Conjunctival swab cytology from a guinea pig: it's elementary!

Three 3-month-old guinea pigs (Cavia porcellus) were evaluated for purulent ocular discharge. Conjunctival swabs were obtained for cytologic evaluation of Wright's-Giemsa-stained preparations. The specimen from the most severely affected guinea pig consisted primarily of karyolytic neutrophils and small lymphocytes. Epithelial cells occasionally were observed that contained intracytoplasmic coccoid basophilic organisms, 0.5-1.5 microm in diameter. The intraepithelial inclusions were most consistent with Chlamydia sp elementary and reticulate bodies. Specimens from the other 2 guinea pigs had a similar inflammatory response, but organisms were not observed. Polymerase chain reaction (PCR) analysis of a conjunctival swab from the most severely affected guinea pig was positive for C psittaci, which also is referred to as Chlamydophila caviae, immunotype 8, formerly known as the guinea pig inclusion conjunctivitis strain of C psittaci. Chlamydial conjunctivitis is a common problem in guinea pig populations, with C caviae being specific for this species. Cytologic identification of elementary or reticulate bodies within epithelial cells is diagnostic for the organism in Giemsa-stained preparations. However, PCR is an important complementary tool when organisms are not observed and for accurate classification of the Chlamydia species.     

Conjunctivitis caused by a swine Chlamydia trachomatis-like organism in gnotobiotic pigs.

The objective of this study was to determine whether a chlamydial strain recovered from growing and finishing swine with conjunctivitis or keratoconjunctivitis could cause the same infections in gnotobiotic pigs. The strain shares biological characteristics with Chlamydia trachomatis. After propagation in Vero cells and preparation of the inoculum (10(7) inclusion-forming units/ml), chlamydial strain H7 was instilled into the ventral conjunctival sac (0.15 ml/sac) of 12 anesthetized 3-day-old gnotobiotic piglets. Four age-matched gnotobiotic piglets were anesthetized and sham infected with uninfected cell culture lysates. None of the principal piglets developed clinical symptoms of conjunctivitis or keratoconjunctivitis. Principal piglets necropsied 7 days postinfection (DPI) had histologic lesions of mild or moderate conjunctivitis; immunohistochemical evaluation revealed chlamydial antigen in conjunctival epithelium. A majority of principal piglets necropsied at 14-28 DPI had histologic lesions of mild conjunctivitis, but chlamydial antigen was not detected by immunohistochemistry. The results indicated that chlamydial strain H7 can cause mild or occasionally moderate conjunctivitis in gnotobiotic pigs, but the conjunctival infection is asymptomatic.     

Prevalence of feline herpesvirus 1, Chlamydophila felis, and Mycoplasma spp DNA in conjunctival cells collected from cats with and without conjunctivitis

OBJECTIVE: To use PCR assays to determine the prevalence of feline herpesvirus 1 (FHV-1), Chlamydophila felis, and Mycoplasma spp DNA in conjunctival cells collected from cats with and without conjunctivitis; to compare results of conventional and real-time fluorogenic PCR assays for amplification of FHV-1 DNA; and to determine whether copy numbers of FHV-1 DNA are correlated with conjunctivitis. ANIMALS: 55 cats with active conjunctivitis, 39 healthy cats that never had conjunctivitis, and 32 cats with a history of conjunctivitis that had been resolved for at least 3 months. PROCEDURES: Samples were obtained by rolling cotton-tipped applicators on the ventral conjunctiva of awake cats treated topically with proparacaine. The DNA was extracted from the swab specimens and assessed in PCR assays to detect DNA of FHV-1 (fluorogenic PCR assay and conventional PCR assay), Mycoplasma spp (conventional PCR assay), and C felis (conventional PCR assay). RESULTS: Overall prevalence rates of FHV-1, C felis, and Mycoplasma spp as assessed by the conventional PCR assays were 6.7%, 3.2%, and 9.6%, respectively. Percentage concordance between conventional PCR and fluorogenic PCR assays for FHV-1 was 92.5%. There were no significant differences among the 3 groups of cats for the mean copy number of FHV-1 divided by the copy number of glyceraldehyde-3-phosphate dehydrogenase. CONCLUSIONS AND CLINICAL RELEVANCE: Mycoplasma spp were the most prevalent organism detected and was associated with conjunctivitis. This study could not confirm that there are increased copy numbers of FHV-1 DNA in cats with conjunctivitis, compared with the copy numbers for cats without conjunctivitis.     

Results of diagnostic ophthalmic testing in healthy guinea pigs

OBJECTIVE-To report values for tear production, central corneal touch threshold (CTT), and intraocular pressure (IOP) in healthy guinea pigs and determine results of aerobic bacterial culture and cytologic examination of conjunctival swab specimens. DESIGN-Cross-sectional study. ANIMALS-31 Healthy guinea pigs (62 eyes) of various ages and breeds. PROCEDURES-Tear production was measured by the phenol red thread tear test (PRT) and Schirmer tear test (STT) before and after topical anesthetic application, CTT was measured with an esthesiometer, and IOP was measured by applanation tonometry. RESULTS-Combining data from all eyes, mean +/- SD PRT values before and after topical anesthetic administration were 21.26 +/- 4.19 mm/15 s and 22.47 +/- 3.31 mm/15 s, respectively, and mean IOP was 18.27 +/- 4.55 mm Hg. Median STT values before and after topical anesthetic administration were 3 mm/min (range, 0 to 12 mm/min) and 4 mm/min (range, 0 to 11 mm/min), respectively, and median CTT was 2.0 cm (range, 0.5 to 3.0 cm). Values did not differ between eyes for any test, but significant differences were identified for PRT values between males and females and between values obtained before and after topical anesthetic administration. Common bacterial isolates included Corynebacterium spp, Streptococcus spp, and Staphylococcus spp. Cytologic examination of conjunctival swab specimens revealed mainly basal epithelial cells; lymphocytes were common. CONCLUSIONS AND CLINICAL RELEVANCE-Results provided information on values for PRT, STT, CTT, and IOP in healthy guinea pigs and on expected findings for aerobic bacterial culture and cytologic examination of conjunctival swab specimens.     

Antigenic Detection of Human Strain of Influenza Virus A (H3N2) in Swine Populations at Three Locations in Nigeria and Ghana during the Dry Early Months of 2014

Since the first detection of human H3N2 influenza virus in Taiwanese pigs in 1970, infection of pigs with wholly human viruses has been known to occur in other parts of the world. These viruses, referred to as human‐like H3N2 viruses, have been known to cause clinical and subclinical infections of swine populations. Due to the paucity and complete unavailability of information on transmission of influenza viruses from other species, especially humans, to swine in Nigeria and Ghana, respectively, this study was designed to investigate the presence and prevalence of a human strain of influenza A (H3N2) in swine populations at three locations in two cities within these two West African countries in January and February, 2014. Using stratified random technique, nasal swab specimens were collected from seventy‐five (75) pigs at two locations in Ibadan, Nigeria and from fifty (50) pigs in Kumasi, Ghana. These specimens were tested directly by a sensitive Quantitative Solid Phase Antigen‐detection Sandwich ELISA using anti‐A/Brisbane/10/2007 haemagglutinin monoclonal antibody. Influenza virus A/Brisbane/10/2007 (H3N2) was detected among pigs at the three study locations, with an aggregate prevalence of 4.0% for the two locations in Ibadan, Nigeria and also 4.0% for Kumasi, Ghana. Transmission of influenza viruses from other species to swine portends serious sinister prospects for genetic reassortment and evolvement of novel viruses. We therefore recommend that further studies should be carried out to investigate the presence of other circulating human and avian influenza viruses in swine populations in West Africa and also determine the extent of genetic reassortment of strains circulating among these pigs. This would provide an early warning system for detection of novel influenza viruses, which could have pandemic potentials.     

Isolation of transmissible Gastroenteritis virus, pseudorabies virus, and porcine enterovirus from pharyngeal swabs taken from market-weight-swine

Pharyngeal swab samples were collected at a central Iowa abattoir from 6,010 market-weight slaughter hogs from September 1, 1979 to August 31, 1980. The swab samples were examined for cytopathic viruses by inoculation of monolayer cultures of continuous line of swine testicular cells. Of the 6,010 swab samples tested, transmissible gastroenteritis virus was isolated from 91 (1,51%), pseudorabies virus was isolated from 431 (7.17%), and porcine enterovirus was isolated from 21 (0.35%). Although all 3 viruses were identified throughout the year, transmissible gastroenteritis and pseudorabies viruses were found more frequently during the winter and early spring. In contrast, porcine enterovirus was detected more frequently during the spring and summer.     

Expression of Mx protein and interferon-alpha in pigs experimentally infected with swine influenza virus

Expression of Mx protein and interferon-alpha (IFN-alpha) was examined by immunohistochemistry in pigs experimentally infected with swine influenza virus. In infected pigs euthanatized at 1 day postinoculation (dpi), the lumen of bronchioles were filled with large numbers of mononuclear cells, small numbers of neutrophils, sloughing epithelial cells, and proteinaceous fluid. Lesions at 3 and 5 dpi were similar but less severe. Alveolar spaces were filled with neutrophils. By 7 and 10 dpi, microscopic lesions were resolved. The immunohistochemical signals for Mx protein and IFN-alpha antigen were confined to cells in areas that had hybridization signal for swine influenza virus. In situ hybridization and immunohistochemistry of serial sections of lung indicated that areas containing numerous swine influenza virus RNA-positive cells also have numerous Mx and IFN-alpha antigen-positive cells. Mean immunohistochemical scores for Mx protein-positive cells were correlated with mean immunohistochemical scores for IFN-alpha antigen-positive cells (r(s) = 0.8799, P < 0.05). These results indicated that Mx protein and IFN-alpha antigen were expressed in the lung from pigs experimentally infected with swine influenza virus, but their biological functions remain to be examined.     

Keratoconjunctivitis associated with Toxoplasma gondii in a dog

A 12-year-old Pug presented with a 3-mm corneal mass OD. The dog was currently being treated for keratoconjunctivitis sicca (KCS) and pigmentary keratitis OU. A superficial keratectomy followed by cryotherapy was performed OD. A histopathologic diagnosis of epithelial dysplasia and suppurative keratitis was made and the lesion resolved. Two months later, a yellow/tan conjunctival mass, diffuse chemosis and conjunctival thickening was discovered OD. Necrotizing conjunctivitis with protozoal parasites was diagnosed with histopathology. Complete blood count and a serum biochemistry panel were normal. Neospora caninum and Toxoplasma gondii titers were negative. The conjunctivitis resolved after a 6-week course of oral clindamycin. Two months later, the patient presented with a similar conjunctival mass OS. Toxoplasma gondii was confirmed as the etiologic agent with immunohistochemical staining. Repeat T. gondii titers were negative. Oral clindamycin was re-instituted. The corneal biopsy was re-reviewed and protozoal organisms were discovered. Three months later, a recurrence was suspected and oral ponazuril was initiated for 28 days. There has been no evidence of recurrence since this treatment. Ocular toxoplasmosis is rare in the dog but reports have included episcleritis, scleritis, retinitis, anterior uveitis, ciliary epithelium hyperplasia, optic neuritis and polymyositis. To our knowledge, this is the first confirmed report of toxoplasmosis causing only corneal and conjunctival disease in the dog. We hypothesize that these localized lesions may be associated with topical immunomodulating therapy for KCS. Toxoplasmosis should be considered as a differential for canine conjunctivitis and corneal disease and has the potential to manifest in one or both eyes.     

Differences in virulence between two strains of Streptococcus suis type II after experimentally induced infection of newborn germ-free pigs

Fifteen newborn germ-free pigs were inoculated with 2 strains, D-282 and T-15, of Streptococcus suis type II. Some pigs also were preinoculated with Bordetella bronchiseptica, which successfully predisposed them to S suis infection. The 2 streptococcal strains were differentiated by muramidase treatment, which released certain high molecular-weight proteins, termed muramidase-released proteins (MRP), from the cell wall of strain D-282, but not from the cell wall of strain T-15. Only strain D-282 (MRP-positive) induced clinical signs of disease and markedly increased neutrophil numbers in pigs. Streptococci were more frequently isolated from fecal swab specimens obtained from pigs inoculated with strain D-282 (MRP-positive) than from specimens obtained from pigs inoculated with strain T-15 (MRP-negative). Both strains were isolated from nasal swab specimens obtained from all infected pigs. Postmortem examination revealed fibrinopurulent meningitis, polyserositis, and polyarthritis in pigs inoculated with strain D-282; this strain was isolated from the CNS, serosae, visceral organs, heart, and joints. Whereas strains D-282 caused several pathologic changes, strain T-15, isolated from the lungs, caused only pneumonia. Both strains were isolated from the tonsils of all pigs. Virulence differed distinctly between the MRP-positive and the MRP-negative strains.     

Conjunctival lesions caused by Moraxella bovis in gnotobiotic calves

Hemolytic Moraxella bovis was instilled into the conjunctival sac of gnotobiotic calves, and conjunctivae were sampled serially after infection. Bilateral lesions developed in seven of eight infected calves. Histologically, M. bovis was first seen within swollen epithelial cells near the lid margins and occasionally within superficial epithelium in other areas. Conjunctival erosions and ulcers were seen in later stages. Scanning electron microscopy showed M. bovis in pits on surfaces of epithelial cells and in erosions on palpebral conjunctivae; lesions were prominent near lid margins. By transmission electron microscopy, M. bovis was seen within swollen epithelial cells near lid margins; many epithelial cells had undergone cytolysis. This study demonstrates that virulent M. bovis can invade bovine conjunctival epithelial cells and cause conjunctivitis in the absence of injurious ultraviolet irradiation or other predisposing environmental factors.     

Treatment of canine nictitans plasmacytic conjunctivitis with 0–2 per cent cyclosporin ointment

Nictitans plasmacytic conjunctivitis, commonly referred to as plasma cell infiltrate of the nictitans or plasmoma, was diagnosed in 12 dogs (23 eyes) on the basis of clinical signs and nictitans conjunctival biopsy specimens. These dogs underwent a clinical therapeutic trial with twice daily 0·2 per cent cyclosporin ophthalmic ointment. Response to therapy was monitored over a six-week period and repeat biopsy specimens were then taken. Significant (P<0·05) reductions between pre- and post trial scores were recorded for: mucopurulent ocular discharge quantity; degree of bulbar conjunctival hyperaemia; areas of nictitans hyperaemia, thickening and depigmentation. Schirmer tear test values significantly increased between the start and end of treatment. Biopsy specimens were subjected to selective detection procedures for plasma cells (methyl green-pyronin staining) and T lymphocytes (CD3 antigen labelling). Mean cell counts showed a significant reduction in plasma cell numbers, but the trend towards reduced T lymphocyte numbers was not significant.     

Postweaning multisystemic wasting syndrome induced after experimental inoculation of cesarean-derived, colostrum-deprived piglets with type 2 porcine circovirus.

Cesarean-derived, colostrum-deprived pigs (n = 23) were inoculated intranasally and subcutaneously with a low cell culture passage of type 2 porcine circovirus. In 11 pigs, a persistent fever that lasted 7-17 days began 12-15 days after inoculation with virus. Additional signs of disease in those 11 pigs included depression (11 of 11 pigs), palpable enlargement of inguinal, prefemoral, and popliteal lymph nodes (11 of 11), icterus (6 of 11), and hyperpnea (2 of 11). The remaining 12 pigs had fever that occurred intermittently for 2-4 days between days 12 and 20 postinoculation. Overt signs of disease in those pigs were limited to palpable enlargement of inguinal and popliteal lymph nodes (9 of 12 pigs). When compared with control pigs of similar age, the average daily rate of weight gain for all pigs inoculated with virus was less over a 2-week period that began 2 weeks post inoculation. At postmortem examination, lymph node enlargement was seen in 14 of 14 pigs euthanized between days 20 and 28 postinoculation. Lymph node enlargement was especially prominent in pigs that developed a persistent fever. Microscopic lesions noted in pigs that developed a persistent fever included cellular depletion in lymphoid tissues; hepatic cell necrosis; and lymphogranulomatous inflammation of lymph nodes, Peyer's patches of the intestine, liver, kidney, and heart. Virus was isolated with varying frequency from nasal, rectal, or tonsil swab specimens, buffy coat, serum, urine, and lung lavage fluid obtained antemortem or postmortem. Virus was isolated from or viral DNA was detected in a variety of tissues obtained postmortem up to 125 days postinoculation. Antibody against type 2 porcine circovirus usually was detected in serum between 15 and 20 days postinoculation; however, antibody against virus was not detected in serum from 4 pigs euthanized 20-24 days postinoculation. Direct contact with pigs inoculated with virus 42 days previously resulted in transmission of virus to 3 of 3 control pigs.     

Detection of transmissible gastroenteritis virus in feces from pigs by reversed passive hemagglutination

A reversed passive hemagglutination (RPHA) method was developed for the detection of transmissible gastroenteritis (TGE) virus in the fecal specimens from pigs. Ovine erythrocytes fixed with glutaraldehyde and treated with tannic acid were coated with anti-TGE virus swine antibodies, which were purified by affinity chromatographic technique linked with purified TGE virus. The RPHA test was done by the Microtiter method. Erythrocytes coated with purified specific antibodies were agglutinated by TGE virus, but not by porcine rotavirus or porcine enterovirus. The reaction was specifically inhibited by antiserum against TGE virus, confirming the specificity of the reaction. A litter of seven 3-day-old pigs was orally inoculated with TGE virus, and fecal specimens were obtained once a day and serum was obtained every 4th day. With the RPHA test, TGE virus was detected in the diarrheal feces; all of the inoculated pigs developed virus-neutralization antibody for the TGE virus. The RPHA test detected TGE virus in feces from pigs with naturally occurring diarrhea. The RPHA test detected TGE virus in 5 of 6 fecal specimens (80%), whereas the positive rate was only 50% (3/6) for the immunofluorescent staining of primary cultures of porcine kidney cells inoculated with the specimens. The advantages of the RPHA method are simplicity, high sensitivity, and rapid to do.     

Porcine reproductive and respiratory syndrome virus: routes of excretion

This study was conducted to delineate potential sites of exit and duration of shedding of porcine reproductive and respiratory syndrome virus (PRRSV). Two experiments of 6 pigs each were conducted. Pigs were farrowed in isolation, weaned at 7 days of age, and housed in individual HEPA filtered isolation chambers. In each experiment, 3 pigs served as controls and 3 were inoculated intranasally with PRRSV (ATCC VR-2402) at 3 weeks of age. In a first experiment, on days 7, 14, 21, 28, 35, and 42 post inoculation (PI), pigs were anesthetized and intubated. The following samples were collected: serum, saliva, conjunctival swabs, urine by cystocentesis, and feces. Upon recovery from anesthesia, the endotracheal tube was removed, rinsed, and the rinse retained. In the second experiment, the sampling schedule was expanded and serum, saliva, and oropharyngeal samples were collected from day 55 to day 124 PI at 14 day intervals. Virus was isolated in porcine alveolar macrophages up to day 14 from urine, day 21 from serum, day 35 from endotracheal tube rinse, day 42 from saliva, and day 84 from oropharyngeal samples. No virus was recovered from conjunctival swabs, fecal samples, or negative control samples. This is the first report of isolation of PRRSV from saliva. Virus-contaminated saliva, especially when considered in the context of social dominance behavior among pigs, may play an important role in PRRSV transmission. These results support previous reports of persistent infection with PRRSV prolonged recovery of virus from tonsils of swine.     

Isolation of Treponema hyodysenteriae from sources other than swine

Fecal samples were collected from animals and environments on 3 swine farms and cultured for Treponema hyodysenteriae. Each farm was a farrow-to-finish operation and, at the time of sampling, swine dysentery was enzootic among 8- to 22-week-old pigs. Pathogenic T hyodysenteriae was isolated from pigs on all 3 farms. On farm A, nonpathogenic T hyodysenteriae was isolated from a sample of lagoon water. On farm B, pathogenic T hyodysenteriae was isolated from a waste-holding pit. On farm C, a dog was observed to be eating feces of pigs that had swine dysentery. The dog was diarrheic and a fecal sample yielded a pathogenic isolant of T hyodysenteriae. Further isolation attempts were unsuccessful after the dog was removed from the infected premises. Isolation of pathogenic and nonpathogenic organisms from waste-holding systems emphasizes the need for cultural techniques in detecting pathogenic T hyodysenteriae.     

STUDIES ON THE EFFECTS OF INFECTIOUS BOVINE RHINOTRACHEITIS VIRUS ON REPRODUCTION IN HEIFERS

Natural venereal infection of heifers with IBR virus resulted in vulvovaginal lesions in 9 of 12 heifers at 3 weeks after introducttion to bulls, and virus was isolated from 5 of the 9 affected animals. The infection was not associated with any effect on conception. Experimental nasal and conjunctival infection of 18 heifers pregnant for 3, 5 or 7 months with a genital strain of IBR virus caused mild rhinitis or conjunctivitis in all inoculated animals and virus was recovered from 14 animals at 8 days after inoculation. No effects on pregnancy were recorded.     

Isolation of transmissible gastroenteritis virus from pharyngeal swabs obtained from sows at slaughter.

A virologic survey was conducted to determine the frequency of transmissible gastroenteritis (TGE) virus infection in farm-raised sows. Pharyngeal swab specimens collected in an abattoir were examined for TGE virus by inoculation onto swine-testes cell culures. The virus was detected in 61 (3%) of a sample of 2,058 Iowa sows after slaughter. All TGE viral isolates, given orally to 2- or 3-day-old pigs, caused acute gastroenteritis and in some cases death. All pigs that recovered from illness had serum antibody to TGE virus.     

Comparison of tissue and fluid samples for the early detection of canine distemper virus in experimentally infected dogs

The clinical utility of various specimens was examined for the early diagnosis of canine distemper (CD). Seven healthy dogs at 17 weeks of age were experimentally infected with a field isolate of canine distemper virus. The RT-PCR was carried out to detect CDV NP gene. Dogs showed mild fever and leukopenia, however, typical clinical signs of CD were not seen through the experimental period. CDV amplicons were detected more, earlier and for longer period in the conjunctival swabs than in the other samples employed. These results suggested that conjunctival swab samples, which are easy to obtain and non-invasive, would be the most suitable and practical specimen for the early antemortem diagnosis of CDV infection.