In vitro antibacterial efficacy of autologous conditioned plasma and amniotic membrane eye drops.

OBJECTIVE: To determine the in vitro antibacterial efficacy of equine and canine autologous conditioned plasma (ACP) and amniotic membrane extract eye drops (AMEED) against aerobic bacteria common to the corneal surface. PROCEDURES: Canine (n = 4) and equine (n = 4) anticoagulated whole blood samples were sterilely collected, pooled for each species, and processed using the Arthrex ACP® Double-Syringe System. Platelet counts were performed on ACP and pooled blood. AMEED were obtained from a commercial source. An electronic medical records search (2013-2022) identified aerobic bacteria cultured from canine and equine corneal ulcers at Mississippi State University College of Veterinary Medicine (MSU-CVM). Ten commonly isolated bacteria for each species were collected from cultures submitted to the MSU-CVM Microbiology Diagnostic Service and frozen at -80°C. The Kirby-Bauer disk diffusion method was used to determine the sensitivities of these isolates to ACP and AMEED. Bacterial isolates were plated onto Mueller-Hinton +5% sheep blood agar and blank sterile discs saturated with 20 µL of ACP or AMEED were tested in duplicate. Imipenem discs served as positive controls and blank discs as negative controls. Zones of inhibition were measured at 18 h. RESULTS: ACP platelet counts were 1.06 and 1.65 times higher than blood for equine and canine samples, respectively. Growth of a multi-drug resistant Enterococcus faecalis was partially inhibited by canine and equine ACP. AMEED did not inhibit growth of any examined bacteria. CONCLUSIONS: Canine and equine ACP partially inhibited E. faecalis growth in vitro. Further studies using varying concentrations of ACP against bacterial isolates from corneal ulcers are warranted.

Development and validation of a quantitative PCR for the detection of Guinea worm (Dracunculus medinensis).

Dracunculus medinensis (Guinea worm) is a parasitic nematode that can cause the debilitating disease dracunculiasis (Guinea worm disease) in humans. The global Guinea Worm Eradication Program has led intervention and eradication efforts since the 1980s, and Guinea worm infections in people have decreased >99.99%. With the final goal of eradication drawing nearer, reports of animal infections from some remaining endemic countries pose unique challenges. Currently, confirmation of suspected Guinea worm infection relies on conventional molecular techniques such as polymerase chain reaction (PCR), which is not specific to Guinea worm and, therefore, requires sequencing of the PCR products to confirm the identity of suspect samples, a process that often takes a few weeks. To decrease the time required for species confirmation, we developed a quantitative PCR assay targeting the mitochondrial cytochrome b (cytb) gene of Guinea worm. Our assay has a limit of detection of 10 copies per reaction. The mean analytical parameters (± SE) were as follows: efficiency = 93.4 ± 7.7%, y-intercept = 40.93 ± 1.11, slope = -3.4896 ± 0.12, and the R2 = 0.999 ± 0.004. The assay did not amplify other nematodes found in Guinea worm-endemic regions and demonstrated 100% diagnostic sensitivity and specificity. Implementation of this quantitative PCR assay for Guinea worm identification could eliminate the need for DNA sequencing to confirm species. Thus, this approach can be implemented to provide more rapid confirmation of Guinea worm infections, leading to faster execution of Guinea worm interventions while increasing our understanding of infection patterns.

Effect of water temperature on frog virus 3 disease in hatchery-reared pallid sturgeon Scaphirhynchus albus.

Ranaviruses are large double-stranded DNA viruses within the genus Ranavirus (family Iridoviridae) that are being detected with increasing frequency among aquacultured and wild fishes. In the USA, multiple sturgeon hatcheries have experienced ranavirus epizootics resulting in significant morbidity and mortality in young-of-year (YOY). Significant economic losses have resulted from repeated outbreaks of frog virus 3 (FV3), the type species for the genus Ranavirus, in YOY pallid sturgeon Scaphirhynchus albus reared at a hatchery within the Missouri River Basin. Water temperature and stocking density are known to influence the severity of ranavirus disease in ectothermic vertebrates. To determine the effect of water temperature on ranavirus disease in hatchery-raised S. albus, we conducted FV3 challenges at 2 temperatures (17 and 23°C) and compared cumulative survival over a 28 d study period. A mean (±SE) survival rate of 57.5 ± 13.2% was observed in replicate tanks of sturgeon maintained at 23°C, whereas no mortality was observed among sturgeon maintained at 17°C. In a second challenge study, we compared the effect of water temperature on disease progression by regularly sampling fish over the study period and evaluating lesions by histopathology and in situ hybridization, and by assessing viral titer and load in external and internal tissues using virus isolation and qPCR, respectively. Results suggest that temperature manipulation may be an effective mitigation strategy that sturgeon hatcheries can employ to minimize ranavirus-associated disease.

The Effect of Maternal Antibodies on Clinical Response to Infection with Epizootic Hemorrhagic Disease Virus in White-Tailed Deer (Odocoileus virginianus) Fawns.

We investigated whether naturally acquired maternal antibodies to epizootic hemorrhagic disease virus serotype 2 (EHDV-2) would protect white-tailed deer (Odocoileus virginianus) fawns against infection and clinical disease following an EHDV-2 challenge. We compared viremia and clinical response in 27-47-d-old, experimentally infected fawns with and without maternally derived antibodies to EHDV-2. Mild to moderate clinical signs were observed in four seronegative (maternal antibody-negative) fawns, which were viremic from 3 to 14 d postinoculation. Individual peak blood virus titers for seronegative fawns ranged from 104.3 to 106.3 median tissue culture infective doses (TCID50)/mL. In contrast, clinical signs were not observed in seropositive (maternal antibody-positive) fawns and a transient low-level viremia (≤102.4 TCID50/mL) occurred in two of six fawns. Our results indicated that the presence of maternally derived EHDV-2 antibodies in fawns prevents or greatly reduces clinical disease and the level and duration of EHDV-2 viremia.

Reference Intervals for Blood Analytes of Adult Aquarium-Housed Russian Sturgeon Acipenser gueldenstaedtii.

Russian Sturgeon Acipenser gueldenstaedtii are an important, critically endangered, roe-producing species. Despite a wealth of knowledge pertaining to other members of family Acipenseridae, there is very limited published information regarding baseline blood analytes in Russian Sturgeon. The objectives of this study were (1) to establish reference intervals for a suite of hematological and biochemical data and (2) to compare plasma chemistry data to two point-of-care (POC) cartridges, tested on the VetScan iSTAT 1 analyzer, that use heparinized whole blood for the assessment of clinically normal, aquacultured adult Russian Sturgeon sedated with eugenol (AQUI-S 20E) at a single institution. Reference intervals are reported. The calculated hematocrit measured by the POC analyzer tended 4-5% lower than the spun packed cell volume, confirming the importance of spun packed cell volume as a reliable measurement of red blood cell mass. Various analytes, notably whole-blood urea nitrogen, glucose, sodium, total carbon dioxide, chloride, ionized calcium, and anion gap, were significantly different by both POC cartridges. This study successfully produced reference intervals for blood analytes in adult Russian Sturgeon under managed care and creates a foundation for future studies into the effects of extrinsic and intrinsic factors and variations of analytical methodologies on blood analytes in this species.

Necroulcerative dermatitis associated with Myxobolus dermatoulcerans n. sp. (Cnidaria: Myxobolidae) in red-bellied piranha, Pygocentrus nattereri Kner (Characiformes: Serrasalmidae), from Peru.

A group of red-bellied piranha, Pygocentrus nattereri Kner, recently imported from Peru exhibited multifocal, cutaneous ulcerations with exposure of the underlying musculature. Skin scrapes yielded moderate numbers of myxospores morphologically consistent with Myxobolus Bütschli, 1882. Myxospores from these fish were morphologically and molecularly distinct from other myxobolids infecting piranha. Myxospores are pyriform to capsular with a rounded posterior and slightly rounded to tapering anterior aspect in valvular view. Myxospore bodies are 14.3-17.8 (mean 16.1) µm long and 7.6-10.3 (mean 8.9) µm wide. Polar capsules are symmetrical, slender, elongate, and measure 7.4-10.2 (mean 9.2) µm long and 2.1-3.7 (mean 3.0) µm wide. Sequence generated for the 18S rRNA gene had no direct matches to any sequence available on GenBank but demonstrated less than 89% nucleotide similarity to various published and unpublished Myxobolus spp. from Piaractus brachypomus (Cuvier) and Colossoma macropomum (Cuvier). This paper provides the morphological and molecular characterisation of Myxobolus dermatoulcerans n. sp. from red-bellied piranha and describes associated pathological lesions.

Is the Mole Rat Vomeronasal Organ Functional?

The colonial naked mole rat Heterocephalus glaber is a subterranean, eusocial rodent. The H. glaber vomeronasal organ neuroepithelium (VNE) displays little postnatal growth. However, the VNE remains neuronal in contrast to some mammals that possess nonfunctional vomeronasal organ remnants, for example, catarrhine primates and some bats. Here, we describe the vomeronasal organ (VNO) microanatomy in the naked mole rat and we make preliminary observations to determine if H. glaber shares its minimal postnatal VNE growth with other African mole rats. We also determine the immunoreactivity to the mitotic marker Ki67, growth-associated protein 43 (GAP43), and olfactory marker protein (OMP) in six adult and three subadult H. glaber individuals. VNE volume measurements on a small sample of Cryptomys hottentotus and Fukomys damarensis indicate that the VNE of those African mole rat species are also likely to be growth-deficient. Ki67(+) cells show that the sensory epithelium is mitotically active. GAP43 labelling indicates neurogenesis and OMP(+) cells are present though less numerous compared to GAP43(+) cells. In this respect, the VNO of H. glaber does not appear vestigial. The African mole rat VNE may be unusually variable, perhaps reflecting reduced selection pressure on the vomeronasal system. If so, African mole rats may provide a useful genetic model for understanding the morphological variability observed in the mammalian VNO. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc. Anat Rec, 303:318-329, 2020. © 2019 American Association for Anatomy.

A morphological, molecular, and histopathological redescription of Henneguya nyongensis Fomena & Bouix, 1996 (Cnidaria: Myxobolidae) infecting the gills of Peter's elephantnose fish, Gnathonemus petersii (Günther) (Osteoglossiformes: Mormyridae), imported from Nigeria.

A Henneguya sp., morphologically resembling Henneguya nyongensis Fomena & Bouix, 1996, was isolated from the gills of Peter's elephantnose fish, Gnathonemus petersii Günther, imported from Nigeria. Plasmodia were located between lamellae and within the gill epithelium, often leading to lamellar fusion. Although slightly smaller, the myxospores from these fish were morphologically consistent with H. nyongensis. In valvular view, spores are elongate, pyriform with a rounded posterior and tapering caudal processes. Myxospore bodies are 9.6-12.3 (mean 11.2) µm long and 4.0-4.7 (mean 4.3) µm wide. Polar capsules are pyriform, elongate, 4.5-5.2 (4.7) µm long and 1.3-1.6 (1.4) µm wide, with a characteristic neck-like structure at the apical end. Sequence generated for the 18S small subunit rRNA gene did not directly match any sequences available on GenBank, but demonstrated 91% nucleotide similarity to an unpublished Henneguya sp. infecting Mormyrus kannume Forsskål. Herein, the description of H. nyongensis is supplemented with new data on histopathology, molecular characterisation, and expanded host and geographical range.

FATAL RANAVIRUS INFECTION IN A GROUP OF ZOO-HOUSED MELLER'S CHAMELEONS (TRIOCEROS MELLERI).

A group of five juvenile Meller's chameleons (Trioceros melleri) experienced 100% mortality over a period of 1 mo due to ranavirus infection. The index case was found dead without premonitory signs. The three subsequent cases presented with nonspecific clinical signs (lethargy, decreased appetite, ocular discharge) and were ultimately euthanatized. The final case died after initially presenting with skin lesions. Postmortem examination revealed thin body condition in all five animals and mild coelomic effusion and petechiae affecting the tongue and kidneys of one animal. Microscopically, all animals had multifocal necrosis of the spleen, liver, and kidney; four of five animals had necrosis of the nasal cavity; and two of five had necrosis of adrenal tissue, bone marrow, and skin. Numerous basophilic intracytoplasmic inclusions were present in the liver of all animals and nasal mucosa of three of the five animals. Consensus polymerase chain reaction for herpesvirus and adenovirus were negative, whereas ranavirus quantitative polymerase chain reaction was positive. Virus isolation followed by whole genome sequencing and Bayesian phylogenetic analysis classified the isolates as a strain of frog virus 3 (FV3) most closely related to an FV3 isolate responsible for a previous outbreak in the zoo's eastern box turtle (Terrapene carolina carolina) group. This case series documents the first known occurrence of ranavirus-associated disease in chameleons and demonstrates the potential for interspecies transmission between chelonian and squamate reptiles.

Partial validation of a TaqMan real-time quantitative PCR for the detection of ranaviruses.

Ranaviruses are globally emerging pathogens negatively impacting wild and cultured fish, amphibians, and reptiles. Although conventional and diagnostic real-time PCR (qPCR) assays have been developed to detect ranaviruses, these assays often have not been tested against the known diversity of ranaviruses. Here we report the development and partial validation of a TaqMan real-time qPCR assay. The primers and TaqMan probe targeted a conserved region of the major capsid protein (MCP) gene. A series of experiments using a 10-fold dilution series of Frog virus 3 (FV3) MCP plasmid DNA revealed linearity over a range of 7 orders of magnitude (107-101), a mean correlation coefficient (R2) of >0.99, and a mean efficiency of 96%. The coefficient of variation of intra- and inter-assay variability ranged from <0.1-3.5% and from 1.1-2.3%, respectively. The analytical sensitivity was determined to be 10 plasmid copies of FV3 DNA. The qPCR assay detected a panel of 33 different ranaviral isolates originating from fish, amphibian, and reptile hosts from all continents excluding Africa and Antarctica, thereby representing the global diversity of ranaviruses. The assay did not amplify highly divergent ranaviruses, members of other iridovirus genera, or members of the alloherpesvirus genus Cyprinivirus. DNA from fish tissue homogenates previously determined to be positive or negative for the ranavirus Epizootic hematopoietic necrosis virus by virus isolation demonstrated a diagnostic sensitivity of 95% and a diagnostic specificity of 100%. The reported qPCR assay provides an improved expedient diagnostic tool and can be used to elucidate important aspects of ranaviral pathogenesis and epidemiology in clinically and sublinically affected fish, amphibians, and reptiles.

EXTENSION OF THE KNOWN HOST RANGE OF INTRANUCLEAR COCCIDIOSIS: INFECTION IN THREE CAPTIVE RED-FOOTED TORTOISES ( CHELONOIDIS CARBONARIA).

The intranuclear coccidian parasite of Testudines (TINC) is an emerging pathogen of tortoises. Three captive red-footed tortoises ( Chelonoidis carbonaria) from an isolated collection presented with multiple acute, nonspecific clinical signs. One tortoise died and was diagnosed with intranuclear coccidiosis on histopathology with confirmation by quantitative polymerase chain reaction (qPCR). In addition to tissues where TINC has been previously described, coccidia were identified in the pineal gland, choroid plexus, and testicular Sertoli cells. The two remaining tortoises survived after treatment with oral ponazuril (20 mg/kg every 48 hr for 56 days) and remained asymptomatic, although not cleared of infection, for 21 months, as the number of coccidian gene copies detected by qPCR was reduced in one tortoise. This report extends the known host range of this parasite to continental South American tortoises, describes new sites of infection by histopathology, and has management implications for this disease.