Innate and adaptive immune response of Rainbow trout (Oncorhynchus mykiss) naturally infected with Yersinia ruckeri.

Rainbow trout is an important fish species for Peruvian artisanal aquaculture, comprising over 60 % of the total aquaculture production. However, their industry has been highly affected by several bacterial agents such as Yersinia ruckeri. This pathogen is the causative agent of Enteric Redmouth Disease, and causes high mortality in fingerlings and chronic infection in adult rainbow trout. To date, the immune response of rainbow trout against Y. ruckeri has been well studied in laboratory-controlled infection studies (i.e. intraperitoneal infection, bath immersion), however, the immune response during natural infection has not been explored. To address this, in this study, 35 clinically healthy O. mykiss without evidence of lesions or changes in behavior and 32 rainbow trout naturally infected by Y. ruckeri, were collected from semi-intensive fish farms located in the Central Highlands of Peru. To evaluate the effect on the immune response, RT-qPCR, western blotting, and ELISA were conducted using head kidney, spleen, and skin tissues to evaluate the relative gene expression and protein levels. Our results show a significant increase in the expression of the pro-inflammatory cytokines il1b, tnfa, and il6, as well as ifng in all three tissues, as well as increases in IL-1ß and IFN-γ protein levels. The endogenous pathway of antigen presentation showed to play a key role in defense against Y. ruckeri, due to the upregulation of mhc-I, tapasin, and b2m transcripts, and the significant increase of Tapasin protein levels in infected rainbow trout. None of the genes associated with the exogenous pathway of antigen presentation showed a significant increase in infected fish, suggesting that this pathway is not involved in the response against this intracellular pathogen. Finally, the transcripts of immunoglobulins IgM and IgT did not show a modulation, nor were the protein levels evaluated in this study.

Local regulation of immune genes in rainbow trout (Oncorhynchus mykiss) naturally infected with Flavobacterium psychrophilum.

Flavobacterium psychrophilum is the etiological agent of bacterial cold water disease (BCWD), also referred to as rainbow trout fry syndrome (RTFS), a disease with great economic impact in salmonid aquaculture. Despite this, to date, not many studies have analyzed in depth how the immune system is regulated during the course of the disease. In the current study, we have studied the transcription of several immune genes related to T and B cell activity in the skin of rainbow trout (Oncorhynchus mykiss) naturally infected with F. psychrophilum in a farm located in Lake Titicaca (Peru). The levels of expression of these genes were tested and compared to those obtained in asymptomatic and apparently healthy rainbow trout. In the case of symptomatic fish, skin samples containing characteristic ulcerative lesions were taken, as well as skin samples with no lesions. Our results pointed to a significant local up-regulation of IgD, CD4, CD8, perforin and IFNγ within the ulcerative lesions. On the other hand, no differences between the levels of expression of these genes were visible in the spleen. To confirm these results, the distribution of IgD+ and CD3+ cells was studied through immunohistochemical techniques in the ulcerative lesions. Our results demonstrate a strong local response to F. psychrophilum in rainbow trout in which IgD and T cells seem to play a major role.

Detection of toxigenic Clostridium perfringens and Clostridium botulinum from food sold in Lagos, Nigeria.

Food-borne diseases contribute to the huge burden of sickness and death globally and in the last decade, have become more frequently reported in Africa. In line with this, food safety is becoming a significant and growing public health problem in Nigeria. Diarrhoea is a common problem in Nigeria and has been reported but there has been little data on the possibility of clostridia as aetiological agents. Clostridium species are ubiquitous in the environment and in the gastrointestinal tract of man and animals and can serve as a marker for faecal contamination. We set out to determine the potential of these foods to transmit Clostridium species. A total of 220 food commodities from six local governments in Lagos State were sampled. Isolates obtained were identified based on cultural, morphological and biochemical characteristics. Toxinotyping was done using multiplex-PCR with primers specific for alpha, beta, epsilon and iota-toxin genes, enterotoxigenic cpe gene and neurotoxigenic BoNt gene. Fifty (22.7%) clostridial species were isolated of which 29 (58%) were identified as C. perfringens. Toxinotyping of the 29 strains showed that 28 (96.6%) were toxin producing C. perfringens type A while one (3.4%) was C. perfringens type D. Two (4%) C. botulinum species were isolated and identified by 16S rRNA sequencing, both harbouring BoNt/A gene. The contamination rates of food with Clostridium species show that food hygiene is a problem and Clostridium species may be a source of food borne disease in Lagos State, Nigeria.

Sialidase production and genetic diversity in Clostridium perfringens type A isolated from chicken with necrotic enteritis in Brazil.

The sialidase activity and genetic diversity of 22 Clostridium perfringens strains isolated from chickens with necrotic enteritis were determined. Sialidase activity was detected in 86.4 % of the strains. All C. perfringens showed a high value of similarity (>96 %), and they were grouped into seven clusters clearly separated from the other reference bacterial strains. From these clusters four patterns were defined in accordance with their phenotypic (sialidase production and antibiotic resistance profile) and genotypic (presence of nanI and nanJ genes) characteristics. Our results showed heterogeneity among strains, but they were genotypically similar, and it is suggested further studies are needed to better understand the pathogenesis of necrotic enteritis.

Co-Circulation of Multiple Coronavirus Genera and Subgenera during an Epizootic of Lethal Respiratory Disease in Newborn Alpacas (Vicugna pacos) in Peru: First Report of Bat-like Coronaviruses in Alpacas.

Coronaviruses (CoVs) infect a wide range of hosts, including humans, domestic animals, and wildlife, typically causing mild-to-severe respiratory or enteric disease. The main objective of this study was to identify CoV genera and subgenera detected in Peruvian alpacas. Lung lavage specimens were collected from 32 animals aged 1 to 6 weeks. CoVs were identified by using RT-PCR to amplify a pan-CoV conserved region of the RNA-dependent RNA polymerase-encoding gene. A nested PCR was performed to identify ß-CoVs. Then, ß-CoV-positive samples were subjected to RT-PCR using specific primers to identify the Embecovirus subgenus. Out of 32 analyzed samples, 30 (93.8%) tested positive for at least one CoV genus. ß-, α-, or unclassified CoVs were identified in 24 (80%), 1 (3.3%), and 1 (3.3%) of the positive samples, respectively. A CoV genus could not be identified in two (6.7%) samples. A mixture of different CoV genera was detected in two (6.7%) samples: one was co-infected with ß- and α-CoVs, and the other contained a ß- and an unclassified CoV. A sequence analysis of the amplicons generated by the PCR identified 17 ß-CoV strains belonging to the subgenus Embecovirus and two α-CoV strains belonging to Decacovirus. A phylogenetic analysis of two strains revealed a relationship with an unclassified Megaderma BatCoV strain. A subgenus could not be identified in nine ß-CoV samples. Our data show a high prevalence and a high genetic diversity of CoV genera and subgenera that infect alpacas, in which the ß-CoV subgenus Embecovirus predominated. Our data also suggest a new role for bats in the dissemination and transmission of uncommon CoVs to alpacas raised in rural Peru.

Enteric Viral Infections among Domesticated South American Camelids: First Detection of Mammalian Orthoreovirus in Camelids.

Enteric infections are a major cause of neonatal death in South American camelids (SACs). The aim of this study was to determine the prevalence of enteric viral pathogens among alpacas and llamas in Canchis, Cuzco, located in the southern Peruvian highland. Fecal samples were obtained from 80 neonatal alpacas and llamas and tested for coronavirus (CoV), mammalian orthoreovirus (MRV), and rotavirus A (RVA) by RT-PCR. Of the 80 fecal samples analyzed, 76 (95%) were positive for at least one of the viruses tested. Overall, the frequencies of positive samples were 94.1% and 100% among alpacas and llamas, respectively. Of the positive samples, 33 (43.4%) were monoinfected, while 43 (56.6%) had coinfections with two (83.7%) or three (16.3%) viruses. CoV was the most commonly detected virus (87.5%) followed by MRV (50%). RVA was detected only in coinfections. To our knowledge, this is the first description of MRV circulation in SACs or camelids anywhere. These data show that multiple viruses circulate widely among young alpaca and llama crias within the studied areas. These infections can potentially reduce livestock productivity, which translates into serious economic losses for rural communities, directly impacting their livelihoods.

Draft Genome Sequence of Yersinia ruckeri Strain FMV-22, Isolated from Diseased Rainbow Trout (Oncorhynchus mykiss) in Peru.

Yersinia ruckeri is a significant and frequent bacterial fish pathogen in Peru. We report the draft genome sequence of strain FMV-22, isolated from a diseased rainbow trout, which consists of 3.84 Mb, with a G+C content of 47.45% and 3,765 protein-coding genes.

Draft Genome Sequence of Aeromonas sobria Strain CHT-30, Isolated from a Diseased Rainbow Trout (Oncorhynchus mykiss) in Peru.

The Gram-negative bacterium Aeromonas sobria is an opportunistic pathogen that affects humans and animals, including fish. Here, we report the draft genome of strain CHT-30, which was isolated from a diseased rainbow trout in Peru. The genome size is 4.91 Mb, with a G+C content of 57.7%, and the genome includes 4,820 coding sequences.

Adhesion and invasion of Clostridium perfringens type A into epithelial cells.

Clostridium perfringens is the causative agent for necrotic enteritis. It secretes the major virulence factors, and α- and NetB-toxins that are responsible for intestinal lesions. The TpeL toxin affects cell morphology by producing myonecrosis, but its role in the pathogenesis of necrotic enteritis is unclear. In this study, the presence of netB and tpeL genes in C. perfringens type A strains isolated from chickens with necrotic enteritis, their cytotoxic effects and role in adhesion and invasion of epithelial cells were evaluated. Six (27.3%) of the 22 C. perfringens type A strains were harboring the tpeL gene and produced morphological alterations in Vero cells after 6h of incubation. Strains tpeL (-) induced strong cell rounding after 6h of incubation and produced cell enlargement. None of the 22 strains harbored netB gene. All the six tpeL (+) gene strains were able to adhere to HEp-2 cells; however, only four of them (66.6%) were invasive. Thus, these results suggest that the presence of tpeL gene or TpeL toxin might be required for the adherence of bacteria to HEp-2 cells; however, it could not have any role in the invasion process.

Adhesion and invasion of Clostridium perfringens type A into epithelial cells

ABSTRACT Clostridium perfringens is the causative agent for necrotic enteritis. It secretes the major virulence factors, and α- and NetB-toxins that are responsible for intestinal lesions. The TpeL toxin affects cell morphology by producing myonecrosis, but its role in the pathogenesis of necrotic enteritis is unclear. In this study, the presence of netB and tpeL genes in C. perfringens type A strains isolated from chickens with necrotic enteritis, their cytotoxic effects and role in adhesion and invasion of epithelial cells were evaluated. Six (27.3%) of the 22 C. perfringens type A strains were harboring the tpeL gene and produced morphological alterations in Vero cells after 6 h of incubation. Strains tpeL (-) induced strong cell rounding after 6 h of incubation and produced cell enlargement. None of the 22 strains harbored netB gene. All the six tpeL (+) gene strains were able to adhere to HEp-2 cells; however, only four of them (66.6%) were invasive. Thus, these results suggest that the presence of tpeL gene or TpeL toxin might be required for the adherence of bacteria to HEp-2 cells; however, it could not have any role in the invasion process.