 1 is reported for the first time in the Eurasian otter showing high infection value in southern Italy. Occurrence of this infection should be studied further to understand its possible pathogenicity and virulence to the fragile and isolate Eurasian otter population which live in southern Italy.     

Presence of Hepatitis E Virus in a RED Deer (Cervus elaphus) Population in Central Italy

Hepatitis E is an acute human disease caused by the hepatitis E virus (HEV). In addition to humans, HEV has been detected in several animal species and is recognized as a zoonotic pathogen. Pigs, wild boar and deer can be reservoir. In this study, we evaluated HEV prevalence in a free‐living red deer (Cervus elaphus) population in central Italy by detecting virus‐specific antibodies and RNA in sera. A total of 35 of 251 red deer sera were positive for anti‐HEV IgG. HEV RNA was detected in 10 of 91 sera examined. Two genomic fragments targeted by diagnostic PCRs in the capsid region were sequenced, both matching with genotype 3 HEV. Overall results confirmed the occurrence of HEV infection in deer also in Italy.     

First detection of Mycobacterium bovis infection in Giraffe (Giraffa camelopardalis) in the Greater Kruger National Park Complex: Role and implications

Bovine tuberculosis (bovine TB) caused by Mycobacterium bovis has become endemic in some wildlife populations in South Africa. The disease has been reported in 21 wildlife species in the country. In this study, we report M. bovis infection in two female giraffes (Giraffa camelopardalis) from two different nature reserves within the Greater Kruger National Park Complex (GKNPC). Mycobacterium bovis was isolated from tissue lesions consistent with macroscopic appearance of tuberculosis (TB) and confirmed by polymerase chain reactions (PCRs), targeting the RD4 region of difference on the genome of the isolates. Spoligotyping and variable number of tandem repeat (VNTR) typing revealed infection of one giraffe with a strain (SB0294) previously not detected in South Africa, while a resident M. bovis strain (SB0121) was detected from the other giraffe. Our work is first to report M. bovis infection in free‐ranging giraffes in South Africa. We have further demonstrated the existence of at least three genetically unrelated strains currently infecting wildlife species within the GKNPC. This finding suggests that the epidemiological situation of M. bovis within the GKNPC is not only driven by internal sources from its established endemic presence, but can be additionally fuelled by strains introduced from external sources. It further emphasizes that regular wildlife disease surveillance is an essential prerequisite for the timely identification of new pathogens or strains in ecospheres of high conservation value.