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The majority of cases of CIRDC are currently believed to be viral in etiology and so antimicrobial administration is often not indicated. Most dogs with clinical signs of CIRDC including mucopurulent nasal discharge maintain normal appetite and attitude and might resolve spontaneously within 10 days without antimicrobial treatment. The Working Group recommends that antimicrobial treatment be considered within the 10‐day observation period only if fever, lethargy, or inappetence is present together with mucopurulent discharges.
If bacterial CIRDC is suspected in dogs with mucopurulent nasal discharge, fever, lethargy, or inappetence but no clinical evidence of pneumonia (eg, crackles or wheezes on thoracic auscultation), the Working Group recommends administration of doxycycline empirically for 7–10 days as the first‐line antimicrobial option (Table 1). Doxycycline is believed to have clinical activity against Mycoplasma. As in cats, doxycycline is well tolerated by dogs and isolates of B. bronchiseptica from dogs are typically susceptible in vitro to doxycycline.60, 66 However, the susceptibility testing studies used an unapproved standard. Optimal duration of treatment for dogs with bacterial causes of CIRDC is unknown and the 7–10‐day recommendation was based on the clinical experiences of the Working Group. Of the 17 reviewers, 15 (88%) agreed with this recommendation and 2 disagreed. One reviewer stated that if there is no evidence of pneumonia and the case is not at high risk of pneumonia (brachycephalic, collapsing airways; immunosuppressed), antimicrobial treatment is not indicated at all. The other dissenting reviewer disagreed with the recommendation because there is no breakpoint data for doxycycline for B. bronchiseptica or Mycoplasma spp. in dogs and so whether the agents are truly susceptible to the drug is unknown.
Additional antimicrobial susceptibility data for secondary bacterial agents like Pasteurella spp., Streptococcus spp., Staphylococcus spp., and anaerobes are needed. For Pasteurella spp. and Streptococcus spp., amoxicillin is usually adequate, whereas strains of Staphylococcus spp. are usually susceptible in vitro to amoxicillin–clavulanic acid. Thus, these antimicrobials are considered by the Working Group to be alternate first‐line antimicrobials for the treatment of secondary bacterial infections in this syndrome if treatment with doxycycline fails or is not possible (eg, it is not well tolerated). However, it should also be recognized that some B. bronchiseptica isolates and all mycoplasmas are resistant to amoxicillin–clavulanate. Of the 17 reviewers, 13 (77%) agreed, 3 reviewers (18%) disagreed, and 1 reviewer was neutral (6%). Reviewers that provided negative comments were concerned that because the concentrations of beta‐lactams in bronchial secretions are unknown for dogs and cats, the use of these drugs could be ineffective if tracheobronchitis without pneumonia was present. Another concern was that use of amoxicillin–clavulanate more likely selects for resistance phenotypes of clinical concern (eg, methicillin resistance in staphylococci).
Inhalational aminoglycoside treatment has been anecdotally mentioned as beneficial for the management of dogs with B. bronchiseptica‐associated CIRDC. However, in the absence of controlled studies for safety or efficacy, the Working Group does not recommend this treatment protocol for dogs with suspected bacterial CIRDC.
The clinical syndrome associated with CIRDC is generally characterized by an acute onset of cough with or without sneezing. Nasal and ocular discharges can also occur depending on the infectious agent that is involved. Fever is uncommon but might be present. The viruses that have been implicated include canine adenovirus 2, canine distemper virus, canine respiratory coronavirus, canine influenza viruses, canine herpesvirus, canine pneumovirus, and canine parainfluenza virus.55, 56, 57, 58, 59 Bacteria implicated as primary pathogens in this complex include B. bronchiseptica, S. equi subspecies zooepidemicus, and Mycoplasma spp.55, 59, 60, 61, 62, 63
Dogs with canine distemper virus infection often have diarrhea and can have mucopurulent ocular and nasal discharge that might be confused with mucopurulent discharges caused by primary bacterial pathogens. Because of its significance to the health of other dogs and for prognosis, the possibility of underlying distemper virus infection should always be considered in young dogs with mucopurulent ocular and nasal discharges, even when other signs of distemper are absent. Infection with S. equi subspecies zooepidemicus should be suspected if cases of acute hemorrhagic pneumonia or sudden death are reported.64
Co‐infections with multiple respiratory pathogens are common in dogs with CIRDC and each of the agents can be harbored by dogs with no clinical signs. Vaccines are available for some of the causes of CIRDC in some countries and include canine parainfluenza virus, canine adenovirus 2, canine distemper virus, H3N8 canine influenza virus, H3N2 influenza virus, and B. bronchiseptica. With the exception of canine distemper virus, the immunity induced by vaccination does not prevent colonization and shedding of the organisms and clinical signs of disease can develop in vaccinated dogs (2011 AAHA Canine Vaccination Guidelines; www.aahanet.org). However, morbidity is generally decreased in vaccinates compared with dogs that are not vaccinated when exposed to the pathogens.
Bacterial pneumonia is a serious lower respiratory tract infection in dogs with substantial morbidity and risk of mortality. Although BP was described in Dogs decades ago, information on the mechanisms leading to the development of the disease still is limited. Factors such as diseases predisposing to aspiration, immunodeficiency, or ciliary dysfunction that lead to impairment of pulmonary defense mechanisms and thereby predispose to the development of BP have been described.3 However, the role of preceding or concurrent infections with CIRD viruses has not been fully evaluated in dogs with BP, although it has been suspected to play a role in the etiology, as reported in humans with CAP.34, 35, 36, 37, 38, 39 Previously, respiratory viral‐bacterial co‐infections mostly have been reported in dogs housed in dense populations, such as kennels and rescue shelters, and bacteria accompanying viruses have been primary CIRD bacteria (B. bronchiseptica, S. equi sp. zooepidemicus, and Mycoplasma spp.).6, 7, 19, 44
Our study indicates that respiratory viruses, primarily CPIV, frequently are also found in dogs with BP, which is caused by opportunistic bacteria. Therefore, it is likely that CIRD viruses can predispose dogs to opportunistic bacterial lung infections by increasing bacterial adhesion, as has been reported in humans.35
In this study, CPIV was the most common viral pathogen detected, which is in accordance with previous reports describing viruses responsible for CIRD in different countries.6, 13, 19 Novel CRCoV was detected in 1 dog with BP, further demonstrating that CRCoV has a worldwide distribution and also may be detected in Northern Europe.
Canine parainfluenza virus was prevalent despite recent vaccination, which can be considered indicative of poor vaccine‐induced antibody coverage against CPIV. In contrast, CAV and CDV were not encountered in dogs vaccinated against these viruses. This finding is in accordance with previous reports. In a longitudinal study on respiratory viruses in a rehoming center in England, CPIV was commonly detected despite regular vaccinations, but CDV and CAV‐2 were not encountered, most likely because of adequate vaccination coverage.13 It remains unknown whether more efficient CPIV vaccines and possible CRCoV vaccinations could decrease the incidence of BP, as has been shown in humans, in whom protection against influenza and respiratory syncytial virus decreased the incidence of secondary bacterial infections.50, 51, 52
Nosocomial infections with respiratory viruses also have been reported in dogs. An outbreak of CPIV was described in an animal hospital and an outbreak of CHV was reported in immunocompromised dogs.53, 54 Because co‐infections with CIRD viruses are shown to be common in dogs with BP, the infection risk needs to be taken into account when treating BP patients in the same premises (eg, intensive care units) with immunocompromised patients.
Dogs with viral co‐infections were significantly heavier than those without virus infection. This finding might be influenced by the structure of the virus‐negative group: All 4 dogs with another predisposing factor for the development of BP were <20 kg (West Highland White Terrier, Dachshund, Spanish Water Dog, and Schnauzer). Dogs with viral co‐infections also were younger than those without viral co‐infection, although this did not reach statistical significance. This finding is not unexpected, because young animals might have insufficient acquired immunity against CIRD viruses.2
Clinical findings, arterial blood gas analysis, and hematology, as well as respiratory sample cytology in both groups were in accordance with previously reported findings in BP and did not differ between virus‐negative and virus‐positive groups.42, 43, 48 On thoracic radiographs, an alveolar pattern in the cranial and the middle lobes was predominant in both groups without group predisposition. Radiographic findings in dogs with BP have been thoroughly reported previously for cases of aspiration etiology.55, 56 In our study, radiographic findings in dogs with BP caused by other etiologies were similar to those reported for aspiration pneumonia. Aspiration etiology was considered unlikely, because none of the dogs with BP had a history of vomiting, regurgitation, recent anesthesia or signs compatible with laryngeal paralysis. Our findings could indicate that an alveolar pattern in cranial and middle lung lobes may be typical for pneumonia, regardless of etiology. On the other hand, aspiration pneumonia might have played a role in some dogs but could not be confirmed or denied based on available history, examination findings, or imaging. We were unable to identify clinical variables to reliably distinguish dogs with BP and viral co‐infection, and PCR testing therefore appears to be required to identify viral respiratory infections in dogs with BP. A similar finding was reported in humans.57
There were no significant differences in the duration of hospitalization (P = .427) or partial pressures of arterial oxygen at presentation (P = .343) between BP dogs with and without viral co‐infection, indicating that viral co‐infections do not appear to cause a more severe course of BP. In dogs, limited information is available on the severity of BP of different etiological origins, and in humans the reports are contradictory. Some studies have shown that mixed infections with viruses and bacteria induce a more severe clinical disease, whereas others have been unable to demonstrate significant differences in disease severity.49, 58, 59, 60, 61
Previous studies reporting microbiological findings in dogs with pneumonia have found growth of a single species of bacteria in 40–74% of cases.42, 43 All of these studies used TTW as a sampling method. Factors that might have influenced the finding of primarily a single species of bacteria in our study may be the use of BAL as a sampling method in majority of cases, compared to previous studies where TTW was used and the widespread use of prior antimicrobial treatments in these dogs.
Novel molecular methods have allowed the rapid testing of several respiratory pathogens simultaneously. Lower respiratory tract samples are considered ideal when diagnosing viral respiratory infections in humans with CAP, and it has been shown that virus‐positive PCR in BALF is associated with respiratory symptoms in humans.62, 63 Naturally, the invasiveness of retrieving BALF, compared to upper respiratory sampling, limits the usefulness of this accurate sample. However, especially when using molecular methods, virus recovery from the upper respiratory tract may be suggestive of virus exposure rather than indicative of an active viral infection.6 We chose lower respiratory tract samples in order to decrease the number of false‐positive results, but a comparison of PCR findings between upper and lower respiratory tract samples would be useful. Underestimation of virus‐positive PCR results may have occurred in our dogs in cases in which viral infection preceded BP and sampling was performed outside of the viral shedding period.
Bordetella bronchiseptica and Mycoplasma spp. were tested using both PCR and conventional culture methods. Polymerase chain reaction was, as expected, able to reliably demonstrate both pathogens in dogs with positive culture results. Additionally, Mycoplasma PCR was positive in 3 dogs with negative culture results. The clinical relevance of these positive results is difficult to interpret, because Mycoplasma spp. are also encountered in the respiratory tract of healthy dogs.64 On the other hand, because Mycoplasma requires special culture methods (and in this study also shipping to an outside laboratory), there might have been dogs in which Mycoplasma culture was falsely negative. Quantitative PCR might have aided in assessing the clinical relevance of these PCR findings.
Respiratory viruses were not detected in control dogs with prolonged BBTB. Bordetella bronchiseptica commonly accompanies CIRD viruses in acute respiratory infections and signs usually are self‐limiting.6, 19, 28, 29 Dogs with prolonged BBTB were considered more likely than those of the general dog population to have been exposed to CIRD viruses previously. Infections with CIRD viruses are self‐limiting within the first weeks, and because all BBTB dogs had prolonged clinical signs, an active viral infection therefore was considered unlikely.7, 9, 11, 65 Consequently, the negative results in the BBTB group are considered to increase the reliability of positive virus PCR findings in dogs with BP.
The most important limitation in this study was the small number of dogs in each group. This decreases statistical power (ie, the possibility of detecting a true difference between groups or reporting a difference that does not truly exist). Additionally, because this study was performed in Northern Europe in household dogs with low infection pressure, the results may not be applicable in all situations.
In conclusion, respiratory viruses, primarily CPIV, were found frequently in lower respiratory samples of dogs with BP and may play an important role in the etiology and pathogenesis of BP. Additionally, clinical variables and disease severity did not differ between BP dogs with and without viral co‐infection.
UTIs are common bacterial infections in dogs. To the best of our knowledge, this is the first case report of a dog with UTI caused by P. acnes.
Ureaplasmas, also belong to the same family as the Mycoplasmas, and are pathogenic bacteria which were initially associated with urogenital tract infections but have also been isolated from pneumonic bovine lungs [10, 68, 75]. The species Ureaplasma diversum has been associated with clinical respiratory disease [75, 76]. As Ureaplasma was found to be present in pneumonic lung tissue samples from calves which died from BRD, this genus may be an important contributor to BRD which is often overlooked.
Pleuritic lesions registered at slaughter ranged from 20.5 to 33.1 % in the four herds. High levels of serum antibodies to A. pleuropneumoniae and P. multocida, either alone or in combination, were seen. Pigs in this study seroconverted to M. hyopneumoniae late during the rearing period (herd B–D), or not at all (herd A), confirming a positive effect of age segregated rearing in preventing or delaying infections with M. hyopneumoniae. The results obtained highlight the necessity of diagnostic investigations to define the true disease pattern in herds with a high incidence of pleuritic lesions.
Mycoplasma was one of the most abundant OTUs present in the post-mortem lung and lymph node samples. It was found to be more abundant among the tissue samples from dairy calves which died from BRD relative to the tissue samples from the clinically healthy, lung lesion-free, H-F calves. This is consistent with previous studies which reported Mycoplasma to be one of the dominant genera in nasopharyngeal swab samples from cattle at feedlot [26, 33], despite being infrequent in cattle at feedlot entry. Additionally, this result is concordant with previous observations which report that Mycoplasma species bovis, dispar and bovirhinis, were identified more often in pneumonic lungs and respiratory tracts compared with clinically healthy lungs and respiratory tracts [25, 62, 68, 69].
Although Mycoplasma bovis is a recognised BRD pathogen and is commonly screened for in veterinary diagnostic laboratories [71, 72], the other major Mycoplasma species are not generally screened. However, they may be responsible for BRD as Mycoplasma dispar is a recognised pathogenic Mycoplasma species, capable of colonising the lower respiratory tract and caused pneumonia when inoculated into gnotobiotic calves. Moreover, it has been cultured from the respiratory tracts of calves presenting with BRD [63, 74] and has been isolated from lavage fluids of calves with recurrent respiratory disease. Furthermore, although Mycoplasma arginine and Mycoplasma bovirhinis did not cause pneumonia following inoculation into gnotobiotic calves, these species have also been isolated from lavage fluids of calves with recurrent respiratory disease and pneumonic lungs [68, 69] and have been suggested to act as co-pathogens which may intensify respiratory disease symptoms [69, 74].
There have been few reports of Propionibacterium infection in animals. Hodgin et al. reported a case of a dog with osteomyelitis and arthritis due to Propionibacterium infection caused by a dog bite. In our case, there was no history of trauma and the route of infection could not be identified. Several factors that predispose to UTI, such as diabetes, Cushing’ disease, and hypothyroidism, were ruled out in this case based on urine and blood analyses. Anatomic abnormalities were not identified by diagnostic imaging. Concurrent infections with viruses, yeasts, fungi, and M. canis, were not identified by serological and microbiological tests. In addition, this patient did not have a history of steroid use. Thus, we could not identify any factors to predispose this patient to P. acnes UTI.
One hypothesis is that this was an ascending urinary tract infection, a common source of UTI, as P. acnes is as a part of the normal flora of the skin and feces. Another possibility is translocation of bacteria from the intestinal tract, given that the patient had concurrent severe diarrhea caused by whipworm infection, which could have damaged the gut/blood barrier. A final possibility is that the whipworm infection may have made the dog more susceptible to anthroponotic infection and the bacteria may have been normal flora from a human in contact with the dog. In this case, P. acnes was not detected in urine via Gram staining. This was also demonstrated in a previous study, and should be taken into account when considering P. acnes infection as a differential.
Our case was diagnosed as emphysematous cystitis (EC), a rare type of UTI, based on several diagnostic imaging techniques. EC occasionally occurs in diabetic dogs but is relatively rare in nondiabetic dogs. EC results from an infection by gas-producing bacteria, including E. coli, Proteus spp., Aerobacter aerogenes, and Clostridium spp.. While P. acnes has not been reported to cause EC in dogs previously, gas production was noted in a human with P. acnes infection. Thus, P. acnes should be regarded as a gas-producing bacterium and a potential cause for EC in dogs.
Our P. acnes isolate was identified as sequence type 53. This type has been isolated from human cases of acne and meningitis and reported in the MLST database. In vitro antimicrobial susceptibility testing showed that our isolate was highly susceptible to most of the antimicrobials tested, except metronidazole, an agent to which P. acnes is consistently resistant. A similar finding was reported for isolates from human cases of implant-associated infection. Thus, our isolate shared sequence type and antimicrobial susceptibility with human isolates. However, the MLST database of P. acnes only contains human isolates, which prevents determination as to whether the bacteria were from a human source. The addition of P. acnes strains from dogs and other animals to the MLST database would be helpful in future epidemiological analyses.
This case’s condition rapidly worsened despite administration of enrofloxacin, to which the P. acnes isolate was susceptible. This case developed urinary retention due to the formation of blood clot, which is a risk factor for pyelonephritis. In addition, in this case, renal function decreased concurrently with the development of urinary retention. Therefore, antimicrobial treatment may have had poor efficacy as a result of the development of acute pyelonephritis.
Mycoplasma alkalescens is a bovine mycoplasma species, which was originally isolated from the nasal cavity of cattle in Australia. Like many other mycoplasmas, M. alkalescens is a normal inhabitant of the upper respiratory tract, but it has also been associated with disease. M. alkalescens has mostly been implicated in mastitis in cattle. It has been isolated from bulk tank milk samples, as well as from outbreaks and sporadic cases of clinical mastitis. Furthermore, M. alkalescens has been isolated from cases of severe arthritis, and its ability to induce joint lesions has been confirmed under experimental conditions. Rosenfeld & Hill isolated M. alkalescens in pure culture from abomasum and lung of an aborted bovine foetus, while Lamm et al. found M. alkalescens in association with otitis in calves. Finally, M. alkalescens has occasionally been found in association with disorders of the respiratory tract.
Recently, 17 bronchoalveolar lavage samples from calves suffering from pneumonia in a single herd in Denmark were submitted, on two occasions, to the National Veterinary Institute for laboratory examinations. The samples were examined for the presence of bacterial pathogens, bovine respiratory mycoplasmas, as well as for the presence of bovine respiratory syncytial virus (BRSV), bovine coronavirus and parainfluenza-3 virus (PI-3). The samples were not examined for infectious bovine rhinotracheitis virus, as Denmark is considered free of this infection. Thereby, eight arginine-metabolizing mycoplasmas were isolated. In the present study the identification of the isolates as M. alkalescens is presented. This is the first report of isolation of this species in Denmark.
Bacteriological examination was performed according to standard laboratory procedures. Examination for BRSV, bovine coronavirus and PI-3 was performed using an indirect sandwich-ELISA assay. Isolation of mycoplasmas was performed according to standard laboratory procedures using a Hayflick's type of medium enriched with arginine, with and without addition of 5% of rabbit hyperimmune antiserum against Mycoplasma bovirhinis. The isolates were filtered through 0.45 μm membrane filters (Millipore), cloned and submitted to serologic identification, which was performed by the disc growth inhibition test (DGI) with 6 mm filter paper discs prepared with rabbit hyperimmune antisera against M. alkalescens PG51T (ATCC 29103; NCTC 10135), Mycoplasma arginini G230T (ATCC 23838; NCTC 10129) and Mycoplasma canadense 275CT (ATCC 29418; NCTC 10152), as well as by the indirect epi-immunofluorescence test (IF) on colonies on solid medium. Molecular identification of the isolates was performed by amplification and sequencing of their 16S rRNA genes by using universal primers.
Laboratory examinations revealed the presence of several bacterial pathogens in 15 of the 17 bronchoalveolar lavage samples, while no pathogenic bacteria were detected in the two remaining samples. Pasteurella multocida, Mannheimia haemolytica, Histophilus somni and Salmonella Dublin were found in three, three, one and one samples, respectively. Mycoplasma bovirhinis was found in five samples, while arginin-metabolizing mycoplasmas were found in eight samples. Two P. multocida, three M. bovirhinis and five arginin-metabolizing mycoplasma isolates were found as sole bacteriological findings in 10 respective samples. From five samples multiple bacterial species were isolated: M. bovirhinis was found once in combination with P. multocida and once in combination with M. haemolytica, while arginin-metabolizing mycoplasmas were found once in combination with M. haemolytica, once in combination with S. Dublin and once in combination with M. haemolytica and H. somni. No viral pathogens were detected in any of the analysed samples.
Further examinations on arginin-metabolizing mycoplasmas showed that all eight isolates could be serologically identified as M. alkalescens and that they were clearly different from the two other arginine-degrading species that are commonly found in cattle, M. canadense and M. arginini (Table 1). The reactions were clear-cut for the DGI tests showing approximately 3–5 mm broad zones of total or nearly total inhibition around the disc. In the IF test some minor cross reactions were noted when three of the isolates and the type strain of M. alkalescens PG51T were tested with anti-M. canadense 275CT hyperimmune serum. The 16S rDNA sequences of the analysed isolates were identical to each other and to the 16S rDNA sequence of the type strain M. alkalescens PG51T (GenBank accession no. U44764;), which corroborated the serological identification of the isolates as M. alkalescens.
Previous studies have shown that H. somni, M. haemolytica and P. multocida are the bacteria that are most commonly associated with bronchopneumonia in calves in Denmark, although they are also part of the normal bacterial flora of the respiratory tract. Also, S. Dublin, which is predominantly found in cattle, is capable of causing pneumonia in calves. Isolation of these bacterial species in the submitted samples most likely indicate their role as the primary cause of respiratory disease in the herd, probably in combination with unidentified environmental factors. In addition to the bacteria, M. bovirhinis and M. alkalescens were also isolated. M. bovirhinis is commonly found as a part of the normal respiratory tract microflora of cattle and is not considered to be pathogenic. M. alkalescens, however, has been found in association with disorders of the respiratory tract but its role as a respiratory pathogen remains equivocal. Experimental infections have demonstrated that M. alkalescens has the ability to colonize lung tissue, but the two strains used in the study apparently failed to produce pneumonia. In the present study, we found M. alkalescens in pure culture in five of the analysed bronchoalveolar lavage samples. This finding is, however, not sufficient to warrant a role of M. alkalescens as a cause of bronchopneumonia, since we also found two samples containing only a non-pathogenic M. bovirhinis, and two samples without any bacterial or viral pathogens, despite the fact that they derived from calves with clinical signs of a respiratory disease. The failure to detect respiratory tract pathogens in these samples may be due to i.e., their absence in a particular disease stadium when sampling took a place or due to antibiotic treatment. Taking into the consideration the overall bacteriological findings of this study, it seems likely that M. alkalescens may have had a role either as a secondary invader or as an opportunistic pathogen rather than suggesting a causal role of the organism in the pneumonia complex.
M. alkalescens is regarded as one of the most common causative agents of mastitis. So, with demonstration of the organism in a Danish cattle herd, a further member of Mycoplasma is added to the group of bovine mastitis-inducing microorganisms in Denmark. The only mycoplasma species isolated from clinical outbreaks of mastitis in Denmark so far has been Mycoplasma bovis, while other mastitis-inducing species, Mycoplasma bovigenitalium and M. canadense, have been isolated only from the respiratory and the genital tract and semen samples. Further investigations are needed in order to determine the prevalence of M. alkalescens in the Danish cattle population and, indeed, to draw a firm conclusions on its importance in disease conditions other than mastitis.
Respiratory illness is traditionally regarded as the disease of the growing pig, and has historically been associated with bacterial infections such as Mycoplasma hyopneumoniae [1–3] and Actinobacillus pleuropneumoniae [4–6]. These bacteria still are of great importance, but the continuously increasing herd sizes have complicated the clinical picture. As the number of transmission events between pigs in a population is equal to the number of pigs multiplied with the number of pigs minus one [x = n * (n − 1)], they will escalate as the herd size increase. Thus, the number of transmission events between pigs will increase with a factor of around four if a population is doubled and with a factor of around 100 if a population is enlarged ten times.
The increased number of transmissions between pigs may increase the influence of other microbes. M. hyopneumoniae and A. pleuropneumoniae are important pathogenic microbes, but co-infections may intensify or prolong clinical signs of respiratory disease [8–11]. It has also been observed that the incidence of respiratory illness may vary with season. Therefore, infections in the respiratory tract of grower pigs have become regarded as a syndrome rather than linked to single microorganisms [11, 13, 14]. This syndrome is referred to as the porcine respiratory disease complex (PRDC). As stated above PRDC is regarded to be dominated by bacterial species, and important primarily pathogenic bacterial species include M. hyopneumoniae [1–3] and A. pleuropneumoniae [4–6]. The frequent demonstration of interferon-α in serum in growers during the first week after arrival to fattening herds [15, 16] suggest that PRDC can be associated with viral infections, and that PRDC can also include the influence of secondary invaders such as Pasteurella spp [17, 18].
When Sweden in 1986 as the first country in the world banned the use of low dose antibiotics in animal feed for growth promotion, some introductory health disturbances were recorded. As a consequence, a strict age segregated rearing from birth to slaughter was implemented in a large scale, which improved health as well as productivity [19, 20]. As seen in Fig. 1, the incidence of recorded pathogenic lesions in the respiratory tract at slaughter decreased during the last decade of the twentieth century. The registrations of pneumonia at slaughter has remained stable at that level since then. In contrast, the incidence of recorded pleuritis at slaughter has continuously increased since the year 2000, as has the clinical evidence of actinobacillosis. Discussions concerning the reason for this increase has included suggestions of introduction of new strains, or mutation of existing strains of A. pleuropneumoniae. However, acute actinobacillosis has in Sweden historically been dominated by serotype 2, and is still dominated by that serotype. Further, Pulse Field Gel Electrophoreses has revealed that strains isolated in the twenty-first century were identical to strains isolated in the 1970s and 1980s. As a consequence, the increase of actinobacillosis and pleuritic recordings at slaughter has merely been linked to the continuously increasing herd sizes with increasing number of transmissions of microbes between pigs, within and between units.
The aim of this study was to validate the presence of A. pleuropneumoniae and M. hyopneumoniae, as well as the secondary invaders P. multocida and Streptococcus suis in pig herds with a high incidence of pleuritic lesions at slaughter.
Pneumonia is a significant cause of morbidity and mortality in calves, both during the pre-weaning period and shortly following weaning. A range of events are linked with increased disease risk, including weaning management, painful procedures, housing systems and ventilation and effective preventive measures have been demonstrated. The management of pneumonia in calves is reliant on a sound understanding of aetiology and of relevant risk factors and of effective approaches to diagnosis and treatment.
Health surveillance of laboratory animals is conducted since adventitious infection is a realistic possibility that can have a significant, negative impact on animal research (9). Although infectious interstitial pneumonia, a prevalent and transient interstitial pneumonia of immunocompetent laboratory rats, had been attributed to a putative virus referred to as RRV, the etiology of infectious interstitial pneumonia has not been established (4,22). In the present study, naive rats housed on 50% soiled bedding from the cages of colony rats with RRV-type lesions were shown to develop lesions identical to those of the colony animals. In addition, P. carinii was found to be an etiologic agent of infectious interstitial pneumonia in a group of immunocompetent laboratory rats. The observations support previous data that demonstrated the transmissibility of the condition. Widespread presence of P. carinii has been documented in the lungs of clinically-healthy, commercially-produced immunocompetent rats (5,23). The infectivity of P. carinii in immunocompetent laboratory animals has also been supported by previous studies (7,8,15,16,19,24). Henderson et al (8) demonstrated that P. carinii infection was transmitted to immunocompetent rats by bedding transfer and direct contact with contagious animals. In addition, Gigliotti et al (16) demonstrated that brief cohousing with P. carinii-infected mice resulted in infected immunocompetent mice. There was active replication of the organisms in the immunocompetent host such that the organism was transmitted to other Pneumocystis-free immunocompetent mice, again with active replication. Similarly, Chabé et al (19) found that healthy host-to-healthy host transmission of Pneumocystis organisms can occur, and that Pneumocystis organisms are able to replicate in the lungs of immunocompetent hosts, indicating that these hosts are a reservoir for P. carinii. The observations of the present study are consistent with these studies.
In addition, the results of the present study provide a histopathological description of the PCP time course in immunocompetent rats. Consistent with previous results (6–8), characteristic lesions of PCP were observed, consisting of multifocal lymphohistiocytic interstitial pneumonia and dense perivascular cuffs of lymphocytes and macrophages around the blood vessels. Foamy eosinophilic exudates containing GMS-positive Pneumocystis cysts were also detected. Gross lesions appeared during week 7, while the microscopic lesions necessary for diagnosis appeared slightly earlier at week 5. The time course of the observations indicated that the lesions were mild at week 5, severe lesions appeared between week 7 and 10 and resolution occurred by week 14 following exposure. This time course is similar to that observed in a previous study by Albers et al (6), but not consistent with that of a recent study by Livingston et al (7) that found that the lesions in rats were first observed at week 3, most severe at week 5 and were resolved by week 7. The reason for this difference in lesion onset, progression and resolution is unknown, but may reflect several factors, including the timing of infection, strain of Pneumocystis, strain of rat and other environmental factors.
It is well-known that immunocompetent rats can be subclinically infected with Pneumocystis spp., but the development of PCP was considered to occur only in immunodeficient rats due to genetic factors (for example, nude rats) or artificial interventions (for example, immunosuppressive doses of glucocorticoids) (5,23,25). Thus, immunocompetent rat colonies have not been routinely tested for Pneumocystis infections. Now that an etiology for RRV-type lesions has been identified, Pneumocystis-specific diagnostic tests, including histopathological examination for the identification of pneumonia and other organisms and PCR assays for the detection of Pneumocystis DNA, can be used to detect infected rats and monitor colonies for Pneumocystis infections. Previously, histopathology was the only diagnostic assay available to detect rats with idiopathic pneumonia, now known as PCP (6). However, high-throughput immunoassays to detect serum antibodies against Pneumocystis and quantitative PCR to quantify Pneumocystis DNA expression may also be used in the testing of rat colonies for this pathogen.
Although the present study documented the role of P. carinii as the causative agent of significant lung pathology in immunocompetent rats, it is unknown whether other Pneumocystis spp. are capable of causing a similar disease. P. carinii and P. wakefieldiae are the two Pneumocystis spp. that have been documented to infect rats used in biomedical research, however, at least three other provisional Pneumocystis spp. have been identified in wild rats (25,26). P. carinii can occur as a monoinfection or coinfection with P. wakefieldiae (5,26). The possibility that other Pneumocystis spp. cause pneumonia in immunocompetent rats cannot be eliminated, thus, excluding all Pneumocystis spp. from rat colonies in which Pneumocystis pneumonia is unwanted is recommended. Further investigation is necessary to address the pathogenic potential of P. wakefieldiae in immunocompetent rats.
It is well established that P. jirovecii infections cause severe pneumonia in immunocompromised humans and are a leading cause of mortality in patients with acquired immunodeficiency syndrome. However, evidence is starting to be amassed associating subclinical P. jirovecii infections in immunocompetent humans with several diseases in infants and adults, including sudden infant death syndrome, chronic obstructive pulmonary disease, asthma, bronchiolitis and other lung conditions (27). The marked, predictable lung pathology in immunocompetent laboratory rats that are naturally infected with P. carinii may provide a possible animal model for P. jirovecii infection in immunocompetent humans.
Limitations of the data presented in the current study should be acknowledged, including the presence of sampling error. The data are merely compilations of results from the sample stream passing through the laboratory and were not selected as representative samples from entire populations. In addition, since the observations are derived almost exclusively from the testing of laboratory rats, typically of a specific pathogen-free health status, the data cannot be construed to indicate that rodents reared as pets or to feed raptors or reptiles are of a similar health status as specific pathogen-free rats. Furthermore, as the sources of P. carinii for the experiments were infected rat lungs and not pure cultures, it cannot be claimed categorically that no other agent is involved in the pathogenesis of interstitial pneumonia. For instance, a virus transmitted with P. carinii may contribute to the disease by facilitating Pneumocystis colonization of the lower respiratory tract in immunocompetent hosts. However, the participation of an infectious agent other than P. carinii in the pathogenesis of interstitial pneumonia is highly improbable for the following reasons. Firstly, routine surveillance of the colony used in the experiments did not detect any known pathogens. Secondly, novel pathogens are infrequently found in long-used and intensively characterized laboratory animal species, such as the rat.
In conclusion, the results of the present study demonstrate that P. carinii, and not a virus, is the causative agent of lymphohistiocytic interstitial pneumonia in a group of laboratory rats. The observations strongly support the conclusion that P. carinii infection in immunocompetent laboratory rats causes the lung lesions that were previously attributed to RRV.
Bovine respiratory syncytial virus (BRSV) is an economically significant pathogen in cattle production, as it is one of the most important causes of lower respiratory tract infections in calves. In dairy cattle, BRSV infection usually occurs in young calves aged between 2 weeks and 9 months. Adult animals with subclinical infection are the main source of infection, since reinfections are common in the herds [1, 4, 5].
BRSV, bovine herpesvirus 1 (BoHV-1), bovine viral diarrhea virus (BVDV) and bovine parainfluenza type-3 (PI-3) are considered primary agents involved in the bovine respiratory complex. Additionally, secondary infection by Pasteurella multocida, Histophilus somni and mycoplasmas contribute to the aggravation of the disease. Clinical signs are characterized by respiratory symptoms, initially with moderated intensity, such as nasal and ocular discharges which can be aggravated leading to pneumonia. However, mainly in calves, an acute and severe onset is also observed, due to maternal antibodies not effectively protect against BRSV infection.
Considering the high prevalence of the disease, several studies determined risk factors involved in the epidemiology of BRSV. In Europe, risk factors were mainly attributed to herd size, herd density, purchasing of new animals, geographic location of the farms, herd type and concomitant BVDV infection [7–11]. Similar studies have also been performed in some Latin American countries and they showed that most of the animals probably have already been exposed to the virus with consequent high BRSV prevalence in cattle herds. In these countries, herd size, age group, presence of bordering farms, herd type and geographic location of the farms were the main risk factors associated with BRSV infection [12–16].
In Brazil, BRSV was first diagnosed in calves in the state of Rio Grande do Sul and some studies have shown that BRSV infection is widespread in Southern and Southeastern Brazil, with high serological prevalence rates [18–20]. Nevertheless, research has not been conducted in order to verify possible risk factors involved in BRSV epidemiology. Due to this, the current study aimed to determine antibody prevalence against BRSV and investigate some risk factors associated with BRSV seroprevalence in herds of an important milk producing region in São Paulo State, Brazil.
The disease is caused by Corynebacterium pseudotuberculosis. There are two basic forms of caseous lymphadenitis, that is, internal form and external form. Most of the affected animals manifest both forms of the disease depending on the multiple factors that are age, physiological conditions, environmental factors, and managemental practices. There is obvious nodule formation under the skin as well as enlargement of peripheral lymph nodes in the external form. The affected lymph nodes along with the subcutaneous tissues are enlarged with thick as well as cheesy pus which may rupture outward spontaneously or during the process of shearing or dipping. The internal form of caseous lymphadenitis (CLA) is manifested by vague signs such as weight loss, poor productivity, and decrease in fertility [3, 148, 149]. For the detection of the causative agent, Corynebacterium pseudotuberculosis, in sheep and goats, a double antibody sandwich ELISA has been developed, which has been further modified for improving the sensitivity. The main objective of developing this test is to detect the presence of antibodies against the bacterial exotoxin. It has been found that six proteins with varying molecular mass ranging from 29 to 68 kilo Dalton (kDa) react with sera from both goats and sheep acquiring infection experimentally or naturally. For classification of the sera with inconclusive results, immunoblot analysis has been found to be valuable [100, 101]. Quantification of interferon gamma (IFN-γ) is essential for accurate diagnosis of the disease for which an ovine IFN-γ ELISA has been developed. The sensitivity of the assay is slightly more for sheep than in goats while the specificity of the assay is higher for goats than for sheep. It can thus be concluded that IFN-γ is a potential marker in order to determine the status of CLA infection in small ruminants. For the diagnosis of CLA, another novel strategy is the employment of PCR for identification of the bacteria isolated from abscesses. The PCR has been found to be both sensitive and specific in addition to its rapidity of detecting C. pseudotuberculosis from sheep that are naturally infected.
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.
The author(s) declare that they have no competing interests.
Pericardial disease is not commonly encountered in cats. Previous studies have reported that the overall incidence of pericardial disease
in cats ranges from 1 to 2.3% [6, 22], with congestive heart failure secondary to cardiomyopathy being the
most common cause in cats. Other causes of
pericardial disease in cats include neoplasia, trauma, peritoneopericardial diaphragmatic
hernia, feline infectious peritonitis (FIP), disseminated intravascular coagulation, renal
failure and infective pericarditis [9, 27, 29]. Infective
pericarditis has rarely been reported in cats, and the reported causes of this disease include
dental infection, pneumonia, abscess, peritonitis, pyometra or idiopathic [14, 17, 19, 22]. In these
cases, the long-term use of appropriate antibiotics could induce clinical remission [14, 19]. However,
fibrinous pericarditis, which is well recognized in cattle, has a poor prognosis and has rarely been reported in cats and dogs [20, 23].
In the present study, we described the clinical and histological findings of a feline case of
fibrinous pericarditis presumably caused by Moraxella osloensis.
A three-year-old spayed domestic short-haired cat, weighing 3.3 kg, presented to a primary
care veterinarian with a one-week history of lethargy, anorexia, dyspnea and weight loss. The
cat was group fed, liver indoors, and had no history of trauma. Thoracic radiography revealed
cardiomegaly and pleural effusion. Due to lack of response to antibiotics, the cat was
referred to the Veterinary Medical Center, Obihiro University of Agriculture and Veterinary
At the time of presentation, salient physical examination findings were tachycardia (224
beats per min), tachypnea (32 breaths per min), lethargy and thin body condition (3/9). The
remaining physical examination was unremarkable. Initial laboratory data revealed the
following abnormalities; anemia (hematocrit, 24.4%; reference limits, 34–51%), neutrophilia
(32.2 × 103/µl; reference limits, 2.3–9.8 ×
103/µl), hypoalbuminemia (2.2 g/dl; reference
limits, 2.5–3.9 g/dl), hyperglobulinemia (5.2 g/dl;
reference limits, 2.6–5.0 g/dl) and low blood urea nitrogen (8
mg/dl; reference limits, 14–28 mg/dl). Feline
immunodeficiency virus (FIV) antibody and feline leukemia virus (FeLV) antigen test (IDEXX
Laboratories, Westbrook, ME, U.S.A.) were negative. Thoracic radiography revealed an enlarged,
globoid cardiac silhouette with a large volume of pleural effusion and dorsal deviation of the
trachea (Fig. 1). Echocardiographic examination demonstrated a thickened pericardium with
mild pericardial effusion and a large volume of pleural effusion (Fig. 2). Following appropriate aseptic skin preparation, thoracocentesis was performed using a
21-gauge needle, and approximately 100 ml of pleural effusion was removed.
Fluid analysis revealed an exudate effusion with a total nucleated cell count of 162,000
cells/µl, consisting of 30% macrophages and 70% degenerative neutrophils,
and a total protein level of 3.8 g/dl. Although the pleural effusion was
submitted for aerobic and anaerobic bacterial culture tests, both cultures eventually returned
negative. Fine-needle aspiration of the thickened pericardium was performed, and cytologic
evaluation indicated a large amount of degenerative neutrophils with fewer macrophages,
lymphocytes and fibroblasts. No neoplastic cells or bacteria were identified. FIP virus
polymerase chain reaction (PCR) performed using the pleural effusion sample tested negative,
and feline coronavirus titer was medium (1:200) (IDEXX Laboratories).
The cat was treated with ampicillin (20 mg/kg IV q12h) and intravenous lactated Ringer’s
solution (3 mg/kg/hr). However, the clinical symptoms did not improve, and thoracic
radiography revealed a recurrence of the pleural effusion on day 6 of hospitalization.
Thoracocentesis was performed again, and approximately 100 ml of pleural
effusion was removed. The cat was hypothermic (35.9°C), and enrofloxacin (5 mg/kg SC q24h),
furosemide (2 mg/kg SC q12h) and benazepril hydrochloride (0.83 mg/kg PO q24h) were added to
the treatment regimen. Despite these measures, the cat developed dyspnea and died on day
Necropsy revealed a large volume of modified transudates cloudy abdominal (total nucleated
cell count of 3,200 cells/µl, and total protein level of 3.5
g/dl) and pleural effusion (total nucleated cell count of 4,400
cells/µl, and total protein level of 3.0 g/dl). The heart
was encapsulated by a pale-yellow, fibrinous substance (Fig. 3). In the cross section of the heart, the epicardium was covered with an approximately
0.5- to 1.0-cm layer of fibrin (Fig. 4). The fibrinous layer adhered to the epicardium strongly and could not be separated
easily. In addition, there was a 1.5-cm abscess adhered to the pericardium and cranial segment
of the left cranial lung lobe. Histopathological examination revealed severe exudation of
fibrin with neutrophil infiltration around the epicardium. In particular, an organized
granulation tissue was noted between the surface of the epicardium and the fibrinous layer
(Fig. 5). Bacterial colonies were also seen in the fibrinous layer (Fig. 6A). Special stains revealed that the bacterial colony consisted of small cocci that were
negatively stained with Gram’s stain (Fig. 6B and
6C). Based on these findings, the cat was diagnosed with fibrinous pericarditis
secondary to bacteria. Aerobic and anaerobic bacterial cultures were performed using samples
from the abscess, pericardial fibrinous layer, pleural effusion and ascites. The aerobic
culture performed using the abscess sample showed the growth of Moraxella
osloensis, a gram-negative coccus, and the anaerobic culture showed the growth of
Bacteroides pyogenes, a gram-negative bacillus. Moraxella
osloensis was also detected in the ascites sample. In addition,
Brevundimonas vesicularis, a gram-negative bacillus, was detected in the
abscess, ascites and pleural effusion samples. Taking into account the morphological
characteristics of the bacteria observed in the fibrinous layer, Moraxella
osloensis was suspected as the causative agent.
A previous report documented the case of cat that developed bacterial pericarditis secondary
to bacteremia from a dental procedure, which was caused by a common oropharyngeal
microorganism, Peptostreptococcus. Another case report described bacterial pericarditis in a cat with pyometra and
hematogenous spread of Escherichia coli. In addition, Enterobacteriaceae and
Staphylococcus spp. infections resulted in septic pericarditis in another
cat. Furthermore, Pasteurella multocida,
Actinomyces canis, and Fusobacterium and
Bacteroides spp. were detected in the pericardial fluid of a cat with
septic pericarditis. In the present report,
Moraxella osloensis, Bacteroides pyogenes and
Brevundimonas vesicularis were detected from the abscess, ascites and
pleural effusion. Moraxella osloensis is a normal commensal of the upper
respiratory tract in humans, and
Moraxella spp. are commonly detected in the healthy feline oral cavity
. Bacteroides spp. have been
isolated from the oral flora of cats, and
Bacteroides pyogenes has been detected in cat bite wounds in a human
vesicularis has been isolated from the external environment. It was reported that Moraxella osloensis and
Brevundimonas vesicularis could induce bacteremia and/or infective
endocarditis in immunocompromised human patients [8,
16, 28]. In
this study, histopathological examination revealed gram-negative, small cocci in the fibrinous
layer. Although Moraxella osloensis was not isolated from the bacterial
cultures of the pericardium in the present case, Moraxella osloensis was
suspected as the causative agent based on the morphologic appearance and gram staining of the
bacteria in the fibrinous pericardium. The cat reported here was not infected with FIV and
FeLV, which are known to cause immunosuppression in cats. On the other hand, a part of the
cat’s left lung was abscessed and adhered to the pericardium, suggesting a possible
respiratory origin of the bacteria. Although the infection course of these pathogens is
unidentified, it was suggested that inflammatory processes in adjacent lung parenchyma might
produce the abscess and cause the spread to the pericardium.
Previously published reports on bacterial pericarditis in cats have shown that retention of
the pericardial fluid is the main constituent, and fibrin exudation is not as severe as seen
in the present case. Based on the necropsy and histopathological examination findings, we
diagnosed this cat with fibrinous pericarditis due to bacterial infection. Fibrinous
pericarditis, which is well recognized in cattle, has rarely been reported in dogs, horses and
pigs [1, 4, 5, 23]. In cats, only
one case of the fibrinous pericarditis caused by FIP has been reported. Feline coronavirus titer of the case was medium (1:200). Therefore, we
suggested that antibody titers measured in the serum be interpreted cautiously, and that
medium titers do not have any value in diagnosing FIP. In addition, many healthy cats exposed to the feline enteric coronavirus have
titers ranging from 1:100 to 1:400. PCR
examination for the detection of FIP using a pleural effusion sample was negative. Compared
with serological tests, PCR provides the obvious advantage of directly detecting the
infection, together with higher sensitivity and specificity in case of cell-free body cavity
effusion [7, 10].
Moreover, the histopathological findings of the pericardium in the present case were not
identical to the typical findings of FIP infection.
The appropriate therapeutic approach for infective pericarditis in cats is unclear because of
the low incidence and small number of published case reports. In general, therapy for
infective pericarditis requires pericardiocentesis for drainage of the purulent effusion and
the administration of antimicrobial agents.
Appropriate antibiotic therapies have reportedly resulted in complete resolution of the
pericardial effusion in two cats [14, 19]. In contrast, pericardiectomy is the treatment of
choice for dogs and cats with evidence of infectious pericardial disease [2, 17, 25]. In human medicine, pericardiectomy is typically
reserved for people who develop constrictive pericarditis. Doppler echocardiography and cardiac catheterization have also been used for
the diagnosis of constrictive pericarditis in cats [18,
25]. In this case, aerobic and anaerobic bacterial
cultures using pleural effusion samples were performed at the time of presentation to
VMC-OUAVM. However, no bacteria were cultured and antibiotic selection based on susceptibility
testing could not be performed. Because empirically administered antibiotics could not improve
the clinical symptoms in the present case, broad-spectrum antibiotics should have been started
immediately. In this case, a large amount of ascites and pleural effusion characterized by
modified transudates were seen at necropsy. It was thought that fluid administration converted
the exudative effusion to a modified transudate. In addition, severe fibrin exudation and
subsequent organization resulted in constrictive pericarditis and induced decompensation and
congestive heart failure. It might be necessary to use cardiotonic agents for the purpose of
improvement of the heart failure, and pericardiectomy might be suitable in such cases.
Effusive-constrictive pericarditis induced by coccidioidomycosis and several neoplastic
pericardial diseases have been reported in dogs [11,
12]. However, these findings were not seen on
histological examination in the present case.
In conclusion, fibrinous pericarditis is a rare in cats, and empirical administration of
antibiotics without surgical management could be insufficient for the treatment of the
fibrinous pericarditis. This report described a rarely case of fibrinous pericarditis in a cat
secondary to bacterial infection. Based on the morphological characteristics of the bacteria,
Moraxella osloensis was suspected as the causative agent.
In the PCR analysis of BALF or TTW fluid, CPIV was detected in 7/20 (35%; 95% confidence interval, 14–56%) and CRCoV in 1/20 (5%; 95% confidence interval, 0–15%) dogs with BP. CAV‐2, CHV, CIV, CDV, or CnPnV were not detected in any of the samples. Dogs with positive virus PCR did not have other diseases predisposing to BP. Respiratory viruses were not detected in dogs with BBTB.
Respiratory samples were retrieved using BAL in 13/20 and TTW in 7/20 dogs diagnosed with BP. BAL was used as a sampling method in all dogs with BBTB. Results from cytology analysis of BALF and TTW fluid in BP dogs are presented in Table 3. Because BALF and TTW fluid cytology did not differ significantly in other variables aside from the percentage of epithelial cells (median in BALF, 0.0; interquartile range, 0.0–0.0 versus median in TTW fluid, 0.7; interquartile range, 0.0–14.7; P = .008), combined results are represented for the 2 sampling techniques.
Altogether, 11/20 dogs with BP had received antimicrobials before sampling. Significant bacterial growth (≥103 colony‐forming units/mL in BALF or TTW fluid) was identified in the primary culture in 11/20 samples (5/11 dogs with prior antimicrobial treatment). A single species was isolated in 8/11 samples, including Pasteurella sp. (1/10), Escherichia coli (2/10), Streptococcus sp. (2/10), Hemophilus sp. (1/10), Mycoplasma sp. (1/10), and Nocardiopsis sp. (1/10), and 2 species in 3 samples, Pasteurella sp. and Mycoplasma sp.47 Intracellular bacteria were seen in 6/11 dogs with significant bacterial growth in primary culture. In 1 dog with a negative primary culture and prior antimicrobial treatment, >2 intracellular bacteria/oil immersion field were demonstrated, and Actinomyces sp. was cultured after enrichment.47 Positive bacterial growth was detected only after enrichment in 4/20 dogs (single species of bacteria, including Streptococcus sp. [1/4], Pasteurella sp. [1/4]), Haemophilus sp. [1/4], and Actinomyces sp. [1/4]), 2 of these dogs had received prior antimicrobial treatment. A negative bacterial culture in airway samples was recorded in 4/20 dogs (2 had received prior antimicrobial treatment), but they showed an acute onset of respiratory signs and had new alveolar densities in thoracic radiographs as well as neutrophilia in BALF cytology. All of these dogs showed a rapid response to antibiotics, and a full clinical and radiographic cure was achieved with antimicrobial treatment. CPIV was detected by PCR in 2/4 and Mycoplasma spp. in 1/4 dogs with negative culture results.
Information on previous CDV, CAV‐2, and CPIV vaccinations was available for all dogs with BP. All dogs with positive CPIV PCR in respiratory samples (n = 7) had been vaccinated against CPIV <12 months previously (median, 6.5 months; interquartile range, 4.5–9.0 months). In total, 12/13 dogs with negative CPIV PCR in respiratory samples were vaccinated against CPIV (median, 13.0 months; interquartile range 2.9–24 months). There was no significant difference in the timing of CPIV vaccination between dogs with positive and negative respiratory virus PCR (P = .152). All dogs participating in this study had previously been vaccinated against CAV and CDV.
Bordetella bronchiseptica PCR analysis was positive in 12/13 dogs with BBTB and 1/20 dogs with BP. All dogs with BBTB and none of the dogs with BP had positive growth of B. bronchiseptica in BALF or TTW fluid.
In addition to Mycoplasma spp. PCR analysis, a culture for Mycoplasma spp. was performed in 15/20 dogs with BP and 4/13 dogs with BBTB. Mycoplasma PCR was positive in 8/20 dogs with BP and 3/13 dogs with BBTB. Mycoplasma culture was positive in 4/15 dogs with BP and 2/4 dogs with BBTB. Mycoplasma PCR was positive in all dogs with BP and BBTB with positive culture results. Additionally, PCR was positive in 3 dogs with negative Mycoplasma culture results (2 dogs with BP and 1 with BBTB) and in 2 dogs with BP lacking culture results.
Respiratory infections in pigs are very important factor affecting the profitability of pig production [1, 2]. Although various bacteria or viruses could induce the respiratory infection separately, it has commonly been caused by coinfection with more pathogens under field conditions [1–3]. The most important infectious agents responsible for infection of the respiratory tract in pigs are: swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV), Pasteurella multocida (Pm), Actinobacillus pleuropneumoniae and Mycoplasma hyopneumoniae [2, 4–6]. Besides, the above mentioned pathogens, the Haemophilus parasuis (Hps) can also be recovered from the lungs of pigs with pneumonia [1, 7–10]. In these cases Hps is often isolated along with other bacterial or viral pathogens and, therefore, the role of Hps in producing pneumonia is not clear [8, 11].
Bacterial pneumonia secondary to influenza is often observed in pigs. SIV is a significant contributor to the respiratory diseases and may predispose to secondary bacterial infection. Hps is an important and common respiratory pathogen of pigs. It can be a primary pathogen or be associated with other diseases such as SIV [3, 8]. It could be also isolated from nasal cavity, tonsils and trachea of apparently healthy pigs [8, 14]. Under favorable conditions, Hps can cause severe systemic infection characterized by fibrinous polyserositis, arthritis and meningitis [8, 11, 14]. Factors leading to systemic infection by Hps have not been clarified to date [9, 14].
Although there are previous reports of experimental reproduction of Hps or SIV infection in conventional pigs, little is known about the effect of concurrent infection with SIV and Hps on the disease severity and inflammatory response in pigs, even if this coinfection is common under field conditions [13, 15–17]. There are also limited data on the role of Hps in the production of pneumonia in the absence of other respiratory pathogens. Furthermore, the kinetics of acute phase protein (APP) response in SIV/Hps co-infected pigs has not been studied to date. As it has been shown for other pathogens, the exposure to several pathogens can lead to a stronger APP response, as compare to single infection [18–20]. Thus, in order to investigate the influence of SIV and Hps coinfection on clinical outcome, both local and systemic inflammatory response as well as pathogen shedding and load at various time points following intranasal inoculation, three experimental infections (Hps- and SIV-single infection, SIV/Hps co-infection) has been performed in the present study. The correlation between local concentration of cytokines and severity of infection (clinical score, lung score) as well as serum APP concentration has been also studied.
HPAI H5N1 viruses have recently caused disease outbreaks in poultry in Malaysia during 2017 and Cambodia, Japan, Korea, and Taiwan during January 2018. Animal disease surveillance and monitoring activities have been routinely performed throughout Asia with a focus on HPAI virus and other transboundary diseases that affect international trade. Processing samples originating from animal sources in a limited biosafety environment could pose a significant risk of LAI and could lead to unintentional release of the pathogen into environment by aerosol transmission when performing laboratory procedures.
In the case of potential pandemic pathogens, such as HPAI virus, existing wild type H5N1 viruses were reported to have a limited ability for human to human transmission and the risk of the viruses crossing species boundaries to become a pandemic threat in humans was considered to be low. However, evidence of a HPAI H5N1 strains, first detected in China in 2008, had undergone genetic re-assortment resulting in new NA subtypes (including N2, N3, N5, N6, and N8) and aggressively spread to birds worldwide (e.g., Asia (mainly H5N6), Europe (H5N8), Middle East (H5N8), Russia/Mongolia (H5N8) and North America (H5N8, H5N1 and H5N2)). These H5Nx viruses are believed to become more transmissible and more stable in the environment and in wild birds than other influenza A viruses. Similar to the H5Nx, H7N9 low-pathogenicity avian influenza (LPAI) first caused human infection in 2013 in China. Since then, the viruses recurred annually and caused 1589 human cases with 616 deaths. It is unclear how mutation and re-assortment occur in nature. Influenza viruses are well known for their fast rate of genetic re-assortment especially when co-infection has occurred. There is a possibility that these viruses could undergo genetic assortment and infect humans leading to a potential pandemic. The viruses that have been circulating in the region persist in the environment and infect a range of hosts posing a risk of generating a potential pandemic strain.
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.
This review summarized LAI reports from the Asia-Pacific region and has examined some of the potential risks associated with laboratory investigations with zoonotic pathogens. Clinical diagnoses and routine disease surveillance and monitoring activities comprise a major workload for health-related laboratories at both public and animal health interfaces in the region. Working with infectious materials on a daily basis poses a risk to laboratory staff health as well as those involved in the collection and transportation of samples. Accidental infection of laboratory staff can occur even in laboratories where strict biosafety measures are employed and enforced as demonstrated by those that occurred in laboratories located in HIC. Reporting of LAIs can be an indication of a biosafety or biosecurity breach that may be caused by technical failures or human errors. Thorough investigations to determine the root cause of LAIs or unintended releases have the potential to improve laboratory biosafety by providing an evidence base to determine risks of such occurrences.
A series of landmark studies of LAI occurrence and cause were performed in the USA by Sulkin and Pike between 1935–1978 with a total of 4079 LAI reports that that concluded that the majority of LAIs were caused by bacterial pathogens with lower numbers of viral and rickettsial infections. In another study, Pike claimed that only 64% of LAI reports were published (2465 out of 3921 cases; data collected between 1935 and 1974); however, these were based mostly on LAIs from research and animal laboratories and did not represent cases in clinical laboratories. In 2017, Byers and Harding stated that, based on their review study, 43% (out of 2308) of LAIs occurred in clinical laboratories and 39% in research laboratories.
Most LAI reports in this study were from developed HIC, where biosafety measures are more likely to be compliant with international standards, and which report LAIs as per national regulatory requirements. Furthermore, there may be an incentive to report LAIs in HICs due to the requirement for biosafety competency of staff coupled with an awareness of potential LAI hazards, and the need to report accidents so that appropriate post-exposure treatment could be provided in a timely manner. In comparison to other parts of the world, the number of LAI reports in the Asia-Pacific was relatively low. This may be due to lower numbers of laboratories in Asia, especially those that would normally be handling high-consequence pathogens, such as high-containment laboratories (i.e., BSL3 and BSL4). Furthermore, the lower number of LAIs may also be due to different reporting requirements for research, diagnostic, or clinical laboratories, and whether they are government or privately-funded, and the fear of stigma. A limitation of this review is that searches were only performed using searchable sources and that the authors did not have access to information such as locally published unofficial reports, or those in languages other than English, which may also have contributed to the low number of LAI reports. Nevertheless, it is likely that there is significant under-reporting of LAIs in the UMIC and LMIC of the Asia-Pacific, given the significant disease risk profile. Under-reporting and lack of recognition of such LAIs could pose risk not only to staff but also the community and the environment.
Working with ministries of health, ministries of agriculture, and other governing bodies within the UMIC and LMIC of the Asia-Pacific region to set up mandatory LAI reporting, as well as accidental release or escape, would provide evidence of capacity gaps and where to focus biosafety resources; however, these data have limitations. The downside of using LAIs reports to determine capacity gaps is that they are an insensitive method of detecting laboratory exposure, because they are based on acute symptomatic infection, while data on asymptomatic infection and host immune response are rarely measured to determine seroconversion status. A survey by Willemarck et al. commented that the majority of LAI reports and publications only identified the most obvious risk group 3 organism LAIs and risk group 2 organisms, which cause milder or asymptomatic infection, were unlikely to be detected as the resulting LAIs would be unknown or unnoticed. Lack of awareness could result in low or no LAI reporting in some laboratories. In the US, a mathematical model determined that the probability of LAI incidents was between 0.1 and 0.5%, which was similar to the probability of an annual pathogen escape at 0.3%, although it is not clear how these data would relate to the Asia-Pacific setting. Therefore, it is important that all laboratory staff are educated in accident and incident reporting, as well as following up cases of exposure to pathogens with post-exposure prophylaxis. Additionally, a registry documenting the near-miss incidents would help to improve laboratory safety.
Results of Asia-Pacific LAIs reports presented here share some characteristics with those previously published. Elsewhere, LAI reports have generally decreased due to increasingly effective vaccines along with better laboratory safeguards; however, the risk of laboratory exposure and escape still remains when working with live agents. A literature review of LAI reports in the USA during 2000–2009 revealed that there was a total of 34 cases with four deaths, due to bacteria (22) and viruses (11) and parasites (1). According to the Monitoring Select Agent Theft, Lost and Release reports in the USA (2004–2010), 11 LAIs were reported with no fatalities or evidence of secondary infection to others. However, it should be noted that these reports were confined to Select Agents and not all possible pathogens. There were six cases of brucellosis, four cases of Francisella tularensis, and one case of Coccidioides immitis/posadasii. A survey in Belgium indicated that between 2007 and 2012 there were 94 LAI cases; 23% caused by Salmonella spp. and 16% by Mycobacterium spp. In 2000, Sewell reported that the most common organism causing LAIs included bacteria (Shigella spp., Salmonella spp., E. coli, Francisella tularensis, Brucella spp., Mycobacterium tuberculosis), viruses (hepatitis C virus, human immunodeficiency virus), and a dimorphic fungus. To date, even though pathogens listed above by Sewell remain the primary cause of LAIs, other organisms including Neisseria meningitidis and vaccinia virus, as well as newly-emerged pathogens with a potential pandemic risk (e.g., SARS, influenza viruses, West Nile virus, and Ebola virus) should also be considered as significant potential pathogens for LAIs.
To reduce the likelihood of LAI occurrences, it is important that each laboratory plan and implement their own pathogen-specific, preventative strategies to improve biosafety and biosecurity. This includes development and application of protocols specific for occupational health and safety incorporating accident reporting and ‘close call’ incidents, and pre/post-exposure serological surveys. When working with pathogens, a risk-based approach should be applied for all biosafety programs focusing on pathogen-based factors. The factors to be considered are routes of infection, infectious dose, quantity and concentration of the agent to determine the most appropriate risk mitigation strategies, such as administrative and engineering controls, and personal protective equipment. Furthermore, annual health checks and vaccinations, post-exposure prophylaxis—including reporting and monitoring for post-vaccination adverse events—and symptom monitoring, are recommended.
In conclusion, clinical and diagnostic laboratories are on the front line for detecting outbreaks of EIDs and zoonotic diseases. Laboratories require strong biosafety measures to protect staff health and prevent environmental contamination with pathogens. The fundamentals of a biosafety program include staff education and awareness to ensure good understanding and implementation of biosafety measures, including risk assessment and control measures. The international community has an important and continuing role to play in supporting laboratories in UMIC and LMIC to ensure that they maintain a safe working environment for the staff, their families, and the wider community.
The respiratory diseases of small ruminants are generally fatal to lambs and kids. The lamb and kid pneumonia are mostly regarded as a complex of disease. It involves interaction of host related factors (immunological and physiological) and etiological agents, namely, virus, bacteria, mycoplasma, and environmental factors [4, 7, 108]. Many times, immunosuppression, malnutrition, and adverse climatic conditions lead to infection due to unusual infectious agents. There are reports on Streptococcus pneumoniae, commensal bacteria of the nasopharynx of animals associated with a majority of cases of morbidity and mortality in young lambs due to pneumonia [7, 13, 153]. Similarly, many other unusual pathogens Haemophilus ovis, Streptococcus spp., Pasteurella spp. [5, 6], M. bovis in sheep [7, 12] and goat, Mycoplasma arginini, and Haemophilus somnus may cause pneumonia. Many time mixed infections are observed. Thus, isolation and identification of such samples are always tedious to perform [7, 9]. The use of monoclonal antibodies based serological tests has simplified the process of early and specific diagnosis of many of these pathogens. Simultaneously, development of molecular techniques like PCR particularly multiplex PCR is very useful for the identification and differentiation of etiological agents from such complex conditions.
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.
Bovine respiratory disease complex (BRDC) is a global problem causing severe economic losses to the cattle farming industry through mortality, loss of production, and treatment costs [1, 2]. It has a complex etiology that involves various pathogens, host factors, and environmental factors. Viruses such as bovine herpes 1 virus (BoHV-1, parainfluenza virus 3 (PBIV-3), bovine respiratory syncytial Virus (BRSV), respiratory bovine coronavirus (BoCoV) and bovine viral diarrhoea virus (BVDV) in conjunction with stress factors have been implicated as causes of respiratory tract infections of cattle by immunosuppression and damage to the respiratory epithelium. A primary viral infection can be followed by an opportunistic secondary infection with bacteria like Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, or Trueperella pyogenes [2, 4, 5], but these bacteria could also act as primary pathogen. In addition it has become increasingly clear that Mycoplasmas are important contributors to BRD, either as primary pathogens or in co-infection [2, 6–9]. M. bovis is the best known Mycoplasma species causing respiratory disease [4, 7], but also M. dispar and M. bovirhinis have been associated with BRD [2, 9–11]. M. bovis has not only been identified as a primary or opportunistic pathogen in BRD in beef cattle worldwide, but it has also been implicated in other clinical manifestations in cattle, such as mastitis, otitis, arthritis, and reproductive disorders. M. bovirhinis and M. dispar are regularly isolated from the nasal cavity of cattle with respiratory disease and are usually regarded as an opportunistic pathogen in respiratory diseases [7, 12].
Bacteriological, serological and histopathological examinations are important tools to detect particular animal-carriers of Mycoplasma, however, these assays are time-consuming, insensitive and can give false positive results. Bronchoalveolar lavage fluid (BALF) from calves with BRD may contain various potential pathogens, but additional antibiotic use in the affected herds can inhibit cultivation and thereby can cause false-negative test results. In BRD, differential diagnosis of these pathogens with rapid turnaround time procedure is essential to implement appropriate treatment and intervention measures in a timely manner. Rapid detection of these pathogens at the early stage of outbreak can contribute substantially to minimize the spread of infection and increase treatment efficiency. Today quick, highly sensitive and species-specific PCRs are used in the diagnosis of Mycoplasma-associated diseases for M. dispar [14, 15], M. bovis [4, 16] and M. bovirhinis in BALF or nasal swabs. Combining a 16S Ribosomal DNA PCR with denaturing gradient gel electrophoresis fingerprinting (PCR/DGGE) enabled the simultaneous detection of mixed Mycoplasma populations, however information about the detection limit in clinical samples is limited. Additionally, a DNA microarray assay was developed for the parallel detection of 37 Mycoplasma species, in which species-specific probes derived from the 23S rRNA and tuf genes were used for species differentiation.
Multiplex real-time PCR could be a promising and practical approach to speed up the differential diagnosis from 1 to 2 weeks for traditional culture to 24 h, with limited expenses. This will make diagnostic testing more accessible for veterinary practitioners and thereby improve BRD diagnosis. This report describes the RespoCheck triplex PCR developed by Central Veterinary Institute (CVI, Lelystad, The Netherlands) for detection of three Mycoplasma species.