Characterization of the immune response against Testudinid herpesvirus 3: new insight

Testudinid herpesvirus 3 (TeHV3) is one of the most lethal viral agents in tortoises worldwide. Although TeHV3 have been extensively studied, only little information is available about host-pathogen interaction. TeHV3 infections in different species of the genus Testudo correlate with various lesions profiles, disease severity and clinical outcome, suggesting the existence of a complex host-pathogen interaction. This might reflect a possible viral-host coevolution (Origgi, 2012).To study the host-pathogen interaction, we previously screened 5.000 clones from a bacteriophage library obtained from the TeHV3 genomic DNA using Testudo graeca seropositive sera. Of the six detected positive clones, only one was confirmed by F.A.C.S. Selected clone was determined to be a concatamer of different TeHV3 genomic fragments including the partial sequence of TE17, UL15, Major capsid protein (MCP), and Glycoprotein B (gB) genes. After complete sequencing of the selected clone, the MCP and the gB were antisenses compared to the phagemid promoter.In order to assess which of the gene fragments among TE17 and UL15 was encoding for the antigenic determinant that was recognized by the anti-TeHV3 tortoise sera, distinct approaches were followed.TE-17 and UL15 fragments were knock out from the original phagemid using the following approaches: a) directed-site mutagenesis, b) molecular cloning, and c) restriction enzymes cloning. All the modified constructs were cloned in two different E. coli cloning vectors (D5α and XL 1-Blue).Transformation of competent cells with the constructs described above did not yield any viable bacteria.Among the different aspects might have influenced transformation success rate, construct size was probably the most relevant (about 9Kb). Furthermore, we could not entirely exclude that genomic DNA editing might have induced mutations in the construct sequence causing toxic effects on the host bacterial cell. Cloning of TE-17 and UL15 gene fragments into different prokaryotic expression vectors is currently under way

Characterization of the immune response against Testudinid herpesvirus 3.

Numerous infectious diseases have been documented in reptiles, however minimal information isavailable concerning their immunological response. One of the most diffuse and lethal reptile pathogenis Testudinid herpesvirus 3 (TeHV3), a Alphaherpesvirinae. All species of tortoises (Testudinide) areconsidered susceptible to TeHV3, however the virus is over represented in the genus Testudo, whichincludes, among others, T. graeca, T. hermanni, T. marginata, and T. horsfieldii, that are popular pets inEurope. Incidence of TeHV3-associated disease is highest right after hibernation (Origgi, 2012).The aim of this work is to partially characterize the immunological response of T. graeca against TeHV3.A bacteriophage library composed of about 5.000 clones containing genomic DNA fragments of TeHV3was produced. Bacteriophages were amplified in a specific strain of E. coli and were screened withTeHV3-seropositive sera from T. graeca. Phagemids were excised from the positive bacteriophages,sequenced, and compare with the TeHV3 genome to identify the encoding genes. Six differentstructural and non-structural proteins have identified as immune relevant. Vero cells where transfectedwith phagemids of the positive clones, to confirm previous results. TeHV3’s proteins expression wasassessed by F.A.C.S using T. graeca seropositive sera. Of all the six selected clones, only that expressingthe partial sequence of the glycoprotein B (gB) showed a positive signal in the F.A.C.S. analysis. Thisresult is consistent with the well-known immunogenicity of gB of other herpesviruses including thoseinfecting humans and with the highly conserved role that gB plays in host-pathogen interaction acrossspecies and evolution (Beals et al., 2016)

Detection of Testudinid alphaherpesvirus, Chlamydia spp., Mycoplasma spp., and Salmonella spp. in free‑ranging and rescued Italian Testudo hermanni hermanni.

Testudo hermanni is included as near‑threatened in the Red List of the International Union for Conservation of Nature, while T. hermanni hermanni is considered endangered in the Italian Red List. Appropriate management of smuggled or seized wild individuals is recommended before their reintroduction into the wild. Accordingly, a health monitoring study was carried out. During 2014‑2016, 133 oral swabs and 121 cloacal swabs were collected from a total of approximately 180 free‑ranging and rescued T. hermanni hermanni from eight different Italian regions to investigate the presence of DNA of Testudinid alphaherpesvirus (TeAHV), Chlamydia spp. and Mycoplasma spp. in the oral cavity, and Salmonella spp. isolates in the cloaca. Mycoplasma spp. was detected in 52 out of 87 (59.77%) of rescued and in 1 out of 46 free‑ranging (2.17%) individuals; 33 out of 53 (62.26%) Mycoplasma spp. positive samples were typed as M. agassizii by PCR. Salmonella spp. was isolated from 45 out of 121 (37.19%) cloacal swabs, typed into 14 serovars, and characterized for complete antimicrobial susceptibility. A significantly different distribution of Salmonella spp. isolates was found in 2016 in comparison with 2014 and 2015, without any difference between free‑ranging and rescued tortoises. All the tested tortoises were negative for TeAHV and Chlamydia spp. These results are considered a baseline information critical to monitor the dynamics of these microorganisms in free‑ranging and rescued populations of T. h. hermanni, and to correctly approach the management of rescued animals and possible relocation programs

Microorganisms detected by bacteriological and mycological examination in samples from dead free-ranging roe deer examined at the Centre for Fish and Wildlife Health, Switzerland, 1958–2014.

<p>Microorganisms detected by bacteriological and mycological examination in samples from dead free-ranging roe deer examined at the Centre for Fish and Wildlife Health, Switzerland, 1958–2014.</p

Malformations reported in free-ranging dead roe deer submitted to the Centre for Fish and Wildlife Health, Switzerland, 1958–2014.

<p>Malformations reported in free-ranging dead roe deer submitted to the Centre for Fish and Wildlife Health, Switzerland, 1958–2014.</p

Number of submitted roe deer per canton and year.

<p>Slightly more than half of the cases were submitted by official game wardens (n = 893) followed by voluntary gamekeepers (n = 259). Considerably fewer cases were delivered by the police (n = 68), hunters (n = 57), private veterinarians or veterinary authorities (n = 48), and cantonal hunting authorities (n = 37). Overall, cantons with official game wardens submitted more animals (78%; n = 1227) than cantons without game wardens (20%; n = 313).</p

Selected gross pathology findings in free-ranging roe deer from Switzerland.

<p>(A) Skull, actinomycosis-like lesions of bacterial origin. A severe proliferative and destructive process is affecting the lateral aspect of the skull. Inset: The right mandibular region is severely expanded by a proliferative inflammatory process similar to that shown in the main figure (both cases were from 2013). (B) Spleen, lymphoma. There is diffuse and severe splenomegaly with multiple nodules expanding within the parenchyma and elevating the capsule. Additional similar lesions were also observed in liver, bone marrow and lymph nodes (1994). (C) Skull, osteomyelitis secondary to traumatic injury. A transverse fracture is associated with a granular appearance of the bone consistent with remodeling secondary to osteo-myelitis. Obvious bone loss and rarefaction is seen in cross section (inset) (1958). (D) Mesenteric lymph nodes, paratuberculosis. The mesenteric lymph nodes are severely enlarged (lymph-adenomegaly), up to 2–4 x 1.5–1 x 2–2.5 cm (2011). (E) Lung, tarsal and metatarsal bones, pyogranulo-matous fungal pneumonia and hypertrophic osteopathy. A large pyogranuloma is expanding, compressing and replacing the lung parenchyma. The cortical bone of the frontal aspect of the metatarsus is diffusely and markedly thickened by a bony proliferation (2010). (F) Inguinal hernia. A large mass covered by normal skin is bulging in the region of the left inguinal space (diameter 46cm). The mass contained small intestine loops and part of the caecum (2011).</p

Performed parasitic, bacteriological and histological investigations over time.

<p>Performed parasitic, bacteriological and histological investigations over time.</p

Selected categories of main diagnoses in percentage of total submitted cases per year.

<p>(A) Parasitic. (B) Bacterial. (C) Traumatic. (D) Undetermined. Dashed horizontal black lines indicate total number of submitted cases. Vertical dotted lines separate time periods with different main pathologists.</p