Hematological and Hematopoietic Analysis in Fish Toxicology-A Review.

Hematological analysis is commonly used to assess the physiological state of fish. It includes red blood cell parameters, white blood cell parameters, and the number of thrombocytes per blood volume unit. Hematological analysis is one of the basic tools (often accompanied by biochemical and histopathological analyses) to assess the influence of organic and inorganic substances on fish. It is, therefore, applicable in both ecotoxicology and pharmacotoxicology. The advantages of this research method are the lack of need for specialized laboratory equipment and low costs, and the limitations are the need for extensive experience among the personnel performing the tests. One of the recommended methods of supplementing routinely determined hematological parameters is assessing the cellular composition and activity of hematopoietic tissue. As there is very little scientific data available on the issue of the effects of xenobiotics on the cellular structure of fish head kidney hematopoietic tissue, filling this gap should be considered an urgent need. Therefore, we recommend conducting research with the simultaneous use of hematological and hematopoietic analysis as reliable and complementary methods of assessing the impact of toxic substances on fish.

Virulence analysis and antibiotic resistance of Klebsiella pneumoniae isolates from hospitalised patients in Poland.

Klebsiella pneumoniae (KP) is a nosocomial pathogen causing difficult-to-treat infections. The presence of virulence genes and antibiotic resistance of 109 KP isolates from hospitalized patients were investigated. Among them, 68.8% were multi-drug resistant (MDR) and 59.6% produced extended-spectrum beta-lactamases (ESBLs). Metallo-ß-lactamases (MBLs) were produced by 22% of isolates (mainly from anus), including 16.5% of isolates producing New Delhi metallo-ß-lactamase (NDM-1). The genes encoding adhesins (fimH-91.7%, mrkD-96.3%), enterobactin (entB-100%) and yersiniabactin (irp-1-88%) were frequently identified. The genes encoding salmochelin (iroD-9.2%, iroN-7.3%) and colibactin (clbA, clbB-0.9%) were identified rarely. Iron acquisition system-related kfu gene and wcaG gene involved in capsule production were identified in 6.4% and 11% of isolates, respectively. The rmpA gene associated with hypermucoviscosity was present in 6.4% of isolates. In 19.2% of isolates magA gene was detected, specific for K1 capsule serotype, while 22.9% of isolates showed K2 capsule serotype. The rmpA, iroD or iroN genes being diagnostic biomarkers for hypervirulent KP (hvKP) were detected in 16.5% of isolates. We found that 55.5% of hvKP were MDR and produced ESBLs, thus hospital KP isolates pose a serious threat to the healthcare system.

The prevalence of virulence determinants in methicillin-resistant Staphylococcus aureus isolated from different infections in hospitalized patients in Poland.

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for hard-to-treat infections. The presence of 19 virulence genes in 120 MRSA isolates obtained from hospitalized patients and genetic relationships of these isolates were investigated. The eno (100%) and ebps (93.3%) genes encoding laminin- and elastin binding proteins, respectively, were ubiquitous. Other adhesion genes: fib (77.5%), fnbB (41.6%), bbp (40.8%), cna (30.8%) encoding proteins binding fibrinogen, fibronectin, bone sialoprotein and collagen, respectively, and map/eap (62.5%), encoding Eap, were also frequent. The etB and etD genes, encoding exfoliative toxins, were present in 15.6% and 12.5% isolates, respectively. The splA, splE and sspA, encoding serine protease were detected in 100%, 70.8% and 94.2% isolates, respectively. The tst gene, encoding toxic shock syndrome toxin-1 was found in 75% isolates. The cna, map/eap and tst genes were the most common in wound isolates and much less common in blood isolates. We identified 45 different spa types, t003 (21.7%) and t008 (18.8%) being the most common. The t003 was the most frequent among isolates from the respiratory tract (35.5%), while t008 in blood isolates (40%). Identification of virulence factors of MRSA is important for evaluation of pathogen transmission rate and disease development.

Hematological and Hematopoietic Effects of Bactericidal Doses of Trans-Cinnamaldehyde and Thyme Oil on Cyprinus carpio Juveniles.

The effects of two potential antibacterial agents of plant origin: trans-cinnamaldehyde (TC) and thyme oil (TO) on the peripheral blood parameters and cellular composition of hematopoietic tissue of Cyprinus carpio were studied. Both phytochemicals were used in the doses based on the bactericidal concentrations against Aeromonas spp. developed earlier in in vitro study. The fish were fed for 2 weeks on a commercial feed supplemented with 10 µl/kg of TC or 20 µl/kg of TO. Groups TC1 and TO1 were fed diets containing phytochemicals daily, while groups TC2 and TO2 every 2 days. Control group and groups TC2 and TO2 on the remaining days were fed plain feed. Peripheral blood and head kidney hematopoietic tissue were sampled from all the fish at the end of the experiment. In all the groups, hematological values were within the reference ranges for the healthy common carp juveniles. However, blood hemoglobin (Hb) concentration and mean corpuscular hemoglobin concentration (MCHC) were significantly lower in all the groups exposed to TC and TO, while MCH in TC1, TO1, and TO2 compared to the control. TC and TO did not affect leukocyte count [white blood cell (WBC)], differential leukocyte count, the oxidative activity of phagocytes [nitroblue tetrazolium (NBT)], or thrombocyte count (Thro). No significant alterations were observed in the hematopoietic tissue. The results showed that TC and TO exhibited no considerable hematotoxic effects and trials of their use in the treatment of fish infected with Aeromonas spp. may be undertaken.

Effects of embryonic exposure to chromium (VI) on blood parameters and liver microstructure of 1-day-old chickens.

Hexavalent chromium (Cr(VI)) has carcinogenic, nephrotoxic, hepatotoxic, and neurotoxic effects. Exposure to Cr(VI) can also lead to hematological alterations and blood biochemical changes. The literature on Cr(VI) toxicity concerns mostly adult forms of vertebrates. In this study, an attempt was made to determine the effect on the developing chicken embryo of Cr(VI) in ovo administration. It was observed that chromium affected the hatchability of chicks in a dose-dependent manner. At a dose from 25 to 250 µg per egg, Cr(VI) resulted in a statistically significant reduction of hatchability. Chromium administrated at lower doses (1.56 and 2.5 µg per egg) caused a statistically insignificant increase of hatchability. However, chromium at a level of LD50 (15.6 µg per egg) or 1/10 LD50 (1.56 per egg) did not cause major changes in hematological parameters or plasma biochemical indices in newly hatched chicks. The same doses did not lead to any histopathological changes in the liver.

Effects of Oxytetracycline and Gentamicin Therapeutic Doses on Hematological, Biochemical and Hematopoietic Parameters in Cyprinus carpio Juveniles.

Hematological, biochemical and hematopoietic effects of therapeutic doses of two antibiotics, oxytetracycline (OTC) and gentamicin (GEN), in clinically healthy common carp juveniles were studied. The fish were divided into four groups: controls 1 and 2 (untreated or injected with 0.6% NaCl solution), and two groups treated with antibiotics (orally with 75 mg/kg OTC four times every two days or injected with a single dose (4 mg/kg) of GEN dissolved in 0.6% NaCl). Blood and head kidneys were sampled from all fish 3 days post-treatments for hematological, biochemical and hematopoietic tissue analyses. No major alterations in the values of hematological and serum biochemical parameters occurred following administration of OTC or GEN. Glucose concentrations were significantly lower in both groups of fish subjected to injections (Control 2 and GEN), while the oxidative metabolic activity of phagocytes increased in the antibiotic-treated groups (significantly in OTC). More alterations were observed in hematopoietic tissue. Immunocytochemical analysis revealed that G caused a significant increase in the rate of cell proliferation (PCNA-positive cells) and an increase in the frequency of apoptotic cells (caspase-positive). The frequency of lymphoid lineage decreased, which was related to a decrease in the abundance of mature lymphocytes in GEN-treated fish. Percentages of neutrophilic lineage were significantly elevated in OTC and GEN groups compared to controls. The obtained results showed no considerable hematotoxicity or hepatotoxicity of therapeutic doses of OTC and GEN to carp.

Effect of manuka honey on biofilm-associated genes expression during methicillin-resistant Staphylococcus aureus biofilm formation.

Methicillin-resistant Staphylococcus aureus (MRSA) are among the most important biofilm-forming pathogens responsible for hard-to-treat infections. Looking for alternatives to antibiotics that prevent biofilm formation, we investigated the effects of manuka honey on the transcriptional profile of genes essential for staphylococcal biofilm formation using qRT-PCR. mRNA from two hospital MRSA strains (strong and weak biofilm producer) were isolated after 4, 8, 12 and 24 h from cells grown in biofilm. Manuka honey at 1/2 minimum biofilm inhibition concentration (MBIC) significantly reduced MRSA cell viability in biofilm. Manuka honey downregulated the genes encoding laminin- (eno), elastin- (ebps) and fibrinogen binding protein (fib), and icaA and icaD involved in biosynthesis of polysaccharide intercellular adhesin in both weakly and strongly adhering strain compared to the control (untreated biofilm). Expression levels of cna (collagen binding protein) and map/eap (extracellular adherence protein-Eap) were reduced in weakly adhering strain. The lowest expression of investigated genes was observed after 12 h of manuka honey treatment at 1/2 MBIC. This study showed that the previously unknown mechanism of manuka honey action involved inhibition of S. aureus adhesion due to reduction in expression of crucial genes associated with staphylococcal biofilm.

Antibacterials in Aquatic Environment and Their Toxicity to Fish.

Antibacterial agents are commonly present in aquatic environment at low concentrations. Terrestrial animal farms, human medicine and aquaculture are main sources of water contamination with antibacterials. Antibiotics were proved to be directly toxic to fish causing oxidative stress, general stress response, histopathological lesions, hematological, metabolic, and reproductive disorders, as well as immunosuppressive and genotoxic effects. Environmentally realistic low concentrations of antibiotics also disturb aquatic bacterial communities causing alterations in fish symbiotic microbiota and induce emergence of antibiotic-resistant pathogenic bacteria by exerting selective pressure on spread of antibiotic-resistance genes.

Blood biomarkers of herbicide, insecticide, and fungicide toxicity to fish-a review.

Pesticides are widely used in the world agriculture, and they may adversely affect non-target organisms, including fish. The present 2000-2019 literature review summarizes hematological and blood biochemical effects of various herbicides, insecticides, and fungicides in fish. The observed changes usually indicate anemia and inflammation, as well as hyperglycemia, hypoproteinemia, increase in cortisol concentration and activities of hepatic aminotransferases that are typical for intoxication and stress. Other changes that are also sometimes observed such as increase in red blood parameters indicate compensatory response. The often-noted symptoms of immunosuppression show an adverse effect of pesticides on immune system and possible immunosuppression. Pathophysiological changes in fish induced by pesticides depend on many factors, such as active compound and its concentration, exposure duration, fish species, environmental conditions, etc. Hematological and blood biochemical parameters appear to be useful biomarkers for evaluation of physiological state of fish exposed to pesticides; however, they are not specific markers of intoxication.

Effect of trans-Cinnamaldehyde on Methicillin-Resistant Staphylococcus aureus Biofilm Formation: Metabolic Activity Assessment and Analysis of the Biofilm-Associated Genes Expression.

The effects of trans-cinnamaldehyde (TC) on transcriptional profiles of biofilm-associated genes and the metabolic activity of two methicillin-resistant Staphylococcus aureus (MRSA) strains showing a different degree of adherence to polystyrene, were evaluated. Metabolic activity of S. aureus in biofilm was significantly decreased in the presence of TC at 1/2 minimum biofilm inhibition concentration (MBIC). Expression levels of the genes encoding laminin binding protein (eno), elastin binding protein (ebps) and fibrinogen binding protein (fib) in the presence of TC at 1/2 MBIC were lower than in untreated biofilm in both the weakly and strongly adhering strain. The highest decrease of expression level was observed in case of fib in the strongly adhering strain, in which the amount of fib transcript was 10-fold lower compared to biofilm without TC. In the presence of TC at 1/2 MBIC after 3, 6, 8 and 12 h, the expression level of icaA and icaD, that are involved in the biosynthesis of polysaccharide intercellular adhesin, was above half lower in the weakly adhering strain compared to biofilm without TC. In the strongly adhering strain the highest decrease in expression of these genes was observed after 3 and 6 h. This study showed that TC is a promising anti-biofilm agent for use in MRSA biofilm-related infections.

Antibacterial Activity of Commercial Phytochemicals against Aeromonas Species Isolated from Fish.

Antimicrobial activities of phytochemicals-trans-cinnamaldehyde (TC), ferulic acid (FA), p-coumaric acid (p-CA), caffeic acid (CA), chlorogenic acid (CHA), Thymus vulgaris essential oil (TO), Eugenia caryophyllus essential oil (ECO), and Melaleuca alternifolia oil (TTO) against Aeromonas species-were assessed. Growth of all Aeromonas salmonicida subsp. salmonicida and almost all Aeromonas sobria strains was inhibited by TC at concentration 0.01 mg/mL, and for most Aeromonas hydrophila strains minimal inhibitory concentrations (MIC) ranged from 0.01 to 0.19 mg/mL. The inhibitory effect of TC against A. salmonicida subsp. salmonicida was comparable to the effect of oxytetracycline, and in the case of A. salmonicida subsp. salmonicida and A. sobria was higher compared to gentamicin. MIC of FA, p-CA, and CA for most strains ranged from 1.56 to 3.12 mg/mL, and MIC values of TO for most strains ranged from 0.39 to 0.78 mg/mL. TO and TC at the concentrations below ½ MIC values used in mixtures exhibited strong synergism. ECO and TC showed synergy in mixture of ⅛ MIC of ECO and » MIC of TC. TC and TO exhibited the strongest inhibitory and bactericidal effect against investigated Aeromonas species, and they are a promising alternative to the use of antibiotics in controlling the growth of these fish pathogens.

Exposure to herbicide linuron results in alterations in hematological profile and stress biomarkers of common carp (Cyprinus carpio).

Phenylurea herbicides such as linuron are commonly applied in agriculture. Common carp juveniles were subjected to 31.5 µg/L of linuron for 14 days, and then to 30 days of purification. Peripheral blood was sampled after 1, 3, 7 and 14 days of exposure and 7, 14 and 30 days of purification and hematological parameters were evaluated: erythrocyte (RBCc) and leukocyte (WBCc) counts, hematocrit (Ht), hemoglobin concentration (Hb), mean cell volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and differential leukocyte count. For evaluation of cortisol and catecholamine concentrations blood was sampled after 3, 6 and 12 h, after 1, 3 and 14 days of exposure, and after 30 days of purification. Linuron caused mainly transient increase in RBCc, Ht and MCV values and increase in WBCc and percentage of juvenile neutrophils. The herbicide caused persistant increase of cortisol and catecholamine concentrations. The results indicate that exposure to low concentration of linuron induced a stress response in common carp.

Haematological effects of 2-phenoxyethanol and etomidate in carp (Cyprinus carpio L.).

OBJECTIVE: To evaluate the effects of handling alone versus handling under anaesthesia with 2-phenoxyethanol or etomidate on haematological parameters in carp. STUDY DESIGN: Prospective, randomized, laboratory experiment. ANIMALS: Seventy-two juvenile carp (Cyprinus carpio) weighing 35.9 ± 10.4 g were divided into six groups of 12 fish. METHODS: Either 2-phenoxyethanol or 2% etomidate were administered to induce deep anaesthesia (0.3 mL L(-1) and 0.6 mL L(-1) , respectively) or deep sedation (0.15 mL L(-1) and 0.3 mL L(-1) , respectively). Fish were handled with and without sedation. Blood was sampled at 1 hour and 1 week post-treatment. Phagocyte oxidative activity [nitrotetrazolium blue reduction test (NBT)] and differential erythrocyte [red blood cell (RBC)] and leukocyte (white blood cell) counts were evaluated. RESULTS: At 1 hour after the induction of anaesthesia, haematocrit (Ht) and haemoglobin (Hb) were increased in fish anaesthetized with 2-phenoxyethanol, and mean corpuscular haemoglobin (MCH) was increased in fish anaesthetized with etomidate. At 1 week, an increase in RBC, erythroblastosis, erythrocyte damage, lymphopenia, neutrophilia, monocytosis and thrombocytosis occurred in both groups. Red blood parameters did not change 1 hour after handling alone, but after 1 week Ht, Hb and mean cell volume decreased, whereas MCH concentration (MCHC) and abnormal erythrocytes increased. Lymphopenia, neutrophilia, monocytosis, thrombocytosis and a decrease in NBT occurred. Fish handled under sedation showed an increase in Hb and MCHC followed by a decrease at 1 week in Ht, Hb and MCH, erythroblastosis and increased abnormal erythrocytes. Lymphopenia and neutrophilia were less pronounced than in fish handled without sedation, but a decrease in NBT was noted at 1 week post-treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Deep anaesthesia with 2-phenoxyethanol or etomidate induced significant haematological alterations in juvenile carp. Deep sedation reduced the immediate immunosuppressive effects of handling but did not eliminate longterm effects. These anaesthetics should be avoided during experimental procedures involving haematological measurements. In contexts that require the short-term handling of carp, these drugs should be used with caution in view of their possible side effects.

The effects of cadmium and copper on embryonic and larval development of ide Leuciscus idus L.

The effects of Cd and Cu on embryos and larvae of the ide Leuciscus idus were evaluated. The ide is an European cyprinid fish, natural populations of which tend to decrease. The ide is also used as a bioindicator organism to evaluate water quality. However, sensitivity of ide early developmental stages to heavy metal intoxication is not known. Fish were exposed to Cd or Cu (100 µg/L) during embryonic, larval or both developmental periods. Survival of the embryos, time of hatching, size and quality of newly hatched larvae were evaluated at the end of embryonic period. Correctly developed larvae from the control and Cd or Cu-exposed groups were transferred to clean water, Cd or Cu solutions (100 µg/L) immediately after hatching. Larval development was observed, and the larvae were photographed. Time of yolk sac resorption, onset of active feeding and swim bladder inflation were evaluated, and the measurements were done on body and swim bladder size. The results showed that exposure of embryos to Cd and Cu significantly reduced embryonic survival and increased frequency of body malformations and death in newly hatched larvae and delayed hatching. Exposure to Cd and Cu during larval period reduced larval survival, growth and delayed development (yolk utilization, beginning of active feeding and swim bladder inflation). Cadmium was more toxic to the ide embryos and larvae than copper. Exposures to metals during embryonic period alone caused adverse impact on larval performance even when larval development took place in clean water. However, exposure of embryos to Cu reduced toxic impact of metal on larvae in continuous Cu exposure compared to the non-preexposed fish, but no such an effect occurred in case of Cd exposure. The results show that even a short-term exposure to Cd or Cu during early development of ide may adversely affect recruitment of this species. Among the measured endpoints, quality of newly hatched larvae (frequency of body malformations and larvae dead immediately after hatching) and swim bladder size were the most sensitive to intoxication with both metals. Embryos were more sensitive to Cu intoxication than larvae, while in case of Cd, sensitivity of both stages was similar.

Cadmium and copper reduce hematopoietic potential in common carp (Cyprinus carpio L.) head kidney.

The effects of cadmium and copper on activity of common carp head kidney hematopoietic tissue were evaluated. The fish were subjected to short-term (3 h, Cd-s and Cu-s) or long-term (4 weeks, Cd-l and Cu-l) exposures to 100% 96hLC50 or 10% 96hLC50, respectively. Head kidneys were isolated weekly from 5 fish of each group for 4 weeks (post-short-term exposure and during long-term exposure). Percentage of early blast cells among the hematopoietic precursors was calculated. Proliferative and apoptotic activity were evaluated using immunocytochemical staining for proliferating cell nuclear antigen (PCNA) and caspase 3, respectively. Hematopoietic activity was calculated as the ratio of proliferating to apoptotic cells. All metal exposures induced an increase in frequency of early blast cells. The frequency of proliferating (PCNA-positive) cells also significantly increased. A considerable and significant increase in the frequency of apoptotic cells was the most pronounced effect of metal exposures. Both short-term and long-term treatments caused similar effects, but in case of Cd exposures, the reaction was more pronounced. All metal exposures reduced hematopoietic potential of fish measured as the ratio of proliferating to apoptotic precursor cell frequency. However, in all cases, hematopoietic activity was higher than 1 showing that the rate of repair of hematopoietic tissue prevailed over destruction.

The effects of heavy metals on embryonic development of fish (a review).

Early developmental stages of fish are particularly sensitive to water pollution. Heavy metals may affect various developmental processes during the embryonic period, which results in a reduction of offspring quantity and quality. Waterborne metals may accumulate in the gonads of spawners and adversely affect gamete production and viability, or exert direct toxic influence upon developing embryos. The egg shell does not fully protect the embryo against metal penetration, particularly during the swelling phase; thus, metals may accumulate in the egg. The results depend on metal concentration and range from developmental disturbances to death of the embryo. Metals disturb various processes of fish embryonic development and affect the development rate. Early stages just after fertilization are particularly sensitive to metal intoxication, when most disturbances and the highest embryonic mortality occur. Waterborne metals also promote developmental anomalies during organogenesis, including body malformations. Heavy metals often induce a delay in the hatching process, premature hatching, deformations and death of newly hatched larvae. All these disturbances result in reduced numbers and poor quality of the larvae, which show small body size, high frequency of malformations and reduced viability.

The effects of anticoagulants on hematological indices and blood cell morphology of common carp (Cyprinus carpio L.).

Hematological parameters (Ht, Hb, RBC, WBC, PLT), erythrocyte size, and osmotic fragility, differential leukocyte count, ROS production in common carp blood collected on three anticoagulants: heparin (10 IU/mL, Na2EDTA (0.1, 0.5, and 1 mg/mL), and sodium citrate (0.3 mg/mL) were compared. Na2EDTA caused partial blood hemolysis in Ht tubes which made Ht measurement impossible, and resulted in high variability of the results. Both, citrate and Na2EDTA increased sensitivity of red blood cells to hemolysis. Na2EDTA also induced erythrocyte anisocytosis and anisonucleosis. Na2EDTA significantly increased ROS production but no effect of anticoagulants on WBC, PLT or differential leukocyte count was observed. The obtained results show that Na2EDTA should not be used for evaluation of red blood cell parameters and erythrocyte morphology, and for ROS production measurement in common carp. Heparin proved to be the most appropriate anticoagulant to use for this species, although Na2EDTA and sodium citrate may be used for WBC and leukocyte differential count evaluations.

The effects of heavy metals on common carp white blood cells in vitro.

The in vitro effects of cadmium, copper, lead and zinc, and various cadmium compounds (chloride, sulphate and nitrate) on common carp (Cyprinus carpio) lymphocyte viability and phagocyte activity, were evaluated. The percentage of dead lymphocytes was determined after Trypan blue staining, and phagocyte activity was measured by using the nitroblue tetrazolium (NBT) reduction test. Lead was the most toxic to lymphocytes--the maximum mortality exceeded 30%, and was significantly higher at 1 microM of lead, compared to the control. The maximum mortality caused by cadmium was below 10%, but was significantly elevated with 5 microM or more of cadmium. Zinc induced lymphocyte mortality from 10 microM, whilst no effect was observed with copper. The incubation of full blood with the three cadmium compounds (at 5mg/l of cadmium) showed that cadmium nitrate and cadmium sulphate were more toxic (over 35% and 25% mortality, respectively) than cadmium chloride (about 15% mortality). This was confirmed by the results of tests on isolated cells--1mg/l of cadmium as nitrate and sulphate increased lymphocyte mortality compared to the control and cadmium chloride. Phagocytic activity was less sensitive to heavy metals than was lymphocyte viability. It was significantly reduced following exposure to 50 microM and 100 microM cadmium, and 100 microM zinc, but no effects were observed with either lead or copper.

The changes in common carp blood after short-term zinc exposure.

Blood zinc level, hematological parameters and blood cell morphology were evaluated in common carp immediately after 3 h exposure to 20 mg dm(-3) of zinc (Zn0), and in 24, 48 and 96 hours after the end of it (Zn24, Zn48, Zn96). Blood zinc level in the non-exposed fish was 8 mg dm(-3), reached a maximum of 20 mg dm(-3) in Zn48, while it dropped to 9 mg dm(-3) in Zn96. Zinc caused a stress reaction in fish indicated by an increase in hematocrit value in Zn0, and elevated plasma glucose level and trombocytosis which persisted until the end of the experiment. Zinc-exposed fish showed an increased frequency of abnormal erythrocytes, and a compensatory release of immature erythrocytes to the blood stream. In zinc-treated fish, leukocyte count initially increased and subsequently decreased significantly below the control level due to a drop in lymphocyte number. Lymphocyte viability was reduced, and abnormal lymphocytes appeared. A decreased count of juvenile neutrophiles, and reduced phagocyte activity also occurred. The results indicate possible zinc-induced disturbances in both specific and non-specific immune mechanisms.