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.     

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.     

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.     

Newcastle Disease Virus in Little Owls (Athene noctua) and African Penguins (Spheniscus demersus) in an Israeli Zoo

Newcastle disease is a contagious and often fatal disease, capable of affecting all species of birds. A velogenic Newcastle disease virus (vNDV) outbreak occurred in an Israeli zoo, in which Little owls (Athene noctua) and African penguins (Spheniscus demersus) were found positive for presence of NDV. Some of them have died. The diagnostic process included: post‐mortem examination, histopathology, real‐time RT‐PCR assay, virus isolation, serology, intracerebral pathogenicity index and phylogenetic analysis. A vNDV was diagnosed and found to be closely related to isolates from vNDV outbreaks that occurred in commercial poultry flocks during 2011. All isolates were classified as lineage 5d.     

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.     

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.     

Elizabethkingia miricola infection in Chinese spiny frog (Quasipaa spinosa)

Elizabethkingia miricola is a Gram‐negative rod which has been incriminated in severe infections in humans. Recently, a serious infectious disease was identified in Chinese spiny frogs (Quasipaa spinosa), in the Sichuan Province of China; the disease was characterized by corneal opacity, the presence of ascites and neurological symptoms. A Gram‐negative bacillus was isolated from the liver, spleen and kidney of the diseased frogs. Experimental infection test revealed that the bacillus could infect the frogs Q. spinosa and the LD₅₀ value was 1.19 × 10⁶ cfu per frog. The isolated Gram‐negative bacillus was identified as E. miricola according to phenotypic characteristics, 16S rRNA and gyrB gene sequence analysis. The isolated strain was only susceptible to florfenicol among all investigated chemotherapeutic agents. Histological examination revealed that E. miricola infection caused pathological lesions to multiple organs and tissues, especially in the liver, brain, kidney. These results confirmed that E. miricola is an emerging pathogen of Chinese spiny frogs.     

Systemic Besnoitiosis in a Juvenile Roe Deer (Capreolus capreolus)

Herein, we report the first incidence of systemic besnoitiosis in a male juvenile roe deer Capreolus capreolus. The animal was found dead in an area where bovine besnoitiosis is endemic and showed cachexia and multiple skin erosions in the metacarpal and metatarsal areas. Moreover, round and elevated white structures suggestive of Besnoitia spp. tissue cysts were also present. Twenty‐eight tissue samples from different anatomical locations were collected for microscopic lesion and parasite detection through histopathology and PCR. Immunohistochemistry was performed to confirm Besnoitia‐positive reaction in the tissue cysts. In addition, the identity of Besnoitia spp. in PCR‐positive tissue samples was also investigated using microsatellite (MS) markers, and the comparison of protein disulphide isomerase gene sequences (BbPDI) of B. besnoiti and B. tarandi isolated from cattle and reindeer, respectively. Besnoitia cysts were detected in the skin (several parts), respiratory and upper digestive tracts, eyes, kidney, liver, testicle, cardiac muscle and lymphoid tissue. Remarkably, the presence of tissue cysts in the brain confirmed the capacity of Besnoitia spp. to form tissue cysts in the central nervous system (CNS). Finally, the Besnoitia species detected showed the same MS genotype as B. besnoiti, and BbPDI sequences from roe deer and two B. besnoiti isolates were genetically identical throughout multiple sequence alignment. Thus, for the first time, there is evidence that roe deer might act as an intermediate host of B. besnoiti. Further molecular analyses and parasite isolations are needed to corroborate these findings.     

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.     

Exposure of free‐ranging capybaras (Hydrochoerus hydrochaeris) to the vaccinia virus

The aim of this study was to evaluate the possibility of free‐ranging animals/hunting dogs as sources of infection in the vaccinia virus (VACV) transmission chain. Serological, cell culture and molecular assays were conducted in 56 free‐ranging animals and 22 hunting dogs. ELISA/neutralizing assays showed that two (2.5%) capybaras (Hydrochoerus hydrochaeris) had anti‐OPV positive antibodies, while all samples tested negative through PCR/cell culture. After being hit by cars on roads, capybaras that exhibited neither clinical signs nor any association with bovine outbreaks had neutralizing antibodies against the Orthopoxvirus, as detected through plaque‐reduction neutralizing tests and ELISA. Evidence exists regarding peridomestic capybaras acting as a source of the virus and serving as a link between wild and urban environments, thus contributing to viral maintenance.