Spatial-temporal risk factors in the occurrence of rabies in Mexico.

Rabies is a zoonotic disease that affects livestock worldwide. The distribution of rabies is highly correlated with the distribution of the vampire bat Desmodus rotundus, the main vector of the disease. In this study, climatic, topographic, livestock population, vampire distribution and urban and rural zones were used to estimate the risk for presentation of cases of rabies in Mexico by co- Kriging interpolation. The highest risk for the presentation of cases is in the endemic areas of the disease, i.e. the States of Yucatán, Chiapas, Campeche, Quintana Roo, Tabasco, Veracruz, San Luis Potosí, Nayarit and Baja California Sur. A transition zone for cases was identified across northern Mexico, involving the States of Sonora, Sinaloa, Chihuahua, and Durango. The variables topography, vampire distribution, bovine population and rural zones are the most important to explain the risk of cases in livestock. This study provides robust estimates of risk and spread of rabies based on geostatistical methods. The information presented should be useful for authorities responsible of public and animal health when they plan and establish strategies preventing the spread of rabies into rabies-free regions of México.

Growth Performance and Fecal Microbiota of Dairy Calves Supplemented with Autochthonous Lactic Acid Bacteria as Probiotics in Mexican Western Family Dairy Farming.

Probiotic supplementation in dairy cattle has achieved several beneficial effects (improved growth rate, immune response, and adequate ruminal microbiota). This study assessed the effects on the growth parameters and gut microbiota of newborn dairy calves supplemented with two Lactobacillus-based probiotics, individually (6BZ or 6BY) or their combination (6BZ + 6BY), administrated with the same concentration (1 × 109 CFU/kg weight) at three times, between days 5 and 19 after birth. The control group consisted of probiotic-unsupplemented calves. Growth parameters were recorded weekly until eight weeks and at the calves' ages of three, four, and five months. Fecal microbiota was described by high-throughput sequencing and bioinformatics. Although no significant effects were observed regarding daily weight and height gain among probiotic-supplemented and non-supplemented calves, correlation analysis showed that growth rate was maintained until month 5 through probiotic supplementation, mainly when the two-strain probiotics were supplied. Modulation effects on microbiota were observed in probiotic-supplemented calves, improving the Bacteroidota: Firmicutes and the Proteobacteria ratios. Functional prediction by PICRUSt also showed an increment in several pathways when the two-strain probiotic was supplemented. Therefore, using the three-administration scheme, the two-strain probiotic improved the growth rate and gut microbiota profile in newborn dairy calves. However, positive effects could be reached by applying more administrations of the probiotic during the first 20 days of a calf's life.

Presence of co-infection between bovine leukemia virus and bovine herpesvirus 1 in herds vaccinated against bovine respiratory complex.

The aim of this study was molecular identification of bovine leukemia virus and possible co-infection with bovine respiratory disease complex (BRDC) viral agents in Mexican dairy herds. We collected 533 blood samples from cattle vaccinated against the BRDC virus in 9 states across Mexico. Peripheral blood leukocytes were removed and genetic material was extracted to detect bovine leukemia virus (BLV), bovine herpesvirus 1 (BoHV-1), bovine viral diarrhea virus (BVDV), bovine parainfluenza virus 3 (BPIV-3), and bovine respiratory syncytial virus (BRSV) infection using polymerase chain reaction. We identified high BLV infection rates in 270 cattle (50.65%). One hundred and thirty-three cows (24.95%) tested positive for BoHV-1, of which 65 samples were positive for both viruses (BoHV-1 and BLV) and 68 were only positive for BoHV-1. Only 4 samples tested positive for BPIV-3 and no sample was positive for BVDV or BRSV. Relative risk and odds ratio analyses did not identify that the presence of BLV infection favors BoHV-1 co-infection in vaccinated herds.

Le but de cette étude était l'identification moléculaire du virus de la leucémie bovine et une éventuelle co-infection par des agents viraux du complexe des maladies respiratoires bovines (BRDC) dans des troupeaux laitiers mexicains. Nous avons recueilli 533 échantillons de sang de bovins vaccinés contre le virus BRDC dans neuf états du Mexique. Les leucocytes du sang périphérique ont été prélevés et le matériel génétique a été extrait pour détecter le virus de la leucémie bovine (BLV), le virus de l'herpès bovin 1 (BoHV-1), le virus de la diarrhée virale bovine (BVDV), le virus parainfluenza bovin 3 (BPIV-3), et le virus respiratoire syncytial bovin (BRSV) par réaction d'amplification en chaîne par la polymérase. Nous avons identifié des taux élevés d'infection par le BLV chez 270 bovins (50,65 %). Cent trente-trois bovins (24,95 %) ont été testés positifs pour le BoHV-1, desquels 65 échantillons étaient positifs pour les deux virus (BoHV-1 et BLV) et 68 étaient uniquement positifs pour le BoHV-1. Seuls quatre échantillons ont été testés positifs pour le BPIV-3 et aucun échantillon n'a été positif pour le BVDV ou le BRSV. Les analyses du risque relatif et des rapports de cotes n'ont pas identifié que la présence d'une infection par le BLV favorise la co-infection par le BoHV-1 dans les troupeaux vaccinés.(Traduit par les auteurs).

Study of the Genetic Expression of Antiretroviral Restriction Factors and Acute Phase Proteins in Cattle Infected with Bovine Leukemia Virus.

The goal of this study was to analyze the genetic expression of antiretroviral restriction factors (ARF) and acute phase proteins (APP), as well as their correlation with proviral and viral loads in cattle with aleukemic (AL) and persistent lymphocytosis (PL). Complete blood samples were collected from a herd of dairy cows, and we extracted genetic material from peripheral blood leukocytes. Absolute quantification of the expression of ARF (APOBEC-Z1, Z2, and Z3; HEXIM-1, HEXIM-2, and BST2) and APP (haptoglobin (HP), and serum amyloid A (SAA)) was performed by qPCR. Statistical significance was observed in the expression of APOBEC-Z3 in BLV-infected animals. We only found positive correlations with a strong expression of the ARF genes in the AL group. The participation of APOBEC (Z1 and Z3), HEXIM-1, and HEXIM-2 was more frequently identified in BLV-infected animals. HEXIM-2 showed active gene expression in the AL group. Although the expression of ARF in early stages of infection (AL) maintains an important participation, in late stages (PL) it seems to have little relevance.

Quantitation of Rabies Virus in Various Bovine Brain Structures.

Bovine paralytic rabies (BPR) is a form of viral encephalitis that is of substantial economic importance throughout Latin America, where it poses a major zoonotic risk. Here, our objective was to utilize a laboratory protocol to determine the relative copy number of the rabies virus (RABV) genome in different bovine brain anatomical structures using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). qRT-PCR quantifies the specific number of gene copies present in a sample based on fluorescence emitted after amplification that is directly proportional to the amount of target nucleic acid present in the sample. This method is advantageous owing to its short duration, reduced risk of contamination, and potential to detect viral nucleic acids in different samples more easily compared to other techniques. The brains of six rabid animals were divided into six anatomical structures, namely the Ammon's horn, cerebellum, cortex, medulla, pons, and thalamus. All brains were identified as positive for RABV antigens based on a direct immunofluorescence test. The same anatomical structures from the brains of four RABV-negative bovines were also assessed. RNA was extracted from each structure and used for qRT-PCR. An assay was performed to determine the copy numbers of RABV genes using an in vitro transcribed nucleoprotein gene. The standard curve used to quantify viral RNA exhibited an efficiency of 100% and linearity of 0.99. Analysis revealed that the cortex, medulla, and thalamus were the ideal CNS portions for use in RABV detection, based on the observation that these structures possessed the highest levels of RABV. The test specificity was 100%. All samples were positive, no false positives were detected. This method can be used to detect RABV in samples that contain low levels of RABV during diagnosis of BPR.

Swine virome on rural backyard farms in Mexico: communities with different abundances of animal viruses and phages.

Domestic swine have been introduced by humans into a wide diversity of environments and have been bred in different production systems. This has resulted in an increased risk for the occurrence and spread of diseases. Although viromes of swine in intensive farms have been described, little is known about the virus communities in backyard production systems around the world. The aim of this study was to describe the viral diversity of 23 healthy domestic swine maintained in rural backyards in Morelos, Mexico, through collection and analysis of nasal and rectal samples. Next-generation sequencing was used to identify viruses that are present in swine. Through homology search and bioinformatic analysis of reads and their assemblies, we found that rural backyard swine have a high degree of viral diversity, different from those reported in intensive production systems or under experimental conditions. There was a higher frequency of bacteriophages and lower diversity of animal viruses than reported previously. In addition, sapoviruses, bocaparvoviruses, and mamastroviruses that had not been reported previously in our country were identified. These findings were correlated with the health status of animals, their social interactions, and the breeding/rearing environment (which differed from intensive systems), providing baseline information about viral communities in backyard swine.

Virulence of infectious pancreatic necrosis virus (IPNV) isolates from Mexico.

Infectious pancreatic necrosis virus (IPNV) causes economic losses in Mexican rainbow trout industry. In this study, virulence and genetic fingerprints of Mexican IPNV isolates was investigated for the first time. Two Mexican IPNV isolates were analyzed in rainbow trout fry and the Sp strain was included as high virulence. One of the Mexican IPNV isolate was obtained from diseased fish and the other from fish without clinical signs. The infection was performed using a standardized immersion. Clinical signs were observed at 4 days post infection in fry group infected with strain Sp, two days earlier than in trout infected with IPNV isolates Mexican. Severe lesions were found in 100% of the individuals of Sp group, but only in 25% of each isolated Mexican group. Results suggest that Mexican IPNV isolates are pathogenic, but less virulent than strain Sp. The amino acid motif residues of both Mexican isolates, corresponded to a subclinical disease. Nevertheless, the accumulated motility observed in the field, suggest that other factors play a role in the virulence of the disease.

Hologenomic adaptations underlying the evolution of sanguivory in the common vampire bat.

Adaptation to specialized diets often requires modifications at both genomic and microbiome levels. We applied a hologenomic approach to the common vampire bat (Desmodus rotundus), one of the only three obligate blood-feeding (sanguivorous) mammals, to study the evolution of its complex dietary adaptation. Specifically, we assembled its high-quality reference genome (scaffold N50 = 26.9 Mb, contig N50 = 36.6 kb) and gut metagenome, and compared them against those of insectivorous, frugivorous and carnivorous bats. Our analyses showed a particular common vampire bat genomic landscape regarding integrated viral elements, a dietary and phylogenetic influence on gut microbiome taxonomic and functional profiles, and that both genetic elements harbour key traits related to the nutritional (for example, vitamin and lipid shortage) and non-nutritional (for example, nitrogen waste and osmotic homeostasis) challenges of sanguivory. These findings highlight the value of a holistic study of both the host and its microbiota when attempting to decipher adaptations underlying radical dietary lifestyles.

Viral Communities Among Sympatric Vampire Bats and Cattle.

Vampire bats are the only mammals known to feed exclusively on blood from other animals, often from domestic cattle. We tested the hypothesis that the adaptation of vampire bats to hematophagy would have resulted in shared viral communities among vampire bats and cattle, as a direct result of historic spillover events occurring due to hematophagy. We analyzed the presence of different viruses in sample populations of sympatric bat and prey populations and searched for shared viruses between taxa. A limited number of DNA viral groups were detected within each species. However, there was no evidence for a shared viral community among the vampire bat and cattle populations tested.

Bats, Primates, and the Evolutionary Origins and Diversification of Mammalian Gammaherpesviruses.

Gammaherpesviruses (γHVs) are generally considered host specific and to have codiverged with their hosts over millions of years. This tenet is challenged here by broad-scale phylogenetic analysis of two viral genes using the largest sample of mammalian γHVs to date, integrating for the first time bat γHV sequences available from public repositories and newly generated viral sequences from two vampire bat species (Desmodus rotundus and Diphylla ecaudata). Bat and primate viruses frequently represented deep branches within the supported phylogenies and clustered among viruses from distantly related mammalian taxa. Following evolutionary scenario testing, we determined the number of host-switching and cospeciation events. Cross-species transmissions have occurred much more frequently than previously estimated, and most of the transmissions were attributable to bats and primates. We conclude that the evolution of the Gammaherpesvirinae subfamily has been driven by both cross-species transmissions and subsequent cospeciation within specific viral lineages and that the bat and primate orders may have potentially acted as superspreaders to other mammalian taxa throughout evolutionary history. IMPORTANCE: It has long been believed that herpesviruses have coevolved with their hosts and are species specific. Nevertheless, a global evolutionary analysis of bat viruses in the context of other mammalian viruses, which could put this widely accepted view to the test, had not been undertaken until now. We present two main findings that may challenge the current view of γHV evolution: multiple host-switching events were observed at a higher rate than previously appreciated, and bats and primates harbor a large diversity of γHVs which may have led to increased cross-species transmissions from these taxa to other mammals.

Detection and phylogenetic analysis of bovine papillomavirus in cutaneous warts in cattle in Tamaulipas, Mexico.

Papillomas occur more frequently in cattle than other domestic animals. The causal agent of bovine papillomatosis is a virus that belongs to the family Papillomaviridae. In Tamaulipas, Mexico, the virus is considered a serious problem and has impeded the export of cattle to the United States, resulting in serious economic losses. Owing to the lack of information regarding the subtypes of papillomaviruses that infect cattle in Mexico, the aim of this study was to determine the subtypes in Tamaulipas. Fifty-two warts were analyzed with the use of polymerase chain reaction (PCR) involving primers that amplify the E7 gene of bovine papillomavirus (BPV). The PCR products were sequenced to differentiate the BPV-1 and BPV-2 subtypes. The sequencing quality was determined with the use of MEGA 6.0 software. Comparison of the Tamaulipas sequences with those of known BPV types by means of the MUSCLE algorithm showed that 53% of the former were BPV-1 and 47% were BPV-2. The distribution of the 2 subtypes in the cattle was homogeneous. This study demonstrated the presence of BPV-1 and BPV-2 in cattle from Tamaulipas and constitutes the first molecular characterization of papillomas in Mexico.

Les papillomes sont rencontrés plus fréquemment chez les bovins que chez n'importe quelle autre espèce domestiques. L'agent causal de la papillomatose bovine est un virus appartenant à la famille Papillomaviridae. Dans l'état mexicain de Tamaulipas le virus est considéré comme un problème sérieux et a empêché l'exportation de bovin vers les États-Unis d'Amérique, causant ainsi des pertes économiques importantes. Étant donné le manque d'information concernant les sous-types de papillomavirus qui infectent les bovins au Mexique, l'objectif de l'étude était de déterminer les sous-types présents dans l'état de Tamaulipas. Cinquante-deux verrues ont été analysées par réaction d'amplification en chaine par la polymérase (ACP) à l'aide d'amorces amplifiant le gène E7 du papillomavirus bovin (PVB). Les produits de l'ACP ont été séquencés afin de différencier les sous-types PVB-1 et PVB-2. La qualité du séquençage fut déterminée à l'aide du logiciel MEGA 6.0. La comparaison des séquences obtenues pour l'état de Tamaulipas avec celles des types connus de PVB par l'algorithme MUSCLE a permis de démontrer que 53 % étaient des PVB-1 et 47 % de PVB-2. La distribution des deux sous-types chez les bovins était homogène. La présente étude démontre la présence de PVB-1 et PVB-2 chez les bovins de Tamaulipas et constitue le premier rapport sur la caractérisation moléculaire des papillomes au Mexique.(Traduit par Docteur Serge Messier).

The evolution of bat nucleic acid-sensing Toll-like receptors.

We characterized the nucleic acid-sensing Toll-like receptors (TLR) of a New World bat species, the common vampire bat (Desmodus rotundus), and through a comparative molecular evolutionary approach searched for general adaptation patterns among the nucleic acid-sensing TLRs of eight different bats species belonging to three families (Pteropodidae, Vespertilionidae and Phyllostomidae). We found that the bat TLRs are evolving slowly and mostly under purifying selection and that the divergence pattern of such receptors is overall congruent with the species tree, consistent with the evolution of many other mammalian nuclear genes. However, the chiropteran TLRs exhibited unique mutations fixed in ligand-binding sites, some of which involved nonconservative amino acid changes and/or targets of positive selection. Such changes could potentially modify protein function and ligand-binding properties, as some changes were predicted to alter nucleic acid binding motifs in TLR 9. Moreover, evidence for episodic diversifying selection acting specifically upon the bat lineage and sublineages was detected. Thus, the long-term adaptation of chiropterans to a wide variety of environments and ecological niches with different pathogen profiles is likely to have shaped the evolution of the bat TLRs in an order-specific manner. The observed evolutionary patterns provide evidence for potential functional differences between bat and other mammalian TLRs in terms of resistance to specific pathogens or recognition of nucleic acids in general.

A novel endogenous betaretrovirus in the common vampire bat (Desmodus rotundus) suggests multiple independent infection and cross-species transmission events.

The Desmodus rotundus endogenous betaretrovirus (DrERV) is fixed in the vampire bat D. rotundus population and in other phyllostomid bats but is not present in all species from this family. DrERV is not phylogenetically related to Old World bat betaretroviruses but to betaretroviruses from rodents and New World primates, suggesting recent cross-species transmission. A recent integration age estimation of the provirus in some taxa indicates that an exogenous counterpart might have been in recent circulation.

Molecular characterization of the VP2 gene of infectious pancreatic necrosis virus (IPNV) isolates from Mexico.

Infectious pancreatic necrosis virus (IPNV) is one of the most important viruses in the Pacific salmon Oncorhynchus spp., Atlantic Salmon Salmo salar, and Rainbow Trout O. mykiss industry. This virus has been shown to produce high mortality among salmonid fry and juveniles, and survivors might become carriers. Since 2000, IPNV has affected Mexican Rainbow Trout culture, resulting in considerable economic losses. In the current study, molecular characterization of the VP2 gene of a number of Mexican IPNV isolates was done and the virus's phylogenetic relationships to IPNV reference strains were investigated. The phylogenetic analysis indicated that Mexican IPNV isolates are closely related to strains from the United States and Canada and that all Mexican IPNV isolates belong to genogroup 1. Furthermore, low genetic diversity was found between the Mexican isolates (identity, 95.8-99.8% nucleotides and 95.8-99.6% amino acids). The result of the analysis of the amino acid residues found at positions 217, 221, and 247 (alanine, threonine, and glutamic acid, respectively) could be associated with virulence, although the expression of virulence factors is more complex and may be influenced by the agent and host factors. The high percentage of identity among the VP2 genes from geographically distant IPNV isolates and the evidence of wide distribution in the country might have been facilitated by carrier trout. This hypothesis is supported by the identification of the amino acid threonine at position 221 in all Mexican isolates, a factor related to the carrier state for IPNV, as reported by other studies.

Viral vaccines for bony fish: past, present and future.

Since 1970, aquaculture production has grown. In 2010, it had an annual average rate of 6.3% with 59.9 million tons of product and soon could exceed capture fisheries as a source of fishery products. However, the occurrence of viral diseases continues to be a significant limiting factor and its control is important for the development of this sector. In aquaculture farms, fish are reared under intensive culture conditions, and the use of viral vaccines has enabled an increase in production. Several types of vaccines and strategies of vaccination have been developed; however, this approach has not reached the expected goals in the most susceptible stage (fingerlings). Currently, there are inactivated and recombinant commercial vaccines, mainly for salmonids and cyprinids. In addition, updated genomic and proteomic technology has expedited the research and expansion of new vaccine models, such as those comprised of subunits or DNA. The objective of this review is to cover the various types of viral vaccines that have been developed and are available for bony fishes, as well as the advantages and challenges that DNA vaccines present for massive administration in a growing aquaculture, possible risks for the environment, the controversy regarding genetically modified organisms and possible acceptance by consumers.

Induction of a protective immune response to rabies virus in sheep after oral immunization with transgenic maize, expressing the rabies virus glycoprotein.

The introduction of exogenous genes into plants permits the development of a new generation of biological products, i.e., edible vaccines. Cereals, especially maize, have been the systems of choice for the expression of antigenic proteins because the proteins can be expressed at high levels in the kernel and stored for prolonged periods without excessive deterioration. The utilization of plant-derived antigens for oral delivery provides an alternative strategy for the control of pathogens in animals compared to the current vaccine administration methods, such as injection. However, there is some doubt about the efficacy of these types of vaccines in polygastric animals due to the features of their digestive system. Here, we report the efficacy of an edible vaccine against rabies evaluated in sheep. Kernels containing different doses of G protein (0.5, 1, 1.5 and 2mg) were given in a single dose by the oral route. Cumulative survival was better in groups that received 2mg of G protein and for the positive control (inactivated rabies vaccine); this observation was supported by the presence of neutralizing antibodies. Animals in the control group died after challenge. The degree of protection achieved for 2mg of G protein was comparable to that conferred by a commercial vaccine. In conclusion, this is the first study in which an orally administered edible vaccine showed efficacy in a polygastric model.

Vaccine production in plant systems--an aid to the control of viral diseases in domestic animals: a review.

Plants have been identified as promising expression systems for the commercial production of vaccines because of the possibility of introducing exogenous genes into them, which permits the development of a new generation of biological products called edible vaccines. The advantages of oral vaccines of this new type are that they induce mucosal, humoral, cellular and protective immunity, they are cheaper, easier to store, distribute and administer, they do not require cold chain management, and some species can be stored for long periods of time without any spoilage and may be administered as purified proteins. Owing to these benefits, plant-produced vaccines represent a valuable option for animal health. The aim of this paper is to present a review of plant-produced vaccines against viruses affecting domestic animals. Some aspects of the feasibility of their use and the immune response elicited by such vaccines are also discussed, as the balance between tolerance and immunogenicity is a major concern for the use of plant-based vaccines.

Expression of rabies virus G protein in carrots (Daucus carota).

Antigens derived from various pathogens can readily be synthesized at high levels in plants in their authentic forms. Such antigens administered orally can induce an immune response and, in some cases, result in protection against a subsequent challenge. We here report the expression of rabies virus G protein into carrots. The G gene was subcloned into the pUCpSSrabG vector and then used to transform carrot embryogenic cells by particle bombardment. The carrot cells were selected in liquid medium, a method previously unreported. The presence of the transgene was verified by PCR, and by RT-PCR. By western blot, G protein transgene was identified in 93.3% of adult carrot roots. The G protein was quantified by densitometric analysis (range 0.4-1.2%). The expressed protein was antigenic in mice. This confirms that the carrot is an adequate system for antigen expression.

Expression of the rabies virus nucleoprotein in plants at high-levels and evaluation of immune responses in mice.

Transgenic plants have been employed successfully as a low-cost system for the production of therapeutically valuable proteins including antibodies, antigens and hormones. Here, we report expression of a full-length nucleoprotein gene of rabies virus in transgenic tomato plants. The nucleoprotein was also transiently expressed in Nicotiana benthamiana plants by agroinfiltration. In both cases, the nucleoprotein was expressed at high levels, 1-5% of total soluble protein in tomato and 45% in N. benthamiana. Previously, only epitopes of the nucleoprotein had been expressed in plants. The presence and expression of the transgene was verified by PCR, Southern, northern and western blots. Mice were immunized both intraperitoneally (i.p.) and orally with tomato protein extracts containing the N protein induced the production of antibodies. The antibody titer of mice immunized i.p., was at least four times higher than that of mice immunized orally. These results were reflected in the challenge experiments where i.p.-immunized mice were partially protected against a peripheral virus challenge whereas orally immunized mice were not. This protection was comparable to that obtained in previous experiments employing different expression systems. Work is in progress to express both G and N proteins in transgenic plants and evaluate protection in mice.