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BPI-3V sometimes cause severe disease as a single agent and can predispose the animal to bacterial infections of the lung. Our results revealed high BPI-3V seroprevalence (47.1%) in the three explored municipalities that indicate most adult cattle have been exposed to this pathogen. These results agree with those by Carbonero et al., who found high seroprevalence values in cattle of Yucatan, Mexico. However, the results obtained in this study differ with those published by Betancur et al., who reported lower seroprevalence values (13.5%) in cattle from Monteria, Colombia. The high seroprevalence of BPI-3V found in this research is in agreement with the ubiquitous nature of the virus and with its worldwide distribution. In this research, the seroprevalence was higher in the age group of >24 months of age (Table-4). This age group was a significant risk factor for BPI-3V transmission (OR=3.5). Possibly, due to the presence of some stress factor in these animals that favors reinfections with or without respiratory signs. In adults, especially BPI-3V, it is subclinical unless it is part of concomitant infections with other viruses and bacteria such as Pasteurella multocida, Mannheimia haemolytica, Mycoplasma spp., and immunosuppressive factors. With regard to the clinical signs, conjunctivitis had a statistical association with the BPI-3V seroprevalence values, and regarding sex, female was a significant risk factor for BPI-3V infection (OR=3.6). This result differs with those published by Betancurt et al., who found no statistical association between BPI-3V infection and sex.
HAstVs are a classic cause of viral diarrhea in children, along with rotavirus, norovirus, sapovirus and adenovirus. Seroprevalence studies indicate that most children in Europe encounter astrovirus before the age of two. Astrovirus-associated diarrhea is not reported in immunocompetent adults, as infection in childhood is considered to confer protective immunity. Additionally, humoral immunity is considered to play a major protective role, along with cellular adaptive immunity. Therefore, immunosuppressed patients and the elderly can also develop astrovirus-associated diarrhea.
In non-immunocompromised individuals, after an incubation period of 4–5 days, an astrovirus infection will induce a mild disease, characterized by mild and short watery diarrhea for two to three days, followed by nausea, vomiting, and abdominal pain, which usually resolves spontaneously. These symptoms are most often milder than a rotavirus infection. Recent seroprevalence studies have indicated that some infections can be asymptomatic as well. As reported for rotavirus and norovirus, astrovirus has also been associated with intussusception in infants. Although virological diagnosis of astrovirus-associated diarrhea is not routinely used in medical practice, it is sometimes used in epidemiological studies in the context of diarrheal outbreaks and surveillance of diarrheal diseases.
An astrovirus infection in immunocompromised individuals may induce gastroenteritis, but it can also lead to severe and sometimes fatal systemic and central nervous system (CNS) infections, as seen in multiple cases of astrovirus-associated encephalitis and meningitis. These reports are associated with newly identified HAstVs that belong to novel species (MAstV 6 and 9). Studies are under way to assess the actual disease burden associated with these novel neurotropic astroviruses in humans. These novel astroviruses are enteric viruses, associated with diarrhea and fecal carriage, but their pathogenicity in the non-immunosuppressed host has not yet been precisely determined, although a case of meningitis in an apparently healthy adult has recently been reported. Therefore, these newly-discovered viruses seem to share some clinical characteristics with enteroviruses, due to their association with diarrhea, but may also induce meningitis and encephalitis in the immunosuppressed patients. For this reason, their detection should now be part of the laboratory diagnostic work-up in patients, in particular those who are immunosuppressed and are diagnosed with meningitis or encephalitis of unknown cause.
In mammals, astroviruses have been reported in piglets, minks and dogs with preweaning diarrhea, but accurate diagnosis is complicated due to the prevalence of fecal shedding in healthy animals, which complicates the interpretation of the results. Therefore, etiological diagnostic is not a routine practice. In mink presenting with the so called “shaking mink syndrome”, and cattle with encephalitis, astroviruses can be tested in necropsy brain samples. Additionally, astroviruses have been associated with severe avian diseases (i.e., chicken diarrhea, duck hepatitis, turkey enteritis, and avian nephritis), and diagnosis can be made in severely affected flocks by reverse transcription polymerase chain reaction (RT-PCR), using necropsy samples in specialized laboratories.
This is the first epidemiological study to assess risk factors for BRSV seroprevalence carried out in Brazil. Even though BRSV prevalence of 79.5% in the animals sampled was similar to that estimated, the prevalence in adult animals was higher than that expected, reaching 87% of samples. In calves, the seroprevalence was lower than that found in adult animals (62.8%) and could be even lower once VNT does not allow the distinction between antibodies from colostrum and natural infection. Thus, this study demonstrated that the prevalence of BRSV antibodies was higher in adult animals, as previously reported in other countries [13, 16].
Adult animals are associated with high seroprevalence of BRSV as consequence of a repeated exposure to the virus infection throughout their life and possibility of reinfections. Similarly, the highest antibody titers were associated with non-vaccinated adult cattle, probably due to the exposure to successive viral reinfections, which results in a booster effect on antibody titers. Other factor related to high antibody titers is recent BRSV infections, which can be confirmed only by paired serology, antibody screening in calves after the period of colostral antibody detection or viral detection by direct methods. As respiratory disease was not reported in half of the herds studied, it is indicative that BRSV infection can be subclinical. This is consistent with previous reports. Herds can remain free of clinical BRSV infection for many years even in areas of high prevalence of the virus.
The presence of other pathogens is also associated with the prevalence of BRSV [8, 11, 14, 16]. This information explains the association of BRSV serological prevalence with the prevalences of BoHV-1 and BVDV-1. The infection by these viral agents is also reported in Brazilian herds, with high prevalences [27, 28]. BVDV infection can cause impairment of the animal’s immune function and thereby decrease resistance to other infections. The synergistic effects of BVDV with other respiratory pathogens have been observed [29, 30]. Thus, health status of the herds may also be affected indirectly by BVDV control measures.
Dairy cattle herds in São Paulo State usually have poor biosecurity measures, such as the lack of quarantine of newly purchased animals, lack of diagnosis of respiratory diseases (particularly for BRSV) and vaccination is rarely performed against these viruses. Therefore, we hypothesized that risk factors for the seroprevalence of BoHV-1, BVDV-1 and BRSV in the studied population likely to overlap.
Despite the logistic regression not confirming “type of calves feeding” variable as a risk factor for high prevalence of BRSV, the Fisher’s exact test detected “natural suckling” as a protective factor. “Natural suckling” would be important as it may be able to reduce the risk of calves becoming infected by BRSV. Weaning can be stressful and results in impaired immune function, which may further exacerbate a BRSV exposure. Suckling reduces the occurrence of diarrhea, prevents the abnormal behavior of cross-suckling of other calves and improves animal health [31, 32]. Prior to the current study there have been no report about “natural suckling” and its relationship with BRSV seroprevalence or its role as a protective factor, therefore, based on the results presented, it has the potential to decrease seroprevalence to BRSV.
Similarities were observed among the results found at the present study and those previously obtained by others conducted in Brazil [18–20]. In Latin America countries, equivalents prevalences of BRSV have also been reported [12, 14–16], as well as difficulties in detecting the risk factors involved in the dissemination of the agent, even using different forms of sampling and analyzing a considerable number of variables. Thus, the dynamics of infection may differ even in a particular country or geographic area.
The high serological prevalence of BRSV found in this study shows the importance to know more about this infection since it is not considered important in the country, mainly due to the lack of diagnosis. The awareness of the risk factors involved in the BRSV dissemination can allow understanding its mechanisms, even though, as in other studies, these factors were not very clear. Thereby, further studies as a complement to the current one should be performed until concrete information has been found.
Two independent reviewers screened titles and abstracts for their relevance. We included publications that mentioned norovirus in the title or abstract but we excluded papers about food (oyster) and waterborne outbreaks, food surveillance or food related experiments, and oyster/seafood surveillance. We excluded papers on murine noroviruses as models. Papers describing norovirus surveillance in wild mice and papers using mice as model for human norovirus were included (Figure 5).
In a second round, we screened the papers for whether they described (1) animal surveillance studies to detect human or animal norovirus by PCR, sequencing or by serosurveillance including negative results; (2) experimental animal infections with human or animal norovirus; (3) human surveillance studies to detect animal norovirus by PCR, sequencing or by serosurveillance including negative results; (4) animal norovirus characterization including molecular assays and genome announcements.
Our results revealed high BRSV seroprevalence (98.6%) in the three explored municipalities that indicate most adult cattle have been exposed to this pathogen. The herd seroprevalence of BRSV (100%) found in this research is consistent with published data of Solis-Calderon et al., Saa et al., and Carbonero et al., who reported a herd prevalence of 90.8% (Mexico), 91.3% (Ecuador), and 95.8% (Argentina), respectively. However, these results differ from those reported by Obando et al. Contreras and Parra, who found lower seroprevalence values in other studies. The individual seroprevalence of BRSV (98.6%) agrees with the findings of Saa et al. who reported 80.4% of seroprevalence in herds of Ecuador. This result also agrees with those of Betancur et al. and Betancur et al. who found 13% and 31% of seroprevalence in dairy cows and calves, respectively, in herds of Montería state, Colombia.
Nevertheless, these results differ from those published by Carbonero et al., who reported 46.6% of seroprevalence in Argentina. The results obtained demonstrate that BRSV is widespread among animals and dual-purpose cattle herds in Cesar department. Probably, after the initial infection occurs in some animals, the virus is rapidly spread throughout the animals, probably by aerosols, particularly in herds without prior exposure to the virus, increasing seropositivity. The several herds in Colombia are not being vaccinated against BRSV and result from this research demonstrates that this virus circulates among the animals and herds from the three municipalities. It would be important to include BRSV in vaccination programs with the aim of controlling infection in this region.
Regarding the age, BRSV infection was observed in both age groups in this research. Although the analysis was not done in younger animals, as reported in the literature, the clinical disease is more frequent in calves. This seroreactivity in adult animals suggests possible reinfections during the course of their life. The result obtained in this research agrees with those reported by Betancur et al., who found no statistical association between infection and age group. Nevertheless, the results obtained differ from those published by Bidokhti et al., who found statistical differences with respect to the age of the infected animals. They demonstrate that after infection with BRSV, the animals will remain seropositive for several years. The older cows were seropositive while the younger cows were seronegative, i.e., there had been no virus circulating for several years. In this study, municipality, sex, and herd size were not a significant risk factor (Table-4). Regarding the clinical signs, animals with respiratory problems (34.9%) and conjunctivitis (38.5%) were found (Table-3). However, there was no statistical association (p>0.05) between seroprevalence values and respiratory signs in tested animals. These results are due to BRSV, which is observed in any age group, but infections that result in severe clinical disease are typically observed in calves. Nevertheless, there was no sampling in calves in this research.
Norovirus|Norovirusses|Norwalk|“smallround-structur”|srsv animal|animals|reservoir|nonhuman|zoonosis|zoonoses|"disease model"
The first “non-classic” HAstV strain characterized was MLB1, the virus was detected in a stool sample from a 3 year old Australian child with acute diarrhea in 1999; the child had previously received a liver transplant. The majority of MLB1 strains characterized to date have been detected in India, Kenya, and Japan with limited detected in the USA, China, Bhutan, Egypt, Brazil, and Italy and prevalence has been reported in the range of 0.2% to 9%. However, a seroepidemiologic study in the USA revealed that primary exposure to MLB1 occurs in childhood and that seropositivity reached 100% by adulthood suggesting the widespread circulation of the virus in the human population. MLB2 viruses were first identified in Vellore, India with the majority of strains subsequently identified in Japan, The Gambia, and Switzerland with limited detection in Turkey, USA, Kenya, China, and Thailand and prevalence reported in the range of 0.3% to 1.5%. MLB2 has been associated with meningitis and other CNS complications and has been detected in immunocompromised children. MLB3 viruses were first detected in India in 2004, with subsequent detection in Kenya and The Gambia and the prevalence in stools ranges from 0.6% to 3.1%.
There is a dual naming system for some HAstV species due to the simultaneous characterization of these viruses by different researchers; these viruses are termed VA/HMO named for VA—Virginia and HMO—Human-Mink-Ovine-like viruses, due to their genetic relatedness to previously characterized mink and sheep viruses. In 2009, VA2/HMO-A strains were detected in children with non-polio acute flaccid paralysis in Nigeria, Pakistan, and India. The prevalence of VA2/HMO-A viruses in stools ranges from 0.3% to 2.3%, with strains also detected in Egypt, Japan, USA, Kenya, and China. VA4 has only been detected in Nepal and the BF34 strain has only detected in Burkina Faso.
Serum samples from 1243 animals belonging to 26 dairy cattle herds were taken and tested by VNT, in which 988 (79.5%) were seropositive to BRSV. Regarding the age group, 87% (767/ 891) of the serum samples were positives for adult animals while the prevalence rate in calves was 62.8% (352/221). Antibodies to BRSV were detected in all cattle herds, with prevalence rates ranging from 40 to 100%. The mean antibody titers for adult animals was 2 to 512, and for calves, 2 to 32. Therefore, prevalence of BRSV both in herds and animals was considered high.
The chi-square test showed association of BRSV seropositivity with “age group” and animals tested seropositives to BoHV-1 and BVDV-1 (Table 2). Nevertheless, the Fisher’s exact test only detected statistical difference with the variable “type of calves feeding” (Table 3). In this case, the relative risk (RR) value was less than one, i.e., the factor “natural suckling” was considered protective. Logistic regression (values of p < 0.2, two-tailed Fisher) did not show significant results, suggesting that the variables analyzed were not risk factors for the high seroprevalence of BRSV in the studied population.
Co-detection of BCV and H. somni at the time of the disease outbreak suggests that these two pathogens contributed to disease pathogenesis. Characterizing the factors that may have contributed to the outbreak described here can help veterinarians, researchers, and producers better understand the risk factors associated with pre-weaning BRD. This information can be used to guide prevention and control strategies to decrease the incidence of BRD and associated adverse animal health issues and production losses.
Astroviruses are non-enveloped, positive sense, single-stranded RNA viruses, with solid capsid shell ~35 nm in diameter (~44 nm with spikes), classified into two genera: mammalian viruses (Mamastroviruses (MAstVs), 19 species recognized by the International Committee for Taxonomy of Viruses (ICTV)) and avian viruses (Avastroviruses (AAstVs), three species recognized by ICTV). The taxonomy does not take into account the species of origin anymore. The genome is 6.8 kb to 7.9 kb in length and harbors a 5′ untranslated region (UTR), followed by three open reading frames (ORFs), namely ORF1a, ORF1b, and ORF2, a 3′ UTR and a polyA tail.
Human astroviruses (HAstVs) are found in four MAstV species (MAstV 1, 6, 8, 9), as summarized in Table 1. MAstV 1 includes the eight serotypes of classic HAstVs (HAstV 1–8), a common cause of viral gastroenteritis in children, targeted by usual diagnostic PCRs. MAstV 1 and MAstV 6 form a monophyletic group, together with astroviruses from cats, pigs, dogs, rabbits, California sea lions, and dolphins. The two other genotypes, MAstV 8 and MAstV 9, are closely related to astroviruses from mink, sheep, California sea lions, bats, cattle, pigs, and mice.
Within genotypes, strains can be grouped by serotypes, based on their antigenicity, although variability is still high, even within the same serotype. For this reason, subtypes are defined, which are well-documented within the eight serotypes of MAstV 1, and most likely also exist for the other species as well. Two species defined by prototypal MLB1 (MAstV 6) and VA1 (MAstV 9) strains have been described and more recently associated to severe cases of encephalitis in immunocompromised patients. High genetic variability (even within each genotype) and concerns regarding capabilities for cross-species transmission are challenges for the definition of adequate diagnostic tools capable of identifying distant strains. Moreover, association between astrovirus infection and disease is still being investigated, as well as the interaction of astroviruses with other enteric viruses in diarrhea physiopathology, all of which suggests that diagnostic tools will need to continue to evolve in the near future.
This stool was collected in 1984 from a 38 month old child presenting to the emergency department of the Royal Children's Hospital, Melbourne, Australia with acute diarrhea and stored at -80°C. Previous testing of this diarrhea specimen for known enteric pathogens using routine enzyme immunoassays (EIA) and culture assays for rotaviruses, adenoviruses, and common bacterial and parasitic pathogens was negative. Additionally, RT-PCR assays for caliciviruses and astroviruses were also negative.
The present study showed that calves infected with BCoV shed viral RNA for five weeks, and harbored viral RNA in intestinal tissues and lymph nodes even longer. Interestingly, contact with these calves three weeks after challenge, when the clinical condition had improved and the calves had seroconverted, did not lead to infection in sentinel calves and virus isolation was not possible from calves shedding viral RNA at this time point.
In concordance with other studies [18, 29], all EG calves became BCoV positive shortly after contact with infected calves and shed viral RNA continuously for two weeks. This supports that introduction of BCoV into a naïve population leads to a high basic reproduction number (R0). R0 depends on the duration of the infectious period, the number of exposed susceptible individuals and the probability of a susceptible individual to be infected. In herds and transportation systems where cattle from different herds are commingled, the risk of virus transmission is high.
The detection of BCoV RNA in nasal swabs from naïve calves in EG shortly after exposure might be due to passive inhalation of virus excreted by the FG, or to virus replication in the respiratory tract. Since the viral load in the nasal swabs from EG exceeded that of FG at D2, the study confirms that BCoV replicated massively in the airways of EG calves already at D2. Fecal shedding started later than nasal shedding which is in concurrence with other studies. Saif and colleagues found that when inoculating calves intranasally, BCoV was first detected in nasal epithelial cells and secondly in feces. In contrast, in calves inoculated orally, fecal detection of BCoV preceded detection in nasal swab specimens. They concluded that the infection route could determine the sequence of infection of the respiratory and intestinal tract. The present study supports that the respiratory route is the most common infection route when calves are naturally infected by direct contact. With indirect virus spread, the fecal-oral route could be more common.
Nasal swabs were more often positive for BCoV than fecal samples in this trial, most likely due to a higher limit of detection for BCoV in feces than in nasal swabs. For diagnostic purposes, nasal swab specimens therefore seem advantageous to fecal samples for virus detection in calves with suspected BCoV related disease.
Moving and commingling are associated with stress, which has been found to affect the intestinal immune system. It is possible that stress increased the BCoV RNA shedding observed in the EG calves after introduction of the sentinel calves. Buying and selling of calves often involve extended transportation and commingling with susceptible cattle. The stress response, and a possible increased fecal shedding of virus, would probably be higher under field conditions.
In the acute stage of the infection, the agreement between positive PCR results and clinical score was relatively high. Three weeks after exposure to BCoV, the clinical signs and detection of viral RNA varied more independently. In an experiment with porcine deltacoronavirus, the severity of the clinical signs did not correlate with the shedding of virus in conventionally reared piglets, only in gnotobiotic piglets. This indicates that secondary pathogens and changes in microbiota are important for disease development and clinical signs. The present study supports that after the acute stage of disease other factors than virus replication are important for clinical signs; for instance secondary bacterial infections.
Although the sentinel calves did not get infected with BCoV, they showed sporadic unspecific signs during the trial, but below the mildest category “mild disease” in the clinical scoring system. Since acclimatization was not possible, the calves changed environment including feeding routines when enrolled in the experiment, which could cause the signs observed. Other infectious agents could also have been present, and if so, most likely less virulent pathogens. Bovine virus diarrhea virus and bovine herpesvirus 1 are not present in Sweden, and the sentinel calves showed no serologic response to BRSV. Co-infection between BCoV and other agents is likewise possible in FG and EG, as is the case under field conditions.
Unlike most enteric viruses, BCoV is enveloped and therefore susceptible to environmental inactivation. One might expect that the conditions in the forestomaches and abomasum would inactivate BCoV and one possibility is that BCoV is transported from the oronasal cavity to the small intestines through the bloodstream. However, viremia was not detected in the present study, and transport of the virus to the intestines appears to have been through the digestive tract. Park and colleagues detected BCoV RNA in serum samples from calves infected with a winter dysentery strain between day three and eight post inoculation. They used nested PCR for detection, which is generally a more sensitive method than RT-qPCR, but also more vulnerable for contamination. Short viremic period or intake of a lower virus dose in naturally infected calves could also explain the negative results in the present study. Inhibition of the RT-qPCR by plasma components was tested and ruled out. Despite the absence of detectable viremia in the present study, BCoV RNA was found in mesenteric lymph nodes at late stages of the infection. Viral RNA must have been transferred in low concentrations in blood or lymph to the draining lymph node, by antigen presenting cells or as free virus particles.
The finding of BCoV RNA in lymph nodes, ileum and colon six weeks after infection indicates coronavirus persistence in calves, however, the importance of this persistence for virus transmission is uncertain. Other coronaviruses are known to create persistent or chronic infections in mice and cats [35, 36]. MERS-CoV is shown to be excreted for more than a month in humans and human coronavirus 229E creates persistent infections in vitro. Although fecal shedding of BCoV RNA was detected five weeks post infection in the present study, the transmission potential at this stage is most likely negligible, as at three weeks post infection.
BCoV VRC were quantified by RT-qPCR, which does not give information on the number of infective particles. The ratio of total to infective particles (T/I) is challenging to establish for BCoV due to difficulties in cultivating virus from clinical samples. In the present study, virus titration showed a T/I ratio of approximately 5 log10. With this high T/I ratio it is not surprising that virus isolation was unsuccessful after D13, when the VRC numbers are decreasing. It also agrees with the sentinel calves not getting infected D21. In contrast, roughly 8.8 log10 VRC were detected per nasal swab and gram feces from the seronegative FG calves that infected the EG calves. With a T/I ratio of 5 log10, each nasal swab and gram of feces contained more than 3.8 log10 infective virus particles.
The high T/I ratio and the failure of virus isolation after D13 could be due to either few infective particles or low sensitivity of the isolation method. Low levels of infective particles could be caused either by high production of defective particles or by neutralizing effect of antibodies. Low sensitivity could be caused by suboptimal conditions in cell culture compared to in vivo (particularly for virus from clinical samples not adapted to cell culture growth), dilution of viral content in the swab, and freezing and thawing of the material. For feline enteric coronavirus, the T/I increased from 3–4 log10 during the first week after infection, to up to 8 log10 28 days post infection, the increase possibly caused by the antibody response.
Few methods are available for studying transmission potential apart from live animal experiments, although ethically challenging and resource demanding. Existing literature is based on experimental studies examining BCoV shedding for 14 [20, 22, 40] to 21 [19, 41] days. To the authors’ knowledge, the present study is the first to study the shedding for as long as six weeks under experimental conditions. In addition, it is also the first to study the impact of this shedding using sentinel calves. Although a low number of calves were used, the results indicate that calves are not infectious three weeks after exposure to BCoV. This information is important and relevant in order to produce scientific based advices on how to avoid introduction of BCoV into herds. Further investigation of calves at different stages of disease is recommended to verify and corroborate these findings. The effect of stress related to transport on viral shedding and infectivity should also be considered.
In the present study, the virus that caused winter dysentery in adult cattle primarily gave respiratory disease in calves. Niskanen et al. also found that BCoV derived from an outbreak of winter dysentery caused mainly respiratory disease in weaned calves, supporting that BCoV is an important cause of respiratory disease in calves [42, 43] and winter dysentery in adults. The economic and welfare consequences of BCoV therefore include the combined effects of neonatal enteritis, respiratory disease in young cattle and winter dysentery in adults. Also considering the high prevalence worldwide, BCoV is an important loss-inflicting factor in the cattle industry.
In this study, C. pecorum was isolated from specimens obtained from 2
calves with diarrhea. In case 1, the bacterium was isolated from jejunum, and C.
pecorum specific omp1 genes were detected from several locations
in the intestines. Additionally, C. pecorum antigens were observed in the
jejunal villi by immunohistochemical staining. Necrotizing enterocolitis due to
Clostridium perfringens infection was also observed. Therefore, we
speculated that C. pecorum might exacerbate the disease caused by
In these 2 cases, it is possible that C. pecorum was either the primary
pathogen or an exacerbating factor causing diarrhea. On the other hand, it is thought that
C. pecorum causes asymptomatic infections. Recently, Poudel et al. reported that asymptomatic endemic C. pecorum infections
reduce growth rates in calves by up to 48%. They considered that the mechanism of growth
suppression by subclinical chlamydial infection was malabsorption of nutrients due to a
local inflammatory response to intestinal mucosal infection. Additionally, despite the
absence of clinical signs, chlamydial infection was associated with reduced serum iron
concentrations and lower hematocrit values, and infected calves were leukopenic. Mohamad and Rodolakis reported that the persistence of C. pecorum strains
in the intestines and vaginal mucus of ruminants could cause long-term sub-clinical
infection which may affect the animal’s health. This may explain the poor weight gain
observed in case 2 after recovery from diarrhea. Further studies on the pathogenesis of
C. pecorum infections are required.
Although the standard method for detecting antibodies to Chlamydiaceae
spp. in animals is still the complement fixation test, our study showed that a neutralization test was also a useful method for
diagnosis of C. pecorum infections. In addition, in the dead calf, it was
confirmed that immunohistochemistry for C. pecorum antigens was also
Genetic and antigenic analysis showed that 22–58 and 24–100 strains were more closely
related to Bo/Yokohama strain isolated from cattle with enteritis than to Bo/Maeda strain
isolated from cattle with pneumonia and Ov/IPA strain isolated from sheep with
polyarthritis. Bo/Yokohama-like C. pecorum strains might cause enteritis
more than other serotypes. However, the 2 isolates showed higher sequence identities to
Bo/Maeda and Ov/IPA strains than to 66P130 strain isolated from cattle with enteritis in
United States. Thus, sequence identities of C. pecorum omp1 gene might vary
according to their geographical background.
The isolation of similar strains from different locations in separate years suggests that
C. pecorum might be spreading among the cattle population in Yamaguchi
Prefecture. An epidemiological study of C. pecorum is being conducted
currently to clarify the seroprevalence and relationship with disease.
In conclusion, this study showed that C. pecorum isolates similar to
Bo/Yokohama might be endemic in Yamaguchi Prefecture and cause enteric diseases in
After YC2014 challenge, piglets in group one, group two and group four showed significant acute diarrhea. Weight gain was reduced, loss of appetite and mental uneasiness persisted, while piglets in group three, group five and group six showed no obvious diarrhea symptoms. Two days after challenge, mortality of group one, group two and group four were 100 %. No mortality or obvious clinical symptoms were observed in group three, group five and group six (Table 1).
Porcine epidemic diarrhea virus (PEDV) is an enveloped, single-stranded, positive-sense RNA virus that is taxonomically classified within the family Coronaviridae, genus Alphacoronavirus. PEDV is the main causative agent of porcine epidemic diarrhea (PED), a devastating enteric disease that is characterized by watery diarrhea, vomiting, dehydration and significant mortality in piglets. Approximately 80 to 100% of PEDV-infected piglets die within 24 h of being infected with virulent PEDV strains, resulting in tremendous economic losses to the swine industry [1, 2].
Since December 2010, a large-scale outbreak of diarrhea, characterized by watery stool, dehydration, and vomiting, with 80 to 100 % morbidity and 50 to 90 % mortality in suckling piglets, has been observed in swine farms in China [3, 4]. Accumulated evidence indicates that this large-scale outbreak of diarrhea may be caused by highly virulent PEDV variants [5, 6]. In the present study, a PEDV strain, YC2014, was isolated from intestinal samples of suckling piglets with acute diarrhea in 2014, the evolutionary characteristics and the immune protective efficiency of YC2014 were also determined.
This study represents the first prospective longitudinal study of the relationship between anti-BCV antibody levels and BCV infections and respiratory disease in nursing beef calves. Two outbreaks of respiratory disease in one research herd resulted in two-thirds of the calves being mass treated for BRD. BCV shedding was found to occur in conjunction with the pre-weaning BRD outbreak. However, when levels of passively or actively acquired immunity to BCV were measured, they were not found to associate with respiratory disease incidence between the three study herds, nor with the development of BRD in individual calves within that herd. Rather, BCV infections were likely common, with many subclinical and some persistent or recurrent infections detected.
The results from this study agree with the results from previous studies in young dairy calves [16, 17]. In these studies, dairy calves shed virus, sometimes repeatedly, in spite of relatively high anti-BCV antibody titers. Furthermore, they observed no statistically significant correlation between maternal BCV specific IgG serum antibody titers and clinical disease or infection by BCV in that population. Similarly, in the study reported here, we found that BCV specific serum IgG did not correlate with respiratory disease; and 8/45 calves (18%) from Herd 2 that were involved in mass treatment were found to be shedding BCV at two or more sample acquisition times. This result supports the idea that persistent or recurrent shedding episodes can occur in the same animal with or without signs of disease.
BCV infection (as measured by virus shedding) was similarly not found to be associated with anti-BCV antibody levels; however, this measurement was hampered by the infrequency of sample collection in our study design (Fig. 1). For example, nearly all of the animals in the Herd 2 mass treatment groups shed BCV between our routine sample collection times. While we did not see differences in anti-BCV antibody abundances in Herd 2 between those calves mass treated for BRD and those from the same herd that remained untreated, it is possible, and even likely, that many untreated calves also shed BCV between sample acquisition dates. Lack of BCV detection in nasal swabs at routine collection times may lead to “false negatives” for BCV infection that mask any influence antibodies may have on virus shedding. Furthermore, the small number of BCV positive individuals found in Herds 1 and 3 (two calves from each herd were shedding BCV at preconditioning processing) hinder our ability to detect with any confidence differences that may exist. More intensive sampling would be required to better determine the association between anti-BCV antibody titers and BCV infection. Other hypothetical reasons why serum anti-BCV IgG abundance has not been found to associate with BCV infection or treatment for disease has been discussed previously [15, 16]. Among these is the lack of knowledge related to the immune correlates of protection for respiratory BCV infections. How cell mediated immunity and other antibody isotypes (such as IgA) contribute to protection from infection and disease are areas that require additional research.
Given that BCV infection is common, it remains unknown why some animals display signs of respiratory disease while others remain sub-clinically infected. To determine whether there were differences in the BCV strains circulating in the three herds, a 1102 nucleotide fragment of spike gene was analyzed. The spike gene encodes the surface glycoprotein that is responsible for attachment to the host cell and is a major neutralizing antigen targeted by the host immune system [34, 35]. The region of the spike gene spanning the hypervariable region and antigenic domain II was selected because it is variable between coronavirus strains and isolates, and variations in this region have been associated with altered antigenicity and/or pathogenicity in other species. No polymorphisms were found in this region between Herd 1 and Herd 2 or between the isolates from subclinical shedding episodes compared to the isolates circulating at the time of the BRD outbreak. Furthermore, only one or two SNPs were found between the isolates circulating in Herd 3 compared to the isolates in Herds 1 and 2. In contrast, up to 35 SNPs and 11 amino acid differences were detected in this same region when these isolates were compared to 14 isolates collected between 2014 and 2017 from USMARC and the University of Nebraska-Lincoln Veterinary Diagnostic Center (unpublished data). Thus, differences in the BCV strains circulating in the three herds are unlikely to account for the major differences in treatment rates observed in this study.
One side effect of respiratory viral infection is the increased risk for bacterial superinfection. While BCV may occasionally produce a clinical syndrome consistent with BRD in the absence of bacterial infection, its involvement, like other respiratory viruses, is generally considered to be a precursor to a bacterial infection, which exacerbates disease [2, 4, 36]. Thus, the presence of co/secondary bacterial infections may help explain the differences in disease severity in BCV-infected calves observed in this study. Using qPCR to look at common bacterial pathogens associated with respiratory disease in cattle, we observed H. somni in high frequency and abundance in the upper respiratory tract of sick cattle but not clinically normal cattle from the same herd collected 2–3 weeks after the outbreak. Furthermore, H. somni was not detected in these calves at initial vaccination, prior to the disease outbreak. Thus, we hypothesize that a potential secondary bacterial infection with H. somni may explain why the cattle in Herd 2 displayed more signs of respiratory disease and subsequently required treatment. Of note, however, within treatment groups there was no difference in the relative abundance of H. somni in the nasal cavity of animals when categorized by rectal temperature taken at the time of sample collection and treatment. This suggests that infection was widespread in the herd, though only 15–30% of the animals were displaying clinical signs of disease at that particular time. Frequency differences of various strains of these bacteria that have different propensities to cause disease may have been present between these populations, but were not differentiated by the assays used. It is also possible that additional pathogens that were not measured in this study could have contributed to the disease outbreak. Thus, unbiased metagenomic approaches are currently underway to determine whether any additional viral or bacterial pathogens were associated with the disease outbreak. How the URT microbiome may have influenced respiratory health is also being examined in these populations.
The rapid dissemination of PEDV across the US in months demonstrated the vulnerability of a concentrated, interwoven swine production system to a highly transmissible novel enteric pathogen. This unprecedented spread was dependent on many factors including the duration and magnitude of virus shedding from infected to naïve pigs. The goal of this study was to better understand the role of pig-to-pig transmission in the US PEDV epidemic by developing a model to characterize the host response to infection with PEDV.
In 4-week-old contact pigs, mild-to-moderate diarrhea was observed for approximately one week post exposure to a PEDV experimentally-infected seeder pig. No other clinical disease was observed in the contact pigs. Within 24 hours-post-inoculation or post-exposure, all rectal swabs were PCR positive demonstrating the rapidity of transmission and the potential for pigs to shed infectious virus. In the pigs “naturally” infected by contact, 13/13 pigs were PCR positive for 9 days and most pigs were positive for 2-weeks-post exposure during which time one or more of the pigs shed an infectious dose of virus to pigs S1 and S2. Like the 13 naturally exposed pigs, S1 and S2 became PCR positive within 24 hours of contact, and seroconverted within 2 weeks post-exposure. The speed by which all pigs became PEDV RNA positive and the potential for pigs to shed infectious virus up to 2-weeks-post infection to age-matched pigs, suggests the basic reproduction number or R0 should be high for this virus.
As would be expected for a virus with a high R0 value, the transmissibility of the virus would be enhanced by large numbers of naïve animals concentrated in small areas. In the case of PEDV, an enteric virus that can be shed in feces in large quantities, it is not a surprise that the virus would heavily contaminate transportation systems that would facilitate its spread throughout the US. Moreover, potential spread of the virus through contaminated feed and by air would allow this virus to circumvent most bio-security practices. Collectively, these factors combined to make a “perfect storm” for spread of PEDV in the US.
Based on negative PCR results and lack of specific antibody, pigs S3 and S4 were determined to have remained PEDV negative despite contact with PG pigs that were intermittently PCR positive. The negative status of S3 and S4 indicates the PG pigs did not shed infectious virus in sufficient quantity to infect age-matched pigs (aged 7 and 8 weeks, respectively). It is not known if younger, presumably more susceptible pigs might have become infected under similar conditions. Likewise, we do not know if the infectious character of the virus being shed changes with time, i.e., the rectal swabs are PCR positive but may not contain infectious virus. For this experiment, the PCR results were defined as positive or negative based on a Ct cutoff of 35, as recommended by the University of Minnesota Veterinary Diagnostic Laboratory. Whether or not this positive/negative cutoff correlates to infectious/non-infectious virus is not known.
Protective immunity to homologous challenge was observed in the PG pigs based on the absence of clinical disease following challenge, and the pigs remained PCR negative from D49 through 63. In contrast, following challenge at D49, the 11-week-old N/C pigs developed an intermittent mild diarrhea beginning 2–3 days post-challenge that lasted for several days. Within 48-hours post-challenge, 4/5 pigs were PEDV PCR positive and all 5 were positive from D52-59. The number of positive pigs reduced to 3/5 positive from D60-62.
Field reports describe age differences in clinical PEDV disease ranging from essentially 100% mortality in neonatal pigs to a moderate to mild diarrhea in older pigs and adults that may include vomiting. The minimal infectious dose, and the minimal lethal dose for different ages of swine are not known. Moreover, there could be considerable differences in the pathogenicity of different PEDV isolates, and innate resistance between genetic lines of swine. For the purposes of this paper, we chose to give a uniform dose to the younger (4 weeks-of-age) and older (11 weeks-of-age) pigs realizing that the older pigs might be less affected due to a relatively smaller dose, and the possibility that there may be inherent resistance to infection as pigs mature. Characterizing the relationship between age, challenge dose, and clinical disease is beyond the scope of this study. Although both age groups did become infected following challenge, back titrations of challenge virus suggests the younger pigs received a larger challenge dose than the older pigs (about 6 log10 vs. 5.1 log10) which limits any interpretation of the potential relationship between age, challenge dose and disease.
In this study, the 4-week-old pigs were more clinically affected when compared to 11-week-old pigs. None of the pigs in this study became clinically dehydrated or succumbed to the infection, and other than showing signs of diarrhea, it would have been difficult to discern any apparent illness if the pigs were housed on slatted floors. We presume this difference is mostly age-related as younger pigs are more susceptible to disease. However, the younger pigs became infected by contact with a seeder pig while the older pigs received a known oronasal challenge of cell culture propagated virus, and it is not known how these different routes of exposure might affect the pig. Although the study was not designed to compare the clinical effect of cell-culture propagated virus challenge vs. natural infection, it is interesting to compare the ELISA OD values for the one seeder and 5 N/C pigs that received an oronasal challenge to the 13 naturally infected pigs (Figure 2). The mean OD of the 6 inoculated pigs was higher than the naturally exposed pigs at each post-inoculation time point (7, 14, 21, and 28 days-post-inoculation). The importance of this trend and whether or not it would be reproducible in subsequent studies is not known. In addition, for the 13 contact pigs, the mean OD value was greater at each post-exposure time point for the PG group when compared to the 4 contact pigs in the SG group. This apparent trend could reflect exposure, albeit a short exposure, to “fresh” virus being shed by S1 and S2 when they were commingled with the PG for 3 days at D7 and 14, respectively. Perhaps this potential exposure affected the development of the humoral antibody response. Under the conditions of this study, an anamnestic humoral immune response was not observed following challenge of the 9 PG pigs. Lack of immunological memory was also observed in a report using the Belgian isolate, CV-777. Given that the N/C pigs replicated challenge virus and developed specific antibody, we are confident the PG pigs received an infectious homologous challenge and had no detectable replication of homologous challenge virus or rise in humoral antibody titer.
The infection of contact pigs exposed to the seeder pig, and the infection of S1 and S2 pigs demonstrated pig-to-pig transmission of infectious virus. In the case of the seeder and contact pigs, transmission happened quickly since all of the contact pigs were rectal swab positive within 24 h of contact to either the seeder pig, or the seeder pig and first contact pig. These results are similar to a recent report using a different US PEDV strain. Similarly, S1 and S2 pigs were rectal swab positive within 24 h of contact with the PG group. Collectively, these results indicate pigs can rapidly become infected and shed infectious virus for at least 2 weeks. However, this study does not provide insight into the relationship between samples that are deemed positive by PCR and the presence of infectious virus in rectal swab fluids. In the case of the seeder pig infecting the 13 contact pigs, the seeder pig was positive from D1-15 and clearly shed infectious virus on D1 since the first contact pig was PCR positive by D2. Whether the 12 remaining contact pigs became infected by exposure to virus from the seeder pig or the first contact pig is unknown, but all were positive 24 h later on D3. All 9 PG pigs were PCR positive on D7 when S1 was commingled and at least one of these pigs shed infectious virus since S1 was PCR positive by the next day. When S2 was commingled on D14, 7/9 PG pigs were PCR positive and at least one was shedding infectious virus since S2 was positive on D15. At D21 and 28, 4 and 1 of 9 PG pigs were PCR positive, respectively, but none of the pigs shed an infectious dose based on lack of demonstrable infection in S3 and S4. The Negative/Positive status of the rectal swab was based on using the Ct value of 35; negative (>35) or positive (<35). This cutoff value was determined by the original designers at the University of Minnesota Veterinary Diagnostic Laboratory and is routinely used by other diagnostic laboratories performing either the original real-time PCR assay, or a modification of the assay (D. Madson, personal communication). Additional studies are warranted to understand the relationship between Ct value and the presence of infectious virus.
There is justifiable concern by the swine industry on how robust this virus is and what efforts are necessary to eliminate environmental contamination. In this study, there were movements of pigs into rooms that had previously housed PEDV-infected pigs. We did not conduct any environmental sampling of the rooms for PEDV contamination prior to use, thus we do not know what potential contamination might have existed in the room from preceding pigs. Each room was cleaned following the removal of previous pigs according to our standard protocol for cleaning the ABSL-2 isolation rooms. During the acute phase of the experiment it was not possible to assess if infectious virus may have been present in the room upon entry of the pigs because the pigs were already positive. Later in the experiment, we did not find evidence for pigs becoming infected upon movement into a room, i.e., S3 and S4 did not become infected when moved from the PG into separate isolation rooms. In addition, these rooms have been used subsequently for non-PEDV pig studies and no PEDV contamination was detected. Based on these experiences, we believe our routine cleaning protocol was adequate to inactivate PEDV contaminated surfaces.
In general, results from this study agree with recent observations by others that have experimentally infected 3- and 4-week-old pigs with US PEDV isolates resulting in the production of mild to moderate clinical disease. Conversely, prior to this study, the longest duration of fecal shedding of PEDV was reported out to 24 days post-inoculation. We detected intermittent viral shedding by PCR in several pigs up to D42 even though clinical signs diminished approximately 7 days post exposure. To our understanding, this is the longest length of PEDV shedding reported in pigs to date. Asymptomatic shedding of PEDV in pigs introduces a higher level of difficulty in the management of the disease throughout the swine industry.
In summary, this study and the work of others demonstrates how easily pigs become infected with PEDV, and may help explain the rapid transmission of virus recently observed in the US. In addition, pigs shed infectious virus for 2 weeks which would help explain how easily the virus was transmitted among farms. The apparent sterile immunity following primary infection suggests there may be value in a consistent feedback program, and it demonstrates potential for a vaccine to help manage the disease.
In the PG group, 2/9, 8/9, and 9/9 pigs were positive for PEDV-antibodies by D7, 14, and 21, respectively. In the N/C group, 2/5 pigs were ELISA positive by D56 (7 days post challenge) and all pigs were positive from D63 to D78. S1 and S2 seroconverted by 7 days post-contact and S3 and S4 remained seronegative throughout the remainder of the study. Average ELISA OD readings for the PG, SG, and N/C groups are depicted in Figure 2.
Bovine coronavirus (BCoV) is an important livestock pathogen with a high prevalence worldwide. The virus causes respiratory disease and diarrhea in calves and winter dysentery in adult cattle. These diseases result in substantial economic losses and reduced animal welfare. One way of reducing the negative consequences of this virus is to prevent virus transmission between herds. Inter-herd transmission is possible either directly via transfer of live animals [2, 3], or indirectly via contaminated personnel or equipment. Measures to prevent virus spread between herds must be based upon knowledge of viral shedding, the potential for transmission to susceptible animals and the role of protective immunity. Several observational studies have been published on BCoV shedding in feces of diarrheic calves and after transportation to feedlots [3, 5–10]. However, relatively few studies on BCoV pathogenesis with emphasis on transmission potential under controlled conditions have been published.
BCoV belongs to the genus Betacoronavirus within the family Coronaviridae, also including the closely related HCoV-OC43, which causes respiratory infections in humans, and the human pathogens SARS-CoV and MERS-CoV [11–13].
BCoV consists of one serotype with some antigenic variation between different strains [14, 15]. Acutely infected animals develop antibodies that persist for a long period, possibly for several years [16–18]. However, the protective immunity is shorter and incomplete. In two experimental studies, infected calves were not protected against reinfection with a different BCoV strain three weeks after the first challenge, but did not develop clinical signs [19, 20].
BCoV is transmitted via the fecal-oral or respiratory route. It infects epithelial cells in the respiratory tract and the intestines; the nasal turbinates, trachea and lungs and the villi and crypts of the small and large intestine, respectively [21, 22]. Replication leads to shedding of virus in nasal secretions and in feces. Important factors for the pathogenesis are still not fully explored, such as how the virus infects enterocytes shortly after introduction to an animal. Viremia has been detected in one study by Park et al.. Clinical signs range from none to severe, and include fever, respiratory signs and diarrhea with or without blood [1, 15]. As the time of infection is usually unknown and laboratory diagnostics are usually not performed, occurrence of clinical signs is the most relevant parameter to relate to viral shedding. The majority of experimental studies have used BCoV inoculation as challenge procedure, which may influence clinical signs and viral shedding, and thereby the transmission potential compared to natural infection. It has been hypothesized that BCoV can cause chronic subclinical infections which could be an important virus source. Kapil et al. documented viral antigen in the small and large intestines of infected calves three weeks post inoculation. Crouch et al. found that ten cows were shedding BCoV-immune complexes in the feces for 12 weeks. It is, however, difficult to establish whether there is true persistence of virus, or reinfection of partially immune animals and whether these animals represent a risk to other animals. There is a lack of experimental studies investigating viral shedding pattern for longer periods than two weeks, with sensitive detection methods. Viral load and infectivity also needs to be determined. This is of high practical relevance, since the farmers need guidance on biosecurity in trade and transport of live animals.
The current study was conducted to fill prevailing gaps in the knowledge on fundamental aspects of BCoV infection. The specific aims were to:study the duration and quantity of BCoV shedding in feces and nasal secretions, related to clinical signs in calves.study the presence of viremia and persistence of virus in lymphatic, intestinal and lung tissue.test the hypothesis that seropositive calves are not infectious to naïve in-contact calves three weeks after BCoV infection.
Immunodeficiency disorders often are associated with cryptosporidiosis, which can lead to chronic malabsorption and weight loss. In a case study of a child with congenital hypogammaglobulinemia, severe vomiting and diarrhea due to cryptosporidiosis, gastric infusion with hyperimmune bovine colostrum from cows immunized with cryptosporidium oocytes resolved the symptoms within a few days and oocyts were no longer found in stool samples after about eight days. Similarly, in a child with AIDS who had severe diarrhea caused by cryptosporidiosis, administration of a commercial hyperimmune bovine colostrum preparation with anticryptosporidial activity improved the diarrhea and eliminated the parasite.
Previously, virus discovery in animals has focused on pathogenic infections or on animals that on the basis of relevance to (re-)emerging viruses are thought to represent a key risk host for emerging virus-associated disease in humans. With the recent advances in the metagenomics field, a substantial increase in studies looking at virus epidemiological baseline levels in different (wildlife) animals has been observed,,,–. Even though ferrets are a very important animal model for a number of human viral infections, not much is known about viruses that naturally occur in ferrets–. This study describes the viral communities in fecal material of ferrets (Mustela putorius furo).
Sequences closely related to known viral sequences were identified, with homology to ferret coronavirus, ferret hepatitis E virus, Aleutian mink disease virus, different avian viruses and murine astrovirus STL1,,. The avian viruses may be a reflection of the diet of these ferrets, which were fed chicken. It is of note that both the viral screening with random amplification and next-generation sequencing and a specific ferret hepatitis E virus taqman assay indicated that household ferrets that are kept as pets are significantly more likely to excrete ferret hepatitis E virus in their fecal material than farm animals in this study. Such a correlation was not observed for ferret coronavirus excretion for which prevalence in both cohorts seemed similar, but not as high as previously reported. Sporadic cases of human hepatitis E virus infections seem to be increasing and several observations suggest that these cases are caused by zoonotic spread of infection from wild or domestic animals–. Follow-up studies into seroprevalence and/or virus prevalence in household ferrets, their owners, and possibly other pets may provide indications for cross species transmission.
We report on the first identification of a kobuvirus, parechovirus, papillomavirus and anellovirus in ferrets. The kobu- and parechovirus belong to the family Picornaviridae that currently comprises 17 genera and many more different virus species. They infect a wide range of mammals and are well-known for their involvement in gastroenteritis. Our phylogenetic analysis revealed that the ferret kobuvirus clustered with bovine and ovine kobuvirus in species Aichivirus B. The close relationship between bovine, ovine, and ferret kobuviruses may indicate past cross species transmission events and subsequent evolution in the separate host species resulting in the distinct types seen today. As was observed for ferret hepatitis E virus but not ferret coronavirus, ferret kobuvirus is significantly more often detected in household ferrets than in farm ferrets by a specific ferret kobuvirus taqman assay, although random amplification and next-generation sequencing suggested the presence of kobuvirus in two additional samples from farm ferrets, which could reflect the presence of another divergent kobuvirus species. Also here follow-up studies into seroprevalence and/or virus prevalence in household ferrets, their owners, and possibly other pets should be performed. An overall kobuvirus prevalence of ∼15% was observed in this study. Two out of 3 animals (67%) with diarrhea and 4 animals out of 36 (11%) without diarrhea were positive by taqman for ferret kobuvirus. Because of the small sample size and the fact that other causes for diarrhea were not excluded, further in depth studies are needed to show that these viruses can cause diarrhea in ferrets.
The ferret parechovirus is the third parechovirus species identified to date. It is highly divergent from human parechovirus and Ljungan virus and may even constitute a new genus in the family Picornaviridae based on the observed sequence diversity. Interestingly, the P3 genome region of MpPeV1, encoding the nonstructural proteins NS3A–3D, is not more conserved than the P1 genome region, encoding the structural capsid proteins, when compared to the corresponding regions of human parechoviruses 1 and 2. This has been observed for Ljungan virus as well by us and others and requires more in-depth studies. Only 1 out of 39 ferrets was positive for MpPeV1, suggesting that the ferret parechovirus prevalence may be relatively low (<2.5%). In humans, parechovirus infections are most common in young children and the prevalence in adults may be low as well. It is of note that the parechovirus-positive animal did not show any signs of gastroenteritis. Low virus prevalence was observed for the identified ferret papillomavirus as well. Papillomaviruses are, however, not typically detected in fecal material or associated with gastroenteritis like parechoviruses. Although recently a full-length papillomavirus genome was characterized from fecal material of a deer mouse as well. Not much knowledge exists on papillomatosis in ferrets as a disease, or the virus(es) causing them. The impact of papillomavirus infections on ferrets in general and the specific role of MpPV1 thus has to be further addressed.
Like many other mammals, it seems that ferrets also harbor anelloviruses. The ferret anellovirus MpfTTV1 is most closely related to the recently identified anellovirus from pine marten. Based on phylogenetic analysis, we propose that MpfTTV1 should be placed in the proposed new anellovirus genus, Xitorquevirus, in analogy to the classification of torque teno viruses in nine genera named Alpha-, Beta-, Gamma-, Delta-, Epsilon-, Eta-, Iota-, Theta-, and Zetatorquevirus, and the proposed five genera Kappa-, Lambda-, Mu-, Nu-, and Xitorquevirus
,,. A closely related virus to MpfTTV1 seemed to be present in one other ferret (Table 1) besides ferret 21, resulting in a virus prevalence of ∼5% in this study. Anelloviruses do not generally seem to be as abundantly present in fecal material as in serum, as evidenced by fecal virome studies in different host species,,,,,,,.
Ferrets are carnivores that are significantly affected by a number of human pathogens and hence are widely used as a small animal model for viral infections–. In addition, they are capable of spreading viral infections to humans, among which influenza A virus and on rare occasions rabies virus–. The characterization of the fecal virome of ferrets provides epidemiological baseline information about pathogens which allows the swift identification of possible sources of future zoonotic infections and their subsequent control. Especially the seemingly interesting correlation between certain viral infections in ferrets and their presence in human households as pets needs to be further addressed. In addition, this study shows that ferrets can be infected with a wide range of different viruses, some of which may influence the outcome in experimental virus infection studies, thus delivering a strong argument in favor of development of specific pathogen-free ferret colonies.
In this study, a new system for simultaneous detection of cattle diarrhea-associated
pathogens was developed. This novel system was designated as a detection system for microbes
from bovine diarrhea by real-time PCR (referred to as Dembo-PCR). Dembo-PCR can detect a
total of 19 pathogens in a single run, including 9 RNA viruses (BLV is targeted as a
provirus), 2 DNA viruses, 6 bacteria and 2 protozoa, within 3 hr.
In 2014, an outbreak of severe diarrhea occurred on a farm in Japan in the winter, and
decrease in milk production was observed in the affected cattle. BCoV is a pathogen that
causes “winter dysentery” and is one of the major infectious agents that causes epidemic
outbreaks in adult cattle. Cattle infected with
this virus occasionally present with severe diarrhea and reduced milk production with
weakening during the winter. In a previous report, BCoV was detected in more than 57.8% of
adult cattle suffering from diarrhea. Our
results showed that BCoV was detected in all diarrheal samples collected in January. Judging
from the results, gel-based PCR and the clinical findings, the epidemic outbreak of diarrhea
in January 2014 was caused by BCoV. In addition, diarrhea occurred after introducing one cow
(No. 6). Therefore, the outbreak is thought to have been caused by cow No.
On the other hand, both BCoV and BEV were detected in samples from cow No. 5 and healthy
cow (No. 7). Some studies have claimed that BEV infection may cause diarrhea in cattle
Conversely, other studies have shown that BEV infection is noncritical in cattle because of
its high prevalence in healthy cattle [1, 5]. The latter opinion agrees with our results showing
that the healthy cow sample was positive for BEV. Therefore, there is a low possibility that
BEV caused diarrhea in the cow. However, the pathogenesis of this virus remains to be
clarified. Additional evaluation of BEV pathogenicity should be conducted.
One calf (No. 8) was affected by diarrhea at 2 months after the first incidence of
diarrhea, although the outbreak of diarrhea in adult cattle had been stamped out. The
results showed that only BToV was detected in this calf. Previous studies showed that BToV
produces mild-to-moderate diarrhea in calves under both experimental and field conditions
The results obtained from Dembo-PCR suggested that diarrhea in the calf in this study was
caused by BToV. However, diarrhea can also be caused by noninfectious agents, such as
environmental factors and the condition of cattle immunity. Moreover, BToV can be detected occasionally in healthy cattle. The association
of BToV with diarrhea in this case was not obvious. A further study of how BToV causes
diarrhea in cattle should be conducted.
In this study, we describe the development and validation of a novel tool for differential
diagnosis of infectious diarrhea in cattle. Dembo-PCR has the advantage of being able to
detect known and unknown diarrheal pathogens. This system will be a powerful tool for
rapidly diagnosing the causes of this nuisance disease.
In this study, we identified a previously undescribed picornavirus present in stool and sewage. Phylogenetic analysis demonstrated that this virus is most closely related to other picornaviruses in the genus Kobuvirus. Based on the criteria established by the picornavirus study group, members of a genus should share > 40%, > 40% and > 50% amino acid identity in P1, P2 and P3 genome regions respectively. Klassevirus 1 shared only 43% amino acid identity in the P3 region and 33% amino acid identity in the P2 region to its closest relative, Aichi virus. Given these observations, and using strictly the percent identity definitions, klassevirus 1 may represent the first member of new picornavirus genus. However, we note that at all loci, bootstrap analysis suggests that klassevirus 1 diverged from an ancestor common to all of the known kobuviruses. Thus the formal classification of klassevirus 1 at the genus level is currently uncertain and subject to further discussion per the ICTV.
Subsequent screening by RT-PCR using primers targeting the 2C region of the genome established that klassevirus 1-like sequences were present not only in Australia, but also in North America and Europe. The presence of klassevirus 1 in the United States was determined by the traditional strategy of screening of individual stool samples. In addition, we also examined raw sewage collected in Barcelona to see if we could detect klassevirus 1. Sewage represents a pooled meta-sample of literally thousands of individual specimens. Known enteric viruses such as adenoviruses, noroviruses astroviruses, and hepatitis A have frequently been tested for and detected in sewage by PCR and RT-PCR. We reasoned that detection of klassevirus 1 in raw sewage would serve as a proxy for its presence in human stool in the population that generated the sewage. Since the exact history of the sewage is poorly defined, it is possible that other waste products, such as animal feces could contribute to the raw sewage meta-genome. Nonetheless, we propose that raw sewage screening from a diversity of sites can serve to rapidly define the geographic distribution of a given virus. The detection of klassevirus 1 in stool and sewage from Melbourne, Barcelona and St. Louis, demonstrates that klassevirus 1 is globally distributed. Moreover, since both the Barcelona sewage and St. Louis stool specimens were collected in 2008, we conclude that klassevirus 1 is currently circulating in the human population.
Whether klassevirus 1 represents a true human pathogen remains to be determined. It is possible that klassevirus 1 is a human pathogen that causes gastroenteritis. It is also possible that klassevirus 1 injures other organs but is excreted through the intestinal tract like poliovirus. Another possibility is that klassevirus 1 is a human commensal virus. Alternatively, klassevirus 1 could represent a non-human virus acquired from dietary exposure. Further investigations are needed to determine if klassevirus 1 is a causal agent of human disease(s). To begin addressing this question, epidemiologic studies including case-control and seroprevalence analyses are needed.
Several authors have tested the efficacy of immunoglobulin preparations with antibody activity against human rotavirus as a means of providing passive immunity to children. For example, children consuming a defatted colostrum preparation from cows immunized against a strain of human rotavirus had no improvement of symptoms when the infection was established (patients admitted to a hospital with rotavirus infection), however the preparation was effective in limiting diarrhea in children when consumed prior to the infection. In another study, cessation of excretion of rotavirus in the stool of infants with acute rotavirus gastroenteritis was correlated with the presence of neutralizing activity in the stool after ingestion of a bovine whey protein concentrate from rotavirus-hyperimmunized cows, although there was not a significant decrease in duration of diarrhea in that study. Other studies have found that treatment of children with hyperimmune bovine colostrum from cows immunized with human rotavirus serotypes reduces the duration and severity of diarrhea due to rotavirus, and can provide significant protection from rotavirus infection.
Enteropathogenic bacteria have also been the target for development of immune milk. Over 80% of childrens’ stools became negative for the E. coli strains used to hyperimmunize the cows that provided the source of immunoglobulin in a bovine colostrum/milk immunoglobulin concentrate consumed by children for 10 days. Interestingly, only one in nine children treated with the immunoglobulin concentrate, and having diarrhea that was associated with E. coli strains which were not used in the immunization of the cows, developed negative stools, underscoring the importance of the bacterial strain-specificity of the immune product. Consumption of a hyperimmune immunoglobulin concentrate with a high antibody titer against a lipopolysaccharide isolated from Shigella flexneri 2a also has been shown to provide protection against a challenge with the same strain. However, no difference in diarrhea or other symptoms in children with stools positive for S. dysenteriae was found whether treated with bovine colostrum from cows immunized against S. dysenteriae or with colostrum from cows not hyperimmunized.
Enterotoxigenic E. coli also is commonly associated with traveler’s diarrhea. Prophylaxis against this infection may be achieved by providing passive immunity with immune milk. A bovine whey protein concentrate from cows immunized with enterotoxigenic E. coli serotypes and consumed 3-times daily for seven days protected all of the adult volunteers from developing diarrhea after being challenged with an enterotoxigenic E. coli strain. In contrast, 90% of the volunteers who received control immunoglobulin concentrate prior to challenge developed diarrhea after the E. coli challenge. Subsequent studies using IgG isolated from bovine colostrum from cows hyperimmunized against specific E. coli colonization factor antigens also have shown protective effects in volunteers challenged with colonization factor antigen-bearing enterotoxigenic E. coli, however other studies by the same group did not demonstrate significant effects of similar milk immunoglobulin products.
Bovine colostrum concentrate preparations derived from cows that have not been hyperimmunized against specific antigens also may provide some benefit via passive immunization for some diseases. For example, a commercial product which is made from large standardized pools of colostrum collected from over 100 cows has been used to treat a number of diseases, including diarrhea caused by diarrheagenic E. coli. Similar preparations from non-immunized cows may provide protection against bacterial toxins that are the cause of diarrhea in AIDS patients. These studies, along with the above mentioned study comparing colostrum preparations from cows immunized against S. dysenteriae or non-immunized cows, demonstrate that bovine colostrum contains significant antimicrobial properties as a result of natural exposure of the cows to antigens of pathogens that may afflict humans.