Detection of HEV in naturally infected swine from central Argentina by an optimized HEV/MS2 duplex RT‐qPCR

Hepatitis E virus (HEV) is currently considered as a global health concern due to the recognition of its zoonotic transmission to humans, mainly from swine, and its association with the development of severe cases of hepatitis in human risk populations. The lack of updated data on HEV state of infection in swineherds of Argentina, and the necessity of robust technologies for its detection in complex biological samples, positions HEV as an emerging issue in public health. Here, we have optimized a RT‐qPCR with internal control for a more precise and accurate HEV RNA detection in swine stool samples. We implemented this optimized molecular tool to analyse the current epidemiological scenario of HEV infection in swine from the core region of commercial activity of Argentina. A total of 135 stool samples were collected from 16 different farms and tested for HEV presence, resulting in 11 positive cases (8.1%). Phylogenetic analysis demonstrated that all of them correspond to HEV genotype 3 and that different subtypes circulate in the region. Moreover, two of the detected strains presented a high nucleotide similarity with a previously identified isolate from human sewage discharges, suggesting the zoonotic transmission of HEV to humans. Collectively, this work provides a better understanding of HEV epidemiology in Argentina while contributes to the improvement of HEV detection technologies.     

First molecular detection of canine herpesvirus 1 (CaHV-1) in the Eastern Brazilian Amazon

BackgroundCanine herpesvirus type 1 (CaHV-1) infects dogs and is associated with neonatal deaths and reproductive, ocular, neurological, and respiratory problems. In Brazil, reports of CaHV-1 have been restricted to the southeast and south regions, particularly in municipalities in the state of Rio Grande do Sul.ObjectivesTo assess the presence and variability of CaHV-1 in canine populations in the state of Pará, North Brazil.MethodsBiological samples from 159 dogs from 4 municipalities in the State of Pará were evaluated using polymerase chain reaction and phylogenetic analyses, with the target being the viral enzyme, thymidine kinase.ResultsCaHV-1 was detected in 13 dogs (8.2%), with 2 animals being from the municipality of Santa Bárbara do Pará, 8 from Algodoal Island, 2 from Salinópolis, and one from Capanema. The study sequences revealed 100% identity among themselves and 64% to 100% identity with the other nucleotide sequences from Australia, Brazil, United Kingdom, and United States, including 100% identity with the 2002 isolate from Australia. The 1996 isolate from France was grouped in a branch that was different from the sequence of this study.ConclusionsThis study presents the first molecular detection of CaHV-1 in dogs from the Amazon region in northern Brazil. The nucleotide identity between the strains and cytosine insertion in the sequences isolated in this study suggests at least 2 strains of CaHV-1 circulating in Brazil (Pará and BTU-1).     

Co-circulation of two genetically distinct viruses in an outbreak of African swine fever in Mozambique: no evidence for individual co-infection

In 1998, domestic pigs originating from villages within a 40 km radius of Ulongwe in the northern Tete Province of Mozambique were held in a quarantine facility for a 3-month period prior to their importation into South Africa. Eight of a total of 25 pigs died within the first 3 weeks of quarantine of what appeared clinically and on post mortem examination to be African swine fever (ASF). Organs were collected and preserved in formol-glycerosaline and the presence of ASF virus in these specimens was confirmed by three independent polymerase chain reaction (PCR) tests. Two gene regions were characterised, namely the C-terminus end of the major immunodominant protein VP72 and the central variable region (CVR) of the 9RL open reading frame (ORF). Results confirmed the presence of two genetically distinct viruses circulating simultaneously within a single outbreak focus. However, despite the pigs being housed within the same facility, no evidence of co-infection was observed within individual animals. Comparison of the two 1998 virus variants with viruses causing historical outbreaks of the disease in Mozambique revealed that these viruses belong to two distinct genotypes which are unrelated to viruses causing outbreaks between 1960 and 1994. In addition, the CVR and p72 gene regions of one of the 1998 Mozambique virus variants (variant-40) was shown to be identical to the virus recovered from an ASF outbreak in Madagascar in the same year, whilst the other (variant-92) was identical to a 1988 pig isolate from Zambia.     

Survey of Actinobacillus (Haemophilus) pleuropneumoniae infection in swine by different methods

Lung and serum samples from pigs that died or were emergency-slaughtered in a pooled, conventional fattening herd were examined to survey Actinobacillus pleuro-pneumoniae infection and to compare the sensitivity of different testing methods. A total of 110 lungs were used for cultural isolation of the agent and direct immunofluorescence (IF) of impression smears. Boiled lung suspensions were tested by coagglutination (Co-A) and agar gel precipitation (AGP). Eighty-seven sera were tested along with lung samples from the same pigs. The lungs yielded a varied bacterial flora most often containing Pasteurella multocida and less frequently Actinomyces (Corynebacterium) pyogenes, E. coli and Salmonella. A. pleuropneumoniae was isolated from 30 lungs: from 22 lungs it grew out in pure culture, from 7 as mixed culture with P. multocida and from 1 as mixed culture with A. pyogenes. The number of positive samples obtained by the different methods was as follows: coagglutination test (with boiled lung suspensions): 63 (57.3%); immunofluorescence: 43 (39.2%); AGP test (with serum): 31 (35.6%); AFP test (with boiled lung suspension): 25 (22.7%). A total of 23 samples (20.7%) were negative by all serological tests and by cultural isolation. Most samples gave positive results by two or more tests while 26 samples only by one test (most often, on 13 occasions, by the Co-A test). The Co-A test detected antigenic components of serotypes that have not been isolated in Hungary so far. This indicates that it is not enough to test one strain from a given lung sample: several colonies must be cultured and serotyped.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence and molecular characterization of Bartonella spp. and hemoplasmas in neotropical primates from Brazilian Amazon

Little is known about the prevalence and genetic diversity of Bartonella spp. and hemoplasmas in nonhuman primates (NHP). The present study aimed to investigate the occurrence of and assess the phylogenetic position of Bartonella spp. and hemoplasma species infecting neotropical NHP from Brazilian Amazon. From 2009 to 2013, a total of 98 blood samples from NHP belonging to the Family Cebidae were collected in the island of São Luís, state of Maranhão, of which 87 NHP were from Wild Animal Screening Center (CETAS) in the municipality of São Luís, and 11 (9 Sapajus sp. and 2 Saimiri sciureus) were from NHP caught in the Sítio Aguahy Private Reserve. DNA samples were screened by previously described PCR protocols for amplifying Bartonella spp. and Mycoplasma spp. based on nuoG, gltA and 16S rRNA genes, respectively. Purified amplicons were submitted to sequencing and phylogenetic analysis. Bacteremia with one or more Bartonella spp. was not detected in NHP. Conversely, 35 NHP were PCR positive to Mycoplasma spp. The Blastn analysis of seven positive randomly selected sequencing products showed percentage of identity ranging from 95% to 99% with other primates hemoplasmas. The Maximum Likelihood phylogenetic analysis based on a 1510 bp of 16S rRNA gene showed the presence of two distinct clusters, positioned within Mycoplasma haemofelis and Mycoplasma suis groups. The phylogenetic assessment suggests the presence of a novel hemoplasma species in NHP from the Brazilian Amazon.     

Outbreaks of attacks by hematophagous bats in isolated riverine communities in the Brazilian Amazon: a challenge to rabies control

Human rabies has re-emerged in Latin America due to bat associated transmission. We present data related to attacks by hematophagous bats in three riverine communities in the Rio Negro basin, Brazilian Amazon. A cross-sectional survey was carried out to obtain demographic and epidemiological data through interviews with 201 inhabitants. A total of 721 bat attacks with bites took place from 2004 to 2006, 238 (33%) reported by residents in Campinas do Rio Preto, 329 (46%) in águas Vivas and 154 (21%) in the community of Malalahá. Incidence density among surveyed inhabitants was 84 attacks/100 persons-years in Campinas do Rio Preto, 249 attacks/100 persons-years in águas Vivas and 81 attacks/100 persons-years in Malalahá. The proportion of surveyed inhabitants bled by bats at least once was 67% (63/94) in Campinas do Rio Preto, 96% (42/44) in águas Vivas and 62% (39/63) in Malalahá. Among subjects bled by bats, the average number of bites was 4.6 ± 4.2 standard deviations (SD) in Campinas do Rio Preto, 8 ± 6 SD in águas Vivas and 4.1 ± 3.9 SD in Malalahá. Regarding houses, 67% (26/39) had animals in the peridomestic environment (chickens and/or dogs) and all were vulnerable to bats due to gaps in the walls and/or in the windows and doors. In 13 dwellings, rudimentary protection against bats through the fixation of fishing nets and/or straw nets to the windows and other openings was observed. Among dwellers reporting attacks, 48% (68/144) received the full post-exposure anti-rabies vaccination protocol with five doses of diploid human cell vaccine, 28% (39/144) received an incomplete schedule (1-4 doses), and 26% (37/144) did not receive any dose. No cases of rabies were reported during the study; however, regular pre-exposure vaccination in the studied populations must be considered.     

Porcine circovirus 2 infection in swine foetuses inoculated at different stages of gestation

The ability of porcine circovirus 2 (PCV2) to replicate and cause pathologic abnormalities in foetuses at selected time points of gestation was examined in this study. Two foetuses were inoculated in utero in each of two sows at 57, 75 and 92 days of gestation, respectively, with PCV2 (1121). The remaining foetuses were left uninoculated to assess whether intra-uterine spread occurred. Twenty-one days after inoculation, the foetuses were collected and examined for gross lesions and for virus and infected cells in different organs. Serum samples from all foetuses were tested for PCV2 antibodies. Virus replication was detected in all inoculated foetuses. Spread to non-inoculated foetuses did not occur. Virus replication was significantly higher in foetuses inoculated at 57 days compared to that inoculated at 75 and 92 days. The heart contained the highest virus titre and highest number of viral antigen positive cells. Gross lesions were observed only in foetuses inoculated at 57 days of age. PCV2 antibodies were detected only in foetuses inoculated at 75 and 92 days. This study shows the ability of PCV2 to replicate in foetuses at different stages of gestation and to cause pathologic abnormalities in foetuses inoculated at 57 gestational days.     

Characterization of Haemophilus parasuis isolated from Brazilian swine through serotyping, AFLP and PFGE

Haemophilus parasuis infection in pigs is characterized by fibrinous polyserositis, arthritis and meningitis. Despite the fact that traditional diagnosis is based on herd history, clinical signs, bacterial isolation and serotyping, molecular-based methods are alternatives for species-specific tests and epidemiological studies. The aim of this study was to characterize H. parasuis field strains from different states of Brazil, employing serotyping and genotyping methods. Serotyping revealed that serovar 4 was the most prevalent (26.1%), followed by serovars 5 (17.4%), 14 (8.7%), 13 (4.4%) and 2 (4.4%), whereas 39% of the strains were considered as untypeable. AFLP with a single enzyme and PFGE were able to type all isolates tested, generating 34 and 20 different profiles, respectively, including untypeable strains. Besides the slightly higher discrimination index presented by AFLP, PFGE with Not I restriction enzyme showed a better correlation with epidemiological data, grouping strains of the same serovar, animal or farm origin. The results indicated AFLP and PFGE as valuable tools for typing H. parasuis isolates collected in Brazil.     

猪群HEV血清抗体调查及一株新的猪HEV ORF2部分基因序列分析

戊型肝炎病毒(Hepatitis E virus,HEV)主要引起人的戊型肝炎,新近研究发现猪在病毒传播中可能发挥重要作用.本研究对我国部分省区HEV感染血清学调查,在被检的1 138份血清中,有666份(57.5%)为HEV抗体阳性,猪群抗体阳性率随着月龄增长而升高.通过RT-PCR方法从一份猪粪中扩增并克隆了HEVORF2 N端主要抗原决定区339bp基因片段,序列分析显示,该段基因与我国人群HEV基因4型毒株核苷酸序列同源性为85.9%,但氨基酸序列完全一致.这一结果提示我国猪群存在广泛的HEV感染,并在一定程度上与人群HEV毒株密切相关.     

Protection of pregnant swine by vaccination against leptospira infection

SUMMARY The protection conferred on pregnant gilts by 2 commercially available Leptospira interrogans serovars pomona and tarassovi bacterins was evaluated. Gilts vaccinated either 3, 6 or 12 months prior to natural challenge with L. interrogans serovar pomona had significantly lower abortion rates (2% vs 69%) and foetal mortality rates (14% vs 57%) than unvaccinated controls. One vaccine was significantly superior to the other and contained approximately twice the number of L. interrogans serovar pomona organisms per vaccine dose. Neither vaccine protected against renal colonisation but vaccination reduced urinary excretion of leptospires. Both vaccines reduced agglutinating antibody response to infection, as measured by the microscopic agglutination (MA) test. This may prevent the detection of a carrier animal by serology. Foetal pigs did not develop specific MA titres. Cultural methods were not reliable in making a diagnosis of foetal infection. Histopathology of foetal liver and kidneys helped in making a diagnosis of foetal infection.     

Transmission of swine pathogens: different means, different needs

There seems to be two main types of pathogens that cause diseases in swine: those that are mainly introduced through direct pig contacts, and those that are often, and in some situations mainly introduced by indirect transmission means. In this review, the mange mite (Sarcoptes scabiei), toxigenic Pasteurella multocida and Brachyspira hyodysenteriae will be used as examples of the first type, and foot and mouth disease virus, Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome (PRRS) virus as examples of the second. It is now clear from various epidemiological studies as well as experimental and field data that aerosol transmission of some swine pathogens plays an important role in their epidemiology. As previous biosecurity programs did not take this factor into consideration, it can at least partially explain why many of these programs suffered frequent failures and why air filtration is now becoming increasingly popular in North America. Identifying and quantifying transmission means should be a priority for every important infectious disease for which it has not been done.     

2011年～2012年中国部分地区猪细环病毒在不同猪群中的流行病学调查及其与PCV2、PPV4或PRRSV的共感染分析

为了解猪细环病毒(TTV)在无明显临床症状的猪群和不同发病猪群中的感染情况,本实验于2011年～2012年期间共采集江苏、安徽、天津、湖北、上海、浙江6个省份的无明显临床症状的母猪血清共488份,发病猪群样品352份,包括发生PMWS的猪血清及组织样品203份,发生PRRSV感染的猪群样品105份,发生流产的初产母猪血清23份,以及流产胎儿组织样品21份.对无明显临床症状的猪群进行TTV1和TTV2的病原学检测,对发病猪群样品进行TTV和PCV2、PPV4、PRRSV共感染检测,并对40条TTV1和32条TTV2的UTR部分基因分群区域进行测序和进化分析.结果显示,在488份无明显临床症状的母猪和种公猪中,猪源TTV1和TTV2感染率较高,分别为41％和67％,二者混合感染率为28％.PMWS发病猪样品中PCV2阳性率高达75％,PCV2和TTV2的混合感染高达61％.在PRRSV为阳性的病料中,TTV2的阳性率高达86％.在初产母猪和流产胎儿中,TTV1和TTV2的感染率,以及与不同病毒的混合感染率均高于健康猪群的感染率.对基因序列的进化分析表明,TTV1主要可以分为8个基因群,TTV2可以大致分为4个基因亚群.本研究为研究猪源TTV的潜在致病力以及分子进化提供了进一步的资料.     

猪戊型肝炎病毒ORF2片段克隆及原核表达

设计套式引物，内套引物含有BamHⅠ／XhoⅡ酶切位点，采用RT-PCR技术扩增猪戊型肝炎病毒（UEV）结构蛋白基因ORF2部分片段，长度为634bp，将其克隆于PGEX-T载体，测序结果表明插入的片段属于戊型肝炎病毒ORF2部分，将该片段插入PproEXHTb表达载体，经酶切鉴定证实获得了重组表达质粒。将重组质粒转化大肠杆菌BL21，经1mmol／L IPTG诱导，得到表达，表达蛋白分子量为27Ku，以包涵体形式存在。Western blot分析表明，该蛋白可以与猪戊型肝炎阳性血清反应，表明该蛋白具有良好的反应原性。     

Experimental infection of domestic swine with Baylisascaris procyonis from raccoons

Six 8-week-old ascarid-naive pigs which were experimentally infected with 72,000 embryonated Baylisascaris procyonis eggs of raccoon origin developed lesions limited to the intestines and liver. Intestinal lesions consisted of multifocal areas of inflammation by macrophages, eosinophils, and lymphocytes in the mucosa and submucosa, in association with Baylisascaris larvae; similar lesions were seen in the mesenteric lymph nodes. Typical white, granulation type, multifocal interstitial hepatitis ("milk-spots"), 1 to 5 mm in diameter, were seen in the livers by 7 days, with resolution by 47 days. Microscopically, these consisted of multifocal areas of marked periportal and interlobular edema, and influx of eosinophils, and large intralobular aggregates of eosinophils. At 47 days, hundreds to thousands of small white granulomas were seen on the serosa of the intestines; microscopically, they were discrete collections of macrophages, lymphocytes, and eosinophils in the submucosa and muscle layers surrounding nonviable remnants of Baylisascaris larvae. Larvae or lesions were not seen in other tissues, including the brain. These experiments indicated that B procyonis will undergo limited migration in swine and can produce typical white spots in the liver. The larvae were killed by cellular reactions in the intestinal wall and liver, and, unlike the situation in most other animals infected with this parasite, no somatic migration or CNS disease occurred after infection.     

Detection of infection with classical swine fever virus in wild boar: a comparison of different laboratory diagnostic methods

The aim of this study was to evaluate two commercially available ELISAs for routine diagnosis of classical swine fever virus (CSFV) in wild boar. For this, 222 tissue samples from wild boar were tested in the ELISAs and the results were compared to those obtained using standard methods. First, frozen spleen sections were examined by direct immunofluorescence, and organ suspensions were prepared and tested for CSFV antigen samples were simultaneously examined with the Chekit-ELISA (Dr. Bommeli AG) and the Herd-Chek-ELISA (IDEXX). From the 222 organ suspensions examined in cell culture 102 were positive for CSFV, while no virus could be isolated from the remaining 120 samples. Taking virus isolation as a standard, the Chekit-ELISAs showed a sensitivity of 97%, and the Herd-Chek-ELISA of 72.5%. Both ELISAs revealed high specificities ranging between 99 and 100%. No correlation was found between false negative results obtained in one or in both of the ELISAs with the positive findings in the immunofluorescence test and in the PLA, nor with the clinical reports. Due to the fact that a big number of samples can be processed in a short time with accurate results, the Chekit-ELISA may be considered useful for routine testing of wild boar samples for CSFV.