Sialodacryoadenitis virus-associated lesions in the lower respiratory tract of rats

Eight- to 10-week-old outbred Wistar rats were inoculated intranasally with 10(2.9) medium mouse lethal infective doses of sialodacryoadenitis (SDA) virus. Sham inoculated control rats and challenged rats were killed at 1 day intervals for the first 8 days, then on days 10, 12, 14, and 20. Typical lesions associated with SDA were seen microscopically in the salivary and lacrimal glands of inoculated rats. In addition, laryngitis, tracheitis, bronchitis, bronchiolitis, and multifocal alveolitis were present during the acute stages of the disease. Viral antigen was demonstrated in epithelial cells lining airways by immunofluorescence microscopy. SDA virus was recovered from the lower respiratory tract from days 2 to 6 post-inoculation (PI). Serum antibodies to SDA virus, but not to Sendai virus or Mycoplasma pulmonis were present in rats tested at day 20 PI. These findings demonstrate that during the acute stages of the disease, significant lesions do occur in the lower respiratory tract of SDA virus-infected rats.     

Sialodacryoadenitis in the rat: effects of immunosuppression on the course of the disease

Eight-week-old outbred male and female Crl:CD(SD)BR rats were treated with prednisolone (PR) or cyclophosphamide (CY) and were inoculated intranasally with sialodacryoadenitis (SDA) virus. The course of the disease was compared with nonimmunosuppressed, SDA virus-inoculated rats of the same stock. Criteria used to compare SDA in the 3 groups, included histologic changes in salivary and lacrimal glands, immunofluorescent microscopy of paraffin-embedded tissues, serum amylase levels, and antibody response. Based on these criteria, there was little detectable difference in the course and intensity of SDA in PR-treated and nonimmunosuppressed rats. In CY-treated rats, there was a delay in the onset of SDA, in the appearance of inflammatory cells in affected glands, and in the reparative process in the salivary and lacrimal glands. Viral antigen persisted longer in CY-treated rats than in PR-treated and nonimmunosuppressed rats. Antibody to SDA virus was not detected in CY-treated rats. The efficacy of immunosuppression by PR and CY was confirmed by the sheep erythrocyte agglutination procedure performed in selected rats. Male and female rats of the same strain were immunosuppressed beginning 4 weeks after inoculation with SDA virus to produce recrudescence of the disease. Histologic examination of salivary and lacrimal glands, immunofluorescent microscopy, serum amylase values, and viral isolation studies did not reveal evidence of reactivation of a persistent viral infection or viral shedding. Based on these studies, there is no evidence that SDA virus may persist as an inapparent infection after recovery from the disease.     

Sequential changes in the harderian and exorbital lacrimal glands in Wistar rats infected with sialodacryoadenitis virus

A sequential light and electron microscopic study of the exorbital and Harderian lacrimal glands was done on 2.5- to 15-month-old Wistar rats exposed to sialodacryoadenitis (SDA) virus. Typical coronaviral particles were readily demonstrated in cytoplasmic vesicles of Harderian and exorbital glands examined at 6 days post-inoculation. Lesions were seen in a relatively high percentage of lacrimal glands in infected animals of all ages, with no obvious age-related variations in the incidence and extent of changes. Lesions frequently persisted for a longer interval post-exposure in lacrimal glands than in salivary glands. The persistence of lesions commonly seen in Harderian glands was attributed, at least in part, to the cytotoxic effects of porphyrin-containing secretions released during the acute necrotizing stages of the disease. The persistence of lesions in some lacrimal glands indicates that they are useful tissues for microscopic examination for the retrospective provisional diagnosis of SDA. Persistent lesions also indicate that normal functions of these glands may be compromised for up to several weeks following outbreaks of SDA.     

Coronavirus infection in the laboratory rat: immunization trials using attenuated virus replicated in L-2 cells.

Sixty-nine specific pathogen-free male Wistar rats approximately eight weeks of age were used to evaluate the efficacy of an attentuated strain of sialodacryoadenitis (SDA) virus in providing protection against infection on subsequent challenge with virulent SDA virus. Fifty-four animals were inoculated intranasally with approximately 10(3.5) median cell culture infectious doses of the 25th passage of SDA virus in L-2 cells. Randomly-selected vaccinated animals were killed in order to evaluate the safety and efficacy of attenuated virus by histopathological examination of the salivary glands, lacrimal glands, and lower respiratory tract, and titration of sera for antibody to SDA virus. At three months and six months postvaccination (pv), animals were selected at random and challenged with virulent SDA virus. Seronegative, age-matched animals were also challenged, and served as controls. In animals examined at six to ten days pv, lesions were absent in submandibular and parotid salivary glands and lacrimal glands, but transient lesions were present in major airways of the lower respiratory tract. In a comparison of the incidence and extent of lesions, and antibody titers in challenged vaccinates and seronegative controls, lesions were minimal or absent in vaccinates compared to challenged naive rats, particularly in animals inoculated at three months pv. In addition, antibody titers in challenged vaccinates were much higher than were postinoculation titers in inoculated controls. In a comparison of lesions in salivary and lacrimal glands in vaccinated and control animals challenged at six months pv, there was a significant reduction in the number of animals without lesions in the vaccinated group (p = less than 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Coronavirus infections in the laboratory rat: degree of cross protection following immunization with a heterologous strain.

One hundred and twenty-one specific pathogen-free male Wistar rats eight to ten weeks of age were used to evaluate the efficacy of Parker's rat coronavirus (PRC) in affording cross protection on subsequent challenge with virulent sialodacryoadenitis (SDA) virus. Sixty-two animals were inoculated intranasally on day 0 and 21 days later with approximately 10(2) median tissue culture infective doses (TCID50) of the tenth passage of PRC replicated in L-2 cells. Animals were selected at random postvaccination to evaluate the safety and efficacy of PRC by histopathology, immunohistochemistry and serology. At three and six months postvaccination (PV), vaccinated and seronegative control rats were inoculated intranasally with approximately 10(3) TCID50 doses of virulent SDA virus. Challenged rats were then killed at 6, 10 and 14 days postchallenge and necropsied. Evaluations were based on lesion indices in lacrimal and salivary glands and respiratory tract, the presence of viral antigen by immunohistochemistry, and antibody response. Lesions were observed in rats killed PV, but in general, they were significantly reduced compared with those present in seronegative animals post-exposure to virulent SDA virus (p < or = 0.05). However, they were still considered to be an unacceptable level for a routine vaccination procedure. Potvaccination antibody titers to rat coronavirus were evident in all animals tested at three or six months prior to challenge with SDA virus.(ABSTRACT TRUNCATED AT 250 WORDS)     

An enzyme-linked immunosorbent assay for the detection of antibody against avian influenza virus

An enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibody to type A avian influenza (AI) virus. The sensitivity and group specificity of the AI-ELISA were compared with those of the agar-gel-precipitin test (AGPT) and the hemagglutination-inhibition (HI) test under conditions of both controlled and field exposure. During the course of temporal experimental infection (0-76 days) of specific-pathogen-free (SPF) chickens with AI subtype Hav9N2, the AI-ELISA was able to detect specific AI antibody as early as 8 days postinoculation (PI), and it measured rising levels of antibody through 35 days PI, at which time the chickens were re-exposed to AI virus. Conversely, AGP tests were negative through 35 days PI, and HI tests began to detect low levels of AI antibody only at 21 days PI. Following a secondary infection at 35 days PI with the same AI subtype, all tests measured rising levels of AI-specific antibody (35-76 days PI). However, the AGP test was positive at only the 7- and 14-day samplings postsecondary immunization. Under field conditions, the AI-ELISA was able to detect serum AI antibody in flocks from which highly pathogenic AI was isolated, but the AGP tests of these sera were negative.     

Gallid-1 herpesvirus infection in the chicken. 3. Reinvestigation of the pathogenesis of infectious laryngotracheitis in acute and early post-acute respiratory disease

Specific-pathogen-free chickens were infected via the trachea when 4 weeks old with 2000 plaque-forming units (PFU) of the virulent Australian infectious laryngotracheitis (ILT) virus strain CSW-1. Titers of ILT virus in the trachea were greatest (10(7.0) PFU/ml in washings, 10(6.0) PFU/g of tissue) 2-4 days postinfection (PI). Infectivity then declined rapidly, to become undetectable by 7 days PI, although highly localized areas of ILT antigen in the tracheal epithelium were occasionally observed by fluorescent antibody staining at 7 and 8 days PI. Tracheal organ cultures established 7 and 8 days PI provided no evidence of latent ILT virus infection at this immediate post-acute stage of pathogenesis. ILT virus was not isolated from peripheral blood leukocytes or lymphoid organs (spleen, bursa, thymus). ILT virus was found in the trigeminal ganglia and/or brain in 14 of 36 chickens (40%) examined between 4 and 7 days after intratracheal inoculation, but it was not in these tissues in five chickens examined at 8 days PI. Virus was also detected at 6 days PI in the trigeminal ganglia in one of five chickens infected by the conjunctival route. These data indicate that the early pathogenesis of ILT (CSW-1) infection frequently involves the tissues of the nervous system. In acute ILT in 4-week-old chickens, interferon-alpha/beta activity was not detectable in serum or tracheal exudates within 14 days PI, but tracheal washings contained significant virus-neutralizing activity by 7 and 8 days PI. In 3-day-old chickens infected via the trachea with 200 PFU of ILT CSW-1, the clearance of ILT virus from the trachea was similar to that observed in 4-week-old chickens, but ILT virus spread systemically to the livers of 20% by 5-7 days PI.     

Experimental rabies in skunks: Immune response and salivary gland infection

Groups of striped skunks (Mephitis mephitis) were inoculated intramuscularly with graded doses of street rabies virus. At various intervals after inoculation, saliva and sera were tested for rabies virus and neutralizing antibodies, respectively. Skunks that developed rabies were killed in terminal stages of the disease and the following examinations were made: titers of virus and antibody in submandibular salivary glands and brain, extent of immunofluorescence in submandibular salivary glands, and histologic examination of various tissues.

Skunks that received inocula containing 4 × 10⁴ to 4 × 10⁵ mouse intracerebral lethal dose₅₀ (MICLD₅₀) had detectable serum neutralizing antibodies by 7–12 days postinoculation; however, most of the skunks that received lower doses (40 to 4 × 10³ MICLD₅₀) did not have detectable serum neutralizing antibodies until clinical signs began. In the salivary glands, slight and extensive immunofluorescence corresponded to high and low titers of tissue neutralizing antibody. Also low viral titers were associated with high tissue neutralizing antibody titers. There was a close correlation between viral titers in right and left submandibular salivary glands.

The results suggest that the immune response can impede the process of infection of the salivary glands resulting in lack of antigen or low amounts of antigen in this tissue. This could occur through interference with centrifugal neural transport of virus and/or neutralization of virus during transfer from neural elements to epithelial cells. Lack of infectious virus or low viral titers in salivary glands containing antigen and high levels of tissue neutralizing antibodies can be caused partly by postmortem virus neutralization (during viral titration).     

Pathogenesis of sialodacryoadenitis in gnotobiotic rats.

The pathogenesis of sialodacryoadenitis was studied in gnotobiotic CD rats inoculated intranasally with the causal virus. Virus replication was detected sequentially in the nasopharynx, tracheobronchial tree, cervical lymph nodes, submaxillary and parotid salivary glands, exorbital gland, and Harderian gland. Acute rhinitis appeared within 2 days after inoculation, and salivary glands had lesions in 4 days. Early changes in salivary and exorbital glands were characterized by necrosis of ductal epithelium, which rapidly progressed to widespread acinar necrosis, marked inflammation, edema and total effacement of glandular architecture. Harderian glands also had massive necrosis of tubuloalveolar units. Repair in all glands was characterized by marked squamous metaplasia of tubuloalveolar units. Repair in all glands was characterized by marked squamous metaplasia of ducts. Neutralizing and complement-fixing antibodies were detected in 7 days, and there was a concomitant decrease in tissue-virus titers. There was no detectable evidence for hematogenous spread of virus or for retrograde infection by way of major salivary ducts.     

Distribution of viral antigen and tissue lesions in persistent and acute infection with the homologous strain of noncytopathic bovine viral diarrhea virus.

Viral distribution and lesions were compared between calves born with persistent infection (PI) and calves acutely infected with the same bovine viral diarrhea virus (BVDV) isolate. Two PI calves from 1 dairy herd were necropsied. The PI viruses from these calves were isolated, characterized by sequencing, and found to be identical. This virus strain, designated BVDV2-RS886, was characterized as a noncytopathic (ncp) type 2 BVDV. To establish acute infections, BVDV2-RS886 was used to inoculate clinically healthy, seronegative calves which were 3 weeks to 3 months old. Nine calves received 10(6)-10(7) tissue culture infective dose of BVDV2-RS886 intranasally. Four additional age-matched animals served as noninfected controls. Infected calves were necropsied at 3, 6, 9, or 13 days postinoculation (dpi). Viral antigen was detected by immunohistochemistry in frozen sections, and lesions were evaluated in hematoxylin eosin-stained paraplast sections. In the PI calves, a wide distribution of viral antigen was found in all tissues and was not associated with lesions. In the acutely infected calves, viral antigen was widespread in lymphoid tissues at 6 dpi but had been mostly eliminated at 9 and 13 dpi. Depletion of lymphoid tissues was seen at 6, 9, and 13 dpi and repopulation at 9 and 13 dpi. In 1 of the calves at 13 dpi, severe arteritis was present in lymph nodes and myocardium. This comparison shows that an ncp BVDV strain that causes no lesions in PI animals is able to induce marked depletion of lymphoid tissues in calves with acute infection. Therefore, the failure to eliminate PI cattle from a herd causes problems not only in pregnant cattle but may also affect other age groups.     

Study of the persistence of Aujeszky's disease (pseudorabies) virus in peripheral blood mononuclear cells and tissues of experimentally infected pigs

The presence of Aujeszky's disease virus (ADV) in peripheral blood mononuclear cells (PBMC) and tissues of experimentally infected pigs was studied. Vaccinated and unvaccinated pigs were inoculated with different doses of Aujeszky's disease NIA-3 strain. Pigs were periodically bled and PBMC were used for virus isolation and PCR detection of virus. Tissues were obtained at the time of death (8 weeks post-inoculation) and used for ADV genome detection by PCR. ADV genome was amplified from PBMC during the acute phase of infection and, in some experimental groups, up to 38 days post-inoculation (PI). The virus was sporadically detected by virus isolation performed from PBMC. In neural tissues, ADV was constantly amplified from the trigeminal ganglia and the olfactory bulb of persistently infected pigs (euthanised 8 weeks PI). In other tissues, the viral genome was rarely detected in lymph nodes and tonsils, and, occasionally, in the bone marrow. Our results indicated that PBMC are not an appropriate source for detecting ADV persistence, since inconsistent results were obtained throughout the experiments. In neural tissues, the olfactory bulb turned out to be as important a target for ADV persistence as the trigeminal ganglia. Viral genome detection in the bone marrow indicated that this tissue may play a role in the establishment of a persistent infection.     

Distribution of the rabies virus in the central nervous system of naturally infected foxes]

Quantitative distribution of rabies virus and the character of immunofluorescence in various parts of the central nervous system [CNS] and salivary glands were investigated in 21 naturally infected foxes. A mean death period was recorded in mice infected with the virus obtained from various parts of the CNS and salivary glands of the investigated foxes. The highest average titer of the virus was found out in the salivary glands - 4.46 log MLD50 per 0.03 ml i. c. The average titer was 2.30 log in the cornu ammonis, 1.99 log in the lobus olifactorius, 1.80 log in the spinal cord of the sacro-lumbar region, 1.73 log in the cortex, 1.71 log in the medulla oblongata, and 1.64 log in the cerebellum. The highest intensity of specific fluorscence was recorded in the thalamus, lobus piriformis, and cornu ammonis. In these regions, numerous round fluorescent inclusion bodies similar to Babes-Negri bodies occurred. Babes-Negri bodies in the cornu ammonis of foxes were proved in 81% of cases. Mice infected with the virus obtained from the salivary glands showed the shortest mean death period - 12.2 days and from the cornu ammonis it was 14.6 days. In virus infection from the other parts of the CNS the mean death period ranged from 16.0 to 17.4 days.     

Ultrastructural features of lesions in bronchiolar epithelium in induced respiratory syncytial virus pneumonia of calves

Ultrastructural changes were observed in bronchioles in acute and repair stages of respiratory syncytial virus pneumonia induced in eight young calves (calf Nos. 1-8) using a bovine strain of respiratory syncytial virus. Five of the calves were Friesians and three were Hereford x Friesians and all were male. Tissues from three mock-infected control calves (two Friesian, one Hereford x Friesian) were also examined. Calves were from 3 to 6 days old at the time of first inoculation, with the exception of calf No. 8, which was 2 weeks old. In the acute stage of the induced pneumonia, evidence of respiratory syncytial virus replication and release was demonstrable in both ciliated and non-ciliated bronchiolar epithelial cells, with the virus-releasing process most obvious at 4 and 5 days after infection. Respiratory syncytial virus infection of bronchiolar epithelium was associated with various changes, including hypertrophy, hyperplasia, and formation of syncytia. Necrosis of epithelial cell structures usually appeared to be preceded by their desquamation from bronchiolar walls. Respiratory syncytial virus infection resulted in considerable damage to the bronchiolar ciliary apparatus. Such damage was seen as early as 1 day post-infection and was still obvious at 10 days post-infection. Neutrophils were closely associated with respiratory syncytial virus infected epithelial cells and evidence of neutrophil fusion with infected epithelial cells was seen. These observations suggest that neutrophils may be involved in killing respiratory syncytial virus infected cells and that neutrophils might play an important role in early antiviral defense against respiratory syncytial virus at a time when antibody levels are low and other cellular defenses are not fully in play. Bronchiolar repair was evident from 6 days after infection and was well advanced at 10 and 13 days after infection.     

Studies on the efficacy of intranasal vaccination for the prevention of experimentally induced parainfluenza type 3 virus pneumonia in calves.

The efficacy of intranasal vaccination in preventing or limiting disease of the lower respiratory tract induced by parainfluenza 3 (PI3) virus was evaluated under experimental conditions, using a commercially available live vaccine containing a temperature-sensitive strain of PI3 virus. In a preliminary study four colostrum-deprived calves were vaccinated intranasally at one week and again at two months of age, and two similar calves were given an intranasal placebo. After the second vaccination serum antibodies to PI3 virus were detected in all four vaccinated calves, but not in the control animals. Seventeen days after the second vaccination all six calves were challenged with virulent PI3 virus, and they were killed six days later. The clinical scores and the extent of pulmonary consolidation were reduced in the vaccinated animals; PI3 virus was detected in the upper and lower respiratory tract of the control calves but in none of the vaccinated calves. In a larger scale study with 14 colostrum-fed calves, seven were vaccinated at one week and again at five weeks of age, and seven were given an intranasal placebo. Two weeks after the second vaccination all 14 calves were challenged with virulent PI3 virus. The clinical scores and lung consolidation were significantly reduced in the vaccinated calves in comparison with the controls. Six days after infection, 10 of the 14 calves were killed; PI3 virus was detectable in the nasal secretions of all seven control calves but in only one of the vaccinated animals, and PI3 viral antigen was detected in the lungs of the control calves but not in those of the vaccinated animals. One of the vaccinated calves had developed a severe clinical response after the challenge, but it had only minor lung consolidation when killed.     

Exacerbation of murine respiratory mycoplasmosis by sialodacryoadenitis virus infection in gnotobiotic F344 rats

To test the hypothesis that sialodacryoadenitis virus infection could exacerbate respiratory mycoplasmosis in rats, four groups of 40 7- to 9-week-old gnotobiotic F344/N rats were given two intranasal inoculations 7 days apart: Mycoplasma pulmonis, then sialodacryoadenitis virus; M. pulmonis followed by sterile culture medium; medium initially, then virus; or two doses of medium. Immediately and 3, 5, 10, and 20 days after the second inoculation, the nasal passages, middle ears, larynges, tracheas, lungs, and salivary and lacrimal glands of four rats from each group were prepared for histologic examination, and the respiratory organs from four other rats were collected for quantitative culture of M. pulmonis and sialodacryoadenitis virus. To test statistically the effect of virus infection on mycoplasmosis lesions, we determined indices of the severity of respiratory tract lesions by subjective scoring. In rats given both organisms, indices of nasal and tracheal lesions were significantly (P less than 0.05) greater at 3 days and after than in rats given M. pulmonis alone, and middle ear, laryngeal, and lung lesion indices were significantly greater at 5 days and after. Rats given both mycoplasma and virus had significantly more mycoplasmal colony-forming units in the nasal passages at 3 days and after, and in the larynges, tracheas, and lungs at 10 and 20 days, than rats given only mycoplasma. These results show that sialodacryoadenitis virus infection can exacerbate respiratory mycoplasmosis in rats under experimental conditions; therefore, the virus probably also contributes to expression of naturally occurring mycoplasmosis.     

Immune dysfunction following infection with chicken anemia agent and infectious bursal disease virus. II. Alterations of in vitro lymphoproliferation and in vivo immune responses.

To determine the functional impact of alterations in lymphocyte concentrations and ratios following infection with chicken anemia agent (CAA) alone or in combination with infectious bursal disease virus (IBDV) on the immune system of young chickens, in vitro lymphoproliferation assays and in vivo responses to vaccination with several common viral agents were assessed at various time intervals post-inoculation (PI). Concanavalin A (Con A), phytohemagglutinin (PHA) and pokeweed mitogen (PWM) stimulation of splenic lymphocytes (SPL) collected from control birds could not be detected until 10-14 days PI. Infection with CAA was characterized by significantly higher PWM stimulation of SPL at 17 days PI and significantly lower PWM stimulation of peripheral blood lymphocytes (PBL) at 14 days PI, compared with uninfected controls. Concanavalin A and PWM stimulation of SPL was significantly increased in birds inoculated with IBDV alone. Lymphocytes harvested from birds inoculated simultaneously with CAA and IBDV had significantly lower responses. Effects on humoral and cell-mediated immunity following CAA and/or IBDV were determined by evaluating vaccination responses to Newcastle disease virus (NDV), fowl pox virus (FPV) and infectious laryngotracheitis virus (ILTV) during the acute phase of CAA infection (2 weeks PI). Vaccination of birds 2 weeks following CAA infection at 1 day of age resulted in decreased protection against NDV (85.7%) and ILTV (7.1%) challenge compared with protection rates in control birds (100% and 53.3% respectively). Infectious bursal disease virus infection was associated with decreased protection against NDV (60%) only. Concomitant infection at 1 day of age resulted in a greater reduction in NDV challenge protection (33.3%), slightly decreased FPV protection (87.5%), increased numbers of persistent FPV vaccination lesions and increased protection against ILTV challenge (71.4%). Vaccination of birds 2 weeks following CAA infection at 2 weeks of age resulted in slightly decreased NDV humoral antibody, development of persistent FPV vaccination lesions (17%) and increased immunity to ILTV challenge compared with control birds (83.3% vs. 66.7%). Chickens inoculated with IBDV alone displayed a more severe depression in NDV antibody titers and only a slight decrease in ILTV protection. Vaccination following concomitant infection at 2 weeks of age resulted in a higher percentage of FPV persistent vaccination lesions (39%) and greatly enhanced immunity to ILTV challenge (100%).     

Foot-and-mouth disease virus (FMDV) experimental infection: susceptibility and immune response of adult mice

Adult mice are susceptible to foot-and-mouth disease virus (FMDV) infection only under some experimental conditions. This paper report the results of pathogenesis studies on 4 different strains of mice (CF1, C3H, NIH-nude, BALB-c/J) infected with the cloned and uncloned 0(1)C strain of FMDV. High virus titers were detected in blood and pancreas 12-24 h after infection (p.i.); these persisted for up to 48 h p.i. in CF1 and BALB-c/J mice and 72 h p.i. in the two other mouse strains. Virus titers observed in other organs were lower than those found in blood. In pancreas, and occasionally in salivary glands, oropharynx, heart and testicles, viral antigen was detected by direct immunofluorescent assay. Circulating neutralizing antibodies appeared in CF1 and C3H mice at 72 and 96 h p.i. respectively, and their titers remained unchanged during the 30-day experimental period. Antibodies against viral infection-associated antigen (VIA) were detected for a shorter period. In animals irradiated with 1 LD 50 (total body irradiation), viremia persisted up to 14 days p.i. and a low antibody response was observed which began at the end of viremia. No differences in the response of mice to cloned or uncloned FMDV were observed.     

Preliminary studies of the development of Anaplasma marginale in salivary glands of adult, feeding Dermacentor andersoni ticks

On each day of feeding on susceptible calves, salivary glands obtained from groups of adult ticks that transmitted Anaplasma marginale were examined for A marginale colonies by use of light microscopy and transmission electron microscopy. On day 8 of feeding, salivary glands were examined, using fluorescein-labeled antibody and methyl green-pyronine stain. Use of fluorescein-labeled antibody consistently revealed small numbers of fluorescent foci in salivary gland acinar cells obtained from ticks that had fed for 8 days. Colonies of A marginale were seen by transmission electron microscopy only in salivary gland acini of male ticks; these colonies could not be identified, using light microscopy, in companion 1-micron plastic sections stained with Mallory stain. Methyl green-pyronine stain, used commonly to detect theilerial parasites in tick salivary glands, did not differentiate A marginale from cytoplasmic inclusions normally found in salivary gland acinar cells.