Skin mucus of Cyprinus carpio inhibits cyprinid herpesvirus 3 binding to epidermal cells

ABSTRACT: Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a mortal and highly contagious disease in common and koi carp. The skin is the major portal of entry of CyHV-3 in carp after immersion in water containing the virus. In the present study, we used in vivo bioluminescence imaging to investigate the effect of skin mucus removal and skin epidermis lesion on CyHV-3 entry. Physical treatments inducing removal of the mucus up to complete erosion of the epidermis were applied on a defined area of carp skin just before inoculation by immersion in infectious water. CyHV-3 entry in carp was drastically enhanced on the area of the skin where the mucus was removed with or without associated epidermal lesion. To investigate whether skin mucus inhibits CyHV-3 binding to epidermal cells, tail fins with an intact mucus layer or without mucus were inoculated ex vivo. While electron microscopy examination revealed numerous viral particles bound on the fins inoculated after mucus removal, no particle could be detected after infection of mucus-covered fins. Finally, anti-CyHV-3 neutralising activity of mucus extract was tested in vitro. Incubation of CyHV-3 with mucus extract reduced its infectivity in a dose dependent manner. The present study demonstrates that skin mucus removal and epidermal lesions enhance CyHV-3 entry in carp. It highlights the role of fish skin mucus as an innate immune protection against viral epidermal entry.     

Feeding Cyprinus carpio with infectious materials mediates cyprinid herpesvirus 3 entry through infection of pharyngeal periodontal mucosa

ABSTRACT: Cyprinid herpesvirus 3 (CyHV-3), also known as Koi herpesvirus, is the etiological agent of a mortal disease in common and koi carp. Recently, we investigated the entry of CyHV-3 in carp using bioluminescence imaging and a CyHV-3 recombinant strain expressing luciferase (LUC). We demonstrated that the skin is the major portal of entry after inoculation of carp by immersion in water containing CyHV-3. While this model of infection mimics some natural conditions in which infection takes place, other epidemiological conditions could favour entry of virus through the digestive tract. Here, we investigated whether ingestion of infectious materials mediates CyHV-3 entry through the digestive tract. Carp were fed with materials contaminated with the CyHV-3 LUC recombinant (oral contamination) or immersed in water containing the virus (contamination by immersion). Bioluminescence imaging analyses performed at different times post-infection led to the following observations: (i) the pharyngeal periodontal mucosa is the major portal of entry after oral contamination, while the skin is the major portal of entry after contamination by immersion. (ii) Both modes of inoculation led to the spreading of the infection to the various organs tested. However, the timing and the sequence in which some of the organs turned positive were different between the two modes of inoculation. Finally, we compared the disease induced by the two inoculation modes. They led to comparable clinical signs and mortality rate. The results of the present study suggest that, based on epidemiological conditions, CyHV-3 can enter carp either by skin or periodontal pharyngeal mucosal infection.     

Prevalence and characteristics of Cyprinid herpesvirus 3 (CyHV-3) infection in common carp (Cyprinus carpio L.) inhabiting three rivers in Kochi Prefecture,Japan

Cyprinid herpesvirus 3 (CyHV-3) causes lethal disease in common and koi carp. Mortality by CyHV-3 disease has not been reported since 2011 in Kochi Prefecture, Japan. Here, we detected and quantified CyHV-3 in common carp inhabiting three rivers in the prefecture to examine if the carp are carriers of CyHV-3 as a source of infection. CyHV-3 DNA was detected in 16.7% (12/72) of brain samples in Kagami River, 3.9% (3/76) of brain and 3.9% (3/76) of gill samples in Monobe River, and 5.1% (4/79) of brain and 1.3% (1/79) of gill samples in Wajiki River. CyHV-3 genotypes identified in the 23 samples were classified as the J genotype A1 that has been found in Japan. The CyHV-3 DNA load did not differ statistically between sampling months, indicating that CyHV-3 has been silent in common carp, unlike Lake Biwa where the annual reactivation occurs in spring. Taken together, our results represented definitive evidence that seasonal changes in water temperature do not affect CyHV-3 activity in carp. Considering that infectious virus was not isolated from CyHV-3 DNA-positive samples, it was suggested that CyHV-3 establishes a latent infection in carp populations inhabiting Kagami River, Monobe River and Wajiki River. Further, the presence of circular or concatameric CyHV-3 DNA was detected in five of 23 CyHV-3 DNA-positive samples. Common carp inhabiting Lake Biwa were reported previously to harbor linear but not circular CyHV-3 DNA. This difference suggested that the CyHV-3 genome may be circularized for long-term maintenance without active viral replication.     

Transmission of Cyprinid herpesvirus-3 (CyHV-3) from goldfish to naïve common carp by cohabitation

Cyprinid herpesvirus-3 (CyHV-3) has spread worldwide and has had a major impact on koi and common carp production. Previous studies on the host range of the CyHV-3 found that fish species other than koi and common carp are fully resistant to natural virus exposure. Recently, CyHV-3 was detected in goldfish (Carassius auratus auratus) that were in contact with CyHV-3 infected koi. In the present study, a specific RT-PCR product was amplified from the viral thymidine kinase gene in gills, intestine and brain tissues of CyHV-3 infected goldfish. This implied that CyHV-3 replicated in these goldfish. Also, in the presence of a stress factor such as temperature fluctuation, the CyHV-3 infected goldfish transmitted the virus to cohabitated naïve SPF common carp. CyHV-3 DNA was detected in the cohabitated naïve carp tissues by PCR. The results of this study demonstrate that goldfish is a carrier for CyHV-3, permit virus propagation, and disseminate the virus to susceptible carp causing the disease.     

Cyprinid herpesvirus 3: an interesting virus for applied and fundamental research

Cyprinid herpesvirus 3 (CyHV-3), a member of the family Alloherpesviridae is the causative agent of a lethal, highly contagious and notifiable disease in common and koi carp. The economic importance of common and koi carp industries together with the rapid spread of CyHV-3 worldwide, explain why this virus became soon after its isolation in the 1990s a subject of applied research. In addition to its economic importance, an increasing number of fundamental studies demonstrated that CyHV-3 is an original and interesting subject for fundamental research. In this review, we summarized recent advances in CyHV-3 research with a special interest for studies related to host-virus interactions.     

Detection of cyprinid herpesvirus-3 DNA in lake plankton

The disease caused by cyprinid herpesvirus-3 (CyHV-3) severely impacts the natural freshwater ecosystem and damages carp and koi farming, however, the pathway of CyHV-3 transmission remains unclear. It is possible that the virus adheres to plankton, which then facilitate viral movement and transmission, and therefore, it is hypothesised that plankton are involved in the disease dynamics. In this study, plankton were collected at eight sites in the Iba-naiko lagoon; we detected and quantified CyHV-3 DNA from plankton samples. The results of the correlation analysis showed a significant positive correlation between CyHV-3 copies and the number of Rotifera, suggesting that CyHV-3 binds to and/or is concentrated by Rotifera. Our results suggest that plankton affect viral ecology in the natural environment.     

Nationwide Cyprinid herpesvirus 3 contamination in natural rivers of Japan

Cyprinid herpesvirus 3 (CyHV-3) disease is a significant threat for common and koi carp cultivators and for freshwater ecosystems. To determine the prevalence of CyHV-3 in Japanese rivers, a nationwide survey of all national class-A rivers was undertaken in the Summer of 2008. The virus was concentrated from river water samples using the cation-coated filter method. CyHV-3 DNA was detected in 90 rivers, representing 90% of 103 successfully analysed rivers. More than 100,000 copies of CyHV-3 DNA per litre of sample were detected in four rivers, higher than that reported during the Yura River outbreak in 2007. For CyHV-3-positive rivers, the log CyHV-3 density was negatively correlated with the water temperature on the sampling date and positively correlated with the suspended solids and dissolved oxygen, which are annually averaged for each river. Our results demonstrate that virus detection using molecular biology techniques is a powerful tool for monitoring the presence of CyHV-3 in natural environments.     

Mode of Transmission of Vibriosis among Ayu Plecoglossus altivelis

Abstract

The mode of transmission of Vibrio anguillarum infection in ayu (Plecoglossus altivelis) and the portal of entry for the pathogen were investigated by the use of various challenge methods. The methods employed were immersion, direct contact, cohabitation, patch contact (filter paper soaked in bacterial suspension placed on part of a test fish for 1 min), and intubation. Based on the results of immersion, direct contact, and cohabitation challenges, it was concluded that water-borne infection is the primary mode of transmission of the disease. The patch contact challenge revealed that V. anguillarum penetrated the host through the skin, fins, anus, and gills. Among those, the skin and anus were determined to be the essential portals of entry for the pathogen. Artificial wounds of the skin surface caused by swabbing or scale removal facilitated invasion by the pathogen. Infection also was established by oral and anal intubations.     

Emergence of fatal European genotype CyHV-3/KHV in mainland China

Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is a highly infectious causative agent to common carp and koi worldwide. The virus is mainly consisted of European and Asian genotype isolates. To date, no European genotype CyHV-3 has been found emerging in the East and Southeast Asian regions. In late March 2011, an outbreak of CyHV-3 disease occurred in Guangzhou City, Guangdong Province, China, resulting in the deaths of approximately 200 large-sized adult koi within four weeks. One moribund koi was sampled for CyHV-3 isolation. Thus, a CyHV-3 was isolated in KCF-1 cells and designated as KHV-GZ11. Abundant mature or immature virions in infected KCF-1 cells were observed under a transmission electron micrograph. In addition, intra-nuclear inclusion body-like structures with masses of virions were also observed. Based on the TK and ORF136H genes, the sequence analyses revealed that KHV-GZ11 is a distinct European genotype of CyHV-3. Moreover, the infectivity experiment showed that KHV-GZ11 was highly virulent to koi. In summary, we are the first to confirm the emergence of fatal European genotype CyHV-3/KHV in East and Southeast Asia. Our study will provide new insight to explore the virus origin and epidemiology, as well as its pathogenicity.     

Surveillance of herpesviruses in koi carp Cyprinus carpio koi and goldfish Carassius auratus cultured in West Bengal,India

Background: Viral diseases create one of the greatest challenges for the rapidly expanding ornamental fish culture sector. In recent years, the host-specific herpesviruses are receiving much attention due to the intensification in aquaculture globally. Cyprinid herpesvirus-2 (CyHV-2, goldfish hematopoietic necrosis virus), and Cyprinid herpesvirus-3 (CyHV-3, koi herpesvirus) are the lethal pathogens of koi carp Cyprinus carpio koi and goldfish Carassius auratus, respectively. In India, West Bengal is the leading state in ornamental fish production and export. Methods: The surveillance of CyHV-2 and CyHV-3 in koi carp and goldfish cultured in West Bengal was carried out between November 2014 and January 2017 as per Office International des Epizooties guidelines. Results: Both fish species were negative for CyHV-3. The CyHV-2 infection was detected in both gill and kidney tissues of apparently healthy and diseased C. auratus from December 2014 to March 2015. Severe necrosis was observed in the gills of infected C. auratus. Coinfection with members of the bacterial genera Aeromonas and Flavobacterium was also observed in the kidney of infected goldfish. The histopathological observations in the kidney of CyHV-2 infected C. auratus demonstrated the pathogenic potential of CyHV-2 and tissue tropism. Conclusions and Clinical relevance: Environmental stressors like low water temperature and the use of wastewater for culture played a vital role in the onset of CyHV-2 infection in the stressed goldfish. The spread of CyHV-2 can likely pose a certain degree of threat to aquaculture especially the unrestricted movement of goldfish. The results of the present study emphasize the necessity of an organized risk assessment to plan for the management strategies on the control and spread of CyHV-2 in Indian aquaculture.     

Reservoirs of Cyprinid herpesvirus 3 (CyHV-3) DNA in sediments of natural lakes and ponds

Cyprinid herpesvirus 3 (CyHV-3) is a lethal DNA virus that infects common carp and koi. It has caused outbreak of the disease within both aquaculture and natural environmental ecosystems. However, there is not enough understanding of the distribution of CyHV-3 in the natural environments, partly because there is no suitable quantification method. In this study, we tested CyHV-3 extraction methods from sediment and then compared its abundance between sediment and water using real-time PCR. Sediment samples were taken from lake and pond, and total viral DNA was extracted using the viral elution method recommended by the US Environmental Protection Agency (manual method), as well as a commercial DNA extraction kit for soil (commercial kit method) before PCR detection. 7 of 12 (58%) and 5 of 10 (50%) sediment samples showed PCR positive signal for CyHV-3 DNA using the manual method and the commercial kit, respectively, and consistent results were obtained from the samples using the manual method between two independent primer sets. The quantification of CyHV-3 DNA in natural sediment using the manual method and external standard virus revealed that its concentration was 1.2×10(4) to 3.3×10(5) copies DNA/kg. The concentration in sediments was 46-1238 times higher than that in water from the same location, suggesting that sediment could act as a reservoir for CyHV-3 in natural freshwater environments. This is the first report of the existence of CyHV-3 in the sediment of a natural lake or pond.     

Mycologic disorders of the skin

Cutaneous tissue can become infected when fungal organisms contaminate or colonize the epidermal surface or hair follicles. The skin can be a portal of entry for fungal infection when the epithelial barrier is breached or it can be a site for disseminated, systemic fungal disease. The two most common cutaneous fungal infections in small animals are dermatophytosis and Malassezia dermatitis. Dermatophytosis is a superficial cutaneous infection with one or more of the fungal species in the keratinophilic genera Microsporum, Trichophyton, or Epidermophyton. Malassezia pachydermatis is a nonlipid dependent fungal species that is a normal commensal inhabitant of the skin and external ear canal in dogs and cats. Malassezia pachydermatis is the most common cause of Malassezia dermatitis. The diagnosis and treatment of these cutaneous fungal infections will be discussed.     

The Histopathological Changes in the Skin of Pigs With Dermatosis Vegetans

Dermatosis vegetans, an hereditary disease seen in Landrace swine, is characterized by defects in the skin, and a giant cell pneumonia. Histological changes of the skin from 12 affected pigs of various ages were described. Lesions of the skin were characterized by acanthosis, hyperkeratosis, parakeratosis, and leukocytic infiltration. In general, the defects followed a definite developmental pattern, which were divided into three stages: (1) the stage of early development, (2) the intermediate stage characterized by an acute inflammatory response, and (3) the stage of regression. The specific role of the eosinophil, a prominent feature in this and other diseases of the porcine skin, remains obscure.     

Rapid Detection of Cyprinid Herpesvirus 3 in Latently Infected Koi by Recombinase Polymerase Amplification

Since the emergence of cyprinid herpesvirus 3 (CyHV-3), outbreaks have been devastating to Common Carp Cyprinus carpio and koi (a variant of Common Carp), leading to high economic losses. Current diagnostics for detecting CyHV-3 are limited in sensitivity and are further complicated by latency. Here we describe the detection of CyHV-3 by recombinase polymerase amplification (RPA). The RPA assay can detect as low as 10 copies of the CyHV-3 genome by an isothermal reaction and yields results in approximately 20 min. Using the RPA assay, the CyHV-3 genome can be detected in the total DNA of white blood cells isolated from koi latently infected with CyHV-3, while less than 10% of the latently infected koi can be detected by a real-time PCR assay in the total DNA of white blood cells. In addition, RPA products can be detected in a lateral flow device that is cheap and fast and can be used outside of the diagnostic lab. The RPA assay and lateral flow device provide for the rapid, sensitive, and specific amplification of CyHV-3 that with future modifications for field use and validation could lead to enhanced surveillance and early diagnosis of CyHV-3 in the laboratory and field.

Received September 14, 2015; accepted April 9, 2016 Published online August 2, 2016     

Colonization of the tonsils of calves with Pasteurella haemolytica.

Tonsils of 10 calves were inoculated with Pasteurella haemolytica (PH) and the degree of colonization was followed by collecting sequential tonsil. wash specimens. Tonsils were colonized for at least 3 weeks after instillation of PH into the tonsillar sinus. Calves with colonized tonsils responded with serum and nasal secretion antibody responses to PH and to leukotoxin. Pasteurella haemolytica was detected in nasal mucus specimens of 2 calves during the week after inoculation of the tonsils, but all other specimens were culture-negative. Infectious bovine rhinotracheitis virus-induced respiratory tract disease 25 days later did not elicit a population increase of PH in the tonsils, and did not elicit shedding of PH in nasal mucus.     

Use of monoclonal antibodies directed against Eimeria tenella sporozoites to determine stage specificity and in vitro effect on parasite penetration and development

Three different hybridoma-produced monoclonal antibodies (Ab) were used to study their reactivities with in vitro developmental stages of Eimeria tenella and their effects on sporozoite penetration and intracellular development. One Ab (designated B10) was stage-specific, whereas the other 2 Ab (designated C3 and E5) reacted with various intracellular developmental stages of the coccidia. The E5 Ab interacted with the cytoplasm of cultured cells that were infected with sporozoites at 24 hours after inoculation. All 3 Ab inhibited penetration to various degrees--the one designated B10 having the greatest inhibitory effect. These 3 Ab also inhibited development of the parasite in cell culture, provided that Ab was continuously present in the cell culture medium. Removal of Ab from the medium allowed coccidial development to continue at about the same rate as in controls. A longer pretreatment time of the sporozoites with the Ab before cell inoculation increased the inhibitory effect with respect to both the penetration and the development of the parasite.     

Experimental Cryptosporidium baileyi infections in chickens and turkeys produced by ocular inoculation of oocysts

Developmental stages of Cryptosporidium baileyi were observed on the conjunctival epithelium of 3 of 14 chicks and none of 7 turkeys following ocular inoculation of oocysts. No clinical signs of disease were observed in chicks or turkeys. All 14 chicks had developmental stages of C. baileyi in the cloaca, whereas 4 of the 7 turkeys were infected at this site.     

A pathogenesis study of foot-and-mouth disease in cattle, using in situ hybridization.

Eight calves were exposed in an aerosol chamber to nebulized foot-and-mouth disease virus. Two control animals were exposed in a similar manner to cell culture media only. Animals were euthanized at intervals and various tissues examined by in situ hybridization using a biotinylated RNA probe corresponding to a portion of the viral gene coding for the polymerase enzyme. By this technique large amounts of viral nucleic acid were found in coronary band, interdigital cleft and tongue as early as six hours postexposure, indicating a very rapid delivery from the portal of entry to the predilection sites for lesion development. This occurred well before the onset of viremia which by virus isolation was not detectable until 30 hours postexposure. The in situ hybridization signal in these tissues decreased in intensity and extent with time to focally positive areas, occasionally surrounding a vesicle. Other epidermal sites not normally thought of as sites for foot-and-mouth lesion development, such as carpus and eyelid, also had some viral nucleic acid detectable at various time intervals. In the lung by in situ hybridization, alveolar septa had viral nucleic acid early in infection (6-18 h postexposure) while later (36-96 h postexposure), the in situ hybridization signal was prominent in alveolar macrophages.     

Experimental infection with BCG as a model of tuberculosis in the brushtail possum (Trichosurus vulpecula)

AIMS: To study the development and progression of lesions produced following experimental inoculations of possums with Bacille Calmette-Guérin (BCG) Pasteur Strain 1173P2 and to compare these with lesions that occurred following natural Mycobacterium bovis infection. METHODS: Possums were inoculated with 5 x 106 colony forming units (cfu) of BCG via the intra-dermal (I/D) route into the dorsum of the neck (n=38) or the left brachium (n=7),orally (n=10), via the endobronchial (E/B) route (n=12), or intravenously (I/V) (n=10, half of which received 5 x 106 cfu and half of which received 5 x 107 cfu of BCG). The possums were humanely killed between 1-4 weeks post inoculation (p.i.), and the nature and distribution of lesions examined grossly and histopathologically. RESULTS: The distribution of lesions following I/D inoculation via either route was similar to that of the natural disease, but there were few lesions in the lung. Endobronchial inoculation resulted in pulmonary disease but produced few lesions outside the respiratory tract. Lesions produced by I/V inoculation were similar in distribution to those seen in terminally ill tuberculous possums. No lesions were produced following oral inoculation. Regression of lesions commenced after 3 weeks p.i. CONCLUSIONS: Although the phenomenon of lesion resolution restricts the use of BCG to the study of early lesion development, it avoids the overwhelming disease induced using M. bovis and thus allows the early phases of the development and progression of tuberculosis in this species to be observed. Intradermal inoculation produced evidence that infection through the skin is associated with lesions in superficial lymph nodes, whereas pulmonary disease was associated with E/B inoculation. The results are consistent with the hypothesis that both percutaneous and respiratory routes are important in natural infection of possums with M. bovis.